CA2183992A1 - Method and reagent for inhibiting the expression of disease related genes - Google Patents

Abstract

Enzymatic RNA molecules which cleave ICAM-? mRNA, IL-5 mRNA, re? A mRNA, TNF-.alpha. mRNA, RSV mRNA or RSV genomic RNA, or CML associated mRNA, and use of these molecules for the treatment of pathological conditions related to those mRNA-levels; ribonucleosides or nucleotides modified in 2', 3' or 5', methods for their synthesis, purification and deprotection vectors containing multiple enzymatic nucleic acids, optionally in chimeric form with tRNAs; method for introducing enzymatic nucleic acids into cells by forming a complex with a second nucleic acid where the complex is capable of taking an R-loop base-paired structure: method for altering a mutant nucleic acid in vivo by hybridization with an oligonucleotide capable of activating dsRNA deaminase, comprising an enzymatic activity or a chemical mutagen. Further are disclosed trans-cleaving or -ligating hairpin ribozymes lacking a substrate RNA moiety, as well as hammerhead ribozymes having an interconnecting loop between base pairs in stem II.

Description

WO 95123225 2 1 ~ 3 g 9 ~ 156 METHOD AND REAGENT FOR INHIBITING THE EXPRES~ION
OF DISEASE RELATED GENES
BackQrQund Qf the Invention This invention relates to reagents useful as inhibitors of gene expression relatir1g to diseases such as i"~lal"",dl~ry or autoimmune disorders, chronic myelogenous leukemia, or respiratory tract iilness.
SummAry of the Invention The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting the ~ s:,iun of disease related genes, ~sL ICAM-1, IL-5, relA, TNF-a, p210 bcr-abl, and respiratory syncytial virus genes. Such ribozymes can be used in a method for 10 treatment of diseases caused by the ~X~ SSiO11 of these genes in man and other animals, including other primates.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be 15 targeted to virtually any RNA transcript, and efficient cleavage has been achieved In vitro. Kim et al., 84 Proc. Natl. ~t!A~ Sci. U~P~ 8788, 1987;
Haselofl and Gerlach, 334 ~¦~L~ 585, 1988; Cech, 260 JAMA 3030, 1988;
and Jefferies et al., 17 Nucleic ~irl~ RQcr~Arch 1371, 1989.
Six basic varieties of naturally-occurring enzymatic RNAs are known 20 presently. Each can catalyze the hydrolysis of RNA pl1o~l1oJit~ l bonds in trans (and thus can cleave other RNA molecules) under physiological concliIions. Table 1 summarizes some of the cha,d.;I~ ,li,s of these ribozymes.
Ribozymes act by first binding to a target RNA. Such binding occurs 25 through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first r~oy~ es and then binds a target RNA
through c~"",lel"~"ldly base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a 30 target RNA will destroy its ability to direct synthesis of an encoded protein.
After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

r~ 156 WO 95123225 2 ~ 8 3 9 9 2 The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its Lldl1sldliul~) sincethe effective col1ce,,l,dliol~ of ribozyme necessary to effect a therapeutic 5 treatment is lower than that of an antisense oligonucleotide. The advantage reflects the ability of the rlbozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.
In addition, the ribozyme is a highly specific inhibitor, with the speclficity of inhibition d~,uell-li"g not only on the base pairing "~e~,l,d~ ", of blnding, 1û but also on the r"eul~d"i:,"~ by which the molecule inhibits the ~ ,iu,l of the RNA to which it binds. That Is, the inhibition is caused by cleavage of the RNA target and so speclflcity is defined as the ration of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage ",ecl~dl)is", is dependent upon factors additional to 15 those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antlsense oligonucleotide binding the same RNA site. With their catalytic activity and increased site specificity, rlbozymes represent more potent and safe therapeutic molecules than antisense oligorlllrl~ûti~ec Thus, in a first aspect, this invention relates to tibozymes, or enzymatic RNA molecules, directed to cleave RNA species encoding ICAM-1, IL-5, relA, TNF-a, p210bCr-abl, or RSV proteins. In particular, applicant describes the selection and function of ribozymes capable of cleaving these RNAs and their use to reduce levels of ICAM-1, IL-5, relA, TNF-a, p210 bor-abl or RSV proteins In various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostlc uses.
Applicant indicates that these ribozymes are able to inhibit ~A,ul~ iull of ICAM-1, IL-5, rel A, TNF-a, p210bCr-abl, or RSV genes and that the catalytic activity of the ribozymes is required for their inhibitory effect.
Those of ordinary skill in the art, will find that it is clear from the examplesdescribed that other ribozymes that cleave target ICAM-1, IL-5, rel A, TNF-a, p210bCr-abl, or RSV encoding mRNAs may be readlly designed and are within the invention.
These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to ~ ,es~;l,le regions of their target mRNAs. The RNA molecules also contain domains that catalyze the , ~ woss/2322s 2183992 r~1/~ 5l~-156 cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing lldllaldliull and protein accumulation. In the absence of the eXI~ aSiOI~ of the target gene, a therapeutic eflect may be observed.
By ~gene~ is meant to refer to either the protein coding regions of the5 cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.
By "enzymatic RNA molecule" it is meant an RNA molecule which has 10 ~",,ul~",~"ldrily in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to :~,ueui~ lly cleave RNA in that target. That is, the enzymatic RNA molecule is able to i"~..",oldcularly cleave RNA and thereby inactivate a target RNA molecule.
This complementarity functions to allow sufficient hyL,Iidi~dlioll of the 15 enzymatic RNA molecule to the target RNA to allow the cleavage to occur.
One hundred percent cu",,ul~ ldrity is preferred, but Culll,ult~ llldlily as low as 50-75% may also be useful in this invention. By "equivalent" RNA to a virus is meant to include those naturally occurring viral encoded RNA
molecules ~csoc;~ d with viral caused diseases in various animals, 20 including humans, cats, simians, and other primates. These viral or viral-encoded RNAs have similar stnuctures and equivalent genes to each other.
By "~o",,ul~",~"ldlily" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for examplke, I looy~ c", type) of base-25 paired i~lelduliùlls.
In preferred ~,llb~di",~lll of this invention, the enzymatic nucleic acid molecule is fommed in a 11d"""~,1,ead or hairpin motif, but may also be fommed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA
(in ~Cso~ on with an RNA guide sequence) or Neurospora VS RNA.
30 Examples of such lld,,,,,,ell,ead motifs are described by Rossi et al., 1992,Aids ~esearch and Human ~etroviruses, 8,183, of hairpin motifs by Hampel and Tritz, 1989 EJioo,ll~-"i~lr~. 28, 4929, EP 0300257 and Hampel et al., 1990, Nucleic Acids ~es. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry. 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell, 35 849, W0 95123225 - - ~ r~ ls6 2183~2 cleavage of RNA. Upon binding, the ribozymes cleave the target encoding mRNAs, preventing l,dl1~1dtiu,, and protein accumulation. In the absence of the ~A,U~ liUll of the target gene, a therapeutic effect may be observed.
By "gene~ is meant to refer to either the protein coding regions of the5 cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.
By "enzymatic RNA molecule" it is meant an RNA molecule which has 10 CUIll,ultlll~llldlily in a substrate binding region to a specified gene targetl and also has an enzymatic activity which is active to :,,ueui~k,~lly cleave RNA in that target. That is, the enzymatic RNA molecule is able to i,~lt"",olecularly cleave RNA and thereby inactivate a target RNA molecule.
This colll~ llalily functions to allow sufficient ll~s,li~ dtiul~ of the 15 enzymatic RNA molecule to the target RNA to allow the cleavage to occur.
One hundred percent culllpltllllelllLdlily is preferred, but culllpl~ l,;a,ily as low as 50-75% may also be useful in this invention. By "equivaient" RNA to a virus is meant to include those naturally occurring viral encoded RNA
molecules associdlt,d with viral caused diseases in various animals, 2û including humans, cats, simians, and other primates. These viral or viral-encoded RNAs have similar structures and equivalent genes to each other.
By "col"plal"t"ldrity" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for examplke, I loou~luc:,, type) of base-25 paired illLuld.,liul~s.
In preferred t""L,o ii",~"ts of this invention, the enzymatic nucleicacid molecule is fommed in a l,d"""ell,ead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA
(in ~c5ou;~ with an RNA guide sequence) or Nevrospora VS RNA.
30 Examples of such l,a"""t"l,ead motifs are described by Rossi etal., 1992, Aids Resr~rch and Hum~n Retroviruses, 8,183, of hairpin n~otifs by Hampel and Tritz, 1989 ~io.,llt~",i~ . 28, 4929, EP 0360257 and Hampel et al., 1990, Nucleic AC`;'IC fles. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 B~ocll~ ;".r~. 31 35 16 of the RNaseP motif by Guerrier-Takada et al., 1983 ~IL 35 84g, .. . . .

~ g ~ S (~ G ~ ~ /G 6) wo 9s/232~s . . j P~ 56 218~3~g~

expressed in eukaryotic cells from the d,~Jpl~J,Ulid~t: DNA or RNA vector. The activity of such ribozymes can be augmented by their release from t~,e primary transcript by a second ribozyme (Draper et al., PCT W093/23569, and Sullivan et al., PCT W094/û2595, both hereby i,,~,oluordl~d in their 5 totality by reference herein; Ohkawa, J., et al., 1992, Nuclelc Acids Symp.
~G 27, 15-6; Taira, K. et al., Nucleic Acids Res.. 19, 5125-30; Ventura, M., et al., 1993, Nucleic Acids Res.. 21, 3249-55, Chowrira et al., 1994 J. Biol.
~h~m,,. 269, 25856 ).
By "inhibit" is meant that the activity or level of ICAM-1,Rel A, IL-5, TNF-a, p210bCr-abl or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of ICAM-1, IL-5, Rel A, TNF-o~, p210bCr-abl or RSV
protein or activity in a cell or tissue. By "related" is meant that the inhibition of ICAM-1, IL-5, Rei A, TNF-o~, p210bCr-abl or RSV mRNA lldilaldliUIl, and thus reduction in the level of, ICAM-1, IL-5, Rel A, TNF-c~, p210bCr-abl or RSV proteins will relieve to some extent the symptoms of the disease or condition .
Ribozymes are added directly, or can be c~",plt"~ed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.
The RNA or RNA COll~,ultX~S can be local!y ddllli~ d to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. In preferred ~"~bodi"~e:"~, the ribozymes have binding arms which are co"~ "~el~ldry to the sequences in Tables

2,3,6-9, 11 j 13, 15-23, 27, 28, 31, 33, 34, 36 and 37.
Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding amms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

WO 95123225 218 3 9 ~ 2 PCT~IB9S/00156 Those in the art will reco3nize that these sequences are s~"l~ /e only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity.
For example, stem-loop ll sequence of hammerhead ribozymes listed in 5 the above identified Tables can be altered (s~h.ctitllti~ln, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can fomm. Similarly, stem-loop IV sequence of hairpin ribozymes listed in the above identified Tables can be altered (s~h~titl~ti~n, deletion, and/or insertion) to contain any sequence, provided a minimum of 10 two base-paired stem structure can form. The sequence listed in the above identified Tables may be formed of ribonucleotides or other nllCleQticles or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
In another aspect of the invention, ribozymes that cleave target 15 molecules and inhibit ICAM-1, IL-5, Rel A, TNF-~, p210bCr-abl or RSV
gene eX,ult:aSiOIl are expressed from Lldllsc,i,ulio" units inserted into DNA, RNA, or viral vectors. Another means of accumulating high cullc~,,l,dlions of a ribozyme(s) within cells is to i"~r,uoldt~ the ribozyme-encoding sequences into a DNA or RNA ~X,~ sioll vector. Tldllsc,i~Jliull of the 20 ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol 1), RNA polymerase ll (pol ll), or RNA polymerase lll (pol lll). Transcripts from pol ll or pol lll promoters will be expressed at high levels in all cells; the levels of a given pol ll promoter in a given cell type will depend on the nature of the gene regulatory sequences (~IIlldl~
25 silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the ap~,.,y,i~ . cells (Elroy-Stein and Moss, 1990 Proc. Natl.
Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 NucleicAcids Res., 21 2867-72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 30 1990 Mol. Cell. Biol., 10, 4529-37). Several investigators have d~lllol1:,L,dl~d that ribozymes expressed from such promoters can function in l"ar"",alial1 cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 90, 6340-4;
L'Huiller et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl.
35 Acad. Sci. U.S.A., 90 8000-4). The above ribozyme lldllS~,I, ' .1 units can be i,,cu,~uld~d into a variety of vectors for introduction into Illdllllll~
cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors ... _ _ _ , . .. . .. ... ... ..... . ... _ . . _, ~ WO 95/23225 21~ 3 9 9 2 r~ 156 (such as adenovirus or adeno-associal~d virus vectors), or viral RNA
vectors (such as retroviral or alphavirus vectors).
Other features and advantages of the invention will be apparent from the following desb,i~liol~ of the preferred ~:"ILo~;",t",l~ thereof, and from 5 the claims Descri~tion Of The Preferred ~illb~
The drawings will first briefly be described.
Drawings:
Figure 1 is a didyldllllllali~ pl~s~,"ldliol~ of the hammerhead 10 ribozyme domain known in the art. Stem ll can be 2 2 base-pair long.
Figure 2(a) is a diay,d"""ali-, Ic:,ultl~,~llldlioll of the l~a"""e,l,edd ribozyme domain known in the art; Figure 2(b) is a ~idyld~llldlic pl~S~llldl;ol~ of the lld"""e,l,ead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 15 2(c) is a similar diagram showing the 11d"""erl,ead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2(d) is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. ~es., 17, 1371-1371) into two portions.
Figure 3 is a diayldlllllldli~ ali~l~ of the general structure of a 20 hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., nis 1,2,3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is 21 base).
Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4-20 25 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' ;"d~l,el~d~"lly is any normal or modified base and each dash l~ s~ a potential base-pairing i"l~ld.lion. These nucleotides may be modified at the sugar, base or pl,o~ dle. Complete base-pairing is not required in the helices, but is 30 preferred. Helix 1 and 4 can be of any size (i.e., o and p is each illd~ ld~lllly from 0 to any number, e.g. 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be WO 95~23225 2 1 ~ 3 9 9,,,2 PCIIIB9S/00156 ..

modified chemically (abasic, base, sugar andlor plloapl~dtu Illodi~;~dliol~s) or replaced with another base without significant effect. Helix 4 can be fommed from two separate molecules, i.e., without a cu""e-;li"g loop. The c~ ,euli"9 loop when present may be a ribonucleotide with or without 5 Illodiri ;dliolls to its base, sugar or plloapl,dl~. q is ~ 2 bases. The con"e ili"g loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases.
refers to a covalent bond.
Figure 4 is a l~,urusu,,~dli~l~ of the general structure of the hepatitis 10 delta virus ribozyme domain known in the art.
Figure 5 is a ruplua~llld~iul~ of the general structure of the self-cleaving VS RNA ribozyme domain.
Figure 6 is a didyld~ Id~i u ~uplua~ d~iull of the genetic map of RSV
strain A2.
Figure 7 is a diaÇ,ldlllllldliC representation of the solid-phase synthesis of RNA.
Figure 8 is a diau,d"""alic representation of exocyclic amino protecting groups for nucleic acid synthesis.
Figure 9 is a diauldlllllld~i-i ru,uluaullldliuil of the du,~Jlut .~.~iUII of RNA.
Figure 10 is a graphical ~u,u~uau~dlioll of the cleavage of an RNA
substrate by ribozymes synthesized, d~ lu~u~ud and purified using the improved methods described herein.
Figure 11 is a schematic lu,uluaull~dliull of a two pot dùplulu,liol7 protocol. Base d~,ululuu~iull is carried out with aqueous methyl amine at 65 C for 10 min. The sample is dried in a speed-vac for 2-24 hours du,uull.li,lg on the scale of RNA synthesis. Silyl protecting group at the 2-hydroxyl position is removed by treating the sample with 1.4 M anhydrous HF at 65C for 1.5 hours.
Figure 12 is a schûmatic ~u,u~USUll:.~ ;ull of a one pot deu,u~u ;~iui, of 3û RNA s~ l,esi ed using RNA phosphoramidite chemistry. Anhydrous methyl amine is used to deprotect bases at 65C for 15 min. The sample is allowed to cool for 10 min before adding TEA-3HF reagent, to the same ~ WO951232~S 2 1 8 3 9 9 2 1 ~1 ~S~,. 156 pot, to remove protecting groups at the 2'-hydroxyl position. The d~plutcluLiull is carried out for 1.5 hours.
Figs. 13a - b is a HPLC profile of a 36 nt long ribozyme, targeted to site B. The RNA is d~urul~L~d using either the two pot or the one pot 5 d~plul~l,liull protocol. The peaks corresponding to full-length RNA is indicated. The sequence for site B is CCUGGGCCAGGGAUUA
AUGGAGAUGCCCACU .
Figure 14 is a graph cu",l.a~i"g RNA cleavage activity of ribozymes d~,ululeul~d by two pot vs one pot dep,ul~,~Liun protocols.
Figure 15 is a schematic representation of an improved method of synthesizing RNA containing phosphorothioate linkages.
Figure 16 shows RNA cleavage reaction catalyzed by ribozymes containing phosphorothioate linkages. I Id"""ell,ead ribozyme targeted to site C is synthesized such that 4 nts at the 5' end contain phosphorothioate 15 linkages. P=O refers to ribozyme without pllo~,ul,orull,iodl~ linkages. P=S
refers to ribozyme with pllo~,ul lo,ull liOdl~ linkages. The sequence for site Cis UCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG.
Figure 17 is a schematic Itl,ul~s~llldliull of synthesis of 2'-N-phtalimido-nucleoside phosphoramidite.
Figure 18 is a diayldlllllld~ n~s~llldliOl~ of a prior art method for the solid-phase synthesis of RNA using silyl ethers, and the method of this invention using SEM as a 2'-protecting group.
Figure 19 is a ~lidyldlllllldtic l~pl~se"ldliu" of the synthesis of 2 -SEM-protected rlll~leosi~l~c and pllo~,ul~ord,"idil~s useful for the synthesis of RNA. B is any nucleotide base as ~x.d"l,l.';'i~d in the Figure, P is purine and I is inosine. Standard abbreviations are used throughout this :,, ' ' :l, well known to those in the art.
Figure 20 is a diagrammatic It:pl~s~,,ldliol, of a prior art method for deprotection of RNA using TBDMS protection of the 2'-hydroxyl group.
Figure 21 is a didyldlllllldli~ ,ul~s~llldliul~ of the dtl,~lul~uliull of RNA
having SEM protection of the 2'-hydroxyl group.
SUBSTITUTE SHEET (~ULE 26) WO 95/23225 ~ r ~ ls6 21839~2 Figure 22 is a ~t.pltes~llldliol~ of an HPLC ChlUllldlOyldlll of a fully d~ u~ d 1 0-mer of uridylic acid.
Figs. 23 - 25 are d;dyldlllllld~ic l~ s~llldlio"rj of hammerhead, hairpin or hepatitis delta virus ribozyme cu" ,i"g self-processing RNA
transcript. Solid arrows indicate self-p,-,~,a~s;"g sites. Boxes indicate the sites of nucleotide sllhctitlltion~ Solid lines are drawn to show the binding sites of primers used in a primer-extension assay. Lower case letters indicate vector sequence present in the RNA when l,ansc,;Led from a Hindlll li.,edli~ad plasmid ~23) HH Cassette, transcript cu"l_i.,;"g the hammerhead trans-acting ribozyme linked to a 3' cis-acting lld,,,,,,ell,ead ribozyme. The structure of the hammerhead ribozyme is based on pllyl~ and mutational analysis (reviewed by Symons, 1992 ~).
The trans ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (UC at positions 50 and 51) at its 3' end. The 3' p~u~as~il)g ribozyme is co",,uri~ad of r-~lcleoti~s 44 through 96. Roman numerals 1, ll and lll, indicate the three helices that contribute to the structure of the 3' cis-actinglla"""ellledd ribozyme (Hertel et al., 1992 Nucleic ~ q Res. 20, 3252).
Substitution of G70 and A71 to U and G respectively, inactivates the ~ld"""~,l,aad ribozyme (Ruffner et al., 1990 Bio~ e",i~lry 29, 10695) and generates the HH(mutant) construct. (24) HP Cassette, transcript containing the hd"""~,l,ead trans-acting ribozyme linked to a 3' cis-acting hairpin ribozyme. The structure of the hairpin ribozyme is based on phylogenetic and mutational analysis (Berz~l Ha"d"~ et al., 1993 FMBO. J
12, 2567). The trans-ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 5 non-ribozyme rlllrl~tid~s (UGGCA at positions 50 to 54) at its 3' end. The 3' cis-acting ribozyme is comprised of n~lrleoti~s 50 through 115. The transcript named HP(GU) was constructed with a potential wobble base pair between Gs2 and U77; HP(GC) has a Watson-Crick base pair between Gs2 and C77. A shortened heli% 1 (5 base pairs) and a stable tetraloop (GAAA) at the and of helix 1 was used to connect the substrate with the catalytic domain of the hairpin ribozyme (Feldstein & Bruening, 1993 Nucleic Acids Res. 21, 1991; Altschuler et al., 1992 su~ra). (25) HDV
Cassette, transcript containing the trans-acting hammerhead ribozyme linked to a 3' cis-acting hepatitis delta virus (HDV) ribozyme. The secondary structure of the HDV ribozyme is as proposed by Been and . _, . . .... , .. _ . . .. _ . . _ _ _ WO 9S123225 = ~ PCTIIB95/00156 218~992 coworkers (Been et al., 1992 Bi~ Ily 31, 11843). The trans-ribozyme domain extends from r~lrleoti~es 1 through 48. After 3'-end p,uce~i"g, the trans-ribozyme contains 2 non-ribozyme nllcleoti~s (AA at positions 49 to 50) at its 3' end. The 3' cis-acting HDV ribozyme is col",u,i~ed of 5 rlll.-leoticles 5û through 114. Roman numerals 1, I!, lll & IV, indicate the Iocation of four helices within the 3' cis-acting HDV ribozyme (Perrota &
Been, 1991 Nature 35û, 434). The ~HDV transcript contains a 31 nr~cleotide deletion in the HDV portion of the transcript (nucleotides 84 through 115 deleted).
1û Fig. 26 is a schematic ,~pl~s~"IdIion of a plasmid containing the insert encoding self-~,uc~i,lg cassette. The figure is not drawn to scale.
Fig. 27 d~",ol1aI,dI~:s the effect of 3' flanking sequences on RNA self-processing in vitro. H, Plasmid templates linearized with Hindlll restriction enzyme. Transcripts from H I~r"~.ld~c,s contain four non-ribozyme 15 nucleotides at the 3' end, N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from N templates contain 220 non-ribozyme r~ leoti~ss at the 3' end. R, Plasmid templates linearized with Rcal restriction enzyme. Transcripts from R templates contain 450 non-ribozyme n~cl~qotide~ at the 3' end.
Fig. 28 shows the effect of 3' flanking sequences on the trans-cleavage reaction catalyzed by a hammerhead ribozyme. A 622 nt internally-labeled RNA (<1û nM) was incubated with ribozyme (1û0û nM) under single turn-over conditions (Herschlag and Cech, 199û Bio~ "~i~Iry 29, 1û159). HH+2, HH+37, and HH+52 are trans-acting ribozymes produced by lldll::>ClipliUII from the HH, ~HDV, and HH(mutant) constructs, respectively, and that contain 2, 37 and 52 extra n~ otirJes on the 3' end.
The plot of the fraction of uncleaved substrate versus time was fit to a double exponential curve using the KaleidaGraph graphing program (Synergy Software, Reading, PA). A double exponential curve fit was used - 3û because the data points did not fall on a single exponential curve, presumably due to varying cu,,~ul,,,~,~ of ribozyme and/or substrate RNA.
Fig. 29 shows RNA self-j ,uc~s~ g in OST7-1 cells. In vitro lanes contain full-length, r~llp,uuessed Ildns~,,i,uI~ that were added to cellular Iysates prior to RNA extraction. These RNAs were either pre-incubated with MgCI2 (+) or with DEPC-treated water (-) prior to being hybridized WO 95/23225 ~ ~ 8 ~ ~1 9 ~ r ~ ~ SG

with 5' end-labeled primers. Cellular lanes contain total cellular RNA from cells lld~ d with one of the four self-p~ucessi~g constructs. Cellular RNA are probed for ribozyme ~,u~:ssion using a sequence specific primer-extension assay. Solid arrows indicate the location of primer extension 5 bands co"~pul,di,lg to Full-Length RNA and 3' Cleavage Products.
Figs. 30,31 are ~lidyld~ ldIi~ l~,ult,sellIdIiulls of self-p,uc~s:~i"g cassettes that will release trans-acting ribozymes with defined, stable stem-loop structures at the 5' and the 3' end following self-p,vc~s~i,,g. 30, shows various permutations of a 11a"""erl,edd self-p,ùces~illg cassette. 31, 10 shows various permutations of a hairpin self-p~uces~i"g cassette.
Figs. 32a-b Schematic l~pl~st~llIdIio~l of RNA polymerse lll promoter structure. Arrow indicates the Ildllsc,i~Iiol~ start site and the direction of coding region. A, B and C, refer to consensus A, B and C box promoter sequences. I, refers to i"'~.",e.lidl~ cis-acting promoter sequence. PSE, 15 refers to proximal sequence element. DSE, refers to distal sequence element. ATF, refers to activating Ildllsu,il.Iiui~ factor binding element. ?, refers to cis-acting sequence element that has not been fully ~,Ilald~ d.
EBER, Epstein-Barr-virus-encoded-RNA. TATA is a box well known in the art.
Figs. 33a-e Sequence of the primary tRNAjmet and ~3-5 transcripts.
The A and B box are intemal promoter regions necessary for pol lll Ird~ . Arrows indicate the sites of endogenous tRNA p~u,~s~ g~
The ~3-5 transcript is a truncated version of tRNA wherein the sequence 3' of B box has been deleted (Adeniyi-Jones et al., 1984 supra). This IIIOdiliCdliUI~ renders the ~ 3-5 RNA resistant to endogenous tRNA
processing.
Figure 34. Schematic It~pl~s~llIdIiull of RNA stnuctural motifs inserted into the ~3-5 RNA. ~3-5/HHI- a hammerhead (HHI) ribozyme was cloned at the 3' region of ~3-5 RNA; S3- a stable stem-loop structure was i"c~"Joldl~d at the 3' end of the ~3-5/HHI chimera; S5- stable stem-loop structures were i,,~,ur~,oldl~d at the 5' and the 3' ends of ~3-5/HHI ribozyme chimera; S35- sequence at the 3' end of the ~3-5/HHI ribozyme chimera was altered to enable duplex formation between the 5' end and a culllpl~ llIdly 3' region of the same RNA; S35Plus- in addition to stnuctural alterations of S35, sequences were altered to facilitate additional SUESTITUTE SHEET (RULE 26) _ _ _ .. _ . , , .... .. . . , . _, , . . ,, . . ,, . ,, . , _ _ .

duplex formation within the non-ribozyme sequence of the ~3-5/HHI
chimera.
Figures 35 and 36. Northern analysis to quantitate ribozyme expression in T cell lines transduced with ~3-5 vectors. 35) ~3-5/HHI and its variants were cloned individually into the DC retroviral vector (Sullenger et al., 1990 supra). Northern analysis of ribozyme chimeras expressed in MT-2 cells was performed. Total RNA was isolated from cells (Chomczynski & Sacchi, 1987 Analytical Biochemistry 162, 156-159), and transduced with various constructs described in Fig. 34. Northern analysis was carried out using standard protocols (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). Nomenclature is same as in Figure 34. This assay measures the level of expression from the type 2 pol lll promoter. 36) Expression of S35 constructs in MT2 cells. S35 ~+ribozyme), S35 construct containing HHI ribozyme. S35 (-ribozyme), S35 construct containing no ribozyme.
Figure 37. Ribozyme activity in total RNA extracted from transduced MT-2 cells. Total RNA was isolated from cells transduced with ~3-5 constructs described in Figs. 35 and 36 In a standard ribozyme cleavage reaction, 5 ~g total RNA and trace amounts of 5' terminus-labeled ribozyme target RNA were denatured separately by heating to 90C for 2 min in the presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCI2. RNAs were renatured by cooling the reaction mixture to 37C for 10-15 min. Cleavage reaction was initiated by mixing the labeled substrate RNA and total cellular RNA at 37C. The reaction was allowed to proceed for ~ 18h, following which the samples were resolved on a 20 % urea-polyacrylamide gel. Bands were visualized by autoradiography.
Figures 38 and 39. Ribozyme expression and activity levels in S35-transduced clonal CEM cell lines. 38) Northern analysis of S35-transduced clonal CEM cell lines. Standard curve was generated by - 30 spiking known concentrations of in vitro transcribed S5 RNA into total cellular RNA isolated from non-transduced CEM cells. Pool, contains RNA
from pooled cells transduced with S35 construct. Pool (-G418 for 3 Mo), contains RNA from pooled cells that were initially selected for resistance to G418 and then grown in the absence of G418 for 3 months. Lanes A
through N contain RNA from individual clones that were generated from the pooled cells transduced with S35 construct. tRNAjmet, refers to the WO 95/23225 218 3 9 9 2 PCT/IB95/00156 ~

endogenous tRNA. S35, refers to the position of the ribozyme band. M, marker lane. 39) Activity levels in S35-transduced clonal CEM cell lines.
RNA isolation and cleavage reactions were as described in Fig.37.
N~i"t:l~cldlure is same as in Figs. 35 and 36 except, S, 5' terminus-labeled 5 substrate RNA. P, 8 nt 5' terminus-labeled ribozyme-mediated RNA
cleavage product.
Figures 40 and 41 are proposed secondary stnuctures of S35 and S35 containing a desired RNA (HHI), respectively. The position of HHI
ribozyme is indicated in figure 41. I,,~ld,,,ol~lar stem refers to the stem 10 structure fommed due to an i"lld"~olecular base-paired interaction between the 3' sequence and the ~,u~ le~e~d~y 5' terminus. The length of the stem ranges from 15-16 base-pairs. Location of the A and the B boxes are shown.
Figures 42 and 43 are proposed secondary structures of S35 plus 15 and S35 plus containing HHI ribozyme.
Figures 44, 45, 46 and 47 are the nucleotide base sequences of S35, HHIS35, S35 Plus, and HHIS35 Plus respectively.
Figs. 48a-b is a general fonmula for pol lll RNA of this invention.
Figure 49 is a diyldlllllldli~, r~p~e:sell~d~iol1 of 5T constnuct. In this 20 construct the desired RNA is located 3' of the i"~ld",ol~cular stem.
Figures 50 and 51 contain proposed secondary structures of 5T
construct alone and 5T contruct containing a desired RNA (HHI ribozyme) respectively.
Figure 52 is a didyldlllllld~iC Itl,Ul~:S~ d~iull of TPZ-tRNA chimeras.
25 The site of desired RNA insertion is indicated.
Figure 53 shows the general structure of HHITRZ-A ribozyme chimera.
A hammerhead ribozyme targeted to site I is inserted into the stem ll region of TRZ-tRNA chimera.
Figure 54 shows the general structure of HPITRZ-A ribozyme chimera.
30 A hairpin ribozyme targeted to site I is cloned into the indicated region of TRZ-tRNA chimera.
SUBSTITUTE SHEET (RULE 26) w0 9sl2322s 2 1 8 ~ 9 ~ 2 ~ .sc .

Figure 55 shows a co""~a~ l of RNA cleavage activity of HHITRZ-A, HHITRZ-B and a chemically synthesized HHI l-d"l",~,l,ead ribozymes.
Figure 56 shows ~ 5ioll of ribozymes in T cell lines that are stably transduced with viral vectors. M, markers; lane 1, non-transduced CEM
5 cells; lanes 2 and 3, MT2 and CEM cells transduced with retroviral vectors;
lanes 4 and 5, MT2 and CEM cells transduced with AAV vectors.
Figs. 57a-b Schematic diagram of adeno-~.ccof:i~t~d virus and adenovirues vectors for ribozyme delivery. Both vectors utilize one or more ribozyme encoding Lldlls,,,i,uli."l units (RZ) based on RNA polymerase ll or 10 RNA polymerase lll promoters. A. Diagram of an AAV-based vector containing minimal AAV sequences ~ ri~ y the inverted terminal repeats (ITR) at each end of the vector genome, an optional selectable marker (Neo) driven by an exogenous promoter (Pro), a ribozyme ~IdllS.;li,UliUI1 unit, and sufficient additional sequences (stuffer) to maintain a5 vector length suitable for efficient packaging. B. Diagram of ribozyme ing adenovirus vectors containing deletions of one or more wild type adenoviorus coding regions (cross-hatched boxes marked as E1, plX, E3, and E4), and insertion of the ribozyme lldlls..,i,uti.,,, unit at any or several of those regions of deletions.
Fig. 58 is a graph showing the effect of arm length variation on the activity of ligated lld"""t"l,ead (HH) ribozymes. Nomenclature 5/5, 616, 7/7, 8/8 and so on refers to the number of base-pairs being fommed between the ribozyme and the target. For example, 5/8 means that the HH ribozyme forms 5 bp on the 5' side and 8 bp on the 3' side of the cleavage site for a total of 13 bp. -~G refers to the free energy of binding calculated for base-paired illl~ld~iliUI~s between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Ann. Rev. Biophys. Chem. 17, 167). RPI A is a HH
ribozyme with 6/6 binding amms.
Figs. 59 and 60 and 61 show cleavage of long substrate (622 nt) by ligated HH ribozymes.
Fig. 62 is a diayldlllllld~iC l~ s~"ldliul) of a lla"""e~l~edd ribozyme (HH-H) targeted against a site termed H. Variants of HH-H are also shown that contain either a 2 base-paired stem ll (HH-H1 and HH-H2) or a 3 base-paired stem ll (HH-H3 and HH-H4).
SUBSTITUTE SHEET (R~LE 26) WO 95123225 2 1 8 3 9 ~ 2 P~l,,., :/~ 156 Figs. 63 and 64 show RNA cleavage activity of HH-I and its variants (see Fig.62). 63) cleavage of matched substrate RNA (15 nt). 64) cleavage of long substrate RNA (613 nt).
Figs. 65a-b is a schematic rt~ul~tnlldli~l1 of a method of this invention 5 to synthesize a full length hairpin ribozyme. No splint strand is required forligation but rather the two fragments hybridize together at helix 4 prior to ligation. The only prerequisite is that the 3 fragment is phosphorylated at its 5' end and that the 3 end of the 5 fragment have a hydroxyl group. The hairpin ribozyme is targeted against site J. H1 and H2 are intermolecular 10 helices fommed between the ribozyme and the substrate. H3 and H4 are i"~,d",ole~ular helices formed within the hairpin ribozyme motif. Arrow indicates the cleavage site.
Fig. 66 shows RNA cleavage activity of ligated hairpin ribozymes targeted against site J.
Figs. 67a-b is a diayldrllllldliC l~Ultl~tllltdliU~I of a Site K Hairpin Ribozyme (HP-K) showing the proposed secondary structure of the hairpin ribozyme substrate complex as described in the art (Berzal H~; Idl 1~ et al., 1993 EMBO. J.12, 2567). The ribozyme has been ass~",bled from two fragments (bimolecular ribozyme; Chowrira and Burke 1992 Nucleic Acids Res. 20 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent i"l,~",ol~cular helix fonmation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme). Left panel (HP-K1) indicates 4 base-paired helix 2 and the right panel (HP-K2) indicates 6 base-paired helix 2.
Arrow indicates the site of RNA cleavage. All the ribozymes discussed herein were chemically sy"ll~esi~d by solid phase synthesis using RNA
phosphoramadite chemistry, unless otherwise indicated. Those skilled in the art will recognize that these ribozymes could also be made l,d,,s.:,i,uliol,a:ly in vitro and in vivo.
Figure 68 is a graph showing RNA cleavage by hairpin ribozymes targeted to site K. A plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-K2 (6 bp helix 2) cleaves a 422 target RNA to a greater extent than the HP-K1 (4 bp helix 2).
SUBSTITUTE SHEET (RULE 26) WO 95123225 2 1 8 3 9 3 2 PCTllB951001s6 To make illLulll~lly labeled substrate RNA for trans-ribozyme cleavage reactions, a 422 nt region (containing hairpin site A) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was lldllsc,iL,ed in a standard lldlls."i,uliu" buffer in the presence of [a-32P]CTP (Chowrira &
Burke, 1991 supra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chlulu~ullll:iaOdlllyl alcohol (25:1), pr~ with isup,u,l,d"ol and washed with 7û% ethanol. The dried pellet was resuspended in 2û
1 û DEPC-treated water and stored at -2ûC.
Unlabeled ribozyme (1,uM) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris HCI pH 7.5 and 1û mM MgCI2) by heating to 9ûC for 2 min. and slow cooling to 37C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37C. Aliquots of 5 ,ul were taken at regular time intervals, quenched by adding an equal volume of 2X ~u"~d"~i~e gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Pl1o~,uho,l,,,agel (Molecular Dynamics, Sunnyvale, CA).
Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary stnucture of the hairpin ribozyme-substrate complex. The ribozyme was dss~",~led from two fragments as described above. The nu" ,~l ,cldlure is the same as above.
Figure 7û shows RNA cleavage by hairpin ribozymes targeted to site L. A. plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-L2 (6 bp helix 2) cleaves a 2 KB target RNA
to a greater extent than the HP-L1 (4 bp helix 2). To make internally-30 labeled substrate RNA for tran~ribozyme cleavage reactions, a 2 kB region (containing hairpin site L) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. The cleavage reactions were carried out as described above.
SUBSTITUTE SHEET (RULE 26) WO 9S/23225 2 1 8 3 9 ~ 2 ~ 156 Figs. 71 a-b shows a Site M Hairpin Ribozyme (HP-M) with the proposed secondary structure of the hairpin ribozyme-substrate complex.
The ribozyme was assembled from two fragments as described above.
Figure 72 is a graph showing RNA cleavage by hairpin ribozymes 5 targeted to site M. The ribozymes were tested at both 20C and at 26C.
To make intennally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 1.9 KB region (containing hairpin site M) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Cleavage reactions were canried out as described 10 above except that 20C and at 26C temperatures were used.
Figs. 73a-d shows various structural " lodiiicdtiul ,s of the present invention. A) Hairpin ribozyme lacking helix 5. N~""~ ;ldIure is same as described under figure 3. B) Hairpin ribozyme lacking helix 4 and helix 5.
Helix 4 is replaced by a nucleotide loop wherein q is 2 2 bases.
15 Nolll~llclal~re is same as described under figure 3. C) Hairpin ribozyme lacking helix 5. Helix 4 loop is replaced by a linker 103"L", wherein L is a non-nucleotide linker molecule (Benseler et al., 1993 J. Am. Chem. Soc, 115, 8483; Jennings et al., WO 94/13688). r~vlllt~ re is same as described under figure 3. D) Hairpin ribozyme lacking helix 4 and helix 5.
20 Helix 4 is replaced by non-nucleotide linker molecuie "L" (Benseler et al., 1993 supra; Jennings et aL, supra). N~.l"~".ildl~re is same as described under figure 3.
Figs. 74a-b shows Hairpin ribozymes containing nucleotide spacer region "s" at the indicated location, wherein s is 2 1 base. Hairpin 25 ribozymes containing spacer region, can be synthesized as one fragment or can be a~e~ d from multiple fragments. Nomenclature is same as described under figure 3.
Figs. 75a-e shows the structures of the 5'-C-alkyl-modified rlll~lP~ti~lP.~ R1 is as defined above. R is OH, H, O-protecting group, NH, or 30 any group described by the pl 1' ' .15 discussed above, and those described below. B is as defined in the Figure or any other equivalent nucleotide base. CE is cyanoethyl, DMT is a standard blocking group.
Other abbreviations are standard in the art.
SUBSTITUTE SHEET (RULE 26) WO 95123225 2 1 8 3 9 9 ~ PCr/~B95100156 Figure 76 is a diayldll~ dLic l~,ult~s~llldliull of the synthesis of 5'-C-alkyl-D-allose n~cle~.ci~iPc. and their phosphoramidites.
Figure 77 is a diayldlllllldlk. I~pl~stlllld~ilJll of the synthesis of 5'-C-alkyl-L-talose rlll~:leoci~lpc and their pllo~ o,d" c - 5 Figure 78 is a ~idyldlllllldlk; I~,ult:st~ dli~ of hammerhead ribozymes targeted to site O containing 5'-C-methyl-L-talo Illodi~i..dliolls at various positions.
Figure 79 shows RNA cleavage activity of HH-O ribozymes. Fraction of target RNA uncleaved as a function of time is shown.
Figure 80 is a ~idyldlllllld~ r~"ldliu" of a position numbered lld"""erl,ead ribozyme (according to Hertel etal. NucleicAcids Res. 1992, 20, 3252) showing specific s~lhctitlltions.
Figs. 81 aj shows the structures of various 2'-alkyl modified rlllclPoti~ips which exemplify those of this invention. R groups are alkyl groups, Z is a protecting group.
Figure 82 is a diayldlllllldliC l~,,ul~s~"ldliu" of the synthesis of 2'-C-allyl uridine and cytidine.
Figure 83 is a ~idyl~lllllldli~ pl~5~ of the synthesis of 2'-C-methylene and 2'-C-difluoromethylene uridine.
Figure 84 is a didyldlllllldlic ,t~ s~"ldlion of the synthesis of 2'-C-methylene and 2'-Gdifluorul"t:ll"~lcne cytidine.
Figure 85 is a ~lidyldlllllldli~; l~pl~S~llldliOIl of the synthesis of 2-~
methylene and 2'-Gdifluoromethylene adenosine.
Figure 86 is a iiayldlllllldlic r~ St:llldliOIl of the synthesis of 2'-C-carboxymethylidine uridine, 2'-Gmethoxycarboxymethylidine uridine and derivatized amidites thereof. X is CH3 or alkyl as discussed above, or another substituent.
Figure 87 is a didyldlll~lldlil~ ltls~llldliul~ of a synthesis of nucleoside 5'-deoxy-5'-difluoromethylpho:".llondl~s.
SUESTITUTE SHEET (RULE ~6) WO95~23225 : r~l/~ s. -1!;6 21839~
Figure 88 is a diay,d"""dlic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylphos,ol1ol1dl~ 3'-phosphoramidites, dimers and solid supported dimers.
Figure 89 is a didyld~ alic representation of the synthesis of 5 nucleoside 5'-deoxy-5'-difluoromethylene triphosphates.
Figures 90 and 91 are didyldlllllldlic ~ sellldli.",s of the syrithesis of 3'-deoxy-3'-difluoromethyl,~llo~ ol~dLas and dimers.
Figure 92 is a schematic l~ s~l .l of sy~ ,e~i~i"y RNA
plloa~ ordllliL~ of a nucleotide containing a 2'-hydroxyl group 10 IllO.Iiri~dliol~ of the present invention.
Figs. 93a-b describes a method for d~ylu~cli~ of oligonucleotides containing a 2'-hydroxyl group modification of the present invention.
Figure 94 is a diayldlllllldLi~ s~ dli~n of a hammerhead ribozyme targeted to site N. Positions of 2'-hydroxyl group sllhstitl~ti~n is 1 5 indicated.
Figure 95 shows RNA cleavage activity of ribozymes containing a 2'-hydroxyl group Illo.liFi- dlioll of the present invention. All RNA represents l,d"""e~ ad ribozyme (HHN) with no 2'-hydroxyl group Illodi~;~dliolls. U7-ala ,t~ s~"ts HHN ribozyme containing 2'-NH-alanine l"o,' ' I at the 20 U7 position. U4/U7-ala ~ sel~L~ HHA containing 2'-NH-alanine ",- "' ,s at U4 and U7 positions. U4 Iys It~,lJl~S6'11~b HHA co"ld;"i"g 2'-NH-lysine Illol ' r~n at U4 position. U7 Iys l~ s~ HHA containing 2'-NH-lysine modification at U7 position. U4/U7-lys I~I,,t,s~"l~ HHN
containing 2'-NH-lysine Illo~'' I at U4 and U7 positions.
Figures 96 and 97 are schematic ,~pl~se"ldti.",s of sy"ll,e~i~i"g (so!id-phase synthesis) 3' ends of RNA with modification of the present invention. B, refers to either a base, modified base or an H.
Figure 98 and 99 are schematic l~pl~llLdli~,lls of synthesizing (solid-phase synthesis) 5' ends of RNA with modification of the present invention. B refers to either a base modified base or an H.
Figures 100 and 101 are general schematic l~pl~llldliol~s of the invention.
SUBSTITUTE SHEET (RU~E 26) wo ss/2322s 2 ~ 2 r~l,~ s 156 Fig. 102a-d is a schematic Itt,u~ Idliol~ of a method of the invention.
Fig. 103 is a graph of the results of the e~.ue~ idyldlllllled in figure 104.
Figure 104 is a diayldlllllldliU l~,ul~S~ d~iull of a fusion mRNA used 5 in the ~,w~,i",~"~ didyldlllllle;i in Fig. 102.
Figure 105 is a didyldlllllld~ ul~sellldlioll of a method for selection of useful ribozymes of this invention.
Figure 106 generally shows R-loop formation, and an R-loop complex. In addition, it indicates the location at which ligands can be 10 provided to target the R-loop complex to cells using at least three differentprocedures, such as ligand receptor interaction, lipid or calcium pl10spl~dI~
mediated delivery, or electroporation.
Figure 107 shows a method for use of self-p~uc~ssi"g ribozymes to generate therapeutic ribozymes of unit length. This method is essentially described by Draper et al., PCT WO 93/23509.
Figure 108 shows a method of linking ligands like folate, carbohydrate or peptides to R-loop fomming RNA.
Ribozymes of this invention block to some extent ICAM-1, IL-5, rel A, TNF-a, p210bCr-abl, or RSV genes t~,~,U1~:55iUII and can be used to treat 20 diseases or diagnose such diseases. Ribozymes will be delivered to cells in culture and to tissues in animal models. Ribozyme cleavage of ICAM-1 11-5, rel A, TNF-oc ,p210bCr-abl, or RSV mRNA in these systems may prevent or alleviate disease symptoms or conditions.
I. Target sites Targets for useful ribozymes can be detemmined as disclosed in Draper et al PCT W093/23509, Sullivan et al., PCT W094/02595 as well as by Draper et al., PCT/US94/13129 and hereby illCol~uoldltd by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those ap; ' ~s and synthesized to be tested in v~tro and In vivo, as also described. Such ribozymes can also be SUBSTITUTE SHEET (RULE 26) optimized and delivered as described therein. While specific examples to animal and human RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below. Thus, the same target may be used, but binding arms suitable for 5 targeting human RNA sequences are present in the ribozyme Such targets may also be selected as described below.
It must be e~dLli.,lled that the sites predicted by the computer-based RNA folding algorithm correspond to potential cleavage sites.
Ha,,,,,,c,,l,ead or hairpin ribozymes are designed that could bind and are 10 individually analyzed by computer folding (Jaeger et al., 1989 Proc, Natl, Acad. Sci" USA, 86 7706-7710) to assess whether the ribozyme sequences fold into the ~JUIUplidl~ secondary structure. Those ribozymes with unfavorable intramolecular i~ ,.dl,liol1s between the binding arms and the catalytic core are eliminated from ccl,sid~r~liu,,. Varying binding arm 15 lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.
mRNA is screened for Acces~ cleavage sites by the method described generally in Draper et al., PCT W093123569 hereby incorporated by reference herein. Briefly, DNA oligonucleotides 20 ,~,t,sel,li"y potential lld,,,,,,ell,ead or hairpin ribozyme cleavage sites are synthesized. A pol~lltlldse chain reaction is used to generate a substrate for T7 RNA polymerase lldilSCIi,UIiOI~ from cDNA clones. Labeled RNA
transcripts are synthesized in vitro from DNA t~r"pldl~s. The oligonucleotides and the labeled trascripts are annealed, RNaseH is 25 added and the mixtures are incubated for the cieaiylldl~d times at 37C.
Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiuy,dpllic quantitation using a phosphor imaging system. From these data, hammerhead or hairpin ribozynme sites are chosen as the 30 most A~es~ible Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences desribed above. The ribozymes are chemically synthesized. The method of synthesis used 35 follows the procedure for nommal RNA synthesis as described in Usman et al., 1987 J. Am, Chem. Soc., 109, 7845 and in Scaringe et al., 1990 WO 95123225 2 1 8 3 9 ~ 2 r~ 156 Nucleic Acids F~es., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, pl~ or~",i,d;~.3 at the 3'-end. The average stepwise coupling yeilds are >98%. Inactive ribozymes are synthesized by substituting a U for Gs and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are sy~ ed in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Ribozymes are also sy"~l ,esi,l:d from DNA
templates using bacteriophage T7 RNA polymerase (Milligan and ~0 Uhlenbach, 1989, Methods Enzymol, 180, 51). All ribozymes are modified extensively to enhance stability by ",odiricd~ion with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-amethyl, 2'H (for a review see Usman and Cedergren, 1992 TIBS 17,34). Ribozymes are purified by gel eleul,ul holtsi~ using heneral methods or are purified by high pressure liquid cl"u",alug,d,ul,y and are resuspended in water.
El~Arnple 1: ICAM-1 Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to ill~ld,,lllldluly or autoimmune disorders. ICAM-1 Function can be blocked therAre~' 'Iy using l,,olloclol~al antibodies. Ribozymes have 2û the advantage of being generally immunologically inert, whereas ~iy~ icanl neutralizing anti-lgG ~ u"ses can be observed with some ~u~oclollal antibody llt:dllll~l~t~.
The foliowing is a brief desc~i,uli~ of the ,ull~l~,;ûloyiudl role of ICAM-1.
The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention .
Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose expression is induced by i"fld"""d~ory mediators. ICAM-1 is required for adhesion of leukocytes to endothelial cells and for several immunological functions including antigen pl~ lldli~l1l immunoglobulin production and cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell recognition and activity during transplant rejection and in animal models of rheumatoid arthritis, asthma and reperfusion injury.

WO 95~13225 P~,1/1L. ' '~ - '' 21839~2 24 Cell-cell adhesion plays a pivotal role in illrlallllllaluly and immune le~bpul1s~s (Springer et al., 1987 Ann. Rev. Immunol. 5, 223-252). Cell adhesion is required for leukocytes to bind to and migrate through vascular ~lidulllelial cells. In addition, cell-cell adhesion is required for antigen 5 pl~s~,,latiun to T cells, for B cell induction by T celis, as well as for the cytotoxicity activity of T cells, NK cells, Il,ollocyt~s or granulocytes.
Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell i~,Ltlia~,liuns (Simmons et al., 1988 Nature (London) 331, 624-627).
ICAM-1 is ~,~prt,ssed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J. Immunol. 137, 245-254). Upon treatment with a number of illrlal~ dlury mediators (li,uu~olyOac~llaride~ ~interferon, tumor necrosis factor-a, or interleukin-1), a variety of cell types (~lldulllelial, epithelial, ~iblulJlat.liu and l1c:lllalupOi~lic 15 cells) in a vâriety of tissues express high levels of ICAM-1 on their surface(Sringer et. al. supra; Dustin et al., supra; and Rothlein et al., 1988 J.
Immunol. 141,1665-1669). Induction occurs via increased llalls~ ut;ùll of ICAM-1 mRNA (Simmons et a/., supra). Elevated ~ ,iu, l is dt,l~1table after 4 hours and peaks after 16 - 24 hours of induction.
ICAM-1 induction is critical for a number of illrldllllllaluiy and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd,1988 Proc. Natl. Acad. Sci. USA
85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331).
Thus, ICAM-1 c,~,ui~siu" may be required for the extravasation of immune cells to sites of i"'' Illlldtiuli. Antibodies to ICAM-1 also block T cell killing, mixed Iymphocyte reactions, and T cell-mediated B cell di~ lllialiOIl, suggesting that ICAM-1 is required for these cognate cell illl~la~iliol1s (Boyd eta/., supraJ. The i""~o,la"~e of ICAM-1 in antigen p~s~lllaliull is ulld~ ,.,o,~d by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell p,ulir~,dliùn (Dang et al., 1990 J.
Immunol. 144, 4082-4091). Conversely, murine L cells require llall~rel.:tiol1 with human ICAM-1 in additiûn to HLA-DR in order to present antigen to human T cells (Altmann et al., 1989 Nature (London) 338, 512-514). In summary, evidence in vitro indicates that ICAM-1 is required for cell-cell illlelduliùl~s critical to illrldlllllldlUry responses, cellular immune l~ ,ses~and humoral antibody responses.

wo 95t23'LlS 2~.8~2 F~~ .S.~/~ 156 By engineering ribozyme motifs we have designed several ribozymes directed against ICAM-1 mRNA sequences. These have been sy"ll,eai~d with modi~icd~iolls that improve their nuclease I~lSia~di~c~. These ribozymes cleave ICAM-1 target sequences in vitro.
The sequence of human, rat and mouse ICAM-1 mRNA can be screened for A..ce74il,1e sites using a compter folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables 2, 3, and 6-9, (All sequences 10 are 5' to 3' in the tables) While rat, mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility.
The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4 - 8 and 10. Those in the art will recognize that 15 these sequences are ,~ a~"l~live only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding anms) is altered to affect activity and may be formed of ribon~lcl~otid~s or other nucleotides or non-rl~lcleotirles Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
The ribozymes will be tested for function in vivo by exogenous delivery to human umbilical vein e:llclul~ l cells (HUVEC). Ribozymes will be delivered by incorporation into liposomes, by ~ull,plexi"g with cationic lipids, by ",i~,~i"je~,lion, or by ~ Jr~:saiol1 from DNA or RNA
vectors described above. Cytokine-induced ICAM-1 expression will be 2~i monitored by ELISA, by indirect immunofluoresence, and/or by FACS
analysis. ICAM-1 mRNA levels will be assessed by Northem, by RNAse protection, by primer extension or by quantitative RT-PCR analysis.
Ribozymes that block the induction of ICAM-1 protein and mRNA by more than 90% will be identified.
As disclosed by Sullivan et al., PCT W094/02595, illCoiyoldl~d by reference herein, ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of ICAM-1 mRNA and protein. The effect of the anti-lCAM-1 ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced . _ _ . .... . . ... ,, _ _ _ _ _ . .

WO 95/23225 '' 218 3 9 ~ 2 26 r~ 56 into joints of mice with collagen-induced arthritis or rabbits with Strf~ptoGocc~AI cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-A~o~ cl vinus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-lCAM-1 ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate ld",~ t~ly and immune responses in these diseases.
ICAM-1 plays a central role in immune cell l~cou,,ilio~l and function.
Ribozyme inhibition of ICAM-1 e~ ssion can reduce transplant rejection and alleviate symptoms in patients with rheumatoid arthritis, asthma or other acute and chronic ill~ldlllll~dluly disorders. We have engineered several ribozymes that cleave ICAM-1 mRNA. Ribozymes that efficiently inhibit ICAM-1 ex~ lc,ssiul1 in cells can be readily found and their activity measured with regard to their ability to block lld,~s~ la"l rejection and arthritis symptoms in animal models. These anti-lCAM-1 ribozymes represent a novel therapeutic for the treatment of immunological or ill~ldlllllldl-~ly disorders.
The therapeutic utility of reduction of activity of ICAM-1 function is evident in the following disease targets. The noted references indicate the role of ICAM-1 and the therapeutic potential of ribozymes described herein.
Thus, these targets can be therapeutically treated with agents that reduce ICAM~ .,essio" or function. These diseases and the studies that support a critical role for ICAM-1 in their pathology are listed below. This list is not meant to be complete and those in the art will recognize further conditions and diseases that can be effectively treated using ribozymes of the present invention.
Transplant rejection ICAM-1 is ~ Jr~ssed on venules and capillaries of human cardiac biopsies with l~ lou;~l evidence of graft rejection (Briscoe et al., 1991 Tl~n~pldll/a~
51, 537-539).
Antibody to ICAM-1 blocks renal (Cosimi et al., 1990J. Immunol. 144, 4604-4612) and cardiac (Flavin et al., 1991rransplant. Proc. 23, 533-534) graft rejection in primates.

WO 95123225 -2 1 8 ~ 9 9 2 P~l, . ,. s ~ i56 A Phase I clinical trial of a r~lùl~oulol-al anti-lCAM-1 antibody showed significant reduction in rejection and a significant increase in graft function in human kidney transplant patients (Haug, et al., 1993Tlal7~lall~aliùll 55, 766-72).
Rheumatoid arthritis ICAM-1 ovel~,ur~ssion is seen on synovial fibroblasts, ~ldu~l,elial cells, macrophages, and some Iymphocytes (Chin et al., 1990 Arthritis Rheum 33, 1776-86; Koch et al., 1991 Lab Invest64, 313-20).
Soluble ICAM-1 levels correlate with disease severity (Mason et al., 1993 Arthritis l~heum 36, 519-27).
Anti-lCAM antibody inhibits collagen-induced arthritis in mice (Kakimoto et al.,1992 Cell Immunol 142, 326-37).
Anti-lCAM antibody inhibits adjuvant-induced arthritis in rats (ligo et al., 1991 J
Immunol 147, 4167-71).
Myocardial ischemia, stroke, and reperfusion injury Anti-lCAM-1 antibody blocks adl,elt",ce of neutrophils to anoxic endul~elidl cells (Yoshida et al., 1992 Am J Physiol262, H1891-8).
Anti-lCAM-1 antibody reduces neurological damage in a rabbit model of cerebral stroke (Bowes et al., 1993 Exp Neurol 119, 215-9).
Anti-lCAM-1 antibody protects against reperfusion injury in a cat model of myocardial ischemia (Ma et al., 1992Circulation 86, 937-46).
Asthma Antibody to ICAM-1 partially blocks eosinophil adhesion to endothelial cells and is ove,~prt,ssed on inflamed airway endothelium and epithelium in vivo (Wegner et al., 1990 Science 247, 456-9).
25 In a primate model of asthma, anti-lCAM-1 antibody blocks airway eo:,i,,u,ul,'' -(Wegneret al., supra) and prevents the resurgence of airway i"~ld"""dliol1 and hyper-responsiveness after cl~xd",~;:,oso~1e treatment (Gundel et al., 1992 ClinExp Allergyæ, 569-75).
Psoriasis WO 95123225 2 i 8 3 ~ ~ 2 ~ 56 ~

Surface ICAM-1 and a clipped, soluble version of ICAM-1 is c"g~tssed inpsoriatic lesions and ~ ssioll correlates with ill~ldlllllldLiull (Kellner et al., 1991 Br JDermatol 125, 211-6; Griffiths 1989 JAmAcad Dermatol20, 617-29;
Schopf et al., 1993 Br JDermatol 128, 34-7).
5 Anti-lCAM antibody blocks keratinocyte antigen pr~se"ldlion to T cells, (Nickoloff et al., 1993J Immunol 150, 2148-59 ).
Kawasaki disease Surface ICAM-1 ~A,urt:~SiOIl correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989Lancet2, 1298-302).
10 Soluble ICAM levels are elevated in Kawasaki disease patients; particularly high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992Arthritis ~heum 35, 672-7; Tsuji, 1992 Arerugi41, 1507-14).
Circulating LFA-1t T cells are depleted (presumably due to ICAM-1 mediated eY~travasation) in Kawasaki disease patients (Furukawa et al., 1993Scand J
Immunol37, 377-80).
FY~rnple 2: IL-5 Ribozymes that cleave IL-5 mRNA represent a novel therapeutic approach to i"~ld"""dtury disorders like asthma. The invention features use of ribozymes to treat chronic asthma, ç~, by inhibiting the synthesis 20 of IL-5 in Iymphocytes and preventing the recruitment and activation of eosinophils.
A number of cytokines besides IL-5 may also be involved in the activation of ill~ldlllllldliol1 in asthmatic patients, including platelet activating factor, IL-1, IL-3, IL-4, GM-CSF, TNF-a, gamma interferon, VCAM, ILAM-1, 25 ELAM-1 and NF-KB. In addition to these molecules, it is dp,ulc~,idl~d that any cellular receptors which mediate the activities of the cytokines are also good targets for intervention in i"~lar""ldl~ry diseases. These targets include, but are not limited to, the IL-1R and TNF-aR on keratinocytes, epithelial and ~lldul~l~lidl cells in airways. Recent data suggest that certain 30 neuropeptides may play a role in asthmatic symptoms. These peptides include substance P, neurokinin A and calcitonin-gene-related peptides.
These target genes may have more general roles in inflammatory diseases, but are currently assumed to have a role only in asthma.
-WO 9512322S _~ 2 1 ~ ~ ~ 9 2 r~ c: 156 Ribozymes of this invention block to some extent IL-5 ~A,UI~ssiu~ and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of asthma (Clutterbuck et al., 1989 supra. Garssen et al., 1991 Am. Rev.
Respir. Dis. 144, 931-938; Larsen et al., 1992 J. Clin. Invest. 89, 747-752;
Mauser et al., 1993 ~). Ribozyme cleavage of IL-5 mRNA in these systems may prevent i"rld"""dlury cell function and alleviate disease symptoms.
The sequence of human and mouse IL-5 mRNA were screened for 1 û accessible sites using a computer folding algorithm. Potential har"",e,l,dad or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 11, 13, and 14, 15. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test eflicacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the d~siy"dlæd type of ribozyme. (In Table 12, lower case letters indicate positions that are not conserved between the Human and the Mouse IL-5 sequences.) The sequences of the cl~ l'y synthesized ribozymes useful in this study are shown in Tables 12, 14 -16. Those in the art will recognize that these sequences are ~t~,ul~sc~llldli\/e only of many more such sequences where the enzymatic portion of the ribozyme tall but the binding amms) is altered to affect activity. For example, stem loop ll sequence of 11d"""e,1,dad ribozymes listed in Tables ~2 and 14 (5'-GGCCGAAAGGCC-

3') can be altered (s~hstit~tion, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form.
Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 15 and 16 (5'-CACGUUGUG-3') can be altered (s~hstitlltion, deletion and/or 3û insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 12, 14-16 may be formed of riborl~l~leoti~lAs or other r~lcleotid~s or non-n~ oti-~es Such ribozymes are equivalent to the ribozymes described spe-.iric~lly in the Tables.
By ~l1y;"ed,i,1g ribozyme motifs we have designed several ribozymes directed against IL-5 mRNA sequences. These ribozymes are synthesized . , , . _, ,, _ _ _ , . . .

WO 95/23225 2 1 8 3 9 9 2 ~ 56 with "~o, - ~s that improve their nuclease resistance. The ability of ribozymes to cleave IL-5 target sequences in vitro is evaiuated.
The ribozymes will be tested for function in vivo by analyzing IL-5 ~,ur~:,siull levels. Ribozymes will be delivered to cells by illCOI,uOIdliull into liposomes, by c~",ule~i"~ with cationic lipids, by Illiclui~ Lliull, or by ex~r~ssion from DNA or RNA vectors. IL-5 e,~.,e~ic." will be monitored by biological assays, ELISA, by indirect immunofluoresence, and/or by FACS analysis. IL-5 mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.
Ribozymes that block the induction of IL-5 activity and/or IL-5 mRNA by more than 9û% will be identified.
Interleukin 5 (IL-5), a cytokine produced by CD4+ T helper cells and mast cells, was originally termed B cell growth factor ll (reviewed by Takatsu et al., 1988 Immunol. Rev. 102, 107). It stimulates UIL' ' dliUII of activated B cells and induces production of IgM and IgA. IL-5 plays a major role in eosinophil function by promoting ~ idliUII (Clutterbuck et al., 1989 ~Ls~ 73, 1504-12), vascular adhesion (Walsh et al., 1 99û
Immunology 71, 258-65) and in vitro survival of eosinophils (Lopez et al., 1988 J. FYn I\A~I 167, 219-24). This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. FYn. Med. 172, 1525-8) The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma:
Several studies have shown a direct cull~ldliull between the number of activated T cells and the number of eo~i"opl,il~, from asthmatic patients vs. normal patients (Oehling et al., 1992 J. Investig. Allergol. Clin. Immunol.
2, 295-9). Patients with either allergic asthma or intrinsic asthma were treated with curLi-O~ltlluicla. The blu~lLIloa~vcolar lavage was monitored for eosinophils, activated T helper cells and recovery of pulmonary function over a 28 to 30 day period. The number of eosi,,ùpl,ils and activated T
helper cells decreased progressively with subsequent improvement in pulmonary function cûmpared to intrinsic asthma patients with no cullico~lt,,uiJ treatment.
Bronchoalveolar lavage cells were screened for production of cytokines using in situ 1, jb, iJi~dtioll for mRNA. In situ I Iybl i.li~dLiol1 signals W0 95123225 2 1 8 J 9 ~ 2 P~ , 156 were detected for IL-2, IL-3, IL-4, IL-5 and GM-CSF. Upregulation of mRNA
was observed for IL-4, IL-5 and GM-CSF (Robinson et al., 1993 J. Allerav Clin. Immunol. 92, 313-24). Another study showed that upregulation of IL-5 transcripts from allergen challenged vs. saline challenged asthmatic 5 patients (Kli~lllld:~V~dllly et al., 1993 Am. J. Respir. Cell. Mol. Biol. 9, 279-86).
An 18 patient study was perfur~ed to determine a ",eul,al,is", of action for co~ o~ uid improvement of asthma symptoms. Improvement was monitored by methacholine responsiveness. A correlation was 10 observed between the methacholine responsiveness, a reduction in the number of eo:,i"opllil~, a reduction in the number of cells expressing IL-4 and IL-5 mRNA and an increase in number of cells ~x,ul~s~illg interferon-gamma.
Bronchial biopsies from 15 patients were analyzed 24 hours after 15 allergen challenge (Bentley et al., 1993 Am. J. Respir. Ce~l. Mol. Biûl. 8, 35-42). Increased numbers of eo~i,lu,ull;'s and IL-2 receptor positive cells were found in the biopsies. No differences in the numbers of total leukocytes, T Iymphocytes, elastase-positive neutrophils, macrophages or mast cell subtypes were observed. The number of cells eA~ul~sa;~lg IL-5 20 and GM-CSF mRNA ~iylli~i~cll~ly increased.
In another patient study, the eosinophil phenotype was the same for asthmatic patients and normal individuals. However, eosinophils from asthmatic patients had greater leukotriene C4 producing capacity and migration capacity. There were elevated levels of IL-3, IL-5 and GM-CSF in 25 the circulation of a~ll""~ , but not in normal individuals (Bruijnzeel et al., 1992 SchwRi~ Med. Wo~ "~cl,r. 122, 298-301).
Efficacy of antibody to IL-5 was assessed in a guinea pig asthma model. The animals were challenged with ovalbumin and assayed for eo:,i"upl ' and the ~ ,uul l:~; Joness to the bl ul)~,l li~co~ iliol l substance30 P. A 30 mg/kg dose of antibody ad",i,l;~ d i.p. blocked ovalbumin-induced increased sensitivity to substance P and blocked increases in brul~ul,oalveolar and lung tissue accumulation of eo~i"opl,il., (Mauser et al., 1993 Am. Rev. Respir. Dis. 148, 1623-7). In a separate study guinea pigs challenged for eight days with ovalbumin were treated with 35 ",ol10cl~11al antibody to IL-5. Treatment produced a reduction in the WO95123225 2183992 r~ tlS6 number of eovi,,opl,', in bronchoaiveolar lavage. No reduction was observed for u"ul,allG-l,y~d guinea pigs and guinea pigs treated with a control antibody. Antibody treatment completely inhibited the development of hyperreactivity to histamine and arecoiine after ovalbumin challenge (van Oosterhout et al. 1993 Am. Rev. Respir. Dis. 147 548-52) Results obtained from human clinical analysis and animal studies indicate the role of activated T helper cells cytokines and eovi,,o,ul,;'s in asthma. The role of IL-5 in eosinophil development and function makes IL-5 a good candidate for target selection. The antibody studies neutralized 10 IL-5 in the circulation thus preventing eovi"o~ Inhibition of the production of IL-5 will achieve the same goal.
Asthma - a pru",' ,~"l feature of asthma is the infiltration of eosinophils and deposition of toxic eosinophil proteins (e.g. major basic protein, eosinophil-derived neurotoxin) in the lung. A number of T-cell-15 derived factors like IL-5 are It,v~,ol~v;ul~ for the activation and ",ai"lai"d"ce of eo:,i"upl,ilr. (Kay, 1991 J. Allergy Clin. Immun. 87, 893). Inhibition of IL-5 tlxpr~vsiol~ in the lungs can decrease the activation of eovi,,upl,.lc, and willhelp alleviate the symptoms of asthma.
Atopy - is u lldld.~ d by the develu~,~",~"l of type I hyp~,vG-I "'~c 20 reactions ' ' - with exposure to certain ~llr;.Ulllll~ dl antigens. One of the common clinical manifestations of atopy is eosinophilia (accumulation of dl,"or"-"y high levels of eosinophiis in the blood).
Antibodies against IL-5 have been shown to lower the levels of eosinophils in mice (Cook et al., 1993 in Immu,,ùpl,d,,,,dGol. Eo~i"uul,;lv ed. Smith and 25 Cook pp. 193-216, Academic London, UK) Paras~tic infect~on-related eosinoph~lia- infections with parasites like helminths can lead to severe eovi,,o,ul ''i? (Cook et al. 1993 ~). Animal models for eovi,,uul~''' suggest that infection of mice for example, can lead to blood, peritoneal and/or tissue eovi,,oul,''' all of 30 which seem to be lowered to varying degrees by dntibodies directed against IL-5.
Pulmonary infiltration eosinophilia- is characterised by accumulation of high levels of eosi"opl,ils in pulmonary parenchyma (Gleich 1990 J. Allergy Clin. Immunol. 85, 422).

WO95/23225 2183~2 P~ lr5'~ 156 L-Tryptophan-associ~ted eosinophilia-myalgia syndrome (EMS)- The EMS disease is closely linked to the consumption of L-tryptophan, an essential aminoacid used to treat conditions like insomnia (for review see Varga et al., 1993 J Invest. Demm~t~-l 100, 97s). Pathologic 5 and histologic studies have ~I,,o~ dL~d high levels of eosinophils and mononuclear ill~ldlllllldluly cells in patients with EMS. It appears that IL-5 and L,d,~:,ru,,,,i,,g growth factor play a significant role in the development of EMS (Varga et al., 1993 ~) by actiYating eosi"opl,"- and other ld"",Idluly cells.
Thus, ribozymes of the present invention that cleave IL-5 mRNA and thereby IL-5 activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible il,dicd~ions. Development of an effective ribozyme that inhibits IL-5 function is described above; available cellular and activity assays are numerous, reproducible, and accurate. Animal models for IL-5 function and for each of the s~gest~d disease targets exist (Cook et al., 1993 ~) and can be used to optimize activity.
FY~rnple 3: NF-~R
Ribozymes that cleave re/ A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. IrlrldlllllldLory mediators such as lipopolysac~l,drkJe (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) act on cells by inducing lldll~uli,uliull of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the l,d,,s,,,i,uliui,al regulator, NF-KB. One subunit of NF-IcB, the relA gene product (tenmed RelA or p6~) is implicated ~,ueuiric~l:) in the induction of illrldlllllldluiy responses. Ribozyme therapy, due to its exquisite specificity,' is particularly well-suited to target intracellular factors that contribute to'disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A or - 30 TNF-a may represent novel therapeutics for the treatment of i, Irld~ ldlui y and autoimmune disorders.
The nuclear DNA-binding activity, NF-KB, was first identified as a factor that binds and activates the immunoglobulin K light chain enhancer in B cells. NF-lcB now is known to activate lldi~ ,tiull of a variety of other cellular genes (e.g., cytokines, adhesion proteins, oncogenes and viral WO 95/23225 2 1 8 3 9 ~ 2 r~ , s . 156 .

proteins) in response to a variety of stimuli (e.g., phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and uio~l~elllical characterization of NF-7cB has shown that the activity is due to a hu~odi~el or h~I~Iu~i"l~l of a family of DNA binding subunits. Each 5 subunit bears a stretch of 3ûO amino acids that is homologous to the oncogene, v-r~l. The activity first described as NF-IcB is a heterodimer of p49 or p50 with p65. The p49 and p50 subunits of NF-KB (encoded by the nf-~cB2 or nf-~cB1 genes, respectively) are generated from the precursors NF-lcB1 (p105) or NF-~cB2 (p100). The p65 subunit of NF-IcB (now 10 temmed Rel A ) is encoded by the rel A locus.
The roles of each specific l,d":,c,i,u~iu"-activating complex now are being elucidated in cells (N.D. Perkins, et al., 1992 Proc. Natl Ar:~l Sci ~1~ 89, 1529-1533). For instance, the h~ udi~er of NF-KBl and Rel A
(p50/p65) activates Ildllsu,i,uIi."~ of the promoter for the adhesion molecule, 15 VCAM-1, while NF-lcB2/RelA heterodimers (p49/p65) actually inhibit Lldl~:,cli,uliùl) (H.B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289 (1993)).
Conversely, h~ uui",e~ of NF-KB2/RelA (p49/p65) act with Tat-l to activate l,d"su,i~.Iion of the HIV genome, while NF-lcB1/RelA (p50/p65) h~Ie,udi",e,:, have little effect (J. Liu, N.D. Perkins, R.M. Schmid, G.J.
20 Nabel, l. Virol. 1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligorl~lrleoticles specifically blocks embryonic stem cell adhesion; blocking NF-KB1 gene ~,ur~sio~ with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 Mol. Cell. Biol. 13, 3802-3810). Thus, the promiscuous role initially 25 assigned to NF-1cB in Ildllsuliluliullal activation (M.J. Lenardo, D. Baltimore, 1989 ~ 58, 227-229) ,~,u,t,~t,,,t~ the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent I-di~sg~";c "knock-out" mice of individual members of the rel family. Such "knock-outs" show few dov~l~pi~,ental defects, suggesting that essential 30 i,dnsl,,i,uliu,,al activation functions can be performed by more than one member of the rel family.
A number of specific inhibitors of NF-lcB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-KB binding sites, and over ~Xp~S~iu11 35 of the natural inhibitor MAD-3 (an llcB family member). These agents have WO 95123225 2 1 8 3 9 ~ 2 ~ IL s ~ . 156 been used to show that NF-KB is required for induction of a number of molecules involved in ill~lalllllldliull~ as described below.
~NF-lcB is required for phorbol ester-mediâted induction of IL-6 (I.
Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993 Mol. Cell. Biol. 13, 6137-46).
~ NF-1cB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 Mol. Ceil. Biol. 13, 6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et al., 1994 J. Ex~, Me~ 179, 503-512) on ~llduLI,-" ' cells.
~NF-KB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al., 1993 supra).
The above studies suggest that NF-lcB is integrally involved in the induction of cytokines and adhesion molecules by illrld""" '~.y mediators.
Two recent papers point to another conl~e~lioll between NF-lcB and ill~ldlllrlld~iUI~. gl~ocu,li.;oid~ may exert their anti-i,,~ld,,,,,,dlury effects by inhibiting NF-lcB. The glu~oc~"iuoicl receptor and p65 both act at NF-KB
binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol.
269, 6185-6192). Glu.,oco,li.;uid receptor inhibits NF-lcB-mediated induction of IL-6 (Ray and ~l~ru"ldi"e, 1994 Proc. NRtl Af~Rrl Sci USA 91, 752-7~6). Conversely, over~ s~ion of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-imm~op,c,-,i,u~t~JIl ~xpeli",~l,L~ de",u"~ d~d direct physical interaction between p65 and the glucùco, li~,ùid receptor (Id.).
Ribozymes of this invention block to some extent NF-lcB ~,u~:s~iull and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of l~ "osis, transplant rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA in these systems may prevent illrldllll~ldluly cell function and alleviate disease symptoms.
The sequence of human and mouse re/A mRNA can be screened for accessible sites using a computer folding algorithm. Potential 11d"""ell,ead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 17, 18 and 21-22. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and WO 9S/23225 2 1 8 3 ~ 9 2 P~,l/1L3~,. 156 ribozymes thereafter designed, the human targetted sequences are of most utility.
The sequences of the ,;I,~",i~.ally Sy.,;:,Hsi~Hd ribozymes uséful in this study are shown in Tables 19 - 22. Those in the art will recognize that 5 these sequences are ~ HsH"ldli~/e only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity and may be formed of ribonllclestides or othar nllcleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described -r~ ''- Ily in the Tables.
By dl)yi"e~,i"g ribozyme motifs we have designed several ribozymes directed against re/A mRNA sequences. These ribozymes are synthesized with Illod;ri~,dliulls that improve their nuclease resistance. The ability of ribozymes to cleave re/A target sequences in vitro is evaluated.
The ribozymes will be tested for function in vivo by analyzin3 cytokine-15 induced VCAM-1, ICAM-1, IL-6 and IL-8 d~ul~ssiun levels. Ribozymes will be delivered to cells by illColt ulc~liull into liposomes, by cu,,,,ule,.i,,g with cationic lipids, by ",ic,ui,l;~vliul~, or by v~lUlHS~ from DNA and RNA
vectors. Cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 t~X,ult:aSiOI1 will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS
2û analysis. Rel A mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.
Activity of NF-KB will be monitored by gel-rHk-,d~liu" assays. Ribozymes that block the induction of NF-KB activity and/or rel A mRNA by more than 50% will be identified.
RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1, ICAM-1, IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be ~Ccssse~ Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with StlHl)Iococcql cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or ad3no-a~so; ~lHd virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-relA ribozyme or a gene construct that constitut~vely expresses the ribozyme may abrogate ill~lclllllllc.lùly and immune responses in these diseases.
... _ ...... .... , . . .,, .,,, .. , .. , _ _ _ _ , _ .

WO 95123225 ~ r~ iS6 218399~

Uses A therapeutic agent that inhibits cytokine gene c~,~,u1~5;,;011, inhibits adhesion molecule t,x~ siul), and mimics the anti-i"~ld"""dl~ly effects of glucocor~iu~idb (without inducing steroid-l~b~u"r,,v~ genes) is ideal for the 5 treatment of i"~ld"""dlury and autoimmune disorders. Disease targets for such a drug are numerous. Target i,,di~dliu,ls and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute il l.li.;dli~l~s, or ex vivo treatment.
1 û Rheumatoid arthritis (RA).
Due to the chronic nature of RA, a gene therapy approach is logical.
Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-~."o~ d virus vectors. For instance, the dp~lu,ulidl~
adenovirus vector can be administered by direct injection into the 15 synovium: high efficiency of gene transfer and UA~ ssi~l1 for several months would be expected (B.J. Roessler, E.D. Allen, J.M. Wilson, J.W.
Hartman, B. L. Davidson, J. Clin. Invest. 92, 1û85-1û92 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple 2û adlllillibLIdliùlls may be necessary. Retrovirus and adenO-abbOCidlt:d vinus vectors would lead to pt""al,~,lt gene transfer and exur~bsiol~ in the joint.
However, p~lllldl)~lll t~A,Ult~bsiOI1 of a potent anti-i,,~ld,,,,,,dluiy agent may lead to local immune deficiency.
'n_~ Obib.
Expression of NF-~cB in the vessel wall of pigs causes a narrowing of the luminal space due to excessive deposition of extracellular matrix components. This phenotype is similar to matrix deposition that occurs subsequent to coronary dlly,iulJldbly. In addition, NF-IcB is required for the - expression of the oncogene c-myb (F.A. La Rosa, J.W. Pierce, G.E.
Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-IcB induces smooth muscle proliferation and the expression of excess matrix ~olllpoll~ b~ both processes are thought to contribute to reocclusion of vessels after coronary ar,yioplably.
Trdl Ib,UIdrlldli~

WO9S/23225 218393~ r~l,.v. ~ 156 3~
NF-KB is required for the induction of adhesion molecules (Eck et al., supra, K. O'Brien et al., J. Clin. Invest. 92 945-951 (1993)) that function in immune l~coy~ JI) and inflammatory ,~bp~l1ses. At least two potential modes of treatment are possible. In the first lldrlspldlllud organs are 5 treated ex vivo with ribozymes or ribozyme eA~ s~iul~ vectors. Transient inhibition of NF-IcB in the lldll~Jldll~d endothelium may be sufficient to prevent transplant-~cso~i~tr~d vasculitis and may ~i~Ulli~iCdlllly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme ~xp,bssion vectors. Reui,l)i6l,l~ would receive the 10 treatment prior to transplant. Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1, VCAM-1 and/or B7 an B7-2) can induce antigen-specific anergy. Tolerance to the donor's l,k.locc,lllydliLilily antigens could result; potentially any donor could be used for any lldllauldllldlioll procedure.
1 5 Asthma.
Granulocyte macrophage colony stimulating factor (GM-CSF) is thought to play a major role in recruitment of eoai"opl~ils and other ill~ldllll~ldloly cells during the late phase reaction to asthmatic trauma.
Again blocking the local induction of GM-CSF and other ill~ldll~ dlùly 20 mediators is likely to reduce the persistent ill~ldl,,,,,dliu,, observed in chronic a~ll""dli~. Aerosol delivery of ribozymes or adenovirus ribozyme t~Aplt:~Siùl1 vectors is a feasible treatment.
Gene Therapy.
Immune responses limit the efficacy of many gene transfer 25 techniques. Cells lldlla~ lud with retrovirus vectors have short lifetimes inimmune cu,,,,u~lt,,ll individuals. The length of eA,u,e~siol, of adenovirus vectors in terminally ui~r~"~idl~d cells is longer in neonatal or immune-co",p~ur"i:,ed animals. Insertion of a small ribozyme _A~ aiOI~ cassette that modulates inflammatory and immune responses into existing 30 adenovirus or retrovirus constructs will greatly enhance their potential.
Thus, ribozymes of the present invention that cleave re/A mRNA and thereby NF-KB activity have many potential therapeutic uses, and there are l~a:,ol)able modes of delivering the ribozymes in a number of the possible ill~i~dliOI1s. Development of an eflective ribozyme that inhibits NF-lcB
. . .

W0 95123225 2 1 8 3 9 9 2 r~ lS6 function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for NF-KB function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity.
5 FYArnple 4: TNF-a Ribozymes that cleave the specific cites in TNF-a mRNA represent a novel therapeutic approach to illlldllllll .y or autoimmune disorders.
Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated leukocytes, that is a potent mediator of i"'' "", .y reactions. Injection of 10 TNF-a into ~x~ llldl animals can simulate the symptoms of systemic and local i"~lar"",alury diseases such as septic shock or rheumatoid arthritis.
TNF-a was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, - 15 1985 Science 230, 4225-4231). TNF-a sllhseq~lently was found to be identical to cachectin, an agent l~b,lJUI ,biL,le for the weight loss and wasting syndrome A~.soc;~l~d with tumors and chronic infections (Beutler, et al., 1985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-a have been cloned and found to be related to TNF-B (Shakhov et al., 1990 20 J. FYr~. Med. 171, 35-47). Both TNF-a and TNF-13 bind to the same receptors and have nearly identical biological activities. The two TNF
receptors have been found on most cell types examined (Smith, et al., 1990 ~ 248, 1019-1023). TNF-a secretion has been detected from monoc~lt,s~l"ac,u~-llages, CD4+ and CD8+ T-cells, B-cells, Iymphokine 25 activated killer cells, neutrophils, astrocytes, el~dull,elidl cells, smooth muscle cells, as well as various non-he:",dlu,~.ui~ tumor cell lines ( for a r.eview see Turestskaya et al., 1991 in Tumor Necrosis Factor. strll~tllre~
Function. and !\l '~A~liblll of Action B. B. Aggar~val, J. Vilcek, Eds. Marcel Dekker, Inc., pp. 35-60). TNF-a is regulated l,al,scri,ulionally and 30 translationally, and requires proteolytic prucess;"g at the plasma membrane in order to be secreted (Kriegler et al., 1988 Cell 53, 45-53).
Once secreted, the serum half life of TNF-a is d~,UlU~illldl~ly 30 minutes.
The tight regulation of TNF-a is important due to the extreme toxicity of this cytokine. I"c,~asi"g evidence indicates that overproduction of TNF-a WO 9S123225 218 3 9 ~ 2 r~ s6 during infections can lead to severe systemic toxicity and death (Tracey &
Cerami, 1992 Am. J. TroD. Med. Hyg. 47, 2-7).
Antisense RNA and Hd"""erl,ead ri~u~y,,,es have been used in an attempt to lower the ~ ssion level of TNF-a by targeting specified 5 cleavage sites [Sioud et al., 1992 1. Mûl. Blol. 223; 831; Sioud WO
94/10301; Kisich and co-workers, 1990 abstract (F~.cFR J. 4, A1860; 1991 slide plC~ dlioll (J, ~ n~yte Biol. sup. 2, 70); December, 1992 poster r~s~ dIiùl) at Anti-HlV Therapeutics Conference in SanDiego, CA; and '`Development of anti-TNF-a ribozymes for the control of TNF-oL gene 10 exp,t,saio~l"- Kisich, Doctoral Diss~lIdIiul~, 1993 University of California, Davis] listing various TNF targeted ribozymes.
Ribozymes of this invention block to some extent TNF- exp,~s;,iul1 and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models 15 of septic shock and rheumatoid arthritis. Ribozyme cleavage of TNF-mRNA in these systems may prevent ill~ldll,,,ldLuly cell function and alleviate disease symptoms.
The sequence of human and mouse TNF- mRNA can be screened for ~ s~ le sites using a computer folding algorithm. I Id"""t.r~,~ad or 20 hairpin ribozyme cleavaye sites were identified. These sites are shown in Tables 23, 25, and 27 - 28. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the 25 rioo2yme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the d~:~iy~ Idl~d type of ribozyme.
(In Table 24, lower case letters indicate positions that are not conserved between the human and the mouse TNF-~ sequences.) The sequences of the chemically sy~ esi~d ribozymes useful in this 30 study are shown in Tables 24, 26 - 28. Those in the art will recognize that these sequences are ~ "IdIive only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop ll sequence of 11al"1"ell,ead ribozymes listed in Tables 24 and 26 (5'-GGCCGAMGGCC-35 3') can be altered (~hstit~ltion, deletion, and/or insertion) to contain any ,, _ _ _ . . ... .. . ... .. . . _ . .

WO 95/23225 - ~ 2 1 8 3 ~ ~ ~ P~ 156 sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 27 and 28 (5'-CACGUUGUG-3') can be altered (sllhstitlltion, deletion, and/or insertion) to contain any sequence, provided a minimum of 5 two base-paired stem structure can form. The sequences listed in Tables 24, 26 - 28 may be formed of riborlllclpoti~lp~c or other nll~lP~ti-les or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables or AAV .
In a preferred ~Illbodi~ of the invention, a lldrls~ JIiun unit 10 ~A~ billg a ribozyme that cleaves TNF- RNA is inserted into a plasmid DNA vector or an adenovirus DNA virai vector or AAV or alpha virus or retroviris vectors. Viral vectors have been used to transfer genes to the intact vasculature or to joints of live animals (Willard et al., 1992 Circulation. 86, 1-473.; Nabel et al., 1990 Science, 249, 1285-1288) and 15 both vectors lead to transient gene ~ApltlbSiull. The adenovirus vector is delivered as l~culllu;lldllL adenoviral particles. DNA may be delivered alone or c~,npl~At d with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the I~Cullluilldlll adenovirus particles are locally ad",i"ib~t"t,d to the site of treatment, e.g., through the use of an 20 injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.
In another preferred e",bodi",t:"l of the invention, a lldl)scliyliull unit ~AI~r~sbi~ 19 a ribozyme that cleaves TNF-~ RNA is inserted into a retrovirus vector for sustained ~xp, ~ ,:,iul7 of ribozyme(s).
By t",~i"e~,i"g ribozyme motifs we have designed several ribozymes directed against TNF-~ mRNA sequences. These ribozymes are synthesized with l~lodi~i~idliolls that improve their nuclease resistance. The ability of ribozymes to cleave TNF-a target sequences in vitro is evaluated.
The ribozymes will be tested for function in cells by analyzing bacterial lipopolysaccharide (LPS)-induced TNF- expression levels.
Ribozymes will be delivered to cells by i"cor~,o,dliol~ into liposomes, by c~",plt,Aillg with cationic lipids, by Illi~,luillj~l,liUl), or by expression from DNA vectors. TNF-a eAl~le:sbiull will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. TNF- mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension .. . . . . .

WO 95/23225 218 3 9 ~ 2 ~PCTJIB95100156 ~2 analysis or quantitative RT-PCR. Ribozymes that block the induction of TNF-~ activity and/or TNF-o: mRNA by more than 90% will be identified.
RNA ribozymes and/or genes encoding them will be locally delivered to ",dc,upl-ayes by i,,lld,u~,ilui1eal injection. After a period of ribozyme 5 uptake, the peritoneal ",a-,upl1ag~s are harvested and induced ex vivo with LPS. The ribozymes that Siyll;riGalllly reduce TNF-oL secretion are selected. The TNF-a can also be induced after ribozyme treatment with fixed strBptococcus in the peritoneal cavity instead of ex v~vo. In this fashion the ability of TNF-~ ribozymes to block TNF-cL secretion in a 1 û localized i"~ld"""dlury response are evaluated. In addition, we will detemmine if the ribozymes can block an ongoing i, " Illlldlu~y response by delivering the TNF-a ribozymes after induction by the injection o~ fixed St~ co~G~c To examine the effect of anti-TNF- ribozymes on systemic 15 i,,~ld,,,,,,dlivll, the ribozymes are delivered by intravenous injection. Theability of the ribozymes to inhibit TNF-a secretion and lethal shock caused by systemic LPS adlllillialld~iull are assessed. Similarly, TNF- ribozymes can be introduced into the joints of mice with collagen-induced arthritis.
Either free delivery, liposome delivery, cationic lipid delivery, adeno-20 ACsociAt~d virus vector delivery, adenovirus vector delivery, retrovirusvector delivery or plasmid vector delivery in these animal model ~ueri~ tS can be used to supply ribozymes. One dose (or a few infrequent doses) of a stable anti-TNF-c~ ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate tissue damage in 25 these i"rld"""dlory diseases.
Ma~lOlJllRSld isolation.
To produce responsive macrophages 1 ml of sterile fluid thioglycollate broth (Difco, Detroit, Ml.) was injected i.p. into 6 week old female C57bl/6NCR mice 3 days before peritoneal lavage. Mice were Illc.i,lldi"ed 30 as specific pathogen free in autoclaved cages in a laminar flow hood and given sterilized water to minimize "spontaneous" activation of ",a~,,upllay~s. The resulting peritoneal exudate cells (PEC) were obtained by lavage using Hanks balanced salt solution (HBSS) and were plated at 2.5X105/well in 96 well plates (Costar, Cambridge, MA.) with Eagles 35 minimal essential medium (EMEM) containing 10% heat inactivated fetal _ .. . . . . . . . ... _ .. .. . _ .... _ .... _ _ _ _ ... .

wo ss/2322s 218 3 9 9 2 r~ 156 bovine serum. After adhering for 2 hours the wells were washed to remove non-adherent cells. The resulting cultures were 97% macrophages as d~ r~ led by ",o"ul~oloyy and staining for non-specific esterase.
T~ ar~ iul~ of ribozymes into l/la~ lagb?~.
The ribozymes were diluted to 2X final Cull~ lldliOI~, mixed with an equal volume of 11nM li,uu~t~cld~ e (Life Technologies, Gaithersburg, MD.), and vortexed. 100 ml of lipid:ribozyme complex was then added directly to the cells, followed illl",~.lidl~ly by 10 ml fetal bovine serum.
Three hours after ribozyme addition 100 ml of 1 mg/ml bacterial 1 û lipopolysaccaride (LPS) was added to each well to stimulate TNF
production.
Quantitation of TNF-o: in mous~ ",a",~hd~
S~,uer~dldllL~ were sampled at û, 2, 4, 8, and 24 hours post LPS
stimulation and stûred at -70C. Quantitation of TNF-a was done by a specific ELISA. ELISA plates were coated with rabbit anti-mouse TNF-a serum at 1:1000 dilution (Genzyme) followed by blocking with milk proteins and incubation with TNF-a collldi"i"g supe",dld"l~i. TNF-a was then detected using a murine TNF-a specific hamster Illu~o~;lollal antibody (Genzyme). The ELISA was developed with goat anti-hamster IgG coupled 2û to alkaline pl~Oa~ dldSe.
Asse~:""~"~ of reagent toxicity:
Following ribozyme/lipid treatment of ,,,a.;,u~l,ay~:s and harvesting of supellld~dlll~ viability of the cells was assessed by incubation of the cells with 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). This compound is reduced by the mitochondrial dihyd,u~ ases, the activity of which correlates well with cell viability. After 12 hours the dbsulbdl~,e of reduced MTT is measured at 585 nm.
Uses The ~Ccoc~ ol~ between TNF-a and bacterial sepsis, rheumatoid arthritis, and autoimmune disease make TNF-a an attractive target for therapeutic intervention ~Tracy & Cerami 1992 ~; Williams et al., 1992 Proc. I\~tl Acad. Sci. USA 89, 9784-9788; Jacob, 1992 J ~lltoirnmun~ 5 (Supp. A), 133-143].

WO 9512322S 2 1 8 3 9 9 2 ~ 77~ 6 Se~tic Shock Septic shock is a c~", " ) of major surgery, bacterial infection, and polytrauma characterized by high fever, increased cardiac output, reduced blood pressure and a neutrophilic infiltrate into the lungs and 5 other major organs. Current treatment options are limited to antibiotics to reduce the bacterial load and non-steroidal anti-i"rld"""dlolies to reduce fever. Despite these II~ J7 in the best intensive care settings, mortality from septic shock averages 50%, due primarily to multiple organ failure and diast""i"dlt,d vascular co~ tion. Septic shock, with an incidence of 10 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-i"rlar"",dlury cytokines which are not normally detected in the serum, such as TNF-, interleukin-1B (IL-1B), ~interferon (IFN~), interleukin-6 (IL-15 6), and interleukin-8 (IL-8). Other non-cytokine mediators such as leukotriene b4, pn,ald~71d"di" E2, C3a and C3d also reach high levels (de Boer et al., 1992 Imm~"Q~ r" ~ ~v 24, 135-148).
TNF-~ is detected eariy in the course of septic shock in a large fraction of patients (de Boer et al., 1992 ;~a). In animal models, injection of TNF-20 has been shown to induce shock-like symptoms similar to those induced by LPS injection (Beutler et al., 1985 Science 229, 869-871); in contrast, injection of IL-lB, IL-6, or IL-8 does not induce shock. Injection of TNF-also causes an elevation of IL-1B, IL-6, IL-8, PgE2, acute phase proteins, and TXA2 in the serum of ~xperi",t",lal animals (de Boer et al., 1992 25 supra). In animal models the lethal effects of LPS can be blocked by pre-ad",i"i~l,dLiol, of anti-TNF- antibodies. The cumulative evidence indicates that TNF- is a key player in the pdll,og~"esis of septic shock, and therefore a good candidate for therapeutic intervention.
Rhe~m~t~irl Arthrifi~
Rheumatoid arthritis (RA) is an autoimmune disease ~lldld~ ri~d by chronic illrldlllllldlioll of the joints leading to bone destruction and loss ofjoint function. At the cellular level, autoreactive T- Iymphocytes and monocytes are typically present, and the synoviocytes often have altered ",og.l,ology and imm~.,o7l~i"i"g patterns. RA joints have been shown to contain elevated levels of TNF-, IL-1 and IL-lB, IL-6, GM-CSF, and TGF-WO 95123225 P~l~, 156 2183~92 B (Abney et al., 1991 Imm. Rev. 119, 105-123), some or all of which maycontribute to the pathological course of the disease.
Cells cultured from RA joints ~,uu~a~eously secrete all of the pro-ld~ dluly cytokines detected in vivo. Addition of antisera against TNF-5 to these cultures has been shown to reduce IL-1c/B production by thes0 cells to ull~ levels (Abney et al., 1991 ~). Thus, TNF-c~ may directly induce the production of other cytokines in the RA joint. Addition of the anti-i"~lar"",dluiy cytokine, TGF-B, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical 10 ~ue~ s clearly d~llloll~lldl~ the production of TNF-, IL-1c/r~, and IL-6 from ",a.ilUpl1dg~s near the callilaga~a""~s junction when the pannus in invading and overgrowing the cartilage (Chu et al., 1992 Br. J.
Rheumatology 31, 653-661). GM-CSF was shown to be produced mainly by vascular endothelium in these samples. Both TNF-~ and TGF-B have 15 been shown to be fibroblast growth factors, and may contribute to the accumulation of scar tissue in the RA joint. TNF-a has also been shown to increase osteoclast activity and bone resorbtion, and may have a role in the bone erosion commonly found in the RA joint (Cooper et al., 1992 Clin.
EXD~ Immunol. 89, 244-250).
Cl;,llilldLiull of TNF- from the rheumatic joint would be predicted to reduce overall illrldlllllld~iU~l by reducing induction of MHC class ll, IL-1c~/B, 11-6, and GM-CSF, and reducing T-cell activation. Osteoclast activity might also fall, reducing the rate of bone erosion at the joint. Finally, 1'i.11il~dlio"
of TNF- would be expected to reduce accumulation of scar tissue within the joint by removal of a fibroblast growth factor.
Treatment with an anti-TNF- antibody reduces joint swelling and the hic~ol-l~icAI severity of collagen-induced arthritis in mice (Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788). In addition, a study of RA
patients who have received i.v. infusions of anti-TNF- monoclonal antibody reports a reduction in the number and severity of inflamed joints after treatment. The benefit of monoclonal antibody treatment in the long term may be limited by the expense and immunogenicity of the antibody.
Psoriasis Psoriasis is an illrldlllllldloly disorder of the skin characterized by keratinocyte hyp~,,u,ulir~,dlio~l and immune cell infiltrate (Kupper, 1990 L
.... . ... .. . . _ . _ .. . ..

WO 95123225 218 3 ~ 3 2 PCT/IB95100156 Clin. Invest. 86, 1783-1789). It is a fairly common condition, affecting 1.5-2.0% of the population. The disorder ranges in severity from mild, with small flaky patches of skin, to severe, involving illrld~ dliùl1 of the entire epidermis. The cellular infiltrate of psoriasis includes T-lymphocytes, 5 neutrophils""a.,~upl~dy~s, and dermal dendrocytes. The majority of T-lymphocytes are activated CD4+ cells of the TH-1 phenotype, although some CD8+ and CD41CD8- are also present. B Iymphocytes are typically not found in abundance in psoriatic plaques.
Numerous hypotheses have been offered as to the proximal cause of 10 psoriasis including auto-antibodies and auto-reactive T-cells, overproduction of growth factors, and genetic pr~ l,us;l;un. Aithough there is evidence to support the involvement of each of these factors in psoriasis, they are neither mutually exclusive nor are any of them necessary and sufficient for the pathogenesis of psoriasis (Reeves, 1991 15 Semin. DemlAt~l 10, 217) The role of cytokines in the pdll,oge"esi:, of psoriasis has been investigated. Among those cytokines found to be a~l10n, -"y expressed were TGF-a, IL-1a, IL-1r~, IL-lra, IL-6, IL-8, IFN-7, and TNF-a . In addition to abnormal cytokine production, elevated ~x,u,~ioll of ICAM-1, ELAM-1, 20 and VCAM has been observed (Peeves, 1991 ~L~. This cytokine profile is similar to that o~ nommal wound heaiing, with the notable exception that cytokine levels subside upon healing. Keratinocytes themseives have recently been shown to be capable of secreting EGF, TGF-a, IL-6, and TNF-a, which could increase pluli~ldliull in an autocrine fashion (Oxholm 25 et al., 1991 APMIS 99, 58-64).
Nickoloff et al., 1993 (J DemmAt~l Sci. 6, 127-33) have proposed the following model for the initiation and ",di"l~"a~,c~ of the psoriatic plaque:
Tissue damage induces the wound healing response in the skin.
Keratinocytes secrete IL-1a, IL-113, IL-6, IL-8, TNF-a. These factors 30 activate the endothelium of dermal capillaries, recruiting PMNs, ",al,,u,ul,ay~s, and T-cells into the wound site.
Dermal dendrocytes near the dermal/epidel",al junction remain activated when they should return to a quiescent state, and subsequently secrete cytokines including TNF-a, IL-6, and IL-8. Cytokine ~X,UIt~:~Siul" in WO 95/23225 218 3 9 ~ 2 r~ 156 turn, maintains the activated state of the endothelium, allowing extravasation of additional immunocytes, and the activated state of the keratinocytes which secrete TGF-~ and IL-8. Keratinocyte IL-8 recruits immunocytes from the dermis into the epidermis. During passage through the dermis, T-cells encounter the activated dermal dendrocytes which efliciently activate the TH-1 phenotype. The activated T-cells continue to migrate into the epidermis, where they are stimulated by keratinocyte-expressed ICAM-1 and MHC class ll. IFN-~ secreted by the T-cells synergizes with the TNF-o~ from dermal dendrocytes to increase keratinocyte pll ' e~ d~iUII and the levels of TGF-r~, IL-8, and IL-6 production.
IFN-y also feeds back to the dermal dt~d~ucyte, ~ ;,,Ldi,,i,,g the activated phenotype and the ill~ldlllllldluiy cycle.
Elevated serum titres of IL-6 increases synthesis of acute phase proteins including c~lll,ul~llle:lll factors by the liver, and antibody production by plasma cells. Increased culll,ulalllt:lll and antibody levels increases the probability of autoimmune reactions.
Al! Itell~dll~,e of the psoriatic plaque requires continued U~,UI~ iUII of all of these processes, but attractive points of therapeutic intervention are TNF-o~ expression by the dermal dendrocyte to maintain activated endothelium and h~rdli"ocyt~s, and IFN-y ex~ s~iuil by T-cells to maintain activated dermal dendrocytes.
There are 3 million patients in the United States afflicted with psoriasis. The available II`~d~ for psoriasis are cullic~ luids. The most widely pl~su,iLed are TEMOVATE (clobetasol prupiol,21~)~ LIDEX
(fluocinonide), DIPROLENE (b~ldll,~ll,asone propionate), PSORCON
(diflorasone diacetate) and TRIAMCINOLONE formulated for topical ip~"- ,. The ",e~;l,a~,;s", of action of co~liu~ ,ui~:, is multifactorial.
This is a palliative therapy because the underlying cause of the disease remains, and upon discontinuation of the treatment the disease returns.
-. 30 Discontinuation of treatment is often prompted by the appearance of adverse effects such as atrophy, telangiectasias and purpura.
Colli.,o:,lt,lui;l~ are not recu~ll"~ ded for prolonged l~dl"~el~l~ or when treatment of large and/or inflamed areas is required. Alternative lltldllllt~
include retinoids, such as etretinate, which has been approved for treatment of severe, refractory psoriasis. Alternative retinoid-based ~l~dllll~ are in advanced clinical trials. Retinoids act by converting _ _ _ _, _ ,, .. , . . ... . _ .... .. .. ~ . =,, .

WO 9S/23225 ~ /lbSS. . 156 2183~2 keratinocytes to a di~ lidl~d state and restoration of normal skin development. Immunosuppressive drugs such as cyl~loaporille are also in the advanced stages of clinical trials. Due to the no"~ ",el;l,d~ "~ of action of corticosteroids, retinoids and immunosuppressives, these 5 l,~d~",e"t~ exhibit severe side effects and should not be used for extended periods of time unless the condition is life-threatening or disabling. There is a need for a less toxic, effective therapeutic agent in psoriatic patients.
HIV and AIDS
The human immunodeficiency virus (HIV) causes several 10 fundamental changes in the human immune system from the time of infection until the development of full-blown acquired immu"ode~ici~,lcy syndrome (AIDS). These changes include a shift in the ratio of CD4+ to CD8+ T-cells, sustained elevation of IL-4 levels, episodic elevation of TNF-a and TNF-B levels, hyp~lyal""~ ",ia, and Iy""~l~u",a/leukemia 15 (Rosenberg & Fauci, 199û Immun. Today 11, 176; Weiss 1993 ~D~
260, 1273). Many patients t,~p~ "ce 8 unique tumor, Kaposi's sarcoma andlor unusual opportunistic infections (e.g. Pneumocystis carinii, cytomegalovirus, herpesviruses, hepatitis viruses, papilloma viruses, and tuberculosis). The immunological dysfunction of individuals with AIDS
20 suggests that some of the pathology may be due to cytokine dysregulation.
Levels of serum TNF-~ and IL-6 are often found to be elevated in AIDS patients (Weiss, 1993 su~ra). In tissue culture, HIV infection of monocytes isolated from healthy individuals stimulates secretion of both TNF- and IL-6. This response has been reproduced using purified gp120, 25 the viral coat protein r~lJoll~iLJle for binding to CD-4 (Buonaguro et al., 1992 J. Virol. 66, 7159). It has also been del"on~L,dl~d that the viral gene regulator, Tat, can directly induce TNF lldll~ Jliol1. The ability of HIV to dir~ctly stimulate secretion of TNF- and IL-6 may be an adaptive l"e~,l,ani:,"l of the virus. TNF-a has been shown to upregulate lldl~ Jli 30 of the LTR of HIV, i"~ asi"g the number of HlV-specific lldllS~ in infected cells. IL-6 enhances HIV production, but at a post-lldnb~ ,t;u"al level, apparently i"~ as;"g the efficiency with which HIV l,dns.,,il,L~ are translated into protein. Thus, stimulation of TNF- secretion by the HIV
virus may promote infection of neighboring CD4+ cells both by enhanciny 35 virus production from latently infected cells and by driving replication of the virus in newly infected cells.
.. , _ _ _ . .. . . .

WO 95123225 21 r~ O~SC

The role of TNF- in HIV ~ has been well e:,ldLl;sl)ed in tissue culture models of infection (Sher et al., 1992 Immun. Rev. 127, 183), suggesting that the mutual induction of HIV replication and TNF-a replication may create positive feedback In vivo. However, evidence for the 5 presence of such positive feedback in infected patients is not abundant.
TNF-a levels are found to be elevated in some, but not all patients tested.
Children with AIDS who were given zidovudine had reduced levels of TNF-a compared to those not given zidovudine (Cremoni et al., 1993 ~ 7, 128). This cor,~ldliol1 lends support to the hypothesis that reduced viral 10 replication is physiologically linked to TNF-a levels. Furthermore, recently it has been shown that the polyclonal B cell activation accori,.l.-d with HIV
infection is due to ",~",~,d,~e-bound TNF-a. Thus, levels of secreted TNGa may not accurately reflect the contribution of this cytokine to AIDS
pathogenesis.
Chronic elevation of TNF-a has been shown to shown to result in cachexia (Tracey et al., 1992 Am. J. Trol~. MP~ Hy~. 47, 2-7), increased autoimmune disease (Jacob, 1992 su~ra), lethargy, and immune suppression in animal models (Aderka et al., 1992 Isr. J. Med. Sci. 28, 126-130). The cachexia associated with AIDS may be A~so~:iAted with 20 chronically elevated TNF-a frequently observed in AIDS patients.
Similarly, TNF-a can stimulate the p~ dLion of spindle cells isolated from Kaposi's sarcoma lesions of AIDS patients (Barillari et al., 1992 J
Immunol 149, 3727).
A therapeutic agent that inhibits cytokine gene ~A~ I~SSioll, inhibits 25 adhesion molecule ~Aprt:,~ioll~ and mimics the anti-i"fla",l"dl~,,y effects of glu~,ocollk;oids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target i"di.ialiol,s and the delivery options each entails are summarized below. In all cases, because of the potential 30 immunosuppressive properties of a ribozyme that cleaves the specified sites in TNF-a mRNA, uses are limited to local delivery, acute illdicaLiOlls~
or ex vJvo treatment.
Septic shock.

WO 9512~i225 218 3 9 9 2 r~l~7~ 6 Exogenous delivery of ribozymes to macrophages can be achieved by intraperitoneal or intravenous injections. Ribozymes will be delivered by i~lccl,uoldliull into liposomes or by c~lllpl~,-il,g with cationic lipids.
Rheumatoid arthritis (RA).
Due to the chronic nature of RA, a gene therapy approach is logical.
Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno~ o~ 1 virus vectors. For instance, the d,U,UI u,UridLe adenovirus vector can be adl"i"isIer~d by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected (B.J. Roessler, E.D. Allen, J.M. Wilson, J.W.
Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple a~i,,i,,i~l,dliu,,s may be necessary. Retrovirus and adeno-~csoci,.l~ virus vectors would lead to pt:""a~ "l gene transfer and eA,ur~s~iull in the joint.
However, p~ a~ uXp~ SiOI~ of a potent anti-i"rlc"""dlury agent may iead to local immune deficiency.
Psoriasis The psoriatic plaque is a particularly good candidate for ribozyme or - 20 vector delivery. The stratum corneum of the plaque is thinned, providing access to the plulil~ lil,g keratinocytes. T-cells and dermal dendrocytes can be efficiently targeted by trans-epidermal diffusion .
Organ culture systems for biopsy ~,ueci,,,e~ls of psoriatic and normal skin are described in current literature (Nickoloff et al., 1993 ~L~).
Primary human keratinocytes are easily obtained and will be grown into epidermal sheets in tissue culture. In addition to these tissue culture models, the flaky skin mouse develops psoriatic skin in response to UV
light. This model would allow delllù~l~lldlioll of animal efficacy for ribozyme llt:dLIll~llt;. of psoriasis.
Gene Therapy.
Immune responses iimit the efficacy of many gene transfer techniques. Cells Lldll~ d with retrovirus vectors have short lifetimes in immune competent individuals. The length of ex,u,~siu,, of adenovirus ~ W095123225 2 1 8 3 9 ~ 2 r~l,lL. `C 156 vectors in terminally di~c,c,,IidIcd cells is longer in neonatal or immune-cu,llylulllis~d animals. Insertion of a small ribozyme ex,ulcssion cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.
Thus, ribozymes of the present inYention that cleave TNF-a mRNA
and thereby TNF-a activity have many potential therapeutic uses, and there are ,casol-able modes of delivering the ribozymes in a number of the possible ill~i~dliol1s. Development of an effective ribozyme that inhibits TNF-a function is described above; available cellular and activity assays are number, reproducible, and accurate. Animal models for TNF-a function and for each of the suggested disease targets exist and can be used to optimize activity.
FYAmple 5 p210bcr-abl Chronic myelogenous leukemia exhibits a characteristic disease 15 course, presenting initially as a chronic granulocytic hyperplasia, and invariably evolving into an acute leukemia which is caused by the clonal expansion of a cell with a less di~clc, -' phenotype a~, the blast crisis stage of the disease). CML is an unstable disease which ultimately pruy,c~ses to a temminal stage which lcsc",l.les acute leukemia. This lethal disease affects approximately 16,000 patients a year.
CI1cllluIllclc-pcutic agents such as hydroxyurea or busulfan can reduce the leùkemic burden but do not impact the life cx~.c~,ldllcy of the patient (ç~
approximately 4 years). Consequently, CML patients are cdll~;ddlcs for bone marrow Ildll:"JldllIdliol1 (BMT) therapy. However, for those patients which survive BMT, disease recurrence remains a major obstacle (Apperley et al., 1988 ~r. J. Hr~r~matol. 69, 239).
The Philadelphia (Ph) ch,ui"osor"e which results from the .lldllslocdliol1 of the abl oncogene from ~ ru~osu~e 9 to the bcrgene on cl,,u,,,os~,,,e 22 is found in greater than 95% of CML patients and in 10-25% of all cases of acute Iymphoblastic leukemia [(ALL); Fourth International Workshop on Chromosomes in Leukemia 1982, ~ç~
Genet. Cyto~enet. 11, 316]. In virtually all Ph-positive CMLs and app,u,~i",d~,ly 50% of the Ph-positive ALLs, the leukemic cells express bcr-abl fusion mRNAs in which exon 2 (b2-a2 junction) or exon 3 (b3-a2 junction) from the major brcakl oi, ,~ cluster region of the bcr gene is spliced WO95/232~ 8;39~2 PCI/IB9S/00156 to exon 2 of the abl gene. I l~;~,t~,.kdr,,,u et al., 1985 Nature 315, 758;
Shtivelman et al., 1987, Blood 69, 971). In the remaining cases of Ph-positive ALL, the first exon of the bcr gene is spliced to exon 2 of the abl gene (Hooberman et al., 1989 Proc. Nat. ~`A~ Sci. USA 86, 4259;
1 Iv;_i~;hdlll,u et al., 1988 Nucleic ~ c Res. 16, 10069).
The b3-a2 and b2-a2 fusion mRNAs encode 210 kd bcr-abl fusion proteins which exhibit oncogenic activity (Daley et al., 1990 ~ 247, 824; ~ lkdl,lpetal., 1990~1~344,251). Thei~ JU~ld~ ofthebcr-abl fusion protein (p210bCr-abl) in the evolution and ",a;"tt,nance of the 10 leukemic phenotype in human disease has been de:lllon~l,dl~d using antisense oligonucleotide inhibition of p210bCr-abl e-~,u,~ ,ion. These inhibitory molecules have been shown to inhibit the in vitro ,u,, ~iC~dliUII of leukemic cells in bone manrow from CML patients. Szczylik et al., 1991 ~i~ 253, 562).
Reddy, U.S. Patent ~,246,921 (hereby incorporated by reference herein) describes use of ribozymes as therapeutic agents for leukemias, such as chronic myelogenous leukemia (CML) by targeting the specific junction region of bcr-abl fusion ~Id~1~c~i,ul~. It indicates causing cleavage by a ribozyme at or near the L,~akp~i"l of such a hybrid ul,lul,losollle, 20 ~.e.;i~i~,..lly it includes cleavage at the sequence GUX, where X is A, U or G. The one example presented is to cleave the sequence 5' AGC AG
AGUU (cleavage site) CM MGCCCU-3'.
Scanlon WO 91/18625, WO 91/18624, and WO 91/18913 and Snyder et al., WO93/03141 and W094/13793 describe a ribozyme effective 25 to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras t:~ul~iol1 in response to extemal stimu~i.
The invention features use of ribozymes to inhibit the development or e,~ ion of a l,dn~rul",ed phenotype in man and other animals by modulating ~Xpl tlaaiOIl of a gene that contributes to the ~ saic l l of CML.
30 Cleavage of targeted mRNAs expressed in pre-neoplastic and l,d,,~ù,,,,ed cells elicits inhibition of the l,~l,b~u,,,,ed state.
The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred a.ll,,i,,i~l,clli~l, protocols can be used, either in vivo a~ dliùi1 to reduce the tumor burden, or ex vivo treatment to ~ WO gS/23225 218 3 9 9 2 r~ .156 eradicate transformed cells from tissues such as bone marrow prior to l ~il l ll~ldl lldliUI~.
This invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associdl~d with development or Illdill~ dllCe of 5 CML. The mRNA targets are present in the 425 nllf~lPotides surrounding the fusion sites of the bcrand abl sequences in the b2-a2 and b3-a2 UII Ibil Idl 11 mRNAs. Other sequences in the 5' portion of the bcrmRNA or the 3' portion of the abl mRNA may also be targeted for ribozyme cleavage.
Cleavage at any of these sites in the fusion mRNA molecules will result in 1 û inhibition of Lldl IsldliUI I of the fusion protein in treated cells.
The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of CML. Such enzymatic RNA molecules can be delivered exogenously or endogenously to afflicted cells. In the preferred hd"""e,l,ead motif the small size (less 15 than 40 rl~cleoticl~s, preferably between 32 and 36 n~lcl~otirl~s in length) of the molecule allows the cost of treatment to be reduced.
The smallest ribozyme delivered for any type of treatment reported to date (by Rossi et al.. 1992 supra) is an in vitro transcript having a length of 142 r~ oticles Synthesis of ribozymes greater than 100 rlll~l~otiries in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by ~ 55kl11 vectors is primarily feasible using only ex vivo t~t:d~lllt:lll`i. This limits the utility of this approach. In this invention, an alternative approach uses smaller ribozyme motifs and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure. Thus, unlike the situation when the hammerhead structure is included within longer ~Idlls~ JlY, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, as well as ~""ul~",~"ldry binding of the ribozyme to - 30 the mRNA target.
The enzymatic RNA molecules of this invention can be used to treat human CML or ,UltlCdllC~IuUS conditions. Aflected animals can be treated at the time of cancer detection or in a prophylactic manner. This timing of treatment will reduce the number of affected cells and disable cellular WO 951232~5 - - r~ 156 2183~92 54 replication. This is possible because the ribozymes are designed to disable those structures required for successful cellular 1 " ~ .dtiUIl.
Ribozymes of this invention block to some extent p21 ObCr-ab ~.,u~ssiu~l and can be used to treat disease or diagnose such disease.
5 Ribozymes will be delivered to cells in culture and to tissues in animal models of CML. Ribozyme cleavage of bcr/abl mRNA in these systems may prevent or alleviate disease symptoms or conditions.
The sequence of human bcr/abl mRNA can be screened for Ar:ces.cihle sites using a computer folding algorithm. Regions of the mRNA
10 that did not form secondary folding structures and that contain potential hd"""e,llead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Table 29 (All sequences are 5' to 3' in the tables). The nucleotide base position is noted in the Tables as that site to be cleaved by the desiy, lal~d type of ribozyme.
15 The sequences ûf the ul":",i~.. "y synthesized ribozymes most useful in this study are shown in Table 30. Thûse in the art will recognize that these sequences are ,~p,~se"lalive only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding amms) is altered to affect activity. For example, stem-loop ll sequence of 20 hammerhead ribozymes listed in Table 30 (5'-GGCCGAAAGGCC-3') can be altered (.Cl~hstitlltinn~ deletion, and/or insertion) to contain any sequenceprovided, a minimum of two base-paired stem structure can form. The sequences listed in Tables 30 may be formed of ribon~lcleotif~fi or other nucleotides or non-r~cleotides. Such ribozymes are equivalent to the 25 ribozymes described s,ue..i~i,,ally in the Tables.
By engineering ribozyme motifs we have designed several ribozymes directed against bcr-abl mRNA sequences. These have been synthesized with Illo.li~iudliolls that improve their nuclease resistance as described above. These ribozymes cleave bcr-abl target sequences in vitro.
The ribozymes are tested for function in vivo by exogenous delivery to cells ~Xur~a~ 9 bcr-abl. Ribozymes are delivered by incorporation into liposomes, by con,,ale,d"g with cationic lipids, by ",ki",i"je~ ,n, or by rt~,iull from DNA vectors. Expression of bcr-abl is monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Levels of W0 95123225 2 1 ~ 3 9 9 2 r~ 156 bcr-abl mRNA are assessed by Northern analysis, RNase protection, by primer extension analysis or by quantitative RT-PCR techniques.
Ribozymes that block the induction of p210bCr-ab~ protein and mRNA by more than 20% are identified.
5 FYRrnple 6: RSV
This invention relates to the use of ribozymes as inhibitors of respiratory syncytial virus (RSV) production, and in particular, the inhibition of RSV replication.
RSV is a member of the virus family paramyxoviridae and is classified 10 under the genus Pneumovirus (for a review see Mclntosh and Chanock, 1990 in Virology ed. B.N. Fields, pp. 1045, Raven Press Ltd. NY). The infectious virus particle is c~"~,uosed of a rlllcleo~ i(l enclosed within an envelope. The n~ eoc3r~id is c~""uosed of a linear negative single-stranded non-sey",erl~:d RNA RC50Ci~lf d with repeating subunits of 15 capsid proteins to fonm a compact structure and thereby protect the RNA
from nuclease dey,addliv". The entire r~llrleoc~l-si~l is enclosed by the envelope. The size of the virus particle ranges from 150 - 300 nm in diameter. The complete life cycle of RSV takes place in the cytoplasm of infected cells and the nucleocapsid never reaches the nuciear 20 compartment (Hall, 1990 in Principles and Practice of Infectious Diseases ed. Mandell et al., Churchill Livingstone, NY).
The RSV genome encodes ten viral proteins essential for viral production. RSV protein products include two structural gly~u,u,~ i"s (G
and F) found in the envelope spikes, two matrix proteins [M and M2 (22K)]
25 found in the inner membrane, three proteins localized in the r~ eo~
(N, P and L), one protein that is present on the surface of the infected cell (SH), and two nonstructural proteins [NS1 (1C) and NS2 (1B)] found only in the infected cell. The mRNAs for the 10 RSV proteins have similar 5' and 3' ends. UV-inactivation studies suggest that a single promoter is used 30 with multiple L,dns~i,ifJlio~ initiation sites (Barik et al., 1992 J. Virol. 66, 6813). The order of l,dns.;,il.ti~ COIl~alJOll~illg to the protein a~siy"",e~l on the genomic RNA is 1C, 1B, N, P, M, SH, G, F, 22K and L genes (Huang et al., 1985 Virus Res. 2, 157) and transcript abundance c~"~ ond~ to the order of gene da~iylllll~lll (for example the 1C and 1B mRNAs are 35 much more abundant than the L mRNA. Synthesis of viral message begins . , _ _ _ _ _ _ _ _ _ _ _ _ _ . . . . .. .

WO 95/2322!i 218 3 9 ~ ~ r~ 07~ 156 immediately after RSV infection of cells and reaches a maximum at 14 hours post-infection (Mclntosh and Chanock, supra).
There are two antigenic subgroups of RSV, A and B, which can circulate simultaneously in the community in varying p~upu~liull:, in different 5 years (Mclntosh and Chanock, supra). Subgroup A usually pltldulllil~dtt:s Within the two subgroups there are numerous strains. By the limited sequence analysis available it seems that homology at the nucleotide level is more complete within than between subgroups, although sequence divergence has been noted within subgroups as well. Antigenic 10 cl~ ld~s result primarily from both surface gly~o~,ul~ " F and G. For F, at least half of the neutralization epitopes have been stably lI,..;,II..;,Idd over a period of 3û years. For G however, A and B subgroups may be related dllli~l1icdlly by as little as a few percent. On the nucleotide level, however, the majority of the divergence in the coding region of G is found 15 in the sequence for the extracellular domain (Johnson et al., 1987, Proc.
Natl. Acad. Sci. USA 84, 5625).
Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract illness during infancy and childhood (Hall, supra) and as such is ~soci~ltd with an estimated 9û,000 I~ - .IS and 450û
20 deaths in the United States alone (Update: respiratory syncytial virus activity ~ United States, 1993, Mmwr Morb Mortal Wkly Rep, 42, 971).
Infection with RSV generally outranks all other microbial agents leading to both pneumonia and bronchitis. While primarily affecting children under two years of age, immunity is not complete and reinfection of older children 25 and adults, especially hospital care givers (Mclntosh and Chanock, supra), is not uncommon. Imm~"ùcu",,u,u",ised patients are severely affected and RSV infection is a major cu", ' 1 for patients u~d~yui~g bone marrow lldl~ lallldliUII .
Uneventful RSV respiratory disease ~ "l~les a common cold and 3û recovery is in 7 to 12 days. Initial symptoms (rhinorrhea, nasal COIl~t:aliull, slight fever, etc.) are followed in 1 to 3 days by lower respiratory tract signsof infection that include a cough and wheezing. In severe cases, these mild symptoms quickly progress to tachypnea, cyanosis, and li~llus:,l,ess and hoc~it~ tion is required. In infants with underlying cardiac or 35 respiratory disease, the ~,uu,~iu,~ of symptoms is especially rapid and can lead to respiratory failure by the second or third day of illness. With WO 9S/23225 218 3 ~ 9 2 r~ ; 156 modem intensive care however, overall mortality is usually less than 5% of Hd patients (Mclntosh and Chanock, supra).
At present, neither an efficient vaccine nor a specific antiviral agent is available. An immune response to the viral surface glycù~ rul~i,,s can 5 provide rH~i~ldl~ce to RSV in a number of experimental animals, and a subunit vaccine has been shown to be effective for up to 6 months in children previously 11u~ with an RSV infection (Tristam etal., 1993, J. Infect. Dis. 167, 191). An attenuated bovine RSV vaccine has also been shown to be effective in calves for a similar length of time (Kubota et a/., 10 1992 J. Vet. Med. Sci. 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in subsequent natural infections with RSV (Connors et aL, 1992 J. Virol. 66, 74~4).
The current treatment for RSV infection requiring 11r~ is the 15 use of aeru~ d ribavirin, a guanosine analog [Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J. Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ud., NY.]. Ribavirin therapy is A~o,.;,.l~-d with a decrease in the severity of the symptoms, improved arterial oxygen and a decrease in the amount of viral shedding at the end of the treatment 2û period. It is not certain, however, whether ribavirin therapy actually shortens the patients' hospital stay or diminishes the need for supportive therapies (Mclntosh and Chanock, supra). The benefits of ribavirin therapy are especially clear for high risk infants, those with the most serious symptoms or for patients with underlying bronchopulmonary or cardiac 25 disease. Inhibition of the viral polymerase complex is supported as the main ",e~l,d"i~." for inhibition of RSV by ribavirin, since viral but not cellular polypeptide synthesis is inhibited by ribavirin in RSV-infected cells (Antiviral Agents and Viral Diseases of Man, 3rd edition. 1990. (eds. G.J.
Galasso, R.J. Whitley, and T.C. Merigan) Raven Press Ltd., NY]. Since 30 ribavirin is at least partially effective against RSV infection when delivered by ae,us~ dliu~, it can be assumed that the target cells are at or near the epithelial surface. In this regard, RSV antigen had not spread any deeper than the superficial layers of the respiratory epithelium in autopsy studies of fatal pneumonia (Mclntosh and Chanock, supra).
3~ Jennings et a/., WO 94/13688 indicates that targets for specific types of ribozymes include respiratory syncytical virus.
. _ _ _ _ . ... . . ...

wo gsl2322~ 21~ 3 9 ~ 6 The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting production of respiratory syncytial virus (RSV). Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals. The 5 invention also features cleavage of the genomic RNA and mRNA of these viruses by use of ribozymes. In particular, the ribozyme molecules described are targeted to the NS1 (lc), NS2 ~lB) and N viral genes.
These genes are known in the art (for a review see Mclntosh and Chanock, 1 990 supra ).
Ribozymes that cleave the specified sites in RSV mRNAs represent a novel therapeutic approach to respiratory disorders. Applicant indicates that ribozymes are able to inhibit the activity of RSV and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described 15 that other ribozymes that cleave these sites in RSV mRNAs encoding 1 C, lB and N proteins may be readily designed and are within the invention.
Also, those of ordinary skill in the art, will find that it is clear from the examples described that ribozymes cleaving other mRNAs encoded by RSV (P, M, SH, G, F, 22Kand L) and the genomic RNA may be readily 20 designed and are within the invention.
In preferred t!",bodi",~:"l~, the ribozymes have binding amms which are c~"lpl~"lt,~ y to the sequences in Tables 31, 33, 35, 37 and 38.
Examples of such ribozymes are shown in Tables 32, 34, 36-38. Examples of such ribozymes consist essentially of sequences defined in these 25 Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding amms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
Ribozymes of this invention block to some extent RSV production and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of respiratory disorders. Ribozyme cleavage of RSV encoded mRNAs or the genomic RNA in these systems may alleviate disease symptoms.

W0 95123~25 21~ 3 91 ~ 2 r~ 156 ~9 While all ten RSV encoded proteins (1C, 1B, N, P, M, SH, 22K, F, G, and L) are essential for viral life cycle and are all potential targets for ribozyme cleavage, certain proteins (mRNAs) are more favorable for ribozyme targeting than the others. For example RSV encoded proteins 1C, 5 1 B, SH and 22K are not found in other members of the family - paramyxoviridae and appear to be unique to RSV. In contrast the ~uludu~ail~ of the G protein and the signal sequence of the F protein show significant sequence divergence at the nucleotide level among various RSV sub-groups (Johnson etal., 1987 supra). RSV proteins 1C, 1B and N
1 û are highly conserved among various subtypes at both the nucleotide and amino acid levels. Also, 1C, 1B and N are the most abundant of all RSV
proteins.
The sequence of human RSV mRNAs encoding 1 C, 1 B and N
proteins are screened for ~ccessible sites using a computer folding 15 algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 31, 33, 34, 37 and 38 (All sequences are 5' to 3' in the tables.) The nucleotide base position is noted in the Tables as that site to be cleaved by the desi_"dLt:d type of ribozyme.
Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845-7854 and in Scaringe et al., 199û
Nucleic Acids Res., 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as ~ lllu~yl~ilyl at the 5'-end, and pilOb,ul1ordl"i~ s at the 3'-end The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for 3û Gs and a U forA14 (numbering from Hertel et al., 1992 Nucleic Acids Res., 20, 3252). Hairpin ribozymes are sy"ll,~bi~d in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-284û). Hairpin ribozymes are also synthesized from DNA
templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified "bivcly to enhance stability by modification with nuclease resistant ... . . ... ... _ . _ .. _ , , _ _ _ groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel eleullupl,o,~sis using general methods or are purified by high pressure liquid ~ lUllldlu~ld,ully and are resuspended in water.
The sequences of the ~ t,llliu~'ly synthesized ribozymes useful in this study are shown in Tables 32, 34, 36, 37 and 38. Those in the art will recognize that these sequences are r~ "~dli~/e only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop ll sequence of 11d"""~,1,edd ribozymes listed in Tables 32 and 34(5'-GGCCGAMGGCC-3') can be altered (sllhstitlltinn~ deletion, and/or insertion~ to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 37 and 38 (~'-CACGUUGUG-3') can be altered (sllhstit~ltion~
deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 32, 34, 36, 37 and 38 may be fommed of ribonucleotides or other n~lcl~otiriPs or non-r~cl~oti~4s Such ribozymes are equivalent to the ribozymes described specifically in the Tables.
By el1~i"eeri"g ribozyme motifs we have designed several ribozymes directed against RSV encoded mRNA sequences. These ribozymes are synthesized with ",- ';" ' )s that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro is evaluated.
Numerous, common cell lines can be infected with RSV for experimental purposes. These include HeLa, Vero and several primary epithelial cell lines. A cotton rat animal model of clx~ dl human RSV
infection is also avaiiable, and the bovine RSV is quite homologous to the human viruses. Rapid clinical diagnosis is through the use of kits designed for the immunoflu~,~scel,ce staining of RSV-infected cells or an ELISA
assay, both of which are adaptable for ~pe,i",~"lal study. RSV encoded mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of RSV activity andlor 1C, lB and N protein encoding mRNAs by more than 90% will be identified.

-W0 95123225 r~ 56 218~9~2 O~timi7irlg Ribozvme Activity Ribozyme activity can be optimized as described by Drapor et al., PCT
W093/23569. The details will not be repeated here, but include altering the length of the ribozyme binding arms or chemically synthesizing 5 ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Eckstein et al., International Publication No.
WO 92/07065; Perrault et al., 1990 ~1~ 344, 565; Pieken et al., 1991 ~j~ 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci.
17, 334; Usman et al., I"L~r"dlional Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which describe various chemical modi~kidliul1s that can be made to the sugar moieties of enzymatic RNA
molecules. All these p~ s are hereby incorporated by reference herein.), Illodi~iudliul~s which enhance their efficacy in cells, and removal of15 stem ll bases to shorten RNA synthesis times and reduce chemical requirements.
Sullivan, et al., PCT W094/02595, illCo,~o,dl~:d by reference herein, describes the general methods for delivery of enzymatic RNA molecules .
Ribozymes may be ad",i"i~ d to cells by a variety of methods known to 20 those familiar to the art, including, but not restricted to, erlc~rclll~tion in liposomes, by iontophoresis, or by ill~,OI~JUldliOI~ into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.
25 Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed Jes.i,i,uliu,,s of ribozyme delivery and ad",i"ial~d~iol1 are provided in Sullivan, et al., supra and Draper, et al.,30 supra which have been ill..ùl,Uuld~d by reference herein.
Another means of accumulating high cùllcelllldliulls of a ribozyme(s) within cells is to ill~UI~JUldl~ the ribozyme-encoding sequences into a DNA
~,urtss~ion vector. Tldl1~uli~ioll of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol 1), RNA polymerase ll 35 (pol ll), or RNA polymerase lli (pol lll). Transcripts from pol ll or pol lllpromoters will be expressed at high levels in all cells; the levels of a given WO 95/23225 1~ r ~ 156 . 21~39~2 pol ll promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic RNA polymerase promoters are also used providing that the prokaryotic RNA polymerase enzyme is expressed in the dU~UlUplidl~ cells 5 (Elroy-Stein and Moss, 1990 Proc. ~f~t~ A~ Sci. U S A. 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res. 21 2867-72; Lieber et al. 1993 Methods Fn7ymol. 217 47-66; Zhou et al., 1990 Mol. Cell. Biol..~10 4529-37). Several illlr~.ti~d~ul~ have d~lllu~ al~d that ribozymes ~u~ 7sed from such promoters can function in l"ar""~ cells (e.g. Kashani-Sabet 10 et al. 1992 Antisense Res. Dev.. 2 3-15; Ojwang et al. 1992 Proc. I~IAtl Acad. Sci. U S A. 89, 108û2-6; Chen et al. 1992 Nucleic Acids Res.. 20 4581-9; Yu et al. 1993 Proc. NAtl At:R~ Sci. U S A. 90 6340-4; LHuillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. NAtl Acad.
Sci. U. S. A.. 90 8000-4). The above ribozyme Irdllscli~uliul~ units can be 15 i"~o"uoldltld into a variety of vectors for introduction into Illdlllll 1 cells including but not restricted to plasmid DNA vectors viral DNA vectors (such as adenovirus or adeno ~sou;rl. d virus vectors) or viral RNA
vectors (such as retroviral or alpha virus vectors).
In a preferred 6",Lodi",e"t of the invention, a lldl~suliuliull unit 20 ~Xu,t,~;,;"g a ribozyme that cleaves target RNA is inserted into a plasmid DNA vector, a retrovirus DNA viral vector an adenovirus DNA viral vector or an adeno-~ d virus vector or alpha virus vector. These and other vectors have been used to transfer genes to live animals (for a review see Friedman, 1989 Science 244, 1275-1281; Roemer and Friedman, 1992 25 Eur. J. Biochem. 208 211-225) and leads to transient or stable gene ex,~ ,siù,1. The vectors are delivered as r~cu",Li"d"l viral particles. DNA
may be delivered alone or culllul~x~d with vehicles (as described for RNA
above). The DNA, DNA/vehicle c~lllpl~ s or the rec~",Li"ar,l virus particles are locally a.l",i"i~ d to the site of treatment e.g., through the 30 use of a catheter stent or infusion pump.
Dia~nostic uses Ribozymes of this invention may be used as diagnostic tools to examine genQtic drift and mutations within diseased cells. The close l`~lldliUll~lliU between ribozyme activity and the structure of the target RNA
35 allows the detection of mutations in any region of the molecule which alters the base-pairing and three-di",~"siu"al structure of the target RNA. By WO 95123225 218 3 ~ 9 2 PCT/IB95/00156 using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function In vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene ~.,u,t7ssiol~ and define the role 5 (esse, "y) of specified gene products in the pluyl~ssiol1 of disease. In - this manner, other genetic targets may be defined as important mediators of the disease. These experiments will lead to better treatment of the disease pluylt:~;ull by aflording the possibility of c~"~i,i"..~ ,al therapies (e.~., multiple ribozymes targeted to different genes, ribozymes coupled 10 with known small molecule inhibitors, or intermittent treatment with Collll,illdliol,s of ribozymes and/or other chemical or biological molecules).
Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA ~o,.i~l~d with ICAM-1, relA, TNF-a, p210, bcr-abl or RSV related condition. Such RNA is detected 15 by d~L~""i"i"g the presence of a cleavage product after treatment with a ribozyme using standard Ill~ o iuloyy.
In a speoific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second 20 ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "non-targeted" RNA species. The cleavage products from the synthetic 25 substrates will also serve to generate size markers for the analysis of wild- type and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions. The presence of cleavage products wili be determined using an RNAse protection assay so that full-length and 30 cleavage fragments of each RNA can be analyzed in one lane of a polyd.i,yl.."l;de gel. It is not absolutely required to quantify the results to gain insight into the ~,ul~SaiOIl of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The ~A,ul~ssiull of mRNA
whose protein product is implicated in the development of the phenotype 35 (i.e., ICAM-1, rel A, TNFoc, p210bCr-abl or RSV) is adequate to establish risk. If probes of cu",pardi,l~ specific activity are used for both lldlls~ Jlalthen a qualitative c~",pari~ùl1 of RNA levels will be adequate and will .

WO9S/23225 2183992 r~l,~r~ lS6 decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared q~ ely or quantitatively.
Il. Chemical Sy"ll,~sis Of p~ Jy~ es There follows the chemical synthesis, d~.,ulæuliu,,, and purification of RNA, enzymatic RNA or modified RNA molecules in greater than milligram quantities with high biological activity. Applicant has d~ ",i"ed that the synthesis of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its preparation.
Specifically. it is important that the RNA pl~o~,ullorc,l"idi~t,s are coupled efficiently in temms of both yield and time, that correct exocyclic amino protecting groups be used, that the ap~u,ulidl~ conditions for the removal of the exocyclic amino protecting groups and the alkylsilyl protecting groups on the 2'-hydroxyl are used, and that the correct work-up and purification procedure of the resulting ribozyme be used.
To obtain a correct synthesis in temms of yield and biological activity of a large RNA molecule (i.e., about 30 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or s~h.stitut~d amide protecting groups, which must be, on the one hand, stable enough to survive the conditions of synthesis, and on the other hand, removable at the end of the synthesis. These requirements are met by the amide protecting groups shown in Figure 8, in particuiar, benzoyl for adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine, which may be removed at the end of the synthesis by incubating the RNA in NH3/EtOH
(ethanolic ammonia) for 20 h at 65 C. In the case of the phenoxyacetyl type protecting groups shown in Figure 8 on guanosine and adenosine and acetyl protecting groups on cytidine, an incubation in ethanolic ammonia for 4 h at 65 C is used to obtain complete removal of these protecting groups. Removal of the alkylsilyl 2'-hydroxyl protecting groups can be accu,,,pl;~l,ed using a tetrahydrofuran soiution of TBAF at room temperature for 8-24 h.
The most quantitative procedure for recovering the fully dep,u~ d RNA molecule is by either ethanol pl~ui,uildliùl~, or an anion exchange cartridge desalting, as described in Scaringe et al. Nucleic Acids Res.
35 1990, 18, 5433-5341. The purification of the long RNA sequences may be ~ W0 95123~25 2 1 8 3 9 9 2 r ~ ~ sG

accomplished by a two-step chlurlla~uylapllic procedure in which the molecule is first purified on a reverse phase column with either the trityl group at the 5' position on or off. This purification is accu",,u'i~ ed using anacetonitrile gradient with triethyl~ "~o~ rn or bicarbonate salts as the 5 aqueous phase. In the case of the trityl on purification, the trityl group maybe removed by the addition of an acid and drying of the partially purified RNA molecule. The final purification is carried out on an anion exchange column, using alkali metal pe~,l,lordl~ salt gradients to elute the fully purified RNA molecule as the d~u,u~up~idle meta! salts, e.g. Na+, Li+ ~tc. A
10 final de-salting step on a small reverse-phase cartridge cu""ul~las the purification procedure. Applicant has found that such a procedure not only fails to adversely affect activity of a ribozyme, but may improve its activity to cleave target RNA molecules.
Applicant has also dalallllilled that significant (see Tables 39-41) 15 improvements in the yield of desired full length product (FLP) can be obtained by:
1. Using 5-S-alkyltetrazole at a delivered or effective cù, ICallllu~iOl1 of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA (oranalogue) amidite during the coupling step. (By delivered is meant that the 2û actual amount of chemical in the reaction mix is known. This is possible for large scale synthesis since the reaction vessel is of size sufficient to allow such manipulations. The term effective means that available amount of chemical actually provided to the reaction mixture that is able to react with the other reagents present in the mixture. Those skilled in the art will 25 recognize the meaning of these temms from the examples provided herein.) The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.
Alkyl, as used herein, refers to a saturated aliphatic hyd,uca,bùll, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 30 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be s~hstitl~ ~ or ur~fi~hstit~tP~I When s~hstit~t~d the ~hstit~tRd group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO2 or N(CH3)2, amino, or SH.
- The tenm also includes alkenyl groups which are unsaturated hydrocarbon groups co,,lc,i,,i,,g at least one carbon-carbon double bond, including 35 straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to ... . . . ... . _ _ _ _ _ _ _ _ _ _ _ . . . . . .... .

WO 9~/23225 2 1 8 ~ 9 ~ 2 P~ 6 ~

7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be c, Ihstitl It~d or url.s~ IhCtitl ItP~ When sl ~hstit~ ~ted the s~ ~hstit~ ~ -' group(s) is preferably, hydroxyl, cyano, alkoxy, =0, =S, N02, halogen, N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups which have an 5 unsaturated hyt lu~ luoll group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be s~hstit~t~Pd or urlcllhctitl~ When s~hstitlltPd the 10 s~hstit~tPd group(s) is preferably, hydroxyl, cyano, alkoxy, =0, =S, NO2 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conj~gated ~ electron 15 system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally sl~hstit'~ The preferred substituent(s) of aryl groups are halogen, l,i~,a!~,l"~t.lyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as 20 described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally sllhctitll~ Heterocyclic aryl groups are groups having from 1 to 3 h~t,,ùdlu,l,s as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable h~t, ludlulll~ include oxygen, sulfur, 25 and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally .511hStitlltP~ An ~amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
2. Using 5-S-alkyltetrazole at an effective, or final, col,c~"l,dli~
of 0.1-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. The time for this step is shortened from 10-15 m, vide supra, to5-lOm.
3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl, propyl or butyl) or NH40H/alkylamine (AMA, with the same preferred alkyl groups as noted for MA) ~ 65 C for 10-15 m to remove the exocyclic ~ WO 9S123~2S 2 1 ~ 3 9 ~ 2 P~ S6 amino protecting groups (vs 4-20 h ~ 55-65 C using NH40H/EtOH or NH3/EtOH, vide supra). Other ~ yl..."i"es, e.g. ethylamine, propylamine, butylamine etc. may also be used.

4. Using anhydrous triethylamine-hydrogen fluoride (aHF-TEA) ~ 65 C for 0.5-1.5 h to remove the 2'-hydroxyl alkylsilyl protecting group (vs 8 - 24 h using TBAF, vlde supra or TEA-3HF for 24 h (Gasparutto et al.
Nucleic Acids Res. 1992, 20, 5159-5166). Other alkylamine-HF
CUIII,UI~ACIS may also be used, e.g. trimethylamine or diisopropylethylamine.

5. The use of anion-exchange resins to purify and/or analyze the fully deprotected RNA. These resins include, but are not limited to, quartenary or tertiary amino derivatized stationary phases such as silica or polystyrene. Specific examples include Dionex-NA100~1D, Mono-Qa~, Poros-Q~.
Thus, the invention features an improved method for the coupling of RNA pho~,ul10rdllliu~ for the removal of amide or s~hstitl~t~d amide protecting groups; and for the removal of 2'-hydroxyl alkylsilyl protecting groups. Such methods enhance the production of RNA or analogs of the type described above (e.g., with s~hstit~' ~' 2'-groups), and allow efficient synthesis of large amounts of such RNA. Such RNA may also have enzymatic activity and be purified without loss of that activity. While specificexamples are given herein, those in the art will recognize that equivalent chemical reactions can be perfommed with the alternative chemicals noted above, which can be optimized and selected by routine ~A,ue~ ldliull.
In another aspect, the invention features an improved method for the purification or analysis of RNA or enzymatic RNA molecules (e.g. 28-70 nucleotides in length) by passing said RNA or enzymatic RNA molecule over an HPLC, e.g., reverse phase and/or an anion exchange .illrUll~alUyld,Ully column. The method of purification improves the catalytic activity of enzymatic RNAs over the gel purification method (see Figure 10).
Draper et al., PCT W093/23569, illC~l~uuld~d by reference herein, disclosed reverse phase HPLC purification. The purification of long RNA
molecules may be acc~",~ "led using anion exchange ulllullldIuyldplly, particularly in conjunction with alkali p~lullluld~ salts. This system may be used to purify very long RNA molecules. In particular, it is advantageous to WO95123225 21839~2 r~.,~. :156 ~

use a Dionex NucleoPak 100~) or a Pharmacia Mono Q(D anion exchange column for the purification of RNA by the anion exchange method. This anion exchange purification may be used following a reverse-phase purificstion or prior to reverse phase purification. This method results in the formation of a sodium salt of the ribozyme during the ~ lullldluyldpl~y.
Repla~,~,,,c,,l of the sodium alkali earth salt by other metal salts, e.g., lithium, magnesium or calcium p~lulll~ldL~, yields the ~o~ ,uùl,.li,,g salt of the RNA molecule during the purification.
In the case of the 2-step purification procedure, in which the first step 1 û is a reverse phase purification followed by an anion exchange step, the reverse phase purification is best acco,,,ulisl,ed using polymeric, e.g.
polystyrene based, reverse-phase media, using either a 5'-trityl-on or 5'-trityl-off method. Either molecule may be recovered using this reverse-phase method, and then, once dt,llilyl~ , the two fractions may be pooled 15 and then submitted to an anion exchange purification step as described above.
The method includes passing the el,zy",dlically active RNA
molecule over a reverse phase HPLC column; the enzymatically active RNA molecule is produced in a synthetic chemical method and not by an 20 enzymatic process; and the enzymatic RNA molecule is partially blocked, and the partially blocked e"~y~"dli~,~lly active RNA molecule is passed over a reverse phase HPLC column to separate it from other RNA
molecules.
In more preferred ~ Odi~ a, the enzymatically active RNA
25 molecule, after passage over the reverse phase HPLC column, is d~urult~-,ltld and passed over a second reverse phase HPLC column (which may be the same as the reverse phase HPLC column), to remove the enzymatic RNA molecule from other c~""uon~"li. In addition, the column is a silica or organic polymer-based C4, C8 or C18 column having 30 a porosity of at least 125 A, preferably 300 A, and a particle size of at least 2 llm, preferably 5 ,um.
Activation The synthesis of RNA molecules may be accu,,,~,lk.l,ed ul,e",i.ially or enzymatically. In the case of chemical synthesis the use of tetrazole as an 35 Qctivator of RNA ,ulloa,ul,oldll " - is known (Usman etal. J. Am. Chem.

wo gslf^~f f s r~ I!;C
~ 218~,9g~

Soc. 1987, 109, 7845-7854). In this, and sl~hfieq~Pnt reports, a 0.5 M
solution of tetrazole is allowed to react with the RNA pll~:~,ullolalllidil~ andcouple with the polymer bound 5'-hydroxyl group for 10 m. Applicant has t dt~ ""i"ed that using 0.25-0.5 M solutions of 5-S-alk~ d,vles for only 5 5 min gives equivalent or better results. The following exemplifies the - procedure.
F~r~le 7: Synthesis of RNA and Ribozymes Usin~ 5-~-Alk~ ll,.f.,l~.c Activating A~ent The method of synthesis used follows the general procedure for RNA
10 synthesis as described in Usman et al., 1987supra and in Scaringe et al., Nucl~ic Acids Fles 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and pho~ o~d",idil~s at the 3'-end. The major difference used was the activating agent, 5-S-ethyl or -methyltetrazole ~ 0.25 M
15 cu"c~i ,l, , for 5 min.
All small scale syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 ,umol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 m coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 IlL of 0.1 M = 32.5 llmol) of ,ull~:,,uholdllliu;:~ and a 40-fold excess of S-ethyl tetrazole (400 IlL of 0.25M = 100 ,umol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colori",~l,ic quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF;
oxidation solution was 16.9 mM Iz, 49 mM pyridine, 9% water in THF.
Fisher Synthesis Grade dGt,~unil,ile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from 30 the solid obtained from Applied Biosystems.
All large scale syntheses were conducted on a modified (eight amidite port capacity) 390Z (ABI) synthesizer using a 25 llmol scale protocol with a 5-15 min coupling step for alkylsilyl protected RNA and 7.5 m coupling step for 2'-O-methylated RNA. A six-fold excess (1.5 mL of 0.1 M = 150 ~Lmol) of .11o~,ul1oldllli~ and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of .. . . . . . . .

wo 9!i/23225 218 3 9 9 2 r~l,~ 5 ~ ~ 156 ~

0.25 M = 1125 ,umol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 390Z, dt,lt""i"ed by colorimetric quantitation of the trityl fractions, was 95.0-96.7%.
Oligonucleotide synthesis reagents for the 390Z: Detritylation solution was 5 2% DCA in methylene chloride, capping was perfommed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF;
oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.
Fisher Synthesis Grade ac~lol,illil~ was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25-0.5 M in acJtol,il,il~) was made up 10 from thô solid obtained from Applied Biosystems.
~vl ul~iliùl~
The first step of the depl ul~ lion of RNA molecules may be a.cu,,,,.,l;Ol~ed by removal of the exocyclic amino protecting groups with either NH40H/EtOH:3/1 (Usman etaL J. Am. Chsm. Soc. 1987, 109, 7845-15 7854) or NH3/EtOH (Scaringe et al. I\lucleic Acids Res. 1990, 18, 5433-5341) for ~20 h ~ 55-65 C. Applicant has determined that the use of methylamine or NH40H/methylamine for 10-15 min ~ 55-65 C gives equivalent or better results. The following e..~" I~ it,s the procedure.
EY~rnDle 8: RNA and Rihn7yme D~ulul~ of EYn~,yclic Amino 20 Prûtecting Groups Usin~ Methylamine (MA) or NH~OH/Methylamine (AMA) The polymer-bound o~igonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or NH40H/methylamine (AMA) e~ 55-65 C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oligoribonucleotide was llclllo~t~lled from the 25 synthesis column to a 4 mL glass screw top vial. NH40H and aqueous methylamine were pre-mixed in equal volumes. 4 mL of the resulting reagent was added to the vial, ellll ' ' u for 5 m at RT and then heated at 55 or 65 C for 5-15 min. After cooling to -20 C, the supernatant was removed from the polymer support. The support was washed with 1.0 mL
3û of EtOH:MeCN:H20/3:1:1, vortexed and the supematant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The same procedure was followed for the aqueous ",~ l..",i"e reagent.
Table 40 is a summary of the results obtained using the improvements outlined in this ~ for base d~,u~ul~uliùn.

W095123~5 . r~, l l56 The second step of the deprotection of RNA molecules may be acco,l,pl;~l1ed by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman e~ al. J. Am. Chem. Soc. 1987, 109, 7845-7854). Applicant has v~ ed that the use of anhydrous TEA-HF in N-methylpyrrolidine (NMP) for û.5-1.5 h ~ 55-65 C gives equivalent or better results. The following ~A~ iri~s this procedure.
FYRmDle 9: RNA and Rihn~yme D~vlvlavliùl~ of 2'-Hydroxyl Alkylsilyl Protectin~ GrouDs Usin~ Anhydrous TE~-HF
To remove the alkylsilyl protecting groups, the ammonia-d~vlulevlt~d oligoribonucleotide was resuspended in 250 uL of 1.4 M anhydrous HF
solution (1.5 mL N-methylpyrrolidine, 750 IlL TEA and 1.0 mL TEA-3HF) and heated to 65 oc for 1.5 h. 9 mL of 50 mM TEAB was added to quench the reaction. The resulting solution was loaded onto a Qiagen 500~1D anion exchange cartridge (Qiagen Inc.) !~ ed with 10 mL of 50 mM TEAB.
After washing the cartridge with 10 mL of 50 mM TEAB, the RNA was eluted with 10 mL of 2 M TEAB and dried down to a white powder.
Table 41 is a summary of the results obtained using the improvements outlined in this ~ ' , for alkylsily! d~ulult:.liu,,.
FYArnple 10: HPLC Purification. Anion Exchan~e ~ mn For a small scale synthesis, the crude material was diluted to 5 mL
with diethylpyrocarbonate treated water. The sample was injected onto either a Pharmacia Mono Q3~ 16/10 or Dionex NucleoPaca~ column with 100% buffer A (10 mM NaClO4). A gradient from 180-210 mM NaClO4 at a rate of 0.85 mM/void volume for a Pharmacia Mono Q~lD anion-exchange column or 100-150 mM NaClO4 at a rate of 1.7 mM/void volume for a Dionex NucleoPac~ anion-exchange column was used to elute the RNA.
Fractions were analyzed by a HP-1090 HPLC with a Dionex NucleoPac~!D
column. Fractions containing full length product at 280% by peak area were pooled.
For a trityl-off large scale synthesis, the crude material was desalted by applying the solution that resuited from quenching of the desilylation reaction to a 53 mL Pllalllld~iid HiLoad 26/10 Q-Sepllalvs~c Fast Flow column. The column was thoroughly washed with 10 mM sodium per-;llloldl~ buffer. The oligonucleotide was eluted from the column with WO 95/23225 2 1 8 3 9 9 2 = ; P~ S,'~ 6 ~
300 mM sodium pe~ ,al~. The eluent was quantitated and an analytical HPLC was run to detemmine the percent full length material in the synthesis.
The eluent was diluted four fold in sterile H2O to lower the salt c~llce,,l,dliu,, and applied to a Pharmacia Mono Qx 16/10 column. A
gradient from 10-185 mM sodium per,l,lo,dl~ was run over 4 column volumes to elute shorter sequences, the full length product was then eluted in a gradient from 185-214 mM sodium p~lullluldl~ in 30 column volumes.
The fractions ûf interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPa,,~: column. Fractions containing over 85% full length material were pooled. The pool was applied to a Pharmacia RPC~ column for desalting.
For a trityl-on large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pl,al",auid HiLoad 26/10 Q-Sepharoseg Fast Flow column. The column was thoroughly washed with 20 mM NH4CO3H/10%
CH3CN buffer. The oligonucleotide was eluted from the column with 1.5 M
NH4CO3H/10% ac~u"illil~. The eluent was quantitated and an analytical HPLC was run to detemmine the percent full length material present in the synthesis. The oligonucleotide was then applied to a Pharmacia Resource RPC column. A gradient from 20-55% B (20 mM NH4CO3H/25% CH3CN, buffer A = 20 mM NH4CO3H/10% CH3CN) was run over 35 column volumes. The fractions of interest were analyzed on a HP-1 09û HPLC with a Dionex NucleoPac~!D column. Fractions containing over 60% full length material were pooled. The pooled fractions were then submitted to manual detritylation with 80% acetic acid, dried down i"""edidlt,ly, resuspended in sterile H2O, dried down and resuspended in H2O a0ain. This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac3 column. The material was purified by anion exchange Clllullldluyld~Jl,y as in the trityl-offscheme (vide supra).
F~Dle 11 Rihn~ymeActivityAssay Purified 5'-end labeled RNA substrates (15-25-mers) and purified 5'-end labeled ribozymes (-36-mers) were both heated to 95 C, quenched on ice and e~ll)"' dl~d at 37 C, separately. Ribozyme stock solutions were 1 ~LM, 200 nM, 40 nM or 8 nM and the final substrate RNA
cùl~ce"~,dliulls were - 1 nM. Total reaction volumes were 50 IlL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCI2. Reactions were ~ WO 95t23225 2 1 8 ~ g g2 P~,l,..,. 5'~-156 initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 IlL were removed at time points of 1 5, 15, 30, 60 and 120 m. Each aliquot was quenched in formamide loading buffer and loaded onto a 15%
t denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a uI,u,ul,ori,,,ayt,r (Molecular Dynamics).
F~tRrnole 12: One Dot dt:ulule~. tiv~l of RNA
Applicant has shown that aqueous methyl amine is an efficient reagent to deprotect bases in an RNA molecule. However, in a time consuming step (2-24 hrs), the RNA sample needs to be dried completely prior to the de~,ul~.liu" of the sugar 2'-hydroxyl groups. Additionally deprute~.liùl~ of RNA s~",ll,esi~ed on a large scale (e.g. 100 umol) becomes ~;l le~ )yil ,9 since the volume of solid support used is quite large.
In an attempt to minimize the time required for d~,ulult~.tioll and to simplify the process of d~luLt~ iol~ of RNA synthesized on a large scale, applicant describes a one pot de~lul~, liol~ protocol (Fig. 12). According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base d~u,u~:uliùn is carried out at 65 C for 15 min and the reaction is allowed to cool for 10 min. D~ulut~uliol~ of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA-3HF reagent.
The reaction is quenched with 16 mM TEAB solution.
Referring to Ei~, hammerhead ribozyme targeted to site B is synthesized using RNA pllo~ul~o~d",a~ chemistry and d~ulul,:ul~d using either a two pot or a one pot protocol. Profiles of these ribozymes on an HPLC column are compared. The figure shows that RNAs dt:u~ut~ult:d by either the one pot or the two pot protocols yield similar full-length product profiles. Applicant has shown that using a one pot d~ulu~e:.1iul~ protocol time required for RNA d~u,ul~u~iull can be reduced col~idt"dbly without cu" ",, u, l li:~il Iy the quality or the yield of full length RNA.
Referring to Fig. 14, hammerhead ribozymes targeted to site B (from Ei9~ are tested for their ability to cleave RNA. As shown in the fi~ure 14, ribozymes that are deprult~ d using one pot protocol have catalytic activity cu,,,uardble to ribozymes that are deprotected using a two pot protocol.

WO 9S/232~5 218 3 3 9 2 74 r~ . IS6 FY~rnDle 12a:1mproved vrotocol fDrthe synthesis of pl1D~uh~ioll~
cont~ininq RNA ~nd ribozymcs usinQ ~-S-AIkyl~ as Activ;7tin~
The two sulfu-izing reagents that have been used to sy"~l,esi~
5 li~opl,osph~,u~l,iodl~s are tetraethylthiuram disulfide (TETD; Vu and Hirschbein,1991 Te~rahedronLetter31,3005),and3H-1,2-be,,~ouitl,iu1-3-one 1,1-dioxide (Beaucage reagent; Vu and Hi,~..lli)~i,l, 1991 supra).
TETD requires long sulfurization times (6ûO seconds for DNA and 36ûû
seconds for RNA). It has recently been shown that for sulfurization of DNA
1 û oligonucleotides, Beaucage reagent is more efficient than TETD
(Wyrzykiewicz and Ravikumar, 1994 ~ioorganic Med. Chem. 4, 1519).
Beaucage reagent has also been used to ~y"ll,e~ us~uhon~ d~t, oligonu~leotids~ containing 2'-deoxy-2'-fluoro Illo~lilicdliu"s wherein the wait time is 1 û min (Kawasaki et al., 1992 J. Med. Chem).
The method of synthesis used follows the procedure for RNA
synthesis as described herein and makes use of common nucleic acid protecting and coupling groups, such as di,l,~l,oA~trityl at the 5'-end, and pll~:,plluld-,,idila:~ at the 3'-end. The sulfurization step for RNA described in the literature is a 8 second delivery and 1û min wait steps (Beaucage 2û and Iyer, 1991 Tt~l~ahe~ 49, 6123). These conditions produced about 95% sulfurization as measured by HPLC analysis (Morvan et al., 199û
Tetrahedron Letter31, 7149). This 5% cu,,~d,,,i,,~lillg oxidation could arise from the presence of oxygen dissolYed in solvents and/or slow release of traces of iodine adsorbed on the inner surface of delivery lines during 25 previous synthesis.
A major improvement is the u$e of an activating agent, 5-S-ethyltetrazole or 5-S-methyltetrazole at a cul~ ldliol~ of 0.25 M for 5 min.
Additionally, for those linkages which are phos,uo~ul~,iodl~, the iodine solution is replaced with a O.û5 M solution of 3H-1,2-b~ udill,;ole-3-one 3û 1,1-dioxide (Beaucage reagent) in acetonitrile. The delivery time for the sulfurization step is reduced to 5 seconds and the wait time is reduced to 30û seconds.
RNA synthesis is conducted on a 394 (ABI) syntheslzer using a modified 2.5 llmol scale protocol with a reduced 5 min coupling step for 35 alkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 IlL of û.1 M = 32.5 ,~mol) of pl~o~,ullc.ldllli.lil~
RECT~FiED SHEET (RULE 91) ISA/EP

WO 951232~5 21 8 3 9 9 2 r~ L~r ~156 and a 40-fold excess of S--ethyl tetrazole (400 IlL of 0.25 M = 100 umol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle.
Average coupling yields on the 394 synthesizer"~ ""i"ed by culolill~
+ quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide 5 synthesis reagents for the 394 synthesizer: detritylation solution was 2%
TCA in methylene chloride; capping was perfommed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF;
oxidation solution was 16.9 mM 12, 49 mM pyridine 9% water in THF.
Fisher Synthesis Grade acetonitrile was used directly from the reagent 10 bottle. S-Ethyl tetrazole solution (0.25 M in ac~tol1il,ile) was made up from the solid obtained from Applied Biosystems. Sulfurizing reagent was obtained from Glen Research.
Average sulfurization efficiency (ASE) is determined using the formula: ASE = (PS/Total)1/n~1 where, PS = integrated 31 P NMR values of the P-S diester Total = integration value of all peaks n = length of oligo Referring to tables 42 and 43 effects of varying the delivery and the wait time for sulfurization with Beaucage's reagent is described. These data suggest that 5 second wait time and 300 second delivery time is the condition under which ASE is maximum.
Using the above conditions a 36 mer hammerhead ribozyme is synthesized which is targeted to site C. The ribozyme is synthesized to contain phosphorothioate linkages at four positions towards the 5' end.
RNA cleavage activity of this ribozyme is shown in Fi~. 16. Activity of the pho:",llorulll;odl~ ribozyme is c~,n,.arable to the activity of a ribozyme t, , . Iacking any pllû~ llorul~liudl~ linkages.
Example 13: Pro~ocol forthe synthesis of 2'-N-pht~lirnido-nucleoside phosphor~rnidite The 2'-amino group of a 2'-deoxy-2'-amino nucleoside is normally protected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supra~Pieken et al. 1991 Science 253 314). This protecting group is not stable in CH3CN solution or even in dry fomm during _ _ _ . _ . . .. ... . . . _ ... . _ . .. . .

WO 95123225 218 3 ~ 9 2 F~,.l~ _.'1-156 prolonged storage at -20 C. These problems need to be overcome in order to achieve large scale synthesis of RNA.
Applicant describes the use of altemative protecting groups for the 2'-amino group of 2'-deoxy-2'-amino nucleoside. Referring to Figure 17.
5 phosphoramidite 17 was synthesized starting from 2'-deoxy-2'-aminonucleoside (12) using transient protection with Matkevich reagent (Markiewicz J. Chem. Res. 1979, S, 24). An i"l~""e.lidt~ 13 was obtained in 50% yield, however subsequent introduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960 Nature 185, 306), desilylation (15), 10 dimethoxytrytilation (16) and phosphitylation led to pl~o~,l,ord",iu~ 17.
Since overall yield of this multi-step procedure was low (20%) applicant investigated some alternative approaches, co~ lc~ dli"g on selective introduction of N-phtaloyl group without acylation of 5' and 3' hydroxyls.
When 2'-deoxy-2'-amino-nucleoside was reacted with 1.05 15 equivalents of Nefkens reagent in DMF overnight with subsequent treatment with Et3N (1 hour) only 10-15% of N and 5'(3')-bis-phtaloyl derivatives were fomned with the major .,~ pc,~e~l being N-Pht-derivative 15. The N,O-bis by-products could be selectively and quantitively converted to N-Pht derivative 15 by treatment of crude reaction mixture 20 with cat. KCN/MeOH.
A convenient "one-pot" procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCUEt3N and resulting in the preparation of DMT
derivative 16 in 85% overall yield as follows. Standard phosphytilation of 25 16 produced pllospll~ldll~idil~ 17 in 87% yield. One gram of 2'-amino nucleoside, for example 2'-amino uridine (US Biochemicals(l~ part #
77140) was co-evaporated twice from dry dimethyl ~UIIIIdlllid~ (Dmf) and dried in vacuo overnight. 50 mls of Aldrich sure-seal Dmf was added to the dry 2'-amino uridine via syringe and the mixture was stirred for 10 minutes 30 to produce a clear solution. 1.0 grams (1.05 eq.) of N-carbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) was added and the solution was stirred ovemight. Thin layer chromatography (TLC) showed 90% conversion to a faster moving products (10% ETOH in CHCI3) and 57 ,ul of TEA (0.1 eq.) was added to effect closure of the 35 ~I,II,~Ii",kle ring. After 1 hour an additional 855 ,ul (1.5 eq.) of TEA was added followed by the addition of 1.53 grams (1.1 eq.) of DMT-CI
.. . .. ..

~ W095123225 ~ 2 1 8 ~ ~ 9 2 r~l,lL ~ C-156 (Lancaster Synthesis~), 98%). The reaction mixture was left to stir overnight and quenched with ETOH after TLC showed greater than 90%
desired product. Dmf was removed under vacuum and the mixture was washed with sodium bi~;dr~olldl~ solution (5% aq., 500 m~s) and extracted 5 with ethyl acetate (2x 200 mls). A 25mm x 300mm flash column (75 grams Merck flash silica) was used for purification. Compound eluted at 80 to 85% ethyl acetate in hexanes (yield: 80% purity: >95% by 1HNMR).
Plloa,ullord,,, ' were then prepared using standard protocols described above.
With phosphoramidite 17 in hand applicant synthesized several ribozymes with 2'-deoxy-2'-amino 1llO~ iOI15. Analysis of the synthesis d~",ol~lldl~d coupling efficiency in 97-98% range. RNA cleavage activity of ribozymes containing 2'-deoxy-2'-amino-U Illo.li~i~,dli~lls at U4 and/or U7 positions (see Figure 1), wherein the 2'-amino positions were either 15 protected with Fmoc or Pht, was identical. Additionally, complete deprotection of 2'-deoxy-2'-amino-Uridine was confirmed by base-c~",~osili~l) analysis. The coupling efliciency of pho~.l,old",i.lil~ 17 was not effected over prolonged storage (1-2 months) at low temperatures.
Protecting 2' Position wlth a SEM Group There follows a method using the 2'-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base d~ ulc:~lioll. At the same time, this group should also be readily removed when desired. To that end the t-butyldimethylsilyl group has been efficacious (Usman,N.;
Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Ac~ds Res.
1990, 18, 5433-5441). However, long exposure times to tetra-n-butylammonium fluoride (TBAF) are generally required to fully remove this protecting group from the 2'-hydroxyl. In addition, the bulky alkyl substituents can prove to be a hindrance to coupling thereby n~esail~i. ,g longer coupling times. Finally, it has been shown that the TBDMS group is base labile and is partially deprotected during treatment with ethanolic ammonia (Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res. 1990, WO g5r23~25 2 1 8 3 9 9 ,~ I _I/IL,','C 156 18, 5433-5441 and Stawinski,J.; Stromberg,R.; Thelin,M.; Westman,E.
Nucleic Acids Res. 1988, 16, 9285-9298).
The (trimethylsilyl)ethoxymethyl ether (SEM) seems a suitable substitute. This protecting group is stable to base and all but the harshest 5 acidic conditions. Therefore it is stable under the conditions required for oligonucleotide synthesis. It can be readily introduced and the oxygen carbon bond makes it unable to migrate. Finally, the SEM group can be removed with BF3-OEt2 very quickly.
There follows a method for synthesis of RNA by protecting the 2'-10 position of a nucleotide during RNA synthesis with a(trimethylsilyl)ethoxymethyl (SEM) group. The method can involve use of standard RNA synthesis co,idilions as discussed below, or any other equivalent steps. Those in the art are familiar with such steps. The nucleotide used can be any normal nucleotide or may be s~hstitl~tPd in 15 various positions by methods well known in the art, e.g., as described by Eckstein et al., I"l~",d~iol~al Publication No. WO 92/07065, Perrault et al., Nature 1990, 344, 565-568, Pieken et a/., Science 1991, 253, 314-317, Usman,N.; Cedergren,R.J. Trends in Blochem. Sci. 1992, 17, 334-339, Usman et al., PCT W093/15187, and Sproat,B. European Patent 20 Application 92110298.4 .
This invention also features a method for covalently linking a SEM
~roup to the 2'-position of a nucleotide. The method involves contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions. In a preferred t",Lodi"~e~l, the conditions are dibutyltin oxide, 25 tetrabutylammonium fluoride and SEM-CI. Those in the art, however, will recognize that other equivalent conditions can also be used.
In another aspect, the invention features a method for removal of an SEM group from a nucleoside molecule or an oligonucleotide. The method . involves collld.,~illg the molecule or oligonucleotide with boron trifluoride 30 etherate (BF3-OEt2) under SEM removing conditions, e.g., in acetonitrile.
Referring to Figure 18. there is shown the method for solid phase synthesis of RNA. A 2',5'-protected nuclectide is contacted with a solid phase bound nucleotide under RNA synthesis conditions to form a dinucleotide. The protecting group (R) at the 2'-position in prior art WO 95/23225 PCT/IB~S/00156 ~ . 2183992 methods can be a silyl ether, as shown in the Figure. In the method of the present invention, an SEM group is used in place of the silyl ether.
Otherwise RNA synthesis can be performed by standard Illc~lllodolu~y.
Referring to Figure 19. there is shown the synthesis of 2'-O-SEM
5 protected n~l~leosides and pl1o~phord",adites. Briefly, a 5'-protected nucleoside (1) is protected at the 2'- or 3'-position by contacting with a derivative of SEM under appluplidle coll~iliolls. Specifically, those conditions include contacting the nucleoside with dibutyltin oxide and SEM
chloride. The 2 ~uiui~ el:~ are separated by ulllullldLu~ldully and the 2'-1 û protected moiety is converted into a phosphoramidite by standardprocedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.
Referring to Fi~ure 20, a prior art method for ~ Iute~ iull of RNA using silyl ethers is shown. This contrasts with the method shown in Figure 21 in 15 which d~,uluL~liull of RNA containing an SEM group is perfommed. In step 1, the base protecting groups and cyanoethyl groups are removed by standard procedure. The SEM group is then removed as shown in the Figure. The details of the synthesis of pl~o~,ulluldlllidiL~:s and SEM
protected n~ P~sides and their use in synthesis of oligor~c~ and 20 subsequent dt~JIu~ iull of F~rnvle 14: Synthesis ûf 2'-O-((trimethylsilyl)ethoxymethyl)-5'-O- Di-methûxytrityl Uridine (2) Referring to FicJure 19. 5'-O-dimethoxytrityl uridine 1 (1.0 9, 1.83 mmol) in CH3CN (18 mL) was added dibutyltin oxide (1.0 g, 4.03 mmol) 25 and TBAF (1 M, 2.38 mL, 2.38 mmol). The mixture was stirred for 2 h at RT
(about 20 25C) at which time (trimethylsilyl)ethoxymethyl chloride (SEM-Cl) (487 ,uL, 2.75 mmol) was added. The reaction mixture was stirred ovemight and then filtered and evaporated. Flash ulllullldluuldplly (30%
hexanes in ethyl acetate) yielded 347 mg (28.0%) of 2'-hydroxyl protected 3û nucleoside 2 and 314 mg (25.3%) of 3'-hydroxyl protected nucleoside 3.
F~mvle 15: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl) Uri~ e (4) Nucleoside 2 was detritylated following standard methods, as shown in FiQure 1~. -WO 95/2322~ 218 3 9 9 2 F~,l/J 156 ~

FYFImr~le 16: Svnthesis of 2~-~((trimethylsilyl)ethQxymethyl)-5~.3l-~Acet Uridine (5) Nucleoside 4 was acetylated following standard methods, as shown in Fieure 19.
5 FYArn~DIe 17. Svnthesis Qf 5'.3'-O~Acetyl Uririine (6) Referring to Figure 19. the fully protected uridine 5 (32 mg, 0.07 mmol) was dissolved in CH3CN (700 IlL) and BF3-OEt2 (17.5 uL, 0.14 mmol) was added. The reaction was stirred 15 m and MeOH was added to quench the reaction. Flash clllullldluy,a~.l,y (5% MeOH in CH2C12) gave 10 2û mg (88%) of SEM dey,ul~ul~d nucleoside 6.
FY~rr~le 18: Synthesis of 2'-~((trimethylsilyl)ethoY~ymethyl)-3'-a Succinyl-5'-~ Dimethoxytrityl Urirline (2) Nucleoside 3 was succinylated and coupled to the support tollowing standard procedures, as shown in Figure 19.
15 FYArnple 19: Synthesis of 2'-~((trimethylsilyl)ethoxymethyl)-5'-~ Di-methoxytrityl Uri~ine 3'-~-Cyanoethyl N.N-diisoDropylrJhoOullol~Jl ~
Nucleoôide 3 was pl)oO,ul,ilylated following standard methods, as shown in Fi~ure 19.
20 FYArnple 2û: Synth~ci~ of RNA Usinp 2'-~.CF \~l Protection Referring to Fi~ure 18. the method of synthesis used follows the general procedure for RNA synthesis as described in Usman,N.;
Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucl. Acids Res.
25 1990, 18, 5433-5441. The phosphoramidite 8 was coupled following standard RNA methods to provide a 10-mer of uridylic acid. Syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 llmol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 IlL of 0.1 M = 32.5 ,umol) of phOs,ullo,a,,,idil~ and a 80-fold excess of tetrazole 30 (400 ,uL of 0.5 M = 200 ,umol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, dt:~t,r",i"ed by colu~i"~riu quantitation of the trityl fractions, were 98-99%.
Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-.

~ wo 9sl2322s 2 1 8 3 9 ~ ~ PCr/lB95~00156 Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.
Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle.
Referring to Fi~ure 21. the homopolymer was base de,uluLt~ d with NH3/EtOH at 65 C. The solution was decanted and the support was washed twice with a solution of 1:1:1 H2O:CH3CN:MeOH. The combined solutions were dried down and then diluted with CH3CN (1 mL). BF3-OEt2 (2.5 ~L, 30 ~Lmol) was added to the solution and aliquots were removed at ten time points. The results indicate that after 30 min dep,u~iliu" is complete, as shown in Figure 22.
111. Vectors E~,u~ lg RiLu~ s There follows a method for ~,ul~aaiull of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a ,iec,~,ased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful for scaling up production of a ribozyme, which may be either modified or ~" " " ~ , In situ or in vitro. Such vector systems can be used to express a desired ribozyme in a specific cell, or can be used in an in vitro system to allow productiuon of large amounts of a desired riboqyne, The vectors of this invention allow a higher yield synthesis of a ribozyme in the fomm of an RNA transcript which is cleaved in situ or in vitro before or after transcript isolation.
Thus, this invention is distinct from the prior art in that a single ribozyme is used to process the 3' and 5' ends of each therapeutic, trans-acting or desired ribozyme instead of plu~daaillg only one end, or only one ribozyme. This allows smaller vectors to be derived with multiple trans-acting ribozymes released by only one other ribozyme from the mRNA
transcript. Applicant has also provided methods by which the activity of such ribozymes is increased compared to those in the art, by designing ribozyme-encoding vectors and the corresponding transcript such that Wo 95/23225 . ~ ^ PCT/11395/00156 2183-9~ ~

folding of the mRNA does not interfere with processing by the releasing ribozyme.
The stability of the ribozyme produced in this method can be enhanced by provision of sequences at the termini of the ribozymes as described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein.
The method of this invention is advantageous since it provides high yield synthesis of ribozymes by use of low cost transcription-based protocols, compared to existing chemical ribozyme synthesis, and can use isolation techniques currently used to purify chemically s~"l~,esi~ed oligor~ leotides Thus, the method allows synthesis of ribozymes in high yield at low cost for analytical, diagnostic, or therapeutic a;, ' :~s.
The method is also useful for synthesis of ribozymes in vitro for ribozyme structural studies, enzymatic studies, target RNA ~ escih:~y 15 studies, l,dns,il,liol~ inhibition studies and nuclease protection studies, much is described by Draper et al., PCT WO 93/23509 hereby illCul,,Joldl~d by reference herein.
The method can also be used to produce ribozymes in situ either to increase the intracellular col,ce"~,dlioll of a desired therapeutic ribozyme, or to produce a cu"~dl~",eric transcript for subsequent in vitro isolation of unit length ribozyme. The desired ribozyme can be used to inhibit gene ~Apl~SSiOIl in molecular genetic analyses or in infectious cell systems, and to test the efficacy of a therapeutic molecule or treat afflicted cells.
Thus, in general, the invention features a vector which includes a bacterial, viral or eucaryotic promoter within a plasmid, cosmid, phagmid, virus, viroid, virusoid or phage vector. Other vectors are equally suitable and include double-stranded, or partially double-stranded DNA, formed by an al~ ,d~ l method such as the polymerase chain reaction, or double-stranded, partially double-stranded or single-stranded RNA, formed by site-directed homologous ~cor,,~i,,c.lio,, into viral or viroid RNA genomes.
Such vectors need not be circular. T,d"s~"il.li.",~l,y linked to the promoter region is a first ribozyme~encoding region, and nucleotide sequences encoding a ribozyme cleavage sequence which is placed on either side of a region encoding a therapeutic or otherwise desired second ribozyme.

WO 95/232~5 218 3 9 9 2 r~ 156 Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA ~xpr~ssion system. The desired second ribozyme may be any - desired type of ribozyme, such as a lld"""t~ ad, hairpin, hepatitis delta virus (HDV) or other catalytic center, and can include group I and group ll introns, as discussed above. The first ribozyme is chosen to cleave the encoded cleavage sequence, and may also be any desired ribozyme, for example, a Te~rahymena derived ribozyme, which may, for example, include an imbedded restriction endonuclease site in the center of a self-lecoy"iliol) sequence to aid in vector constnuction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector.
When the promoter of such a vector is activated an RNA transcript is produced which includes the first and second ribozyme sequences. The first ribozyme sequence is able to act, under ap~.rupridl~ conditions, to cause cleavage at the cleavage sites to release the second ribozyme sequences. These second ribozyme sequences can then act at their target RNA sites, or can be isolated for later use or analysis.
Thus, in one aspect the invention features a vector which includes a first nucleic acid sequence (encoding a first ribozyme having i"lld",olecular cleaving activity), and a second nucleic acid sequence (encoding a second ribozyme having i~L~ ùlecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA
transcript encoded by the vector. The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have i"lt:l",olecular cleaving activity.
As discussed above, the first ribozyme can be chosen to be any self-30 cleaving ribozyme, and the second ribozyme may be chosen to be any desired ribozyme. The flanking sequences are chosen to include - sequences l~:coy"i~d by the first ribozyme. When the vector is caused to express RNA from these nucleic acid sequences, that RNA has the ability under a~,rul,,idl~ conditions to cleave each of the flanking regions and thereby release one or more copies of the second ribozyme. If desired, several different second ribozymes can be produced by the same vector, or WO 95123225 2 ~ 8 3 ~ ~ ~ r~l,~. 156 several different vectors can be placed in the same vessel or cell to produce different ribozymes.
In preferred ~ L;o~i,,,t:,,L,, the vector includes a plurality of the nucleic acid sequences encoding the second ribozyme, each flanked by nucleic 5 acid sequences l~coy,li~d by the first ribozyme. Most preferably, such a plurality includes at least six to nine or even between 60 100 nucleic acid sequences. In other preferred embodiments, the vector includes a promoter which regulates expression of the nucleic acid encoding the ribozymes from the vector; and the vector is chosen from a plasmid, 10 cosmid, phagmid, virus, viroid or phage. In a most preferred l:"l~odi",~l,l, the plurality of nucleic acid sequences are identical and are arranged in ssquential order such that each has an identical end nearest to the promoter. If desired, a poly(A) sequence adjacent to the sequence encoding the first or second ribozyme may be provided to increase stability 15 of the RNA produced by the vector; and a restriction endonuclease site adjacent to the nucleic acid encoding the first ribozyme is provided to allow insertion of nucleic acid encoding the second ribozyme during construction of the vector.
In a second aspect, the invention features a method for formation of a 20 ribozyme t,,~ s:,iun vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a single-stranded DNA encoding a second ribozyme, as discussed above. The single-stranded DNA is then allowed to anneal to form a partial duplex DNA which can be filled in by a treatment with an ap~ur~prial~ enzyme, 25 such as a DNA polymerase in the presence of dNTPs, to form a duplex DNA which can then be li~qated to the vector. Large vectors resulting from this method can then be selected to insure that a high copy number of the single-stranded DNA encoding the second ribozyme is il,~,o",ordled into the vector.
In a further aspect, the invention features a method for production of ribozymes by providing a vector as described above, expressing RNA from that vector, and allowing cleavage by the first ribozyme to release the second ribozyme.
In preferred ~",I,odi",~"l~, three different ribozyme motifs are used as cis cleaving ribozymes. The hammerhead, hairpin, and hepatitis delta ~ WO95/23225 218 ~9 92 P~ r i~;6 virus (HDV) ribozyme motifs consist of small, well-defined sequences that rapidly self-cleave in vitro (Symons, 1992 Annu. Rev. E3iochem. 61, 641).
While structural and functional Ji~ es exist among the three ribozyme motifs, they self-process efliciently in vivo. All three ribozyme motifs self-5 process to 87-95% Golll,ul~Iivl~ in the absence of 3' flanking sequences. In vitro, the self-processing constructs described in this invention are biy"i~icanlly more active than those reported by Taira et al., 1990 supra;
and Altschuler et al., 1992 Gene 122, 85. The present invention enables the use of cis-cleaving ribozymes to efficiently truncate RNA molecules at 10 specific sites in vivo by ensuring lack of secondary structure which prevents plUC~bi"g.
Isolation of Thera~eutic RibQzyme The preferred method of isolating therapeutic ribozyme is by a ChlUllldlVyld~JlliC technique. The HPLC purification methods and reverse HPLC purification methods described by Draper et al., PCT WO 93/23509, hereby i~n,or,uu~dI~d by reference herein, can be used. Alternatively, the attachment of c~"")ler"e"ldry oligonucleotides to cellulose or other ~II,ur,,d~uy,dphy columns allows isolation of the therapeutic second ribozyme, for example, by llybliJi~dIiul1 to the region between the flanking 20 arms and the enzymatic RNA. This hybliJi~aIiol~ will select against the short flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybliJi~dIioll can be acco",~ l,ed in the presence of a chaotropic agent to prevent nuclease degradation. The oligor~lcl~oticles on the matrix can be modified to minimize nuclease 25 activity, for example, by provision of 2'-O-methyl RNA oligonucleotides.
Such IlloJi~i~,dIivl~s of the oligonucleotide attached to the column matrix willallow the multiple use of the column with minimal oligo d~:yldJdtivll. Many such ",odi~iua~iolls are known in the art, but a chemically stable non-reducible ~I,oJi~icdIiù,~ is preferred. For example, phosphorothioate 30 IllO~ .dliVI)s can also be used.
The ~,.,u,essed ribozyme RNA can be isolated from bacterial or eucaryotic cells by routine procedures such as Iysis followed by guanidine isothiocyanate isolation.
The current known self-cleaving site of Tetrahymena can be used in 35 an alternative vector of this invention. If desired, the full-length _ _ _ _ _ WO 95/23~2S 2 1 8 3 9 9 2 P~ IS6 Tetrahymena sequence may be used, or a shorter sequence may be used.
It is preferred that, in order to decrease the superfluous sequences in the self-cleaving site at the 5' cleavage end, the hairpin normally present in the Tetrahymena ribozyme should contain the therapeutic second ribozyme 3' 5 sequence and its complement. That is, the first releasing ribozyme-encoding DNA is provided in two portions, separated by DNA encoding the desired second ribozyme. For example, if the therapeutic second ribozyme I~.,o~"iIioll sequence is CGGACGA/CGAGGA, then CGAGGA is provided in the self-cleaving site loop such that it is in a stem structure l~coy~ d by 10 the Tetrahymena ribozyme. The loop of the stem may include a restriction endonuclease site into which the desired second ribozyme-encoding DNA
is placed.
If desired, the vector may be used in a therapeutic protoco! by use of the systems described by Lechner, PCT WO 92/13070, hereby 15 incorporated by reference herein, to allow a timed expression of the therapeutic second ribozyme, as well as an ~,u,u~up~ shut off of cell or gene function. Thus, the vector will include a promoter which d,u~lupridI~ly expresses enzymatically active RNA only in the presence of an RNA or another molecule which indicates the presence of an undesired organism 20 or state. Such enzymatically active RNA will then kill or harm the cell in which it exists, as described by Lechner, id., or act to cause reduced ~r.ul~sSiOII of a desired protein product.
A number of suitable RNA vectors may also be used in this invention.
The vectors include plant viroids, plant viruses which contain single or 25 double-stranded RNA genomes and animal viruses which contain RNA
genomes, such as the picornaviruses, myxoviruses, paramyxoviruses, hepatitis A virus, reovirus and retroviruses. In many instances cited, use of these viral vectors also results in tissue specific delivery of the ribozymes.
E~ ole 21: Design of self-p,u.,~.c.,~;,,~ r~.c.CPtta~
In a preferred ~",~oui,ll~llI, applicant compared the in vitro and In vivo cis-cleaving activity of three different ribozyme motifs-the hrll"",e,l,edd, the hairpin and the hepatitis delta virus ribozyme-in order to assess their potential to process the ends of transcripts in vivo. To make a direct cu,,,,ua-i~oll among the three, however, it is important to design the ribozyme-containing I-dnsu~i,ut~ to be as similar as possible. To this end, WO 95/23~25 - I~,I/~. . 156 21839~2 all the ribozyme cassettes contained the same trans-acting hammerhead ribozyme followed i"""e.lidl~ly by one of the three cis-acting ribozymes (Figure 23-25). For simplicity, applicant refers to each cassette by an abbreviation that indicates the d~.. ,a~,~a", cis-cleaving ribozyme only.
5 Thus HH refers to the cis-cleaving cassette cun'Gi";"g a har"",~lllead ribozyme, while HP and HDV refer to the cassettes containing hairpin and hepatitis delta virus cis-cleaving ribozymes, respectively. The general design of the ribozyme cassettes, as well as specific dir~ ces among the cassettes, are outlined below.
A sequence predicted to form a stable stem-loop structure is included at the 5' end of all the lldi,s~ Jt~. The hairpin stem contains the T7 RNA
polymerase initiation sequence (Milligan & Uhlenbeck, 1989 ~/lethQds Enzvmol. 180, 51) and its cu",~ ",t:~l, separated be a stable tetra-loop (Antao et al., 1991 Nucleic Acids Res. 19, 5901). By illuul~uldlillg the T7 15 initiation sequence into a stem-loop structure, applicant hoped to avoid nonproductive base pairing interactions with either the trans-acting ribozyme or with the cis-acting ribozyme. The presence of a hairpin at the end of a transcript may also contribute to the stability of the transcript in vivo. These are non-limiting examples. Those in the art will recognize that 2û other ~",bo~i",~"~:, can be readily generated using a variety of promoters, initiator sequences and stem-loop structure Collll,illdliul~ generally known in the art.
The trans-acting ribozyme used in this study is targeted to a site B
(5'- CUGGAGUCIGACCUUC 3'). The 5' binding amm of the ribozyme, 5'-25 GAAGGUC-3', and the core of the ribozyme, 5'-CUGAUGAGGCCGAAAGGCCGAA-3', remain constant in all cases. In addition, all ~IdllsCii~ also contain a single nucleotide between the 5' stem-loop and the first nucleotide of the ribozyme. The linker nucleotide was required to obtain the same activity ~n vitro that was measured with an 30 identical ribozyme lacking the 5' hairpin. Because the three cis-cleaving ribozymes have different requirements at the site of cleavage, slight l ,ces were unavoidable at the 3' end of the processed transcript. The junction between the trans- and cis-acting ribozyme is, however, designed so that there is minimal extraneous sequence left at the 3' end of the trans-35 cleaving ribozyme once cis-cleavage occurs. The only differences between the constructs lie in the 3' binding arm of the ribozyme, where ... . ... ... .. . ... ..

WO 95123225 1 ~ JS6 21839~2`

either 6 or 7 n~leot~ s 5'-ACUCCA(+/-G)-3', ,~""~I~",e"ldly to the target sequence are present and where, after processing, two to five extra rlll~leotkl~s remain.
The cis-cleaving lld"""~,l,ead ribozyme used in the HH cassette is 5 based on the design of Grosshans and Cech, 1991 ~. As shown in Figure 23, the 3' binding arm of the trans-acting ribozyme is included in the required base-pairing i"~ s of the cis-cleaving ribozyme to form stem 1. Two extra r~cleotides UC, were included at the end of the 3' binding amm to fomm the self-p,ocesDi"g lld"""~,l,ead ribozyme site (Ruffner et al., 1990 surra) which remain on the 3' end of the trans-acting ribozyme following self-,u,uct~ i"9.
The hairpin ribozyme portion of the HP self-,~"ucessi,lg construct is based on the minimal wild-type sequence (Hampel & Tritz, 1989 supra~. A
tetra-loop at the end of helix 1 (3' side of the cleavage site) serves to link 15 the two portions and thus allows a minimal five r~lclsotidAs to remain at theend of the released trans-acting ribozyme following self-p,-,cessi"g. Two variants of HP were designed: HP(GU) and HP(GC). The HP(GU) was constructed with a G-U wobble base pair in helix 2 (As2G .s~hstit~tion;
Fi~ure 24). This slight ~ of helix 2 was intended to improve 20 self-pl.,~ssi"g activity by promoting product release and preventing the reverse reaction (Berz~l Hc,.ldll~ et al., 1992 Genes & Dev. 6, 129;
Chowrira et al., 1993 ~iocl,e",i~lly 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing illl~ ,liù~ls in helix 2 (U77C and As2G sl~h.stitl~tion; Figure 24). Another ",o~i~icdli~n to 25 discourage the reverse ligation reaction of the hairpin ribozyme was to shorten helix 1 (Fiaure 24) by one base pair relative to the wild-type sequence (Chowrira & Burke, 1991 BjUUI~ DIIY 30, 8518).
The HDV ribozyme self-processes efficiently when the nucleotide 5' to the cleavage site is a pyrimidine, and somewhat !ess so when adenosine is 30 in that position, No other sequence requirements have been identified upstream of the cleavage site, however, we have observed some decrease in activity when a stem-loop structure was present within 2 nt of the cleavage site. The HDV self-plu~DDi"g construct (Fiq 25) was designed to generate the trans-acting hammerhead ribozyme with only two additional 35 nucleotides at its 3' end after self-pluces~i"g. The HDV sequence used here is based on the anti-genomic sequence (Perrota & Been, 1992 supra) WO95123225 r~l,~s~o ~s6 218~92 8~
but includes the Illo~ d~iul-s of Been et al., 1992 (Biochemistry 31, 11843) in which cis-cleavage activity of the ribozyme was improved by the s~hstit~ ~ti~n of a shortened helix 4 for a wild-type stem-loop (Figure 25).
To prepare DNA inserts that encode self-processing ribozyme 5 cassettes, partially overlapping top- and bottom-strand oligonucleotides (60-90 nucleotides) were designed to include sequences for the T7 promoter, the trans-acting ribozyme, the cis-cleaving ribozyme and appruplidl~ restriction sites for use in cloning (see Fi~. 26). The single-strand portions of annealed oligonucleotides were converted to double-10 strands using Sequenase(~) (U.S. Biu~ ",;~,als). Insert DNA was ligatedinto EcoR1/H~ndlll-digested puc18 and ~d~ ""~ed into E. colistrain DH5a using standard protocols (Maniatis et al., 1982 in Moler:lllAr Cloning Cold Spring Harbor Press). The identity of positive clones was confirmed by sequencing small-scale plasmid plt~,ualdlio,)s.
Larger scale preparations of plasmid DNA for use as in vitro nscri,ulioll templates and in lldnsdl;liol~s were prepared using the protocol and columns from QIAGEN Inc. (Studio City, CA) except that an additional ethanol pr~ Jilal;~l~ was included as the final step.
EY~mple ~ RNA Processin~ in VitrQ
T,dl1scri,ulio,l reactions containing linear plasmid templates were carried out essentially as described (Milligan ~ Uhlenbeck, 1989 Supra;
Chowrira & Burke, 1991 Sur ra). In order to prepare 5' end-labeled d~Scli,~, standard transcription reactions were carried out in the presence of 10-20 ,uCi [~-32P]GTP, 200 IlM each NTP and 0.5 to 1 u9 of linearized plasmid template. The .ol~cl:lllldLiull of MgCI2 was maintained at 10 mM above the total nucleotide col~c~llldliol~
To compare the ability of the diflerent ribozyme cassettes to self-process in vitro, each construct was lld"s~;,iL,ed and allowed to undergo self-processing under identical conditions at 37C. For these c~",,uali:,u"s, equal amounts of linearized DNA templates bearing the various ribozyme cassettes were transcribed in the presence of ~-32P]GTP to generate 5' end-labeled l~dllS~"i,U~:`. In this manner only the full-length, ullpluc~ssad transcripts and the released trans-ribozymes are visualized by a~lurd.lioyldpl~y. In all reactions, Mg2+ was included at 10 mM above the nucleotide Col,Ct"llldliol~ so that cleavage by all the ribozyme cassettes _ _ . _ _ . _ _ _ _ . . . . . ... ... .. . .... .. _ .. . . . = .. . . ... _ _ _ _ would be supported. Tldl,su,iulioll templates were linearized at several positions by digestion with different restriction enzymes so that self-pluces~ g in the presence of increasing lengths of dU...l:~lltld~ll sequence could be compared (see Fir~. 26). The resulting ~IdllS(.;li,U~ have either 4-5 5 non-ribozyme n~cleoti~s at the 3 end (tlindlll-digested template) 220 rlllcleoti~l~s (Ndel digested templates) or 454 n~r~leotidr~s of do~ d", sequence (Rcal digested template).
As shown in Figure 27, all four ribozyme cassettes are capable of self-u~ùCeSS;~lg and yield RNA products of expected sizes. Two nucleotides 10 essential for hammerhead ribozyme activity (Ruffner et al., 1990 ~) have been changed in the HH(mutant) core sequence (see Figure 2~) and so this transcript is unable to undergo self-p,uc~ i"g (Eis~)- This is evidenced by the lack of a released 5' RNA in the HH(mutant) although the full-length RNAs are present . Comparison of the amounts of released 15 trans-ribozyme (E19~) indicate that there are ~ llu~s in the ability of these ,i u~y",~s to self-process in vitro, especially with respect to the presence of do-~"~ a", sequence. For the two HP constructs, it is clear that HP(GC) is more eflicient than the HP(GU) ribozyme both in the presence and in the absence of extra dU.. l~ d~ sequence. In addition7 20 the activity of HP(GU) falls ofl more dramatically when du...,~ am sequence is present. The stronger G:C base pair likely contributes to the HP(GC) constructs ability to fold correctly (and/or more quickly) into the productive structure even when as much as 216 extra nucleotides are present do~ L,~d",. The HH ribozyme construct is also quite eflicient at 25 self-pluces~ g, and slightly better than the HP(GU) construct even when dU.. ~:,LI~:dlll sequence is present.
Of the three ribozyme motifs, the presence of extra do.;i~:,L,~a~"
sequence seems to most affect the efliciency of HDV. When no extra sequence is present d~ LIl:dlll, HDV is quite efficient and self-processes 30 to appr~Jxill,al~ly the same level as the HH and HP(GC) cassettes.
However when extra .lu...~l,uall, sequence is present the self-~uce~ g activity seems to decrease almost as dldrlldlic~lly as is seen with the (sub-optimal) HP(GU) cassette.

WO95/23225 _ ~ _ r. ~ 01!i6 21839~2 FY~rnple 23: Kinetics of self-Drocessin~ reaction Hindlll-digested template (250 ng) was used in a standard transcription reaction mixture c~lldi~ y. 50 mM Tris-HCI pH 8.3; 1 mM
ATP GTP and UTP; 50 IlM CTP; 40 uci [a-32P]CTP; 12 mM MgC12; 10 mM
5 DTT. The l~ans-;~i,ulion/self-processing reaction was initiated by the addition of T7 RNA pOly",t"dse (15 W~LI). Aliquots of 5 l11 were taken at regular time intervals and the reaction was stopped by adding an equal volume of 2x lu""d",kle loading bufler (95% ru~ldll~ide, 15 mM EDTA, &
dyes) and freezing on dry ice. The samples were resolved on a 10%
10 polyacrylamide sequencing gel and results were quantitated by Phosphorlmager (Molecular Dynamics, Sunnyvale CA). Ribozyme self-cleavage rates were determined from non-linear least-squares fits (KaleidaGraph, Synergy Software,Reeding PA) of the data to the equation:
(Fraction Uncleaved Transcript) = kt (1-e kt) where t rc~prt~S~ time and k ,t,,ur~s~"l~ the unimolecular rate constant for cleavage (Long & Uhlenbeck 1994 Proc. N~tl Acad. Sci. USA
91 6977).
Linear templates were prepared by digesting the plasmids with H~ndlll so that ~d~s~i,iyt~ will contain only four to five vector-derived n~cle: '~ at the 3 end (see Figure 23-25). By comparison of the unimolecular rate constant (k) dt,l~r",i"ed for each construct, it is clear that HH is the most efficient at self-~uces~ g (Table 44! The HH transcript self-processes 2-fold faster than HDV and 3-fold faster than HP(GC) lldi ~ CI i,ula. Although the HP(GU) RNA undergoes self-p,uces~i"g, it is at least 6-fold slower than the HP(GC) construct. This is consistent with previous observations that the stability of helix 2 is essential for self-p,uce~i"g and trans-cleavage activity of the hairpin ribozyme (Hampel et al. 1990 ~; Chowrira &
Burke 1991 ~). The rate of HH self-cleavage during lldns~ ion measured here (1.2 min~1) is similar to the rate measured by Long and - . 3û Uhlenbeck 1994 supra using a HH that has a different stem I and stem lll.
Self-,l~,u~es~i"g rates during ~,al)s~;,i,uliùn for HP and HDV have not been - previously reported. However, self-processing of the HDV ribozyme-as measured here during l~dns~ is ~iy~ dl~lly slower than when tested after isolation from a denaturing gel (Been et al., 1992 ~). This decrease likely reflects the difference in protocol as well as the presence of 5' flanking sequence in the HDV construct used here.
_ .

W0 95123225 218 ~ 9 912 r~l,~ 156 FxArnple 24: Fffect of ~ blladlll se~uences on trAns-cleavA~l~ in vitrQ
Transcripts containing the trans ribozyme with or without 3' flanking sequences were assayed for their ability to cleave their target in trans. To this end, llailsuli,u~a from three templates were resoived on a preparative 5 gel and bands col-t:a,uol~di,lg both to p~ucessed trans-acting ribozymes from the HH lldlls.,,i,ut;oll reaction, and to full-length HH~mutant) and ~HDV
lldl~UIi,Ut:l were isolated. In all three lldnS-;li,ula the trans-acting ribozyme portion is identical-with the exception of sequences at their 3' ends. The HH trans-acting ribozyme contains only an additional UC at its 3' end, 1 û while HH(mutant) and ~HDV have 52 and 37 n~cleotirl~os respectively, at their 3' ends. A 622 nllcl~otkle, internally-labeled target RNA was incubated, under ribozyme excess conditions, along with the three ribozyme lldlls~ ,ts in a standard reaction buffer.
To make internally-labeled substrate RNA for trans-ribozyme 15 cleavage reactions, a 622 nt region (containing hd"lll,~ ead site P) was sy"ll,eai~d by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was lldl)sc,il,~d in a standard transcription buffer in the presence of [-32P]CTP (Chowrira &
Burke, 1991 ~). The reaction mixture was treated with 15 units of 20 ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol (25:1), precipitated with isopropanol and washed with 70% ethanol. The dried pellet was resuspended in 2 DEPC-treated water and stored at -20C.
Uniabeled ribozyme (1~LM) and internally labeled 622 nt substrate 25 RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 1û mM MgCI2) by heating to 9ûC for 2 min. and slow cooling to 37C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37C. Aliquots of 5 ,ul were taken at regular time intervals, 30 quenched by adding an equal volume of 2X ~ulllldlllid~ gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Pl,os~l,orl",a~ ) (Molecular Dynamics, Sunnyvale, CA).
The HH trans-acting ribozyme cleaves the target RNA ap,uluAillldl~ly 1 0-fold faster than the ~HDV transcript and greater than 20-fold faster than W0 9512322!i ~ ; 2-18 3 ~ ~ 2 r~l,~ 156 a3 the HH(mutant) transcript (Figure 28). The additional r~ leotid~s at the end of HH(mutant) form 7 base-pairs with the 3' target-binding arm of the trans-acting ribozyme (Figure 23). This interaction must be disrupted (at a cost of 6 kcal/mole) to make the trans-acting ribozyme available for binding 5 the target sequence. In contrast, the additional nl~cleoticlPs at the end of ~HDV were not designed to fonm any strong, alternative base-pairing with the trans-ribozyme. Nevertheless, the ~HDV sequences are predicted to form multiple structures involving the 3' target-binding arm of the trans ribozyme that have stabilities ranging from 1-2 kcal/mole. Thus, the 10 observed reductions in activity for the /`HDV and HH(mutant) constnucts are consistent with the predicted folded structures, and it reinforces the view that the flanking sequences can decrease the catalytic efficiency of a ribozyme through nonproductive ill~ld~liulls with either the ribozyme or the substrate or both.
15 FY~rnple 25: RNA self-pluces~illg in vivo Since three of the constructs (HH, HDV and HP(GC)) self-process efficiently in solution, the affect of the l,,d,,,,,,alidl~ cellular milieu on ribozyme self-processing was next explored by applicant. A transient siu,~ system was employed to investigate ribozyme activity in vivo. A
20 mouse cell line (OST7-1) that constitutively expresses T7 RNA poly",e,dse in the cytoplasm was chosen for this study (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA 87, 6743). In these cells plasmids containing a ribozyme cassette du.;~la~l~dlll of the T7 promoter will be transcribed efficiently in the cytoplasm (Elroy-Stein & Moss, 1990 supra).
Monolayers of a mouse L9 fibroblast cell line (OST7-1; Elroy-Stein and Moss, 1990 supra) were grown in 6-well plates with ~ 5x105 cells/well.
Cells were lldll~ d with circular plasmids (5 ug/well) using the calcium pl~o~uhdl~-DNA pr~ ildliu" method (Maniatis et al., 1982 supra). Cells were Iysed (4 hours post-l,d,~ ,lion) by the addition of standard Iysis buffer (200 ,ul/well) containing 4M guanadinium isothiocyanate, 25 mM
sodium citrate (pH 7.0), 0.5% sarkosyl (Chomczynski and Sacchi, 1987 Anal. Biûchem. 162, 156), and 50 mM EDTA pH 8Ø The Iysate was extracted once with water-saturated phenol followed by one extraction with Clll~lu~ullll.i:,odlllyl alcohol (25:1). Total cellular RNA was ~ i,uildl~d withan equal volume of isopropanol. The RNA pellet was resuspended in 0.2 WO 95/23Z25 -- 218 3 ~ 9 2 r~ 15C

M ammonium acetate and l~ul_uiuiIalad with ethanol. The pellet was thenwashed with 70% ethanol and resuspended in DEPC-treated water.
Purified cellular RNA (3 ug/reaction) was first denatured in the presence of a 5' end-labeied DNA primer (100 pmol) by heating to 90C for 5 2 min. in the absence of Mg2+ and then snap-cooling on ice for at least 15 min. This protocol allows for efficient annealing of the primer to its complementary RNA sequence. The primer was extended using Superscript ll reverse L,ai,sc,iuLdse (8 U/ul; BRL) in a buffer containing 50 mM Tris-HCI pH 8.3; 10 mM DTT; 75 mM KCI; 1 mM MgCI2; 1 mM each 10 dNTP. The extension reaction was carried out at 42C for 10 min. The reaction was l~ dL~d by adding an equal volume of 2x ~UIIIIdlll;~.le gel loading buffer and freezing on crushed dry ice. The samples were resolved on a 10% polydulyldllli~e sequencing gel The primer sequences are as follows: HH primer 5'-CTCCAGmCGAGCm-3'; HDV primer 5'-15 AAGTAGCCCAGGTCGGACC-3'; HP primer, 5'-ACCAGGTAATATACCACMC-3'.
As shown in Figure 29. specific bands co"~uo"ui"g to full-iength precursor RNA and 3' cleavage products were detected from cells transfected with the self-u,uues~i"g cassettes. All three constructs, in 20 addition to being L~a,,s~ ,iu~iu~ally active, âppear to self-process efliciently in the cytoplasm of OST7-1 cells. In particular the HH and HP(GC) constructs self-process to greater than 95%. The overall extent of self-p,uc~ i"g in OST7-1 cells appears to be strikingly similar to the extent of self-proces~i"g in vitro (Figure 29 aln Vitro +Mgcl2ll vs. "Cellular").
Consistent with the in vitro self-processing results the HP(GU) cassette self-processed to approximately 50% in OST7-1 cells. As expected L,dn~f~uLiu" with plasmids containing the HH(mutant~ cassette yielded a primer-extension product cbll~uol1dil,g to the full-length RNA
with no detectable cleavage products (Fi~ure 29). The latter result strongly suggests that the primer extension band co"~pOI"li"9 to the 3' cleavage product is not an artifact of reverse lldl1S.;liUliUll.
Applicant was ~ ol~u~",ed with the possibility that RNA self-p,uc~s:,i"g might occur during cell Iysis, RNA isolation and /or the primer extension assay. Two precautions were taken to exclude this possibility. First, 50 mM
EDTA was included in the Iysis buffer. EDTA is a strong chelator of divalent ~ WO 95123225 218 3 ~ ~ 2 F~~ i56 metal ions such as Mg2+ and Ca2+ that are necessary for ribozyme activity. Divalent metal ions are therefore unavailable to self-pluces~il,g RNAs following cell Iysis. A second precaution involved using primers in the primer-extension assay that were designed to hybridize to essential 5 regions of the ,u,uce:~si,lg ribozyme. Binding of these primers should - prevent the 3' cis-acting ribozymes from folding into the cu~lrurllldli essential for catalytic activity.
Two t:~,Utllilll~llt~ were carried out to further eliminate the possibility that self-p,uuesai,,y is occurring either during RNA ,u~ualdliol,s or during 1û the primer extension analysis. The first experiment involves primer extension analysis on full-length precursor RNAs that were added to non-lldl l:,rtlult~d OST7-1 Iysates after cell Iysis. Thus, only if self-p,uce~:,i,,g is occurring at some point after Iysis would cleavage products be detected.
Full-length precursor RNAs were prepared by ~IdllsuliLillg under conditions 15 of low Mg2+ (5 mM) and high NTP Cùl)c6111,dliol1 (total 12 mM) in an attempt to eliminate the free Mg2+ required for the self-p, U~eS:~il ,g reaction(Michel et al. 1992 Genes & Dev. 6, 1373). The full-length precursor RNAs were gel-purified, and a known amount was added to Iysates of non-~d~ ulecl OST7-1 cells. RNA was purified from these Iysates and 2û incubated for 1 hr in DEPC-treated water at 37 C prior to the standard primer extension analysis (Fi~ure 29. in vitro "-MgCI2" control). The predominant RNA detected in all cases COIIt:a,uOlld:, to the primer extension product of full-length precursor RNAs. If, instead, the purified RNA containing the full-length precursor is incubated in 1û mM MgCI2 prior 25 to the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (Fi~ure 29. in vitro 'l+M9cl2ll control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-,u,uces~illg, even though the RNA in question is inherently able to 30 undergo self-cleavage.
In a second e,~,udri",e:,ll to dt:lllull~lldl~ lack of self-,u,ocds:,i"g during work up, i"l~",~''; labeled precursor RNAs were prepared and added to non-lld"~r~u~d OST7-1 Iysates as in the previous control. The internally-labeled precursor RNAs were carried through the RNA purification and 35 primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-plucessi"g. By this analysis, the . ~ . . . . . ... . _ .. _ . . . . . _ _ _ WO 95/23225 218 3 9 ~ 2 r~ 156 vast majority of the added full-length RNA remained intact during the entire process of RNA isolation and primer extension.
These two control experiments Yalidate the protocols used and support ~,, ' ,L'~ conclusion that the self-plc.ce~:,i"g reactions catalyzed 5 by HH, HDV and HP(GC) cassettes are occurring in the cytoplasm of OST7-1 cells.
Sequences in figures 23 through 25 are meant to be non-limiting examples. Those in the art will recognize that other el"~odi",~"l~ can be readily generated using techniques generally known in the art.
In addition, those in the art will recognize that Applicant provides guidance through the above examples as to how to best design vectors of this invention so that secondary structure of the mRNA allows efficient cleavage by releasing ribozymes. Thus, the specific constructs are not limiting in this invention. Such constructs can be readily tested as 15 described above for such secondary structure, either by computer folding algorithms or e"" i, 'Iy Such constructs will then allow at least 80%
c~ lioll of release of ribozymes, which can be readily clt,ltr",;"ed as described above or by methods known in the art. That is, any such secondary structure in the RNA does not reduce release of the ribozymes 20 by more than 20%.
IV. Ril..,~ s EYnressed by RNA F~ly~ a.q~ lll Applicant has determined that the level of production of a foreign RNA, using a RNA polymerase lll (pol lll) based system, can be si5~ ki~"l1y enhanced by ensuring that the RNA is produced with the 5' terminus and a 25 3' region of the RNA molecule base-paired together to form a stable intramolecular stem structure. This stem stnucture is formed by hydrogen bond i~ d~iliOIls (either Watson-Crick or non-Watson-Crick) between rll~clestides in the 3' region (at least 8 bases) and complementary n~leoti~s in the 5' terminus of the same RNA molecule.
Although the example provided below involves a type 2 pol lll gene unit, a number of other pol lll promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell 29, 3-5), 5S RNA (Nielsen et al., 1993, Nucle~c Acids Res. 21, 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 C~ll 22, 405-413), U6 snRNA (Gupta and Reddy1 1990 WO 95123225 2 1 8 ~ 9 ~ 2 P~ l/~ rl~ 156 Nucleic Acids Res. 19, 2073-2075), Yault RNA (Kickoefer et al., 1993 J.Biol. Chem. 268, 7868-7873), tel~"~erase RNA (Romero and Blackburn, 1991 Cell67,343-353), andothers.
The construct described in this invention is able to accumulate RNA to 5 a ~iy~ k,dlllly higher level than other constructs, even those in which 5' and 3' ends are involved in hairpin loops. Using such a construct the level of t:A~,r~::,siol~ of a foreign RNA can be increased to between 20,000 and 50,000 copies per cell. This makes such constructs, and the vectors encoding such constructs, excellent for use in decoy, therapeutic editing 10 and antisense protocols as well as for ribozyme formation. In addition, the molecules can be used as agonist or antagonist RNAs (affinity RNAs).
Generally, applicant believes that the intramolecular base-paired interaction between the 5' terminus and the 3' region of the RNA should be in a double-stranded structure in order to achieve enhanced RNA
t 5 accumulation.
Thus, in one preferred ~",Lod~",el,L the invention features a pol lll promoter system (ç,g~, a type 2 system) used to synthesize a chimeric RNA
molecule which includes tRNA sequences and a desired RNA (e.~., a tRNA-based molecule).
The following eAt:ll, "" this invention with a type 2 pol lll promoter and a tRNA gene. Specifically to illustrate the broad invention, the RNA
molecule in the following example has an A box and a B box of the type 2 pol lll promoter system and has a 5' temminus or region able to base-pair with at least 8 bases of a co"ll l~",t:, lldly 3' end or region of the same RNA
molecule. This is meant to be a specific example. Those in the art will recognize that this is but one example, and other ~ odi~ l;, can be readily generated using other pol lll promoter systems and techniques generally known in the art.
By "terminus" is meant the terminal bases of an RNA molecule, ending in a 3' hydroxyl or 5' phosphate or 5' cap moiety. By "region" is meant a stretch of bases 5' or 3' from the terminus that are involved in base-paired lcuiliO115. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from WO 95/23225 2 1 8 3 9 ~ 2 ~ 156 the other end of the molecule provides a useful molecule able to be expressed at high levels.
By "3' region" is meant a stretch of bases 3' from the terminus that are involved in intramolecular bas-paired interaction with complementary 5 nllr-l~ot~ in the 5' terminus of the same molecule. The 3' region can be - designed to include the 3' terminus. The 3' region therefore is 2 0 nucleotides from the 3' terminus. For example, in the S35 construct described in the present invention (Eig~l the 3' region is one nucleotide from the 3' terminus. In another example, the 3' region is - 43 nt from 3' 10 terminus. These examples are not meant to be limiting. Those in the art wlll recogni2e that other 6"~bo ii,~ can be readily generated using techniques generally known in the art. Generally, it is preferred to have the 3' region within 100 bases of the 3' temminus.
By DtRNA molecule" is meant a type 2 pol lll driven RNA molecule that 15 is generally derived from any recognized tRNA gene. Those in the art will recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences 20 which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol lll lldlls~,,i,ulk,".
By "chimeric tRNA molecule" is meant a RNA molecule that includes a pol lll promoter (type 2) region. A chimeric tRNA molecule, for example, 25 might contain an i"l,dl"olecular base-paired structure bctween the 3' region and culllpl~ llldly 5' terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol lll promoter boxes. Thus, such a foreign RNA may be provided at the 3' end of the B box, or may be provided in 30 between the A and the B box, with the B box moved to an app,~prial~
location either within the foreign RNA or another location such that it is effective to provide pol lll lldi~sc,i,utioll. In one example, the RNA molecule may include a 11d"""~,1,ead ribozyme with the B box of a type 2 poi lll promoter provided in stem ll of the ribozyme. In a second example, the B
35 box may be provided in stem IV region of a hairpin ribozyme. A specific example of such RNA molecules is provided below. Those in the art will w09sl23~s 2 1 8 3 ~ 9 2 ~ b75 C[I56 recognize that this is but one example, and other e"~bo,i;",~"iD can be readily generated using techniques generally known in the art.
By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.
By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA illl~ldcliu~,s and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev.
Biochem. 60, 631-652). By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J.American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first l~coyl,i~s and then binds a target RNA
through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-~A,ur~Dsioll of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV
RNA (Sullenger et al., 1990 Cell 63, 601-608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
By "therapeutic editing RNA" is meant an antisense RNA that can bind to its cellular target (RNA or DNA) and mediate the Illo.liri~,dlion of a specific base.
By Uagonist RNA" is meant an RNA molecule that can bind to protein receptors with high affinity and cause the stimulation of specific cellular pathways.

W0 95~23225 . r~ 156 218~99~

By "antagonist RNA" is meant an RNA molecule that can bind to cellular proteins and prevent it from performing its normal biological function (for example, see Tsai et al., 1992 Proc. Natl. Acad Sci. USA 89, 8864-8868).
In other aspects, the invention includes vectors encoding RNA
molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this RNA.
Thus, the invention features a l,di,s~"iL,ed non-naturally occuring RNA
molecule which includes a desired therapeutic RNA portion and an illlldrllol~cular stem formed by base-pairing illL~ld~:liul~s between a 3' region and c~ uler~ dly n~lcl~ot~ s at the 5' terminus in the RNA. The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs.
In preferred ~Illbo~i"~ L~, the 5' terminus of the chimeric tRNA
includes a portion of the precursor molecule of the primary tRNA molecule, of which 2 8 n~cleoticlec are involved in base-pairing interaction with the 3' region; the chimeric tRNA contains A and B boxes; natural sequences 3' of the B box are deleted, which prevents ~ndoyt"~ous RNA ,u,ucessi"y, the desired RNA molecule is at the 3' end of the B box; the desired RNA
molecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group conai~li"g of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and ~I,ldgû,,i~l RNA; the molecule has an i,,lld,,,ùlecular stem resulting from a base-paired interaction between the 5' terminus of the RNA and a colll,.)l~ lldly 3' region within the same RNA, and includes at least 8 bases; and the 5' terminus is able to base pair with at least 15 bases of the 3' region.
In most preferred ~,,,LJOdjlll~ , the molecule is t~d~ ,,iL,ed by a RNA
polymerase lll based promoter system, e.g., a type 2 pol lll promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol lll promoter separated by between 0 and 300 bases; DNA
vector encoding the RNA molecule of claim 51.

WO 9S/23225 ; 2 1 8 ~ 9 9 ~ PCT/IB95/00156 In other related aspects, the invention features an RNA or DNA vector encoding the above RNA molecule, with the portions of the vector encoding the RNA functioning as a RNA pol lll promoter; or a cell containing the vector; or a method to provide a desired RNA molecule in a cell, by 5 introducing the molecule into a cell with an RNA molecule as described above. The cells can be derived from animals, plants or human beings.
In order for RNA-based gene therapy app~a~ s to be effective, sufficient amounts of the therapeutic RNA must accumulate in the a,u~.ruplia~ " IlAr C~ dlllll~llL of the treated cells. Accumulation is 10 a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerase ll (pol ll) and RNA pûly",~,dse lli (pol lll) based t~X~ a~ ll systems have been used to produce therapeutic RNAs in cells (Sarver & Rossi, 1993 AIDS Res. &
Human Retrovirus~s 9, 483-487; Yu et al., 1993 P.N.A.S.(USA) 90, 6340-15 6344). However, pol lll based e~ ssiûl1 cassettes are II,e~ .lly moreattractive for use in ~,.~,,t,~si"g antiviral RNAs for the following reasons.
Pol ll produces ",essel~y~r RNAs located exclusively in the cytoplasm, whereas pol lll produces functional RNAs found in both the nucleus and the cytoplasm. Pol ll promoters tend to be more tissue restricted, whereas pol 20 lll genes encode tRNAs and other functional RNAs necessary for basic "hollcekA~ping" functions in all cell types. Therefore, pol lll promoters are likely to be expressed in all tissue types. Finally, pol lll ~Idlls~ from a given gene accumulate to much greater levels in cells relative to pol ll genes.
I"~ IAr accumulation of therapeutic RNAs is also d~.elld~"l on the method of gene transfer used. For example, the retroviral vectors presently used to a-;c~,"",li~,.l stable gene transfer, integrate randomly into the genome of target cells. This random i,,ley,dli~.l1 leads to varied s:,ioll of the llall~ d gene in individual cells c~"" ~i~i"g the bulk treated cell population. Therefore, for maximum effectiveness, the llall~tell~d gene must have the capacity to express therapeutic amounts of the antiviral RNA in the entire treated cell population, It:gal~l~ss of the illL~ylaliO~I site.

WO 95/23225 -- 2 1 8 3 3 ~ 2 1 ~I,~i t-1s6 `-Pol lll System The following is just one non-limiting example of the invention. A pol lll based genetic element derived from a human tRNAjmet gene and termed ~3-5 (Fiq. 33; Adeniyi-Jones et al., 1984 supra), has been adapted 5 to express antiviral RNAs (Sullenger et al., 1990 Mol. Cell. Biol, 10, 6512-6523). This element was inserted into the DC retroviral vector (Sullenger et al., 1990 Mol. Cell. Biol. 10, 6512-6523) to ac.,~",pl;;,l, stable gene transfer, and used to express antisense RNAs against moloney murine leukemia virus and anti-HlV decoy RNAs (Sullenger et al., 1990 Mol. Cell.
10 Biol. 10, 6512-6523; Sullenger et al., 1990 Cell63, 601-608; Sullenger et al., 1991 J. Virol. 65, 6811-6816; Lee et al., 1992 The New Biologis~ 4, 66-74). Clonal lines are expanded from individual cells present in the bulk population, and therefore express similar amounts of the therapeutic RNA
in all cells. Development of a vector system that generates therapeutic 15 levels of therapeutic RNA in all treated cells would represent a significant advancement in RNA based gene therapy ",o,' ' Applicant examined hammerhead (HHI) ribozyme (RNA with enzymatic activity) t~,ult5s~iul1 in human T cell lines using the ~3~5 vector system (These constructs are termed "~3-5/HHI"; Fia. 34). On average, 20 ribozymes were found to accumulate to less than 100 copies per cell in the bulk T cell populations. In an attempt to improve ~plb~5iull levels of the ~3-5 chimera, the applicant made a series of modified ~3-5 gene units containing enhanced promoter elements to increase llails~ uliul~ rates, and inserted structural elements to improve the i,,l, -" ll~r stability of the 25 ribozyme llal~s.,li~, (~). One of these modified gene units, termed S35, gave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original ~3-5/HHI vector system.
Ribozyme accumulation in individual clonai lines from the pooled T cell populations ranged from 10 to greater than 100 fold more than those 30 achieved with the original ~3-51HHI version of this vector.
The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and a"la~o~ l RNAs. Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such 35 as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral _ _ . : ,.. _ . _.. _ _ _ : .

WO 95/232~5 2 18 ~ ~ 9 2 r~ s6 vectors, including but not limited to, other stable gene transfer systems such as adeno-A~so~ d virus (AAV; Carter, 1992 Curr. Opin. Genet. Dev.
3, 74), as well as transient vector systems such as plasmid delivery and adenoviral vectors (Berkner, 1988 BioTechn~ques 6, 616-629).
As described below, the S35 vector encodes a truncated version of a tRNA wherein the 3' region of the RNA is base-paired to Cu~ )le~ Ldly n~lrlPot~ As at the 5' terminus, which includes the 5' precursor portion that is normally processed off during tRNA maturation. Without being bound by any theory, Applicant believes this feature is important in the level of ~,ur~asiul~ observed. Thus, those in the art can now design equivalent RNA molecules with such high ~A,ur~ssiull levels. Below are provided examples of the Ill~ lodolo~y by which such vectors and tRNA molecules can be made.
~3-5 Vectors The use of a truncated human tRNAjmet gene, termed ~3-5 (E~;
Adeniyi-Jones et al., 1984 supra), to drive ~Xult:a:~iOII of antisense RNAs, and subsequently decoy RNAs (Sullenger et al., 1990 supra) has recently been reported. Because tRNA genes utilize internal pol lll promoters, the antisense and decoy RNA sequences were expressed as chimeras containing tRNAjmet sequences. The truncated tRNA genes were placed into the U3 region of the 3' moloney murine leukemia virus vector LTR
(Sullenger et al., 1990 supra).
RAc~-Paired Str~lrtllres Since the a3-5 vector culllbilldliull has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cioned ribozyme-encoding sequences (temmed as "~3-5/HHI") into this vector to explore its utility for ~,~,ul~a~ therapeutic ribozymes. How~ver, low ribozyme accumulation in human T cell lines stably transduced with -. this vector was observed (E~)- To try and improve accumulation of the 30 ribozyme, applicant illColluoldl~d various RNA structural elements (Fi~. 34) into one of the ribozyme chimeras (~3-5/HHI).
Two strategies were used to try and protect the termini of the chimeric Lrdl~scli,ul:, from exonucleolytic d~ylt~ddliul~. One strategy involved the illCul,uoldliull of stem-loop structures into the termini of the transcript. Two wo ss/2322s 2 1 8 3 9 ~ 2 I~ 156 such constructs were cloned, S3 which contains a stem-loop structure at the 3' end, and S5 which contains stem-loop structures at both ends of the transcript (Figure 34). The second strategy involved modification of the 3' terminal sequences such that the 5' terminus and the 3' end sequences 5 can form a stable base-paired stem. Two such constructs were made: S35 in which the 3' end was altered to hybridize to the 5' leader and acceptor stem of the tRNAjmet domain, and S35Plus which was identical to S35 but included more extensive structure formation within the non-ribozyme portion of the ~3-5 chimeras (Figure 34). These stem-loop structures are 10 also intended to sequester non-ribozyme sequences in structures that will prevent them from interfering with the catalytic activity of the ribozyme.
These constructs were cloned, producer cell lines were generated, and stably-transduced human MT2 (Harada et al., 1985 supra) and CEM (Nara 8L Fischinger, 1988 supra) cell lines were e~ld~ ,l,ed (Curr. Protocols Mol.
15 Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). The RNA sequences and structure of S35 and S35 Plus are provided in Figures 40-47. ~
Referring to Figure 48. there is provided a general structure for a chimeric RNA molecule of this invention. Each N i"de~.el,d~"~ly 11~ 5~
none or a number of bases which may or may not be base paired. The A
20 and B boxes are optional and can be any known A or B box, or a consensus sequence as ~ in the figure. The desired nucleic acid to be expressed can be any location in the molecule, but preferably is on those places shown adjacent to or between the A and B boxes (designated by arrows). FiQure 49 shows one example of such a structure in which a 25 desired RNA is provided 3' of the illlld"~ol~,ular stem. A specific example of such a construct is provided in Fi~ures 50 and 51.
FY~rnple 26: Cloning of ~3-5-Ribozyme Chimera Oligorlllcleoticles encoding the S35 insert that overlap by at least 15 n~lcleotidQs were designed (5' GATCCACTCTG~i I G I I ~; I G I I I I I GA 3' 30 and 5' CGCGTCAAAAACAGAACAGCAGAGTG 3'). The oligonllclPoti~lQs (10 IlM each) were denatured by boiling for 5 min in a buffer containing 40 mM Tris.HCI, pH8Ø The oligon~rleotides were allowed to anneal by snap cooling on ice for 10-15 min.
The annealed oligonucleotide mixture was converted into a double-35 stranded molecule using Sequenase~ enzyme (US Biochemicals) in a _ ~ _ : . .. . .. _ .. _ . .. . . _ _ .

WO 9S12322S 2 1 g 3 9 9 2 r ~ 156 buffer containing 40 mM Tris.HCI, pH7.5, 20 mM MgC12, 50 mM NaCI, 0.5mM each of the four deoxyribonucleotide Ili,uho~ " 10 mM DTT. The reaction was allowed to proceed at 37C for 30 min. The reaction was stopped by heating to 70C for 15 min.
The double stranded DNA was digested with d,l~plOplid~ restriction endonucleases (BamHI and Mlul) to generate ends that were suitable for cloning into the ~3-5 vector.
The double-stranded insert DNA was ligated to the ~3-5 vector DNA
by incubating at room temperature (about 20C) for 60 min in a buffer containing 66 mM Tris.HCI, pH 7.6, 6.6 mM MgCI2, 10 mM DTT, 0.066 uM
ATP and 0.1U/Ill T4 DNA Ligase (US Bi~ui,~",i..~ls).
Competent E. coli bacterial strain was transformed with the l~c~"lLi,)dllI vector DNA by mixing the cells and DNA on ice for 60 min.
The mixture was heat-shocked by heating to 37C for 1 min. The reaction 15 mixture was diluted with LB media and the cells were allowed to recover for 60 min at 37C. The cells were plated on LB agar plates and incubated at 37C for ~ 18 h.
Plasmid DNA was isolated from an overnight culture of l~culllLil~dllt clones using standard protocols (Ausubel et al., Curr. Protocols Mol.
20 Biology 1990, Wiley & Sons, NY).
The identity of the clones were d~ d by sequencing the plasmid DNA using the Sequenase~) DNA sequencing kit (US Biochemicals).
The resulting l~c~lllbilldllI ~3-5 vector contains the S35 sequence.
The HHI encoding DNA was cloned into this Q3-5-S35 co,lldi";"g vector 25 using Sacll and B~mHI restriction sites.
F~rnple 27: Northern analysis RNA from the transduced MT2 cells were extracted and the presence of 1~3-5/ribozyme chimeric lldllSCli,Ul~ were assayed by Northern analysis (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY).
30 Northern analysis of RNA extracted from MT2 transductants showed that Q3-5lribozyme chimeras of d,uulu,ulidl~ sizes were expressed (Fig. 35.36).
In addition, these results demonstrated the relative differences in accumulation among the different constructs (Fi~ure 35.36). The pattern of WO 95123225 ~ 218 3 9 9 2 PCTIIB9~/00156 ~,urt:ssioll seen from the ~3-5/HHI ribozyme chimera was similar to 12 other ribozymes cloned into the ~3-5 vector (not shown). In MT-2 cell line, ~3-5/HHI ribozyme chimeras accumulated, on average, to less than 100 copies per cell.
Addition of a stem-loop onto the 3' end of ~3-5/HHI did not lead to increased ~3-5 levels (S3 in Fig. 35.36). The S5 construct containing both 5' and 3' stem-loop structures also did not lead to increased ribozyme levels (Fi~. 35.36).
yly, the S35 construct ~-UI~sbiUII in MT2 cells was about 100-fold more abundant relative to the original ~3-5/HHI vector lldl)suli,uls (Fig. 35.36). This may be due to increased stability of the S35 transcript.
EYArnple 28: Cleava~e activity To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T
cells were incubated with a labeled substrate containing the HHI cleavage site (Figure 37). Ribozyme activity in all but the S35 constructs, was too low to detect. However, ribozyme activity was detectable in S35-transduced T cell RNA. Comparison of the activity observed in the S35-transduced MT2 RNA with that seen with MT2 RNA in which varying 20 amounts of in vitro transcribed S5 ribozyme chimeras, indicated that between 1-3 nM of S35 ribozyme was present in S35-transduced MT2 RNA. This level of activity c~ ,uu,,d~ to an intracellular conce"l,aliull of 5,000-15,000 ribozyme molecules per cell.
FYArnple 29: Clonal vAriAtir~rl Variation in the ribozyme ~X,u~ iOI~ levels among cells making up the bulk population was d~l~""i"ed by ~el)e~ 9 several clonal cell lines from the bulk S35 transduced CEM line (Curr. Pro~ocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (Fi~ure 38 and 39).
All the individual clones were found to express active ribozyme. The ribozyme activity detected from each clone correlated well with the relative amounts of ribozyme observed by Northern analysis. Steady state ribozyme levels among the clones ranged from approximately 1,000 molecules per cell in clone G to 11,000 molecules per cell in clone H (Fig.

wo 95123225 ~ 1 8 3 9 9 ~ 6 ~). The mean accumulation among the clones, calculated by averaging the ribozyme levels of the clones, exactly equaled the level measured in the parent bulk population. This suggests that the individual clones are - ,~,ur~st",ld~ive of the variation present in the bulk population.
The fact that all 14 clones were found to express ribozyme indicate that the percentage of cells in the bulk population ~ s~i"g ribozyme is also very high. In addition, the lowest level of ~X,u~ iù11 in the clones was still more than 10-fold that seen in bulk cells transduced with the original ~3-5 vector. Therefore, the S35 gene unit should be much more effective in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.
FYArn~¦e 30 StAhi¦i~
Finally, the bulk S35-transduced line, resistant to G418, was p,~,pogdL~d for a period of 3 months (in the absence of G418) to determine if ribozyme e~ ,r~;ol, was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme e:x,u,~5:,iu" after 3 months. This difference probably arose from cells with varying amount of ribozyme t:~,ul~sSiOIl and exhibiting different growth rates in the culture becoming slightly more prevalent in the culture. However, ribozyme expression is apparently stable for at least this period of time.
Exam~le 31: ~esi~n And construction of TR7-tRNA Chimera A ~Idlls~.li,UIiol1 unit, termed TRZ, is designed that contains the S35 motif (Fi~ure 52). A desired RNA (e.g. ribozyme) can be inserted into the indicated region of TRZ tRNA chimera. This construct might provide additional stability to the desired RNA. TRZ-A and TRZ-B are non-limiting examples of the TRZ-tRNA chimera.
Referring to Fi~. 53-54. a hd"""e,l,ead ribozyme targeted to site I
- 30 (HHITRZ-A; ~19~) and a hairpin ribozyme (HPITRZ-A; Fig. 54), also targeted to site 1, is cloned individually into the indicated region of TRZ
tRNA chimera. The resulting ribozyme trancripts retain full RNA cleavage activity (see for example Eig~). Applicant has shown that efficient WO 95/23225 ~ 218 3 9 9 2 F~,l,. 'I 156 r~X~ 5:,kJl~ of these TRZ tRNA chimera can be achieved in ~l~a~
cells.
Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated region of TRZ-5 tRNA chimera to achieve therapeutic levels of RNA expression inr l ldl l ~ cells.
Sequences listed in Figures 40-47 and 50 - 54 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily 10 generated using techniques known in the art, are within the scope of the present invention.
FYAmDle 32: RibQzvme ~x,~"~s~i.." in T cell lines Ribozyme ~ asion in T cell lines stably-transduced with either a retroviral-based or an Adeno-~cso~ d virus (AAV)-based ribozyme 15 ~Xp~55iO11 vector (Figure 56). The human T cell lines MT2 and CEM were transduced with either retroviral or AAV vectors encoding a neomycin slelctable marker and a ribozyme (S35/HHI) expressed from pol lll metj tRNA-driven promoter. Cells stably-transduced with the vectors were selectivelyt expanded medium containing the neomycin antibiotic 20 derivative, G418 (0.7 mg/ml). Ribozyme ~ si~l~ in the stable cell lines was then alalyzed by Northern analysis. The probe used to detect ribozyme l,dnsc,i,ul~ also cross-hybridized with human metj tRNA
sequences. Refering to Figure 56, S35/HHI RNA accumulates to significant levels in MT2 and CEM cells when transduced with either the retrovirus or 25 the AAV vector.
These are meant to be non-limiting examples, those skilled in the art will recognize that other vectors such as adenovirus vector (Figure 57), plasmid DNA vector, alpha virus vectors and the other derivatives there of, can be readily generated to deliver the desired RNA, using techniques 30 known in the art and are within the scope of this invention. Additionally, the L,d":,..,i~,lion ~Inits can be expressed individually or in multiples using pol ll and/or pol lll promoters.
References cited herein, as well as Draper WO 93/23569, 94/02495, 94/06331, Sullenger WO 93/12657, Thompson WO 93/04573, and Sullivan WO 9S/2322S ?~ 1 ~ 3 ~ ~ P~IL ~ .SG

WO 94/04609, and 93/11253 describe methods for use of vectors decribed herein, and are i".io,~ordled by reference herein. In particular these vectors are useful for adl"! ,k.l,dlion of antisense and decoy RNA
molecules.
- 5 FY~ le 33: Lig~ Ribozymes ~re ~ active The ability of ribozymes generated by ligation methods, described in Draper et al., PCT WO 93/23569, to cleave target RNA was tested on either matched substrate RNA (Fi~. 58) or long (622 nt) RNA (FiQ~ 59. 60 and 61).
Matched substrate RNAs were chemically synthesized using solid-10 phase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res.
18, 5433-5441). Substrate RNA was 5' end-labeled using [~32p] ATP and polynucleotide kinase (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley & Sons, NY). Ribozyme reactions were carried out under ribozyme excess conditions (kCatlKM; Herschlag and Cech, 1990 Bio~ ",i~L~y 29, 10159-10171). Briefly, ribozyme and substrate RNA were denatured and renatured separately by heating to 90C and snap cooling on ice for 10 min in a buffer containing 50 mM Tris. HCI pH 7.5 and 10 mM M9CI2-Cleavage reaction was initiated by mixing the ribozyme with the substrate at 37C. Aliquots of 5 1ll were taken at regular intervals of time and the reaction was stopped by mixing with equal volume of ~uilllalllkle gel loading buffer (Curr. Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley &
Sons, NY). The samples were resolved on 20 % polyacrylamide-urea gel.
Refering to~iç~, -~G refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA
(Turner and Sugimoto, 1988 ~). RPI A is a HH ribozyme with 6/6 binding amms. This ribozyme was synthesized chemically either as a one piece ribozyme or was sy"ll ~e~ d in two fragments followed by ligation to generate a one piece ribozyme. The kcatlKM values for the two ribozymes were ~o~ aldLle.
A template containing T7 RNA polymerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone. The target RNA (containing HH ribozyme cleavage sites B, C and D) was ~Idlls.~ ed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during llalls~ n by including [a-32P] CTP as one 35 of the four ribonucleotide llif l1osyhdl~s. The lldll~ lioll mixture was WO 9512322~ 2:18 3 ~ 9 2 1~11~ ' ~ - ISG

treated with DNase-1, following ~Idi~S~;li,UliOI1 at 37C for 2 hours, to digestaway the DNA template used in the lldllsc,i,uli~,. RNA was pl~i,uit..~d with Isoplu,udllol and the pellet was washed two times with 70% ethanol to get rid of salt and r~lci~otidsc used in the lldl1sc,i~uliull reaction. RNA is 5 rssuspended in DEPC-treated water and stored at 4C. Ribozyme cleavage reactions were carried out under ribozyme excess (kCatlKM) conditions [Herschlag and Cech 1990 ~. Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90C for 2 min in the presence of 50 mM Tris.HCI, pH 7.5 and 10 1û mM MgCI2. The RNAs were renatured by cooling to 37C for 1û-20 min.
Cleavage reaction was initiated by mixing the ribozyme and target RNA at 37C. Aliquots of 5 ,ul were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on a sequencing gel.
15 FYArnl~le 34: ~Id"l",~,l,ead ribozymes with 2 2 base-~aired stem ll are ytjCally active To decrease the cost of chemical synthesis of RNA, applicant was interested in cl~ .,lll;,l;,lg whether the length of stem ll region of a typicalhammerhead ribozyme (2 4 bp stem ll) can be shortened without 20 dt~ a";,~y the catalytic efficiency of the HH ribozyme. The length of stem llwas sy~l~llldliu.,lly shortened by one base-pair at a time. HH ribozymes with three and two base-paired stem ll were chemically synthesized using solid-phase RNA ~ ,ulluldlllidi~ chemistry (Scaringe ~1., 1990 .su~ra).
Matched and long substrate RNAs were synthesized and ribozyme 25 assays were carried out as described in example 33. Referring to fiqures .
~ 63 and 64. data shows that sl,o(l~";"g stem ll of a hammerhead ribozyme does not siy"i~;~,d"lly alter the catalytic efficiency. It is applicant's opinion that l1al"",e,l,ead ribozymes with 2 2 base-paired stem ll region are catalytically active.
30 FYAml~le 35: Synthesis of ~tAIyti~Ally active hAi~in ribozymes RNA molecules were bll~lll;C~.I'; synthesized having the nucleotide base sequence shown in Fiq. 65 for both the 5' and 3' fragments. The 3' fragments are pho~,ul~u,;l~ ,d and ligated to the 5' fragment essentially as described in example 37. As is evident from the Figure 65. the 3' and 5' 35 fragments can hybridize together at helix 4 and are covalently linked via WO 95/23225 2 ~i $ 3 ~ 9 ~ r~ . 156 GAAA sequence. When this strUcture hybridizes to a substrate, a ribozyme-substrate complex structure is formed. While helix 4 is shown as 3 base pairs it may be fommed with only 1 or 2 base pairs.
40 nM mixtures of ligated ribozymes were incubated with 1-5 nM 5' 5 end-labeled matched substrates (chemically sy,l'lesi~d by solid-phase synthesis using RNA pho~ or~"~iJil~ chemistry) for different times in 50 mM Tris/HCI pH 7.5, 10 mM MgC12 and shown to cleave the substrate eflici0ntly ~Fig.66).
The target and the ribozyme sequences shown in Fi~ 62 and 65 are 10 meant to be non-limiting examples. Those in the art will recognize that other t~",~o ii"~"ts can be readily generated using other sequences and techniques generally known in the art.
V. Consl~ Y of H~irpin ~ yl~
There follows an improved trans-cleaving hairpin ribozyme in which a 15 new helix (i.e., a sequence able to form a double-stranded region with another single-stranded nucleic acid) is provided in the ribozyme to base-pair with a 5' region of a separate substrate nucleic acid. This helix is provided at the 3' end of the ribozyme after helix 3 as shown in Fi~ure 3~ In addition, at least two extra bases may be provided in helix 2 and a portion 20 of the substrate co~ ," ii"g to helix 2 may be either directly linked to the 5' portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atleast one base. By trans-cleaving is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself 25 in an i"11~ 01ecular cleavage reaction.
By "base-pair" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example I loo~ ell type) of il ~ ti~l ls The increase in length of helix 2 of a hairpin ribozyme (with or without 30 he!ix 5) has several advantages. These include improved stability of the ribozyme-target complex ~n vivo . In addition, an increase in the ~t~cog"ili-,l1 sequence of the hairpin ribozyme improves the specificity of the ribozyme. This also makes possible the targeting of potential hairpin flECr~F~EG SHEET ~
ISA/EP

WO 95/23225 2 1 8 3 ~ ~ 2 . ~ . 156 ribo~yme sites that would otherwise be irlA~ceccihlq due to n~ o,i"g secondary structure.
The increase in length of heiix 2 of a hairpin ribozyme (with or without helix 5) enhances trans-ligation reaction catalyzed by the ribozyme. Trans-5 ligation reactions catalyzed by the regular hairpin ribozyme (4 bp helix 2) isvery inefficient (Komatsu et al., 1993 Nucleic Acids F~es. 21, 185). This is attributed to weak base-pairing i"~rdc~iu"s between substrate RNAs and the ribozyme. By increasing the length of helix 2 (with or without helix 5) the rate of ligation (in vitro and in vivo) can be enhanced several fold.
Results of expeli",~"~ suggest that the length of H2 can be 6 bp without ~ "ili~,anlly reducing the activity of the hairpin ribozyme. The H2 arm length variation does not appear to be sequence dependent. HP
ribozymes with 6 bp H2 have been designed against five different target RNAs and all five ribozymes efficiently cleaved their cognate target RNA.5 Additionally, two of these ribozymes were able to sllccqccflllly inhibit gene 55iUII (e.g., TNF-o~) in Illdlllllldlidll cells. Results of these ex~,~,i",~"l~
are shown below.
HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNA in a sequence-specific manner, however, the rate of the cleavage 20 reaction is lower than those catalyzed by HP ribozymes with 6 bp H2.
FYArnr~le 36: 4 and 6 hACP ~lAir H~
Referring to Figures 67-72. HP ribozymes were synthesized as described above and tested for activity. Surprisingly, those with 6 base pairs in H2 were still as active as those with 4 base pairs.
25 Vl. Chemical l\l~Jl~i~,c.llo OligonuclP~otides with 5'-C-Alkyl ~r~
The introduction of an alkyl group at the 5'-position of a nucleoside or nucleotide sugar introduces an additional center of chirality into the sugar moiety. Referring to Eig~, the general structures of 5'-C-alkyln~lcleotidPc 30 belonging to the D-allose, 2, and L-talose, 3, sugar families are shown.
The family names are derived from the known sugars D-allose and L-talose (R1 = CH3 in 2 and 3 in Figure 75). Useful specific D-allose and L-talose WO 95123~25 P~, I/LI.~5 156 O 21839~12 nucleotide derivatives are shown in Figure 76. 29-32 and Figure 77, 58-61 respectively.
This invention relates to the use of 5'-C-alkylnucleotides in oligon~lclPoti~es, which are particularly useful for enzymatic cleavage of 5 RNA or single-stranded DNA, and also as antisense oligor~ leotide~ As the term is used in this ~r~ ti~ln, 5'-C-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 5'-C-alkylnucleotide c-""~ el~l~. replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or 10 single-stranded ~CO9~ iOII sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave i"~-di"olecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.
Also within the invention are 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides.
Such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of 20 the 5'-C-alkyl group may reduce binding affinity of the oligonucleotide containing this rllOui~i~,dliu~, if that moiety is not in an essential base pairforming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of 25 less consequence. Thus, for example, if a 5'-C-alkyl-c~",ld;"i"g molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in 30 the synthesis of such n~cleotides and oligor~ leoti~Ps (examples of which are shown in the Figures), and to methods for their synthesis.
- Thus, in one aspect, the invention features 5'-C-alkylr~ lPn~ Pc, that is a nucleotide base having at the 5'-position on the sugar molecule an alkyl moiety. In a related aspect, the invention also features 5'-C-35 alkyln~l~leotides, and in preferred e,llbodi",~"~ features those where the nucleotide is not uridine or thymidine. That is, the invention preferably 21~3~

includes all those n~lcleotk~Pc useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above. In preferred embodiments, the sugar of the nucleoside or nucleotide is in an optically pure form, as the talose or allose sugar.
Examples of various alkyl groups useful in this invention are shown in Fi~ure 75. where each R1 group is any alkyl. These examples are not limiting in the invention. Specifically, an "alkyl" group refers to a saturated aliphatic hy~,-,.,GI~oll, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be 5~hCtitl~tPd or url.ellh~ctitllterl When 8l,h5titl,tgd the s~hstitlltpd group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO2 or N(CH3)2, amino, or SH. The term also includes alkenyl groups which are unsaturated hy~ucarLJo~ groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be 5llhstit~ltPrl or uncllh~titlltP-I When sllhstit~ltPd the sl~hstit~te~ group(s) is preferably, hydroxyl, cyano, alkoxy, 2û =O, =S, NO2, halogen, N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated h~ cGIl~oll group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be sllhctitlltPd or unsuhstit~lt~rl When sl~hstitllted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, =O, =S, NO2 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groUps. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated 7r electron system and includes carbocyclic aryll heterocyclic aryl and biaryl groups, all of which may be optionally sl~hctit~l ~ The preferred substituent(s) of ary~ groups are halogen, ~ ldl.~ ,; lyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring _ . _ .. : . . .. . .

~10 95~2322S 218 3 9 9 2 r ~ IS6 atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally sl Ih5titl ItP~ Heterocyclic aryl groups are groups having from 1 to 3 h~l~rud~u",s as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable l~ ' "~ include oxygen, sulfur,5 and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl - pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
1 û In other aspects, also related to those discussed above, the invention features oligor~ oti~f~s having one or more 5'-C-alkylnucleotides; e.g.
enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic 15 molecule with at least one nucleotide having at its 5'-position an alkyl group. In other related aspects, the invention features 5'-C-alkylnucleotide b~JIldlt~s. These lli,UIlOb,UIldl~s can be used in standard protocols to form useful oligorlllclRotirl~s of this invention.
The 5'-C-alkyl derivatives of this invention provide enhanced stability 20 to the oligonulceotides c~r~ g them. While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.
In another aspect, the invention features a method for conversion of a protected allo sugar to a protected talo sugar. In the method, the protected allo sugar is contacted with triphenyl pl~ob,ul,i"e, diethylaz~dk,a,Lu,~ylate, and ~I~illub~ oic acid under inversion causing conditions to provide the protected talo sugar. While one example of such conditions is provided below, those in the art will recognize other such conditions. Applicant has found that such conversion allows for ready synthesis of all types of - nucleotide bases as ~t"" ' ' ~ ~' in the figures.
While this invention is applicable to all oligorl~ lPotid~s, applicant has found that the modified molecules of this invention are particulary useful for 35 enzymatic RNA molecules. Thus, below is provided examples of such WO95/23225 = ' ~ 2183~2 r~ s .-156 molecules. Those in the art will recognize that equivalent procedures can be used to make other molecules without such enzymatic activity.
Specifically, Fiaure 1 shows base numbering of a hammerhead motif in which the numbering of various nl~r,lest~ s in a l1d"""~ ad ribozyme is 5 provided. This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims.
Referring to Figure 1. the preferred sequence of a hallllllelllead ribozyme in a 5'- to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 5'-C-alkyl 5llhctit"~?~ rl~lrlPoticles10 that maintain or enhance the catalytic activity and or nuclease resistance ofthe har"",e,l,ead ribozyme is described. Substitutions of any nucleotide with any of the modified n~cleotides shown in Fi~ure 75 are possible.
The following are non-limiting examples showing the synthesis of nucleic acids using ~'-C-alkyl-substituted phospho,dl"idil~s and the 15 syntheses of the amidites.
EYArn~le 37: Synthesis of I lallllllt~ ad Rihr~ymes CorltAirlin~ 5'-~Alkyl-n~cl~otides 8~ Other Modified Nur,l~otides The method of synthesis would follow the procedure for nommal RNA
synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.;
20 Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. NucleicAcids Fies. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and ,ullo~lJlloldllliJiL~s at the3'-end (compounds 26-29 and 56-59). These 5'-C-alkyl substituted 25 phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 intron catalytic nucleic acids, or into antisense oligor~cleoti~s They are, therefore, of general use in any nucleic acid structure.
FYArrl~le 3~. Methyl-? ~-o-lsoproDylidine-6-Deoxy-~-D-Allf~fllrArlr~cirl~o (4) A suspension of L-rhamnose (100 9, 0.55 mol), CuSO4 (120 g) and conc. H2SO4 (4.0 mL) in 1.0 L of dry acetone was mixed for 24 h at RT, then filtered. Conc. NH40H (5 mL) was added to the filtrate and the newly formed pl~ ildl~ was filtered. The residue was ,.,"ce"l,dlt,cl in vacuo, coevaporated with pyridine (2 x 300 mL), dissolved in pyridine (500 mL) 35 and cooled to 0 C. A solution of p-toluenesufonylchloride (107 9, 0.56 -WO 951'L3'115 2 ~ 8 3 9 ~ ~ F~ 156 1t7 mmol) in dry DCE (500 mL) was added dropwise over 0.5 h. The reaction mixture was left for 16 h at RT. The reaction was quenched by adding ice-water (0.5 L) and, after mixing for 0.5 h, was extracted with ~ loruiu~ (0.75 L). The organic layer was washed with H2O (2 x 500 mL), 10% H2SO4 (2 x 300 mL), water (2 x 300 mL), sat. NaHCO3 (2 x 300 mL), brine (2 x 300 mL), dried over MgSO4 and evaporated to dryness. The residue (115 9) was dissolved in dry MeOH (1 L) and treated with NaOMe (23.2 9, 0.42 mmol) in MeOH. The reaction mixture was left for 16 h at 20 C, neutralized with dry CO2 and evaporated to dryness. The residue was suspended in .,I~lolufu"" (750 mL), filtered, col,~ dl~d to 100 mL and purified by flash l,lllUllldlU,U,ld,UIly in CHCI3 to yield 45 9 (37%) of compound 4.
FY~rnple 39: Methyl-2.3-alsoi~lù,u~ e-5-Gt-Butyldiphenylsilyl-6-Deoxy-~-D-Allofuranoside (5).
To solution of methylfuranoside 4 (12.5 9 62.2 mmol) and AgNO3 (21.25 g, 125.0 mmol) in dry DMF (300 mL) t-butyldiphenylsilyl chloride (22.2 9, 81 mmol) was added dropwise under Ar over 0.5 h. The reaction mixture was stirred for 4 h at RT, diluted with CHCI3 (200 mL), filtered and evaporated to dryness (below 40 C using a high vacuum oil pump). The residue was dissolved in CH2CI2 (300 mL) washed with sat. NaHCO3 (2 x ~0 mL), brine (2 x 50 mL), dried over MgSO4 and ~Vd,l~Oldl~d to dryness.
The residue was purified by flash Clllullldlug,d~l,y in CH2CI2 to yield 20.0 9 (75%) of compound 5.
FY~rnDle 40: Methyl-5-~t-Butyldiphenylsilyl-6-Deoxy-~-D-Allofuranoside ~6~. .
Methylfuranoside 5 (13.5 9, 30.6 mmol) was dissolved in CF3COOH:dioxane:H20 / 2:1:1 (vlvlv, 200 mL) and stirred at 24 C for 45 . m. The reaction mixture was cooled to -10 C, neutralized with conc.
NH40H (liO mL) and extracted with CH2CI2 (500 mL). The organic layer was separated, washed with sat. NaHCO3 (2 x 75 mL), brine (2 x 75 mL), dried over MgSO4 and evaporated to dryness. The product 6 was purified by flash ~ UllldlU~U,ld~-lly using a 0-1û% MeOH gradient in CH2CI2. Yield g.o g (76%).

WO 951232~5 2 1 ~ 3 9 ~ ~ PCT/IB95/001~6 FYArnDIe 41: Methyl-2 ~-di-~Ben~r,yl-~-~t-Butyl~i~nhenylsilyl-6-Deoxy-~-D-AIlofurArloside (7).
Methyifuranoside 6 (7.0 g, 17.5 mmol) was coevaporated with pyridine (2 x 100 mL) and dissolved in pyridine (100 mL). Benzoyl chloride (5.4 9, 38.5 mmol) was added and the reaction mixture was left at RT for 16 h. Dry EtOH (50 mL) was added and the reaction mixture was evaporated to dryness after 0.~ h. The residue was disso~ved in CH2CI2 (300 mL), washed with sat. NaHCO3 (2 x 75 mL), brine (2 x 75 mL) dried over MgSO4 and evaporated to dryness. The product was purified by flash 10 ~ ulllaluyla,ully in CH2C12 to yield 9.5 9 (89%) of compound 7.
EYArnple 42: 1-~Acetyl-7 :~-di-~b~ yl 5 ~t-~ut~lui~ "~ ;lyl 6-Deoxy-~-D-AllofurArlose (8).
Dib~ ual~ 7 (4.7 9, 7.7 mmol) was dissolved in a mixture of AcOH
(10.0 mL), Ac2O (20.0 mL) and EtOAc (30 mL) and the reaction mixture was 15 cooled 0 C. 98% H2SO4 (0.15 mL) was then added. The reaction mixture was kept at 0 C for 16 h, and then poured into a cold 1:1 mixture of sat.
NaHCO3 and EtOAc (150 mL). After 0.5 h of vigorous stirring the organic phase was separated, washed with brine (2 x 75 mL), dried over MgSO4, evaporated to dryness and coevaporated with toluene (2 x ~0 mL). The 20 product was purified by flash ulllullldluuld~Jlly using a gradient of 0-5%
MeOH in CH2CI2. Yield: 4.0 g (82% as a mixture of ~ and ~ isomers).
FY~rnple 43: 1-~2'.3'-di-~Ben~yl-~'-~t-~tvlr~inhenylsilyl-6'-l~eoxv-~-D-AIlQf~lrArlosyl)~racil (9).
Uracil (1.44 9, 11.5 mmol) was suspended in mixture of 25 hexameth~/ldi~ild~dne (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT, evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of acetates 8 (6.36 g, 10.0 mmol) in dry CH3CN (100 mL), followed 30 by CF3SO3SiMe3 (2.8 9, 12.6 mmol). The reaction mixture was kept at 24 C for 16 h, cu~ a~t~d to 1/3 of its original volume, diluted with 100 mL
of CH2CI2 and extracted with sat. NaHCO3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO4, and evaporated to dryness. The product 9 was purified by flash clllullldluy,d,ully using a gradient of 0-5% MeOH in CH2CI2. Yield:
35 5.7 g (80%).
, . . .. . .. . .

WO95/2322~ 39 ~ P~,l/ll,,~l 156 .

FY~rnple 44: N~Ben7nyl-1-(2'.3'-Di-~Ben7nyl-5'-~t-Butyldiphenylsilyl-6'-Deoxy-~-D-Allofuranosyl)Cytosine (10).
N4-benzoylcytosine (1.84 9, 8.56 mmoi) was suspended in mixture of htl,.d",~l~,yldisilazane (100 mL) and pyridine (50 mL) and boiled under 5 reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH3CN (100 mL), followed by CF3SO3SiMe3 (4.76 g, 21.4 mmol). The reaction mixture was boiled 10 under reflux for 5 h, cooled to RT, co",,e"lldl~d to 1/3 of its original volume, diluted with CH2CI2 (100 mL) and extracted with sat. NaHCO3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO4 and evaporated to dryness.
Purification by flash chromatography using a gradient of 0-5% MeOH in CH2CI2 yielded 1.8 9 (55%) of compound 10.
FY~mDIe 45: N~-Ben7nyl-9-(2'.3'-di-~Ben7nyl-5'-~t-Butyldiphenylsilyl-6'-Deoxy-3-D-Allofuranosyl)adenine (11).
N6-benzoyladenine (2.86 9, 11.88 mmol) was suspended in mixture of h~dlll~ .yldi~ild~dl)e (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (7 h) occurred, and then for an additional 20 hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 9, 5.6 mmol) in dry CH3CN (100 mL) followed by CF3SO3SiMe3 (6.59 9, 29.7 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, col,ce"~,dl~d to 1/3 of its original volume,25 diluted with CH2CI2 (100 mL) and extracted with sat. NaHCO3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO4 and evaporated to dryness. The product 11 was purified by flash ulllul~cllu~u,~d,ul~y using a gradient of 0-5%
MeOH in CH2CI2. Yield: 2.7 9 (60%).
FY~rnple 46: N2-lsobutyryl-9-(2l~3l-di-~Berl7nyl-5!-~t-Butyldiphen 30 6'-Deoxy~-D-Allofuranosyl)guanine (12).
- N2-lsobutyrylguanine (1.47 9, 11.2 mmol~ was suspended in mixtureof ~ dll..,;~.~ldi:~ild~dlle (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (6 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and 35 coevaporated with dry toluene (2 x 50 mL). To the residue was added a W095/2322S : 218 3 9 9 2 ~ 15G

solution of of acetates 8 (3.4 9, 5.3 mmol) in dry CH3CN (100 mL~ followed by CF3SO3SiMe3 (6.22 9, 28.0 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, con..~"l,d~d to 1/3 of its original volume, diluted with CH2CI2 (100 mL) and extracted with sat. NaHCO3 (2 x 50 mL), 5 brine (2 x 50 mL) dried over MgS04 and evaporated to dryness. The product 12 was purified by flash chromatography using a gradient of 0-2%
MeOH in CH2CI2. Yield: 2.19 (54%).
E~rnple 47: N~-Ben7~yl-9-(Z'.3'-di-~ben7nyl-6'-Deoxy-B-c-Allofur~no syl)adenine (15).
Nucleoside 11 (1.65 9, 2.0 mmoi) was dissolved in THF (50 mL) and a 1 M solution of TBAF in THF (4 mL) was added. The reaction mixture was kept at RT for 4 h, evaporated to dryness and the product purified by flash clllullld~uyldp~ly using a gradient of 0-5% MeOH in CH2CI2 to yield 1.0 9 (85%) of compound 15.
5 EY~rn~ile 48~ en~nyl-9-~2'.3'-di-0-Ber~yl-5'-0-DimQthoxytrityl-6'-Deoxy-B-D-Allofur~nosyl)-adenine (19).
Nucleoside 15 (0.55 9, 0.92 mmol) was dissolved in dry CH2C12 (50 mL). AgN03 (0.34 9, 2.0 mmol), dil"~ xytii.yl chloride (0.68 9, 2.0 mmol) and sym-collidine (0.48 9) were added under Ar. The reaction mixture was 20 stirred for 2h, diluted with CH2C12 (100 mL), filtered, evaporated to dryness and coevaporated with toluene (2 x 50 mL). Purification by flash ~I,Iullld~oy,d,uhy using a gradient of 0-5% MeOH in CH2CI2 yielded 0.8 g (97%) of compound 1g.
FY~mDle 49: N~-Benzoyl-9-(-5'-~Dimethoxytrityl-6'-Deoxy-~-D-Allo-25 furanosyl)adenine (~3).
N~Cleogi-le 19 (1.8 9~ 2 mmol) was dissolved in dioxane (50 mL), cooled to 0 C and 2 M NaOH (50 mL) was added. The reaction mixture was kept at 0 C for 45 m, neutralized with Dowex 50 (Pyr+ form), filtered and the resin was washed with MeOH (2 x 50 mL). The filtrate was then 30 evaporated to dryness. Purification by flash cl~rùlt,d~uy,dul,y using a gradient of 0-10% MeOH in CH2CI2 yielded 1.1 9 (80%) ûf 23.

WO 95123225 ~ 3 9 9 ~ r~ J, -~6 F~Arn~le 50~ Benzoyl-9-(-5'-~Di~ uxytrityl-2'-~t-butyldimethylsilyl-

6'-Deoxy-~-D-Allofuranosyl)adenine (27).
Nucleoside 23 (1.2 9, 1.8 mmol) was dissolved in dry THF (50 mL).
Pyridine (0.50 9, 8 mmol) and AgNO3 (0.4 9, 2.3 mmol) were added. After 5 the AgNO3 dissolved (1.5 h), t-butyldimethylsilyl chloride (0.35 9, 2.3 mmol) was added and the reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted with CH2cl2 (100 mL), filtered into sat.
NaHCO3 (50 mL), extracted, the organic layer washed with brine (2 x 50 mL), dried over MgSO4 and evaporated to dryness. The product 27 was 10 purified by flash ul,ru",dluy,dp.l,y using a hexdl~es.EtOAc / 7:3 gradient.
Yield: 0.7 9 (5û%).
FY~rnDle 51: N~-Benzoyl-9-(-5'-GDimethoxytrityl-2'-Gt-butyldimethylsilyl-6'-Deoxy-~-D-Allofuranosyl)adenine-3'-(2-Cyanoethyl N.N-diisovropyl-phU~vl~o,d,,,iJilt,) (31).
Standard phosphitylation of 27 according to Scaringe,S.A.;
Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield.
E~ArnDIe 52: Methyl-5-~p-N~I ubtl~ I~ùyl-2.3-Glsuvl u~ ,e-6-deoxy-B-L-T~ furanoside (5) Methylfuranoside 4 (3.1 9 14.2 mmol) was dissolved in dry dioxane (200 mL), p-l,iL,ube,,~oic acid (1û.0 9, 60 mmol) and triphenylphosphine (15.74 9, 60.0 mmol) were added followed by DEAD (1û.45 9, 60.0 mmol).
The reaction mixture was left at RT for 16 h, EtOH (5 mL) was added, and after 0.5 h the reaction mixture was evaporated to dryness. The residue was dissolved in CH2CI2 (300 mL) washed with sat. NaHCO3 (2 x 75 mL), brine (2 x 75 mL) dried over MgSO4 and evaporated to dryness.
Purification by flash cl"u"~dluy~d,ul~y using a ll~Ad,les~EtoAc 19:1 gradient yielded 4.1 9 (78%) of compound 33. Subsequent debenzoylation (NaOMe/MeOH) and silylation (see preparation of 5) led to L-talofuranoside 34 which was converted to IJllo~,ulloldlllidi~s 58-61 using the same m~:ll,odol~gy as described above for the pl~pa,dlioll of the pho~,ul~o,dr" " - of the D-allo-isomers 29-32.
The alkyl sllh~titlltqd n~cleot~ s of this invention can be used to form stable oligor~l~cleotides as discussed above for use in enzymatic cleavage WO 95123225 21 8 3 9 ~ 2 P~ ~ h 156 or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.
A~ dlioll of such oligon~cl~otides is by standard procedure. See Sullivan et al., PCT W0 94/ 02595.
The ribozymes and the target RNA containing site 0 were synthesized, d.3~ulul~u~d and purified as described above. RNA cleavage assay was carried our at 37C in the presence of 10 mM MgC12 as described above.
Applicant has sllhstitlltPd 5'-C-Me-L-talo nu~!lQoti~C at positions A6, A9, A9 + G1û, C11.1 and C11.1 + G10, as shown in Figure 78 (HH-01 to HH-05). HH-0 1,2,4 and ~i showed almost wild type activity (Figure 79).
However, HH-03 cle""~lial~dl~d low catalytic activity. Ribozymes HH-01, 2, 3, 4 and ~ are also extremely resistant to deyldddlio~ by human senum nucleases.
Oligonucleotides with 2'-Deoxy-2'-Alkylnucleotide This invention uses 2'-deoxy-2'-alkylrl~lcleotides in oligor~cl~otides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligor~ leoti-ies. As the term is used in this ~rplic~tion, 2'-deoxy-2'-alkylnucleotide-containing enzymatic 2û nucleic acids are catalytic nucleic molecules that contain 2'-deoxy-2'-alkylnucleotide c~l"~unt:~,L~ replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded ~ucoy"il;oll sequences. These molecules are able to cieave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave i"l,d""~l~cularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.
Also within the invention are 2'-deoxy-2'-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligorlllrleotif4c Contrary to the findings of De l~ "a~k~l et al. applicant has found that such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence o~
the 2'-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an 0ssential base pair W0 95l23225 ~ ~ ~ 3 9 ~ ~ P~ 56 forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stabiiity may make the loss of activity of less consequence. Thus, for example, if a 2'-deoxy-2'-alkyl-~o"ldi"i"g 5 molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such mo i;fi,,dliulls. The invention also relates to novel i"lt:""ed;dlt,~ useful in the synthesis of such r~lcleoticles and oligorlll~l~oti~es (examples of which 10 are shown in the Figures), and to methods for their synthesis.
Thus, in one aspect, the invention features 2'-deoxy-2'-alkylnll~leoticlss that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred ~",bo.li",e~ features those where the nucleotide is not uridine or thymidine. That is, the 15 invention preferably includes all those n~ laotid~Ps useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above.
Examples of various alkyl groups useful in this invention are shown in Fi~ure 81. where each R group is any alkyl. The term "alkyl" does not 20 include alkoxy groups which have an "-O-alkyl" group, where "alkyl" is defined as described above, where the O is adjacent the 2'-position of the sugar molecule.
In other aspects, also related to those discussed above, the invention features oligor~lcleoti~ss having one or more 2'-deoxy-2'-alkylrlll~lPoticlPs 25 (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by fomming the enzymatic molecule with at least one nucleotide having at its 2'-position an alkyl group. In other30 related aspects, the invention features 2'-deoxy-2'-alkylnucleotide o:,ul,dles. These ~ llO~ lldl~s can be used in standard protocols to form useful oligorll~rlPotirles of this invention.
The 2'-alkyl derivatives of this invention provide enhanced stability to the oligon~ Potides COII~dill' 19 them. While they may also reduce 35 absolute activity in an in vitro assay they will provide enhanced overall wo ssl23225 ~ r~ 156 activity In vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.
In another aspect, the invention features 11d"""e,1,ead motifs having 5 enzymatic activity having ribon~rlPoti~les at locations shown in Figure 80 at 5, ~ 8, 12, and 15.1, and having s~hstitllted ribon~lcleotides at other positions in the core and in the substrate binding arms if desired. (The term "core" refers to positions between bases 3 and 14 in Figure 80, and the binding amms correspond to the bases from the 3'-end to base 15.1, and 10 from the 5'-end to base 2). Applicant has found that use of riborl~cl~oti~i~os at these five locations in the core provide a molecule having sufficient enzymatic activity even when modified rlucleoti~l~s are present at other sites in the motif. Other such Cullll,il~dliùl~s of useful ribor)l Icl~oti-t~s can be dt,l~z""i"ed as described by Usman etal. supra.
Figure 80 shows base numbering of a 11dll""ell,~ad motif in which the numbering of various n~rleotides in a l1d,l""~ ad ribozyme is provided.
This is not to be taken as an indication that the Figure is prior art to the pending claims or that the art discussed is prior art to those claims.
Referring to Figure 80 the preferred sequence of a 11d"""~,1,ead ribozyme 20 in a 5'- to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGMA. In this invention the use of 2'-Galkyl s~lhstitl~t~d n~rleoti~le~
that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Although .s~lhstit~ltions of any nucleotide with any of the modified nllcl~otides shown in Figure 81 are 25 possible and were indeed synthesized the basic structure co",,.,osed of promarily 2'-O-Me r~ leotides weth selected sllhstit~ltions was chosen to maintain maximal catalytic activity (Yang et al. Biochemistly1992, 31 5005-5009 and Paolella eta/. EMBOJ. 1992, 11, 1913-1919) and ease of synthesis, but is not limiting to this invention.
Ribozymes from Figure 80 and Table 45 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17 all of the modified ribozymes retained at lease 1/10 of the wild-type catalytic activity. From Table 45, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 55, data nût shown). The order of most agressive nuclease activity was fetal bovine ~ wo ~sl23~s . ~18 3 9 ~ 2 PCT/IB9S/00156 serum, > human serum >human plasma > human synovial fluid. As an overall measure of the effect of these 2'-sllhstitlltions on stability and activity, a ratio B was calculated (Table 45). This 13 value indicated that all - modified ribozymes tested had significant, >100 - >1700 fold, increases in 5 overall stability and activity. These increases in B indicate that the lifetime of these modified ribozymes In vivo are sig"i~i~ar,lly increased which should lead to a more pronounced biological effect.
More general sllhctitlltions of the 2'-modified n~lcleoti-iPs from Figure 81 also increased the t1/2 of the resulting modified ribozymes.
10 However the catalytic activity of these ribozymes was decreased > 1 0-fold.
In Figure 86 compound 37 may be used as a general intermediate to prepare derivatized 2'C-alkyl phosphoramidites, where X is CH3, or an alkyl, or other group described above.
The following are non-limiting examples showing the synthesis of nucleic acids using 2'-C-alkyl 5~hCtitlltPd pl1oa,ullo,d,,,kli~s, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance.
FY~m~le 53: Synthesis of I la~ ad Ribozvmes Containino 2'-Deoxy-2'-Alkvlr~lcleotides & Other 2'-Modified N~lcleotides The method of synthesis used generally follows the procedure for normal RNA synthesis as described in Usman,N.; Ogilvie,K.K:; Jiang,M.-Y.;
Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 1fl 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and pl~o~ )old",idiIt,s at the 3'-end (compounds 10, 12, 17, 22, 31, 18, 26, 32, 36 and 38). Other 2'-modified pl~o~,l,ol~",kliI~s were prepared according to: 3 & 4, Eckstein et al. International PublicaUon No. WO 92/07065; and 5 Kois et al.
N~lcleQsides & N~ oti~l~s1993, 12, 1093-1109. The average stepwise coupling yields were ~98%. The 2'-sllhstitlltPd phospholdi"i.li~t:s were incorporated into hammerhead ribozymes as shown in Figure 80.
However, these 2'-alkyl 9~ tPd pllO~ llOldll ' ~ may be ill(,~llJoldI~d not only into 11d"""~ ad ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group ll intron catalytic nucleic acids, or into antisense WO 951232~5 ~ 2 1 8 3 ~ ~ 2 , ~ ,. 156 oligorl~lcl~ti~les They are, therefore, of general use in any nucleic acid structure.
EY~rnvle 54: Rihn~yme Activity Assay Purified 5`-end labeled RNA substrates (15-25-mers) and purified 5'-5 end labeled ribozymes (-36-mers) were both heated to 95 C, quenched on ice and eqll'' dl~d at 37 C, separately. Ribozyme stock solutions were 1 mM, 200 nM, 40 nM or 8 nM and the final substrate RNA
CùllC~Illldliul-s were ~ 1 nM. Total reaction volumes were 50 mL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCI2. Reactions were 10 initiated by mixing substrate and ribozyme solutions at t = 0. Aliquots of 5 mL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each time point was quenched in ~r"~d",kle loading buffer and loaded onto a 15%
denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a pllo:"~l,u,i",a~r (Molecular Dynamics).
1~ FY~mple 5~: ~C tz,hility Assay 500 pmol of gel-purified 5'-end-labeled ribozymes were plt~.;ipildl~d in ethanol and pelleted by centrifugation. Each pellet was resuspended in 20 mL of a~ ul lidl~ fluid (human serum, human plasma, human synovial fluid or fetal bovine serum) by vortexing for 20 s at room temperature. The 20 samples were placed into a 37 C incubator and 2 mL aliquots were withdrawn after incubation for 0, 1~, 30, 45, 60, 120, 240 and 480 m.
Aliquots were added to 20 mL of a solution containing g5% ~ui"~d",ide and 0.5X TBE (50 mM Tris, 50 mM borate, 1 mM EDTA) to quench further nuclease activity and the samples were frozen until loading onto gels.
2~ Ribozymes were size-fractionated by electrophoresis in 20%
acrylamidet8M urea gels. The amount of intact ribozyme at each time point was quantified by scanning the bands with a phosphorimager (Molecular Dynamics) and the half-life of each ribozyme in the fluids was determined by plotting the percent intact ribozyme vs the time of incubation and 30 ~LId,uoldliu,, from the graph.
EY~rnvle 56: 3l~5l-~(T~Lld;~n! ~uLJyl-r~icil~y~rle-1 .3-dLyl)-2l-~phenoxyth e~rbonyl-Uridine (7) To a stirred solution of 3',5`-O-(tetraisopropyl-disiloxane-1,3-diyl)-uridine, 6, (15.1 g, 31 mmol, synthesized according to Nucleic Acid . , _ . . . _ . _ _ _ .

~ilVO 9SIU225 ~ PCTIIB95~00156 3~92 Chemistry, ed. Leroy Townsend, 1986 pp. 229-231) and dimethylamino-pyridine (7.57 9, 62 mmol) a solution of phenylclllo,ull,iul~oformate (5.15 mL, 37.2 mmol) in 5û mL of acetonitrile was added dropwise and the reaction stirred for 8 h. TLC (EtOAc:hexanes / 1:1) showed di:,a,u,uea,dl~C~
5 of the starting material. The reaction mixture was evaporated, the residue dissolved in cl~l~ru~ul"" washed with water and brine, the organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel with EtOAc:hexanes / 2:1 as eluent to give 16.44 9 (85%) of 7.
1û Example 57: 3'.5'-O-(T~l,disop,upyl-disiloxane-1.3-diyl)-2'-C-Allyl -Uridine To a refluxing, under argon, solution of 3',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)-2'-O-phenoxyllliocd,uo,,yl-uridine, 7, (5 9, 8.û3 mmol) and allyltributyltin (12.3 mL, 4û.15 mmol) in dry toluene, benzoyl peroxide 15 (û.5 9) was added portiu,l~ ,G during 1 h. The resulting mixture was allowed to reflux under argûn for an additional 7-8 h. The reaction was then evaporated and the product 8 purified by flash ullrull~dluyldplly ûn silica gel with EtOAc:hexanes / 1:3 as eluent. Yield 2.82 g (68.7%).
ExamDle 58: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine (9) A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry tetrahydrofuran (THF) was treated with a 1 M solution of tetrabutylammoniumfluoride in THF (3.7 mL) for 1û m at room temperature. The resulting mixture was evaporated, the residue was loaded onto a silica gel column, washed with 1 L of chloroform, and the desired d~ u~ d compound was eluted with chlorofor~ dllol / 9:1. Appropriate fractions were combined, solvents removed by evaporation, and the residue was dried by coevaporation with dry pyridine. The oily residue was redissolved in dry pyridine, dimethoxytritylchloride (1.2 eq) was added and the reaction mixture was left under anhydrous conditions ovemight. The reaction was quenched with methanol (20 mL), evaporated, dissolved in chloroform, washed with 5% aq. sodium L~icdruond~ and brine. The organic layer was dried over sodium sulfate and evaporated. The residue was purified by flash UllldLuyld,ully on silica gel, EtOAc:hexanes/ 1:1 as eluent, to give 0.85 g (57%) of 9 as a white foam.

WO 95123225 ~18 ~ 9 ~ 2 r ~

FY~mple 59: 5'-O-DimethQxytrityl-2'-C-Allyl-Uridine 3'-(2-Cy~oethyl N.N-diisopropylphos~horArni~it~) (10) 5`-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 9, 1.12 mmol) was dissolved in dry .I;.;I,lolul"~ll,al-e under dry argon. N,N-Diisopropylethyl-5 amine (0.39 mL, 2.24 mmol~ was added and the solution was ice-cooled.
2-Cyanoethyl N,N-diisopropyl~ ,upl~0~,ul,o,dl"i.lil~ (û.35 mL, 1.57 mmol) was added dropwise to the stirred reaction solution and stirring was continued for 2 h at RT. The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture 10 was conc~"l,àl~d in vacuo (40 C) and purified by flash ulllull,aloyld,ùlly on silica gel using a gradient of lû-6û% EtOAc in hexanes containing 1%
triethylamine mixture as eluent. Yield: û.78 9 (90%), white foam.
FY~rnOIe 6û 3~s~-o-(T~ icl~ uvyl-r~ y~rle-l~3-diyl)-2~-c-Allyl-N4:
Acetyl-Cyti~line (11) Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol) and ~llo~,ul~oluus oxychloride (0.86 mL, 9.11 mmol) in 5û mL of anhydrous acetonitrile. To the resulting suspension a solution of 3',5'-O-(Ielld,~o~rupyl-disiloxane-l ,3-diyl)-2'-C-allyl uridine (2.32 g, 4.55 mmol) in 30 mL of acetonitrile was 2û added dropwise and the reaction mixture was stirred for 4 h at room temperature. The reaction was uul~c~ al~d in vacuo to a minimal volume (not to dryness). The residue was dissolved in clllo,u~ul,,, and washed with water, saturated aq. sodium bicdllJùl)al~ and brine. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo~ The 25 resulting foam was dissolved in 50 mL of 1,4-dioxane and treated with 29%
aq. NH40H overnight at room temperature. TLC (ulllolu~u,~ l,a~lol /
9:1) showed complete conversion of the starting material. The solution was evaporated, dried by coevaporation with anhydrous pyridine and acetylated with acetic anhydride (0.52 mL, 5.46 mmol) in pyridine 30 overnight. The reaction mixture was quenched with methanol, evaporated, the residue was dissolved in ol~lu~uru~l, washed with sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, evaporated to dryness and purified by flash ulllulllaluyla~ull~ on silica gel (3% MeOH in chloroform). Yield 2.3 9 (90%) as a white foam.

W0 9SI23225 ~ r~ 56 FY~mPIe 61: 5'-aDimethoxytrityl-2'-C-AllYl-N--Acetyl-Cytidine This compound was obtained analogously to the uridine derivative 9 in 55% yield.
FY~rnDle 62: 5'-O-Dimethoxytrityl-2'-C-allyl-N4-Acetyl-Cytidine 3'-(2-5 Cyanoethyl N.N-~ UUlUU~JIlOb,UIlOld~ ) (12) 2'-O-Dimethoxytrityl-2'-C-allyl-N4-acetyl cytidine (0.8 9, 1.31 mmol) was dissolved in dry di~ lu~u~ al~e under argon. N,N-D;;~op,u,uylûthyl-amine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.
2-Cyanoethyl N,N-diisopropylul,loru~ullobpl,ù,d,,,: " (0.38 mL, 1.7 mmol) 10 was added dropwise to a stirred reaction solution and stirring was continued for 2 h at room temperature. The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture was co"c~r,lrd~ed in vacuû (40 C) and purified by flash ~;lllullldt~yldplly on silica gel using cl~lorulurlll:ethanol / 98:2 with 2%
15 triethylamine mixture as eluent. Yield: û.91 9 (85%), white foam.
FY~mple 63: 2'-Deoxy-2'-Methylene-Uridine 2'-Deoxy-2'-methylene-3',5'-O~ "u,u~uy; " ne-1,3-diyl)-uridine 14 (Hansske,F.; Madej,D.; Robins,M. J. Ttl~al/e~luil 1984, 40, 125 and Matsuda,A.; Takenuki,K.; Tanaka,S.; Sasaki,T.; Ueda,T. J. Med. Chem.
20 1991, 34, 812) (2.2 9, 4.55 mmol ) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and cul)c~ dl~d in vacuo. The residue was triturated with petroleum ether and chromato3raphed on a silica gel column. 2'-Deoxy-2'-methylene-uridine (1.0 9, 3.3 mmol, 72.5%) was eluted with 20% MeOH in CH2C12.
25 EY~Dle 64: 5'-GDMT-2'-Deoxy-2'-Methylene-Uridine (15) 2'-Deoxy-2'-methylene-uridine (0.91 9, 3.79 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was 30 collc~"l,dl~d in v~cuo and the residue taken up in CH2CI2 (100 mL) and washed with sat. NaHCO3, water and brine. The organic extracts were dried over MgSO4, col,ce"l,dled in vacuo and purified over a silica gel column using EtOAc:hexanes as eluant to yield 15 (0.43 9, 0.79 mmol, 22%).
. _ .. . . . .

WO 95/~3225 218 3 3 ~ 2 PCTlll~95/00156 ~

EYArn~le 6~: 5~-~DMT-2'-Deoxy-2'-Methylene-Urir~i~e 3'-(2-Cy~noethyl N.N-t{iic~nroDv~ullo~vllolrl~ ) (17~
1 ~(2'-Deoxy-2'-methylene-5'-~dimethoXytrityl-~-D-ribofuranosyl)-uracil (0.43 9, 0.8 mmol) dissolved in dry CH2Clz (15 mL) was placed in a 5 round-bottom flask under Ar. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diiso-propylcl,lu,u,uhospl~ulaillidi~ (0.25 mL, 1.12 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 C). The product (0.3 g, 0.4 10 mmol, 50%) was purified by flash column ~I~IUllldlu~ld,Ully over silica gel using a 25-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.42 (CH2CI2: MeOH /15:1) EY~rnple 66: 2'-DeQxy-2'-DifluQromethylene-3'.5'-G(T~t,,~ uvyldisilox ~ne-1.3-diyl)-ur~ e 2'-Keto-3',5'-O-(Ielli~oplu~uy' 'i~ ' ~e-1,3-diyl)uridine 14 (1.92 g, 12.6 mmol) and triphenylul~o~jul,i,~e (2.5 g, 9.25 mmol) were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 C. A wamm (60 oc) solution of sodium cl~lulu, "" )~,f~a~ . in diglyme (50 mL~ was added (dropwise from an eql~ " dli"g dropping funnel) over a period of ~1 h. The 2û resulting mixture was further stirred for 2 h and cu",,e"t, _' in vacuo. The residue was dissolved in CH2CI2 and ul~lu~llaluyldull~d over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(t~l-.,;.,~p,u,uyl~ ilu,~dne-1,3-diyl)-uridinQ (3.1 9, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.
FY~rnDIe 67: 2'-Deoxy-2'-Difluoromethylene-Uri~ e 2'-Deoxy-2'-methylene-3',5'-a(t~lldi~u,~,u~yldi~iloxane-1,3-diyl)-uridine (3.1 9, 5.9 mmol) dissolved in THF (20 mL) was treated with 1 M
TBAF in THF (10 mL) for 20 m and co~ l..t~,d in vacuo The residue was triturated with petroleum ether and chromatographed on silica gel column.
2'-Deoxy-2'-difluoromethylene-uridine (1.1 9, 4.0 mmol, 68%) was eluted 30 with 20% MeOH in CH2C12.
EY~DIe 68: 5'-GDMT-2'-l:)eoxy-2'-Difl~ romethylene-Uridine (16) 2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (1.42 9, 4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture ~ wo sst2322s ~ 9 ~? ~ r~ 156 was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was collc~"lldlt~d in vacuo and the residue taken up in CH2CI2 (100 mL) and washed with sat. NaHCO3, water and brine. The organic extracts were dried over MgSO4, collc~ dl~d in vacuo and 5 purified over a silica gel column using 40% EtOAc:hexanes as eluant to yield 5'-O-DMT-2'-deoxy-2'-difluoromethylene-uridine 16 (1.05 9, 1.8 mmol, 45%).
E~rnple 69. 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine 3'-(2-Cyanoethyl N. N-diisopropylpho~jul lo, dl I ~i~it~) (18) 1 0 1-(2~-Deoxy-2~-difluoromethylene-5~-o-dimethoxytrityl-~B-D-ribofurano-syl)-uracil (0.577 9, 1 mmol) dissolved in dry CH2CI2 (15 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.36 mL, 2 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diiso-propykil,loluplloOpl~ordlllidil~ (0.44 mL, 1.4 mmol). The reaction mixture 15 was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 C). The product (0.404 9, 0.52 mmol, 52%) was purified by flash ~;llrUllldlUUld,U~ly over silica gel using 20-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. R~
0.48 (CH2CI2: MeOH /15:1).
20 Example 70: 2'-Deoxy-2'-Methylene-3'.5'-O-(T~II d;Oil~JI u~uyldisiloxane-1.3- diyl)-4-N-Acetyl-Cytidine 20 Triethylamine (4.8 mL, 34 mmol) was added to a solution of POCI3 (0.65 mL, 6.8 mmol) and 1,2,4-triazole (2.1 9, 30.6 mmol) in ac~lv"il,il~ (20 mL) at 0 C. A solution of 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldi-25 siloxane-1,3-diyl) uridine 19 (1.65 9, 3.4 mmol) in aG~:v,,iL,il~ (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h. The mixture was co~..ell,l,d~d in vacuo, dissolved in CH2CI2 (2 x 100 mL) and washed with 5% NaHCO3 (1 x 100 mL). The organic extracts were dried over Na2SO4 cûl~ce"l,dl~d in vacuo, dissolved 30 in dioxane (10 mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and CUllc~ dl~d in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (3 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.
NaHCO3 (5 mL). The mixture was collce"l,dl~d in vacuo, dissolved in 35 CH2CI2 (2 x 100 mL) and washed with 5% NaHCO3 (1 x 100 mL). The .

WO 95/2322~ , . 2 1 8 ~ 9 ~ 2 ~ 6 organic extracts were dried over Na2SO4, collce"~,al~d in vacuo and the residue ~ ullldluyldplled over silica gel~ 2'-Deoxy-2'-methylene-3',5'-O-di~oplupyldi~ilo~.dlle-1~3-diyl)-4-N-acetyl-cytidine 20 (1.3 9, 2.5 mmol, 73%) was eluted with 2û% EtOAc in hexanes.
FYArnple 71: 1-(2'-Deoxy-2'-Methylene-5'-O-Dimethoxytrityl-~-~-ribo-furArlosyl)-4-N-Acetyl-CytQsine 21 2'-Deoxy-2'-methylene-3',5'-~(l~ldi~o~Jruuyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 2û (1.3 9, 2.5 mmol) dissolved in THF (2û mL) was treated with 1 M TBAF in THF (3 mL) for 2û m and cul~celllldltld in vacuo. The 1 û residue was triturated with petroleum ether and ulllullldl~yld,ulled on silica gel column. 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (û.56 g, 1.99 mmol, 8û%) was eluted with 1û% MeOH in CH2CI2. 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (û.56 9, 1.99 mmol) was dissolved in pyridine (1û mL) and a solution of DMT-CI (û.81 9, 2.4 mmol) in pyridine (1û mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was CJllCt:lllldl~d in vacuo and the residue taken up in CH2CI2 (1ûû mL) and washed with sat. NaHCO3 (5û mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO4, conc~lllldl~d in vacuo and purified over a silica gel column using EtOAc:hexanes / 6û:40 as eluant to yield 21 (û.88 9, 1.5 mmol, 75%).
EYArrl~le 7~ 1 -(2~-Deoxy-2~-Methylene-5~-o-Dimethoxytrityl-~B-D-ribo-furArlosyl~-4-N-Acetyl-Cytosine 3'-(2-Cyanoethyl-N.N-r~iic~ro~ylphocphor-amidite) (22) 1-(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-,B-D-ribofuranosyl)-4-N-acetyl-cytosine 21 (0.88 g, 1.~ mmol) dissolved in dry CH2CI2 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.8 mL, 4.5 mmol~ was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropyl~,llk~lupllo~,ulloldll "~ (û.4 mL, 1.8 mmol). The reaction 3û mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After lû m the mixture evaporated to a syrup in vacuo (40 C). The product 22 (0.82 g, 1.û4 mmol, 69%) was purified by flash ullnJlll~.~uyld,ully over silica gel using 50-70% EtOAc gradient in hexanes, containing 1%
triethylamine, as eluant. Rf 0.36 (CH2Clz:MeOH 120:1).

~ WO9512322S -- 218~2 r~ 56 F~rnple 73: 2'-Deoxy-2'-Difluoromethylene-3'.5'-O-(Ttll~ uulu~,yl Y~e-1.3-diyl)-4-N-Acetyl-Cytidine (24) Et3N (6.9 mL, 5û mmol) was added to a solution of POCI3 (0.94 mL, 1û mmol) and 1,2,4-triazole (3.1 9, 45 mmol) in ac~tùl,il,ile (20 mL) at 0 C.
5 A solution of 2'-deoxy-2'-difluoromethylene-3',5'-0-(tt:l,diso~lul.yldisilox-ane-1,3-diyl)uridine 23 ([described in example 14] 2.6 9, 5 mmol) in act,l.,"il,ile (20 mL) was added dropwise to the above reaction mixture and left to stir at RT for 4 h. The mixture was col ,c~"lldlud in vacuo, dissolved in CH2CI2 (2 x 100 mL) and washed with 5% NaHCO3 (1 x 100 mL). The 10 organic extracts were dried over Na2SO4 conc~"l,dlt,d in vacuo, dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture was stirred for 12 h and COllcelllldl~d in vacuo. The residue was az~o~,uped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (5 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.
15 NaHCO3 (5mL). The mixture was conce"lldl~d in vacuo, dissolved in CH2CI2 (2 x 100 mL) and washed with 5% NaHCO3 (1 x 100 mL). The organic extracts were dried over Na2S04, cor,c~,"l,dled in vacuo and the residue cl~rullldlu~,lapl~ed over silica gel. 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisil~"~dl-e-1 ,3-diyl)-4-N-acetyl-cytidine 24 (2.2 9, 3.9 20 mmol, 78%) was eluted with 20% EtOAc in hexanes.
F~ rn~le 74: 1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytrityl-~-D-ribofuranosyl)-4-N-Acetyl-Cytosine (25) 2'-Deoxy-2'-difluul u" ,ull ~y l~.)e-3',5'-O-(I~I~ disulJl u~Jyldisiloxane-1,3-diyl)-4-N-acetyl-cytidine 24 (2.2 9, 3.9 mmol) dissolved in THF (20 mL) was 25 treated with 1 M TBAF in THF (3 mL) for 20 m and col,cel,l,dlt,d in vacuo.
The residue was triturated with petroleum ether and ~,I"u",~.`v,,d~,l,ed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 9, 2.8 mmol, 72%) was eluted with 10% MeOH in CH2CI2. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 9, 2.8 mmol) was dissolved in 30 pyridine (10 mL) and a solution of DMT-CI (1.03 9, 3.1 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was conc~lll,dl~d in vacuo and the residue taken up in CH2CI2 (100 mL) and washed with sat. NaHCO3 (50 mL), water (50 mL) and brine 35 (50 mL). The organic extracts were dried over MgSO4, conce"L,dled in wogsl~ 2~3~2 r~ L 5~~156 vacua and purified over a silica gel column using EtOAc:hexanes / 60:40 as eluant to yield 25 (1.2 9, 1.9 mmol, 68%).
FY~rnple 7~ (2l-Deoxy-2l-Difluoromethylene-5l-o-Dimethoxytrityl-~-D
rih~f~r~nosyl)-4-N-Acetylcytosine 3'-(Z-cyA~oethyl-N.N-rliicnpropylDh 5 phor~idite) (26) 1 -(2'-Deoxy-2'-difluoromethylene-5'-~dimethoxytrityl-,~-D-ribofurano-syl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in dry CH2CI2 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.5 mL, 2.9 mmol) was added, followed by the dropwise addition of 2-10 cyanoethyl N,N-diisopropyk,l,luru~uho~ullold,,,iJil~ (0.4 mL, 1.8 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL).
After 10 m the mixture was evaporated to a syrup in vacuo (40 C). The product 26, a white foam (0.52 9, 0.63 mmol, 65%) was purified by flash L;llrulllcltu~ld,ully over silica gel using 30-70% EtOAc gradient in hexanes, 15 containing 1% triethylamine, as eluant. R~ 0,48 (CH2CI2:MeOH / 20:1).
FY~mDIe 76: 2'-Ketû-3'.5'-O-(T~lr~ ,u,u, u,uyl(~ e-1.3-diyl)-6-N-(4-t-Butylberl7nyl)-Adenosine (28) Acetic anhydride (4.6 mL) was added to a solution of 3',5'-O-(tetraiso-propyldisiloxane-1,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (Brown,J.;
2û Christodolou, C.; Jones,S.; Modak,A.; Reese,C.; Sibanda,S.; Ubasawa A.
J. Chem .Soc. Perkin Trans. /1989, 1735) (6.2 9, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was col,c~lllldLed in vacuo. The residue was taken up in EtOAc and washed with water. The organic layer was dried over MgSO4 and 25 co~,cel,L,dlt:d in vacuo. The residue was purified on a silica gel column to yield 2'-keto-3',5'-O-(tull~ opn py' " ' ~e-1,3-diyl)-6-N-(4-t-butylben-zoyl)-adenosine 28 (4~a 9, 7.2 mmol, 78%).
Fx~m~ple 77: 2'-Cleoxy-2'-methylene-3'.5'-~(T~II diso~)rul~yl~ e-1.3-diyl)-6-N-(4-t-Bul~lu~ ,yl)-Adenosine (29) Under a pressure of argon, sec-butyllithium in hexanes (11.2 mL, 14.6 mmol) was added to a suspension of triphen~""el~,~"ul~o~ ol~ium iodide (7.07 9,17.5 mmol) in THF (25 mL) cooled at -78 C. The ll~llloy~eous orange solution was allowed to warm to -3û C and a solution of 2'-keto-3~5~-0~ ldiSO~IU~JlJi~ilu~dl~e-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine _, _, . . . .. .. .. ... ,, _ ,, V10951232'25 f~ 83~ P~ IS6 28 (4.87 9, 7.3 mmol) in THF (25 mL) was l~dlla~ d to this mixture under argon pressure. After warming to RT, stirring was continued for 24 h. THF
was evaporated and replaced by CH2C12 (250 mL), water was added (20 mL), and the solution was neutralized with a cooled solution of 2% HCI.
5 The organic layer was washed with H2O (20 mL), 5% aqueous NaHCO3 (20 mL), H2O to neutrality, and brine (10 mL). After drying (Na2SO4), the solvent was evaporated in vacuo to give the crude compound, which was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc / 7:3 afforded pure 2'-deoxy-2'-methylene-3',5'-o-(tetraiso-10 propyldisiloxane-1 ,3-diyl)-6-N-(4-t-buly~ l)-adenosine 29 (3.86 9, 5.8 mmol, 79%).
FY~rnple 78: 2'-Deoxy-2'-Methylene-ô-N-(4-t-Butylbenzoyl)-Adenosine 2'-Deoxy-2'-methylene-3',5'-O-(It~ u~luyyldisiloxane-l,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (3.86 9, 5.8 mmol) dissolved in THF (30 mL) 15 was treated with 1 M TBAF in THF (15 mL) for 20 m and col,c~"l,dlt,d in V2CUO. The residue was triturated with petroleum ether and ~,IllUllld~UUld~ ed on a silica gel column. 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (1.8 9, 4.3 mmol, 74%) was eluted with 10%
MeOH in CH2C12.
20 Fx~rnple 79: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine (29) 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75 9, 1.77 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI
(0.66 9, 1.98 mmol) in pyridine (10 mL) was added dropwise over 15 m.
25 The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The miXture was c~c~lllldL~d in vacuo and the residue taken up in CH2CI2 (100 mL) and washed with sat. NaHCO3, water and brine. The organic extracts were dried over MgSO4, cul~ce,,lldl~d in vacuo and purified over a silica gel column using 50%
30 EtOAc:hexanes as an eluant to yield 29 (0.81 9, 1.1 mmol, 62%).
Example 80: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine 3'-(2-Cyanoethyl N.N-diisopropylullu~plloldrllidil~) (31) 1 -(2'-D~oxy-2'-methylene-5'-O-dimethoxytrityl-,B-3-ribofuranosyl)-6-N-(4-t-butylbenzoyl)-adenine 29 dissolved in dry CH2CI2 (15 mL) was placed WO 95/232~5 218 3 9 ~ 2 I~ 156 ~

in a round bottom flask under Ar. Diisopropylethylamine was added, followed by the dropwise addition of 2-cyanoethyl N, N-diisopropyl~ loru,ullo~,uhord~ ila. The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was 5 evaporated to a syrup in vacl~o (40 C). The product was purified by flash chlu",d~ùy,ap~ly over silica gel using 30-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant (0.7 9, û.76 mmol, 68%). R~ 0.45 (CH2CI2: MeOH / 20:1) FYArn~le 81: 2'-Deoxy-2'-Difluoromethylene-3'.5'-O-(TPl,~ n~ uv~ldisilox-10 ane-1.3-diyl)-6-N-(4-t-Butylben7~yl)-Adenngi~e 2'-Keto-3',5'-O-(Itlll..;~vpluuyldibiluxdl1e-1 ,3-diyl)-6-N-(4-t-butyl-benzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenyl,.,l,us,vl,i,,e (2.9 g, 11 mmol ) were dissolved in diglyme (20 mLl, and heated to a bath temperature of 160 C. A warm (60 C) solution of sodium 15 chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 mL) was added (dropwise from an ellll "~alill~ dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and ~.o"c~, Ill..~,d in vacuo. Theresidue was dissolved In CH2C12 and ~illlullldlvyldplled over silica gel. 2'-Deoxy-2'-difluolu",PII, jlcne-3',5'-~(tt,l~ ,v,u,ul,yldiailu~d,1e-1,3-diyl)-6-N-20 (4-t-butylbenzoyl)-adenosine (4.1g, 6.4 mmol, 64%) eluted with 15%
hexanes in EtOAc.
ExamDle R:)~ 2~-Deoxy-2~-Difluu~ plll)rlQlle-6-N-(4-t-Butylbell7~lyl) Adenosine 2'-Deoxy-2'-difluolu,,,~l~,ylone-3',5'-~(l~ o,u,uuyl.li~ilvi.d~le-l,3-25 diyl)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 9, 6.4 mmol) dissolved in THF
(20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and COllCPII11dlt:d in vacuo. The residue was triturated with petroleum ether and ~illrUllldlU9ldl~11ed on a silica gel column. 2'-Deoxy-2'-difluoromethyl-ene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol, 77%) was eluted 30 with 20% MeOH in CH2C12.
FY~ le 83: 5'-aDMT-2'-D~nYy-2'-Difluoromethylene-6-N-(4-t-Butyl-ben7lyl)-Adenosine (30) 2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 9, 4.9 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in ...... , . , .. _ , . , . _ _ .. _ .

WO 951~3~5 2 1 8 3 ~ g 2 1 ~ ~. 156 pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was collc~"l,clL~d in vacuo and the residue taken up in CH2CI2 (100 mL) and washed with sat. NaHCO3, water and brine. The 5 organic extracts were dried over MgSO4, collce,,lld~d in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as eluant to yield 30 (2.6 g, 3.41 mmol, 69%).
FYArn~le 84: 5'-O-DMT-~'-Deoxy-2'-Difluoromethylene-6-N-(4-t-Butyl-ben~nvl)-Adenosine 3'-(2-Cyanoethvl N~N-diiso~roDylphosphoramidite) 1 0 (32) 1 -(2'-Deoxy-2'-difluoromethylene-5'-adimethoxytrityl-,~-D-ribofurano-syl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mmol) dissolved in dry CH2CI2 (25 mL) was placed in a round bottom flask under Ar.
Diisopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the 15 dropwise addition of 2-cyanoethyl N,N-diisopropyl.,l,l~,upllo~,ulloramidite (1.06 mL, 4.76 mmol). The reaction miYture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 C). 32 (2.3 g, 2.4 mmol, 70%) was purified by flash column ..lllullldluyld~Jlly over silica gel using 20-50% EtOAc gradient in 20 hexanes, containing 1% ~ ll"rl.."~i"e, as eluant. Rf 0.52 (CH2CI2: MeOH /
15:1).
FYArnDle 85: 2'-Deoxy-2'-Methoxycarbonylmethylidine-3'.5'-O-(Tetraiso-propylJ;,;lvxane-1.3-diyl)-Uridine (33) Methyl(triphenylphosphoranylidine~acetate (5.4 g, 16 mmol) was 25 added to a solution of 2'-keto-3',5' O~ opru~.yl disiloxane 1,3-diyl)-uridine 14 in CH2CI2 under argon. The mixture was left to stir at RT for 30 h. CH2CI2 (100 mL) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCI. The organic layer was . washed with H2O (20 mL), 5% aq. NaHCO3 (20 mL), H2O to neutrality, and 30 brine (10 mL). After drying (Na2SO4), the solvent was evaporated in vacuo to give crude product, that was Gll,ull,dloyldplled on a silica gel column.
Elution with light petroleum ether:EtOAc / 7:3 afforded pure 2'~deoxy-2'-ù~ycarbonylmethylidine-3',5'-a(L~lldi~ul~lupy~ xalle-1~3-diyl) uridine 33 (5.8 9, 10.8 mmol, 67.5%).

WO 95/23225 218 ~ ~ g ~ r~l,~ s ~ -156 Fx~rngle ~6: 2'-DeQxy-2'-Methoxy~rbonylmethyli-line-Uridine (34) Et3N-3 HF (3 mL) was added to a solution of 2'-deoxy-2'-methoxy-carboxylmethylidine-3',5'-O-(t~ld;.,oplupylJi~ilv~-d,~e-1,3-diyl)-uridine 33 (5 9, 9.3 mmol) dissolved in CH2CI2 (20 mL) and Et3N (15 mL). The 5 resulting mixture was evaporated in vacuo after 1 h and ~I,,u,,,dluy,dpl,ed on a silica gel column eluting 2'-deoxy-2'-methoxycarbonylmethylidine-uridine 34 (2.4 9, 8 mmol, 86%) with THF:CH2CI2 / 4:1.
F~rrlple 87: 5'-~DMT-2'-Deoxy-2'-Methoxy~ o"~""~ll,y~ ne Uri~lin~
2'-Deoxy-2'-methoxycarbonylmethylidine-uridine 34 (1.2 9, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of DMT-CI (1.5 9, 4.42 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was collc~, Itldl~d in vacuo and the residue taken 15 up in CH2CI2 (100 mL) and washed with sat. NaHCO3, water and brine.
The organic extracts were dried over MgSO4, cu~ d in vacuo and purified over a silica gel column using 2-5% MeOH in CH2CI2 as an eluant to yield 5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 35 (2.03 9, 3.46 mmol, 86%).
2û E~rn~le 88: 5'-~DMT-2'-Deoxy-2'-Methoxy~:~rbonylmethylidine-Urirline 3'-~2-cy~noethyl-N.N-fI~ r~uv~lvl,u;,,ullu,,..,~ .) (36) 1 -(2'-Deoxy-2'-2'-methoxycarbonylmethylidine-5'-O-dimethoxytrityl-~-D-ribofuranosyl)-uridine 35 (2.0 9, 3.4 mmol) dissolved in dry CH2CI2 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine 25 (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisoprop~ l,lorupl~o~,ul~u~d~idil~ (0.91 mL, 4.08 mmol).
The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture was evaporated to a syrup in vacuo (40 C).
5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine 3'-(2-30 cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1.8 9, 2.3 mmol, 67%) was purified by flash column ClllUllldlU"ld~lly over silica gel using a 30-60% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.44 (CH2CI2:MeOH / 9.5:0,5).

WO95123ZZS ~ g~2 r~ a 156 E~Arnole 89: 2'-Deoxy-2'-Carl uxy,ll~ll,ylidine-3' 5'-0-(T~l,di~u~uru,uyldi-nyAne-l~3-diyl)-uridine 37 2'-Deoxy-2'-" ,c,ll ,ùxy~arLu, ,ylmethylidine-3',5'-O-(lu~ op, u,uyldi-siloxane-1,3-diyl)-uridine 33 (5.û 9, 10.8 mmol) was dissolved in MeOH
5 (50 mL) and 1 N NaOH solution (50 mL) was added to the stirred solution at RT. The mixture was stirred for 2 h and MeOH removed in vacuo. The pH of the aqueous layer was adjusted to 4.5 with 1N HCI solution, extracted with EtOAc (2 x 100 mL), washed with brine, dried over MgSO4 and concel "~ dled in vacuo to yield the crude acid. 2'-Deoxy-2'-10 carboxymethylidine-3',5'-O-(tetraisopropy~" ' ne-1,3-diyl)-uridine 37 (4.2 9, 7.8 mmol, 73%) was purified on a silica gel column using a gradient of 10-15% MeOH in CH2C12.
The alkyl .sllhstitl' ' rl~leotides of this invention can be used to fomm stable oligor~l~cleotides as discussed above for use in enzymatic cleavage 15 or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.
Adlllilli:,lldliol, of such oligonucl~oti~Qs is by standard procedure. See Sullivan et aL PCT WO 94/02595.
Oli~on-lcleotideS with 3' and/or 5' Dil lalUl l~vl l~
This invention synthesis and uses 3' and/or 5' .lillalùpll~:,ullul)dltl-, e.g., 3' or 5'-CF2-pl-o~,ul1u,,dle-, ~hstitllt~d nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule.
As the term is used in this application, 5'- and/or 3'-.lil,alo~ ,,ul~ol~dL~ nucleotide containing ribozymes, cl~ùkyli~u~ymes (see Usman et al., PCT/US94/11649, i,,c~l,uordl~d by reference herein), and chimeras of n~lClQoti~es are catalytic nucleic molecules that contain 5'-and/or 3'-dihalopl10~,ul1ol~dle nucleotide cu",,u~ "l~ replacing, but not Iimited to, double-stranded stems, single-stranded "catalytic core"
sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA or DNA transcript. This invention concerns 21839~ --nucleic acids formed of standard nucleotides or modified nucleotides, which also contain at least one 5'-dihalopl,o~,uhonal~ and/or one 3'-dihalJ~ o~pl1uliale group.
The synthesis of 1-O-Ac-2,3-di-O-Bz-D-ribofuranose 5-d-~+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-O-isopropylidene-13-D-ribofuranose 5-deoxy-5-dihalomethyll,llo~uhondle is described (e.g., for the difluoro, in Figure 87). Col~d~llsd~iùl~ of this suitably derivatized sugar with silylated pyrimidines and purines affords novel nucleoside 5'-deoxy-5'-dihalomethylphosphonates. These i"l~""e~ial~s 10 may be i,,co,~uordl~d into catalytic or antisense nucleic acids by either chGmical (conversion of the nucleoside 5'-deoxy-5'-dihalomethyl,uhoa,ul ,o~ idleS into suitably protected ,ul lo~,ul ,ù, dl I lidi~as 1 2a or solid supports 12b, e.g., Figure 88) or enzymatic means (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into their 15 lli~ lloa,ulldl~s, e.g., 14 Figure 89, forT7 lldi~a.,l,: ,).
Thus, in one aspect the invention features 5' and/or 3'-dihalonucleotides and nucleic acids containing such 5' and/or 3'-dihalonll~leoticles The general structure of such molecules is shown below.
o o (R3O)2PCX2 ~5 RZ ~EI (R30)2PCx2 ~EI
R2 Rt CX2 R~ CX2 Rt (R3O)2P= o (R3O)2P = o where R1 is H, OH, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, or car~u~ial~; each R2 is separately H, OH, or R; each R3 is separately a phosphate protecting group, e.g., methyl, ethyl, cyanoethyl, p-25 nitrophenyl, or ~I~lu~uul~a~yl; each X is separately any halogen; and each Bis any nucleotide base.
The invention in particular features nucleic acid molecules having such modified n~leotid~s and enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleoside 5'-deoxy-~'-30 dihalo and/or 3'-deoxy-3'-dihalophosphonates by condensing a ~ wo 9s/23~s 2 1 8 3 ~ 9 2 ~ /lL ~ I 156 dihalopllo:"ull_lldle-containing sugar with a pyrimidine or a purine under conditions suitable to form a nucleoside 5'-deoxy-5'-_il,dl_ui~o~ onate and/or a 3'-deOXy-3'-~ alo,ul,ob,ullu,~dle.
Phosphonic acids may exhibit important bioiogical properties .. 5 because of their similarity to phosphates (Engel, Chem. Rev. 1977, 77,349-367). Blackburn and Kent (J. Chem. Soc., Perkin rrans. 1986, 913-917) indicate that based on electronic and steric collsi_eldliulls _-fluoro and _,_-difluorometh~ llo~l llolldl_s might mimic phosphate esters better than the co~ on~ ~9 phosphonates. Analogues of pyro- and triphosphates 1, where the bridging oxygen atoms are replaced by a difluoromethylene group, have been employed as substrates in enzymatic processes (Blackbum etal., N(~leo~id~ & N~tcleotide~ 1985, 4, 165-167;
Blackburn et al., Chem. Scr. 1986, 26, 21-24). 9-(5,5-Difluoro-5-phosphonopentyl)guanine (2) has been utilized as a multisubstrate analogue inhibitor of purine nucleoside phosphorylase (Halazy et al., J.
Am. Chem. Soc. 1991, 113, 315-317). Oligon~lcleotides containing methylene groups in place of pllos~ ho ';__'_. 5'-oxygens are resistant toward nucleases that cleave pllospho, - r linkages between phosphorus and the 5'-oxygen (Breaker et al., Biochemistryl993, 32, 9125-9128), but can still form stable cu,,,~ xes with complementary sequences. H~i"-",ar", etal. (Nucleic Acids ~es. 1991, 19, 427-433) found that a single 3'-methyi_.lepl~ospllonate linkage had a minor influence on the C~ lllldliull of a DNA ~ctamer double helix.
.

WO 95123225 - 218 3 9 9 ~ PCT/IB95/00156 ~

8 R 8 --~ N
O - P--X--P - O--P - O~N N
OH OH

(Ho)2opcF2~ - N3~i ocF2L

One common synthetic approach to a,a-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive substrate by diethyl (lithiodifluoromethyl)~ ,ul,o~ (3) (Obayashi et al., Tetrahedron Lett. 1982, 23, 2323-2326). However, our attempts to synthesize nucleoside 5'-deoxy-5'-difluoro-meth~ ospllolldL~s from 5'-deoxy-5'-iodonucleosides using 3 were unsuccessful, i.e. starting compounds were quantitatively recovered. The reaction of nucleoside 5'-aldehydes with 3, according to the procedure of Martin et a/. (Martin et a/., Tetrahedron Lett. 1992, 33, 1839-1842), led to a complex mixture of products. Recently, the synthesis of sugar a,a-difluoroalkylphosphonates from primary sugar triflates using 3 was described (Berkowitz et a/., J. Org.
Chem. 1993, 58, 6174-6176). Unfortunately, our experience is that nucleoside 5~-triflates are too unstable to be used in these syntheses.
The following are non-limiting examples showing the synthesis of nucleoside 5'-deoxy-5'-difluoromethyl-phosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon WO 95/2.722~ 2 ~ 8 3 9 g 2 ~ ~ 156 these examples. These examples d~",onalldl~ that it is possible to achieve synthesis of 5'-deoxy-5'-difluoro derivatives in good yield and thus guide those in the art to such equivaient methods. The examples also indicate utility of such synthesis to provide useful oligonllcl~oti-les as described above.
Those in the art will reco3nize that useful modified enzymatic nucleic acids can now be designed, much as described by Draper et al., PCT/US94/13129 hereby i,,col,uordl~d by reference herein (including drawings) .
FYAmple 90: Synthesis of Nucleoside 5'-Deoxy-5'-difluoromethylulloa,ul~ul Idl~a Referring to Fig. 87, we sy,lllleai~ed a suitable glycosylating agent from the known D-ribose a,u-diflUOlu~ Oa,ullOl~dl~ (4) (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842) which served as a key intermediate for the synthesis of nucleoside 5'-difluoru",~ o~ullolldlt~s.
Methyl 2,3-O-isopropylidene-,B-D-ribofuranose a,a-difluoromethylphosphonate (4) was synthesized from the 5-aldehyde according to the procedure of Martin et al. (retrahedron Lett. 1992, 33, 1839-1842) (Figure 87). Removal of the isopropylidene group was ac~""uLlled under mild conditions (12-MeOH, reflux, 18 h (Szarek et al., T~llal7edl~l7 Lett. 1986, 27, 3827) or Dowex 50 WX8 (H+), MeOH, RT
(about 20-25C), 3 days) in 72% yield. The anomeric mixture thus obtained was benzoylated with benzoyl chloride/pyridine to afford the 2,3-di-O-benzoyl derivative, which was subjected to mild acetolysis conditions (Walczak etal., Synthesis, 1993, 790-792) (Ac2O, AcOH, H2SO4, EtOAc, 0C. The desired 1-O-acetyl-2,3-di-O-benzoyl-D-ribofuranose difluoromethyll.l,ospllùl,al~ (5) was obtained in quantitative yield as an anomeric mixture. These derivatives were used for selective glycosylation of silylated uracil and N4-acetylcytosine under Vorbruggen conditions (Vorbruggen, Nucleos~de Analogs. Chemistry, Biology and Medical ,4p, .' " )s, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F3CSO2OSi(CH3)3 as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1-ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron WO 9~ 2~ = . 218 3 9 ~ 2 PCT/IB95/00156 Lett. 1987, 28, 3623-3626 and ~ ,r~llces cited therein) (SnC14 as a catalyst, boiling ac~lul/iLIil~) to yield ,B~n~lrleosides (62% 6a, 75% 6b).
Glycosylation of silylated N6-benzoyladenine under the same conditions yielded a mixture of N-9 isomer 6c and N-7 isomer 7 in 34% and 15%
yield, respectively. The above ntl~leotidPs were ~ s~flllly deprotected using trimethylsilylbromide for the cleavage of the ethyl groups, followed by treatment with ammonia-methanol to remove the acyl protecting groups.
Nucleoside 5'-deoxy-5'-difluoromethylphosphonates 8 were finally purified on a DEAE Sephadex A-Z5 (HCO3~) column using a 0.01-0.25 M
1 û TEAB gradient for elution and obtained as their sodium salts (82% 8a; 87%
8b; 82% 8c).
Selected analytical data: 31P-NMR (31p) and 1H-NMR (1H) were recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H3PO4 and TMS, respectively. Solvent was CDCI3 unless otherwise noted. 5: 1 H
~ 8.07-7.28 (m, Bz), 6.66 (d, J1,2 4-5, aHI), 6.42 (s, pH1), 5.74 (d, J2 3 4 9~
,~H2), 5.67 (dd, J3 2 4-9, J3 4 6.6, ~H3), 5.63 (dd, J3 2 6.7, J3 4 3.6, H3), 5.57 (dd, J2 1 4-5~ J2 3 6.7, aH2), 4.91 (m, H4), 4.3û (m, CH2CH3), 2.64 (m, CH2CF2), 2.18 (s, ~Ac), 2.12 (s, Ac), 1.39 (m, CH2CH3). 31p 0 7.82 (t, JP F 105.2), 7.67 (t, Jp F 106.5). 6a: 1H ~ 9.11 (s, lH, NH), 8.01 (m, 11H, Bz, H6), 5.94 (d, J1~ 2~ 4 1, lH, H1'), 5.83 (dd, Js 6 8.1, lH, H5), 5.79 (dd, J2',1' 41, J2',3' 6-5, 1H, H2), 5.71 (dd, J3',2' 6.5, J31,416.4, 1H, H3'), 4.79 (dd, J4~ 3. 6.4, J4~,F 11.6, lH, H4), 4.31 (m, 4H, CH2CH3), 2.75 (tq, JH~F
19.6, 2H, CH2CF2), 1.4û (m, 6H, CH2CH3). 31P ~ 7.77 (t, Jp F 104.0). 8c:
31 p (vs DSS) (D2O) ~ 5 71 (t, Jp~F 87.9).
Compound 7 was deacylated with Illc:l~lalJc' ammonia yielding the product that showed ~max (H2O) 271 nm and ~min 233 nm, confirming that the site of glycosylation was N-7.
FY~rnple 91:Synthesis of Nucleic Acids Cont~inin~ Modified Nucleotide Cont~ining Cores The method of synthesis used follows the procedure for normal RNA
synthesis as described in Usman et dl., J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe et al., Nucl~lc Aclds Res. l990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and pllo~,uhold",idiles at the 3'-end (Figure 88 and Janda et al., Sclence 1989, 244:437-440.) These , _ _ _ _ _ _ , . . . . _ WO 95~23~25 ~ - - 2 1 8 3 g 9 2 PCT/IB95/00156 14!;
nucleoside 5'-deoxy-5'-difluoromethyl~ llo~ dl~s may be incorporated not only into 11d"""ell,ead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 introns, or into antisense oligor~cleoti-lec They are, therefore, of general use in any nucleic acid structure.
5 F~rnple 92: Synthesis of ~Aodified Tri~lloalJIldl~
The lli~ oa~ derivatives of the above n~ eoti-la,s can be formed as shown in Fia. 89, according to known procedures. Nucleic Acid Chem., Leroy B. Townsend, John Wiley & Sons, New York 1991, pp. 337-340;
Nucleotide Analogs, Karl Heinz Scheit; John Wiley & Sons New York 1980, pp. 211-218.
Equivalent synthetic schemes for 3' .lillalO,UllOa,UllU~ld~s are shown in Figures 90 and 91 using art l~coy,,i~d nu",~"~,ld~ure. The conditions can be optimized by standard procedures.
The nucleoside dihalophosphonates described herein are 15 advantageous as modified nt~!leoti~as in any nucleic acid structure, e.g., catalytic or antisense, since they are resistant to exo- and endonucleases that normally degrade unmodified nucleic acids in vivo. They also do not perturb the normal structure of the nucleic acid in which they are illC~l~oldl~d thereby Illdillldillillg any activity ACco~ t~ d with that structure.
20 These compounds may also be of use as ",ol)o",e,~ as antiviral and/or antitumor drugs.
Oli~onucleotides with Amido or Peptido Modi~i~,dl;~l, This invention replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and 5' 25 portions of the sugar of a nucleotide may be sl~hctit" d, or the phosphate group may be sllhstitl~tad with amido or peptido moieties. Generally, such anucltotid=hasth=g=n=r=l~trutul=~hownlnFolmulalù=low:

wo s~/2322s 2 18 3 ~ 9 2 r~ 6 B
~J,~, R2 0~
O ~
FORMI ll ~ l The base (B~ is any one of the standard bases or is a modified nucleotide base known to those in the art, or can be a hydrogen group, In 5 addition, either R1 or R2 iS H or an alkyl, alkene or alkyne group containing between 2 and 10 carbon atoms, or hydrogen, an amine (primary, secondary or tertiary, ~, R3NR4 where each R3 and R4 i"d~p~,~d~,llly is hydrogen or an alkyl, alkene or alkyne having between 2 and 10 carbon atoms, or is a residue of an amino acid, i~, an amide), an alkyl group, or 10 an amino acid (D or L forms) or peptide containing between 2 and 5 amino acids. The zigzag lines represent hydrogen, or a bond to another base or other chemical moiety known in the art. Preferably, one of R1, R2 and R3 is an H, and the other is an amino acid or peptide.
Applicant has recognized that RNA can assume a much more 15 complex structural form than DNA because of the presence of the 2'-hydroxyl group in RNA. This group is able to provide additional hydrogen bonding with other hydrogen donors, acceptors and metal ions within the RNA molecule. Applicant now provides molecules which have a modified amine group at the 2' position, such that siyllirk,d"~ly more complex 20 structures can be formed by the modified oligonucleotide. Such modification with a 2'-amido or peptido group leads to expansion and ~ iull~ of the side-chain hydrogen bonding network. The amide and peptide moieties are ~po~1sible for complex structural formation of the oligonucleotide and can form strong c~"~ s with other bases, and 25 interfere with standard base pairing il.t~ s. Such illl~lr~ ce will allow the formation of a complex nucleic acid and protein cùllylu,,,e,dltl.

W0 95123225 . . . , P~ 156 218399~

Oligon~ eoti~-Rs of this invention are ~iy~ a~lly more stable than existing oligon~cleotides and can potentialiy form biologically active bioconjugates not previously possible for oiigor~ leotirl.oc They may also be used for in vitro selection of unique aptamers, that is, randomly 5 generated oligor~ RoticLRs which can be folded into an effective ligand for a target protein, nucleic acid or polysac~,l,a,i.l~.
Thus, in one aspect, the invention features an oligonucleotide containing the modified base shown in Formula 1, above.
In other aspects, the oligonucleotide may include a 3' or 5' nucleotide 10 having a 3' or 5' located amino acid or aminoacyl group. In all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard Illo.li~iCd~iùl~s can be made. For example, an "O" may be replaced with an S, the sugar may lack a base (i.e., abasic) and the pho:".hd~ moiety may be modified to include other s~hstitllti~ns (see 15 Sproat, supra).
F~rnDle 93: General Drocedure for the vl~,a,,,li~,., of 2'-aminoacyl-2'-deoxy-2'-aminonucleoside conjugates.
Referring to Ei9~. to the solution of 2'-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or D-) amino acid (1 mmol) in methanol 20 [dimet~,y;'~.""d",i~t: (DMF) and tetrahydrofuran (THF) can also be used], 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) [or 1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to 50 C from 3-48 hours. Solvents are removed under reduced pressure and 25 the residual syrup is ~ ullld~Uyld,ul~ed on the column of silica-gel using 1-1û % methanol in diul,l~,u,,,~l,al-e. Fractions containing the product are col~ct~ ldI~d yielding a white foam with yields ranging from 85 to 95 %.
Structures are confirmed by 1H NMR spectra of conjugates which show correct chemical shifts for nucleoside and aminoacyl part of the molecule.
30 Further proofs of the structures are obtained by cleaving the aminoacyl protecting groups under dp~luplidIe conditions and assigning 1H NMR
for the fully de:,ulu~ d conjugate.
Partially protected conjugates described above are converted into their 5'-O-dimethoxytrityl derivatives and into 3'-pl~O~ ldllli~ilt,s using 35 standard procedures (Oligonucleotide Synthesis: A Practical Approach, .. . ... .. .. .. . .. . .. . . ... . .. .. _ .. . .

WO95/23225 . 21~33~2 ~ ( 156 M.J. Gait ed.; IRL Press, Oxford, 1984). Incorporation of these pho~pl10rd" " ~ into RNA was performed using standard protocols (Usman et a/., 1987 supra).
A general dt",rut~ io" protocol for oligon~cl~otides of the present 5 invention is described in Fig. 93.
The scheme shows synthesis of conjugate of 2'-d-2'-aminouridine.
This is meant to be a non-limiting example, and those skilled in the art will recognize that, variations to the synthesis protocol can be readily generated to synthesize other nucelotides (e.g., adenosine, cytidine, 10 guanosine) and/or abasic moieties.
FY~rnple 94: RNA cleavage by h~"",e~ ribozymes cont~inin~ 2'-aminoacyl IllQvi~i~dlivl,s.
Hammerhead ribozymes targeted to site N (see Fig. 94) are synthesized using solid-phase synthesis, as described above. U4 and U7 15 positions are modified, individually or in cv,,,Li,,dlivll, with either 2'-NH-alanine or 2'-NH-lysine.
RNA cleava~e assay in vitro: Substrate RNA is ~' end-labeled using [y_32p] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace 20 amount (S 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90C for 2 min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37C for 10 min in a buffer containing 50 mM
Tris-HCI and 10 mM MgCI2~ The reaction is initiated by mixing the 25 ribozyme and substrate solutions and incubating at 37C. Aliquots of 5 1ll are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X ~UI~dr~iv~ stop mix. The samples are resolved on 20 % denaturing polyacrylamide gels. The results are quantified and pt".;~"lage of target RNA cleaved is plotted as a function of 30 time.
Referring to Fi~. 95, hd"""erl,ead ribozymes containing 2'-NH-alanine or 2' r~l I Iy~.i"e Illuvifkialivlls at U4 and U7 positions cleave the target RNA efficiently.

WO 9SI2:32~S 21~ 2 r~ 156 Sequences listed in Figure 94 and the Illo~ liulls described in Fi~ure 95 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (base-s~h~titl~tions, deletions, insertions, mutations, chemical modifications) of the ribozyme and RNA containing 5 other 2'-hydroxyl group ll,odi~i~,alic,l~s, including but not limited to amino acids, peptides and ~,I,ol~ lul, can be readily generated using techniques known in the art, and are within the scope ûf the present invention.
FYArn~ole 95: Aminoacylation of 3'-ends of RNA
1. Referring to Eig~ 3'-OH group of the nucleotide is converted to 1 û succinate as described by Gait, supra. This can be linked with amino-alkyl solid support (for example: CpG). Zig-zag line indicates linkage of 3'0H
group with the solid support.
Il. Fltlpaldliùl) of aminoacyl-derivAti7~r! solid ~ ,o.~rt A) Synthesis of O-Dimethoxytrityl (O-DMT) amino acids Referring to Eis~. to a solution of L- (or D-) serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4,4'-ui",~ oxy"ityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25C) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was par~iLiol,ed between 5% aq.
20 NaHC03 and ~iul llolulll~; ldl ,e, organic layer was washed with brine, dried (Na2SO4) and CullC~lllldldd In vacuo. The residue is purified by flash silicagel column chromatography using 2-10% methanol in di~,l,lolu",~ll,àl~e (containing û.5 % pyridine). Fractions containing product are combined and ~ùl~c~ dlt~d in vacuo to yield white foam (75-85 %
25 yield).
B) P,~valaliull of the solid su,oport and its deriv~ti~ti~ n with amino acids Referring to Fig. 97, the modified solid support (has an OH group instead of the standard NH2 end group) was prepared according to Haralambidis et al., Tetrahedron Lett. 1987, 28, 5199, (P denotes 30 aminopropyl CPG or polystyrene type support). O-DMT or NH-~,,ollu,,,~,,uxytrityl (NH-MMT amino acid was attached to the above solid support using standard procedures for derivatization of the solid support (Gait, 1984, supra) creating a base-iabile ester bond between amino acids wo 95/23225 T ~ 1156 21839~2 1~0 and the support. This support is suitable for the construction of RNA/DNA
chain using suitably protected nucleoside pl~oapl1o,dr"i~ s.
FY~mrJle 96: Aminoacyl~ti~1n of 5'-en~l~ of RNA
1. Referring to Fig. 98. 5'-amino-containing sugar moiety was 5 synthesized as described (Mag and Engels, 1989 Nucleic Acids Res. 17, 5973). A",il~oacyldlion of the 5'-end of the monomer was achieved as described above and RNA phosphoramidite of the 5'-aminoacylated monomer was prepared as described by Usman et d/., 1987 supra. The pl~oayl1oldll~iui`~ was then ill~ Joldl~d at the 5'-end of the oligonucleotide 10 using standard solid-phase synthesis protocols described above.
Il. Referring to Eig~. aminoacyl group(s) is attached to the group at the 5'-end of the RNA using standard procedures described above.
Vll. R~ .i.i"g ~ienetic MutatiQns !\~d;f~ l;oll of existing nucleic acid sequences can be achieved by hol"ologous l~cor,,Li.,dli~n. In this process a transfected sequence recombines with llo",ologous ~;I"ulllOsol"al sequences and can replace the endogenous cellular sequence. Boggs, 8 I .:~,",d~;o"al J. Cell Cloning 80, 1990, describes targeted gene Illodi~i-idlioll. It reviews the use of 20 l1ol"ologous DNA l~c~ Li,ldlio~, to conrect genetic defects. Banga and Boyd, 89 Proc. Natl. Acad. Sci. U.S.A. 1735, 1992, describe a specific example of in vivo site-directed mutagenesis using a 50 base oligonucleotide. In this methodology a gene or gene segment is essentially replaced by the oligonucleotide used.
This invention uses a col",ul~",~"lary oligonucleotide to position a nucleotide base changing activity at a particular site on a gene (RNA or genomic DNA), such that the nucleotide modifying activity will change (or revert) a mutation to wild-type, or its equivalent. By reversion or change of a mutation, we refer to reversion in a broad sense, such as when a mutation at a second site which leads to functional reversion to a wild type phenotype. Also, due to the deg~eldcy of the genetic code, a revertant may be achieved by changing any one of the three codon positions.
Additionally. creation of a stop codon in a deleterious gene (or transcript) is defined here as reverting a mutant phenotype to wild-type. An example of WO 95123225 ~ = 218 3 9 9 2 PCT/IB95/00156 this type of reversion is creating a stop codon in a critical HIV proviral gene in a human.
Referring to Figures 100 and 101. broadly there are two a,u,u,ua~:l,es to causing a site directed change in order to revert a mutation to wild-type.
5 In one (Fig. 100) the oligonucleotide is used to target RNA specifically.
RNA is provided with a co",u16",~"ldry (Watson-crick) oligonucleotide sequence to that in the target molecule. In this case the sequence modifying oligonucleotide would (analogously to an antisense oligonucleotide or ribozyme) have to be continuously present to revert the 10 RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying oligonucleotide. The transient nature of this approach is an advantage, in that treatment could be stopped by simply removing the sequence modifying oligonucleotide (as with a traditional drug).
A second approach targets DNA (Fiq. 101) and has the advantage that changes may be per",al,~"lly encoded in the target cell's genetic code. Thus, a single course (or several courses) of treatment may lead to p~""ant"l reversion of the genetic disease. If inadvertent ~ u~osu~al mutations are introduced this may cause cancer, mutate other genes, or 20 cause genetic changes in the germ-line (in patients of reproductive age).
However, if the base changing activity is a specific methylation that may modulate gene ~,u~s~iol~ it would not necessarily lead to germ-line transmission. See Lewin, Genes.1983 John Wilely & Sons, Inc. NY pp 493-496.
Co",pl~",~"ldry base pairing to single-stranded DNA or RNA is one method of directing an oligonucleotide to a particular site of DNA. This could occur by a strand di.,,ulac~ el~l ",e.,l,ani~", or by targeting DNA
when it is single-stranded (such as during replication, or lldl~s~,,i,uliu,,).
Another method is using triple-strand binding (triplex formation) to double-stranded DNA, which is an established technique for binding poly-pyrimidine tracts, and can be extended to recognize all 4 rl~ ' See Povsic, T., Strobel, S., & Dervan, P. (1992). Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation.
J, Am. Chem. Soc. 114, 5934-5944 (1992). Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, A.V. & Federova, O.S. Design and targeted reactlons of oligonucleotide derivaUves 1-366 (CRC Press, Novosibirsk, ~ . , _,, _ _, _ _ _ _ wo 9512322s 2 1 8 3 ~ P~ 56 1993) describe conjugation of reactive groups or enzyme to oligor~lclPotidec and can be used in the methods described herein.
Recently, antisense oligorlucl~otides have been used to redirect an incorrect splice into order to obtain correct splicing of a splice mutant globin5 gene in vltro. Dominski Z; Kole R (1993) R~s~uldliui1 of correct splicing in id pre-mRNA by antisense oligor~ leoti-les Proc Natl Acad Sci 90:8673-7. Analogously, in one preferred embodiment of this invention a C~lll~,l~lll~llldly oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by 1 0 antisense.
In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence.
This can be accû~ l;Olled by activating an endogenous enzyme (see Figure 102), by appropriate positioning of an enzyme (or ribozyme) 15 conjugated (or activated by the duplex) to the oligonucleotide, or by dplJIul~lidl~ pobi~iUililly of a chemicai mutagen. Specific mutagens, such as nitrous acid which ded",i"~lts C to U, are most useful, but others can also be used if inactivation of a hammful RNA is desired.
RNA editing is an naturally occurring event in l"ar"",alial1 cells in 2û which a sequence modifying activity edits a RNA to its proper sequence post-l~dlls..,i~liona'ly. Higuchi, M." Single, F., Kohler, M., Sommer, B., and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GluR-B: A
base-paired intron-exon structure ~ rl"il,es position and efficiency Cell 75:1361-1370. The machinery involved in RNA editing can be co-opted by 25 a suitable oligonucleotide in order to promote chemical ",o~"' 'i. 1.
The changes in the base created by the methods of this invention cause a change in the nucleotide sequence, either directly, or after DNA
repair by normal cellu~ar ",~uI,a~ ",~. These changes functionally correct a genetic defect or introduce a stop codon. Thus, the invention is distinct 30 from techniques in which an active chemical group (e.g., an alkylator) is attached to an antisense or triple strand oligonucleotide in order to chemically inactivate the target RNA or DNA.
Thus, this invention creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base.

WO 951t3ttS ~ 156 2~ ~3~

This includes correcting a sequence instead of inactivating a gene but can also include inactivating a deleterious gene.
Thus, in one aspect, the invention features a method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic 5 acid molecule. The method includes contacting the nucleic acid molecule in ViVQ with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After fommation of the duplex or triplex molecule a base modifying activity ..II~ll,;c~''y or enzy."dli~.~lly alters the targeted 10 base directly, or after nucleic acid repair in vivo. This results in the functional alteration of the nucleic acid sequence.
By "alter", as it is used in this context, is meant that one or more chemical moieties in a targeted base, or bases, is altered so that the mutant nucleic acid will be functionally different. Thus, this is distinct from prior 15 methods of correcting defects in DNA, such as homologous l~col"Li" ~, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the l~d"D~ d nucleic acid. This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead 20 of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.
By "functionally alter" is meant that the ability of the target nucleic acid 25 to perform its normal function (i.e.., lldi~SCli,uliull or lldllDldliUIl control) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.
By "mutant" it is meant a nucleic acid molecule which is altered in 30 some way compared to equivalent molecules present in a normal individual. Such mutants may be well known in the art, and include, molecules present in individuals with known genetic d~fi~i~".,ies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal ~ .rt:sDion of a 35 gene, such as cancer, II,dlass~",ia's and sickle cell anemia, and cystic WO 95123225 218 3 9 ~ 2 r~ 156 fibrosis. It allows modulation of lipid ",~ld~Gli~ to reduce artery disease, treatment of integrated AIDS genomes, and AlDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a Uwild type" phenotype and/or genotype. For deleterious 5 conditions this involves altering a base to allow ~ ssiùl~ or prevent ~ yl~ iOIl as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant (i.e., non-human gene) to a wild type (i.~., no production of a non-human protein). Such ".- J'" 'ion is p~i~u""ed in trans rather than in cis as in 10 prior methods.
In preferred ~,,,L,oui,,,u,,l:,, the oligonucleotide is of a length (at least 12 bases, preferably 17 - 22) sufficient to activate dsRNA dedllli"ase kL
vivo to cause conversion of an adenine base to inosine; the oligonucleotide is an enzymatic nucleic acid molecule that is active to 15 cl)er";call~ modify a base (see below); the nucleic acid molecule is DNA or RNA; the oligonucleotide includes a chemical mutagen, e,g" the mutagen is nitrous acid; and the oligonucieotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to 5-methylcytosine.
In a most preferred ~IllLJodj,,,el,,L, the invention features correction of a mutation, rather than inactivation of a target by causing a mutation.
Using in vitro directed evolution, it is possible to screen for ribozymes with catalytic activities different than RNA cleavage. Bartel, D. and Szostak, J. ~1993) Isolation of new ribozymes from a large pool of random 2~ sequences. Science 261:1411-1418. Using these methods of in vitro directed evolution, an enzymatic nucleic acid molecule, or ribozyme that mutates bases, instead of cleaving the pl1os~l,odi,~ , backbone can be selected. This is a convenient method of obtaining an enzyme with the appropriate base sequence modifying activities for use in the present invention.
Sequence modifying activities can change one nucleotide to another (or modify a nucleotide so that it will be repaired by the cellular machinery to another nucleotide). Sequence modifying activities could also delete or add one or more nllcl~otides to a sequence. A specific t~ L)odi"~el~l of 3~ adding sequences is described by Sullenger and Cech, PCT/US94/12976 _ _ _ , _ . , , , . , _ , ,, . , ,, . _ _ _ WO 95123225 218 3 ~ ~ ~ r~ 156 hereby i"col,uord~d by reference herein), in which entire exons with wild-type sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 - 3).
Thus, in another aspect, the invention features ribozymes or 5 enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule. Applicant is the first to determine that such molecules would be useful, and to provide a d~-s~,li,uliu~ of how such molecules might be isolated.
Molecules used to achieve in situ reversion can be delivered using 10 the existing means employed for delivering antisense molecules and ribozymes, including liposomes and cationic lipid culllul~ s~ If the in situ reverting molecule is c~,l,,uosed only of RNA, then t~Xpl~5::,iOIl vectors can be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes ~,u~ssio~ vectors.
15 There are several advantages of using such an ~,.pl~:,sio,~ vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that already express that gene. Furthermore, the corrected gene would be properly regulated by its natural lld~ li,uliolldl promoter. Lastly, reversion 20 can be used when the mutant RNA creates a dominant gain of function protein (e.g., in sickle cell anemia), where correction of the mutant RNA is necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.
F~do~enous Mammalian RNA i-diting System It was observed in the mid-1980s that the sequence of certain cellular RNAs were different from the DNA sequence that encodes them. By a process called RNA editing, cellular RNA are post-transcriptionally modified to a) create a translation initiation and l~llllilldLioll codons, b) enable tRNA and rRNA to fold into a functional COllf~lllldliOI~ (for a review see Bass, B. L. (1993) In The RNA World. R. Gesteland, R. and Atkins, J.
eds. (Cold Spring Harbor, New York; CSH Lab. Press) pp. 383-418). The process of RNA editing includes base ",o~iiiiudlion, deletion and insertion of n~l~lPot~ c Although, the RNA editing process is widespread among lower eukaryotes, ver,v few RNAs ffour) have been reported to undergo editing in WO 95/23225 . . . - 2 1 8 3 ~ 9 ~ P~ 156 ~

mammals (Bass, supra). The pr~dc""i.,à"l mode of RNA editing in mammalian system is base ",~di~i,,àlion (C ~ U and A ~ G). The ",e,,l,d"i~"" of RNA editing in the Illdlllll -'' 1 system is p~stlllt~t~td to be that C~U conversion is catalyzed by cytidine d~all,i~,a~,e. The "~e,,11d"i,,", 5 of conversion of A~G has recently been reported for glutamate receptor B
subunit (gluR-B) in rat PC12 cells (Higuchi, M. et al. (1993) Cell 75, 1361-1370). According to Higuchi gluR-B mRNA precursor attains a structure such that intron 11 and exon 11 can form a stable stem-loop structure. This stem-loop structure is a substrate for a nuclear double strand-specific 10 adenosine deaminase enzyme. The deamination will result in the conversion of A~l. Reverse Llai~sc,i,uliol1 followed by double strand synthesis will result in the ill~OI,UUld~iUIl of G in place of A.
In the present invention, the t_"do~u,~"~us dt3alllillase activity or other such activities can be utilized to achieve targeted base IlloJi~i.;dliul~.
The following are examples of the invention to illustrate different methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific ,~",bodi""~"t~, of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims.
2û FY~rnple 97: ExDloitin~ r dsRNA d~ v~,"de"l A~ rline to Inosine converter.
An endogenous activity in most ",ar"",alial1 cells and Xenopus oocytes converts about 5û% of adenines to inosines in double stranded RNA. (Bass, B. L., & Weintraub, H. (1988). An unwinding activity that 25 covalently modifies it double-stranded RNA substrate. Cell, 5~, 1089-1098.). This activity can be used to cause an in situ reversion of a mutation at the RNA level. Referring to Figures 1û2 and 104, for de,,,ol1~l,dlioll purposes a stop codon is incorporated into the coding region of dystrophin, which is fused to the reporter gene luciferase. This 30 stop codon can be reverted by targeting an antisense RNA which is long enough to activate the dsRNA d~d",i"ase, which converts Adenines to Inosines. The A to I transition will be read by the ribosome as an A to G
transition in some cases and will thereby functionally revert the stop codon.
While other A's in this region may be converted to l's and read as G, 35 converting an A to I (G) cannot create a stop codon. The A to I lldl1siliu,~s ~ WO 95123225 ~ 1 8 3 ~ 9 2 P~ 156 in the region surrounding the target mutation will create some point mutations, however, the function of the dystrophin protein is rarely inactivated by point mutations.
The reverted mRNA was then translated in a cell Iysate and assayed 5 for luciferase activity. As evidenced by the dramatic increase in luciferase counts in the graph in figure 103, the A to I transition was read by the ribosome as an A to G transition and the stop codon has successfully been reverted with the Iysate treated complex. As a control, an irrelevant non-complementary RNA oligonucleotide was added to the 10 dystrophin/luciferase mRNA. As expected, in this case no translation (luciferase activity) is observed because of the stop codon. As an additional control, the hybrid was not treated with extract, and again no liu~ (luciferase activity) is observed (Figure 103).
While other A's in the targeted region may have been converted to l's 15 and read as G, converting an A to I (G) cannot create a stop codon, so the ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a dystrophin protein made from an mRNA reverted by this method should retain full activity.
The following detail specifics of the methodology: RNA
oligonucleotides were synthesized on a 394 (ABI) synthesizer using ob~ uldllliu;~u chemistry. The sequence of the synthetic c~lllplt:lllell~dly RNA that binds to the mutant dystrophin sequence is as follows (5' to 3'):
CCCGCGGTAGA ~ ; I (iGAGGCTTACAG I I I I CTACAAACCTCC
CTTCMA (Seq. ID No. 1) Referring to Figure 104. fifty-nine base pairs of a human dystrophin mutant sequence co"~ci"i"g a stop codon was fused in frame to the luciferase coding region using standard cloning technology, into the Hind lll and Not I sites of pRC-CMV (Invitrogen, San Diego, CA). The AUG of luciferase was deleted. The sequences of the insert from the Hind lll site to the start of the luciferase coding region is (5' to 3'):
GCCCCTGAGGAGCGATGGAGGCCTTGMGGGAGGmGTGGAMA
CTGTMGCCTCCAGMAGATCTACCGCGG (Seq ID No. 2) W09S123225 21839~2 r~ 5,'~ 156 ~

This corresponds to base pairs 3649-3708 of normal dystrophin (Entrez ID # 311627) with a Sac ll site at the 3' end. This plasmid was used as a template for in vitro lld~ ClilJIiUII of mRNA using T7 polymerase with the manufacturers protocol (Promega, Madison, Wl).
Xenopus nuclear extracts were prepared in 0.5X TGKED bufler ~0.5X=
25mM Tris (pH 7.9), 12.5% glycerol, 25 mM KCI, 0.25mM DTT and 0.05mM
EDTA), by vortexing nuclei and resuspended in a volume of 0.5X TGKED
equal to total cytoplasm volume of the oocytes. Bass, B.L. & Weintraub, H.
Cen55~ 1089-1098 (1988).
The target mRNA at 500ng/ul was pre-annealed to 1 I,,ic,u,,,olar C~ dry or irrelevant RNA oligonucleotide by heating to 70C, and allowing it to slowly cool to 37C over 30 minutes. Fifty nanograms of mRNA pre-annealed to the RNA oligonucleotides was added to 7ul of nuclear extracts containing 1mM ATP, 15mM EDTA, 1600un/ml RNasin and 12.5mM Tris pH 8 to a total volume of 12ul. Bass, B.L. & Weintraub, H.
supra. This mixture, which contains the dsRNA dear";~,dse activity, was incubated for 30 minutes at 25C. Next, 1.5ul of this mixture was added to a rabbit reticulocyte Iysate in vitro lldllSIdliOII mixture and translated for two hours according to the manufacturers protocol (Life Technologies, Gaithersberg, MD), except that an additional 1.3 mM magnesium acetate was added to ~UllI~ dl~ for the EDTA carried through from the nuclear extract mixture. Luciferase assays were perfommed on 15ul of extract with the Promega luciferase assay system (Promega, Madison, Wl), and I~""i"esc~l1ce was detected with a 96 well lu"~;,lu",~ l, and the results are displayed in the graph in figure 102.
FY~ le 98: Base chan~in~ activities The chemical synthesis of antisense and triple-strand forming oligomers conjugated to reactive groups is well studied and characterized (Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, A.V. & Federova, O.S. Design and targeted reactions of oligonucleotide deriv2tives 1-366 (CRC Press, Novosibirsk, 1993) and Povsic, T., Strobel, S. & Dervan, P.
Sequence-specific double-strand alkylation and cleavage of DNA
mediated by triple-helix formation J. Am. Chem. Soc. 114, 5934-5944 (1992). Reactive groups such as alkylators that can modify nucleotide bases in targeted RNA or DNA have been conjugated to oligorlll~le~tirl,~s ~ W09S~2322S 21839 9 2 r~ ,r l56 Additionally enzymes that modify nucleic acids have been conjugated to oligon~lcleoti~lRs (Knorre, D.G., Valentin, V.V., Valentina, F.Z., Lebedev, A.V. & Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993). In the past these 5 co"; I~AtRd chemical groups or enzymes have been used to inactivate DNA or RNA that is specifically targeted by antisense or triple-strand illL~ld~ s. Below is a list of useful base changing activities that could be used to change the sequence of DNA or RNA targeted by antisense or triple strand il~ld~;liul~s, in order to achieve in situ reversion of mutations,10 as described herein (see figure 100-104).
1. Deamination of 5-methylcytosine to create thymidine (pel~urllled by the enzyme cytidine dedl";"ase (Bass, B.L. in The RNA
World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).
Also, nitrous acid or related compounds promote oxidative deamination of 15 C to be read at T(Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.). Additionally hydroxylamine or related compounds can transform C to be read at T (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.) 2. Deal"i"dlion of cytosine to create uracil (performed by the enzyme cytidine ded",i"ase (Bass, B.L. in The F~NA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993) or by chemical groups similar to nitrous acid that promote oxidative ded,,,i,,dliu,, (MicrobialGenetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.) 3. Dedlllilldlioll of Adenine to be read like G (Inosine) (as done by the adenosine ddd",i"dse, AMP dedl"i"ase or the dsRNA d~d",i,ldlillg activity ( Bass, B.L. in rhe FINA World (Cold Spring Harbor Laboratory - Press, Cold Spring Harbor, 1993).
4. Methylation of cytosine to 5-methylcytosine 5. T,d,,:,~u,,,,i,,g thymidine (or uracil) to 02-methyl thymidine (or O2-methyl uracil), to be read as cytosine by alkynitrosoureas (Xu, and Swann, T~l,dl,e.l,u,~ Letters 35:303-306 (1994)).

WO 95123225 . - 218 3 9 9 2 r~I,~;s ~-156 6. T~dll~rullllillg guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978) which can be done with the mutagen ethyl methane sulfonate (EMS) Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.

7. Amination of uracil to cytosine (as perfommed by the cellular enzyme CTP synthetase (Bass, B.L. in The FINA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).
The following are examples of useful chemical Illo,liri.;dliol~s that can 10 be utilized in the present invention. There are a few preferred straightforward chemical ",o~l; ;c~llions that can change one base to another base. Appropriate mutagenic chemicals are placed on the targetting oligonucleotide, e.g., nitrous acid, or a suitable protein with such activity. Such chemicals and proteins can be attatched by standard 15 procedures. These include molecules which introduce fundamental chemical changes, that would be useful illd~pel~del,l of the particular technical approach. See Lewin, 9~1983 John Wilely & Sons, Inc. NY
pp 42-48.
The following matrix shows that the chemical ~ difi~idliul1s noted can 20 cause transversion reversions (py,i",idi"e to pyrimidine, or purine to purine) in RNA or DNA. The transversions (pyrimidine to purine, or purine to pyrimidine) are not preferred because these are more difficult chemical l,d,,,rur,,,~lions. The footnotes refer to the specific desired chemical transformations. The bold footnotes refer to the reaction on the opposite 25 DNA strand. For example, if one desires to change an A to a G, this can be acc~",t,'i~ ed at the DNA level by using reaction #5 to change a T to a C in the opposing strand. In this example an A/T base pair goes to A/C, then when the DNA is l~ d, or mismatch repair occurs this can become G/C, thus the original A has been converted to a G.
ISR matrix Reverted Base Mutsnt bsse A T(U) C G

~ WO 95123225 _ 2 1 8 3 ~ ~ ~ r_.,.. J.~ 156 1~;1 A ~ TIdl~s~;aiull ¦¦Transversion ¦¦DNA5,3/RNA3 ¦
T(U) ¦Transversion ¦¦- ¦¦DNA~/RNA7 ¦¦Transversion ¦
¦Transversion ¦¦RNA2/DNA6 ¦~ Transversion ¦
G¦DNA6/RNA6 ¦¦Transversion ¦¦Transversion ¦¦-Ded",i,ldliul1 of 5-methylcytosine to create thymidine.
2 D~a,,,i,,c.liull of cytosine to create uracil.
3 Dea",i"dliu" of Adenine to be read like G (Inosine).
4 Methylation of cytosine to 5-methylcytosine.
5 T,d,~Fu,,,,i,,g thymidine (or uracil) to O2-methyl thymidine (or 02-methyl uracil), to be read as cytosine (Xu, and Swann, Tt:lldl,e~,u,~
Letters 35:303-306 (1994)).
6 Tldlla~ullllillg guanine to 6-O-methyl (or other alkyls) to be 10 read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, ~21:77û-778 (1978)).
7. Amination of uracil to cytosine. Bass supra. fig. 6c.
In Vitro Selection Strateqy Referring to Figure 105. there is provided a schematic describing an 15 approach to selecting for a ribozyme with such base changing activity. An RNA is designed that folds back on itself (this is similar to applua~,l,es already used to select for RNA ligases, Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences.
Science 261:1411-1418). A degenerate loop opposing the base to be 20 modified provides for diversity. After incubating this library of molecules in a buffer, the RNA is reverse l,dl,s~;liLed into DNA (that is, using standard in vitro evolution protocol. Tuerk and Gold, 249 Science 5û5, 1990) , and then the DNA is selected for having a base change. A restriction enzyme cleavage and size selection or its equivalent is used to isolate the fraction 25 of DNAs with the ap,urupridl~ base change. The cycle could then be repeated many times.

WO 95/23225 . 2 1 ~ 3 ~ ~ 2 F ~ ~, r4'~ 6 The in vitro selection (evolution) strategy is similar to approaches developed by Joyce (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635-641; Joyce, G. F. (1992) Scientifir American 267, 90-97) and Szostak (Bartel, D. and Szostak, J. (1993) ~i~ 261:1411-1418; Szostak, J. W.
(1993) I~ 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein, each member contains two domains: a) one domain consists of a region with definod (known) nucleotide sequence; b) the second domain consists of a region with deg~l~6,d~ (random) sequence.
The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (the region flanking the mutant nucleotide), 2) c~", " ~ dly DNA (cDNA) synthesis and PCR dll, "" I of molecules selected for their base modifying activity, 3) introduction of restriction endonuclease site for the purpose of cloning. The degel ,e, d~l~ domain can be created to be completely random (each of the four nucleotides 1~ lu~r~:s~ d at every position within the random region) or the de~dl1~,dcy can be partial (Beaudry, A. A. and Joyce, G.F. (1992) Science 257, 635-641). In this invention, the degenerate domain is flanked by regions containing known sequences (see Figure 105), such that the d~gel1tlldL~
domain is placed across from the mutant base (the base that is targeted for Illodi~iudliùl~). This random library of nucleic acids is incubated under conditions that ensure folding of the nucleic acids into col~rUlllldliuns that facilitate the catalysis of base IllO~ .dliUII (the reaction protocol may also include certain cofactors like ATP or GTP or an S-adenosyl-methionine (if methylation is desired) in order to make the selection more stringent).
Following incubation, nucleic acids are converted into c~",, !: "d"ldly DNA
(if the starting pool of nucleic acids is RNA). Nucleic acids with base modification (at the mutant base position) can be separated from rest of the population of nucleic acids by using a variety of methods. For example, a restriction endonuclease cleavage site can either be created or abolished as a result of base Illo.li~icdliclll. If a restriction endonuclease site is created as a result of base ".~r'' -~:1, then the library can be digested with the restriction endonuclease (RE). The fraction of the population that is cleaved by the RE is the population that has been able to catalyze the base IIlO~iri.;dlio,, reaction (active pool). A new piece of DNA (containing 3~ oligonucleotide primer binding sites for PCR and RE sites for cloning) is ligated to the temmini of the active pool to facilitate PCR ~ liri~dlioll and subsequent cycles (if necessary) of selection. The final pool of nucleic acids with the best base modifying activity is cloned in to a plasmid vector wo 9s~ 2 1 8 3 ~ ~ 2 ~ 56 and ll~llaivl",ed into bacterial hosts. Fecol"vil,d"l plasmids can then be isolated from lld~ ur",ed bacteria and the identity of clones can be determined using DNA sequencing techniques.
Base modifying enzymatic nucleic acids (identified via in vitro 5 selection) can be used to cause the chemical Illo~i~iudlivl~ in vivo.
In addition, the ribozyme could be evolved to specifically bind a protein having an enzymatic base changing acltivity.
Such ribozymes can be used to cause the above chemical mo~ idliol1s in vivo. The ribozymes or above noted antisense-type 10 molecules can be administered by methods discussed in the above "ced art.
Vlll. Adllli-.i~l.alion of Nucleic Acids Applicant has dt,l~""i"ed that double-stranded nucleic acid lacking a lldll~ liUliUII l~ signal can be used for continuous t~wr~a~ivl1 of 15 the encoded RNA. This is achieved by use of an R-loop, i.e., an RNA
molecule non-covalently ~so~;; ,l~d with the double-stranded nucleic acid and which causes localized denaturation ("bubble" formation) within the double stranded nucleic acid (Thomas et al. 1976 Proc. Natl. Acad. Sci.
~ 73 2294). In addition applicant has determined that that the RNA
20 portion of the R-loop can be used to target the whole R-loop complex to a desirable intracellular or cellular site, and aid in cellular uptake of the complex. Further, applicant indicates that t~,~pr~sbiùn of enzymatically active RNA or ribozymes can be :jiylli~i~dlllly enhanced by use of such R-loop cu",ul~xes.
Thus, in one aspect the invention features a method for introduction of enzymatic nucleic acid into a cell or tissue. A complex of a first nucleic acid encoding the enzymatic nucleic acid and a second nucleic acid molecule is provided. The second nucleic acid molecule has sufficient c~,,,vlc:,,,e,,larity with the first nucleic acid to be able to fonm an R-loop base pair structure under physiological conditions. The R-loop is formed in a region of the first nucleic acid molecule which promotes t~Xvlt::,aiul~ of RNA from the first nucleic acid under physiological conditions. The method further includes contacting the complex with a cell or tissue under .. .. _ _ _ _ _ wo gs~ 2 1 8 3 ~ 91 2 P~ . IS6 conditions in which the enzymatic nucleic acid is produced within the cell or tissue.
By ~complex" is simply meant that the two nucleic acid molecules interact by i"l~""ol~cular bond formation (such as by hydrogen bonding) 5 between two c~",,ul~",~"~dry base-paired sequences. The complex will generally be stable under physiological condition such that it is able to cause initiation of lldl1sc,i,uliùl~ from the first nucleic acid molecule.
The first and second nucleic acid moleculès may be fommed from any desired nucleotide bases, either those naturally occurring (such as 10 adenine, guanine, thymine and cytosine), or other bases well known in the art, or may have Illo~i~i.;dlk~s at the sugar or phosphate moieties to allow greater stability or greater complex formation to be achieved. In addition, such molecules may contain non-nucleotides in place of rl~lcleotidec Such rllodi~i~dliulls are well known in the art, see e.g., Eckstein et al., I,,~er,,dliùl,al Publication No. W092/07065; Perrault etaL, 1990Nature 344, 565; Pieken etaL, 1991 ~i~n~, 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et aL, International Publication No. WO 93/15187; and Rossi et aL, I~ ,d~iol,al Publication No. WO 91/03162, as well as Sproat,B. European Patent Application 2û 9211029~.4 which describe various chemical Illodi~iG~iùlls that can be made to the sugar moieties of enzymatic RNA molecules. All these pu' ' ~s are hereby incorporated by reference herein.
By "sufficient c~lllplt~ lldlily" is meant that sufficient base pairing occurs so that the R-loop base pair structure can be formed under the 25 ap,ulUplidl~ conditions to cause IlGIIaUI;~UI;UII of the enzymatic nucleic acid.
Those in the art will recognize routine tests by which such sufficient base pairs can be determined. In general, between about 15 - 80 bases is sufficient in this invention.
By "physiological condition" is meant the condiffon in the cell or 30 tissue to be targeted by the first nucleic acid molecule, although the R-loopcomplex may be formed under many other conditions. One example is use of a standard physiological saline at 37C, but it is simply desirable in this invention that the R-loop structure exists to some extent at the site of action so that the ~.,u,t,~iuil of the desired nucleic acid will be achieved at that 35 site of action. While it is preferred that the R-loop structure be stable under WO95~23225 ~2~ 3~ r ~ 5.'l~156 those conditions, even a minimal amount of formation of the R-loop structure to cause ~Ayr~s,ioll will be sufficient. Those in the art will recognize that measurement of such eA~ ssi~l- is readily achieved, especially in the absence of any promoter or leader sequence on the first nucleic acid molecule (Daube and von Hippel, 1992 Science 258, 1320).
Such 6Apr~ssiol~ can thus only be achieved if an R-loop structure is truly formed with the second nucleic acid. If a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that ~di~SU,i~ lion is enhanced.
In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by fomming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or lldl~Cli~J~iOII termination signal such that once t~A,Ul~;~aiUil is initiated it will continue until the first nucleic acid is degraded.
In another related aspect, the invention features a method in which the second nucleic acid is provided with a lo~ factor, such as a protein, e.g., an antibody, transferin, a nuclear lo~Rli~Rtion peptide, or folate, or other such compounds well known in the art, which will aid in targeting the R-loop complex to a desired cell or tissue.
In preferred t""boui",e"L~, the first nucleic acid is a plasmid, ~.g., one without a promoter or a ~Idll:,C,i~,ti~ ""i" .~ signal; the second nucleic acid is of length between about 40-200 bases and is formed of ribonuclQotides at a majority of positions; and the second nucleic is covalently bonded with a ligand such as a nucleic acid, protein, peptide, lipid, carbohydrate, cellular receptor, nuclear localization factor, or is attached to maleimide or a thiol group: the first nucleic acid is an .r~s~ion plasmid lacking a promoter able to express a desired gene, e.g., it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNA/DNA heteroduplex;
no promoter or leadQr region is provided in the first nucleic acid; and the R-loop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular lldl~sc,i~Jt;ol, machinery.
In other preferred ~IllI.o ii",~"~, the first nucleic acid encodes one or more ~n~y",d~i., nucieic acids, 6.g., it is formed with a plurality of W0 95/23225 `' 218 3 ~ ~ 2 r~l,.D, 5'~-156 i"lld",ol~;ular and i"~:r",olecular cleaving enzymatic nucleic acids to allow relsase of therapeutic enzymatic nucleic acid In vivo.
In a further related aspect, the invention features a complex of the above first nucleic acid molecules and second nucleic acid molecules.
5 R-loQD comr~lex An R-loop complex is designed to provide a non-i"~ dli"g plasmid so that, when an RNA polymerase binds to the plasmid, llallsulilutiol~ is continuous until the plasmid is degraded. This is achieved by hybridizing an RNA molecule 40 to 200 r~ leoti~'e~ in length to a DNA e"u,~s~
10 plasmid resulting in an R-loop structure (see fiQure 106). This RNA, when conjugated with a ligand that binds to a cell surface receptor triggers i"l~",ali dliol1 of the plasmid/RNA-ligand complex. Formation of R-loops in general is described by DeWet, 1987 Methods in ~7ymol. 145, 235;
Neuwald et al., 1977 J. Virol. 21,1019; and Meyer et al. 1986 J. Ult. Mol.
15 Str. Res. 96, 187. Thus those in the art can readily design o~ult:xes of this invention following the teachings of the art.
~ lulllu~la placed in retroviral genomes have not always behaved as planned in that the additional promoter will serve as a stop signal or reverses the direction of the poly",e,~:,e. Applicant was told that creation 20 of an R-loop between the promoter and the reporter gene increased the transfection efficiency. Incubation of an RNA molecule with a double-stranded DNA molecule, c~" ,i"g a region of CUlllpl~lllt:ll~drity with the RNA will result in the formation of a stab~e RNA-DNA hetroduplex and the DNA strand that has a sequence identical to the RNA will be displaced into 25 a loop-like structure called the R-loop. This ~i~plac~",e"~ of DNA strand occurs because an RNA-DNA duplex is more stable compared to a DNA-DNA duplex. Applicant was also told that an 80 nt long RNA was used to generate a R-loop structure in a plasmid encoding the B-~ ctl sid~e gene. The R-loop was initiated either in the promoter region or in the 30 leader sequence. Plasmids containing an R-loop structure were ",i.,ui"j~led into the cytoplasm of COS cells and the gene ~ul~ssiol1 was assayed. R-loop formation in the promoter region of the plasmid inhibited e~-prt:~siol1 of the gene. RNA that hybridized to the leader sequence between the promoter and the gene or directly to the first 80 35 nucleotides of the mRNA increased the ,~ siol~ levels 8-10 fold. The .. . . . . _ ... .. . . _ _ _ .

WO 95123225 21~ 3 ~ g ~ 5, r 1~6 proposed mechanism is that R-loop formation prevents nucleosome assembly, thus making the DNA more accessible for l,dnscli,ulioll~
Alternatively, the R-loop may resemble a RNA primer promoting either DNA
replication or lldllsc,i,uliol1 (Daube and von Hippel, 1992, supra!.
, 5 One of the salient features of this invention is to generate R-loops in 5::i;011 vectors of choice and introduce them into cells to achieve enhanced ~,~,u,~ssiol1 from the ~,u~ siul~ vector. The presence of an R-loop may aid in the recruitment of cellular l~dns,;,i,utiol, machinery. Once an RNA poly."~,ase binds to the plasmid and lnitiates l,d"s."i,uliun, the process will continue until a l~llllil IdliUIl signal is reached, or the plasmid is degraded.
This invention will increase the ~,ul~Saiun of ribozymes inside a cell. The idea is to construct a plasmid with no lldllsuli,u~ioll l~llllilldliUIl signal, such that a transcript-co"ldi" ,g multiple ribozyme units can be generated. In order to liberate unit length ribozymes, self-pl-,c0s~illg ribozymes can be cloned du...lalltldlll of each therapeutic ribozyme (see fi~ure 107) as described by Draper supra.
Rnd Tar~etin~
Another salient feature of this invention is that the RNA used to generate R-loop structures can be covalentiy iinked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, etc.). Specific ligands can be chosen such that the ligand can bind seiectively to a desired cell surface receptor. This ligand-receptor illl~ldl~liUI~ will help internalize a plasmid containing an R-loop. Thus, RNA is used to attach the ligand to the DNA such that loc '~ ol1 of the gene to certain regions of the cell is achieved. One of several methods can be used to attach a ligand to RNA.
This includes the i"co"uo,~ ", of deoxythymidine Cullldillillg a 6 carbon spacer having a temminal primary amine into the RNA (see figure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 J. Biol. Chem. 269, 3198-3204). The RNA CUllldillill9 a 6 carbon spacer with a terminal amine group is mixed with folate and the mixture is reacted with activators like 1-(3-Dimethylaminopropyl)-3-ethylcarbodi;",ide hy.l,u.;l,loride (EDC). This reaction should be carried out in the presence of 1-Hydroxybel~ul,id~ùl~ hydrate (HOBT) to prevent any u~ ~de~ dLle side reactions.

WO 9S/23225 2 1 8 3 ~ 9 ~ Lf~,lllL . IS6 The RNA can also be derivatized with a heterobifuctional crosslinking agent (or linker) like succinimidyl 4-(p-malt,i"~idopll~"yl)butyrate (SMPB). The SMPB introduces a maleimide into the RNA. This maleimide can then react with a thiol moiety either in a 5 peptide or in a protein. Thiols can also be introduced into proteins or peptides that lack naturally occurring thiols using succinylacetylll~ioA,~ P
The amino linker can be attached at the 5' end or 3' end of the RNA. The RNA can also contain a series of ri~cleoti~es that do not hybridize to the DNA and extend the linker away from the RNA/DNA complex, thus 10 increasing the ~Ci,i. ' "~y of the ligand for its receptor and not interfering with the hybridization. These techniques can be used to link peptides such as nuclear l~ ,n signal (NLS) peptides (Lanford et al., 1984 ~1137, 801-813; Kalderon et al., 1984 Cell 39, 499-509; Goldfarb et al., 1986 ~1~3Z, 641-644)and/or proteins like the ~Idl~bftlilill (Curiel et al., 1991 15 Proc. i~lAtl Af!Ri-i Sci. USA 88, 8850-8854; Wagner et al., 1992 Proc. i~lAtlAcad. Sci. U~ 89, 6099-6103; Giulio et al., 1994 Cell. Si,~nAI 6, 83-90) to the ends of R-loop forming RNA in order to facilitate the uptake and lOf.AI;,.II;OI1 of the R-loop-DNA complex. To link a protein to the ends of R-loop forming RNA, an intrinsic thiol can be used to react with the maleimide 20 or the thiols can be introduced into the protein itself using either illu~ ldli~ or succinimidyl acetyl II,iuac~tdli- (SATA; Duncan et al., 1983 AnAI Biochem 132, 68). The SATA requires an additional dep,uL~ ;liùn step using 0.5 M hydroxylamine.
In addition liposomes can be used to cause an R-loop complex to be 2~ delivered to an applui~liali~ intracellular cite by techniques well known in the art. For example, pH-sensitive liposomes (Connor and Huang, 1986 GArLcer Res. 46, 3431-3435) can be used to facilitate DNA lldll:lfi-i~liull~
Calcium i,llo,iul,dld mediated or ~ ui o,dliun-mediated delivery of the R-loop complex in to desired cells can also be readily acomplished.
30 In vltro Selection In vitro selection strategies can be used to select nucleic acids that a) can form stable R-loops b) selectively bind to specific cell surface receptors. These nucleic acids can then be covalently linked to each other.
This will help i"ti~",ali,~ the R-loop-containing plasmid efficiently using 35 receptor-mediated endocytosis. The in vitro selection (evolution) strategy is , _ . : .. . .. _.. .. : _ _ .

~ WO 95/23225 2 1 8 3 ~ ~ ~ r~"~ r ~l -156 similar to aFJ,urua~J-es developed by Joyce (Beaudry and Joyce, 1992 Science 257, 635-641; Joyce, 1992 Scientific American 267, 9û-97) and Szostak (Eartel and Szostak, 1993 Science 261:1411-1418; Szostak, - 1993 TIBS 17, 89-93). Briefly, a random pool of nucleic acids is 5 synthesized wherein each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence.
The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (a specific region of the double strand DNA), 2) lû cu~ dly DNA (cDNA) synthesis and PCR dlll~ of molecules selected for their affinity to form R-loop and/or their ability to bind to a specific receptor, 3) introduction of a restriction endonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides ~pl~se~,L~d at every 15 position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 ~ 257, 635-641). In this invention, the degenerate domain is flanked by regions containing known sequences.
This random library of nucleic acids is incubated under conditions that ensure equilibrium binding to either double-stranded DNA or cell surface 2û receptor. Following incubation, nucleic acids are converted into cu,~"ul~",t",ldry DNA (if the starting pool of nucleic acids is RNA). Nucleic acids with desired ~I,ald~ lk,s can be separated from the rest of the population of nucleic acids by using a variety of methods (Joyce, 1992 ~Q~. The desired pool of nucleic acids can then be carried through 25 sllhse~lent rounds of selection to enrich the population with the most desired traits. These molecules are then cloned in to app~u~lidl~ vectors.
Recu",l.i"dll~ plasmids can then be isolated from l,dll~ull"ed bacteria and the identity of clones can be determined using DNA sequencing techniques.
30 O~her ~",l.~li"~ a~ wl~hin ~h~ f~llowlng clalms wo g5123225 ~ 2 18 3 ~ 9 2 ~ L,5,~ 56 Chdtdl,Ltli~lius of Ribozvmes Group I lntrons Size: -200 to ~1000 rl~rl~otides Requires a U in the target sequence i~ idLtly 5' of the cleavage site.
Binds 4-6 r~clPoti~es at 5' side of cieavage site.
Over 75 known members of this class. Found in Tet~ahymena ll,e""o~ila rRNA~ fungal illitO~IIc~ id, ulllulupldaLa~ phage T4, blue-green algae, and others.
RNAseP RNA (M1 RNA) Size: -290 to 400 n~l~lF~tidPc RNA portion of a ribonu.,l~o,u,uLti" enzyme. Cleaves tRNA precursors to form mature tRNA.
Roughly 10 knûwn members of this group all are bacterial in origin.
Hammerhead Ribozyme Size: -~3 to 40 n-lcleoti~iPs Requires the target sequence UH immediately 5' of the cleavage site.
Binds a variable number nucleotides on both sides of the cleavage site.
14 known members of this class. Found in a number of plant pdLllo~t"s (virusoids) that use RNA as the infectious agent (Figures 1 and 2) Hairpin Rit~ozyme Size: -50 nucleotides.
Requires the target sequence GUC il~ ddidL~ly 3' of the cleavage site.
Binds 4-6 n~ otid~s at 5' side of the cleavage site and a variable number to the 3' side of the cleavage site.
Only 3 known member of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot vinus, arabis mosaic virus and chicory yellow mottle virus) which uses P(NA as the infectious agent (Figure 3).
Hepatitis Delta Virus (HDV) Ribozyme Size: 50 - 6û nucleotides (at present).
Cleav.age of target RNAs recently dtlllul1~LrdLtd.
Sequence requirements not fully dtL~Illlill~d.
~inding sites and structural requirements nol fuLly cl~l~""i"~d, although no sequences 5' of cleavage site are required.
Only 1 known member of this class. Found in human HDV (Figure 4).
N~urospord VS RNA Ribozyme Size: ~144 nucleotides (at present) SUBSTITUTE SHEET (RULE 26) WO 95123225 218 3 9 9 2 r~ 6 Cleavage of target FiNAs recently d~rllclla~ ed Sequence requirements not fully determined.
Binding sites and structural requirements not fully determined. Only 1 known member of this class. Found in Neurospola VS RNA (Figure 5).
r SUBSTITUTE SHEET (RULE 26) 2183~1~r~
WO 9sl2322s . PCTrlBssrools6 Table 2 Human ICAM HH Target sequenca nt. Position Target Sequences nt. Position Target Sequences 11 CCCCA~ C G~^UG 386 ACCGLW A C;JGG;!C'J
23 CrGaGCl7 C c~criGc~r 394 CUGG~r C CAG~ACG
26 ~rccr C r~Lcu 420 C~CCC,_J C CC_uC~Ju 31 0co~rr A Ca~G 425 C~iCC_CJ C riL'C~AG
34 uG~crr c Ar~rsG 427 CCCC}CJ U G~GCC
40 rcAG~s rr GCA~CCrr 450 ~G~ACCU r~ ~CccrsAJc 48 GCAACCU c AGCCUCG 451 G~CCW ~ C_CUACG
54 ucAGccr c GcL7ArrGG 456 U~rAcccu A C^~GCC
58 CCUCG~U A uGGcrrcc 495 CCAACCU C ACCGrGG
64 ~GGCJ C CCA~ 510 UGcrGcu C CGrrCGC~
g6 CCGCACrS C CrGGUCC 564 CUG~GOE'r C ~CGACCA
102 i7CCrSG~ C CUGCrrCG 552 G~GAG~17 C ACCAr,W
108 UcCrsGCrr C GGG^~C 607 AGccA~ri rr UCL'CG'.TG
115 CGGGG~ C r~rrccc 608 GCCAA~Tr r CL~GUGC
119 GC~W ~ CCC~GGA 609 ccAA~l~rr C UCGUGCC
12 0 c~srr C CCAGGAC 611 AA~ C GL-GCC C
146 CAGACArr C uGuGrrcc 656 G~GCL~;r rr L~C
152 UC~;UGi7 C CCcCrrcA 657 AGCL~ ;7 G~ACA
158 ucccccu C AaAAGuC 668 AACPCCU C GGCCCCC
16; CAAAAGU C A~CCrrGC 677 GCCCCCJ A CCAGCUC
168 AA~as C CrGCCCC 684 ACCaGCU C CaGACCU
185 GGAGGCU C CG~G 692 CAGACCU U U~rSCC~rG
209 AGCACCU c C~GrrGAC 693 AGACCW u Gr~ccrsGc 227 CCCAAGU U GWGG~C 696 ccrs~Gs C CL~GCCAG
230 AAGwGrr i7 GG~A 709 AGCGACU c CCCCACA
237 UGGGCArr A GAGACCC 720 CACAACU U GDC~GCC
248 ACCCCGU ~ GCC~AAA 723 AACULW c ACCCCCC
2s3 GWGCCU A AAAAGGA 73s CCCG~ C C~GAGG
263 AAG&aGu rr Gc~rccuG 738 GGGrsccu ~ GAC~rGG
267 AG~uGcrr C CUGCCUG 765 ccGrsGGlT C uGuriccC
293 AaG~GU A UGAACUG 769 GGUCUGU rs CCCL~GGA
319 AGAaGAU A GCCAACC 770 GrrCUGW C CCL'GGAC
335 ArrGrrGcrr A r)ucAAAc 785 GGGCUG~7 r CCCAGUC
337 GUGCrSAU U CAAAC~G 786 GGCG~SU C CCA~CU
338 UGC~AW c AAacrrGc 792 ~ UCCCAG~ c UCGGAGG
359 GGGCAGU C AAcaGCU 794 , . CCAGrrcu C G~GGCC
367 AaCAGcU A AaACCW 807 CCCAG~U C C~CCUGG
374 AAAACCU U CCUCACC 833 CA~i rr GAACCCC
375 Aaaccw c CUCACCG 846 CCACAG~ c ACCUAUG
378 ccwcc~ C ACCGUGU 851 G~rCACCU ~ r~Ac SUBSTITUTE SHEET ~RULE 26) WO 9S~23225 173 218 3 ~ 9 2 r~ 156 863 ~ACGal~J C CJGCUCG _ 1408 O~aGAU C WGPGGG

8 6 6 GACUCC0 U CUCGGCC 1410 GAGAIJI~ TJ GAGGGCA
867 ACUCCU7 C ~CGGCC~ 1~21 GGCA~ CU A CCI~ UGU
869 UCCUUCU C GSC-AAG 142S CCUACCtJ C UG[JCGGG
881 Aa~ C AG~JCAGU 1429 CCUCUGU C GGGCCAG
885 CC~J C AGl ~iUGA 1444 GaOEaCU C A~GGGGA
- 933 G~JGcaG~J A AW~17GG 1455 GGGAG~,~J C ACCCGCG
936 C~G~ A C~GGGG~ 1482 AUGUG'~IJ C UCCCCCC
978 ~.~C"~U C ~ 1484 G~,~J C CCCCCGG
980 AG'.'AUCU A C~17UU 14g3 CCCCG~,--.J A IJGAGAUU
986 U~ U UCCG^~-5 1500 AUG~G~U U GUCAUCA
987 ACA';CW IJ CCGGCGC 1503 ~aDUGU C AUCAUC~
988 CA~UU C CGG^GCC 1506 ~U C A~G
1005 ~IJ U C~.,A 1509 1~7CAU C ACIJGUGG
1006 CGUGal.'U C UGaCGAA 1518 CUG~JGC,U A GCAGCCG
1023 CAGAG-U C UCAGAAG 1530 CC~ C A~AAUGG
1025 GAGG~JCU C AGAAGGG 1533 CAGUC~ A AUGGGCA
1066 CCACCCU A GAGCCAA 1551 CaGGCC~J C AGCACGU
1092 Al~'IJ IJ CCZiGCCC 1559 AGCACGU A CCUC~U
1093 IJGGGGW C CAGCCCA 1563 CGUACCU C ~JAUAACC
1~25 CCC~ C ~u~ GA 1565 UACCUCU A II~ACCGC
1163 CGC~'-tJ U CUCC~^ 1567 CCtl~U A ACCGCCA
1164 GCAG~W C IJCCUGCU 1584 GGAAGAU C AA~aAAU
1166 ~^UU~ C l,:u~JCU 1592 A~GAAAU A CA~ACUA
1172 I~'CCI~G~ C ~JGCAACC 1599 ACAG~J A CAa~GG
1200 GCCaGW U AUi~. 1651 CACGCCtr C CCUGAAC
1201 CCA~JU A l;~CaA 1661 UGAACCU A UCCCGGG
1203 AGCU~AU A CAC~AGA 1663 AACCUAU C CCGC,GAC
1227 C-wAGCU U CGUG~JCC 1678 AGGGCCU C WCCUCG
1228 G.^,aGCUU C G~D~J 1680 GGCCUC~J U CCUCGGC
1233 ~GU C CUGUA~G 1681 GCCUC~ C CUCGGCC
1238 GUCC~GU A XGCCCC 1684 UCWCCU C GGCCWC
1264 GAGGGAU U G~CGGG 1690 UCGGCW U CCCa~AU
1267 GG~GU c CGGGA~A 1691 CGGCC~U c CCA~AW
1294 AGA~aAo U CCCaGCA 1696 WCCCA~ A WGGUGG
1295 GaAAAW C CCaGCAG 1698 CCC~ U GGUGGCA
1306 Gca~aCU c C~A~GUG 1737 AAGACAU A UGCCAUG
1321 cca~GCU U GGG~A 1750 ~W~ A C~CCAC
1334 A~CCCAU U GCCCGAG 1756 UACACCrJ A CCG~CCC
1344 CCGAGC~ C AaG~GlJC 1787 AGGGCAU U GUCC~CA
1351 cAaGuGJ C UAaAGGA 1790 GCAWGU C c~c 1353 AG~ A Aa~G 1793 WGUCCU C AGUCAGA
1366 ~J U UCCCACU 1797 CC0~ C AGPIJACA
1367 GX~CW u Ccca~G 1802 GJCaGAU A CAaCAGC
13 68 Gca~w c CCACUGC 1812 ACAGCAU u UGG~GCC
'380 UGCCCalJ C GGsGaAu 1813 C~GCA~JU U GGGC,CCA
1388 GGGG~ C AGJGACU 1825 CCAUG-uU A CC~GCAC
1398 'u-GACUGU C AC~CGAG 1837 CACACCU A AaACACU
1402 UGUCaC~ C GAGAUCU 1845 AAACACU A GGCC~CG
SUBSTITUTE SHEET (RUL 26) WO 95/23225 2 1 8 3 9 9 ~ 156 174 ~i 1856 CAI~ C UGaI~LTG 2189 ~5 5 Gæ~u~u'C
1861 AUCVGaIJ c ~JGW~VC 21g6 ~ W C II~DUA5G
1865 GAUC~15 A G~JCA~ 2198 AGt~UC5 U ~UWA
1868 CD~ C AC~GAC 2199 GUGU~IIU 5 UAI'GUAG
1877 CA~JGACU A AGCCAAG 2200 U~15 5 A5GUAGG
1901 CAaGACu C AAGACAU 2201 G~ A ~AGGC
lgl2 ACA5GAU 5 GAt;GGAU 2205 U~W A G;~C~U,A
1922 UG~aUGU 5 ~aa~5 2210 G~GG U A ~AAC
1923 G~I~U ~ Aa~5C5A 2220 ~,~CAI:/ A GGCCUCU
1928 UU~A~ C ~JAGCC5G 2224 CA~U C ~7C5GGCC
lg30 AAAG~C5 A GC:l~U 2226 rL~l U C ~ u~C
1964 GAG1~C~5 A GC5CC~C 2233 C5GGCC5 C AC~GC
lg83 ~GGACAU A CAAC5GG 2242 CG5aGC5 C CCAG-'JCC
1996 G~GAaA~J A CUGAaAC 2248 , UCCCU~J C CaLWCA
2005 ~;GAAAC5 U GC5GCCU 2254 ~a~,w C ACAU.7CA
201 3 GC,UGCCU A ~lUCGGUA 2259 G~aCAlJ 5 C~AGGUC
2015 5GCCUAU U GGGUAUG 2260 UCACAI~U C AAI~A
2020 AUUGGG5 A I~GCUGaG 2266 ~JCAAG,U C ACCA~U
203g ACAGAC5 U ACAGAAG 2274 ACCahW A CA~W
2040 CAGAC~U A CAGAah`~ 2279 G~ 5 G~GG
2057 UGGCCC5 C C~UAGAC 2282 CAGtlLW A CAGG~JG
2061 CCUCCA5 A GACA~,W 2288 5ACAÇGU U a~cacu 2071 CAD~W A GCal7CAA 22gl AG~7 A C~CDGCA
2076 G~;5 C AAAACAC 2321 A~aAI~5 C Aa;iUGGG
20g7 CC~CAC5 5 cct~a; 2338 UG~U ;7 C1~7GG
2098 CA~C~U C C;~LCGG 233g GGGa~ C UC~Dl7GG
2115 GCCAGCU U GGGCACU 2341 GAC~JC5 C A~l'GGCC
2128 C~W C UACaG;~C 2344 WC5CAU U GGCCAAC
2130 GC~JC5 A CUGACCC 2358 CCUG.--C5 5 UCCCCAG
2145 CaACCCU 5 GA5GAUA 2359 CUGCW5 U CCCCAGA
2152 UGAITGA5 A UGUAUUU 2360 UGCCt~UU C CCCAGAA
2156 GAUA~W A W~WC 2376 GAGI~GAU 5 WUCUAU
2158 UAUG~A-5 U 5AWCAU 2377 AGl7GAW IJ WCI~IJC
2159 A~[IAW U AUl,~lU 2378 GUGaIJClU 5 lJCUaUCG
2160 UG~UUU A WCAUUU 237g UGA13~U 5 C5AUCGG
2162 UAWllAlJ U CAW~lJ 2380 GAWI7t3;5 C UA~JCGGC
2163 AW~UJ5 C AWUGW 2382 W~UCU A UCGGCAC
2166 UAWCAU U IJGWA~7U 2384 W5C5AU C GGCACAA
2167 AWCAW V GWA~5 239g A~ A ~WGGAC
2170 CAI~WW U AlJ~WAC 2401 GCa~AU A UGGACl7G
2171 AWUG~ A UUUGACC 2411 GAC5GG5 A A5GGWC
2173 WG5~5AU U WACCAG 2417 UAAOGGtJ U CA~GGU
217g UGWA5-5 U UACCAGC 2418 AAUGGU;J C ACAGGU5 2175 GWAW~ 5 ACCAGCU 2425 CACA~ U CAGAGAI:J
2176 WAuuuLJ A CCAGCUA 2426 ACAGGW C AGAGAW
2183 ACCAGCU A III~WG 2433 CP~.U U ACCCAGU

2186 AGCUA~5 5 AWGAL~5 2448 GAGGCCU U AWCCUC
2187 G~DW A WGAG5G 2449 AGGCClJ5 A WCC~CC
SUBSTITUTE SHEET (RULE 26) ~ WO 95123225 5 2 1 8 3 9 ~ 2 . ~ 1/~ c, 156 2451 GCCUaAU ~ ccucccu 27s0 UA~JW A GACAAGC
24s2 cc~ C C~7U 27s9 ~AG~7 C ~CGCUCU
24ss ~WU~7 C ccuuccc 2761 AAG7CU C G~U
24s9 ccu~ccu U cccccc~ 2765 UCUCG U C ~;GUCACC
2460 cucccuu c CCCCCAA 2769 G7C~ c ACCCAGG
2479 GACACCU U UGUUAGC 2797 G~AU C AUG^UUC
2480 A~C~ U GuuaGcc 2803 ~ U CACUGCA
2483 c~uuu~u U AGCCACC 2804 CAUGGW C ACUGCAG
2484 CUWGW A G^~CCU 2813 CJ~ C u[~ccu 2492 GCCACCU c CCCACCC 2815 GCAGUC~ a GACCUW
2504 CCCACAU A CAUU~U 2821 UaGACCU U uaGGGcu 2508 CAUACAU U UCUGCCA 2822 ~G~ ~ ~GG~UC
2509 ~ACAW U C~AG 2823 GACCUW U G~^7C~
2510 ~ACAUW c UG~ 2829 WGGGCU c ~GAU
2520 CCAG~GU U CACAA~I~ 2837 Aa~7GAu C cscccAc 2521 CAG~W C ACAAUGA 2840 UG~UCC~ C ccacc~c 2533 ~7 C A~7C 2847 CCCACC~ C AGCCUCC
2s40 CAGCGGU C A~GUC~ 2853 ~CAGCC-.J C CUGAG~A
254s G~7 C UGGAC~ 2860 cc~ ~ GCUGGGA
2568 AGG^~AU A UGCCCAA 2872 G~acCAU A G~7CAC
2s79 CCAAGCU A ~CCWG 2877 A~GGCU C ACAACAC
2s8s ~AUGCC~ U G~CU 2899 GGcaAAu U IJ~WW
2588 GCCUUGU C c~w 2900 GCAAAW U GAUUWa 2591 ua~77ccl~ C ~GUCCU 2904 AUt~U U uwuwu 2593 Gl7CCUCU U GUC7GU 290s UWGAIW U ~-www 2s96 cucuu~ C C~ 7WG 2906 ua~u~u U ~uww 2601 GUCCUG~ u UGC~UW 2907 uGAuaw ~ awww 2602 IJCCU~iUU U GCAI~WC 2908 GAUWW ~ ~uu~u 2607 UWGCAU ~ ~CA~G 2909 AUWUW U uwww 2608 ~A~W ~ CA~7GGG 2910 rwww U Uuuuuu~
2609 UGCArJW C A~GGA 2911 ~uwuw a ~www 2620 GGGAGCU ~ GCA~AU 2912 W;JWW 1:~ WUUWC
2626 WGCA~7 A WGCAGC 2913 wuaaw a 2628 GCACUAIJ U GC~7C 2914 awww U wuucaG
2635 UGCAG.7 C CACUWC 2915 Ut~U~JW 17 WUCAGA
2640 CIJCCA~ u UCCUGCA 2916 IJWWW U UUCA~AG
2641 UCCAGW u CCUGCAG 2917 UUUWW ~ UC~GAGA
2642 CCAGW~ c C~Gl7 2918 WUWW IJ CAGAGAC
2653 CA7GAU c ~7CC 2919 WUWW C AGAGACG
2659 ~CAGGGU C C~GCAAG 2931 ACGCGGU C ~CGCAAC
2689 CCAPGGU A WGGAGG 2933 GGGGUCa C GCAACAU
2691 A~GG~ U GGAGGAC 2941 GCAACAU IJ GCCCAGA
2700 G:~ C CCUCCCA 2951 ccaGa~7 U CCUWGU
2704 ACt~ CCU C CCAGCW 2952 CAGACW C C~7G
2711 ccca~u U l~;GAAGG 2955 ACWCCU ~ UGUGWA
2712 CCAGCW u G^~AGGG 2956 CUUCCI~J U G~G
2721 G~G~ C A~CCGCG 2961 W~7 U ~AU
2724 GGGUC~U C CG~7 2962 WGUGW A G~AUA
2794 UGUGU~,7 A UG~GUAG 2965 UG~ U ~A~AAG
SUBSTITUTE SHEET (RULE 26) WO 95/2322S . ~ 218 3 ~ ~ 2 r~ 6 2966 G31~U~ IaA~C
2969 A~ ~ ~æ5~,~
2975 u~ I~aC
2976 ~AA~.~r 1~ C~7 2977 A.aG~ C l~:aACUG
2979 GC~ C ~JGCC

SUESTITUTE SHEET (RULE 26) ~ WO 9S/23225 177 2 r 1,.. c :156 Table 3 Mouse ICAM HH Targe~ Sequence nt. Position Target Sequence nt. Position Target Sequence 11 CC~,GU C 2cC~;uliG 367 AAugGCU u cA~CCcg 23 C GuGgt~ u C~7 374 gAAsCCU U CCUgcCC
26 u~5~ C r,~l,'cu 375 ~gCCUU C C~7scCCc 31 CliCt~GCU c ClJCcaca 378 CU~ACC~Al7 C ~CCG^~IW
34 UuCUcaU a ~C~Gl7cG 3B6 A~ ~ uUc uuU
40 sCAc~cU IJ GuAsCC!J 394 C_GC,ACU u uc~uCu 48 agcA~J C AG~CI~gG 420 CACaCu~ C CCCcCcg 54 ~gcGCCU C GugAUGG 425~ CaCCCcU C ccaGC~
58 CaUgcCU u UaG~L^~CC 427 CasCUCU c aGCAGug 64 cAcccClJ C CCAGCAG 450 AGgACCU c ACCCUgC
.6 CucugClJ C ClJGC,cCC 451 GAAaCcU u uCClJuuG
102 UgCca~ a Cu~gG 456 UUACCCU c ~AGCc Cu 108 cuCl~GCU C cuGC,CcC 495 CLAcC~ C ACCGUGu 115 uGGLuCU^ C UGcUCCu 510 ~UGCU^ C CG~JGGGG
119 GgaalJC,U c æCC~ 5O'4 CUcAGW A uCCa)ACC
120 CUClJGcU C CugGccC 592 GAaAGAU C ACaugGG
146 CA~,uCslJ C c~cuUCC 607 AGCC~AU U IJCUCa~G
152 I~:t~ C agCCaCu 608 GCCAal~J U CUCaû'GC
158 UCCu-~u~7 u A~AAacC 609 CCAAllUU C lrcaû~Gcc 165 CAgAPGU u gUuuUGC 6~ 1 AAUU-u'CU C alJGCCGC
168 AAGcCuU C C~CCC 656 aAGCUG~J U UGA~g 185 GGuGGgU C CGUGCaG 657 AG~ GAGcugA
209 scCACuU C Cûc~gC 668 cgagCC^~ a G~CC
227 CagAAGlJ IJ G~uuGC 677 G~ACCUCU A CCAGCcu 230 A~UW l^J uuGCucc 6a4 uuCAG~U C CgGuCCU
237 UGI-~GCU5 u GAGAaCu 692 CsGACu^r ^~ cGauCUu 248 AaCCCaU c uCCUAAA 03 AGgaCcU c acCCUGC
253 cc^~GCCU A AggAaGA 696 CCUgUuU C CUGCCuc 263 AgGGuuU c uCUaC~G 709 gGCGsCU C CaCCuCA
267 AGg~GCU C C~GCCUa 720 uAC~ACU U ulJCAGCu 293 A~CI ~GU u U~-AgCUG 723 Aa~uu C AGCuCCg 319 AGgAGAU A CUgA-yCC 735 aCCaG~l:J C CtJgGAGa 335 cU~GCU u ~-,agAAC 738 uGGgCCU c GLGa~GG
337 GUcCaAU U CAcACUG 765 CaGlJcGU C cGclJuCC
338 zGC~gl~U u gAgC~Ga 769 GGcCu~u ~7 uCC-u~Gcc 359 GuG~-AGU C suCcGCU 770 uUurJ-GcU C CCU~a 785 GGcCUGU U uCCuGcC 1353 . AGU,ggl:l c gAaGgUG
786 GccrJGuu u CCuGcC~ 1366 UaaCAgU c UaCzACU
792 UggagGU C ~CGC,AaG 1367 aGCACcU c CCCACcu 794 CugGgCU u GC,AGaCu 1368 GuACUsU a CCACUcu 807 CLCyGzU a uACCUGG 1380 UGCCCAU C GGGGugg 833 CAaAGcU c GAcaCCC 1388 GGzGAcU C A~JGgCU
846 CCcugGlJ C ACCguUG 1398 UGgC~ C ACagaAc 851 GagACCU c UacCAgC 1~02 UGUgc~_U u GA.-AaCU
SUBSTITUTE SHEET ~RULE 26) WO 95/23225 - 2 1 8 3 9 3 2 P~ 156 863 AgCcACU u CclJCl7gG 1408 gCGAGAU C gr~ GG
866 Ga~gcc~ U Cc~cCr 1410 GAGgUCU c GgaaGg,g 86~ AuUCgW u cCGGaSA 1421 ccCACCU A CuUuUGU
869 ~7CuUcC~ C augCAAG 14Z5 aCUsCCU u ç~aGaG
881 AuGGCuU C AacCcGU 1429 uCtJCUaU u GccCCuG
885 CCUugGU a g2u~GUGA 1444 GA~gClJ C AgGAGGA
933 c!JauAaU c AlJuC~7GG 1455 GGaAIlW C ACCaGga 936 uAatJcAU u CDG-uGc 1482 A~uUGuU u lJgC~CCC
978 '~aACagU C I~CU 1484 cUG~UCU u CCuCauG
980 ~Cag~lJ A C~;J 1493 CL~guGcU u UG~GA2c 986 UACAaC17 ~ ~'uCaG u 1500 AUGA2A'U c aUggUCc 987 ~CA2CW ~7 uCaGCuC 1503 gGacUaU 2 AUCAUuc 988 CAa~uuu u CaG uCC 15û6 WaUguU u AUaACcG
lOOS ACcaGAU c CUGgaGA 1509 cuAcCAU C ACcGUGu 1006 uG_GAgU C ~GAA 1518 ucaUG~ c cCAGgCG
1023 ugGAGv~a C ;7CqGAAG 1530 CuauA2U C AUucUGG
1025 GAGGlJCU C gGAa"GG 1533 ugGUCA'U u gUGGGCc 1066 CCACuCl7 c aAaauAA 1551 CAuGCCU u AGCAgcU
1092 AcuGGaU c uCAGgCC 1559 AGCACcU c CCcaccU
1093 UGG_ccU u CAGCCaA 1563 CuUAugU u IJAUAACC
1125 CCCAaC'J C uUcuUGA 1565 IlAuguuu A U~ACCGC
~163 CG2LA~ U ClJuulJGC 1567 ug~7u~ A ACCGCCA
1164 GaAGCW C 17uu~JGCU 1584 GAAAGAU C AgGAuAU
1166 ~UCU u u~lJ 1592 Ag&AuAU A CAaguUA
1172 UCCUGuU u aaaaACC 1599 ACA~gulJ A CA,gaAGG
1200 cuCuGClr c c'J,cCACA 1651 CcCaCCU C CC~7GAgC
1201 ~7CuGCW u IJgaACag 1661 g2AaCCU u UCCuuuG
1203 ACUWLU u CACcAGu 1663 AacctJuu C CuuuGAa lZZ7 GvuAcaU A ('vUWgC 1678 Ar~G~CCU C agCCUgG
lZ28 GaAGCW C uUutJgCU 1680 aOECaC~ U CCUCuGg 1233 WCGUu17 C CgGaqaG 1681 GCCaCW C CUCuGgC
lZ38 GUg0GtJ A ~GuCCu 1684 aCWCCl7 C uGgC~Jgu 1264 GAaGGgU c GllgCa_G 1690 cCGGaCU U uCgAUcU
1267 uGAqaGU C uGGGgAA 1691 Ct;G~ u CgAUcW
1294 AGgArJAU a CugAGCc 1696 U5CCCA~7 c gqG~UGG
1295 GAggqgU C uCAGCAG 1698 C~7gAUAU ~ cc~GGag 1306 GCAGACU C ugAAaUG 1737 qAGACcU c UaCCAgc 1321 g~ c _GGAGgA 1750 gGCgGC0' c CACCUca 1334 A~CCCAU c uCCuaAa 1756 ~7AagCCU u CCuGCCC
13~4 _uGAGCU C gAGaGUg 1787 gaGaCAlJ U GUCCcCA
1351 ugAaUGU a ~aAguuA 1790 GCaWGU u CUCuaau 1793 Ug~JCC~7 C gGcugGA 2173 IJlJagagu U WACCAG
1797 CacCAGU C AcA~aA 2174 UagagW IJ UACCAGC
1802 2cCAGAU c CuggAGa 2175 _gagWtJ U ACCAGCU
1812 ACuGgAU c UcaGGCC 2176 gagWUtJ A CCAr.CUA
18'3 CA~UU U acccLCA 2183 ACCAGCU A W~AWG
1825 CCAcGcU A CCUcugC 2185 CAGCUA~7 U UAWGAG
1837 CAugCCU u u~gCuCc 2186 AGCUU~r U AWGAGU
1845 cgAgcCU A GGCCACc 2187 GC0~ A WGAGlJa SIESTlTUTE SHEET (RULE 2') W095123225 179 2183992 P`~ ~ [156 1856 CggaCulJ u cGA~ 2189 UAU~UAU ~ GAGUacC
1861 Aca~GAU a ~CCAGUA = 2196 caAcUcU u cUCgA~G
1865 cAcu~J A Gc'"CAg 21g8 gc2GcCU c ~Ju 186a CaccAGiJ C ACAU_Aa 2199 GcclJCUl:J a IJgUullAu 1877 CAUGcCU u AGCagcu 2200 UcUucc~ c ~i7GcAaG
1901 uAA2ACU C AAGggAc 2201 aagUU[,U A IJGUcGvC
19~2 ~UagU a GAUc7gU 2205 Wt~7 c GCCcugA
1922 UG2AUGU ~ uAAG;~a 22' 0 GgAGaCU c AgUGgc~
lg23 uGAlJGcU c AgGUaUc 2220 c~cgCAI:~ u G~tXtJCJ
1928 Ur,~ u UuaCCaG 2224 CacA~U 7 UCCAUCC
lg30 ~gACJuU u aCCaGcU 2226 UgG71,~J C 2~CCsC
1964 GA~U u GuCCCca 2233 CJGaC~'J C c~lGGAGg 1983 ~L~AU A C~AgUua 2242 uGt~U 7 sCgG_CC
l9g6 aGGAgAU A CUGAgcC 2248 UauCca~ C C~i7ccCA
2005 IJGgAgCU A G~gGaCc 2254 UCCA uU C A~AcUsA
20~3 G~Uauul:J A WGaGl~A 2259 aUCACAU l:J CAcG.,Ug 2015 ;JGCCcAl:J c G~7ugG 2260 UCACAIJJ C AcGGUgc 2020 gsl~GGuU c UuC~7GAG 2266 ggAAuGU C ~CCAGGa 2039 gCuGgCU a gCAGAgG 2274 ACCAG_~ c CuGgaGa 2040 CuGACcU c CuGgAGg 227g GaAggGU c GiJgCAaG
2057 IJGcuCC~ C CAcAucC 2282 aAGcUGU u u~,77GctJG
2061 CuaCCAU c acCgUGU 2288 UAuAaGi7 U alJggcCU
2071 CAcuUG~ A G._cl~CAg 2291 caGUgG~ u CuCOi;Cu 2076 G~GCcU C AgAgC A 2321 gMAGAU C ~C~G
20g7 CaACu~J U CuUGAuG 2338 UGaGAClJ c Clrgcc~-G
20g8 CACAC~U C CcccCcG 2339 GaaACcU u ~CcWuG
2115 G-CAGCU c GGagsA~ 2341 GACcUClJ a ccaGcCu 2128 CaGCUaU u UAu~GAg 2344 WucgAU c uuCCAgC
2130 cC~JuU c CUGcC~ C 2358 CCcagCU c 17CagCAG
2145 CAACu~U U cuUGAUg 2359 CUGCuW U gaaCAGA
2152 UauUaAU u UagAgW 2360 7aCCU~lJ C CuuuGAA
2156 uu~7AUGU A W~'a 2376 agGr,'GgU U cWCUga 2158 gAUGUAU U UAW2AU 2377 gGUGgW c i~JCUgag 215g AUGi7AlJl:J U AUCaA~U 2378 agGg~W c IJCUAcuG
2160 ~W A WaAlJDU 2379 ~GcU~W c ucAlJaaG
2162 ~ U 2AUCUAg 2380 aAgWCU a IJglJCGGC
2163 AUgl7AW u AWaaW 2382 zWcUCJ A UuGcCcC
2166 acWCAU ~ cucUAW 2384 aUcCagU a GaCACAA
2167 AUguA~U U aWAaW 2399 MaCACU A UgUGvAC
2170 uA~aU U AaW~Ag 2401 aagCUgU u ~GagCtJG
2171 AgWGW u Ugc~JcCC 2411 uACUGGI~ c AgGaUgC
2417 gAAlX;GU a CAuAcGU 2691 MuCJcU c cGAGGcC
2418 Ac~;GaU C uCAGGcc 2700 GAaGcCi7 u CCt~gCCc 2425 CAugGGU c srAGi~GuU 2704 gAcCuCU a CCAGCcU
2426 AuuaaW u AC;AGuW 2711 CCCAGCU c UcagcaG
2433 uAGAGuU U u~Gc 2712 gagGucU c GGaAGGG
2434 AGAGuW u aCCAGcu 2721 GAAGvC,U C gUgCaaG
2448 GAaGCCU U ccUgCcC 2724 GC,uaCAU a CGuGG-Gc 2449 AAGCCiW c cUgCcCC 2744 gGUGgGU c cGUGcAG
SUBSTITUTE SHEET (RULE 26) WO 95/2~22S 21 ~ 3 ~ ~ 2 F~l,.. ,,~,.'~ 156 2~51 GCCUgutJ U CClJgCCU 2750 UA~T~UaU u GAgLIAcC
2452 CCUguW C CUgCCUc 2759 cCgcaClJ u liCGaUCU
2455 gAagCCU u CClJgCCC 2761 AgGacC~ C aCcCUGc 2459 CCaCaCO U CCCCCCc 2765 ~uUuG~--~J C IJGcCgCu 2460 C~CaCU~ C CCCCCcg 2769 ~gUCl~W C AaaCAGG
2479 GAgACClJ c UaccAGC 2797 aUGaAAU C AlJGGUcC
2480 uCACCg5 U G~JgAuCC 2803 UC~ CcagGCg 2483 CCaa1'GU c AGCCACC 2804 ggU~sU C cgUGC~G
2484 CWUuUU c aCCAguc 28-~3 CUcCgG'U C cr~,~CCc 2492 agCACCU C CCCACCu 2815 2cAGUCcr a cAaCUW
2504 CC~^ACcU A CuWWgU 2821 cUG~C~ c c'JGGagg 2508 uAUcCAU c calJcCCA 2822 gGAgCcU c c^~u 2509 uUAgAgU U uUaCCAG 2823 ugCCJl,'U a Gc ~CcC~
2510 UAgAgW U UaCCAGc 2829 ct,'GGaCU a uAaUcAU
2520 Cuuu~GU U CcCAAlJG 2837 AgGUGg~J u CUuC~g~
2521 CAGcaU0 u ~CccUcA 2840 UGAgaCU C C ~gCCUg 2533 IJGAugCO C AGgualJC 2847 CCaAugU C AGCCaCC
2540 CAGCaG~ C cgc~g~G 2853 gCAGCCrJ C ulJauGUu 2545 GUgcTJW a UGGuCctJ 2860 gCcaAGU A aCt,'GuGA
2568 guGaAgU c UGuCaAA 2872 GGACCuU c aGCcaAg 2579 auA~GuU A ~'GgCcUG 2877 uUccCCU a c~-AuCAC
2585 cugGCaU U G~uCUCO 2899 cGgAcul:J U cGAUcW
2588 GCa~GU u CUCOaaU 2900 uUAAuW a GAgWW
2591 UgG~ClJ C ~JgcUCCO 2904 AcUOcAU U cUclJaW
2593 clJuCOuU IJ GQ~Gc 2905 cUUcAW c '~cUaWg 2596 CUu~7 u Ccc~aUG 2906 WGAUgU a l'~Wa~'~Ua 2601 acCgUW a UuCg~U 2907 UGuaUt~U a ~U~AWW
2602 UCCaGcU a cCAUccC 2908 GAagcl',U c Wt'~UgcU
2607 cUcGgAlJ a U~cCUGG 2909 AgcWcU U WgcUcU
2608 caGCAgU c CgCOGuG 2910 UgUaWU a WaaW~
2609 gG~AUgl:J C ACcaG,,A 2911 Ug~aWW a WaaWU
2620 ~GGAcCU c aCcCUgc 2912 WgWcU c UaaUg~'C
2626 WuCgaU c WcCAGC 2913 WWcUcU a cUgqtrCA
2628 GCACacU U GuAGCcU 2914 UgcWW c UcaUaAG
2635 UuCAGCO C CgGOccu 2915 aWWaW a a~ AGA
2640 sgCCUGU U ~JCCUGCc 2916 '~JaWcgU U UcCgGAG
2641 cCCAGcU c uCaGCAG 2917 aWcqW U cCsGAGA
2642 CCUGUW C CUGCcUc 2918 WcgUtiU c CgGAGAg 2653 uAcUGqU C AGGaUsC 2919 Wcl:/caU a AGgGuCG
2659 gaAGGGU C gUGCAAG 2931 usGaGW C UCG5AA5 2689 CuAAUG17 c IJccGAGG 2933 GaGG~J C GsAAggg 2941 GaqACAU ~ GUCCccA
2951 CCAc5CO a CCUclJGc 2952 CAGQgl:J C CscUG~JG
2955 AgUgaC U c UGUGUcA
2956 uWCCW U GaaUcAa 2961 UcUG~W c AGccAcU
2962 aUWaW u aWAAUu 2965 lruugAatJ c AAUAAAG
SUBSTITUTE SHEET ~RULE 26) W0 9S12322S 181 218 3 S 9 ~ r~ 156 2966 GclJgGclJ A gcAgAGg 2969 ~cW ~ AAR~GU
2975 UAgAGu~J U UacCasC
2976 gAgGg~ U C~uAC~
2977 AA(~glJ u l~gAsCJG
2979 uC~WCU C uAu~;CC

SUBSTITUTE SHEET ~RULE 26) WO 95/23225 - 218 3 9 9 2 F~ r ~ ~ 156 Table 4 Human ICAM HH F~ibozyme Sequences nt. Position Ribozyme Sequence 11 CAGCOEJC rrr~TTr~~r^rra7 T rr7-rr,AA AC~JGG~;G
23 AGCAGAG rl7~ rrrr`A~lr~rrr-~A AGrLTcAG
26 ~ ~Tr.Arrr-~r^~aarr-rrr~ AGGA5~
31 CGCt~GAG ~nATlrArr,--rraAArrrmaA AGCAG~G
34 CAA~JT rT~r~rTr~ r,r- r~aar--~rrr~7l ~CA
AG~TCC ~:~TTr~rTr----aAAr--,~rr 48 CGAl;G~ rTT~ ~rTrArr,Trrr~,AArrrmAA AG~,~^
'4 CCA~GC rTTraTTr~ rrr~TAr,~ AA AGG--~,GA
~8 r~GaGCCA rTr~rTr~rrrrr~r~ar_~rr~A AGCGAGG
o4 Ct,l~Ct3t;G rr~r~rTrAr--rra ~Arr7crrA A AGCCArJA
96 GGacr--AG rTr~TTr.Arr7rrrA~Ar~rrrrAA AG~;CGG
1~2 m~CAG rrr~Tr~rr,~rrrAA~rrrrr,AA ACCAr,Cla, 108 GAGCCCC rTTnATTrArrrrra~rr~crr7AA AGraGGA
1~5 GGGA,ACA rTTrATTr~r,r,rrr~ ~Ar~r.--rrAa AGCCCCG
11 9 ~iTCCUGGG rrTrATTr~ ~rrrr~ r~ 7~r~rrrr~ A ACAGA(;C
120 GUCCUGG rTTr.ATTr~r-~rrr~arrrrr~A AACaGAG
14 6 GS~CACA rrTr.~T Tr` rr--rn~ A Ar--,rrrA A A~Gt7a;G
152 r~GGGG rrTr.ATTr~ rrr`aarrrrr`'` ACACaGa 158 GA~UDIJCT rr~Tr.ArTr~rr~ r~aArrr-rr.Aa ACGGGGA
16~ GCAGGArJ rr~X~TTf~rrrrr~ r-rrr~A ACG~OUG
168 GGGGCAG rrr,~TTr~rrrr~AAAr,r,rrr,AA ~T
135 CAGCACG rTTr~rT~Arrrrra ~Arr--cnAA AGCCCTCC
209 GUCACAG rur'~`'-crrr'~'rrrrrAA AGGGrX~T
227 GCrCAAC rTr~rTr~Grrr~rT~r~r7rrr~ ACGIJGGG
230 ua~ccc rr~7r~rTr~ rrrr~Arrrrr~ ACAAS~,T
237 GG5UCIJC rr~Tr-~TTr`"rTrrr~ Cr'rrr`~ A~;C~CA
248 17UUAGGC ~r~rTr~-rrrr~r--rrr~A ACG~ T
253 UCCGUG~T rrn~TTr~--rrrAAArrrrr~r AGGCAAC
2Q CAGGAGC ~TTr~TTr~Arrrcr~ r~r~rrr~AA ACUCCGU
267 CA~GCAG r-r~-r~rTrArrrcr~ A~rrrrrAA AG~T
293 CAGr~TcA rrTr` rTrAr,r,rrr~ a Ar~rrrr~ '~ ACACCUU
319 GGGrJGGC rrr.ATTr~-rrrr~ aTir~rcrrAA AIJC~CCT
335 rUGrG;~A rr,Tr~TTnAr,rrrrAAArr~crr~A AGCACAIJ
337 CA~1U~,TG rrr-ATTr~GrrrrAAArrrrr~AA AW~CAC
338 GCAG~T r7rArTrArrrrr~'ArrrrrA~ AaLTAGCA
359 ArC~U~,T rrXATTrArrrrr~r--rrr.AA ACUGCCC
367 A,A~T rTT~ATTr.Arrrrr.AAArrrrrAA AG~UrT
374 GGuGaGG rrTr~Tr~r~rr~ rrrrrAA AG~Ut,T
375 CGGUCaG rTxATTr~rr~rrrAA~r~rrrr~AA AACG~T
378 ACACGGrJT rTTr,ATTr,Ar~rrr~AAr-,rrr,A~ AGGAAGG
3 8 6 AG~,TCCAG rTxarTrArrrrr~ ~ ~r-rrrA ~ ACACGGrT
394 CG~JC~JG r~TTrATTrAr~rrrAAAr~r~rrr~AA AGt~CCAG
420 AAGAGGG rTXATXArrrrrAA~rr,TrrrAA AGGGGUG
425 C~JGCCAA c~Arrrrr~rr,rrrTa AGGGGAG
SUBSTITU E S;EET IRULE 26) W095123215 183 218 3 ~ 9 2 r~l~.D5~ 56 427 GGa~CC rnr.~rrr~rr.rrr.~rrrrr~ A~aGGGG
450 r,W~ ,7 rnr.~r~ r~.~^rr.~a~rrrrr~ Ar~U
451 CG~iGG rnr~Tlr.~rr,m:~rrr~ AaGGUUC
456 r,~cG rrrr.T,rr.~rrrrr.~rr.m:~ AG~'llaA
495 CCACGGt~ r~rJr~rTr~r-r-rrr7~ r~rr.~ AGG~G
510 CCCCaCG rrTr~TTr~r~rrrr~ rr-rrr~ AGCa~.
564 ~CGtJ rrr~rrr.~rr~rr.~Ar~ rrrJ~ ACC~ aG
532 CCAI~IJ rrr~rTr.~r.~rrr.~rr.~ u~ AUW~C
607 C~CG~ rrTr~Tlr~rrJrrr~ r~r~^rrz~ A~
608 GcacG~ rnr.~rTr~rr~rrr~ r~r~rr~r~ A~GC
609 r~a rr,~r.~rTr.~r~rrr~ r-r-rrr.~ Aa~UGG
6~ ~ GCGGCAC rrr~ r~rrrr~rrrrr3~ AG~AAU~
656 G~CO~ rr~rTr.~rrrrr.~rr~ AC~CUC
657 UWUC~C rnr.~Tlr~rr,rrr~Ar.. rrr.~ Aa~CtJ
668 GGGGGCC rnr.~Trr.~rr,rrr.~A~rrrrr-~ AGG~U
677 Ga~ GG rnr.~rTr~rrrrr~Arr,rrr.~ AGGGGt;C
684 AGG~ UG rTTr~ArTr~r~r-rrrA-AArrrrr7lr AG~;GU
692 CAGGBCA rnr.ArTr.ArrrrrAA~rrrrr~ AGGUCUG
693 Gr LGGAc rnr~-Tr.Ar~rrrr.~AArrrrr=AA AaGGcrcu 696 C~GCAG rrrr.ArTrArrrrr~AAAArrrrr.AA AC~aAGG
709 ~7GGGG rrrr~rrr~rJrrrrAA~rrrrr-AA AGr3r~
720 GGcr~GAC rnr.ATTr~ rr~rrrAAArrrrr.AA AGr~G
723 GcGGGaJ rnrz~nrArrrrr.-AAAr~rrrr~ ACAa~Cr 735 CCCCCAG r7r.ArTr.Arr~rrr~A~r~r,rrr.A-A ACCCGGG
738 CC~CCUC r7r.Arrr.Arrrrr~rrrrr.A-A AGGACCC
765 GGGAacA rnrPTlr~rrrrr-~A'~r-rrrr-AA ACCACGG
769 lJ-ccaGGG rr,r.~rTr.Arrrm~-AArr~rr~ ACAGACC
770 rrJccAr~ rnr'`'rr-~rGrrr` A~r-,rrr~A AACAGAC
7 85 ~r,aC~GG rrr.ATrr.Ar~rrrr.A A Ar.rrrr~ ~ ACa~CCC
786 A~GG rr)TT-ArTr~Ar~rrrr-~Ar~r-r-rrr~-A-A Aa~AGCC
792 ccrJccGA rTJr7~T~rrrrr~AAArrrrrAA AcrJGGGA
794 GGCCUCC rTTr.ATTr.~rrrrrA-AAr~rrr.-AA Ar3~GG
807 CCAGGUG rTTr~-Tr~-Ar-~rrrA~l~r~rrrr.AA ACCrJGGG
833 r~GGGUuc rTTr.-ArTr.P,rr,rrrAAArr,rrrAA ACC~
846 ca~7 rrTr~-Anr~-Ar~r7rrr-A~Ar~r7rrr-~A ACUG~G
851 G~GCCA rTJr.ArTr.ArrrrrAAArrrrrAA AGG~GAC
863 CGA~;AAG rnr~Tr.~rrrrrAa Ar~r~rrr.AA AGUCG~
866 GGCCGAG rrrr.ATTr.-ArrrrrA-AArrrrr.-AA AGGAGUC
867 CrGGccr,A rnr.ATlr.ArrrrnAA~rrrrr.-AA Aa~Cr 869 C~ GCC rTTr.ATTr.Ar~rrrAAArrrrr.AA AGAa.~,GA
881 Acr~ rnr~rTr~-Arr~rrr~-AAAr~r~rrr~A AGGCCUtl 885 Crc-A~Ir rrrr~Tr.Ar~r~rr.-AA-Ar~rrrr.AA Acr~G
933 ccaGcrAl7 rnr~r.Arr,rrrAAArrrrr.A-A ACUGCAC
936 r,rCCcc AG rr,rr.~TTr-ArrrrrAAArrrrr~ r Acro~
978 ACCUGUA r~TTr.~TT,--Arrrrr.-AA~r--,rrr.A~ ACrGGrJcA
980 AaAGcuG rnrATTrArr,rrr~r~r~rrrAA AGAcrGGu 986 CGCCGGA rrJr~TT~Ar~rrrr~AAAr-r~rrrAA AGCr~G~A
987 GCGCCGG rTrATTrArrrrr.AAAr~rrrr.AA AaG~ccrGu 988 GGCGCCG rTT~ArlrArrrrrAAAr~rrAA AaAGccrG
SUBSTITUTE SHEEI (RULE 26) WO 9~il23225 - 2 1 8 3 9 ~ 2 A ~J~ 156 1005 IJCGUCPG ~ r~rrr~r~^rrr~ ~IJCACGI~-1~06 IJUCGUCA rrr.;~nrA~r,rrr~r~rrr-AA AAUCACG
1023 r~, rt~r~anr~arr~AAr~r7rrr-~A AC0C~JG
1025 CCC~J r~r~anr.~r~rrrr7~ rrrrr~ AGACC~JC
1066 ~C rrr,,An,r.~rrrrr~7~r~rrrr~A Ar~G~
1092 GGGCI~GG rr7t~ r,rrr~ rrrrr~ AC~ CCa8 1093 I~G r~ ~rrrAAArrrrrAA AACC.CCA
1125 l~GCAG rrTr~lr~r~rrrrr~ rr~rr~A ~ UGGG
1163 GcArGAG rr~anr~ rrCr~AA AG--tJGCG
1154 ~GGA rrJr~ rrr~rr.rrr,AA Aa~i.~JG~^
1'55 AG~G rrTr~ r~rrr~ r~-rrrAA AG.~AG.^U
1172 r~GcA rnr:lT~Arr~rrr~AAArî~rrAA AGCaGGA
200 ~JG~IJ rr~r.~T~.Ar-'`rrr~aAAt~A~ ~GGC
1201 ~ ~rr, r~:Arr.rrr.AAAr~r~t:AA ~aGG
1203 I~G rnrAnr~ rrAAArr,,~.~. A~
1227 GGACACG rrJr~TTr~ar~rrrr7~ r^~rrrAA A~CCC
1228 AGGACAC rrTr~ r~rrrrAAArGrrr,DD AAGC.UCC
1233 CAUACAG rrJr ~TTr-ar-^~rrr-AAArr~rrr`` ACACGAA
1238 GGGGCCA fTTr7~rTr.arrTrrr~ r-rTrrr~ ACAGGAC
12 54 cccrGAc rrTr.AnrArrrrr~ ~ ~ Grrrr~ A AUCCC~C
1257 UtJt7CCCG rrTr~TIr.Ar^~rr~r.AAArr.rrr.AA ACAAt7CC
1294 UG~ rTTr~TTr~Arr-rrr~AAD~r,r~rrr~Dl Ar~U~7 1295 C~CtJGG rrTr~Anr~r~r~rrr~AAAnrrrr~D Aa~C
1305 CACAUUG rTTr~TTr~rr~rrr7~D~rrrrr~ AGT7Ct7GC
1321 rJt7CCCCC rrTr~ ^r~ rr~ Drrrrr~A AGCC~7GG
1334 Ct~XGGC rr~ ATTrAr~rrrr~DDr^rrr.AA A~; ;Gt7U
1344 GAC1~T rrTrDTTr.Arr-~rrr`~'`rrcrr`D AGCUCGG
1351 rJCcrJ~ rTTrATTrArrrrr~ rrrrrAA AC ~t7G
1353 CAUCCtJU rrJr,~nr~rr~rrr~ rr~rrr~DD AG~CaCt7 1356 AG~7GGGA rrJ~ Anr"rr~rrr-AA~r-r-rrr`'` AGUGCCA
1367 CAGI ~GG rrJr~Anr~ rrrr~ D 71 rrrrr~ ~ ~aAGUGCC
13 68 GcAr,uGG rUr-~nr-~r~r~m:A A DrrrrrA A AAAGUGC
1380 AUUCCCC rTr~:Drr~rr.AAArr~^rr.AA Al,~;GGCA
1388 .a~uc~.cu rrJr~ Arrrrr~ rrTrrr~AA AUUCCCC
1398 C~CGaG;7 rrTr~rTr~ rrrrr~ D~rrrrrD D ACAGIJCA
1402 AGAUCUC ~rrJ~Anr-ArGrrr`"~r~r~rrr.aD AG~ACA
1408 CCC~JCAA crTr~TTr~rr,rrrADArr,~rr,~A AUC~JCGA
1410 UGCcCrJC rrTr~TTr~r-^~AAArr7rrrAa A~C
1421 ACAGAGG rrTr~ rrrr~ rr,r,rr~ ~ AGGr.~cc 1425 CCCGACA rTTrDnrDrrrrr~`DrGrrr.AA A~UAGG
1~29 CUGGCCC rrTr.ATTr.~rrrrRAAAr~rrrrAD ACAGAGG
1444 UCCCCUt7 rTTrAnr.Arr,rrr.A~Ar~rTrrrAD AG~,TC
1455 CGCGG,GU rrrATTrAr~rrrn7DDr,rrrr~ ACCUCCC
1482 GGGGGGA rrTr~Anr,~rrrr~ ~ ArrcrrAA AGCA~U
1484 CCGGGGG rnrAn,nArrrrr~DDr~rrr.~A AGAGCAC t 1493 AAUCUCA rTTrArTrDrrrrr.D~Arrrrr.AA ACCGGGG
1500 UGA~7GAC rTT~r-Anr~Arr-rrr~DDrr~rr~D AUC~,~AU
1503 r~lGAt7GA~7 rur7~aT~T~r~Ar-rrrr~DT~rr~rrr~D ACaAUCU
1506 CAGUGAU rT7r.ArTr~rr~rrr.DADrr~rrriA A~aQA
SUE~ST~TUTE Sl IEET (RULE 26~

wos~l23ns 1~35 2 P ~ 5 ;156 1509 CC~ rrJr-~TTr~r~rrrçaAAr-r~-rr-~ AUGA~A
1518 CGGCDGC rrlr~r7rarr,rrr.a~Arr,rr~r A~ ACCAC~G
1530 rxa~WA~ rnrArlr~Arr~'~7~r-r~ ACUGCGG
1533 UGCcr l~u rr~r-~ r~Arrrr~.AAar~rrrr.;;~ AU~CUG
'551 ACG~JGCU rr~r~Ar~ rrr.A~rr~r.AA ~GCr-uG
1559 AllaGaGG rnr~rArrrrr,AAArrrrr~ DGCU
1563 GG~A rnr~ r~r,~rrr~ r-r~rr-~A AGGUAcrV
1565 r~CGGUUA rrTr~T~r~r~ ;aaArr~V~~ AGar~
1567 ~GGCGC,U r~~Tr~ Arr~r~r~rrr~aA ~aGG
158~ A~U~UU C~7r'~Arrrrr-~Ar-rrrr-AA A~U~CC
15g2 U~UC~ r~rarr-rr~rr~r~ A~C~U
1599 Cr~GL-UG r~nr~T~rArr,rr~ r~rrÇ~A AG~GU
1651 r,r~; r~r~rrrAr~ AAAr~rrrr`` ACGC~G
1661 CCCr;GGA rrr~r~r~ rr,A~rr,crrA~ AGG~CA
1663 GGCCCGG rrrr~ rr~Y;A~Ar--rrrAA A~;G~U
1678 cr~A rnr~ Arr~rrr~A~r~r7rrrJ~ Ar~GCCC~
1630 r,x~GG rrrr~ Arrrrra~Arr~AA Ar~Gcc 1681 ~GCCGa~3 rrrr~ rrrrrAAArrrrr~aA Aa~aGGC
1684 GaA~CC rr~rr rrrAA~rrrrr.Ai AGGaaGA
1690 A~IJGGG rr~rAr~rarr;rrrAA~rr,rrr~A AGt~CCGA
1691 Aa~JAuGG rr~ rrrrr,AAArrrrrAA AaGGCCG
1 696 CCA~ A rrJr-ArTr~Ar~r,~maAArr~ AA ArJ~ GGAA
1698 IJGCCACC rr~Tr~rmArr~rrn~ar~rrrr~aA A~GG
1737 CA~3GCA rrTr~rTr~r~r-^rnAAAr~r-rrr~Aa AL~C~
1750 Gr~;GUG rrr~rrrArr"~rnAAArr7~rrraA AG~GCA
1756 GGGCCGG rrTr~rTr~ rrrA~Ar~r~rrr~Aa AGWGUA
1787 U~GGAC r~rTr~rTrarrrrr~rr,rrr,aA A~CU
17gO GA~rAG r~rTr~rTr Arrrr~r~rr~rrnAA ACAA~C
1793 r~,T rrrr~rrr-~rr7rrr-AaAr~rrrr~AA AGr~aCaA
1797 ~GUWCU rrrr~rTrarr7rrrAAArr,rrr~, AC~G
1802 GC~G rnr~r~ Arrrrr~ rr~rrr~A ArL~GAC
1812 GGrCCCA rrTr~rTnArrrr~r~rrrrrA~ Ar 1813 r,JGGCCCC rrTr~rrnArr7rrna7~r~rrrrrAa AA~G
1825 G~AGG rrTr~rTrArr,rrr~AArrrrrAA ACCU7GG
1837 AGUG~tJ rr~r~rTr-~rrrr~r~7~Ar~r~rrr~A Ar~G
1845 CG~GCC rrr-~rTrArrrrnAAArrrrrAA A~a~rT
1856 Ca~UCA rrr~rTr~r~rrrr7~r.rrrrAA Ar~ GUG
1861 GaCClACA rrTr~rmAr~rrrraaArrrrraA AU~U
1865 AI~G~C rrr~rTr-ar~r-rrr~r`r-rrn~A AcaGal7c 1868 GD~T rr~r~rTr.arrrrr.AA~rr~`~ ACtlaCAG
1877 CUt~;GCU rTTr~TrAr~r~rrrrrAA AG~
1901 Ar~U rrTnar~rr7rrnAAAnrrrrAA AGrJC~
1912 Ar,~C r~TTn~rTnArr,rrr~rrrrnaA Ar~
1922 .~ cT~u~ rTTn~rTnAnr~aAar;rrr~ ACal7CCA
1923 U~ rrTr~rTrArrrrnaAarr,rrra~ aUCC
1928 CA~GC~ rrTnatTrar~r~rrr~rrrrr.a~ AC~AA
1930 AI~GGC rrTr.aTTr.ArrrrnaaArr7rrrA~ Ar~
1964 GUGr~GGC rnraT~rar~rrrAA~rrrrr,AA AIJGUCUC
1983 CCAGr~JG rrr~rr~Ar~r~rrr~Aa~n-~rrr7~ AIJGUCC~
SUBSTITUTE SHEET (~ULE 26) W0 95/23225 - 218 3 3 ~ 2 r~ 156 1996 G~C~Ç rTTr~r~~ r~^rr~ Ar~ rrr- ~ AU13tJCCC
2005 ~^ rnr~.TTr' ^r~rr~3 ~r~-r-~A A~CA
2013 U~CCC3a rnr~ATTr?~ rrr3AA~^-rrr~ c 2015 C~CCC rnr31Tr.Arr^rr3AA~-rrrr3A A~Gca 2020 Ct~CaGCa rnr3nr:3r^rrr.333r,r~3~ ACCC.3A~J
2a~9 CUCC~J rr~ rrr~ t r~3 AGUCUGU
2040 UCWCUG rTJr3TT~"^^rrr3~3 -rrr~
2057 ~;UC1~3 ~-n,r.rr~-^.crr-~ar.. ^~ a ~GGGcca 2061 ~C~.,C rur'~Ar^rr^~r^~rr3A A~
2071 UU~UGC rlJr~rTr~ r^r~33~rr-r-rr~AA ~aCA~
ao76 GIJG~ r~ r-r ~A~a~r----rr-3~ At~'lJAC
2097 CG8'Ca~G rTTr~3r-^-- r r~'3ar~r~rrr~AA ~C., 2098 CCGU~ rr;r-3nr:Arrr-^rAA~rr~rrr~3 A~,~;G
2115 ~CCC c,nr~rr.^aaAr rrrAA AGCUGGC
2128 G'~A t~Tr~ ^rrrA~ -~rr.a A ,~
2130 GW~JCAG rnr~ Ar^rrr~3ar^rrr,Aa AGACAG;
2145 ll~tJC/UJC rrrr.37~ArrTrrr.A3Arrrrr.AA AGGgJw 2152 aAA~JACA rn,r~r~3r~ -,rrr~ 7C~IJCA
2156 ~r,aAUAAA rnr.3TTr~^,rrnAA3r^rrnaA ACa~C
2158 ~.a~ rnr~TTr.3rr,rrr~r~rrr3A Ar~a.
2159 ~AD~aAU rrTrAnr.Arr,~^rn~r,r,rrrn3A AAUACArJ
2160 AAAD~A rnr.3TTr.ArrrrrAaArr^rrAA AaA~aca 2162 ~a~G rnr~T-~cr~rrAAArrrrr-~A Ar~A
2163 A,A~ r~TTr3TTrar-rrrr~ r~r~r~mAa 3AUMAU
2166 AAUAACA rnrpTTr~r~rrr~3~r~33 Ar~A~TA
2167 ~ aA~AC rr,r.3T~r~ ^^,.^rr.3 ~r~r~rrr.3 A A,AIJGaAU
2170 GUA,AaA~ rnn~TTr.Arrrrr~rr~rrr,AA AC3AArJG
2171 GG~MA rrJ~ Anr'^r.Crr'3~r.r~AA A,ACMAU
21?3 Cr.~G~A rr,Jr~3r^^rn337r^rrr~ AUAACAA
2174 GC~A r7r-3TT'-'~r-rrr`'`'~' A~AACA
2175 AG~GU rr,Tr~rr.Ar~rrrr~r^rrrAA AA~AAC
21?6 ~GG rr~^r~:3A~r-rrr.~A MAADAA
2183 CAAI~AA rn,r~ rrrr~AArr^rr~ AGClJGG8T
2185 rFcaAUA rT7r~rTr~^rrrr~3~r~rrrnAA AUACCUG
2186 Acuc~aA~? rnr~ATTt~r^~Grrr~r~r~r~rr733 M~GCU
2187 c3~cucaA rrJr~3TTrAr5~rrrr~A3r~rrrrAA AAAUAGC
2189 GAr~7C rnr.ATTr`' r,rmA33r-rrrr.AA AUA3,AUA
2196 CAUMAA rTTraTTrAr~r,~AA3r.~.~--rrAA ACACUCA
2198 UACAILAA rTTr.AT,r3rrrma3Ar-rm33 AGACACU
219g crJAcAUA rrTr.ATTrArr,rrr.aAArrrm3A AAGACAC
2200 CCUACAlJ crTr~rTr~cr,cm~ 3~r~r,~ ~ AAAGACA
2201 r,~ ~nr~3nr~A~rrr~7Ar~r-rm3A A,A~AGAC
2205 UI~CC rTTr3rTr~rr~rr~AAr~-,rrr~ ~ ACA~AA
2210 GUucAm? rTTnATTn3r~rr~rnAAAr~rrrrAA AGCCUAC
2æo ~AGACC rr,Tr~TrAr-~rrr~ ' ~r ,rr~A3 AUGucca.
2224 Gr~cc3~ rT7n3rTr-Arrrrr~r-,rrr~ ACCUAUG
2226 G~GGCCA rTJr~ArTrAr~rrrr~'~'r--,r~rr~ AGACC~A
2233 G~ccn-u rrJnAnr~Ar^^rr~A3~r~rrrr~AA AGrcc,3G
2242 r~G~GG rnr.3TTr~ ^~rrrr~ A Ar.~^rrrAA AGCUCCG
SUBSTITUTE SHEET (RULE 26~

W0 95l23~5 1~ J. . ? 156 ~ 187 218~9~
2248 7JG~IJÇ r~rr~7rArrrrrT~r~r~rrrT7 AC~GGA
'254 7~AAUGL7 r~T7rA~rr~rAA~r^rrr.~a ACArJÇÇA
2259 ÇA~L7Ç Gr~ 7r~ rrr~T~r~ rrr.A~ AI~JÇAC
2260 UGACCU~7 rr7r.Anrarrr~rr~r~rrrr.AA AA~,A
2266 Ar-c~GGL7 rrr~7Tr~ rrrr~ r~r~rrrAA ACCU~ÇA
2274 ACAA~L7G rr7r~7r.~rrrrr~rrrrrAA Accr~
2279 CC~C rl~ Arrrr~~ rrrrr~A AClJGt7AC
2282 CAACC~7Ç rnr.Anr.Arrrrr.A~arrrrr~" ArW~, 2288 ~WAC rrlr~T7rAr~rrrr~rrrrr.AA ACCUGt~A
2291 ~GL7G r-nr~77r~r~rrrr~AAAr-rrrrAA ACAACC~7 2321 rCcal~UU rr7r~T7r~r~rrrr9A~r~r~rrr~AA AIJCU~L7 2338 CAAtJGA~; rnr~lr3rrrrrA~r~.. ^rr.AA Ar~CCCA
2339 CC~7GA rnr~T7r~rr^rr~ t rrrr~A AA~7CCC
2341 rJGCcAAT7 r77r~-7r.~rrrr~r~ ~rrrrrAA AçAAr~L7c 2344 rJ~ çCC rnr~T7rArrrrr-'~rrrrrAA A~MA
2358 cr~ÇGGGA rrlr~T7rArrrrrAAArrrrr.AA A(;ÇCAÇG
2359 77CXÇÇÇ r77rAnr~- rrr.AAArr~r.A~ AAGÇCAÇ
2360 7~7ÇÇÇ r~77r~rr:Arrrrr7~ r~rrrr-AA AaA~GCA
2376 AUaG~AA rr7r.Anr~~rrrr,AAArrrrrAA AU~L7C
2377 Ç~AA rr7r~T7r.Arrrrr~ rrrr~A AAlJCACL7 2378 CÇA~A rnrAT7r.Arrrrr-~AArrrrr.~A AAAI.7CAC
2379 ccr-TA~G rr7rAnrArr~ rrrrr~A A1~AAI7CA
2380 r~CCGaL7A rTTrAr7r-Arrrrr~ "rrrr-AA AAAAAL7C
2382 r,L7çCcGA rTTrT~7r.ArrrrrAAArrrrr.AA AÇ~AA
2384 ~L7GL7ÇCC rnr.AT~_-rrrAA~rr~AA AI~AA
2399 Ç~7CCAUA rr7r:u7r.Arrrrr.AAArrrrr.AA AÇUGCU~7 2401 CAÇUCC~. r77rA77rarrrrr~l~7rrrrr.~ AU~L7ÇC
2411 r~ACCA77 rr7r~Ar~:AAA-rrrrT~ ACCA~C
2417 ACCUGL7Ç rr7r.Ar7r~-rrrr.AAA~rrrr.AA ACCAWA
2418 AACCr7GL7 rr7r.A77~.Arr~rrrAAArrrrr.AA AACCA~.7 2425 AT~7Ç rr7r~r7r~Ar~rrrr~AAArrrrrA~ ACCL7GL7G
2426 AADCL7CU rnr.~nr.A~rrrr7AAA~rrrrAA AACCL7GL7 2433 A~ÇÇGL7 rr7r~T7rArrrrr7AAA~rrrr.AA A~7C~CUG
2434 CAC~7ÇÇÇ rr7r~AT7r~rrrrr7~ rrrrr~AA AA~7CL7CU
2448 r~aGGaAt7 rr7rAr r.Arrrrrr 7~ rrrr-AA AÇÇCCUC
2449 r~aGGAA rrr.~77r.~rrrrr.AA~rrrrr~2` AAGÇCCt7 2451 AGÇGAGG r77r~T7r~rrrrr7}~AAr~rrrr~AA AUl~AGG.^
2452 AA~ÇGAG rrrAnrArrrrrAA~rrrrrAA AAILAAGG
2~55 ~rGG~AÇÇ rr7rTT7r.Arrrrr.3AAr~rr~r.AA ~}WA
2459 7JGGGGÇÇ rnr.Ar7r.ArrrrrAAArrrrr.AA AÇÇGAGG
2460 ~OGGGGG rnr~ -rrrrAAArr,rrrAA AAG;GGAG
2479 GC~ACA rrrATlr~ rrrr ~rrrrr.AA AGGUGUC
2480 GGCL~AC rnr~T~ rrrrAAArrrrr.AA AAGG~GU
2483 G~ rrr~ATrr~-r7rrr~AAAr~rrrr~A3 ACAAAGG
2484 AGGUGGC rrlr ~r~:Arrrrr.AAArrrrrAA AACaAAG
2492 GG~GGG rrl~nr~-rrrr.~AArrrrrAA ArJGUGGC
2504 A~AAUG rr7rAnrArrrrr'AArrrrr.AA A~ 7GGG
2508 I~GCaGA rrr.3nr.~rrrrrAAArrrrr.AA AUGU~7G
2509 CLTGGCAG r7r3rrArrrrr3'ArrrrrAA AAIJGUAU
SUBSTITUTE SHEET ~RULE 26) WO 9S/23225 2 1 8 3 ~ ~ 2 J ~ 5'I 156 .: 188 2510 ACUJCGrA rrsr~-srAr.r^rr~-~r"~~rrAA Aa~G~
2520 CAUUGUG rr7~ ATlr~rr~r~r~rrr~ AC~G
2521 sJCAUUGts rrsr.~sTrArrrrr.T~r^~AA AA~G
2533 GACCG-U rrsr~-Tr~rr^rr~AAAr~_^rr.AA AGD~scA
2540 ~r~GACAU rrr~TTr,~Ar~r~rrr```r-r-rr-AA ~CGCUG
2545 auG~cca rsTr~Tsr.~r^rrr~AAAr-~rr., ACA~aC
2568 ua~;t;Ca rrsr`~r~r~r^rr`~`r-,rrr~ s 2579 CAAGGCA ru,r~s~rTr~rrrr~ r--~r~rr~aA Ar~C~
2585 A~r~;GAC ~s~Tr~rr~.aA~r ^rrAa ACGCaUA
2588 AC~AC-AG rssr~ssrAr-^rraAArr~aA ~CaACGC
2591 A~r.;~a rrJrArlrTr-^^rr~r---^^r~aa AG;3~AA
2593 ACAG~C rr~r.aT,rArrrrr~-~r-^rraA A~C
2596 CAAACAG rr,r~-Tr~rrrrr~r,-~rrr,AA ACAAGAG
2601 AAA~ ssr.Assr.Ar~rrr~`r^r~ CA~C
2602 GAAA~SGC rl~rAnr~^~rrrr~rrrrrA~ Aa~
2607 CC~'GA rr~ sr~^r~AAArrrrra~ AIJGC.1~AA
2608 CCCAGUG r,r~r AS~r.~rr,rrr~r^~ A~ AAUGCaA
2609 UCCCAr~s rrlr--Tr-~rrrrr~r~rcrr-AA MA~GCA
2620 AUAGUGC r,r,rAssr~^^rrr~ rr~rrî~AA AGC~sccc 2626 ~r~cuGcAA rrsr~AsTr~Ar-r~rrr~AAarr^-^rr~AA Ar~CAA
2628 r~;CUGC rrTr~AsTr~Arr~rrr~ rr-^maA A~GC
2635 GaAACUG rrp asTr.Ar~rrrr.AAAr^,rmAA AGrOGCA
2640 UGC;~ rrsr7Assr~rr~rrr~`r--~rrr-a~ ACUGGAG
2641 r~r,~G rnrarsrTr-^TrrrAAAr~r~rr`Tl AAa~GA
2642 AC~G ~rnr~AsTr~^~r~:```rr-^ma~, AAA~G
2653 G.,ACCC~s rsT~rTar~r~r~r~crrT~rrrrr~aA A~UG
2659 r~UGCAG rrTr~assr~Ar~rrrr~AAAr~rrrr~AA ACCCUI;A
268g CCrlXAA rTTr,Ar~r~rr~rrr~r-~rrr~ ACC~JIJGG
2691 ~r7~sCC~sCC rssr~T~^~r,rrr.A~Arrrrr`` A~s 2100 UGGGaGG rnr~ASSr~-r-rr~rrrrr.~A A~Ct~C
2704 AA~G r~rr~Tsr~Arrrrr~-~r~ rrr~ A~s 2711 CCDUCCA rrSrASSrArrrrr`~rrrrnAA AGcl~sGGG
2712 CCCru~sCC rsTr.Aslr~Arrrrr~rrrrr.A~ AAGCDGG
2721 CGCG&aOs rrSrASSr~^~rrrr~r^~rrr.AA ACCCUUC
2724 ACACG,CG rUr-Assr~Arr~rrr-~aArrrrr-AA AUC-ACCC
2744 CruACACA rlJr~TSr-ArrrrrT~rr~ ACACACA
2750 GCUUGUC rnnAs r~rr~rrr~`~rrrrr~` ACaCAc~.
2759 AGAGCGA rnr~srArrrrr~rr~:AA AGC~,T
2761 ACAr,AGC rslt:As~r.Arrrrn~ rrrrr.AA AGAGC~s 2765 GGIJCACA rnr~-srArrrrr~rrrrr~ AGCGAGA
2769 CCUGGG~ rnr~Tsr7ArrrrrT~r~ ACAGaGC
2797 GAACCAU r~sr~Arr~:AAAnGrrrAA ASJt,SGCAC
2803 1~ rsTr-~sr.ArrrcnT~'T~r~rrrr`' ACCAUGA
2804 c~r~s rnr~AssrArrrrr~rr,rrr,A~ AA~G
2813 AGGUCAA rU(~sTr-Ar-rrrr.AAarrrrr.AA ACUGCAG
2815 AAA~--,UC rsJnArsr-Ar~c~ r~rrrr~ GC
2821 AGCCCAA rrsr~-sr~r~rrrr~rr,rrrAA AG~CAA
2822 GAGCCCA rnrArsr~^~rrrr~rr,rrr~Aa AAa~CA
2823 UGAGCCC rs~srAsTnar~rrr~Tr-rrr7Ar~ AAAGGUC
SUESTITUTE SHEET (RULE 26) ~ W0 95123225 l89 ~ 2 1 8 ~ ~ ~ 2 2829 AI~:aC~ rnr.ATTr.arrrrr.~a~r.rrra~ CCC
2837 G~;GGaG rnr~ Arrrrr~ rr~ a AIJC~
2840 r~aGGuG~ rTTr"TTr.arrrrr~a~r-^rr~ AGG~.
2847 GGa~ rnraTTr.Arrrrr~rrrrr~aa AGa~GG
2853 r~aG rTT;aTTr~arrrrr-aa~rr7rrr~Aa 2860 ~JCC~C r~rT~ arrrrr~ rrrrrAA Ac:Jc.a~G
2872 r,~CC rnr3T~rr~rrr a~ar "rr.A~ auG~c 2877 r~u rnrATTr.arr~a~arr~ a 3~;CC~
2899 AAaAr~lCP. rT~ TTrArr,r~r~ rrrr-~A ~CC
2900 AAaA~ rnr~Tr.Arrr~Aaar~r-rraA ~GC-C
2904 AAaaAAA rTr~TTr~r-rrrr~a~r--~r~-~az~ ~.U
2905 AAA~A rTTr7ATTrArr,~ar~rrr.~
2906 ~AAaAAA rTTrAT~arrrrraa~rrr~ aA ~A.AU~aA
2907 AAaA~AA rTrr~lTTr~arrrrr~r~ a AA~A~JC~
2908 AAAAAAA rrJG,arTr~arr,~r~aAarr~ ~AA~'C
2909 AAA~ rrTrarTrar.r,Crr-a~Arr~A AAAAAAU
2910 AAAaAAA rrT~ ~rTrarrrrr~ rrr^rr~A ~.AAAA~
2911 AAAA~AA rnr~Trarrrrr~~arrrrr~a AAaA~AA
2912 GaAAAA~ Tr.p~Trr~r^,m~ rrrrr~ A l~.AAaAaA
2913 ~a~A rnr~Tr.Arr^rr~Aa A~aAAAA
2914 CD~AAAA rn~r~T~ ar~crr~ rr~rrr~Aa AAA.a~A~
2915 UCOGaAA rTTr~a-Tlr ar~rrrr~a~r-rr~r~ A.a.A.
2916 C~ ;aA rnraTlr Ar~rrrr~ rrrrr~
2917 rJclJccR~a rnrArTr.Arrrrr.Aaarr.rrr~A ~.A~A
2918 Goca~ rT~r~arTrArrrrr~7~arrr~rr~A ~.A.a.
2919 CG~U rnr.~rTr.arrrrr.AaAr^rrr~a ~AaAA~
2931 GUUG~A r~7r~TT~ arrrrr~a7~r,r~rrr~a 2933 AIX;U~C rnraTT~Arrrrr.aAarrrrr.~A A~aCCCC
2941 r~Jc~GGc rnr7~TTr~arrrrraA~rrrrr~a ~ CC
2951 A~ G rTTrArTrarrrrr~-~rrrrr~ Ai~C~G
2952 c~aA~G r~r~rTr~Arr7rrr7~7larr~r~aa AA~JC~
2955 U~A~Ca rnr~rTr.arr,rrr~a~rrrr~a ~AAGU
2956 C~A~C r~r.aTTrarrrrra~arr.. ^rr,a~
2961 A~ rTTr~Tr~rrrrr.A~arr.rrr.A~ A~.c.aa~
2962 UAt~t~AC rnr~aTT~ ar~rrr~ar^~rrr~Aa ~ aCAA
2965 CU~UO rnra~Tr~ar~r~crr~7~Arr~rr~a~ A~.a~a 2966 GC~J rur~Tr.Arrrrr.~a~rrrrr.~a A~AC
2969 AAAGC~ rnr.aTTr.Arr,~rr.~A~rrrrraa A.r.~T
2975 G~ rnr.aTTr.~rrrrr~ rrrrr.AA A~C~
2976 AGWG~G rTTr~ arrrrra~rrrrraA AA~C~.7 2977 CA~G~ rnr~aT~r~rrrraa~r-r~^rr~a AAa~CW
2979 GGC~ rTTr~7Tr.ar.rrrr.aaAr^rrraa A~aa~aGc ~UBSTITUTE SHEET ~ULE 26) wo ~5123~5 2 1 8 ~ 9 9 2 P_ l/~,~l 11s6 - ': 190 Tahle 5 Mouse ICAM HH Ribo2~ Sequanca nt. Position Ribozyme Sequence 11 C~ A~,m~A~A~ Acca~GG
;!3 A~ CT~TT~Ar~ r~rr~ A ACC~
~ ~--f~ r~ AG~ACC~.
;1 ~ ~.Tlr,A~.~r~.AAA~ r-3A aGU~G
34 Ct;ACCC17 t~'T~Arrrrr''~7r--~r~:' AUCa~A
A~JAC t~ ATTr~rrfrr AAAf~r^rr~A A~UGrJGC
48 CCAS~J c~A~ Arr~AAA~ rmAA A~J
54 Ca~JCAC rrlr:Arl2'~ r~AAA~-~.A~ AGGCCC~
~8 GGaGc~ ' ~ Il.AI.~,I I-~AAt r~AA AG
64 C~G ~nr~TTrArr - rf:AAAr~ A~ aCGGG~
96 GGGCCaG ~ArTr~ r~ ~r'f~A~ ~ CaQAG
102 CCAGCaG f~ ~r,rcrAA ACUCGCa 108 G5GCCAG rnr,ATTnAr~r~rrnAAArrrrrA~ AGCAGAG
115 AGGA~GCA rnnATTr.Pr~r,rrnAAAr7rrrnAA Ar-AACCA
119 ~7CCCGGGr rr7r-1TTr~GrrrAAAr-rrrr.AA ACADt7CC
120 GG5CCAG rrTnATTnArrrrr7~ r-rcrr~7~ Ar,CAGAG
146 GGAAt;CG rrTrATTr-Arrfrr~ rr-rrr- ~ AC~-,ACt7G
152 AG17GGCtJ r~Tr~rTrTcrrrnAA7~rrrmA~ ACACacA
158 r~t;~ rT~r~Tr~,frr' ~rrrrr.~A ~ACAGGA
16~ GCA~AAC C.~Ann;~rrfrr`''rrrrr'~ AC.~7Ct7G
168 G5r~GcAG rnr''Tr`r~r-rrr"''rC~rr`" AA~GCDt7 185 CUGC1~CG rrTr~rTr~~r--rr~7~--rrr`r~T~ ACCCACC
209 GCCAGAG rnnATTnArrrrr`'~r'fr'AA AAGt7G5C
227 GCAAAAC rnr~r.A~rr~r~rr~ ACt'U(~G
230 GGAGC~A rnr~TTr~-rrrr.~AArrrrr A~ ACAACt~t7 237 AGCt7Ct7C rnr~TnArr~rr~7C~r~rnAA AAGCaCA
248 1~t7t7AGGA rnn~TTrArrrrr.AAArrfr,.AA AUG~7t7 253 ~CtJt7CC.7 rTTr~TnArnrr~ r,-rrrAA AGGCAGG
263 CAGt7AGA rTTr~ rTr7l--r~rrrAA~rrrrnAA AAACCC~7 267 UAGGCAG rnnATTr~rrfrr~AATr~r~rrr~ AGCCCCT7 293 CAGCXA rTlr`'Tr`-`'~rrr'`~7'Cr`rrnAA ACAGCt~t7 319 G5CtJCAG rnnATTr.Arrrrr~T~r~r7~rrn~ At7Ct7CCT7 335 Gt7t7Ct7CA rnnATTr`~rr~rrr~AAAr-r~CCr' '~ AGCACAG
337 C~GUGt7G rT nATTrAr,r~rrnAAArrrrnAA Al}U~AC
338 UCaGC~7C rTTnATTnArr~rrr.AIArr.rrn~ AACAr,Ct7 35g ~5CGGAC rTTr~ATTrArrrrr~AArrrrr.AA AC.~CAC
367 CGGGtJt7G rTTr.ATTr.Ar.~--rrr.AAAr,r,rrr,~,A ~CU~7 374 GGGCaGG rTJnATTrArrrrrAAAr,rrrr7 ~ AG5Ct~UC
375 GGC,GCAG Cl7nATTr~rrrrr~AAArr~rrr~AA AA~GC~
378 ACACGGt7 rTTr~rTr~ r.,rrnAAAr--rrr.AA AUGGtLAG
386 A~.P.CGAA rT~r.~T~r.Arrrrr~AAArr,rrr.AA ACACGGU
394 AG~UC,GA. rT,nATTnArrrrr.~AArrrrr~,A AGUCCGG
420 Cr~G5GG r~Tn'T~-~rrrrn~AArfirrr.~ A~GUG
425 CtX;CUGG rT~ r-,r-~rrTrrrA~Arr,rrr.~ AGGCGUG
SUBSTITUTE SHEET (RULE 26) W095123225 l91 P I/IL3'J~ 156 427 ra~UsC~,T rrrrArTr~ ~r.rr~r'~ ~ ,rrrA A AG~GC~.TG
450 GC~;GGU rr~r.arrr~-r,rrr.Azl~.r,rrr~A A~GrTcrrfT
451 r~AA~ rrTrArrr~r~r,~~rr~lr-,rrr.AZ~ ~C
456 a~G~L7 rnr~rTr ~r rrrAAAr~rr.AZL ~A~'L
495 ~LracG~u rrTrATTr~~ rrr.AAAr~rrrr~T. A3~
510 CCCC~ r3rTr.Arr~-rr.AA~r,rr~ A AGC~CA
564 5r,~T~,5A rrTr~rr~r~~rZ~AArr~mAA rLCc~5 592 C_CA~ rr r~ rrr.AAArrrrrAA A~C
607 Cal~L5A rnr~rTr.ar~rrr.AAAr,r~rrrAA
608 sra~A,G rrrr.ArTr. Lr-^rrr~ ~ z r,r,rrr~ ,C
609 G;;CA~GA rrrr~Arrr~Arr~rr~ 2lAr~r7rrr~az~ AAAD~
6}1 r~CGGC~U rrTr~-rr~-rrrr.aAAr-r-rrr.aA A~
656 CA~ rTTr.AT-,rArr~rrr~AAAr~rrrr~A ~U
657 ~C rnraTTrA~rrrrZiAAr~rrrr~AA AA~
668 55~GGC'' rTTr~Tr.Arr,rrr.A AArr,rrr.~LA AGGC~
677 A~5 rnr~TTrArrrrraAArrrrrAA ~-C
684 i~;QLCC5 rTTr.~TTrArr~r~rr.AZ~Arr^rr,AA AGC~r~a3 692 M~alJCs rnrATTr.ar-rrr.AA~rr,rrr~ 7~ .aA~CCs 693 5r~ rnrZ~T~Ar.~:rrrAz~Ar,r,~rrAA A~GU~
696 sAr~ s rn zATTr~^rrrrT T T~ rrrr~ ~LCAGG
709 I',GAG~,~G rnrATTrzLrr,rrrAaT.-~r~rr~7~ A5CCGCC
720 AGCI~a~ rrTr.z~TTr~Lr-rrrT. ~Ar~rrrr-, Zl AG~A
123 C5GA~J rr;rAT~Ar-rrr.AAAr~rrrr.AA ~AA~
735 IJCUCCa~; r;r3TTr.Arrrr~.AAArrrrrAA AU~JGG~
738 CCAIJC~C rTTrZLTTrArrrrrAz~ArrrrrAA ~GGCCCA
765 55A~;CG rrJr~ATTrAr---~rrr~AAz~r~rrrr~ AC5~JG
769 5GCAGGA rnr~Tr~^rrrrAAAr~rrrr.Z A ACAGGCC
770 U~TCCA~GG r~ATTr~--rrrrAAArr,rrrAT~ AGcaA-a-A
785 GGCaGGA rnrATTr'` -rrrr.AAArr7rrr~ ~ ~LCAGGCC
786 AGGCAGG rnrATTr.Arrrrr.a~ ~r,rrrr,AA AZ~7LGGC
792 C~JUCCGA rnr.ATTrArrrrr.AAArrrrr~ ACCUCCA
794 AGUCUCC rnr~z~TTr~rrrrr~AAAr~rrrAA AGCCC,Ar.
807 cc-a~GuA r~ATTr.ArrrrrAAArrrrr~AA ~L~JCCG~LG
833 ~C rTTr~ATTr~Arr~rrr`~r~r~rrr`~ AGCIJ~JG
846 C,Ai~CGGU rnrATTrArrrrr,AAArrrrrAA JLCC,AGGG
851 G~;G~A rnrATTrArr~rrrA7~r~rrrr~AA AGGUC~
863 CCa~aGG rTTr.ATTrArr,rrr~A~rr~rrr.AZ~ AG~U
866 GGGCAGG rn~rATTr.Arr,~.AAArr,rrr,AA AGGC~7C
867 UCI ~CCGG rnrATTrArrrrrAAArrrrrAA AacG-aAu 869 C~-AU rnr.ATTr LrrrrrAAArrrrr.AA AGG~AGA
881 AC'.~C.~ rnrATTr.~rr,rrrA~r-,rrrAA AZLGCCAU
885 T.~CCUC rnr~Z~TTr~Arr~rrrAAArrrrrZ A A~FLGG
933 ccAL--aA~J rnr.ATTr.ar~r~rrr,AA~rrrrr~ AUUA~AG
936 Gc~LccaG rrTr ATTr.Arr.~~rr.AAArrr~rrAA ~UGAIJUA
978 AGUUGUA rnrATTr.Arrrrr~ rrrrrA~ ACD~UA
980 AaA~UG rnr ATTr~r~rrrr~AAArr7rcr~AA AGAC~J
986 ~5;CUG~A rnr.ATTrAr~r rrAAArrrrrAA AGUUGUA
98~ r~,G~ rTTrATTr~rr7rrr~z ~ Arr.rrrAa A~Gr.~r,~T
588 r~G~G rr,~r~ATTr~r~,~~rrAa~rrrr~A ,~G~ r-SUBSTITUTE SHEET (RU~E 263 wo 95l2322s ~ ~ 218 3 9 9 2 r~ -ls6 1005 ~UCCAG rrTr~ArT~r~r,rrr.Daarrrrr~a A~GGU
1006 WCCCCA rnrAnr.ArrrrrAAAr~rrrr~T~ AC~CA
1023 CWCCGA rrTr.arrr.ArrrrrT T` 7~r`rr^rr` D ArcUCCA
1025 cccwcc rn,rArTr~rrrrr.aADrr,rrr~ a Ar=,ACcuc 1066 r~ww rrrr~ArTnAr-rrrnAAArr~rrraa ~G
os2 GCCC~GA rrTr ArTr Arrrrr.AAAr ,rr^~ D A~CCAr,u 093 ~GGCUG rnrarTr.Arrrrr~ADar~,rrr.~A Ar~ ccA
1125 UCAAGa.~ rrTr7Ar r.Drr~:AAArrrrr~ a AGWGGG
1163 GC,A-~-AAG rnr.~rrr.Arrrrr.aAar~rrrAA AGCUa~, 1164 AGCAAAA rrTnATTr~ArrrrrD7~r~r~rrr~A AAGCWC
1166 AG~C~ rrrr.ArTr~arrrrr~ r~rrr~ Ar~C~
~72 G~uu~u rnrArTr-Arr~rnAAArrrrra~ AACAGGA
200 U~GGAG r~rTrarTr~rrrrr,ADArrrrrAA AGCAGAG
1201 C~GWCA rrr~ArTnArrrr~rrrmaA AAGCAGC
L203 ACUGGUG rrTr~arTrArrrrr~7~rrrrr~7~ AAAAA~
227 GCACACG rrr.ATTr.ArrrmAaArrr~rr.~a A~GUACC
1228 AGCA,D~AA rnr7ATT~ r,rrr~ --rrr~aA A,A~;CWC
1233 C~CUCCG rnr~ArT~^r~rr.~Darrrrr.AA AAA~GAA.
238 AGGACCA rnranr~rrrrr~ rr,rrrAA ACaGCAC
264 CWGCAC rnr~rrn~r~rrrr.aAarrrrraA ACCCUUC
267 WCCCCA rnrArTr~rrrrr~ rr~rrDA Ac~;~a 294 GGC~CAG rrTr~TTr~-rrrr~7~Dr~Grrr~A~ AUCUCCU
295 CUGCUGA rnr.ATTrarrrrr~rr~rrr.AA ACCCCUC
306 C~UWCA r,nr.AnrAr~rrrrAAAr~rrrr~a AGUC~GC
1321 UCC~JCCU r~nr~rrrrr~ r~-rrrAa AGCC~UC
334 UU~AGGA rrT~TTr.Arrrrr~7~r^rrr.AA A:UGG~
344 CAC~CUC rnr~rrArrrr~naAArrrr~ ~U
1351 ~AACWA r~rTr~rrr.ArrrrrDAArrrrr.DA ACauUcA
1353 c~ccwc rrr~TTrDrrrrr~Dr~rrmaD ACCCDC~r 366 AGWGUA rrr~rrrArrrr~ rrraA AC~UA
367 AGGUGGG rrTrATTr.arrrrr.AAarrrrr.aA AGGK;CU
368 AGAGUGG r~nr~TTr~^rrrr.DAArrrrr.AA Ac~AC
1380 CCACCCC rnr.ArTnArrrmAAArrrrnaA A~GGCA
13 88 AGCCD CU rTr~r~arrrrnD AArrrrr-a a AG~CUCC
1398 G~r rn~rrnDrrrrr.AAArrrrr`7~ AcasccA
1~02 A~CUC r~r~rrr.~rrrm~AArrrrnAA A~GCACA
1408 ccucccc rTT~TTr~arr~rrr~D~rr~rrr~Aa A~CUmC
1410 cccwcc rnr~TTr~arr~rrnaAAr~rrrnaA AGACC~C
421 ACAAAAG rTTr~rnarrrrr7~7~Dr~-rrrr~AA AGGUGCG
425 CUr3ACC rTTr~rrr.Arrrrr~DT~^rrmaA AGGCAGU
429 Ca~;GGGC rnr~TTr.Arrrrr.AAArrrrnDa AUAGAGA
444 uccuccu rnr~ArTr~r~r~rrr~AAAr~rrrr~aa AGCCWC
455 UCCUGGU rTTnArTr~Anr~rmaAAr7rrrnAA ACauUcc 1482 GGGAGCA rTTr.ATTr.arrrrnAAArrrrr.aD AACaACU
1484 CaUGAGG rrr~TTnArrrrr-AaAr-r~rrnDa AGAACaG
1~93 GWCUCA rnr~rrnArrrrr.AAArrrrnAD AGCACAG
1500 GGACCPU rTTr~TTnDrrrmaAArrrrr.Aa AUUDG~U
1503 G~IIGAU rTTnArTrDrrrrn~AArrrrrAA AUAGUCC
1506 CG~WAU rTr~TT-Z~rrrrr~7~7lrrrm~D AACAUAA
SUBSTITUTE SHEET (RULE 26) ~ WO 95/~3225 193 2 1 8 3 ~ 9 2 r ~ 6 1509 1~C~CGGL7 rnr.Arr.Ar~rrrr~AArr~rr:AA Ar~Gr7-A-G
1518 CG~GG rnr~r7r~rrrrr~rrrrr-~A ACCAUGA
1530 rca~a,aU rr7rArrAr-rrrAA~rrrrr.~A ~AG
1533 CGCCCAC rnr.~rr.~rrrm~rrrrr.~A ~X;aCCA
1551 ~-~7 rnrArr~Arrrm~A~r~rrr~A~ G
1559 AG~GGG rnr~r~rr~ r ^rr~A AGG~rGcr.7 1563 G~,~ r~TrArr~r ~ r~AAr rr~ AC~aAG
1565 =~ r~ uA rrr-Anr~rr-rrr.~Arr~:~A Ai~ACAUA
1567 ~^CC~7 rnr~rrAr~ rm~Arrrrr.~ ~aAAr 1584 A~L7CC~7 rr7rArr~r~ rm~rrrrr.~A A~CU~7C
1592 7~,'G rr7r~77CArrrrrA~rr~ .~A A7~L7cr ~-7 1599 r-r-r3u~-7G rnr~rr~Ar~^rrrA~Arrrrr~A AACU~7 1651 Gcr.~G r77cArr~r~rrc~Ar~A AGGUGGG
1661 CAA~GGA r77r.Arr~rrrrr.~Arrrrr.~ ~;GU~7C
1663 7~ AAG rnrArC~rrrrr.A~Arrrrr.~ AaA~7 1678 CCA~;GCU rnr~rrAr~rrrA~Arrrrr~ AGG~cr7 1680 CCa~GG rnr~rr~rrrrr.~AArr~r~ AGUGGC~7 1681 GCCh~G rn,r~T7rArrrrC~Arr~.. ~ AA~7GGC
168~ AcAGcca r77r~ rrr~ -~r~A AGS~AGU
1690 ~CGA rT~r~rr~r^~ r^^rr~A AGt}CCGG
1691 Aa~aUCG rTTr.~rr.~rrrrr~ ` ~r^~rrr.A~ A~CCG
1696 CC~C rT~rATTrArrrrf~ rr~rrr.A~ AUGGG~
1698 CUCCaGG rTTr.Arr~r,^rr.~Arrrrr~ 7~ A~33Cm 1737 GCUGWA rnr.ATTr~r~rrr.~A~rr~ AGG,UCUC
1~50 r~aGGT3G rrJ~:ATTr.~rrrrr~ r-rr^rr.A~ AGCCGCC
1756 GGr~CaGG rT7r~rTr~rr^m~Ar^rrr~ AGGC~T3C
1787 rGGCGAc rTTr~rr~rrrrr~rr~ ~ ArJWCT3C
1790 AuuaGaG rnrArr.~rr~rrr.~rrrrr.AA ACAATJGC
1793 T l CaGcc rTrr~rr~rr~rrr~ rr,rmAA AGGAr~CA
1797 r~33W rTTr.ArrArr.rrr~'r~rrrr.~ AcrJGGT3G
1802 ucr3ccAG rTlr:ArrArrrm~Ar-rrrr~ ' AT3CT3GW
1812 GGCCr3GA rnr~TTrArr~(~ r-r-rrr~ AUCCAGU
1813 r,JGaGGW rTTr.~rTr.~rr~rm~Ar-^,rrr-A~ AA}TGcr3G
1825 GCAGAGG rTTrATTrArrrrr.~Arr,rrrA~ AGCG~GG
1837 GGAr,CT3A rnr.~rTr-~rGrrrA~Arr-rrr`' AGGCAT3G
1845 Grr3rGCC rnr~rr~rrrrr~r^rrr`~ AGGCT3CG
1856 A~GAUCG rTTr,~rr.~rrrrr~rrrm~ A,AWCCG
1861 UACrJGGA rTTr.~rTr.~rrrm~rrrrrA~ AUCAUW
1865 cr3GaGGC rTTrArr.Arrrrr.~rr,rrr.~ ACA~WG
1868 rr3Uar3GU rnr-rTr.~rr,rrr.A~rrrrr.~ Acr3G~3G
1877 AGcr3rcu rTTr~rTr~rrrr~ r-r-rrr~ ~ AGGCAUG
1901 wccr~u ,rrTr.Anr~rr,rrrAA~rrrrr:~ A~r~r~A
1912 ACUGAUC rT3rTirTr.~rrrrr.~AArr,rrrAA ACUAT3Ar3 ~922 r~a~ ,rrTr~ArTr~rr~rrr~ r~r~rrr~ ACArJr3CA
1923 GAI~CCT3 rT3rArTrArrrrr~rr,rrr~A AGcauca 1928 Cr3GGUAA rnr.~nr~rr~rrr7`~rr^rr~ Acr~aA
1930 AGcr3GGT3 rTTr.~rTr~rr~rrr-A~rrrrr.~A AAAcr3cu 1954 T,-GGGGAC rnr.;~rrr~rrrrr~ r ^rrT~ ~LWCUC
1583 UAaCUUG rT3r~rTr~r~r-^rr-~ r--~rrr~r~ ~.u~UCCU
SUBSTITUTE SHEET (RULE 26) wo 9sn3225 2 1 8 3 9 9 ~ P~ s6 1996 r~aG ,rrTrPrTr~rrrr-~ r,rrrr~ rccLr 2005 r~GUCC~r,C rrTrATTÇA,rrr~rrP~Arrrrr~A AGcr-rccA
2013 r~ AA ,rrTrPrTr~ r~rr~ ~P~rrrrr.~ aAUAGc 2015 CCACCCC rrTr.~rrr.~r ,r~~ a ~rrrr--~ r. .~U~r,GGCA
2020 CL~A rrr~A~ r~r~r~r-r` ~ar--r ,.~ r~CCACC
2039 CCUCIJGC r~rTr.ArTr--rr-~~arrrrr.~ ~-C~GC
2040 CCUCCAr, rrTÇArTçA(--,rr-~r-~r,~,A ~
2057 r~Ga~G ~rrTr~ATTr~r ~~~r,~ ~.r.~ A,r,~CA
2061 ~cAcG~--~r rrTr~Ar~r--~r-r~ r---rrr~
2071 CL'CAr~C rrr Arrr~-r~rrr~r~r-rrr~rl ~.aAOE~
2076 r~rcrr rrr.~rTr~Arrr-r~p, AAr,r,rrr, ~ "~AC
2097 CArJc~ rrTr~-TrArrrrr~ r,r,rr--AA A~
2098 CGGt~GGG rnr ~rTr.~rrrrr~ rrrr~r~A AA~
2115 ALrccucc rrrr-TTr~Ar~-rrr~ r,r~rrçp A A~r,CUGGC
2128 c~JcA-A~rA rrJr~TTr~r!r~rrr~T~r~r,~rr~A a;~G~G
2130 GaCG~G rrTr.:~rTr--rr.rr.a~Arrr~rç~A ~AACA~r7ç
2145 CAUCAAG rrTÇ:~rTçArr~rr~ ~r,r,,rrr~ A~WG
2152 AA~JCtIA rrJr.ArTr.~çrrrÇ~ ~- r,r~ Ç~A A~A
2156 r,laA~AA rrTr~rTr~rr~rrr`~arr.rr~.AA ACAU~A
2l5a AIJUaAUA r~r,Tr~TTr.Ar~rrr~r~rrrÇ~A A~CAIJC
2159 AA~JuMLr rrJr~TTr~rrrrr~--rrrrrr~
2160 Ap~A rrJr.ArTÇ~rrrrr~Tlrrr-rç~A AAAr~
2162 Ct~J rrTr~rTç3r~rrrr~rr~rrçAa ALTA-AAlrA
2163 aAl~Alr r~ -rrrrAAArrrrr~ AA~CAU
2166 AAL~AGaG7 rrTçArTçArrrrçAAArrrrç~ AUr,;~r 216~ Aa.~r rrpq~rTr.~rr~rrA~rrrrr.r~A AATTACAU
2170 CL'AA~A~r rUÇArTr~rrrrr'`~'rr~rrÇ~ AL'AAAUA
2171 GGGaGc~ rrTrArTr~rrrrr~ rrrrÇ~A AA~aAccr 2173 cLTGGrLAA rrTr,ArTç~rrrrr~--rrrrç~ ACL'CUAA
2l7g ~r,c~ r~TTr~Arrr~-r~rrrT 7 i\rr,rrr~ ~ ~aAC~rA
2175 ~ GGCr r~nnArTr~ rrrr~rrr~rÇ~A AAAC~CU
2176 L'Ar~G frp~ArTr~-rrrrÇ~arrrrr.~A AAAACUC
2183 CAA~AA rTTçr~rTr~ r,rrr~T~rrrrr~ ArvCUGGU
2185 CUCAAIJA rTTÇArTrArrrrr~ rr,rrrAA AT~GCtJG
2186 ACL'CAAU rrTçArTr,ArrrrrAAprrrrr3A AAT~GCU
2187 UACL'CAA rTTÇArTr~~r~rrr~ rrrrç~ A~ATJAfC
2189 r~C rTp:ArTrArrrrr~ ~ ar.rrrr.AA AT~AAUA
2196 CAUCAaG rTTr~T~rrr~Arr7rrr~33ArrrrrT~ Ar~uG
2198 AA~A~AA rrTç3rTçArr~rrr.AAAr~r,~~rr.AA ACGCDGC
2199 AUA~ACA rrrçArrçArrrrç3 ~ 3r~r~rrrA A AA~r,AGGC
2200 cLTor~cau rrTÇAr7ÇArrrrrAAArr,rrr.AA A~AAGA
2201 ~r~CCGa~A rrTrArTr.Arrrrr~-~r-rrç3A AAa~ T
2 2 05 UCAGGCC rrTr.ATTr3 rrrrç~ A A rrrrç ~ A ACAUAAa.
2210 AGCCACU rrTr~Tr.Arr~rr~7~7rrrrÇ~A AGUCLTCC
2220 AGAGA~AC rrrr~rTr~Arrrrr~AA3rrrrç33 AlrGccAr, 2224 r~ATJGGA rT~r~ATTçArrrrçAAArrrrr~ ' ACCL~G
2226 ~r~cr,Gcr~r rrTr~rrç~rrrrr~rrrrç~r~ ArATJCCA
2233 CCUCCAG rrrç~rTç~r~~rr.~.~r~-rrr~- AC~,GCAG
2242 ~r,~rCCGC rTJr~rT~r-Arrrrç~ ~ ~f~-rr-~ ~Cr~
SUBSTITUTE SHEET (RULE 26) W095123225 ~~ 2183 ~I,~ ei- 1~6 2248 UGGGAUG rnr~ATTr~Ar-r~rrr~AAAr~r7rrr~AA AUGGAUA
2254 UCAGO~T rTJr.ATTr,ArrrrrAAArrrrrZA AAOUGGA
2259 CACCG~JG rTTr.ATTr.~rrrrrAAArrrrr,AA A~JGUGAU
2260 r~cAccG~7 rrTr.3TTr.~rrrrr.~AArr rr~A AA~TGA
2266 TJCCoGGU rr~r~T~r~rrrrrA~Arr~ A Ar~oucc 2274 ~lCUC~ rrTr~Arr^rr.~AArr~rrrAA AUCUGCU
2279 COUGC~C rnr.ATTrAr~rrrr.A~ArrrrrA~ ACCCOUC
2282 CT~C~ rTTr.:~TTrArrrrr.A~Ar~ rrr,AA ~aCAGCO~T
2288 AGGCCA~T rTTr.ATTr.Arrrrr.AAArr,rrr~A AC.~TAUA
2291 AGCA AG rTTrATTrAr~--,rrr.AAAr~rrr.zA ACCACUG
2321 CC~T rTTrzTTr.Ar~.rrrAAAr.r--~rr.AA AUC~JtiUC
2338 CAGGCaG rnr.ATTr.ArrrrrAAArr.rrrAA AGUCUCA
2339 CAA~ rnr~zTTr1~rr~rrr~ r~Grrr-lA AGWUUC
2341 Ar~GCUGG rTTr.Anr.Ar--,rrrAAArr,rrrAA AGAGGUC
2344 GCI~AA rrTrAnr.Arrrrr.AAArr~rrr.AA Ar~ A-A
2358 WGC~GA ror AnrArrrrr~AAAr~Grrr~AA Ar~cuGGG
2359 UCO~OUC rrJt Anr~Ar~r,rrr~AAArr~rrr~AA AA~GC~G
2360 ~P AAG ror AnrArr7rrr.AAArrrrr-~ ~ AA~;GOU
2376 '~}AAG rnrATlr:Arrrrr~ rrrrr~A ACCACW
2377 WCAaA~A rnr~nr~rrrrr~ r~rr~T~ AACCACC
2378 CAGUAGA rnr.ATTrArrrrrAAArrrrr~ ~ AAACCCU
2379 COUAIJGA rnrATTrArrrmAAArrrrr,AA AA~ACCA
2380 GCCGACA rrTr.ATTr.Arr~rrrAAZrrrrr`7` A~AACOU
2382 GGGGCAA rnr.ATTrArrrmAAArrrrr.~A AGAGaA~T
2384 T~WGUC rnr~Tr.T~rrrmAAArrrrr-~ AC~;GA~J
239g CJCCACA rTTr.ZTTr.Arrrrr~ rr,rmAA AGOG~JOU
2401 r~CU~ rnr.Anr~rrmAAArrrrr~ AcaGcoU
2411 GCaUCCLT rTTr.Anr.ArrrrrAAArr,rr--.AA ACCAGUA
2417 Am~JAUG rTTr.Anr.ArrrrrAT~Ar~rrrr.AA ACCA~TC
2418 r~ JGa rTTr.lTTr.Prrrrr.AAAr rrrAA AUCCAGU
2425 AACCCIJC rTTrAnrArrrrr~ rr,rrrA~ ACCCAUG
2426 AA~O~ rnr.AnrAr~r,rrr.ZAArrrrr.AA AAI~LAAU
2433 GCO~JA rnrAnrArrrmAAArr,rrr.ZA AA~ OCTA
2434 AGCUGG~r rnr~TTr.Arrrrr.AAArrrrr.AA AAa~CU
2448 GGGCaGG rnrAnrArrrmAAArrrrrAA AGGCOUC
2 4 4 9 GGGGCAG rTTr. A T Tr.A r~r~rrr.A A ~ rr~rrr.A A AA~GCOU
2451 AGGCAGG rrTrAnr.Arrrrr.AAArrrrr.zA AACAGGC
2452 GAGGCAG rTTrATTr.Arrrrr`~rr,rrr.AA AAACAGG
2455 GGGCAGG r~T~ATTr~Ar~rrrr~AAAr~rrrrAA AGGCOUC
2459 GGGGGGG rrTrAnr.Arrrrr.AAArr,rrr.AA AGUGUGG
2460 CGGGGGG rrTr~TTr.Arrrrr.AAArrrrr,AA Aa~0~3UG
2479 GCO~'ITA rTTrAnrArrrrr.AAArrrrr.AA AGGUC~TC
2480 GGAUCAC rnrATTrArr~rr~ rr,rrrAA ACGGUGA
2483 GG~GGCtT rrp~ATTr.Arrrrr.AAArrrrr.AA ACAOUGG
2484 Gar - uGGt~T rTTrAnr.Arrrrr~Arrrrr.AA AAAAAAG
2492 A~GUGGG rnrAnr.Arr.rrr.AAArrrrr,AA AGG~U
2504 ~a~AAG rTTrznr-Ar-rrrr-AAArrrrr-~l ~;GOTGGG
2508 UGG~AUG rnrAT~lrrrr-.~AArrrrr,~l AUGCA~Z
2509 co~r~z CUr,AUrZr-^rrr~lA~r~rrrr'~ ACrn~aA
SUBSTITUTE SHEET ~RULE 26) WO 95/23225 ,~ 2 1 8 3 3 9 2 PCI/11795/00156 .~ 196 2510 GCUG`-~JA rr,TrATTr~-rrrr.AAArr~rrrAA AA~D ua 2520 c~7rJGGG rTTr~TTr~arrrrr~r~rrrr7~7~ AcAaAAG
2521 l~a~ rr,Tr.ATTrar-^rr.Aaarr7rrr.~A AAr~G
2533 GAUaCC~ rTT~ATTr.~rr,rr~AA~rrj,rrr 2540 CACAGCG rTTr7ATTr.Ar^rrriA~r~rrrr.aA AC~;G~G
2545 AG;~CCA rrTr~rrrrr~ r-"-rr,AA ACA~CAC
2568 r1UUGaCA rTTr,ATTr.Ar-rrr~r^-rr~ ~C
2579 CA~7CCA rTTrjarTr~rrrrr~ ar-7rr~AA A;~U
2 5 85 AG~ac rrTraTTt-arrrrr.a a ~rr~ UGCCaG
2588 AUt~G rTTr~ ~r~ ^rrrr~ 3 ~r~^rr~ 71 AGU.IJGC
2591 ~ rr~rATTri~rr7rrr~ rr~ rr.aA A;~ACCA
2593 GcA~aGC rrT~iATlrar^~rrr~r^rrraA ~aAA
2596 CAU;~GGG rTTrATTr~^rrrraAArrrrr~ AC~AAAG
2601 AA~A~A r,T~ATTrArr-rrr-AAArrrrr-AA A~a.C--GU
2602 GGG~17GG rrTr~TTrAr~rrrr~r~rrrr,AA AG~GA
2607 CC.~ rrTr~AT~Ar~r~rrr~aAArr-rrr-AA A~CCGaG
2608 C~-G rTr~Tr-Ar~^~rrr~Arrrrr.aA ACUG~G
2609 UCCUGGU rTTrT~nr ArrrrrAA~r^jrrr~ a~cc 2620 GC;~GGCU rTTr~aTTr.Ar~rrrr~A~rr~rrrAA AGG~
2626 GCliW~ rTTr.AnrArrrrr.~AAr,rrr~ AUC~AA
2628 AGGCUAC rTTr~nr.~rrrrr.aA~rr-rrAA A~GC
2635 A~GaccG rrTr~Arrrrr~ rrrr~a AGCuGaA
2640 GGC~ nr~TTr.~rrrrr~ rr,rrr.AA A~GGCC
2641 cr~ p, rrTrATTr~arrrrr~A~rrrrr-`~ Ar,CuGGG
2642 GAGGC~G rnrAnr~rr~rrr.aA~rrrrr~A AAAcaGG
2653 GCU3CC~J rTTr~lTTr~-~r~rrr~ r~r~crraA ~CCA~A
2659 CUO~C rTTr.ATTrArrrrr.aAAr~rrrAA ACG~C
2689 CCUCGGA rTTr`~TrArrrrr~ ~ar~ rrr.AA A~ A~G
2691 GGCCUCG rrTr~rTr.Arr~Aa~r-~:AA AGACAW
2700 GGGC~GG rTJr~TTr~r~rrrr~T.~r,rr-rr.AA AGGC~C
2704 ~GGCUGG rrTr~TTr~r~rrrr~ rr~rr-~ ~ AGAGGUC
2711 CUGCUGA c~rT~r~rrrrr~ rr~rrrAA AG.-UGGG
2712 CCCWCC rTTraTTr.~rrrrr.AAAr~ rrrAA AGACCtJC
2721 crJuGcAc rrTr.ATTriAr~rrr~ rrrrfiT~A ACCC~C
2724 GC~ aCG rTTr.ATT~Arrrrr~ r--~rrr.AA AUGUACC
2744 Cr~TGCACG rr~Tr~TTr~ rrr~A~r----~ ACCCACC
2750 Gr~aCuc rTTrAnr.Arrrrr.AAArrrrr.~A AU~AA~A
2759 A~CGA rTJr~TTrArrrrr~ rrrrr~ ~ AG~CCGG
2761 GCAGC~T rnr.ATTr.Arrrrr.a~rr.rrrA~ AGGUCCU
2765 AGCGGCA rTTr.~TTr~-rrrr~rrrrr7~ AGCAAAA
2769 CC~ T rTJr~rJr~r--~rrr~Ar-r~rrr~AA ACaGACU
2797 r~ACCAU rrJr.~nr~ rr~rr~ ~ ~ -rrr~ r~ ~ Au~ucAI r 2803 CGCCUGG rrTr.ArJr~--rrrrAAArr~rr~iAA ACCAUGA
2 8 0 4 CUGCACG rTTr~AJTr ` - ~r7rrr~A A A r-rrrr~A ~ ACCCACC
2813 GGGTJCAG rTTr~TTr~ rrr~r rrrr~T ACCGGAG
2815 ~AGWG rrTrATTr~rr,rrr'`7~Arr~rrr.AA AGAC~
2821 CCUCC~G C~ r-r~rrr~T~r--rrr~ AG-UGLG
2822 ~,rjUCC~, cu~L-r~r~--rrr-a-~r---rr-~ AG~--CC-2823 ~ C~GC rTTr~T~r~r-rrr- ~-r-r~-r,AA A,~C~
SUBSTITUTE SHEET (RULE 26) WO 951~ 5 197 21 8 3 9 9 r~ [ 156 2829 ArJ~UA rTJr.ArTr.Arr,rrr.AAArrrrr.AA AGUCrAG
2837 UCAGl~AG rTTrATTr~rrrrr.AAArr,rrr ~ ACCACCU
2840 CA~-CAG rrTrArTrArrrrrAAArrrrr~T AG~ CA
2847 GG~1GGCU rTTr.ATTr.Ar~rrrr.AAArrrrr.AA ACauuGG
* 2853 ~AA rrTr~ATTr~r-r-rrr-AAAr~r-~W AA AGGCUGC
2860 UrACAGU rr,r.~TTr.Arrrrr.AAAr.,rrr.AA ACU~vGC
2872 C~Gcu rTTr~ATTr'~ rrrr.AAArrrrr.~A AAGGUCC
2877 r~GAr,JGG rr,Tr.ATTr.Arr~,.~rr.AA AGCG-vAA
28g9 AAGAUCG rrrATTr.arrrrrAAAr,~ rrr~A AA~JCCG
2900 AA~C rT~Tr-ATTr~Arr~rrr~AAArrrrr~AA AAArJuAA
2904 A,AIJAGAG rTTr.ATTr.Ar~rrrr.AAArrrrrAA ArJG~ r 2905 ra~AGA rTTr.ATTr.Arrrrr~rrrr.AA AA~AG
2906 UAA~AA rTTr.ATTr.ArrrrrAAArr,rrr.AA ACAUCAA
2907 AAA~lAA rTTr~-Tr~ArrrrrAAArr~rrr~AA AAAUACA
2908 AGCAAAA rrp~ArTrArrrrr~ ~ rrrAA A,Al~Ct7UC
2909 AGAGCAA rTTr.ATTrArrrrrAA Ar~rrrrA~ Ar~
2910 AAAUUAA rTTrArTrAr~rrr~ rrrr.~A AAArJAcA
2911 A,A~IJ~LAA rTTr.ArTrArr~rrr.AAArr~rr~ ~ AAAUACA
2912 r~UA rrJr~ArTr~Ar~r~rrr~r~-~rrr~AA A~A
2913 rJGAccAG rrTr.ArTr.Arrrrr.AAArr-rrr.~A AGAGAA~A
2914 C~Ar~GA rTTr.ATll Ar~r-rrr~AAArr~rrr~3 AAA,AGCA
2915 UC~AAU rTTr.ArTrArrrrr~AAArrrrr.AA A,A,~AAA~
2916 ClJCCGGA ~`TTr.ArTr.Arr~:AAArr~rrrAA ACGAAUA
2917 UCt7CCGG rT~r:ATTr~ rrrrAAArr,rrrAA AACGAAU
2918 CU~UCCG rrTr.ATTr.Ar~r~,--rrrr.AA A,AAcGaA
2919 CGACCC~J rrTrATTrarrrrr~a~ rrr~A ArJGAGAA
2931 C~l7CCGA rr,TrATTrAr~r,rrr.AAAr~rrrr~AA ACCUCCA
2933 CCC~CC rrTr.ATTrArr,rrrAAArr,rrr~ AGACCIJC
2941 rJGGwAc rrTr.ATTrAr~r~rrrAaar-rrrrAA A~JC
2951 GCAGA"G rrTrATTr.ArrrrrAAArr~rrrAA Ar~CGUGG
2952 CaCAGCG rTTrATTrArrrrr~ ~rrrrAA ACUGCUG
2955 rJGAcAcA rTTrATTrArrrrrAAArrr~rrAA AGUCACU
2956 rJUG~wuc rnr~ATTrArr,rrrAAAr~r,rr~:AA A,A~AAA
2961 A~JGGC~ rTTr~ATTr~Arr7rrr~AAAr~r~AA ACACAGA
2962 AA~AA~ rTTrArTrArrrrraAArrrrrAA AA,DACAU
2965 I.uuu~U rTTr.ArTrArrrrrAA~rrrrrAA AU~AA
2966 CC~CUGC rTTrATTrArrrrr~ rrAA AGCCAGC
2969 AA,AACDU rTTrArTr.ar~rrrr.AaAr~rrrr.AA AWGAr~U
2975 GCUr,GUA rrTrATTrAr.,--rrrAAAr~r,rrrAA AACIIC~A
2976 AGI~AG rTTrATTrArrrrr.AAArr,rrr~ ~ AACCCUC
2977 r,a~C~CA rTTr.ATTr.Arrrrr.AAArrrrr.AA ACAGC~
2979 GG~IIA rTTr.ATTrArrrrrAAA ~ rr~A AGAArJGA
SUBSTITUTE SHEET (RllLE 26~

2183~32 woss/2322s , ~ - r~l,~,s,~-ls6 --_ -C~ O
~ c_ ~ e~ ~ o O ~_ ~ o ~ ~ O O
SUBSTITUTE SHEET (RULE 26) W0 9St23225 2 1 8 3 ~3 9 2 r -J~ 156 , ,, _ _ ". ,~ _ _ _ _ _ .. . _ ' ~
S - ; ~

C.;
C~
F
C. o C. .5 ~ 33~
.I - .- r ~
C ` .
. . . s , . . ~
" ~
SUBSTITUTE SHEET (F~ULE 26) wo 95/23225 - . ~ 2 1 8 3 9 9 2 -- -- ~ ~ S ~
C ~ 3~
~ s ~., C CC
~ ' sS ~
- ~ - s sr~ s ~ n ~ O s~
2 ~ --I ~1 ~1 ,~
SUBSTITUTE SHEET (RULE 26) WO 95123225 2 1 8 3 9 9 2 r ~ s ~ . 156 Table 9: Rat ICAI~ HX Rlbozyme TPrget Seque~lce 2t. ~ ~ g~t ~ nc~ 2t. ~1 ~ t S~
Pol:ltio:: Posltlo-1 1 G~CC.~ ~ C~CaClJGA 394 ~ C5~
23v~ C CUCC~ 420 v~CCCCU C C^~CGCA
26Gaaa~CU C UtJCCUCOU 425 C--.I~GGFU 1~ C~.vCCACC
31CC~UGCU C C~G~CCU 427 ~7CCC5GW ;J ~CA
34CUGaACCU C A~C 450 A~ACCU C ~IJC~v 40CUCaACGU A CA~;CCCC 451 v~v~J C CCAGGC
48v~CU C v CCUv. v 456 C~ GG~--W C ~.~A
54CCCCvCCU C CC~GCC 495 vCCaCCA~J C ~WWA
~8CCv~GCCU U I~IU;C~ICCC 510 ^~;GC~ C c~G;aA
64CaAl~;GCU IJ CAaCCCG~ 564 v~aAA~ U CCaACCAC
96CCUCUvCU C C~JGGIJCCU 592 vv--~G~A~J C ACCl~GGvA
102 C~JCC~;GU C C~;C 607 v~A~ U ~C~.~CC
108 v~ tJGCU U G~ 608 A~-~Ca~ 0' CCI=W~
115 ~JCCtll~m7 ~ ~WCCCA 609 CCCAAUW C ~C5U
119 Ga~aaJW c CCCA~C 61 1 C~AUWCtJ C Al.~JCA
120 OEIUOE~ C CCCGG~;CC 656 ~W 1~ C~ADv ~6CCAGAC~7 U v~ACUCC 657 ~ C ~GaA~GU
ACCCGGCU C C}~7CAA 668 G~aaJGCU C ~C~'CW
158 A~ WU C ACGAG~JCA 677 GCACCCCtJ C CCA~.
165 1~7 A CUtJCCCCC 684 AGG~7 C C~U
168 G~AGCC7 C CDGCCO'CG 692 CCAG~C~7 ~J GGAi~CUCC
185 GGGKaGlUJ C CGK;t~G 693 CGG~J C G~l UUCC
209 CAGCCCCU A A~JGACC 696 GCCUli;~TW C C~GCC~7 227 GACCAa~J A ACCG~GAA 709 CAGCA~7 A CCCCUCAC
230 CAAGC~7 U GIJGGGAGG 720 CtJACAAC~ IJ ;I~GCUC
237 Ci~aaG~7 C GaCACCCC 723 CAA~W C ~7CCCA
2~8 GGCCCCCU A CCUUA5GA 735 C~JCCDG7 C C;;G,7CGC
253 CACUGCCU C AGUGGaGG 738 IJCCUGCCU C GGG~GGA
263 GAGCC~AU U UC~IJGC 765 AC~JGUG.7 U ~IGi~GAACU
267 G~iAGCCl~U C CUGCC~JCG 769 UCUC~7 IJ CCCU~
293 GAAGCtJ.7 U CAAGClJGA 770 CUUGUGDU C CC,~aAG
319 CGGaGGAU C ACl~AAt GA 785 AGGCCU~ ,7 IJ ~CC~JGCCU
335 ACUOE7GCU U UGAGlia7 786 GGCCUC~W ;7 CC~7C
337 ~UGCUA~ A UGGUCCUC 792 CUCCUG~,7 C CUGCJCGC
338 A~GCtJCW C AAGCU~G 794 UCCU~;C7 C ~C
359 CACGCA~,7 C C~U 807 GCI~CaGi~lJ A U~J8CUGGA
367 CAA~7 U CAACCG~7 833 CCUGG~7 U GGaG2~7A
374 ~JACCC7 C ACCCAC~7 846 CUG2~A7 1:~ ~G
,,375 AG~AGCCU U CCUGC.7C 851 GCUCAC7 ~J U~C~7 378 ACCCacC~ C ~GG-,7A 863 CaAUGG.7 U C~ACCG~7 386 CGCO~h7 U UUGGAGW 866 CCAI~CW C C~aCA
., SUBYITUTE SHEET (RULE 26) WO 95/23225 ., ` ` 2 1 ~ 3 9 ~ 2 P~ 156 867 GACCaCCU C CCCAC~A 1421 GGGCaCaU C CCCCAi~GC
86g C:ul.:UUCC~7 C ~OEa.AG 142s ACCC~CCU C ClJC~;GCU
881 AWGGC~U C AaCCCWG 1429 U~ A GCCV~GG
885 GACCA~W A AC~aA 1444 A.GAA~-U C ~G
933 ~ C G~JCCCaC 14S5 GGGaGW~} C ~CCaGGGA
g36 GC~.GaG~ U UO;;DGI7CA 1482 AGGWACU U CCCCCAGG
978 ~tJGaGa~ C ~ 1484 AC~'-t7CU U CCUC~GC
980 G~Ah~CU A C~W 1493 CC~;GGGC,U U G~a~A
g86 C~U~ U ~C 1500 CGUGa~AU l:J al~A
g87 ~.aA~ ~ ~lCC 1503 GaAAaUGU U CC~ACCAC
988 ACaAl:D~ U CAGCI7CCC 1506 ~GGU~U A AU~1~7GG
1005 ~UCGD~ C G~;GCG~C 1509 Gcca~Ca~J C AC~U~A
1006 GUGGGAGU A ~JCA~GG 1518 GUCC~JGGU C GCCG~JW
1023 CCGGAGW C ~AGG 1530 ACC~GC~W C AUM~JGU
1025 GG1~;WCU C A~ 1533 C~G~ U GCGGGC~U
1066 ccua~ ~r G~JC AA 1~51 G~JGGCCCU C ~JGCUCWA
1092 AG~ C IJC~GA 1559 I~GaA~ C CCDGD~.
1093 AS~aAlJ C CA~;CCCC~ 1563 ~CC~( CU U ~ CCCA
1125 CCCCAACU C IJUGCCCa8 1565 ~u:.acc~ A IJG~CCGCC
7163 ACGACG--~J U C~GCU 1567 AC~ U ACCGccaG
64 CGACGaW C T~tlC;~lJC 1584 AGGMGau C AGGal7Ula 7 66 ACGC~J U 1~ 1592 C~ A cA~UaC
1172 C~ C ~CGGC~ 1599 UACAAGW A CAG~
1200 ~IJCCA~U C ACaC~aA 1651 CCCCGCC~J C CCt~-C
1201 ~ C ~JCCa~ 1661 Ct.~U U GCCC~,U
1203 GGGC~ C CAI ACGUC 1663 G~ C MUGGaCA
1227 ~AACU C CADG~ 1678 G~CU C GGCC~;GG
1228 G.-GGGCDU C Gt~A~J 1680 GGG~7C~:;J C CA~JC
1233 CD~ C CI~G~ GC 1681 GGCCDWU U CCCGCCtJC
1238 ~ A ~G~C 1684 CIJG~GU A GACC~C
1264 G;~GAU C A~l GGGU 1690 CCCCAI CD A CA~7 1267 GUCa~ IJ CAA~ 1691 CCGGa~ U CGa~UC
1294 Ca~3al~ IJ ~ 1696 C~JCCUGW C CUGGUCGC
1295 AG~;GGW C l~ GA 1698 ~I:aGa~ A CCDGGaG~
1306 At;Ca~;A~ C UaaCalJGC 1737 GA~AIJ U CACGGUGC
1321 AaCA~ C ~GGGa~AA 1750 G~CCA~ A cAccuaua 1334 G~CGU ~ cccaG~c 1756 CCUC~ C C~JCCU
1344 I~GGUOEU C A~AUCC 1787 GA~aACCu C OECCUGGG
1351 ucAcGcca A AGa~ 1790 GaCAC~JGU C CCCaaC~JC
1353 UAOEA~CU C MCAA~GG 1793 AuOEaCCU C ACCaOE-AC
1366 AGGG~ 1:7 CCCCCAGG 1797 ~JCCCUG~ U MaMCCA
1367 GG~W C CCCCAGGC 1802 GCUCAQ~ A UACCUGGA
1368 GAUGGU~ C CCGC~OEC 1812 A~ C UOEGGAM
1380 CUGCCUAU C GGGAU~ 1813 OEGGGC~ C GUGAUCGU
1388 UOE-AG~CU A AaJGGaaG 1825 OECAC~AU C AcuGuGaa 1398 CaOE~ C AcAGGacA 1837 ACCCACCU C ACAGGGaA ~`
1402 CD~ J GaGAAcuG 1845 AGaGGACU C OEAGGGOE
1408 UaCGuGA~ C GD;~GcGac 1856 CCCCUAAU C UG~CCUGC
1410 CGA~c[a~ C GAGlJGGaC 1861 CAUGUGCU A ~GGUCC
SUESTITUTE SHEET (RULE 26) WO95~23225 218 3 9 ~ 2 r~l,.L s -156 .
1865 ~CCGv~ A GACACI~G 2198 GaA~77 C cG~7ca l86a liC~ C Al~ aA~ 2199 AG~ A Ca5GCCAG
1877 i~CaG~7 lJ C'-CCCaGG 22C0 GGG~UU C CCCC~^
1901 C3~Aa~ C Aa~CA 2201 GGG7C~7 C CaG2~,~C
1912 G~ C AaliGGaCA 2205 1~7 C Ai~C~G
1922 iilJG~7 5 i~UA 2210 I,~GaÇA~ A ~17G;a5G
1923 XGACG.7 C ~CCU~UAG 2220 GAL~C7 C G~7GC~, 1928 GC'~,~J ~ 5ACC5G;~ 2224, ACAl!i~lJ 5 CC'~ACC5U
lg30 ~GaC~J A AC5GG~G 2226 C~ C ~
1964 ~ J G5~57C;~^ 2233 ~7~J C ;~Sa~CJ
1983 G~ C GC-CC~æGG 2242 ~a~7 C ~GJ
:996 ~^rJ C 5~CAAG----'J 2248 Cl,'CC~i~7 C C;~7C^~
2005 A~7 ~7 A~7A 2254 A5CCA~IJ C ACacOG~A
2013 Cû.~''~7 A ~wGaUG 2259 G~JCACAU U CACGG~J'GC
201 5 C~ C GGGa~7 2260 ~WWU C ACG~7 2020 ~JGA~ A CCC5~.7AC 2266 A5c~Ga5 A UA~U
2039 CGGaGG~ C AC~GA 2274 GAGC~7 ~J AAI I~D~A
2040 CCt~ GJ C C~GAw~J 2279 GGA~U C A~CGG ,7 2057 C~iGG5CCJ C C~A~7 2282 AC~GWAU U ~aD~U
2061 G~7CCP~ ~C~7A 2288 GCCC,'W7 C C~JCCa~G
2071 AI~ACUt;G~J A GCC5r~AGv 22gl CAGGa5A~J A C~AC
2076 ; GI~aGC--J C A~A 2321 GGAAAGA~J C AUACGGv7 2097 C~A~J U ~7 2338 ~7U C ~JCC~;G
2098 C~J C C~rwArw7 2339 Gw7AC~ C CC~aGGC
2115 ~'CCGA~7 A ww7CC~G 2341 GGv~C57 C GG~7CA
2128 AG;JGC5G5 A CrA~'GAUC 2344 C~7Cv7 A GACCt;CUC
2130 GCC5G5~iU C .7GCC~J.7 2358 CCCI~C-~J C C5CCCa~A
2145 C_aA~7 IJ G5a;~aDW 2359 CC~17CCA5 C CCAC1~G~A
2152 ~JCGaG~ C 5ACA7~iU 2360 C5~ C CC5--^AAG
2156 ~GaCAG~ A li~JGA 2376 G~7 C
2158 5~WG~W 5 ~ 2377 GACllUC.7 5 7Ct~JA
2159 GA~DU IJ A5U~IWUC 2378 GC5G~J C ll~iUCaCGA
2160 A~ A ~A 2379 C~7C~J C C~UGCG
2162 ~DU~7 A CC5~ 2380 U~05.7 5 5CA~Av7 2163 ~ A A~5C~ 2382 AliUU~UU C ACGAr~lCA
2166 5GA5G5A~J U ~U 2384 5AUCCC~7 A GACa~ aAG
2167 GAI~ U AU~ JC 23g9 UAMI~7 A UG~GACG
2170 G~ ~ AU~UCAGA 2401 5G~7AU A 5w~C7C
2171 C~ IJ A~7A 2411 CAAT~U~J.7 C AUGC~lUCA
2173 5,7GC~AU A 5w7CC5C 2417 A5CAGGAIJ A 5ACaAW~J
2174 IJC~JCt~ A Ce~ u 2418 UCA~ C ACAGAA~7 2175 A~UCa~ C ACr~7CA 2425 5C~;aAU U CAGAWUC
2176 GA~AA~ U CCA;~CCAC 2426 CC5GGrw7 U GG~.7A
2183 UGAC1~GD5 ~ Ul~Al~A 2433 5CAGAG5~ C
2185 AC~ 7A~J IJ 5;~1J~7 2434 CGvArwAU C ACAaACGA
2186 CAG5U~DU U AIJ~7A 2448 UGAa~;J A Cll~JCCCCC
2187 AG5a~J A ~AC 2449 GAaGCC5~J C r,~;CCUCG
2189 lit}~WUAU U GAG5ACCC 2451 wCr~UWU U CCtrC~ JC
2196 C~ACh~7 U AlJrJl~WUG 2452 GCCIJ~ C C~CCC~J
SUBSTITUTE SHEET (RULE 26) W0~5~23225 -~ - 2 1 8 ~ P~l"l 5 2455 AC~l aJ ~ CCU~J 2761 CGGAC~J C ~;AUCWCC
2459 C~C~--CU C CUCCCACA 2765 C~J C UGCGCC0 2460 CCIJACC~W IJ iiullcCC~iA 2769 U~A~ U ACCCC~JGC
2479 I~ACCU A UCACCGCC 2797 CG~JGaAAU U WG:~TCAA
a480 G~JCGCCG~J U GUGa~JCCC 2803 CUC~I~--U ~ CAa~;AAC
2483 ACC17JI;W U CCCAAUGU 2804 UCA~U C AG~AACU
2484 CC~J~ C CC~Gl7C 2813 GC~CCCA~J C CD~CCCU
2492 GaCCaCC~I C CCCA~ C~JA 2815 CGGaC~ C GAUC'JUCC
2504 ACCUAC~IJ A C~ CUA 2821 CCCGACCU C CUI~GaG',-tJ
2508 AC~W U CC~laC0tJ 2822 llaf~ ,~CUCC
2509 CU3i~ C C ~CC~ 2823 C~t7DU C AG~CC"~
2510 G~JCCA~J A C~CCt~;U 2829 UCGGUGCU C AG~--~UCC
2520 ACCG~J ~ CCCAAUGl7 2837 CACaGGGU A CWCCCCC
2521 CCD~7Gt;U C CCAAI~UC 2840 GC~CCCCU C CC~GCGCA
2533 ACAGCaI~7 U ACCCC~A 2847 UCA~LI C ACCCACCU
2540 ~'-IJ C AG~.~CC 2853 UaCGa~ 'J CC~ OAG
2545 AGGCaGC~ C CG~ 2860 I~JGU lJ CCC~J~3GAA
2568 C~ U ~GUC~5 2872 GGGCCtJGU C GGU~C~
2579 CClJGCaaJ U UGCCC~GG 2877 UGGACUCU C CC~CACC
2585 CD~GU A GACCIJC~JC 2899 AGGCaGC~ C CGG~a 2588 ~7GCC!JCCU C CCAC~GCC 2900 GGCIJGACU U CCU~U
2591 C~ C ~CGa~ 2904 GaAcUGcu C ~JCCU~
2593 ~1:= A CCCC~ J 2905 GGCUGAa~ U CC[~7CK U
2596 CUCCD~J C CUWUCGC 2906 GU~;aUGU A U~A
2601 ~JGUIi;"U;W A ~JCCUC 2907 CU~CUCUU C CUCU~C~:
2602 G;JCa7GGU C GCCG~U 2908 . UGU~WAU U II~DUP,AW
2607 GUGGGAGtJ A ~GG 2909 GAalllGCU C ~IUCCUCW
2608 CUlJl~aGCU C CCGr~GGGA 2910 AC~UCCW C UCUP~3~1AC
2609 ~ A AC~QaDG 2911 UUCCWCU C ~7AI~IACCC
2620 ~CilG~U C IJGa~G~ 2912 a~JAwu A WAAWCA
2626 C;~C~I A GlJ~ 2913 ~X;D~ C G~CCCA~
2628 1~ U ~GCaCC 291~ GDal~ U AAWCAGA
2635 U~G~ C CAal~CAC 2915 . ~I3U A A~ AGaC
2640 G~J A ~ICCAIJCCA 2916 CDCWCCU C U~GCGAAC
2641 CCCCACCU A CPIIU~ 2917 CUUCC~J U GCGA~;AC
2642 GCCK;~7 C C~CC~IJ 2918 AU~ C aCGAG;JCA
2653 CCACaCGU C AGGWGW 2919 W~JD~JGU C aJ~
2659 A;~aAGGW C C~JGCaAGC 2931 GaU~ C CCGCtJGCC
2689 AC~GW C C~GaAGCU 2933 ~CC C CCA~CC
2691 UCaGG.^a7 A AGAGGACU 2941 CA~aCW C CCCCAGGC
2~00 Al~ U CCCCCAGG 2951 aCCAUGCU U CCUCIJGAC
2704 GACCACCtJ C CCCACCtJA 2952 CCGGI~ J U CGAU~C
2711 CCC~ACCU U AGGAAGGU 2955 UG.~7~JCCU C UGACAUGG
2712 CC~ A GGAAGGUG 2956 CUWCC~U U GAAUCaAU
2721 GG~AA~ C A~ACG~3GU 2961 WUD~;;JGU C AG~'G
2724 AaGAlJC~W A CGGG~U~7G 2962 UWt~UAW C WUCCCAG
2744 GGG~U C CGUG~AGG 2965 C~GaAlJ C AAUAAAGU
2750 GUCCC~GU IJ UAaAaACC 2966 UGGAAGCU C WCAAGCU
275g GACGaACU A UCGAGUGG 2969 GaAlJCAA~J A AAGUUUUA
SUBSTITllTE SHEET (RULE 26) W095/23225 218~992 r~l,~ 5~ 156 2975 ~ C ~C~
2976 ~ C CL~
2977 t~A~CU U CaA

SUBSTITUTE SHEET (RULE 26) W0 95l~3225 : r~ 56 Table 10: Rat ICAM ~F Ribozyme Ser~uences rt, R~lt ~ R~ao2y2 ~ c~
~oJ~ tio~
11 r7C,a~,7G c~ ~TTr~r^rrr~ r^~rr~ c 23 r~ rTTr~arTr~rrrrr`~`r~ aA~7Ca~--26 ~aA rnr.anr~r-7~ r^~ ~,7C
;l A~G r~r~TTr~r--~rr~T ~r-rrr~ G
34 GUaI~iDK7 rrTn:~TTr~T~r-^7crr~ar~r-rr~ G
Gæ~7G c~r~TTr~ r--~rrr~ ~ .U C5~G.~
48 CCCAGGCC r~r~7r~z~nr~rrr~r^~rrr~ ~GW~C
54 GGaJC~G rrTr~r7r~rr~ rr~ GCGGGG
58 GCGaGC~ rnr~TrJ~r5~ r~rrrr`~ AGGG~CGG
64 ACGG~.7G ~ r~r~ CC.~,7G
96 AGGacc~G rnr-:~nr-l~r~rccy-~r~r~rrr~a ~Ga~
102 G G~CQG rnr~TTr~^r~rrra~r-7~-rr~ .~Ca.~aG
108 AGC~,7CCCC rnr~TTr`--rcrr~ r~r7rrr~ cc 1 1 5 ~a rTTr~ TTr7Ar~ rr~` ~ A~X;~
119 GAI~iGGG rnr~TTr~rrrr~ r~rA~rr~ ~ A!.G~7C
120 GGCCCGGG, rnr~TTr~^r~r~ rr,r~ ~ ;wa~C
146 G~CIJCC r~-Trar^~rrr~ 7 ~rrrrr~ u~ uw 152 ~ j; rnr.7~TTr~ rrr~rr,~r-:~ aGCCGG~7 158 ~C~7 rnr~-Tr~r~-~rrr~r^rrr~ APaGAAhT7 165 G5GGGaaG rr7r~TTnPrr~rr3~^^rr.r,~ AC~ TT7CA
168 CGaGGCAG r ,nr~TTr.~rr~rrr~ ` ~r^,rrr~ ` AAr~;CrTT7C
185 CC~ACG ~ r^rrr~ ~ AT7CCACCC
209 r~T7 rrJr~TTr~rr~ ~ T r^~rrnP ~ ~G5GCUG
227 TJ~t7 rrr~-Tr~-~cr~ rrrrr~ AC~TC
230 CC17CCCAC rrr~TTr~ r~rr~ .G0;7C
237 GGG5DGT7C "~ 7~rrcrr~
248 T~AGG rT7r~TTr~r~r~rrr~Tr~ Ar~GGGGCC
253 CCUCCACT7 rTTr~r~çrrr~ rrr~P a~T7G
2~3 GCU7GAGA I u~ rr,rrr~` AT3UGGCUC
267 CGAGGCAG rTTr-aTTr~rr,rrr~rrlCrr-Pa AAGGCUT~
293 TJ~;CUt,TG r~Tr~TTr~^r~rrr~ ~rr7rrr~ GAGCUUC
319 TJCG~G17 rnn~rTr`rrrrr`"`rrCrr`~ A~TCC17CCG
335 AG~A rT7r~TTr~rrrrr~rrrrr~a~ AGCaCaG~T7 337 G~GACCA cnnPnr~Prr7rrr~ rr~rrr~ ~ A~A
338 CDCaGC~7 rnnPTTn~r~rrrr~ r~rrn~ AA~r-AGC~7 359 ~CCGAG rrTr~TTr~rrrn~r~rrrra~ ACT7GCGT7G
367 ACGGGUT7G rTTr~TTr`^~r-rr~ rrrrr`' AGCCATJT7G
374 A~r~W~7 rTTr~-Tn~r~rrrr~rrrrr~7 ~G~GG~A
375 GAGGCAG,G rnnPTTr~ rrr~ rr~ ~ - rrrrra~ A~r~ 7 ~`
378 TJACCCUG~7 rnr7~T7r.~rr~rrr7P~Prrrrr~ AGG~TC-GGt7 386 AGC~TCCAA rTJr,,nr~rrrrr~r~ ArACAGC,G
SUBSTITU L SHEET (RULE 26) W0 95123225 2 1 8 3 9 9 2 P`~ 1'76 394 C~Ca.G rnr.D7Tr~rrcrnDaDrrrrnDa AGC
420 ~JGG rnr~rTr~r-r~-AADrrrnRDA AGGG~
425 GG~ rnr~r7r~Drrrrr'~`rr~rrr-~D AGCC0;G
427 UGOE~ rnrDTr~rr~-rrrrr,,a,~
450 CGCIU~ ~ rrrrr.a~ ALG~UC~
451 GCC~;G ~ ,U,~Il- .. ,,." A~r~rrnDA AA~CC
456 UGW~GCa rnr~DnrDrrr~r--rr~
4g5 'J~ rSTr,,,Dnr~A~r'``r-~?` ;'DIXUGC-C
510 ~ r~ 7Tr~r~r~rrrC~r~
564 G~G r~`rTr~r~~r~r~rr~
592 ~JCC0G.~ rnr~ Tr~r~rrr~Dr-rrn~ .U~ CC
607 Gca~ rnr7A7~TnDn~ r-rrnDA ~.D~,'&;~,~C
608 AI~CWG~ ,.", "". .~r. . ~ rr~r-~ M.~5CU
609 Aaa a~ TTr7~r~7r~r rr'C~rr~--6~1 UG~GC~J rnr,A,~Tr~rrrr=DDDrrrrr:aA A~
65 6 r~hD~ rr 657 ~, ,, ~", , ", .. ~ rrcrr~T~ AA~, 668 AAGaGG~ rnr~rTr~rrrrr~ rrrrr~
677 '.~GCGCDG~, rtTr~TTnarr~r~rrrrr?~
684 AAAGtJCCC, rrTr~rTr~---rrr~rrrrr~ ~GCCU
692 GGaG~c~ rr~r~ ~ ~JGG
693 Ga;~AG~c ( I) ~ Il.~u.. ~ rr~ ~CCG
696 AG~G rnr~TrA~r~rrfrr~ ~GGC
709 G=~GG C~nr~7Tr~--;crrA~-Irrm:AD AaAU~UG
720 GA~ ~TTr~rrrr~ DaArrrrr'~ AGD;JGU~G
723 ~a~CU rnr~-Tr~--rrrr~rr~r.AA AAAA~G
73!; GCG~CAG r~r~rl~rrrrr~rrrrr~
738 ~CCPCCCC rnr~TTrDrrrrr~aAarr~rrr~ ~ A~Gc.a~a 765 A~ C~ TTr~rrrrr~rrrrr~ AGC
769 ~a C~G l l ~ I T - A7.r 7rrr~ ~ IU:aCaiiG~
770 C~ICCA~, ". ~.~. .. r, " ~rrrrr~ AAl aCaAG
785 AGGCaGt~ r~r~Tr~'rr~rrrA~rr~cY`r~A A~ U~GCCU
786 GAJ~;G ~ . " ...... ,," A~rrJ~ aA AA~CC
792 G~G rtTr~Tr.arrrrr7~7~rrrrr`~ A~Ca~G
794 GAI~JC~ I.~r.l.~ ADrrrrr-~
807 I~ _ 7rrr~ GC
833 I~G~JCIJCC rTTr~Tr`rrrrr'~rr~r~ ~G
846 CAA~ rr~rrr~ ~ ~DG~G
851 A(;CI~iC~ rnr~TrDrrrrr~rr~rr~ JGAGC
863 AaæG~G r~Tr~ rr-rrr.AA Ai;CCal~'G
866 ~WcaGaG rnr~ rrrrr~rrrrr~AA ~G
867 ~GGCGGG r~nr~rrrrr~r~r~--rr~A ~ 3GGUC
869 C~lUCG~A rnr~Tr~rrrrr~ ~r--7~ aG
881 cAa~ rnr~ Drr~r~ --rrr~
885 Ut~ rnr~tTrArrrrr.AAArrr~.AA A~C
933 a~AC ~r~ --rrrr~r~rrrrAA AA~
936 ~aA rnr~7Tr.Arrrt~AAArrrrr.AA A'J~IJGC
978 Aa~ rnrATTr~ rrr~'rrrrnAA A~JCCCAA
980 AAA~ "-~AAnrr~-r.AA ~.GA~CtTC
SUBSTITUTE SHEET (RULE 2~) WO95123225 ; 2183992 r~ -156 986 GA~A rr~Anr~rsrrr-~r-^rr~, AG~
987 GG;~ rnr~Trarssrr~rr~r.a~ ~.aCD~
988 GG5aGC~ rTTr-~T7r-r~ rr7~a~rs7rrr~
1005 G~Cr~Ccac rnraTT~ r~Grrra~rssrr~
1006 CC~;WG~ rr7r-T~rr~srr~a~r~r~rrr:u~ U L7Ccr~C
1023 CC~Ot~ rnr.aTTr~rrsrr~_r~rrrr~
~025 CCCC~JaJ crT~ Tr~rs.rrr:~arrsrr~ 7CC
1066 U~aC rnr1~TTr~r-^srr~ ~r,r,,~
~Og2 ~Ga rTTr~ ^~r~r~rr^rr~ ~CCCC~.C~7 1093 ~a~UGGcr~ rnr~ r^,~ r~rr^rr.~ ~ A~C~,7 1125 ;U;~AC~ rr7r-~ -^.rrr~arr^rr~
'16 3 A~;CaAa~ r~r~TTr~rrsrr~ rs~ ;CGCCW
"64r~icbA~ rnr-~r~rsrr~T"rrr^r~
1'66 C~EACCaA r77rpT7r~rr~^rr~r~rsrr~a ~G~7 ~'72 aG5CC'-~ crTr~Arr~rr~rr~
1200 ~;CaGDW r~7r~--rrrr~rr~:AA AADUG~7 1201 C~ Ga r77r~n~--r~rrr~a~r~rrr~a AA5CCC~
1203 r~Am~UG rn~r~7r~ rrr~r-r~r:~A ~GaAGCCC
~27 ;~ rnr~Ar-^srr``~r-^sn-`~ A~A
1228 ACGalJCaC rrr-~r~-rrrr~rS~ A~^
1233 GCGP~cCaG rnr~nrarsrr~ r-srr.aa A~
1238 r~CCA rnr~-lr-r-~rrr~,arrSrr~
1264 ACCCGU;~L7 rn~r~r---^~rr^-~r~rsr~r a~ A~Z~C7CC
1267 CaD~tJG rnranr ar .rrr~rrsrr~ Ar~C
1294 C.~,l~ca, Ill n l, ,~ I, .~ar^~rr---~a A~Ja'Cl;G
295 UC~Ct~ cnr--~ r^,rrr~ ~ ~rs~rrr~ a A,rCCC,~ 7 1306 r~xD~ rnr~r~r~rrr~ar~r~r~a ~GD~;C~7 1321 ~CCC~ cT7r~ -r~a~--rrr~ ArD~W~7 1334 GC~7CC~ r ~ ~crr~ a ACGaA~C
1344 r~ cc.~7 rnr~-7r~r`~ rrrrr'~ A~
135~ Jcc~7 cnr~rcJ-rr~ ~ ~rrsrr~,~A A,r,G-CU~, 1353 CC~7 C~7r~7r~Ars~ccr~ a ~rrrrr~ Ar~:DGCt~a ~ 366 CC~;GGGG rnr`l7r`~Crrr` ~ `CCsrr` ` AW~CCL7 1367 G~= C~n~r~T7r~arss~ r^frr`~ AAG~CCC
13 6a uGC~CGG rr7r~A~:Arsrrr~ rr-~ ACACCAi7C
1380 ACC~7CCC rnr~-anr~r~rsrr~r~rr-~rr~a A~
1388 C~IJccaG~7 rnr~aT7r~-~rrr ~ ~-rrr~a AGt;WC~
1398 7~GL7CCUGt.7 r77r~7rarrsrr~rsrrr:~ ACaGCCAG
1402 C~tJUCUC rnr~rrsrr~a~r--r--rr-~
1408 G~C rnr~r~-~rsrr-`~r-rsrr`~ AD~,CGaA
1410 G~ DC rnr~7r~~ srr~a~r~rcr~ A~G~G
1421 r,cC~;GGG rnr~~lr~ --rrr- ~ ~r-rr~rr` '~ A~CCC
1425 ~-caG;~G r7Tr~-~7r- r-rr~arrr-rr~ ACWGG.. ~.7 '.
1~29 CCt,~GC rnrAT7r~rrsrr.~ ~ ~rrsrr~ ~ ~
14~4 C~,CC~'CCU r~7r~T7r.arrsrr~Tarrsrr.~a AGCCWC~7 145- ~7CCC~ 7 r~nr~7rArrsrr~a~arCsrr~a ~ CUCCC
1482 CC.~ GGGG rnr~T7r~ar~srr~7lr~r~rrr~ ~CCC~r 1~84 GC~A5~ rn~ru7rrArrsrr~r-r~rrr-~ ~7 1493 Ui~c rTJr~aTTr~r~r7-rrr`7``~-srr~1~ ACCCC~G
SUBSTITUTE S~-EET (RULE 26) WO 95123225 209 2 1 8 3 ~ ~ 21 ~11~ . ~
1500 ~CWX~ rnr~TTr~-rrrr~7.rrrrr~ AlJt~ACG
1503 G~GG r~nr~TTr~--rrrr.. ~a~r--~:a7. ACA~C
1506 CCAACa~ rn~r~T~r~^rr~-larrrrr~ AIJGACCCA
1509 UaC~CAGU rnr~ ar-r-r~rr~rrrrr~ A~,.~GC
1518 AcaA~GC rTTr~rArr~ rrrrr~ ~ ACCAGGAC
1530 ;~ rrTraTTr~rrrrr`'~`r~rma~ ~CCC~;GU
1533 AAGCCCGC rTTrATTr~--r~rrr~ar~rrrr~a ~AG
1551 ~JACGaGC~ rrJr~pTTr~3rr~rrr~r-r-rrr~ AG~r,GCCAr 1559 ID~GG rrJr.ATTr.Arr~rrr~ r,r;rr~r~ ~ }~CCCA
15a3 IJGGG.~ACA rnr~ rr~^~7~r-r~rrr~A ;~GGA
1565 GG GGUAA rr.Tr`~^-rr^`~r~rr^rr:AA AG5~1G;~A
1567 CUGGCGG;J rn~r~ Tr.Ar~rrrr~ar--,rrr~ A;JAGGCIG~I
1584 I~I~IJCCU rrlr AT~rarr~rrrPA7~rr,rr~AA A~TC~CCU
15g2 G;laA~G rrTr.ATTr.Arrrrr~rrrrr~:~ A~JCC~G
1599 GCC~tJC!JG rrT~r`7Tr.arrrrr``arr,rrG~A AA~liCWA
1651 GG.^D; aGG crlr prlr~^r~ --r~rrr~l AG~GGG
1661 ACC1~GGGC CrTr7~TTr~Arrrrr~ rr7rr~r~Tl AAa~cAG
1663 ~JCCA~ rrTr3TTr~r-r,rr~ ar-rcr~:AA A~DG~C
1678 CCCAGGCC rTJt:ATTr,Arr~rr~AAPr^,rrr,AA AGW~C
1680 GACC~G~rG rnr~TTr~^r~rrr.AAArrrrr,AA AGMGCCC
1681 G~GCAGG cnr,ATTr,Prr~7~ r~rrrrAA AP~GGCC
1684 GA~C C~:pTmpr~rrrr~ r^rrr-AA ACGaGCAG
1690 A~G ~nATTr~r^rrr`7l~r~r7rrr-A3 AGG~JGG;;G
1691 GA~a~JCG CTTr~pTTrpr-r~ rr"-r~:p3 AAaJCCGG
1696 G~GaCCAG rnr~Tr1~r^rrr~ rrrrr~ ACQGGAG
1698 ~JCUCCaGG r~ Tr~rrrrr~77rrrrrAA AIJA~JGA
1737 GCACCGUG C~Tr~ rr~rr~A3 A;~C
1750 A.3~JG rr~Tr~rrrrr''~rrrrrAA A.3~C
1756 AG^~:C~G rTTr~ Tr.Arrrrr~AAArrrrr~ Ar~CAGAGG
1787 CCC~GGCC r~Jr-~Tr~r~rrr~T~ - ~rrr~ AGG~C
1790 GA~;Cl~GG ~ u~ rrrrr~ ACi~ C
1793 G~ C~ rnr~r7.rrrrr7.~rr~rr~T. aGGaCCAU
1797 ~C~ r-rlr~TTr~r^~ a~rr~rrr~ A~ al;GGA
1802 UCC,a~7A rnr~'Tr~rr~rrr~ AUCOGaGC
1812 UUUCCCCA rnr~ rrrrr-.~,rrrrr~ ACUC~GUU
1813 ACGA~JCAC I ll~ r~rrr~ A~ CCGC
1825 l~a~ rnr~TrPrr~rr~ rr~rrr`~ AI~G;JGGC
1837 ~ Jw rnr.aTTr~r~r~r~-~7lrr~rr~7 p~GG~
1845 GCCCC~JCC rnr~rTr3r~r~rrr~ rr~rrr~ PGrJCCUCtJ
1856 GCA~ A rnr~Tr~ar~r~rrr~ r~r~rr` ~ A~XGG
1861 GGA~I~ rnr~TTr~r~r,crr`7~`rr,~rr~pA pGCa~a~UG
1865 COI~WGUC ~ rrrrr~AA ACCGGAUA
1868 AU0~ r,nr~tTr.3rr~rrr~''r-^rrr`'` AC~JCGUG1~
1877 CC~7GGGGG rnr~Tr~^r~rrr~rr,rrr,3A AGi~CUGU
1901 ~CDU rT7l~aTTr~3rr~rrr~ r~r~rrr~ A~AG
1912 UGUCCaDU rr~ 3rTr~3r-rcrr~rr~rrr~7l AT~uuc 1922 Uac~A~ rTTr~r~^r,rrr~rrr~AA PC~UACAU
1923 C~aaAGGU rnr~rr3rr~rr~ rr~rrr~ ~ P~CGUCCA
1928 UCC,~GGI~ rrTrArTr~ rr~rrr~ 3~r~-~rrr-a ~ p~ AGC
SUE;STITUTE SHEET (RULE 26) WO 95123225 : . , 2 1 8 3 ~ 156 ~930 CA~ rrTr~r~rrrfr~rr~A
1964 ~Ga~aC c~lTTr~r--rrr~a~rrrrr~AA
1 sa3 CCC~GCC rT7r-~T7r~--rrrr` ~ ` rrCmA 2~ A~Jc 1996 i~ c~r-Tr.3T~r~~r-~r.. ,crr~ A~.7CCA
2005 ~ r~7r~TTr~Arr~a~rrr~:~3 ~CC~LT
2Q13 CAlJCCcGa rrTr3TT~--Ar~rr~rr~
2015 ~CCA~7CCC rrTr~TTr~rr~l-rr.~AArr~ AllaG~CaG
2020 ~ ~TTr~ar- - ~`aarrr~``
2039 ~J rrTr~TTr,3r-~r~r~`
2040 ~ JCC~G rrTr~T7r~r^~r~arrrrr~ b~.GG
2057 I~C~,Ca~lUC r~ TTr~3r ~rr~3aarr~rrr~
2061 ~ rrTr~TTr~ rrt~ rrrrr~T. A~C
2071 CCUGI~ GC rrTr~TTrAr~r~rrr~a~rCrr~a ~AG~7 2076 t~ rrTr~TTr~rr-~ rrrrr~ ~;G~.a, 2097 ACalJC~C rrTr~lTTrarr~a~rr~r~7~ AGa~GG
2098 d~ ca5 rrTr~TTr~ar-r7m-~a~--rr~
2115 CA~;GaCcc ~ " .,, A a ar~rrr~
2128 r~G rrTr~TTr~Arrrcr~ rrrrr-aA Aca~
2130 AÇ~G rrTr~TTr.~rrrrr```rrrr,r`~ AAA~-C
2 l 45 ~C aD~A~c rrTr` rTr` cr~r~ ~ ~ rrrrr~ a AG~G
2152 A;U~G~ rT7r~ r--~ a ~rrrrr~
2'56 ~aA~AA ~ T
2158 AAI;~A~ ", ,~ rrrrr~
2159 r~A~ r~Tr~ rrrr~rr.rrr.~A A~C
2160 I7G~A, ". ~, j. ,.r,~ arrrrr.aa a.
2162 ~A~:AA~ ' ')' l ~, . T a~r~ a ~.A~
2163 cU~7r,.~A~7 rrTr~7r~ rrrr~a~~rCr~
2166 ~aA~, ~,. A~l........ '~. 1 ~ na-~rrrrr, 2167 r~All~a~T ~ l fl " .. , T A ~ Tl~r~` ~` AAriaCUC
2170 ~ rr7r~ Tr~ rrrrr~ aAA~C
2171 a~ rrTr~'Tr~r--rrr~a~rrrrr~ AAU~
2:73 r~ rrTr~r~rrrr~ ~~rrr~
2174 AGC~GGGG rrTr~Tr~--r~ rrfw~ a .~.ADa.G
2175 J~T rr7r~rrrrr~rr~!~a A~
2176 r~G rl~r~ rr,~"~,.rrrrr~ Aca~c 2183 ~AIIP~ l Ul~ ., . Aaarrrrr,AA Ai~CCC0~
2185 ~ ," ,j, ".A7.~r~rrr~A ArlaAC~T
2186 ~C~CaAL7 r~Tr~r~rrr~a~rrrrr,aa AA~A~
2187 r~ aA rr2t aT7r~rrcrr7~rrrrraa 2189 r~GG~c~Jc r~Tr~ rr~crr~ rrrrr~
2196 Ca~AA~T ~ "- ,~ r~r~:AA ACC~ AG
2198 ~;aCC~G rTJr~ rrfrr~rrrrraa A~ uc 2199 Ct~GCU7G rrr~lTr~--rrr~ r4rrf:aa ~p~CL7 2200 GCCt~GGG rrTr~ r~r~rrr~rr~rrr.~a P~a~uaccc 2201 GACC~G rrTr~Tr~rrrrr~a~rr~rrr,~ .~aAGCCC
2205 CAG~JGGCOT rrTraT~ rr~rrr~a~r~rrr~7~ A~ aC, 2210 CA~TCCAOE,T rr~Tr-~TTr`~r~rrr~`a~r~crr~ CC,A
2220 CCCAGGCC rr3l'!`'7r`'r7~``~rrcrr-A~ P~G~C~C
2224 ~G rrTraTTr.ar~r~rrr~rrrrr.~a A~.~GIJ
SUBSTITUTE SHEET (RULE 26) W0951U22~ 218 ~ ~ ~ ?~ c 2226 I~WCGCC~ A~r~rr~^r~r~ ~r^.rr~ AGGuCcaG
2233 A~7uu~u~iu rnrAnr-Ar~r--rrr~7~ il.A~
2242 A_I~C~ rnr~ r ,rrr'` ~ :'r-~ Ar~JGDW
2248 r,CG~SCaG rnr~ r rrr~ r-rrrr~
2254 U~W~U rnr7lTTrarrrrr.aaArr~ AAI~
225g ,r,~aCCG~ rr,~n~r.Ar~rrr.A~r-.rrrA~ ac~c 2260 A~ rnr~ r^7rr~r~ r-^rrra~ .a.a~r.;JG;~u 2266 ~ rr~r~rr,rrra~Ar~ XC~IJ
2274 l~nJ rnr~nr~r^r~rr~ r^,rrr.~ ~JGC~JC
2279 ACCCr~U rnr~Tr~r^^rra~r^,rrr~a~ CC
2282 ACWU~ rnr~7lr`rrrrr`7~r^r--rr~ A~ACUGU
2288 raUU~G rnr~ ar~rrr`'l7~r--~:~a AC_~;GGC
2291 ~r~A~ rnr~r.r^rr~rt;rrr.aA ~
2321 ACCC~alJ r~nr7~ r~--rr~r,;~r~r~ iUCC
2338 CC~GGa rnr~ rr-rrr~ r------r~r~ ~ ~CCC~A
2339 GCC~;G~7 rrTr~TTr~rrrrr~r~r,rrr~ Aa~,CCC
2341 ~CaCC rrTr.~r--rrr~7rrrrr~ AraGGCCC
2344 ~r~aGaGGt7C l~TTr~ rrrrr~7rrrrrAA ACGa~
2358 ~JGt~, rnr-Anr~rr~r~r7~rrr~ 8GC~GG-7 23~9 U~GU~7 rnr~Tr.Ar rrr~ ~ ~rr,~r~rraA Al;5;G~G~
2360 Ct7t~; rr~ Tr~rrr~a~r~rrrr~AA ~.aGLCaA~
2376 AA~GAA rrTruTr~rrrrr'~'rr,C'rr-A~ AOEaG~t7C
2377 ~.a~ rt~r Anr~r--~ rr~rrr~ ~ PC~
2378 UC~AA rr7r~Tr~r--,rrr~rr~rrr~ AAAI.~;C
2379 CGCaAGA~ rnr~Tr-~rr~rr~rrCrr~ ~aG.~CaG
23 8 0 ACIJCGWA rr7r~ ~Tr~ r.r~r~ ~ ~ r~r~rrr~ 71 A~aA~
2382 UGaCJC~ rt~Tr~rrrrr~rrrrr~ AU
2384 ~uuw~iuc rnr~ Tr~rrrrr~Arrrrr~a ACC~r,Gat7A
2399 CGt7CCac~ rnr~rr,rrr~rrrrr~, 7~t7A
2401 ,r~ rnr~ATTr~r7rrr~`1`r~rrrr`` A~
2411 ~CA~J rr7r~rr~-~r~rrr`~rrrrr-~A 7~GUa~G
2417 AACWGt7A rnr~Trl~rr7rr~ rrrrr~ 7~ C5GA~J
2418 AG~ Gt7 rlY''~Arrrrr`~'r-rrrr`` AAGCAt7~ra 2425 ~r,~ rnr.Anr~r.r,rrr~r- rrr~ 7~A
2426 13aaJC~CC rnr~-Tr.Ar~rrr~ rr,rrr.~A ACCCCA; G
2433 AA~Gl~A rnr~TTr.~rrrrr~ rrrrr~
2434 UCGtlUt~Gt7 rT7r.~nr~ 7~rrrrr~, AtJC_t7cc~r, 2448 r~GGG~ rnr.AnrArrrrr~rrrrr`' A~C~JU~
2449 CGa~ac, rr7rAnr~rrr~rr-~ rr~r~7~ AA~CC5UC
2451 ~r~G rr7r.Anr~~r.rm~a~rr~`' AAS.~GCC
2452 AGaGGCaG rCr Anr~rr-rrr~rrr~rr~"~ AAA.~GC
2455 AA~ AAAGG rrTr.ATTr~~r,rm~r~rrr~ A~aA~
2459 ur~G rTTr.Anr.Arrr~rr~ r-~`" ACGCaGGG
- 2460 Ut~GG~AC rrTr,ATTr~Arrrrr~rrrrr.~A AA~i;G
2479 ,r,Gc~r,OELAA r~r~Anr.~rrrrr~rr-rrrAA AG.
2480 r~AC rrJt ATTr~rrrrr~rr~rrr~ ~CGAC
2483 A_aUUCGG Cnr~ Arr~rr~rrr~ Acaa~;GU
2484 r~ rTr.ATTr~ rrrr~AArr~rm~ AAC~G
2492 II~GGG rTlr:Anr.ArrrrrAAArrrrrAA AC~7u~U~
SUBSTITUTE SHEET (RU~E 26) WO 95/23225 ~ ' - 2 1 8 ~ 9 ~ 2 P~ l/~ h. IS6 2504 ~ rnr~TTr~rr~r~ r 7 rr AA A13 2508 ~AGG~ rnc~T~ rr~ AA ADGl7 2509 Aaa~7 rnrA7l~Ar~rr~rr~rrr7A7~ K~7 2510 .~A~ rnr~TTr~r~r7rrr~ ~C
2520 A=~G rn~r7ATlr:Ar~r~r~ rrrrr`7l AG~A.
2521 Ga~GG rn~r~ rr~ 7 ~rr~rrrAA ~A~ aAA~
2533 I:JG;~ rnc~T~r.Ar~r~r~ rr~rr`~
2540 G;~ ~ rrf~, ~ ,~
2545 ~AAGD~, ". ~1.. ~.~7~ r^~ ~ UGC~
2568 CUG~ rnr~ rr7m~ rrrr~r77~A i~JCUC~7 25~9 CCl~ f~nC~T~r~rrrrr~ rC~Y:A~ 7 2585 G~G~C rnr~T7r.1~rr7rrr~ rr~rrr.:~1~ .a~GAG
2588 GGC~G ~ rr~rrr~ 7~ AGG
2591 C~I GCaa ~ -A~ rr~rrr~ aAG~7 25g3 ~ G~ rnr~Ar~rr~`7 A.U~.5a~
2596 GCG.a= C~ 7 ~rr~y~r~
2601 GaGG~ca r ~ r ~ rr 2602 A~A~XGC c~TTr~rc^rrT~7~ilrfir~ l A~C
2607 CaJGG~a ~ rrrrr=AA ~CIJccc~c 2608 11CCC~Ca7 r~7r~TTr~rr~'~r~Crr~
2609 C~ CaG~
2620 AAI ~ rnr~TTr~rr~r~ rm~
2 62 6 ~GCu;cac l ~ rrcrr~ ~ ,a~G
2628 GG;~C~ rnr~TTr~r^rrr~rr~r~
2635 G~aAlJ[~ CrTr~ r~r~`~ ~I7C~Ga 2640 ~JG;~ rnr~TTr~ rr~
2641 AAI~G~G r~ al~rr.rrr`~ AK;~7 2642 Al;a~C~G rrTr~tTr~rr~r,r~rrr~ aGGC
2653 ~ ~ I l, , I, .. r. ~ lrrrm~
2659 GW~hG rnr~TTr~rrcrr~ ACC0U~
2689 AGC~7 rrTr~TTr~rrC~rr~~r~
2691 A5D~ rnr~ Tr~rrrrr~rr~r~ AGaX~
2~00 CCU~7 rr~Tr~Tr~rr~rrr~ rr~rr~
2704 ~ rrTr~TTr~ rrr~rrrcr~a~ A~C
2711 A~ rnr~ rrrr~rr~ra~ AGG;~GG
2712 CA~ C~ C rrTr~Tr~r~ r~rcr~
2721 ACC~W rur~TTr~rrrrr~ rrr~ OI G~CC
2724 CAAACC~ rnr~-Tr~rr~rrr7l~rr~ CUU
2744 CCL~7 rTTr~TTr~rr~rr~ rrrrr~ JCC~aCCr 2750 GGD~a ~ U.171~ rr~`l~ ~A5~G~C
2759 CCA~ CcGA rrTr~T~r~rr~rrrrr~ UlJCGUC
2761 G0~C ~lr 1~ ~rI A~rr~rrr~
2765 A~r~GCCGCA rrTr` Tr`^rCr~Y~.A~Tlrrrrr~ caA3AG
276g GCA~;GGG~J rQr~rTr~cr~rrr-~rrrrr~3A A~ar~3,A
2797 ~U~C.~ rTTr~Tr~r~r.~rcr~rrrrr~ CG
2803 r~C~aG rQr3nr.~rrrrr~rrrrr`~ ~UG~7 2804 Ar~JGGT rUrr-3nr~Gr~rr~ CCrrr~ AaGC~
28~3 AGGr~G rUr~TTr~rrC~ 3A3r~rrrr~ A~GC
2815 r~G~WC rrTrAnr~^~r~crr~ r~rrr3A A.3aGUCCG
SUBSTITUTE SHEET (RULE 26J

W0 95123225 2 1 8 ~ 9 9 2 P~l/.L, .o~ I5C

2821 ACIJCC~G rrr~ATTt-~rrrrr~?lAr-,rrr~ XaGG
2822 GGAGC~JGA rnrArrGPrr-^rr7~''r-^rrr`AA AAG~
2823 ~ rr~;~arr^rr`` AAaA~
2829 G~Ccu rr~arrr.ar^rrr~ ~r~rr~.. ~ ~cacCGA
2837 GGGGGAAG rrJnArrr~rr,rrr.AAAr^,r~:AA ACa U~G
2840 rx,~GG ~T~r~rr^~ rrrrraA ;U~GC
2847 A~,~^,U rrJr~-Tr:~r~r-rr~r~ Ai~AA
2 8 r 3 CUI~CGG r~Jr.~rrr~ r^^rr~ ~ ~ rr"^rr~ ~ .a.GaDCG~
2860 ~JU~G ~ rrp~rr.rrr~a Aaca~Ga 2372 ~^ACC rrTr~T~r~rr~ çr~ ~GC^C
2817 GG~^~;GG ~ çrrr~
28g9 AaaG~ rr~r~rAr^rrr~ ' ~rçr~rnA ~ A~GC~
2900 A~G~G ~rrr~r^~ .r~r~ ~CA~C
2904 AA;a~ rnr~ ^.. ~AA AGCaG~C
2905 A~JaG~ r~.. r, ~7~,. r^rr~a ~^C
2906 t~ crr~r- r~ çr,~ ~ ~AC
2907 CGCaA;aG ~trr~ rrjcrr~a~rrrrr~ JaGcAG
2908 ~PIJ~ c~r~rr~rr~rrrrr~rrrrr~AA
2909 AA~ cnr~ rrrrr`~r^~ra~ ~5;~ aGD~
2910 GU~ ~rrr~r~r~rrr~ rr~r~rr~Aa 2911 GG~ crJr`~`r~rr~rrrrr`~ AGaA~G.
2912 r,r~A~A cnr-~r~ r^~r~ ~ Aa.
2913 C~5G~ rr~rlr~rr~ AAAr-^-^rr~
2914 ~JCUG~ rrp!~rlr~rrrrr~a~r-r,rrr-A~ AC
291; C.~ rTr~rrr.3r~r,cr~ r^rrrA3 AA~
2gl6 CU;~ GCaA crr~rrrDrr~ ~ ~çr~r~ ~ Ar~WAG
2gl7 WCU~JCGC C~n~rAnr~r-^~t:AAAr~rr~AA ~Gp~AG
2gla r~ CW rrP~~r^~rr~r~r~rrr~ AA~GaaA~
2919 C~ rnr~rr~rr,crr~'~rrrrr`` A~ca~AA
2931 GGW~CW rnr~rrr~rr,~ ç~-rrr~ c 2933 GG~.JGCCGG C~nr~ rrrrr~ r~rrrr~A~
2941 GCC~JGGGG rnr~ rr~ ~ ~rr~rrr~ ~ ApG~G
2951 G~JCAGaGG rrTr~rrr~r^rrr~ rr~rrC~" ~;CaD~
29S2 GAAG~CG rr~ çr~ r^rrr~3 Aa~CCGC
2955 CCADG~ rnr~lr~rrrrr~ r^f~ AGG
2956 AUW~C cr~r-Anr~rrrrr~çr~rrr~
2961 CA~ rnr~rrr~^r~r^~rr:~ ACAl PAPA
2962 cr-~G;aAc rrr~^rrrr~rrrr~r~~~ AA~ aCA
2965 ~ rnr~rrr~rr~rr~``r,G7rrr`` A~AAG
2966 AG~UGaA rrlr~rrr~rr,rr~ r^rrr7AA AG^UUCC~
2969 r~AA~ ~^r,r~ ~r`'` A~C
2975 A~ WGAA rnr`~lr`Crrrr`~'Çrrr~Ç`` AGCOtJCCA
2976 CAGGUGaG rrlr~rrr~ r,rrr~7-~rr~nA~ ACCAUaD~
2977 rJ~c~JuG ~ rrrrr~r^~A A~C
SUBSTITUTE SHEET (RULE 26) WO95123225 214 r~ L,r,. .S~; ~
Table 11: Huma~ HH Target SequeIlce sLt. ~11 T~--~ut g~qu~nc~ lt. }~ T~ t S~ nc~
Po~ tion PO~J t~ on 8AIJGCaCU U ~JGC 245 AAGAa.~7 C IJWCaGG

9~ ~ C~lGGCC 247 Ga.AA~J tJ UCaGG~
10Gca~U~ C ~ 248 A~AUC~U l7 CaG~
12~UI~ J ~xaAa 249 AAI~C~J~ C ~J
13CWI~J ~ GCcaAAG 257 ~GGGa.~U ~ GGCa~ aC
36~aACGSJ IJ ~C.~GC 273 GGal~G17 C ~AA~'GJ
37a~ J CA;aAaCC 291 ~XGGGU P. Cl~
38AacG~ C ~G~ 305 MA~aCU :~ ~iUCaAaA
56GGAI~CJ U CU~J17 307 A~ac~ U C~AA~
57G~ C I~C~U 308 Ga~;aW C ~aAA~A~
63~ IJ UGaG~ 316 AAA.~ ~ GIJCC~
64COWi~ ~ Ga~G 319 Aaa~G~ C CU~
69IJ~iUG~ U 13~GC 322 U~ JCCU U AAllaA~G
70u~al~U IJ GCl~GCI I 323 ~JCCW ~ ~Pi~A~.
74GI~D~ A GClJCl~l;G 326 CC~ A ~
78G.~GClJ C WGGaGC 334 A~GAAAU ~ CAWGAC
8013~ U GGa~ 338 AAI~ACAlJ U GACGGCC
glGCUGCCJ A CGWllalJ 380 G&aGaGlJ A. AACCA~IJ
97~I:G~ A lJGCCA17C 388 AACCAAU U CCUI~GAC
104 A17GCCAU C CCCACa~3 389 ACCAAW C C~QACU
116 CAGaAAU U CCCACAA 392 AUJ~J A GACl!aCC
117 AQaAaW C CCA~AG 397 Ct~CU A CC~A
130 AGUGCW U GGOlaAA 409 CAA0~7 ~ ~JGGU
145 G~ia~J U GGCaCliG 4la AAGiiGW U CllU~'G
155 CA~CU IJ ~IC111~C 411 AG~aw c WG~.~W
156 ACUGCW U Cl7ACUCA 413 AG~lUt~U IJ G~,~
157 c~ alW C tlaC0~ 419 WGWW A AUGAACA
159 GCI~lJClJ A C~CG 437 AGIJGGAU A ~;AAA
162 U~ C AUCG1lAC 440 GGatlaA~J ~ GaA1~U
16~ ~aCUCAU C G~AC~ 447 AGAAAGU U G~GAC5A
171 UCGaACU C UGC~W 454 UG~CU A ~aClJGGU
179 UGCl~GUJ A GCCAA~ 462 A~COGGU U UGU~CA
192 UGAGACU C IJG~ 463 AC~GW IJ G~
200 ~;AU ~ CCllGWC 466 G~aWGU U GCAGCCA
201 GaCGAW C CUGWCC 479 CAI~AGaU U WGGAGG
206 WCCC~GU U CCOGlJAC 480 AAAGAW IJ t 207 UCCUGW C Cl~ 481 AAGAWW U GG~aG
212 aUCCUGU A CA~AAA 497 AGG~U U ~GC
216 UG~17 A aAaUJCA 498 GGACAW U UAC~^A
222 UMAA~ C ACCAACIJ 499 GACAI~W U ACl7GCAG
SUBSTITUTE SHEET (RULE 26) WO 95~23225 215 ~ 9 ~ g 2 r~l~ 156 500 ACAI~ A C17GC~G~ 684 ~3WU~
531 AMGaW C A~ 685 AC~ IJ C~7 538 CA~;GCCU ~J APD~lCC 686 CWDt~J C ~A
539 A;CC~ A A~WCA 688 ~C~ IJ A~;aAC
542 CC~ IJ I~A 689 1~ A
543 C~ iJ ~CaA~ 691 ~U ~J llaAaJ~A
544 ~IU~ IJ C~ alJA 692 IJCD~ 17 Aa~ WhA
545 W~ C Aal~aDaA 693 : C~W A AC~AC
549 ~llJCaA~ A ~7A 697 ~la~ IJ A~
5~1 ~A~ A Al~ihAC 698 ~A~ A ACAl~tJCU
354 Al~ 1~ ~JC 703 l~ca.a~ ~J Ct~OElaAA
535 1~l3MDlJ ~J A~A 704 W~ C l;~AAA
536 A~A~ A ACWt ~lG 708 Al~ A AaAI~WC
560 ~ 3aACU 17 CA~GGG 715 A~A~GI.7 C I~Gt~AC
561 I~.A~ C AGaGGGA 719 IJG~3W IJ A~laA
573 GG~aA~ A Aa~ 720 G~ A AC~A~
577 AGD~ A WlI:aGG 724 G~A~ IJ AU~A
579 ~Aa~ ~J IJC71GGCA 725 l~aAa~U A A~
580 AAAIIPD~ lJ CAt;GC1~3J 728 Al:l~ A GtiU~7A
581 A~U~ C A~A 731 ~aa~J A ~.
588 CAGGCh~ A Cl~aCAC 733 Al~ ~J ~AAA
597 ~ 3CCaGA 734 lla~ IJ AliGaAA~J
598 Ga~ 11 GC~aGaA 735 A~aDW A l~Gaaa~G
611 AAA~ A AMI~C~ 745 AAa~ ~J AaGaA~U
616 A~Aa~ U C~lD11AaA 746 . Aal~ A AG~A~U
617 ~aAAa~ C I~MAA~ 752 I~aA(:~A~ ~J ~GUa.AA
619 AaA~ ~J AAAA~ 753 AaGAA~U ~J GG~.aAU
Q0AaD~ltJ A AaA}D~A 757 AUl~;W A Ah~lJ
625 ~.AAa~ A ~CA 761 GG~AA~ ~J AÇt~U7~
627 AAAU~ A l~ hGA 7Q G~aAA~ A Gtl~ 7A
Q9AalD~IJ IJ ~DA 765 AA~ A I~W
630 A~Dl7 ~J CAf;a 767 U~AIJ ~J ~A
631 ~DW C AGallUJC 768 ~ ~ A~
636 ~;a~ A ~a.AU 769 AÇ;~ U A ~
638 C~;a~IJ C A~ADCA 771 ~DCI~ IJ ~aADWU
644 IJCI~aaU C A~Gaa~ 772 A~ 17 A~UWIJA
647 GP~ IJ Gaa~ 773 ~ A ~U
633 I~AG~ A l~:ClJ 778 i~MDW IJ A~W~Gtl 633 G~ J ~JCC 779 ~ A 13~WG
656 A~ ~XUCCA 783 G~U U ~,uwu~u 637 A~8~U IJ CC~7CC~\G 788 G~WW IJ Ct~A
638 G~)U C C~Ui;G 789 WG~ C I~ADaAA
661 ~ C ChGGC~A 791 Gr~W~ A Al~aAAAC
672 GC~AAAU U GaliPDaC 794 llEl~W A A~ACAAA
676 Aa~AU A ~U 803 C~AAAAIJ A GacaACU
678 UOGa}~ A CUl~
681 Al~ U U0~
682 u~alU l:J ll~UA
SU6ST1TUTE SHEET ~RULE 26) 218~32 woss/2322s ~ ~ . r~l,.~ c~ls6 Table 12: ~u~a~l IL 6 ~IEI Ribozyme Sequences ~t. ~ R~zy3- S~ c~ ~
~!oa ~ ~clo~ -8 G~aAAGa rrlrarr~ ar ~' AG~C~IJ

10 ~GGCa~ ~T~_ r----~ . ~ r ~r~ ,'GC
rr~ ~~rr~ r--~ A ~.a~
13 C~GGC rrlrar~ ~--rr~r,--g~--rr~
3 6 ~r~ ----r_r~ rr~~rr` 7~ .WCU
37 r~ CUG ,rrT~ .ar.~--.rrr,~ ~ ~r --r~ ~C
38 ~ r~T~ r- .~
56 .aA~ cr~ TTr~_r~.~ r--J~r` ~ A~CC
57 aAU~ ,rrJr,anr~~rr~ r--~:a~ c 63 ~,~ ,rrlr3~ ,r ~rr~ a A~C.
64 caaA~C cr~narr._r~ r~ JG~æ;
69 GC~JACsA rnranrarr~`~7`r--sr~ ~ ACUC~aA
AGCU~GC rnr~ rssrr~ rsrrr~ ~ AaaJcAA
74 CAAG~GC rnr"TTr.~rssr n~ rs~ AGC~AAC
78 ~r0~caA r~7r~rr~arr~l~``'r--rr`~ A 0aGC
rl~C C ~ ars~r~
91 AU~CG r~naTTr.arsrrr~r-7rrr-~a Ar~ GC
97 r5U7GGCA rnr~TTr~ars~sr~r~ r-~aa ~
104 C~UGGG rr7r~r~ ` - Srr` ~ 'rS~ ~ A~--AU
116 Ut7GUt~GG r~ r` rsrr` ~rSrrra~ Al~CaG
1 ' 7 C~;G rrJr~ nr~ -s~s~ rr` ~ ` r~rrr~ ~ AUJtl~JCU
130 I~JCIU:C rn~ s,~rr~rr~ Al~GC~
145 CAGUGCC cn~--~arsrrr~ rrrrr~ ` Ar~Tcl7c 155 ~raG~GA r~r~TTr.~rs.^rr` ~rs~`~ AGC~
156 ~G rnr~TTr.~r.rSr~ rrr~`- AaG~
157 AII;aa~A cnr~ arssr~ A~UU~
159 Cra~aG rr7r~TT~rSrrr~rs~ ~;AAAGC
152 ~IUW r~rs~-rr~ r--rmaa A~G~A
165 AGaG[lUC rr~ rsrrr~rrsrr~ A~A
171 AIJQ~CA rnr~TTr~ar~ rrcrcaa A~CCA
179 CAI7~GGC cnr~TTr~ rsrrr~ ~ T rsm~ 7 A~CA
192 A~JCCIJCA ~rrJr~TTr~rrrr~ rrraA A~7CA
200 GAaCaGG C~Tr`~7lrs~ rrcrr~ A;Jcc~JCA
201 GGaACAG rrJr~r--rrr~a~r--~srr~a AAI~CCUC
206 GUI~CaGG r~ nr` TTr` rs--rr-~ a ~rssrr~ ~ A~A
207 IJG~C~ r~nr~TTr~rs-rrr~ r-ocrr~ AU~GA
212 U~UU~JG rnr~ -r~-rr~ ~ ~ r-srr~ a ACA~A
216 ~U r~TTr~TTr-~s~rrr~ rrra~ ~CA
222 AGWI~ rn,r~-s,rrr~rs~rr.aa A~
245 cC~aA rTTr~rTr~ ssrr~ r^Srra;~ AU~ UU
SUBSTITUTE SHEET ~RULE 26) W095t232~5 218 3 9 9 2 ~ l~ s . 0.SG

247 UCrCUGA rTlr:aT~ r~rrr~arr7rrr~ AGAC~C
248 ~; C~ r~rr~rrr~i~a~r~-rm~ AaGaaaa 249 AUaCCClJ rT~ 7r~rr,rrr~Ar~r,r~lr~a AM,~a 257 GU~;CC cnr~rrr~r-r7rrra7~r-r~rrr~ ADU~
273 Ar~G~r rTTrT~Tl-~rrrrr~a~r,r,rrr~a AC~UCC
291 ~ rT~r.T~T~r.~rr,rmaP.~rr~rrr.l~ ACCCCC~J
305 U~U~AA rnr.~TTr~rr,rrr~-.rr,rrr~, AGr~
307 ~r~r,~ G rnrJU~r~r,rrr~r~r,rrr~ AlIli~U
308 AG~UU rnr~nr~rr.rrr~r~rrm~ AA~C
3' 6 I~GAC rnr~ rrrrr~ r--,rrr.~l~ AGUU~U
319 U~AG ~ r ~ a ar ,rrr~ ~ ACAA~
322 C~tlUW rnr~T~r~r~r,rrr~ r-7rrr~a Ar~aA
323 ~a rnr~u~r~r--rr~ r~r~rrr~ AaGGACA
3 2 6 AUU~ ~rnr,~,T~r~ rrrrr~ a a rr,~--rr` ~ AIJI~AGG
334 G~aA~G rr~r~T~r~rr7rm~ r~r7r~
338 GGCCG~C rrTr~rrr~rr~r~ rr~ AuGaAlJc 380 All~G~ rnr~ rr~ rG~--rr,T~ ACtJCaCC
388 r~G rr~aTTr~rrr~ rr~rrr`` AI~J
389 Ar~G ~ 1 3S2 GG;~ C rnr~rr.~rr~rrr~ rr,~ma~ AGG~
397 I~;G rnr~nr~r~T,~r~r~rrr~ Ar~ JAG
409 ~A cnr,T,T~rrr~ar,r7rrr.~ AC17CWG
410 CACCAAÇ ,~,,, ,,., .I j_.a~rr~rrr.~ A~CaC~
4~ CC,iA ~ ,~r ~a~-r rrr~ A,~A
413 WAC~CC rnr~TTr~rr7rrr~ r~r-rma~ AGAa~
419 I~WCCal~ rnr~rr~r-rrr~7r--rm~ AC~CC~A
437 W~U ~rnr.~TTr~rrrrr.~T~_~rr,rrr~A AIJCCACU
440 A,a~C . ". Al~ ar,r,rrr~7. A.~UA~CC
447 ~JCUC rr~.7,\7~ rrrrr~a7r-,rrr.~a A~7U~U
454 ACCdGW rnr~rrr~rr7rrr~7~rr~rm~a ~CCA
462 1~ rn~~rr.arrr~'l'rr.~ ACC~
463 C~AC cnr~rr~rrrrr~arrrr~r~7 AACCAaJ
466 ~JGGC~;C ~ r~rrrr~_ Ar~aACC
479 rClJC~aA ~ll~ a~rr~rrr~a A~UG
480 ~JCCUXA rnr~_TTr~rrrrr~7~rr~rm~a AA~UUU
481 C~CC rnr-~Tr.~r~rrrr~rr,rr~r~ AaA~CW
497 CCaG~A rnr~u~r~r~rrr~r-~rrr~a A~CU
498 ~A rnr~7.~rrrrr~arrrrr.~ A~CC
499 C~ rnr~rr~rrrrr~`rr~rrr`a A.~WWC
500 AC~G rT~.aTn ~r.r~ rr,~rrraa AaAAIJGU
531 Ah~CCCtJ rnraT7r~rr,rrr~-~rrrrr~7,~ ACr~
538 GAAAAW rT7~r~TTr-arrrrr~ rr~rma7~ A~r,GCC~7 539 ~7GaBAA~ rnr~ rr~rrr~aarr~rma~ A~G~æC~J
542 I~IGaA rnr~aTTr~rr7rrr~r~r-rrr~ AWAaGG
543 AUAI~A rTTr~-7r~rrrrr~rr7rrr,p~T~ AA~AG
544 ~ rnr~rr~r~rm~arrrrr7~ A,~A
545 Ut~u7AuiJ rnr~ T~rrrr~ar~rrrr.~, AAAAUUA
549 U~AAWA rnr~TTr~r~rrr~ ~ 7 r~r~rrr~ aAA
551 G~AA~ rT~.7lT7r.~,rrrrr~ar.r~rrr.a~ AUUJUGA
SUBSTITUTE SHEET 'RULE 26) .~ .. 2183~92 wo 9sn3225 - ~ 218 ~ ' C ~156 554 G.~.A~. rnr;Anr~r~rrrr-~Arr,rrr-.~ A~U
555 ~ rnr~mr~r,rr~AAArr~^rrAA AA~
556 c~aAGcr rr~AT~rrrrr.AAArr~rrrAA AAAl~
560 CCCD~ rnr~An~r--rrr~ r.~ AG~,AA
561 ~JCCCl~J /~ ~ rr~rrr~r~-rrr-.7~ A1~.A
573 AAAI~ rnr~ r~rrrrr~ r-r-~rr~` ` AC~CC
577 CCI~AA rr~ rrrrr~ rr,rrr~
579 UGCC~ rnr.Anr~r~rrrr~ ` `rr~r~ ` .
580 ~GCCD~; r~ r^.rrr~ ~ ~rr.rrr.AA A
581 WUJGCC~ rnr~l~A~r~rr~ rrrrr~ A.aA~DU
588 GUGa:~G r~ c~r^rrr~r~r-rrr~ ~CC~
9? tJC~;GG~. rnr~r~rr7m-~a~r--7rrr~
S 9 8 ~-- rr~ Tlr ~ rr7~ ?~ 7 rr7rrr, A A AAWG~C
611 AGaA~ rnr~ rr7~rrr~a~rrrrr~
616 ~U~.AG r~ r~Ar~rrt~r:A~r--~r~A Al~al7 617 AI~Ut~A rlJr~Tr~rr.rrr~r~r.rrr~ AA
619 ~ rnr1~T7r`-r~ `rr7rrr-AA ~;
620 ~J rnr~ r~ r~
625 ~A~.~A cnr~rrr~rr7crr~ rr7rrr~ A~WaA
627 ~aAA r~ mArrrrr~ rrrrr~ A~
629 ~Ga rr~ ~r~r-rrr~AAArrrrr~ A~J
630 ADalJCUG I lr A~ rr~rrr~ AA~U
631 GA1~7 ~7r.Anr.~rr~aAAr,rrr~A~ AAA~A
636 A~JaClJGA ~lll "'A "' '.~ ~ AaArrCrr~ AIJC~aA
638 UGaWW r~nr~TTr~r~r^~rrr~ ~CUG
644 CUlrCA~IJ rrlr`'~`rr~rr~r--rrr~ AU~ 7GA
647 AUAC~JC cr,-,r~r~r.~rrr~r--rrr.AA A17GADUC
653 A~AA rrr:2~r~:~r-rrrr?~ ~ ~rrrrr` ~ AC~CaA
655 GGAGGAA, c~r~ tr~r--,rrr~r-~rr~ A~C
656 ~A crlr:Anr~-rrrr~rrrrr~ AAllaa.
657 CO;;GaGG rnr~r~ ~r,rrr~ r~rrrr~AA AAA~C~
658 CC17GGaG rnr-Anr`r~rr`~rr~rrr`~ A~ JAC
661 U~ rnrAnr~rrrrr~'~rrr~~r7~A Ar~AAA
6?2 ~C rnrArir~ rGr~rr~ ~ ~rr~r-l ~ AIW~7CGC
676 A~WA rnr.Anr~rr,rrr`'`rr~r~'rA~ A~
678 AAA~AAG rrTr~Tr~rrr~rr~r~rrrr.Z~A A~JCaA
681 A~AAA rnr~ r~ ~rrrrr~ rr7rrr~ ~ AGua~
682 ~AGAAA rnr`~r~r~rr--rr~ r,rrrr~ Aa~
683 A~aAGaA rnr~ rr~rrr~rr7~ AA~JAU
684 A.~AAGA rnr~ Arr~r~ r~r~A AaAAG~
685 AAU~AG rnr~rrrrr~r~rrr.AA AAAAA~
686 ~AU~AA rur~Tl~At-r~r~ rr~a3 A~AAAAG
688 G~ r~lr~TmArr,~rr~ rrA`rr~` AGaAi~AA
689 AG~AA r~r~rrrrr~r~r.rrr~ AA~aAAA
691 UA~ rur~r~Gr~rr~ rr"~rr` ` A~aA
692 UU~AG~J r~7r.~nrArrrrr.~rrrrr.~A AAT~AGA
693 G~AGU rrlr~T~r.Arrrrr-~T.rr~rr.~A AAAI~AAG
697 Gi~U r~ ArrrrrA~rrrrr~ AGU~AA
698 AGAPDGU rnr~T~r~Ar~r~rr~AAAt r7crrAA Aa~LAA
SUBSTITUTE SHEET (RULE 26) WO 95123225 219 218 3 9 9 2 1 _ll~,JI~ G 156 703 ~aG r~ -~rr~ ~ ~r 704 ~c~ca l U~ 3 ~ -rr~
708 Gal:a~ rr~ rr:~rr,rrr~ ^rcr~ ~Atr 715 G~AC~ rrT~r7r~rrrr~rr1~A A~
7~g r3~U rr~r.~rR~r~rr,~ r~rrr~71 AcaG~cA
720 A~AG~J rr~ r~rr.
724 ~ t~-rr~ a~
725 AUa~ rr~ r-~r`~ AAG~
728 II~AA~ rr r~ ar~rrr~A A~
7,~ r~7cr_~ -AA AA~.C;~
735 CA~I~ rrT~ ~rrr~
745 i~AIJ~ rnr~ rrr,:~A A~J
746 Aa~ rr~ r~rr-rrr~ A
752 ~.C~ `r~rrr~r--~crr~ r--~rm~3 A~
753 A~CC rr~ rl~--crr~a~ r~r-rrr~aa Aa~U
757 A~ ~ r~rrr~a~r'r~`` A~X~
761 Aa~tJ rr r-~r~ rrr~ ~m aa Al~
7Q ~aI~S crlr~rr~crr~ -7rrr aa Aa~C
765 .liMI~ rnr~r~rrrrr~rrrrr.a~ Ar~ADtJ
767 ~.h~ ~rrJr~ ~rrr~ frr-Z~a A~aA
768 A~ rrlr~rlr~rcrr~rrrma~ AA~A
769 rU~A rrrr~rr~~rrrr~''r~rrr`~ AaA~IJ
771 Aa~ r, ~ r?~~ ~ rr~ rrrrr-772 t~Al A~ r~r~rlr~rr~rrr~--rma~ AaDMA~
773 A~ ~rr~r~,nr~ ~f rr.aa AAA~AA
778 AcaAca~ rrJr~r~ rrrr~ r-rrr~ ACPD~A
77g C~aAa. rrK~rrr~ rry~ A~WA
783 A5;~C rrl~`rl~r"~rrr```~.. ~:r~` ACaL1aAC
788 ~G rrlr~rr~r~
789 ~ a rrr~r~ r~r--fmazl J~.ACa~.A
791 G;~ r~rr ~r~r~ _r,~` ~ ~ ~` ` AGaAC~
794 ~CW~J rr~ r?~~rCcr~rrrr~
805 AG~JWC rrTr~rTr~arr~rrr~ rrrr~a A~D~G
-SUBSTITU~E SHEET (RULE 26) wo 95,123225 2 1 8 3 ~ ~ 2 ~ ~11~ 5 Ic~c Table 13: ~ouse lI,o HE Ribozyme Target Sequence 3t.EUI ~ g--t S-~IU~ C~ lt. ~ ~!a g~t S-CU-3 Po~ I t~ 03 Pos_t 1 o--8cG uClllJ c ClJ[lt~GCu 253 AGGGgc'J A GaCAuAC
11uC'JUcClJ ~ UGCuSaA 259 '7csAC~U a C~C-aAgA
12ClJ~CCW IJ GCus~aiG 269 G2AC-AaU C aAACUG'J
36GAAg clJ ~ CAGAGuC 269 G2AGAa~ c AAaCugU
36GcAgAcU u cAsAGJc 269 G~7aAU c aA~crJçU
3~MsccpU C AGAGuCA 287 uGGGGG~ A Cl;WGGA
43IJcaG~J c A~aspA 301 AAh~CU A llUCcAAA
58GGAD;ætJ U C~ acl:J 301 aAAugCU a u~aA
59GAIJGCW C VGCAcW 303 AUGCuAU u CCaAaAc 59srA 7Gc~ c uGcAcW 3 03 AugClJAI~ U CcMAAC
66CGGCAcU U Ga~J5lJu 304 u~-,CUAW C CAAaACC
a2~gAcucU c aGcVGUG 315 AACcl-W C aWAA}lA
91GclJç~W c uSgGCCA. }18 c~GUCaU U aAUAaAG
~12ugt~ U CCCAugA 319 I,WCaW A AUAaAGA
113gG~AW C CCA~ 322 CaWAA~ A A~A~
141Ga~ U GaCa~:aG 330 A~aAAU A CAlJlJ'GAC
141GAçACcl7 ~7 GaCAcas 334 AaInU~ 13 GACCGCC
158çUCcgCU C AcCGA~C 334 AAU~C~U u GACcsCC
167cCGASCU C ~JGu~GAc 384 A5sCAsU ~ CCUsG~
196~GclJ U CC ~WcC 385 SSC~ C CUsGAuU
197GaGGcW C C~JcCC 393 Ct~sGALU A CC~--aA
197c~AGGCuU c CUGuCcC 405 C~AGAGU U cCUUGW
202WCCWJ c CClJ~cLC 406 AaGaGw c CUUG~G
202wcaw c CcllAcuc 409 ~UUcC~J U G~WsA
206UGUCccU a cuC UAA 481 UccCAA~ u I~AsWA
212~ ~ aAAaUCa 482 cAcAAW U APgWaA
212UacuCAU A AaA~IJCA 483 A~ AAUW A AgWaAa 218lJaa~l:~ c aCcAGCU 483 AcalWuU ~ aGll~AAa 218~aAAAAU C ACCAgCL7 4gS AAA~UgU c AA~AU
218UaAAaAlJ c acCAgCU 553 G~GuuU c C213hlJAU
232ua~ U GGaGMA 557 uuUcCaU U UauaWW
241S~AGAAAIJ C WWCAGG 564 W~uAuU u alJslJCCU
241S~A~AaAU c I~UucAGG 564 WAuaW u Aus7UcCl7 241~7a~AaAU c WUCAGG 565 uaUADW A ugUCCuG
241~AsAAAU c waQ~ 565 UP.UAuUl; a UgUCcUs 243~aAAucl:J ~ UCAGgGg 569 WuAUGU c cUWaGU
243Gi~AUCU U UCAGGGg 569 ~ l.~a c crJwasJl:r 2~4A~AlJCW U CAGGGsc 613 AAAGuGU u uaaCCW
245~AUCDW C AGGGgcU 61~ AAsUGuU u aPCcWW
SUBSTITUTE SHEET (RULE 26) W0 95123225 Z21 2 1 8 3 ~ 9 ~ 56 620 ~ACc~ u uU~¢UAU 1407 ccaguaU A CUcCAGg 793 caAG~C~ u UGuGcA~J 1407 ccAgU~ a CUCCAGG
816 C~Gag~J a ~aJCcc 1410 gaUU2CU C CAG~AA
818 G~gullAU A CUCCCUC 1434 AUgCOUU IJ al:JuUaAU
825 AClJcCcU c CccC~CA 1434 aUgcUu~J ~J Al~ Au 825 aC~JccCJ c CcCclJCz 1434 al7gcuUU u AuU~AU
839 AuCcuc{J ~ cG~GC~ 1435 ~gC~;J A ~utJ2AUt~
840 uCcuc~U c G~C~u 1435 ugcU~ a u~AaW
863 c~ U cC~GCu 1 438 OuaUA~ U AAuUcus~
864 AAgl~U c CU~GCug 1438 uU~aU ~ AAUucUg 864 AaGt~AW c caçgCug 1439 UC1~13~7 A AUucUgtJ
913 ~AaCJCtJ U GGucCzG 1443 UUU2AuU c ~GuaAGa 917 ~cUucgt~ c CA~LGG 1447 AUUCU~ A AgA~u 957 WagcA~J c ClJWcUc 1458 ugaUca~J z l~UA
960 G''AuccU u ~c~JcCuA 1458 ugWcAI~ A u~WA
960 Gca~JcC~J u uC~JCc~a 1460 UucA~ u AUaUAuç
962 AlJcCwlJ c IJCc~JaGC 1461 1~cA[7AuU A l~aUGA
975 gcccCW u AgAIJAgA 1463 AUAu~ ~ ~lhIJGAug 987 aGaUGAlJ A cwAA~G 1475 AuGgAW c aG~AgU
990 ~auaCJ u A~ugaclJ 1479 AaUcaGU A AglJaAaU
1000 ~ c ~h~uGA 1483 aGuAA~ u AAUA~W
1027 CgggGCU U cC~gClJC 1483 aGl~U ~ AatJA~r 1034 UCC~Gc~ C ClJaUcuA 1484 G;~asUt7 A a~[JaUA
1037 ~gc~JCcU A Uc~J 1487 ag~UAA~ a WuAu~IA
1039 c~}ccuAU c UAACWC 1487 Ag~lUAaU A l~aaua 1039 c~JCd~alJ c UAACWc 1489 ~WaU U uAuUAcA
1041 Cct!AUcU A ACWcAa 1489 UUAaLLaU u JAWaCA
1051 WcAAuU IJ AauAccC 1489 UUAaUAU U IJAWacA
1148 uGAcWW u c~lJGJ 1490 U~AUaW u ~uUAcAc 1213 G~gGaU u WGZ~aa 1490 UAa~W U Al~uAcAc 1213 gc~GGAU u ulJg~AA 1490 ~Aa~W l:J Al~UacAc 1214 cugGP.W IJ UG~A 1491 A~PDW ~ uuaCAcg 1215 ugG~W ~ G~AG 1491 AAUAUul:J a i:JuAcAcg ~34 gG5U~ c ~Jccul~GC 1491 A~rla~ A UuAcAcG
1236 G~UTc{J c cu~7GCAG 1491 Aa~ClJ A ~acAcG
~'~7~ ugGGCC~ U AcWc~JC 1494 A~JuUAW a C~cgUAU
1276 gGG~ W A cWcUCc 1502 cACGUa~ A JaauAUu 1280 CWAcW c ~Ccg~Jg~ 1502 cAcg~ a ~IJaW
1298 UgAACW a AGA cA 1507 AUAlJAaU a ~c:7aaU
1310 gcAAh~ a a~ACcA 1509 AUA~uAU U CUaAuAA
1310 GCaAAgU a aA~Acca 1509 aU~UaU U C5AAUAA
1310 G aA~sU a AA~JAccA 1510 ~aAuAw C ~aAuAAa 1350 AaA~ A AAAUgglJ 1510 UAAuAW C ~aauAAA
1358 AAAUGt;tJ tJ ggGAuglJ 1510 UA'AuAuU c UaaUAAA
1370 UgUuaW C AGg~C 1510 UaaUaW C UAAI~AA
1375 ~U~U A UCAGggU 1512 aUaUJCU A AUA~C
1377 CAGgl}A17 C AGgglJCA 1515 UUCt~A~ A AAgC1~gA
1383 UCaGggU C AcUG~AG
1405 cccCAgtJ U ~UcCA
SUBSTITUTE SHEET (RULE 26) WO 95/23225 2 1 8 3 9 ~ 2 - P~ .'l 156 ~ ~ A "
r r r~
~_, L
SUBSTITUTE SHEET (RUL~ 26) L3'`i 156 ~ WO 95123225 223 2 1 8 3 9 ~ 2 , , , . . . , ".
.

g o -SUBSTITUTE SHEET (RULE 26) W0 95/23225 2 1 8 3 ~ ~ ~ r~ r ls6 ~~ f ~ ~ ~ f ~ ~ ~ f. A' .-- f. f ., ..
~1 . _ .
f ~ rr . .. ~ . f ,~ ~ , _ C
C
,5 E~
SU3Sr~TUTE SHEET (RULE 26) WO 95/23225 ~ 218 3 9 9 2 r~ 156 Table 17 Mouse rel A HH Target sequence nt. Position HH Target Sequence nt. Position HH Target Sequence 19 AAIJGGCU a c CaGSA 467 cCAGGCU ~ cugu~JCs 22 aGCUCcU ~ cG~cG~G 4 69 aaGCc~I7 u AGcCAGC
26 CclJCcaU u GcG~ACz 473 UuUgAGU C ~aGauCAs 93 G~uCUW ~ uCCCCl;C 481 ~GCGaAI~ C C~acCA
94 h~WU u CCCC~ 501 ~CCCC5 ;J uCAcGUU
100 ~uCCCCI~ C A~C~C 502 ~C~_C~U u C ~UC
103 CCC~IJ C ~uCCc ~ 508 UuC~cGU U CCIl/WAG
105 CUCAUCU U uCCcuCA 509 LC~l7 C CilPllAGA
106 ~ua3u u CCcuCAG 51'~ cGllUCC;J A UAGAgGa 129 CAGG"uU C ~iGgCCu 5~4 l:~CC~J A GagGAGC
138 GGgCCu~J A ~ 534 CC~CU A uGi~Cu~JG
148 ~ G~ C A~cGA~LC 556 ~CGcCJ C li~JCC
151 AÇ~I AU c GAaCAGC ~61 C~IJ ~ CC~GUG
180 A~CGal7 U CCGCrJAu 562 UCUGCW C CA~7GA
181 UGCG~U C CGCUAuA 585 ~ u AGcCAGc 186 llUCCGCU A uAAzUGC ~98 GCCCCU C CuCC~Gz 20g GGCGCtJ C ~GCGGC 613 CcCCUG~ C Cl7~uCaC
217 GCaG~AlJ u CCuGGCG 616 CGWCCU c uCzCAUC
239 CACAGAU A CC~CCAA 617 gucCCW C C~ agCC
2 62 CCACCAIJ C ~AGaUCA 62 0 CCUI,'CCJ C i~sGaug 268 UC~aGAU C Aa~XCU 623 UCCUsc~ u CC~l;CtJc 276 AAI;GGCU A CACAGC~a 628 A~'CCsAU u ~U~;AuA
301 UucGaArJ C UCCC~7GG 630 CCsjAUu~ 'J 'u~AAc 3 03 CGaAUCU C cca(~C 6~ 1 CgA~ rurJlJ IJ GauAAcC
310 CCC~GIJ C ACcaAGG 638 ~CcAU u G~7GuuCC
323 GGcCCCtJ C C~Ccug~ 661 CCGAGCU C AACWCU
326 uCCaCCJ C ACCGGCC 667 ~AGAU C tiG CGaG
335 CCGGCC~ C AuCC~A 687 CGgAAClT C t~GGgAGC
349 AuGAzaJ ~ G~gGGgA 700 GC.. GCCU C G WGGGG
352 AGaUc~J c GaAcAGc 71; AUGAGAU C UUCu~J~7C
375 GA~J ~ CIJAUGAG 717 G~AUCU U C~lJgC17G
376 AUGuc~ C IJccGg G 713 AGalJCW C u~ rW
378 GGCUaC17 A l~GaGGCU 721 U~cUCCC c C uUGcG
391 CO~cCU C UGCCC~G 751 AaGA~ U G~WW
409 GCaGuAU C CAuAGcU 759 GAG-~JG;J A ~iU~CG
416 CCgCAGU a ~7CCAuAg 761 GG~U U UCACGGG
417 CALAGc~ U CCAGAAC 762 G~G~;J IJ CACGGA
418 AuAGcW C CAGAACC 763 ~UW C ACGGGAC
433 I;GGGgAU C CaWt~JG 792 CG~CU C CW~UCu 795 GG.--UCCU lJ WCuC~A 1167 GAUGAGU U ULCCCCC
796 G~ CW IJ l~ AAG ll6a ~UG~U ~ uCCcCCA
797 C~C~uuu U CuCAAGC 1169 ~ u CCcCCAU
798 ~JCCU~W C uC~AGCU 1182 AUGcUW U ~CCaUCa 829 ~AU IJ GrJ~GWCC 1183 UGcUGUC z CCaUCaG
SUBSTITUTE SHEET ~RULE 26) WO 95/23225 - ~ 2 1 8 3 9 9 2 1 ~ s 156 834 AUOG~JW IJ CCGGACu 1184 GGccccU C CUcCUGa 835 U~ UU C CG-ACuC ~187 GUc~CutJ c CUcaGCc 845 GaCuCC~ C CglJACÇC 1188 DU~CCaU C ~GGGCAG
849 CCtJCCglJ A CGCcGAC 1198 GGsAGutJ u AGuCuGa 872 cCA~ C CD~uCG 1209 CAGcCCU a caCCUUc 883 UuCGaGU C IJCC~^ 1215 cuGGCC~ ~ aGCaCCG
885 CGaGUCU C cAl~caG 1229 GGuCCCU u CCucAGc 905 G-GGCCU ~ CuGAuCG 1237 CCCAscU C CUGCCCC
gO6 CGGCCDU C uGPuCGc 1250 CCAÇcCU C CA~C
919 GcGAGClJ C AG~GC 1268 CCCaGCU C CuGCCcc 936 AUGGAgU U CCAGlJAC 1279 CCAUGGU c cCuuCc~
937 ~ C c~acu 1281 ~GgcU C A~gcG
942~CaGU A Cu~A 1286 AUgAGuU u UccCCCA
953GCCucAlJ c CAcAuGA 1309 CuCCUGU u CgAGUCu 962AGA~ C GcCACCG 1315 cCCC~ u CDi~aCCC
965CasU~cU u gCCaGAc 1318 CAGUuCU A CCCCgG
973ACCG~ GaaGaGA 1331 gGGuCCU C CcCAGuC
986 GAgACc~J u c~A~;agu 1334 Cuu~hlCC C AaGC~Ga 996 AGGACc~ A ~ACC 1389 ACGCUOEJ C g&~GCC
1005 GaGaCC~ ~ CA~G~Gu 1413 C~ U ~GAi7GcU
1006 AÇACCUU C AAGa~.~A 1414 ~GDU ~ GA~JGc~G
1015 AGAh~Arr C AI~AGA 1437 GGGGCCU ~ GCUUGGC
1028 GAAGal~J C CDU~Aa 1441 CC~ Ir GGCAACA
1031 GAG~CU ~ ~aùGG 1467 Gg2~iu~u ~ CACAGAC
1032 AG~i U CAauGGA 1468 gaGWUU C AC~GACC
1033 Gl~CC~J C AauGGAC 1482 CUGGCAIJ C uGUg&aC
1058 CCGGCCU C C~CcCG 1486 CuUCg;~ a GggAACU
106~ UaCACCU u GAucCAa 1494 GACAACU C aGAGUDU
1072 ÇgCGuA~ U GCtX ~JGC 1500 ~CaGA;~ IJ ~CAGCAG
1082 UGUGCCtJ a CCCG~Aa 1501 CaGAalJ 1:7 CAGCAGC
1083 aaGCCDU C CCGaAGu 1502 2GAGDt~U C AGCAGCU
1092 CG~AaCU C AaCC~ 1525 gGuGCaU c CCD~Gu 1097 Cl~Aaccr U Ca~JCCC 1566 A~ A CCCUGAa 1098 ~ C ~WCCCC 1577 UGAaGCU A UAaCUCG
1102 CWaJGU C CCCAAGC 1579 AaGCUA~r A ACUCGCC
1125 CAGCCCU A caCCDUc 1583 UA~ C GCC~JgGU
1127 GCC2~AIJ a sCcWAC 1588 CUOICCU A GaGAggG
1131 cAUCCCU c agCacCA 1622 CCCaGCU C C~GCcCC
1132 AcaCCW c cCagC~T 1628 ~CCUGCU u CggUu~G
1133 UCCaUdJ c CagCul7C 1648 CGGGGCU u CCCAaUG
L137 WCACulJ u AgCgCgc 1660 c~GaCCcr C u7ccCAG
1140 cC2gCalJ C CCUcAGC 1663 cuClJgCU U cCAGGuG
1153 GCACCA~ C AACU~UG 1664 uCC~CW c CAGGuGA
1158 A~CAACU u UGAnGaG 1665 CUCgcW u cGGA~gU
1680 GAaÇAC~ U CDCC~rCC
1681 AaGACW C UCCUCCA
1683 GAC~ C C~CCAW
1686 WCO'CCC C CAWGCG
1690 CCUCCA~ U GCGGACA

SUBST~TLITE SHEET ~RULE 26) WO 95123225 227 r~l/lL 'I '' 1705 ~Gal:~ C ~UC
1707 G~CU C uG uC~Ju 1721 Ul~ C AGa~G
172~ G~alJ C AG.~CtJ
17.1 Al~ C CCaaCGu 1734 ~JCC~ A~uGc~J
1754 C~GuS7CU C CCaAGAG

SUBSTITUTE SHEET (RUL~ 26) WO 95123225 218 3 ~ ~ 2 r~l, ~ I )iSG

Ta~le 18 Human re/A Hl~ Target Sequences nt. Position HH Target Sequence nt. Position HH Target Sequence lg AAI 1~ C G~JCWUA 467 GC~ A I~UCA
2a GGCUI:GU C ~ JAGUG 469 AGGC~ C A~^
26 CG~ tJGU A G~rGCaCG 473 ~.DC~ C AGC--CA~
93 ~GU 17 CCCCCIJC 4al i~u C CAGACCA
94 A~ C CCCCCCA S01 iUCCC:lJ 'J CCA~G~iU
100 UCCCCCIJ C A~JCllUCC 502 ACCCC;i;J C CA~iC
103 CCCW~ C ~ CGG 508 CCC~a~;J '.J CC~G
105 C~ 17 CCCGGCA S0g CCAaG~ C C~I~GA
106 UCa~JCUU C CCGGCaG 5~2 ~'C~J ~ ~A
129 C~GCC17 C ~GGCCCC 514 I~JCC~ aGc 138 GGCCCCU ~ ~ 534 G;~J A CGACCJG
148 UG~ C A~GC 556 XCGGC'J C ~C~JCC
151 J U GU;CAGC 561 C~JC~--U ~7 CCAG--7~G
180 AI~CGCU U CCGa~aC 562 I~Ct~'U C CACWGA
181 ~3CGC017 C CGCOACa 585 G~alJ C Al;GCAGG
186 ~JCCGCIJ A CaAG~JGC Sg8 G.,CCCCU C CG~;C
204 G~ C CG~C 613 C~CC~JG;7 C C~CCX
217 GCAt;CAU C cclUæCG 616 C~tJCCU IJ CC~UC
23g CaCllGAU A C~aCCAA 617 UG~ C cucaucc 262 CCA~IJ C AAGaucA 620 CC~aJ C AIICCCAU
268 ~AGaU C AIWGGCU 623 I~CCI~W C
276 A~J A Q12aGGA 628 A~TCCCAU C UU~A
301 UGCGCalJ C lJCCCtJG5 630 CCC~UCU U ~JGACaAi:r 303 CGC~ C CCJGGUC 631 CCAI~t~U U G~CAAUC
310 CCCUGGU C ACCMG5 638 ~AU C G;JGCCCC
323 GS~a~ CCU C ClJCaCCG 661 CCGBGCa C AaGAlJCU
326 CCCIJCCU C ACCGGCC 667 ~U C ~3CCG~
335 CCGGCCJ C ACCCCCA 687 CGi~AA~ C ~C
34g ACQGCU IJ G~aA 700 G~GCCU C G~--'~
352 AGCCCGtJ A GGAaAG5 715 AUGAGAU C ~UCC~C
375 GA~1GGClJ l7 Ct~G 717 G.~ 1~ CCUACX
376 AUG~J C UAI~;5 718 AGAUC~U C C~laCtJGU
378 GX~CrJ A IJG~ 721 ~ CCU A C~'WGUG
3gl C~5CU C UGCCCG5 751 AG;a~AU ~ G~J
40g GCDI;CYW C QCaGDU 75g Ga~JGU A ~ C5 416 CCACIIGU ~ UcCAGaA 761 G5tJG~ ~7 IJCIIC--WC~
417 CaQGUtT ~7 CCIIGAAC 762 G;~DtJ U CACGGGA
418 ACAGUtlU C C~aACC 763 ~DW C ACG5GAC
433 UGGGaW C QG;JGlJG 792 C13AGGCU C C~JI~UC5 795 G5CUCCU U UUCGQA 1167 GAI,~.7 U l.'CCCACC
796 GCal CUU IJ UCGQAG 1168 ~A~7 U CCCACQ.
797 CUCCt~ U CGC~AGC 116g I~Ga~ C CCACCAU
7g8 UC~uuuu C GQI~a7 1182 A~GCJG;J U ~iC UUCJ
82g UG5CC~U U GUGUUCC 1183 I;GGliG~ ~ CCUUCU-5 834 AUUG~W U CCG5ACC 1184 G~IU C CIJ~'GG
SUE;STITUTE SHEET (RULE 26) ~ WO 95/23225 229 2 18 3 9 9 ~ r ~ 56 835 ~ C CGGACCC 1187 G~JUCCU U Ct7GGGCA
845 GaCXW C CCCh GC 1188 I~UtJCCUU C UGGGCaG
849 CC~CCCU A CGCAGAC 1198 GGCAGAU C AGCCA~;G
872 GCAGGCU C C~WGCG 1209 CAGGCCIJ C GGCCaUG
883 K CGUGU C ~JCCA~JGC 1215 IJCGGCW U GGCCCCG
885 CGUGUCU C CAI~CaG 1229 GGCCCCU C CCCAAGU
905 GCGGCCU IJ CCGACCG 1237 CCCAAW C C~--CCC
906 CGGCCUU C CGACCG~3 1250 CCAGGaJ C CAGCCCC
919 G~'-lJ C AG~7GAGC 1268 CCCUGGJ C CAGCCAU
936 A~GGaA~J ~ CCAGliaC 1279 CCU~GGtJ A ~JCAGCt/C
937 ~ C CAG~JACC 1281 AT,~ C AGCUC,'G
942 ~CCAGU A CC~;GCCA 1286 AUCAG'IJ C ~GGCCCA
g53 GCCA~UJ ~ CaGACGA 1309 CCCCUW C CCAGUCC
~62 ~CGAU C G~CACCG 1315 UCCC~W C C~CCC
965 CGAIJCGU C ACCGGUJ 1318 CAGat W A G.--CCCAG
973 ACCGGAIJ U GAGGaG~ 1331 AG~CCU C CUCAGGC
986 Gi~aAl w A AAAI~GAC 1334 CCCUCC~ C AGGCUW
996 AG&~ A17 A UG~GaCC 1389 ACGCUG~ C AGaGGCC
1005 GA~ U CAAGAGC 1413 Ct~GCaOET U UGaIJGaU
1006 AGa~ C~ C A7~GAGCA 1414 UGCAGUU U GaDGaIJG
1015 AG~W C A~ 1437 GGGGCCU IJ G~GGC
1028 GAAGAGU C C~JClJCAG 1441 CCtJC~ W IJ GGCAACA
1031 G~CCtJ lJ ~ 1467 GCUGt~W U CACAGAC
1032 AGtrC~l;J ~ CAGCGGA 1468 CI~UGI~U C A~ACC
1033 GUC0Ua C AGCGGAC 1482 C~GCAU C CWCGAC
1058 CCGGCCU C CACCD~G 1486 CAIJCCW C GACAACU
1064 uccacw C GaCGCAU 1494 GAGAACU C CGAG~
1072 G~CGCAU U GCI~-- 1500 UCCGAW U UCAGCAG
1082 UGUGCW U CCCGCAG 1501 CCGAGUU ~ CAi~AGC
1083 G~;C0U C CCGCaGC 1502 CGAGUI~ C AGCAGCU
1092 CGCAGCU C AGC~CU 1525 AGG~W A CCOWG;;
1097 Cl~ U Ct7WCCC 1566 AUGGAG~ A CCCUGAG
1098 IJCAGCt~T C ~UCCCC 1577 I~GU;GW A UAaCUCG
l l 02 C~JCI~W C CCCAAGC 1579 ~ A ACtlCGCC
112~ CAGCCCU A UCCCt/t;U 1583 UAI~ACU C GCC~7AW
1127 GCCC~JAU C CCD131JAC 1588 CUCGCCU A WGACAG
1131 ~JAUCCCtJ IJ ~; G~JCA 1622 CCCAGCU C C~7GCCCC
1132 AlJCCC~ltJ U ACWCAIJ 1628 UCC~JGCU C CACUGGG
1133 ~CCCI~U A cwca~c 1648 CGGGGCtJ C cccaAUG
1137 Ut;~W C Al~ 1660 AUGGCC~J C C~JCAG
1140 ACGUCA~J C CCt~AGC 1663 GCC~J IJ UCA~G
1153 GCA~ C~UJ C AACtll\l~G 1664 CCU~ U C~GGAGA
1158 AIJC~ A IJGI~GAG 1665 CUCCU~ C A~
1680 GA~J U Cl~CtJCC
1681 AaGAC~U C UCCO~XA
1683 GA~J C CUCCA~U
1686 UUCUCCIJ C CAI~JGCG
1690 CCUCCAU U GCG'`ACA
1704 A~GACU U C~JCAGCC
SUBSTITUTE SHEET (~UlE 26) WO 95/23225 2 1 8 3 9 ~ 2 r~ 156 ' 230 1 1~05 U~ C UCAGCCC
1707 ~7 C AOECCC~
1721 GCa~ C ~IJCaG
1726 G~JCaG~i7 C ~:C~
1731 A}~ C C~AGGG
173~ AGCtJCCU A A~J
1754 Cl~GCCCU C CCCA~

SUESTITUTE SHEET (RULE 26) ~ WO 95/23225 231 21~ 3 S 9 ~ r~ 156 Table 1~
Mouse rel A HH ~ibozyme Sequencs nt. HH Riboz~me Sequence Sequence 19 ~JCC~JWG r~rTr.aT~nAr.~^rrr~ 7 ~r^~` ~ AGCCAIlU
22 C i~cr i~CG rrT~ Tr~ar~r~^rr~rr~.^r~ AGG
26 l~GL7CCGC rrTRarTr~r~rr~aar^rr rAa A~GG
93 G~GGGGA rrTrarTr~ rrrra a ACAG1U.7C
94 UGUX;GG rnr~rTnarrrr~rrrrr~ AAC~sAU
100 GaAA~ t~rTr.AT7nar^-rna~arr~raa AGGGGA,a, 103 AGGG~AA rrTr~arTr~rr-rr^~7~ar~r~rrr~ A~0GGG
105 I~=L7A rnrarTr~arrr~r~r.Aa~r~r,cr`r`` Ar~aUt7AG
106 r~G rnrarTr`^^^rr~'`"-^rrr`` AA~IJGA
129 Ar~GCCCA rrT~ rr`~ `rra~ AAGCCIJG
138 CI~CC~ r~rTr.~-rTrarrrma~arrrrr~ AAGGCCC
148 G~(G;UJ r~TT~rrrr~nrrr~r~7~ ~CCA
151 GCD~C rrr~`~Tr;~^^rrr`7~ rrrn~A A~ .7 180 Al.7AGCGG rnnaTTr.Arrrrr~r~^,rmAA ATJCGCAU
181 ~iI~GCG rnraTTrar~rrrr`~r~rrr~ AAUCGCA
186 GCADO~A r`T,7r`~ arrrr'~ rrr~a AGC~3GAA
204 GCCCGC~r rTTr.aTTr~Ar.rrr~r~Arrrrr.aA AGCGCCC
217 CGCCAGG rTTr~ rr` ~ A~C~JGC
239 ~GG rrTr`~Tranrrrr`~ r~`~ AUCU2t.7G
262 1.7GAI7C~J rn~ Tr`'7rrr~`r~`-rr`~ ~UGWGG
268 A~lJtJ rrT^`-7narrrrr`~ rr.aa AIR3D;JGP.
276 O'CC~JWG rnr`~Tr,Arrrrr`7larrrrr~ AGCCA~
301 CCAGGGA rnr`'T'~`'`^-rrr-aaArr~rCr~aA A~CG.~A
303 Ga~GG rnnATTr,~rrrrr~ AGa~ CG
310 CC~tJGGU rnr`~7narr~^~ rrr~ ACCA~GG
323 ~CP.~L7 rrTr~Tr-ar~r~rrr~ ^^rr~ ~ A(~7CC
326 GGCCGW rrJr~TTn~r~rrrr~ r~rrrna~ AGWGGA
335 UGUGGW rTTr`'Tr,Pr-rrrr`''`'r-rCr-Aa Ar.;GCCGG
349 TJCCC~aC rnr~-7r.arrrrr~ rr,r~ 7 352 GC~JC rrT~TTr~^rrr.~r~rrr,r~l~ AUGAUCU
375 Cr,JC~7 rrTr~Tr.arrrrr~ rr~-rr~ ~ AGCCA~JC
376 CUCCGGA ~ rrrr.~A Ar~ccAl7 378 ACCCUCA rnr~ATTr.Ar^rrr~ ~ ~r~rrr~ T~ AG~GCC
391 C~GGCA rTTr~rTrarrrrr~ rrrr~raa AGWCAG
409 ~ C~UG rT~Tr7~rr,rrr~ r~rrrraA AUA~GC
416 CUA~7GGA rrJr ~TTr~ r~ r~rrr~7Aa AC~CGG
417 WUCUGG r~TTraTTr~ r,~:AAar~rr~rr~a Ar~l7G
418 GGUrJCUG rnr-aTTr~ r~rr~a~r~r,~rr~ AAGC~7 433 CaCACUG rnr.~TTr.arGrrr~rr~ AUCCCCA
4 67 CGAACAG rrJr.aTTr~ ^^rrr~ ~ ` rrrrr~ 7~ AGCCUGG
4 69 GCUGGcl r rTTr~TTr~p rr-rrr~ ~ ~ r~r~rrr~ ~ AU~3U
473 CUGAUCU rTTraTTrarrrrraAarrrrr~A ACO~AA
481 uGGr~ G rnr~Tr~r~rrraaarrrrraa ADUCGCU
SUBSTITUTE SHEET ('AULE 26) W0 95123225 2 1 8 ~ ~ 3 ~ /~ . C ISC
t . 232 sal AACG~GA frTr7~ -rrr~ r-,r 502 f~AA~G frTn~rTr~r~-"~--ff~
508 C~GG frTr~ f~ r~r~f~
503 ~UIaG fr7~ r~ 'r~`~' AACG~JGa 512 IJCCI~ frTf.~TTf.ar--,fff ~7 ~rr~,~A AG~;aACG
514 GC~rcc~7c CrTr~TTr~ r~
534 CAA~ fr~Tr~ f~``~f.frr'~ AGrJCCfC
556 f~GaA~Ca f~aT7f,Ar .rff.AAArr~rr~
561 CACCI~G frr~rTr~ fff~ ~frf~ A~G
562 ~G frTr~ -f,t~l~AAff, '85 f~G~'lJ frJf~Tr~--,rrr~a~f~ rrr~r~
538 ~aGGaG frTf~Tf~f.~;--fr`~ar--fff~ GGCCC
613 f~a~aG frTr`~Aff~'f~ .rrf:~
616 G~G~ rTTr~TTr-Arf7r~rf-7~ f-rf~A~ AGG.a~
617 GGC~G~G frTrATTf~r ~:AAAr ~` ~ 3GGac 620 CU~ frT''`~`~--fff`~f~7Crr:AA ACGaA~
623 GA~IGG frTr`~ frf~ AA A~Ga 628 ~D::AAA frTfATTr~r---r-rf~
630 G~laD~ frTf ATTr` rr~fff ` 7. ~rf~ff ~ A AaAUCGG
631 GG~iL7C frTrAT7r~r,~frr~ ~rrrff,AA AA~aDCG
638 f~ACAC rnrAT7r-~ rrrfr~ ~ 7, rr~` ~ AI~GCC~
667 Ar~wclw fl7r~rTr~r--rffAAAfrfffAA AGCIJCGG
667 ClJCGGCA fnr~rTr~rr~rf~rrrfr~ AUC~GA
687 r~ frTr~r~rrcrf~ f~rrfr` A A~OUCCG
700 CCCCACC fr,7r~r~,~rfr~f,r,ffr~ AGGc-A~c 715 f,CAAGAA fTTr~ ~rrff~7r~f~rrr~ ~ .1i.U~
717 r aGcaA~ fnf~-Tr~rrrrr~r.~r,rrr~A Ar~UCUC
718 A~A~;CaA frTr~TTr~rrrrr~rffrr~ a~7 721 CGCaAIJG frTr~ rfr r~ ffrrr.A~ AGGAfAA
751 ACA~ CtJC ~rTr~rr,~ 7rrrrr~ A~l;tJCUt~
759 CWGaAA fnr~Tr~rr7rrf~r-~`~ AC.'~UC
761 CCCGrJGA fr7r~TTr~rr~rrr~ffrf~ Aliacacc 762 ~,'CCCGX f~Tr~TTr~ r~rrr~rrrrr`~ AAUACAC
7 63 f ~,7CcCGa f rTr` 'Tr` rrrrr` ` ~ rf f f r` ~ a AAI'ACA
792 AGAaA~ rrr.ATTr,Arfrfr~rrrrf.~ AGDCG
795 ~U~A frTr~rTfArrrfr~Arrfrr~-. A~CC
796 CaUl~at;7A fnf~ATTf~ rr~rf~ rr~f-rr-~ AA.. ~;GAGC
797 GCU~JGAG fnr~TTr~rrorr~ r~rrrr~ .Tl}U~
798 AGa~GA frTr~7Tr~rr~crf~ rr~rrf~ AAAAGGA
829 GGAACAC fnrATTf~rrffr~AAr.rrrf~ ~ AUGGCrA
834 AGUCCGG ( ~ rr,frr,AA ACA~U,U
835 f,a~7CCG frTf.ATTf.AfrCrf~'~r~'rfrA~ AACaCAA
845 GCGUA~ G fTTrArTn`rrrfn~7.rrrfr,A~ Af,G~UC
849 f,~JCGGCG frTr~rTr~rr~rfr~rfrrfA~ ACGGAf,~.
872 CGA~LCAG fnr~rTr~rfrfr~ rr~rff~AA AGCC~G
883 GCA~ fnr.~rrr~r~frfr` ~r~f,--rn~A ACUCGAA
885 C~G fTr~7Tf~r~f7rrr~ r~r~ffr~ AGACUCG
905 CGAUCAG frJr ArTr~r~f~rrr~ rr7rr~r~ AGGCCGr 906 f,CG~WCA frrATTf.~rf,ffr.~AArfrrf~ AAf,GCfG
SUBSTITUTE SHEET (RULE 26~

WO95123225 218~ P~ ~ -15C

91 9 GCU~ ~ rr~ 7l ar~r~
936 G~ at~r~r~r,~ ~ Cau g37 ~ah ~DTTr7Dr~ r~
94Z ~AG ~TTr~ ~r~
9!~3 IJCA~G ~rJ~TTr`~r'~ar 7~rrDa ADGaGGc 962 CGG;~ arr~ AIJC~CU
965 G~GC ~ DTTGpm~r~ r,~--~DD Aû~D~-G
973 ~ U~JC rTTr~ prr~ r-r~
986 ~G f~~ r--~`~ CUC
r 996 G~ r~.lr Dr--~ ar ~ AGGrJCcU
1005 ulJaJ~ ~r~TTr~Y;rrr~r-~ G~UC
~ 006 ~.CU~J ~ r~r~ D~^^rrr~
1028 liDGAAAG ~ arr~Dl~ UUC
1031 CCU~ ~ ~J~ rr"~ ~ Ga2uc 1032 ~JCC.a,wG ~TnaT~.r~r.D~ AAG~
1033 G~ C~ ~T~ rr,~a7~rr~~7l AAA~C
lOg8 CGGG~7UG 1~ `'1' '~1 ~`7'r^~`~ A2GCCGG
1064 ~C ~ f ~ rr~r~.
1072 GCal ~;C ~ u~ r~7~ UGCC
losa ~ Tr~r~r~ r~t--r~-^~ ~GGCa.
1083 Al U~JC~ ~DTlr Dr~ rrr~DD
1092 AG~A~ rTT^' Tr' rr~r.~r~`~ aOE~ G
1097 GGGa~ t~!r~ Tr~ rr~ AG~aG
1098 GGG0sA ~DTT^~rl~a~rr~`~ AA(3UCG~
1102 G~GGG ~~ r~ r--t-rr~ aP~
1125 GaaGG~G ~TTr~rr~ r-,~
1127 G~aaGCC , ~ ~rrrD D ~GC
131 I;GG~ l7lr~'rrrr~a~rrf-rr~
~132 .a~ nr.DTTr~rr~r~r~rDD AAGG~W
1133 ~ v~ r -f ~ rr,~
1137 GCGCGC~ rr~rr~D~ ~PG~AA
1153 CAAAGa~ rTTr~DTTr~r~arrr~rr.DD PæGUGC
58 C2QIJC~, rTJ~ TTr~rr~r~
1167 G~;;aûGaa rTT~ ~r~rr~fjD~ A~C
1168 ~ ~`~D(ir~rr^rr~ AAc~U
1169 ~UGGGCG rTT~DTTr~r~YY3DDf_r~ AAaC~.
1182 I~UJGSU rTJraTT'-`r~```'-^-rr"`` Aa~
1l83 C~JG~G rTTf~ rrrrr~rr~r~ AP~
1184 T~ca~G rrr~TTr~ ^^~r` r^^r~ a A5GGGCC
1187 G53JGaG rTTr~TTr~rrrrr~ rrCW'`a AaGGGaC
1188 C~JGCCC~J rTTiDTTr~r^f~-rrrr~ WK~.A
1198 U~ r~rDTTr~^^~DD~r,r~ M~CCC
1209 ~G f~rDTT~rrrr~ 'rrrr~A A;GCUG
1215 CGGt~GC[J rT-~r~prT~`~rr~ rrrrDa AGG~
1229 G^~uGaGG rTJ'r:~TTr`^^^rr~DA~-~DI~ ,~CC
'~237 G~GG; aG (~ TTr~rrrrr~rr~rrr~D ~GCDGGG
1250 G.aGCCUG r~J TTr-Drrf^rrDDI~r~r^rr~D AG5C~G
SU8STITUTE SHEET lR~lLE 26) 218~9~2 W0 ~5123~ .. ,7~ 56 1'68 r~G ~r~r^rrr~aAr~^rr~T~ A~GGG
1279 A~GaAGG rnr~ r~^rr~T~ r~
1281 CGCal~3' rnr~7~r~r^rr~A~r^~rrr~a AGCCCAC
1286 '~GGGGa rrTr~7~r~r,rrr- ~r^r~ AA AA~ r 1309 AI~AC~G t~TTr~7rr~r^r~rr~aTr^~rr~ AC~GGA;
13~5 aiG~ rnr~;rr.~r.Gm~T~rr~rrr'~
1318 CCGGGG~J rrr~ r~a~ar-~
1331 Ga~ rnr~rr.a~7rrr`'~rr~rrr`a AGGaccc 1334 ~ ,7 ~nr.~T~ ^,r,rr~r^^rr~
~38g GG~CC "- ~"~ r ^rr`' A~
1413 ~a rnr aTTr~r.. ^T~"~r,rrr~
141~ CA~ JC ~scAT~rr.~ 7.~r^~,AA .aAa,~A
1437 GccPa~c rnr?7~,r rT^^^-. ~ ar^r~ T. A~GCCcr 1441 ~rWGCC r~T~:T~Tr~rrrrr~a~r:r^rr,~A ~GC3 1~67 GUa~G t'nr`7T~'`rr~r.AAArST~:A~ ACaC~JCc 1468 rGUC~,~ ATrr~rr~ r~ r^~rrr~AA AAcac~C
1~82 GrJCc~Ca r~ Trr~ r~rrraAA~Sr~r.AA A~CC.~G
1486 A~rccc ,r~r~,~r,~a.. rrrrr~a ACCG~A~
1494 ~AAa?CU r l lr ~" ~ r,rT rrAa AG~C
150Q CUGC~a rnr~?~ rrrr~ rr7~AA A~.
1501 GC~G rrrr~Trr~rr~a~r-r-rrr~ AA~v'G
15Q2 A~C~7GCU rnr~ ^,rrr~AAr:rrr~a AA.a~,~cu 1525 A~ aC~ rnr~7rr~r~r:rr~ r~^7rrr~ ~aCC
1566 U~GG rnr~rrr~-r,r~-r.AA~r:~ rr~ CC~
1577 CG~ rnrAT?r~.rrraA~r.r:rr-~A AG
_579 GGcGA~r r~TTr~rr~Ar~rrr~?AAAr-~-~rrr~A ,a.
1583 A~ ;GC rnr~7rr~Ar:T~rr-~ar^~rrr~
1588 CCC~C rT?r~ r7^cr~7~arr~rrra7~ ~h?
1622 GCGGCAG rrrr.aT?r~ r~r,rr~ a~rrrrr~ a .;U~CUGGG
1628 Cr~aCCG r~.~Trr~rrr,r~7r:r^rr~, 1648 C~GG cn~ ~T7r~rr~7~r-- ~r~ GCCCCG
166Q C~WGCA ~ " ~ rrrrr~T il~;G~G
1663 CaCCtJGG r~ rrrr~ar^,rrrAA A~C.a.GaG
1664 UCASXUG r~ 7?r~rrrrr~ ~ ~c:~rrr?~ T. AAGCA~
166; ~Cc8?ccG rrr~7?r~r~7l~r7r-~r~T AAG~G
168Q GGaGG~ rT?r~7?r~r~rrr~r~T~r~Gr~ UC
1681 ~JtæAG~ r~?r~ r.~ rrr.AA AA~
1683 A~G~G rnr~rTr~r~rrr~r:~rrAI AGaa~.?C
1686 CGCaAUG r~ T?r~ rrrr~7r^rrr~ AGGA~AA
1690 UGCCCGC rnr~ATTr7~-::rraAAr ~ ~GG
17Q4 AGCACaG rrrr~rrr~rr7~rr~a-~rrccr~T AGUCCAU
1705 GAGCaGA rnr~nr~^:~r-T~ar-r-crr-A~ AA~CCA
17Q7 AAEaGCA ~`T rr"TTr~ ~ ^,rrr.~ ~ ~r^rrr~ 7~ AGAAGUC
1721 C~v~GaD~ rT~7Tr~A~x~rrr~AAar~rrrr~ ACD~
1726 AGGAG-^U rrrr~Anr~rrccr7a~r-r~ rr~A~ AUC~aC
1731 Accv~al; r~rr~rr~rr,rcna~Tir~r-^rr~A AGCUGAU
1734 AGCACC~ r~ AT'r~'-:rrrAA~r~rrrr`7 AGGAGCU ..
17~4 C~CUUGG rrJr`Ur`rr-rrr~ r~rTrrrA~ AGCACUG
SU~vSTlTUTE SHE~T (RULE 26) WO 95123225 ~ i 8 3 9 9 ~ r~1~1. s C156 ~ 235 Table 20 Human re/A HH Ribo2~yme Sequences nt. Position HH Riboz~lrme Sequences 19 Ui~CAGAC rnr~r. arrrrr~rrrrr,a~ A~cca~
22 CA~ rnr~ rrrrr~Arrrrr~ Acr~Gcc 26 cr,~GC~C r~nr~rr~rrr~ 7~rrrr~ ~ Aca~aCG
93 r~ GGG rr~r~-~rarr~ ,. 7~rrrrraA AC~JC
r 94 ~IGAGGGG r~nr~ATlr~rr^rr~ rrrrr~rl A~caG~
100 GQ~A~ rnr ~nr~rrrrr`'l~r,r,rrr~`-A~ AGGC~GGA
CC=A rnr~rrrrr~ rrrrr ~a AUGaGGG
10~ ~;GCCr~G rnranr~rrrrr.~A~r rrr~ AG~DGa~;
r,G r~narJ~:ar~r~rr~r aA~rrrrrA~ A~uGA
rrrrr~r-rrr~ AGGa ~G
c~c~ rnr~Anr:arr~rr-~arr7rrr~-aA AGGGGCC
148 GCIJC~UJ r~nr.~ rr~ r.. -rrr.~ JCUCCA
151 GCIJGC~JC rnr~rr~rr~ rr~rrr,A3 A~;Zwc~r 180 G~C;`G rr~r~r.~rrrrr~,rrrrr,AA A~C5a~.U
GCG rnr-arTt:arr7rrr~ r-~rr a~ A~.
186 GrAC~UG rur~Arrrrr~rr~rrr~a AGCcGaA
204 GCCCGC~-7 rnranr~rrrrr~arrrrr7AA AGCGCCC
2~7 cGrxDGG rnr~ Ir~rr.~r~r--m--7- A~;C~7GC
239 U~ ;G rrr~ rrrr~rrrrrr-~ A.~GUG
262 ~w rnr~rr~:aAArrrrr.~ A~ GG
268 A~ rr~r~r:Ar~r,rrr~rrrrr~ AUC~GA
276 ~CCUWG rr~r~r-~r~ rrrrr,~ AGCC~
301 CCAI~GGa. rnr`~r`rG~W""'rrrrr'" AUGCGCa 303 GACCAGG rnrDT~rrrrr~ rrrrrl~ AGAD~CG
310 CClJ~ rnr~Tr~ rr.m~a Ac--aGGG
323 cr~AG rr~r~rr~rrr~AAArrrrr~ AGGGvrCC
326 GGCCGGU rnr`~Tr~rrrrr~ rrrrraD AGG;~GG
335 1~ rrr~Anr ar---~r~`~rr~rr~ AGGCCGG
349 I~AC rnranrAr~rrrr~ rrrrr~ AGCUC~
352 CCt~ C rr~r~ :Ar--,m-~ rrrr~:3a Ar~CU
375 CUCAT~G rrr~rTr-~r~rrrr~ rr,t~rr~ ~ AGCCalJC
376 Crl:JCAUA rnr~rTrAr~rrrr.. ~ ` ~rr,rrr~ ~ AaGccarLJ
378 AGCC~7CA rrrr.Dr~rarrrrr~rrrrr~ AGAAGCC
391 CCGGGCa rnr~rJr~rrr~rr~ r-rrr~ ~ AGCtJQG
409 Aa~JGUG cnr~-lr;Arrrrr~7.rr,rrr~ A~.~GCAGC
416 ~tJC~ rnr~r~rAr~rrrr`~`rrrrr-~A A5 ~GG
417 GrJUC17GG rnr~r~rr,CrrA~Arr~r~ AaC~wG
418 G~r~WCUG rnr~TTr~Arrrrr~AArrrrr~ AaACUW
433 CACACUG rrTr~ rrrrr~ rr~rrr~ ~ ADl7CCCA
467 U~a rrrAnr ~rrrrr~A~rrrrr`~ AGCCUGC
469 GCUGACU rnr-~nrar~rrrr-~Ar-^~rrr~2~ A7~aGcrU
473 A~JGCGCU rnrATTrArrrrr~ rrrrr~ ;AUA
481 IJGr~ L7G rnr ~T7r7Arrr~rr~ r~rrrr`~ AUGCGCU
501 AacWGG rrJl ~T7r-~r~r~rrr-~ rrrrr~A AGG~
SU~STITUTE SHEET ~RULE 26) 2183~9~
WO95/23225 --- P~ r15 ' ~ 236 502 GAAC~ rr~AT~r~- ~a~ rrAA AAG
508 ~ rnr~rrr~--r7-rr~rr~
509 ~,G rnr~rrr~ AAArrrrrA~ AA~G
512 r~7c~, rrJranr~ rr-rrrAA A~
51~ GCtLrr~C rrr~r~r~Ar~rrrr~A1~r---rrr~AA ~G~A
534 C~;G;i~G rrJ~ Ar-r~rr~ ~ ~r-~7~ ~ A~UCCCC
556 G~aa~ rnr~rn~~r~7m~a~ rrr AA ~GC~ca.
551 cac~G 1 1~ ;111. .. f ,~. .a~mAA ~Ca~
;62 UC~ JG rr~r~rrr~ ^,.-rr~r-rrr7;~
~as CC~ 7 rrr~rr Arrrrr~ rrr~
Sg8 G~æCG rr~r~rrr7Ar ~A~ rrr~7~ a.~;GGGCC
613 GA~ r rrr~ 7rr~ AA ;~,Ca~GCG
616 G~GAGG rrr~rr~Ar-rrr~
617 G0DG~G rr~r`r~- `rrrr` l ~ ~7~AA .
6ao ~GGGUJ ~-r;r~rrr~Ar~r--rr~Aa~rr,~ A~7 623 i~A~ r~r~rrrArr~ r,~AA A~GGp, 6a8 T~CaA~ rrr~r-~a~r-r-rrr~ AD~7 630 A~ rnrar~ r~7~
631 &al~: rr~Anr~ rr~ rrr~ ~ A.aG.~G
638 GaGGCAC rrlr ATr~rrrr~ rr--rr~ WCA
661 7u~A~ rrrr,ArTrArr.Crr~r~rrr~' AG~JCGG
667 C~-A rrr~rrr~r~rrra~rr~rr~ T. A~A
6a7 GCtJGCC~ rrR AT n~r-rrr~ ~ ~ r~Trrr~ ~ 7~WWCG
700 CrCCaCC ~ a aC~Crr~ ~ AGU;C
715 G~GGAA rrTr~ --~rrr~ ~ ~rr~rr~ a 717 C~Gr7AGG rrTrATlr~-r,rrr~ rr~rrA A A~a~c 8 lU:~ rTTr.ATr~ rT~rr`~`rr~:AA 7~A~ U
721 CA~AG rrlt AT~r~r~r7rrr~AAArr,cmAA ~aAGA
751 ACa~Xr7C rrTrAT~---rrr~ ~rr~ ~ A~a7 759 CGt~aAA rTTr-~rrr~ r-,.-rr~ ~ ar~ ' ~ACCOC
761 CCCG~7GA rr7rAT~ -~r~rrrrrAA A~C
76a r7CCCGrJG rr,~r.3TIr~rf~r~ AiU~ aC
763 GrJCCCG~J rnr~rr~!~rrrr~ rr~ U~A
792 CGAA~G rrr~rrr~-~-f~ rrr~AA AGCC~JCG
795 ~r.7GC~A ~~trr~rr~--rrr~arrrrrA~ AG;;a~CC
796 C~JGCG~ rr~ rrr~ rrr~ rrT~ AAGG
797 WJtiGCG rTrr`'rrArr7rC r` ' 7 r~r-rrr` ' AAaGGAG
798 7U~IJGC rr7r~T~3rr~r~r~r~:AA 7.~AaA~
829 G;~aACaC rnr~ATlrAr~r,Crr~rrr~r~ At~GCCA
834 G~ 7CCGG rtrr~rr~--r~rrr~r~ ACa~AiU7 835 rGG~CCG rr7r~TTr~Gr~r--c~r~ AA~CAA
8g5 GCGUAGG rTrr~rrnAr~r7rrr~ar~r~ 7~3GGG~JC
849 Gtl~7GCG rTTr~rr~-rrrnA~crrr~ 7~GGGaGG
872 CGt~G rT~TrnArrrrr~r~;rrr~ 7~ GC
883 GCAi7GG7A rT7r~rr~ rrr~''r--rr~ T~ GCA
885 r~,~GCA~7G rTTnATlr~-rf-rr~ r~rrr~ A~CACG
905 CG~CGG rTrnATrr~rrrr~rr-rr~ ~GGCCGC
906 CCGGT7CG rr7r~ --r~crr~t;rrrr~ 7.~AGGCCG
919 G~ACtl rrnATT3rrrrnAAArrrrr~ 7~CU~cr SU~STITUTE SHEET IRULE 26) WO 95123225 - 2 1 ~ ~ 9 ~ 2 P~ IS6 936 WACIJGG rni~ Tr.arrrrr~AAr~rrrraA ,a,~
93~ GOE~G rT~ Tn~ ^--rr~33rr~--r~
942 ~GCaGG rnr~ r--^rr~A:~Ar~G~ AC~ A
953 IJCGUC0~ rnnArTr~rr^rr~ r^rrnAA ~I;CUGCC
962 CGG~GAC rnr.ATTr~r^rrr.~r-r,~^rC~ A17CWCtJ
96~ AIJCCGW t~rTr~p~TTr~r^^rr~rr^rr~ ACG~7CG
973 iJCUCCruC rnr~ATTr~l~-^rrr~r-~r~r~r~a ATTCCG~7 986 WCCru~T rn2-`'Tr`rrrrr~3A~ r~AA ACGT~rTc 996 G~ll:A crTr.ATTr~--^rr~.r~rrr~ aA Al,'7C~T
1005 GCUC~JUG ~l~:ATTr~rr^rr~3~r~r~ A ~v7CuC
1006 ~U ~rlr:ATTr~ ^rr~rr-^rr~A~ AA~7crJ
1015 T~U~U ~ _ 7r 'l .;, a ~rr^rr~ ~ Aru-G~7Cr.T
1028 crU~a~ Tr~ r~^rr~r^~rrC~ AC~UC
1031 CrGCUGA rrTr~aT7nArrrrr~3~rrrrc~a3 AGGaa,c 1032 17CCGCUG ~ AArrrrr- 71 A
1033 G~CCG.7 t~ATTrArr^rr~ rr~ r~ ~ A~,C
1058 CGAGGIJG t~Tr~TTr.~rr,rrr~37~r-rrrt~ AGGCCGGr 1064 A~GCGUC rTTr~rrr~rrrr~ rr-~ , 1072 r,c~,CAGC rr~ TTr~rrrrr-~3~rrrrr.AA .al~GCWC
1082 CUGCGG,G rTTr~TTnArrrrr~rr~rrr~;~A A~r~GCACA
1083 GC.~;CGG r~Tr.~TTrArr~rn~Arr,rrrAA ~;G~C
lOg2 AGAa~ rn~TTr~rrrrr~ r^rrr.AA AG~;~
1097 G^~ rnr.ArTr.Arrrrr.AAArrrrn,~A AG^CJGaG
1098 GGG5aCA rnr~ATTr~rr~rcr~3rr~A A,A~
1102 GC~;GG mTr.~TTr~rrrr~7lrrrrr.~A ACA~AG
1125 ~aGGGA rnr~.Arr,rrr~ ~ ~r~Gcrr~ 3 ~GGGCC,~G
1127 WAAAGG t~rTr~Tr.Arrrrr.AAArrrrnA~ A~;~^,GC
1131 UGa~ rnraTTr~r~m~r~ rrra~ AGGGAIIA
1132 AUGACW rT~ Tlr~^rrrr~r^,rrr~ ~
1133 GA~CG rnr.~Tlr ~rrrrr.a~r^rrraA Aa~GGGA
1137 CAGGGATT rnr~TTnArr~rrrrn~ ACWAa~
1140 GCUCAGG ~v~ rrrcraA A~aCW
~153 Cal3aGUU rTTr.ATTr~^r,rrr~3~rrrrr7aA AIJGWGC
1158 CVC~.7CA rnr ~TTn~rrrCr~ ~ ~rrrrr'~ ~ AG;~GA17 1167 GWGGGA rrr~TTr~Arr7rrr~rrrrr7AA Acr~aUC
1168 U(~GUGGG rrJr~TTr.~rr^rr~ rrnAA A,A~C~UT
1169 AUGG~GG rnr~rTr~rrrrr~ rr-rrr~a~ Aa~UCA
1182 Ar~aAGGA rT~:ArTnArr~'~r^Crr~ ACACCAU
1183 CAGAaGG rTTnATr~^rrrr~r^,rrr,a~ AACaCC~
1184 CCAGAA~ rnr~ATTr~^rrrr~rrrrr~ AaaCACC
'187 UGCCCaG rnr.ATTr.Arrrrr`~r~r.Crr`' AGGAAAC
1188 CIJGCCCA rrlr aTTr~rrrrr~ ~ ~rr-rrr~a 1198 ccur~Gcu rr ATTr.~rrrrr~ rrrrnA~ AUCUGCC
1209 CAAGGCC r~ ATTr~rrrrr~3Arrrrr~Aa AGGCCUG
1215 CGGGGCC rT~rAr~n~rrrrr~rr.crr.~A AGGCCG~
1229 ACWGGG rTTr~rTr~r~rrrr~rrrrn~A AGGGGCC
1237 GGGGCAG r~.ArTr.Arrrrr~'rrrrr3A ~UGGG
125û GGGGCUG r7rArTnArrrrrAaarrrrrAa AGCCUGG
1268 AT~GCUG rnr.ATTr~rrrr.Aaarrrrr~ AGCAGGG
SUBSTITUTE SHEET (RULE 26) 2183~92 W0 95/23225 ~ , i 156 1279 Ga~ rnr.~nr~rrrrr~r--rcr~a .~cra~
1281 Ca~ rnr~nr~rr~rrr~arrrrr~a ~aCc;~u 1286 ~:GGC~ rnra~arrrrr~r~rrr.~ AGC~JG~U
1309 GG~æG cnra~r.ar~rrrr~ rr~r:a.. ~ AC;3GGGG
L315 r~GGc~G rnr~r-r~ r--rrr._A ArU~
1318 CIJGGGGC rrr~TTr~ar~r~rrr~ r~r~a Ar~G
1331 r,CCt~ç rrTr~^rrrr~ r~r~ ~ Ar~ J
1334 Aca~ 7.rrrrr.:~A AGGa~GG
1389 GGCC~J rnr~ rr~7.~:~rr~rr~ AC;~
1413 A~ rnr~rrr~"~rr7rrr`' Ar.~GCaG
1414 ca;~ a~c rnr~rarrr~r~ ~ ~r~rr~ ~ AA
14;7 GCC~AGC rn~arrrrr~a~r^,r~r~ GCCC~
1~41 ~GCC rnr~l~TrJ~r~r~-.r-,~^rr,~ Ar~G
1467 r~ rnr~ rrrrr~a~r^rrr`a ACA~^
1468 GG~;W rrr~ rrrrr~rr~ AA~G
1482 GUCGa~G rrJr~'mAr'^-rrr`''r~ ACGCCaG
1486 Al~WC rnr~r.~rr~rrr~r^rr~ ACGGalJG
1494 A~, ~ rrrcr~7~ AG;~GUC
1500 u~uw~ rr~r~ r~ a~ A~
lSCl GCUG~G ,, .~rrrrr~a AACU~GG
1502 ArvCUGCJ rrr~nr~rrrrr~ rrrrr~a aAA~,, 1525 CC~aLGG rnrar7r~rGrrr~ r-7c~rr~l ~ ~7GCCG
1566 C~7CAGGG rrr~rrr~r~rrrr~ rrrrr~ CUCCPJ
1577 CGAG~ rnrarrr~r^rrr~rrrrr`~ AGCa~Ca 1579 GG~7 rnr~rrr~ar~;rrr`a7~r^rr~aa A~Ca7 1583 ACOI~GGC rnr.~r7r~arrrrr~ ~ ~ rr7rrr~a ;~ AG~P.
1588 C~ JCaC Crp-~rrr~ar~rrr~rr~^rr~a AGGrGAG
1622 G~a~;CaG rnr~-7rarrrr~r~7lrr,rrraa AGC~JGGG
1628 CCCA~ rnra7~r~rr~rrr~arr^rna~ A~
1648 CA7~;GG rnr~ rr~ rr~ ~ AGCCCCG
1660 C~ AAG rrrr~-rr~^~rr~rr~r~ AGGccaL7 1663 C~JCCUGa rnrarr~r^~~~'rrrrr`' AGGAG5C
1664 7~CUG rnr~-~r~^rrr~-~rr~r~a AAGGa~
1665 AJC~CCC ~r:~rr~rr~"rrr~rraa AaA~GAG
1680 G.^iiGAG rnr~7u7r~^rrr~r~rrrrr~ AGU~C
1681 ~GGa~a rnra~7~ arr~ ~ ~r~r~r~rr~ ~ AAGlJclJl7 1683 A~7GGAG rnr~ rrrrr~ ~ A~7C
1686 CG~G rnr~nr arrr~rr``1`rrvrrr`~ ACGaGaA
~690 7JGt7CCGC rnr~r~r~rrrrr7l~r~rr^rr'~ ADW~GG
1704 GGcuG~G rr7r~-7rarr~r~`~ AG~ CU7 1705 G~ rr7r~-rr~^r~ rr~rrr` ~ AA~CA
1707 C~7 rrrr~r~r~Arrrrr~`r~r~ AGAA~7C
1721 Ctl~7 rnr~rrr~ rGm~ aGC
~726 A~7 rnr~rrr.arrrrr`~r-Grrr.aa AU¢~
1731 CCC~G rrr~rlrArr~:aAArr;rrr~a AGCCGA77 1734 ACCCCC~7 rrr~Tr~r~rrr~7lar~r7rrr~a~ ~;GaGCU
1754 C~JCUGGG l~rr~r7r~rrr~ r~r7rrr~a .~GGcaG
SUBSTITUTE SHEET (RULE 26) wo9s~ s 239 2183 ~ r~ ta- 1~6 ~D T
A' ~ ~ ~' ~T ~T ~ ~ ~ ~ T ~ ~ ~
E
- o ,._ ~, ,..-, .....
.C , ' .;
. . _ C
~ ~ c, æ ~ O~
8 _ SUBSTITUTE SHEET (RULE 26) W095/2322S . 2 1 8`3 9 ~ 2 ~ s6 '; `,.:
tD
t~
tD
tn , . .
t G
~'QZ ~ 8~
.c I ~. 1 . , I , tD ~ ~ ~ t` ~ ~ ,, ,, ,, ~ ~ ~ 2 o C
~UESTITUTE SHEET (RULE 26) WO 95l23225 218 3 9 9 2 r~

Table ~3: Euman TNF~ E[~ Riboz~ne Target Sequence ~t. ~ ~!~rg-t S~ c-~ 3t. r~ T~ ct Se~uc~cu Po~ t~ o:~ ~?o~lt ~ o~
28GG~GG~J ~J C~CC
29GCa(;OE~ C ~C~ CU 321 ~ SaU C AI3~;U~
31A~CU C ~CCIJCU 32~ A~CAU C 1~7CSCGA
33G~Cl~U 1:~ CC~ UCA 326 AUC~ IJ CtJ~AAC
34UtJC~U C C~JaJCAC 327 r~ r C ~JCGaACC
37T;~3mJ C ~ 329 ~17CliU~ C GaACCCC
39~JCCUCU C Al =~ 3~2 A~C~ ~ GCCCA~G
44criw3w A C~CCC 361 CCC~GU ~ G~;CaA
58Ca~GGCU C C~CC~C 364 ~W A GCaAACC
6~CCACCC~ C ~ICCC_ 374 ~aACCCU C AaG.~&a 67ACC~CtJ C ~JG 391 GG~','O C CAOEIGGC
69CC~JCV C CCCDGGA 421 AI~XCU C C~GCCA
106 GCal~ C CGGGA~G 449 G.~ a. ACCaGCU
136 A~''GCU C CCC~A~;X 468 G~JGCC~3J C AG;~Gt;GC
16~ C~;GGCU C C~GCC~ 480 ~CCUGU A CC~WC
177 CGG131~aJ IJ Gl~( C~JC 484 IJG~ACCU C ~CU
180 UGC~JGU U CCllCaGC 487 ACCUCA~J C ~ACUCCC
181 G~,uu C CUCAGCC 489 c~ 7c~ A CUCCCAG
184 Il~;WCCU C AGC0CU 492 AuCQa~J C CCaGG.7C
190 ~CCU C IJ~:CtJ 499 CCCAGGU C C~WCA
192 ~;C0~ U C~CWC 502 AGG~,~ C I~AGG
193 G.^aJCW C UCCWCC 504 GI~CU~ U CAAGGGC
195 CUCtl~J C C~G 505 ~aJ~CW C AAGGGCC
198 uucuu.:u U CCCGUJC 525 IJGCCCC~J C CACCCAU
199 XUCCW C C~IJCG 538 ADG~ C cucaccc 205 I~:C~QaU C G~JGGCAG 541 I~ ICC~T C ACCCaCA
226 CC~U C ~C~GCC 553 A~caU C AGCCGCA
228 ACGC~CU U C~;CC~ 562 GCCG~--AIJ C GCCG~CU
229 CGCUCW C UGCC~7GC 568 UCG-CGU C ~JCCDaCC
243 C~GCi~ U ~IG 570 GCCGUCU C CI~ACCAG
244 ~ i:J G~A 573 GUC~JCCU A. CCAGACC
253 GAI~J C GGCCCCC 586 CCAa~3G;J C AaCCucC
273 GAaGAG~J C CCCCAGG 592 IJCaACClJ C CUC~G
286 G&Gaccu C ~CO~IAA 595 ACC~JCCU C ~JCUCCCA
288 GaCCUCU C tJCl~aAUC 597 C~JCCUCU C 17GCC~C
290 CCUCUClJ C UAa;JCAG 604 CIJGCCAU C AAR;~GCC
292 ~JCUC~CU A AUCAGCC 657 CCCUGGU A UGAGCCC
295 CUC~AU C AGCCClJC 667 AC; CCaU C ~ITCCGG
302 -AGCCCU C ~CCCA 669 cccalJcu A IJCt;GGGA
SUBSTITUTE SHEET (RULE 26~

WO 95123225 , =. = 2 1 8 3 9 9 2 p , 671 CaIJC~U C ~2aGG 960 ~VU C A~,TG
682 GAG'IJ C ~^ 1001 Ai~CCACU A A~UC
684 G--~JCU U CCa~--UG 1007 UAi~AlJ ~J C~G
685 GGaJC~ C C~-l;GG 1008 AAGaa~U C AaAaJGG
709 ACCGACU C AGCG UG 1021 GCGGCC~ C CA~ACU
721 CUGA~alJ C AAIJCGGC 1029 CAGaAClJ C i~ GGG
725 GAlJCi~ r C GG3XG1~ 1040 GGGG.--CU A CACCU~U
735 CCCGaCU A ~ac 1046 I~GCU ~J UG;~IJCCC
737 CG~a~J C ~0 1047 AC~--UU l~ UCC--U
739 ACaal~CU C Ga0UUG 1051 C~IJ C C--UG;~
744 t ~JCGPCU ~ 1060 C5~ C R;&aAl~'C
745 UCG;I~ ~J GCCGA~;U 1067 C '~ C ~C
753 GCCG;U~U C TJGa~G 1085 GG~GCCU ~ 1.~3G;JUCU
763 GW~ C ~CUC~G 1086 GAGCCI~ ~J G~CUG
765 CA!;GD~ A C~GGG 1090 C~ ~J Cl;GGCQ
768 GUCOZU tJ U ~LiC 1091 ~GG~ C U~CaG
769 ~ ~ G--~CA 1~3 CAGGAaJ IJ GAGaaGa 775 ~ C A~CCC 1124 AA~aCCtJ C ~C0hGA
778 G~ aU IJ GCCCC~J ~129 C~7CACCU ~ G~G
801 CGaACaU C CAal Ct~J 1135 ~AAU U G
808 CC~ACC[J U CCCAaAC 1151 ~:CU U ;~--CUU
809 CAACCUU C CCAaACG 1 1 52 G~--:.alXDU ~ GGCC~JC
8ao AACGCCU C CCCUGCC 1l58 W~^,CCU ~ CCU~l7 833 CCCCAalJ C CC~IJ 1159 AGGCCt~U C CUCUC~iC
837 AaIJCCCU 17 ~lltlACC 1l62 CC~CCJ C UC~CCAG
838 AlJCCCtJtJ U ADIJACCC 1164 ~UCCallJ C ~CC~GAU
839 UCCC~lJ A ~ACCCC L166 CC~IJCU C CAG~UGU
841 cc~au l:J ACCCCCU 1174 CAG;IIJGU U l;CCAGAC
842 Cl~ A CCCCCGC 1175 ~7 U CCAGA~J
849 ACCCCCU C CUlJCaGA 1176 GAlJGlWtJ C CAGACllU
852 CCC~ U CAGACAC 1183 CCAGACU U CCU~GAG
853 CCUI ClJtJ C AGACACC 11 84 CACa~U C CUU~3AGA
863 A~XCU C A~IJCU 1187 ACUIJCC~J U G~C
869 IJC~.A~ C lltJCDGGC 1208 CaG CCU C CCCAUGG
871 Aa~ CC( l7 U C~GCI;C 1224 GCCAGCU C CCUC~IJ
872 ACC~JCUU C ~CA 1228 GCi~CCtJ C UAUCWAIJ
878 I~I:UGGCU C AAa~AGA 1230 17CCCtJC5 A UUUIWGU
890 AGaGaA17 ~ GGGI~J 1232 CC~JC~UJ U UAu~iuuu 898 G;~J IJ AGGGUCG 1233 C~C~ IJ AUGW1~8;
899 GGGGC~ A GCWCGG 1234 IJCUAWU A IJWUrJGC
904 ~ C GaaACCC 1238 WUAUGU U ~GCaCUU
917 CCAAGCU IJ AG~ 1239 WaUG~ U GC~CU~JG
918 CaAGC017 A GMI DW 1245 U~J IJ G~alJUA
924 llaGaACU l:J l~aGCaA 1251 WWGAIJ U
925 AG~ U U AaGCaAc 1252 IIWGAW A l~-tJAI~
926 G.~U A AGCAACA 1254 I~UAU U ~AUua~U
945 CACCACU U CGAaACC 1255 GAI;IJADU U ~AI~U
946 ACCACt~lJ C GAAACC~J 1256 AU0AWU A Wl~DUlJA
959 C~JGGGAU ~ CAGGaAlJ 1258 UAW~AIJ U Al,'UUa~U
SUBSTITUTE SHEET (RULE 26) WO 95/23225 218 3 9 9 2 F~,-, r ~ 156 1259 A~ A I~W 1440 ~;~.uuuuu 17 ~AAAUAU
lZ61 ~iDUA~J U ~ ~ 1441 G~WW A tl~A~U
lZ62 ~w3aw IJ AI~IJU 1446 U(~aAAAU A UaA~JG
lZ63 AT~U A ~A 1448 A~ A~GAI~
lZ65 l~tJAU IJ ~JA~J 1449 AAAUAIJU A ~JC:G~J
1266 AI/U~ U AUllai~ 1451 AIJAWAU C ~JGa~A
1~67 U~J A ~ 1456 A~PIJ U A~W
lZ69 UAl~J U A~ 1457 UCUGAW A AG~3~iGUC
lZ70 Al~ A ~ 1461 Au~ AA
lZ72 ~I~ IJ ~U 1464 A.A~J C ~aA
1273 ~ ~ AI~WtJ 1466 G~GCtJ A ~AC;~IJG
lZ74 A3~ A ~J 1479 I;~IJ ~ ~GAC
lZ76 1~7AIJ U ~ac 1480 G.--~.~IJ U G~-GACC
lZT7 ~AW U A~ACA 1494 CAa~JGJ C AaJCAW
lZ78 ~1~ A TJ;~JACAG 1498 ~ACU C A~'G
lZ80 ~ IJ UAC~W 1501 C~U IJ GC~GG
1281 ;UJ~ IJ ACaGaUG 1512 GaGGCClJ C ~'CCC
1282 ~ A Ca5;~1JGA 1517 C~JC~ C CCC~GGG
1294 ~W A ~ 1528 AGGGa~;U IJ G~5 1296 AAIX~7A~ U UA~Gv 1533 'v-;JCG~ C ~AUC
1297 A~l7 U A~GGv 1537 uvu~u A A~GGCC
12g8 UG~JtJ A l~lJIX GGA 1540 Cl~AU C v~CUAC
1300 ~UAU U I~;GGaGA 1546 12CGGCCU A C~l;UCA' 1301 Al~ U GGG;U~ 1549 GCCUAC~I A ~'G
1315 CCGGG~-U A IJCCUG-v 1551 C~CtJA~J U CI~G5GGC
1317 G-OElaU C C~JGGGGG 1552 UAC~WU C ~v 1334 CCAI~IiGU A GvAa:~JG 1566 GaGaAAU A AaG~,~'G
1345 G~GCCU U v~ AG 1572 UaA.~GU U G~PGG
1350 CU;~;aJ C AvACal~-G 1576 GGl~liGC-17 U AGG~AAG
1359 GACa~J ~ I~UCC~JG 1577 G.~U A GG~AAGA
1360 ACAI!WI~ U UCCWGA
1361 CAI~U ~ CCG~A
1362 A~tJ C CG~JGMA
1386 GAaCAAU A ~W
1393 A~ U CCCPDW
1394 GG0WU C CCa~WA

1414 Ct~GCC~J C 1~3CC~
1422 ~CCU U C~'GA
1423 -~JGCCt~lJ C llU~
1 4~5 GCCUCCtJ IJ I~A
1426 CC~IlJC~ U UGAI~
1427 CJtJCUCU U vAl~AUG
1431 UtJUt!CAU ~7 AUG~ltJOlJ
1432 U~U(~U A ~JG~7U~
1436 AUUAUGU U ~I~UaAA
1437 ~/UAu(.uU U UUaMAA
,_ 1438 I~J U UUAAAAU
SU~STITUTE SHEET qULE 26) 21~9~2 wo ss/2322s - : ' r~ s6 Tal~le ~g: ~Iuma~ nm~rh~ Riboz~e Sequences ~t. ~ ozym- g~ 3c~
ç~o~ t~o~:
28 GG~A~G rnr~r.~r ,rrr~7 r~rrrr~
9 AK~.AG,~ rnr~ rrrrr~rrrrrAA AA~GC
31 A;~ rnr-~rr~rr~rrr''7~r~-rr 33 r~.GG rnr~ r rrr~ rrrrrAA AG
34 G~ r~ir~r-~ r~ r rrr~
37 ~G~ r~r~rarr,~-rr-~r,rrr.~A AGG.
39 ~W rrr~-r.arrrrr~ ~rrr~ A~GaA
44 GGG~A~ rnr`~r~r~ a~rr.rrr~
58 Gaa;~ r~r~r~rr~rrr~rrrrr~ AGCCG~
;~;GAa~ rrr~rrrrr-~a~rrrrr~ A~;GWGG
6~ CaGGGGa rnr~ arr.. -r~ r~rrrr~
69 IJCCaGGG r~r~nr`rr~`~'rrrrr,2~ A~aGaGG
106 CGrJCCCG rr~r~r~rr,~rr,~rr~
136 I~XiGG rur Anr~r--rr~rr~ rr~
165 CCGC~:~ rnr~ rr~rrrrr.~A AGC-CU~;
177 GA~;G~ rnr~-rr~-rrrr~r~ rrr~ A~;CaCCG
180 ~r,C~G rr~r~Arr~r~rrr~'~r~rrrr`~ A.C.~AGCa 181 r,GC~ rrJraTr~r~rrr~a~rrrrr`' 7,~ACaaCC
184 At~GG'.17 rnr~ Tr~-r,rrr~ r,rrr.AA A~r,&aAcA
190 A~G.aG~ r~r~r~-~rrrr~r~rrrr~ JGA
192 GaAG~ rnr~-r.arr~rrr~ar~t;rrr,AA A~A13GC~
193 r~ rr~r~ r~rrrr~ar~r~rrr`' AAG~GC
195 CAK~AG rTTr~ -r,rrr~ ~ ~rrrrr~ ~ 7,~.GaAG.~G
198 ~r~D= rr7r~Tr~rrrrr~''rr~rrr`' AG=A
199 C~r,U~G rrT~r~-Tr~-r,rrr~,r,rrrr.A~ AAaaGA
205 C~GCCaC rnr~TTr~-rr,rr~rr,rrr.~A A~GGA
226 ~r,GcaGp~ r~r~AT~r~Ar~r~rrr~r~r~rrr~
228 CAGGC~G r,nr~T~ ~rr,~:~A~r~A A~;CG~
229 r~aEGC,a rT7r~ATTr-^-r~rrr~ r~r~rrr`~ AAG.7~CG
243 CaaJCCA rrTGATJr~-r~rrr~AAAr~r~rr~ A~r,~
244 ~7r~AC7CC rrJr;AT~r~Ar~rrr-~r--~rrr~a~
253 ~r,GGGGCC rr~r ~TTr~-r-~rr~rrrrr~ A 7~7C
273 CCCGGGG rTTr~ rrrrr~ arrrrr,AA AC~
286 ~GaGA r~7r'TT~ rrrr'''rrrrr~ 7CCC
288 Gal~a r~r ATT~rrrrr~rr~a~ AGA~C
ago Ct~AI~ rTr~aT~rArr~rrr~'rrrrr'' AGAGA~G
292 GG.-'OGAIJ rnr~ATlr Ar--~rrr~ ~ arrrrr~ ~ ~GP~AGA
295 ~r~;GGct7 rTTr.ATrArrr~rr~rrrm~A A~a.G.a.G
30~ UGGGCCA rnr~ATrArrrrr~rr~rrr~ ~
SUB. iTUTE SHEET (RULE 26) W09s~23225 245 ~ 39'~P~ S~
321 AGaaG~ ~TT~7aT~.~r~rr~rrrr~ CO~C
324 IJCG.~ uT~ rr~rn~ ~r~
326 G~ ~ TT~.~ r r~A~ ~ r~a AG~
327 GG~ nnr~TT~ rr~ r--t~ auGa 329 GGGG~JC t~TTrl~TTn~r-rrr~A~rrrr! ~ ~ AGAAGa~
352 CU~G5GC ~ n~ rr~ r~n~ A~GCU
- 361 ~C ~r~-Tn~r-~ a~
364 GWC~C ~ rTr.~ a~r~`~
374 UCA~"UU ~ r--~
391 GCC~G ~r^~ I~CC
421 ~-caG ~F~ rty~rrrrr ~ A
449 3U;C~ ~ r--r~ r--. ~ iUx ucuC
468 GCCC~ IJI,.~ A~rrr--, a~ AIJGGC,aC
480 01;G.~G rrTn3TTr`~ r--~
484 A~U rrTnATrr~---r~ rrrrt~:~a A~a-487 GGGaG~ TTnA~-rr~ rr,~ a AUGaGW
489 C~ ( ~, A. N.~ AII~ AGa~aG
492 G~ rr~` ~ A~.G
499 l~aA~ ~TTr.~rTr~rrr~~
502 CC~'GaA ~ rrrrr~ ~ AGG
504 GCCCU~ rr~ rr~rr` ~ ~SaGGaC
505 ~;GCCCU~ T ~` rrrrr` ~ ~--na A AAca~
525 AI~GGsUG rnr~ r,m~ a 538 G;;W~ ~ ~ ll " N.I~ 7 rr~ ACC~
541 ~W;GU (~-rTr~rr~ -r~
553 ~CGGC~ ~TTG~TTf-~tr~ nr~rrr-~a ~UJGG~W
562 A~aCGGC ~:~rTr~ r~ A .alJ GGC
568 r,G~GGa rnr~TTr.Arrrrr~ rr~rr-aa A! GG~a 570 C~G~IAG rrlr ATTr~rrrrr~ r-r-~a ~:GGC
573 GOEJCtJGG rrrr.~Trr~--rCrr` ` ` r~rrr,~ A AGGAGAC
586 G~ U I l ll;;~ rr~rr~ ACC~
592 CAGi~GAG rnr~ r-crr~rr~rr~
595 ~GGca~ rrTr~Tr.~r-~rr~ r~:l~ Ar~G~GG;J
597 GA~ rrTr~7r`'`r~rrr~ rrrrr`~
604 GG~JC~U rrrr~Tr:arrrrr~ rr~rrr~ A~GCaG
6~7 GGG~-,UCA rrr~ ar~rrrr`T~rr~rrr-~ AccaGGG
667 CCAGAl:~ rrrr~ r~r.rrrra~rrrrr~ ATJGGGCU
669 UCCCU~A rTr7~ rrrrr~ rr~r~ a Ar~,y~;;G
671 CCUCCC~ ~ " n7l~r~ ~ A~G
682 GC.~a rTr~TTr~rrrr``~r~~rrr`~ ACC~UC
684 CAGC~JGG rrTr~TrrArr~ rrJ~ ~ A,r,aCCCC
685 CCAGC~JG rrrr~rTr.~rrrrr~ ~ 7Irrrrr~ ~ AAGaccc 709 CU~CGCU rnr~TTrAr-~crr~ 7Ir~r~rrr-~ Ar~,r 721 GCCGlUn7 rrr~r~rr~r~rrr~ ~CAG
725 UC5GGCC rrr~ r~r~r-rr~lrrrrr~?~ A~JGAUC
735 G~JCG.U~ rTTr~TTr~rrrrr~ r-rrr1~ a~ GGG
737 .aA~ rnt-~TTr~-rcr~a~rrrrr~ A~CG
~39 CAAAGUC CrJr~TTr-:rr~rrr~ r~rrrr~A~ Ar~
744 CUCGGCA rrr-~,TTr~rr.rrr`~rr,rrr`'l A~;UCGAG
SUBSTITUTE SHEET (RULE 26) 21839~2 WO 95/23225 , ~ 246 1 ~ 5. ~ ~ 156 745 A~tJCGGC rr~Tr~-~rrr'~ rr~^r~ CG~
753 CXCCCA rrrarT~ rrr~ar-~--a~ crGc 763 CAaAG~ rnraT~r' r~rr~ CC~^C
765 CCCAAaG ~a~~~rrr~ ar~ ;aCCJG
768 CiUJCCCA rnranrar^rrra~a~r~r~ C
769 U~CCC rnrar?~ ,rrr~ ^rrr~ a ~
775GGGCa~ rnraT~ ar~rrr~ ~r~r~ CCCAA
778~ aGGGC rr~rrrrr~ ^~r a;~ JCC
801I~AGW~ rrr.a~ ,rrr~ .u~uuCG
808r~GG rnr~ --r,rrr~ r^^-~ ~GG
809CGlWC~G ( ~ Aar~ .a~,G
820 GGCaGGG rr3~ rrr~ ~^^r^~
833 A~aAAGG ~ rr.aa ~GGGG
837 GGU~ rr~raT~arrrrr``'l'^~rrr~:~ aGGGa~
838 GGG~ r n~ ~^.~.aa .~ GGa~
839 r~A rn~ rr~ aa AA~
841 A~ rnr~r.~rr~ r~ A.~AGG
842 Ga5G~ ~ " ~ r.~ a~
849 ~Jc~aG I I r ~ rr 852 r~JC~JG rr~r`~Tr.~rr~rrr.a~r-~::~a AGGaGGG
853 r~GDWC~ rTTr~r~ rr~ rrr~A ~GG.~G
863 .a~a~7 rrr.aTlt:Ar~r~rrr~A~Ar--~rrr-a~ ;\~U
869 GCCl~ . rnr~aTTr~Ar~l rrraaAr~r~ AGWUG~
871 r~CcaG ~rlr~arr~r~rrrr~Aa A~,~
872 I~GCCa rTr~r~G-rrt aA~r-r rr~^~aA aA~J
878 ~:UO~U rrJr.aT~r.Arr~:a:~ar.m::~A ;~.G.^CaGA
890 A~GCCCCC rrrATTr.Arr~rr~ -rrrA ~
898 cr~accc~ r~r~AT~ar-r-rrr~ rrr~ AGCCCCC
899 ccr~ccc rTrAT~narr~rr~ r rr~ CCC
904 GGGDt/CC CrJ~TTr-Arr~`~r~rrr~:a ACcccaA
917 AAa~Clr ~rr~rrr~rrrr~ G
918 AAi~C rnr.aTlr arr~ ~~r~ U~G
924 ~ rn,r.aTT~ r~ r~ A ~u;;Jccca 925 G~UGC~J r-TJr3~TTr.ar~r~ r~
926 ~GC~T ~`TSC''-~r'-'-~rrr`~ rrr.~a ~ ~C
945 GWUtJCG rTT''~GArrrrr`~ a A~G~GI~G
946 A~JC rTTr ~T~r.arr~rrr~ rrr~
959 AIJ~CCUG rTr~ arr~rrr~ r7t~rr~ A~cCaG
960 CA~JCCtJ rnrAT~ ~rrrr~ rrrr~ iCCCA
1001 ~ rTTr:~Trr.Arrrr'`~`~'-^rrr`~ ~.GUG~
007 CAGWW rnr.ATTrArrrrr~a~r7rrr~
1008 Cc~ ~ "~ ~7~ --^~r.AA .aA~IUcucT
1021 A~JCtJG rnr''Tr,~arr~`'`--GrrraA PGGCCCC
1029 CCCCA~U rnr`~ rr~7~rr7rrr~
1040 AAa~ rrJr.aTr`'~r-rrr`'`'~rrr-~A AGGCCCC
1046 GGGAUCA rnr.~TT/~arrrrr~ r^7rrr.a;~ ~
047 A~GA~C rTTr7ATTr`rrC~rr`~r~-rr`~ AAGC~GU
~051 I~G rnrAnr.~rrrrr.~A)~rr~rr~a A~CaAAG
1.060 GAUUCCA rrJ~TTrar~r7rrr~ r7~rr~ ~ A~.~caG
SUBSTITUTE SHEET (RULE 26) W0 9512322S ,~ ~ ~ 3 9 ~ 6 1067 G~CC?. crK~ rrrrr7~r^,rrr~ AUtJCC~G
1085 A~Ca rr~ r^rrr~r^,rrr~ AGGCUCC
1086 C?~ACC ~rnt~ ^rrra~r^rrr~ A~;GCUC
1090 ~GCCAG r~ rrrr~r-^rr~ ACaU~aG
1091 CUGGCC~ " ~"~ arrrrr-~A .~AccaAA
1.3 ~CUC cnr:~r~^rrr~ a ~^rrr~ ~ AG~;XUG
~174 ~ rrr~r~ ~aar-~,~rr~ AGGUCtW
3129 cLal~3uc 111_~ a~/ rrrr-aA AGG~GAG
1135 ~C ~ K ''' ~ A~ rr,~^rr~a AWUC~
1151 ~AGGCCIJ ~;~a~ r~rr~ rr~ Ar~CCA
1152 ~r~ GCC r~r~ rr-rr~ r^^rr` ~ AaGGccc 115~ AGa5l~GG r~ ~r~rrr~7~ar^crr~ ~U;GCCUA
1159 ~r,a~;aGAG rnr~arr~r''\~ r^rr~ aAGGCCU
1162 C~ cnr~r~rr,r-rr~ r~^rrr~
1164 A~ r l ~ 7 r^--rr~ A
1166 Ar~CUG rr~ ~r~r~-~rrr~r~r-~r~ ~G~GG
1174 GCC!JGGA rnr~ rrr?~ ^rrr`a ACAIJCUG
1175 A~GG ~rT~n~ r~rr~a~ rrr~
1176 .aA~ ,,, ~ r~rr.~A AAA~C
1183 C~CaAGG rrr~Tr~rrrrr~r~-rrr~A A~C~GG
1184 ~ aAG rnr~Tr~rr~arr^rr~ AGUCUG
1181 WWC~C rnr~TTr~rrrrr~ r^rrr~ ~ A~;GaAGU
1208 CCA~JGGG r l l~ rrm:aA AGGGCUC;
~24 Ar~GG rnr~TTr~rr7rrr~rrrrraa A~GGC
1228 A~AAI~ rTTr~TTr~r~Grrr~ ~ ~r~ rrr-a a A~GGlGC
1230 AC?~AA rTTr` Tr`rr~ r^rr~r~ Ar~.
1232 AA~C41~ r~TTr~Tr~rrrrr~rrrr~a ~GAGG
1233 CAAA( ~U C~ rG~ rrrrraA AA~G
1234 r~,AACA c~lr-lr~Grrr~r-rrrr~-l ~Aa~Ga 1238 AAGCG~ " ~ Aaa~r~A A~AA
1239 CAAGUGC cTTr-aTTr~rrrcr-~A~r-^~rr~ a~A
1245 ~C CTT aTTr~rrrrr~r~rr~ caA
12Sl AA~AAU Cnr-ATr~r~r~crr~ tr-rrr~7~ AUCa~A
1252 ~AA rrTr~r~rrrrr~a7~rr~rrr~ aCA
1254 AA~ ~", n ~ ,, r [ ~rrrrr.aa AL~C~
1255 AA~ rnr~Tr~r~rrr~'`r'-~rrr`~ AAUhAUC
1256 I~AAI~A rTlr:ATr~r~ r~-rrra~ AA
1258 AU~A~ cTr~Tr-Arrrrr~ r^-rrr-AA U~aA~
125g AAU~AA rnr-~Tr~r^-m~ ar-r~rcr~7~ AAIiaAAIJ
1261 Atl~A C~T~Tr~rr~rr~ rrrrrAA AU~A
1262 A?~ cnr~rrrrr~``r~r-rrr-~A AAllaAUA
1263 I~A~aAA cnraTTr~--rrr~rrrrriA AaA~
1265 A~ r-rrATTr~r~rrr~`r-^~rrr`` A~AAUA
1266 AaAl~aA~ rTlr:ATTr~r~ rrr~ AA~
1267 II~A rTJr~ ^rrr~ r^c~aA AAAUaAA
1269 AAr~A~U rTR~~Tr~rr^rr~ rr~rrra~ A~AAU~
270 AaAu~AA cTTr-~TTr~r~r-^rr-AAAr^rrr~ AA~AA~
~, 1272 AIlaAAIl~ rTTrATTr~r^rmaAArr,rrrA~ Au~ aA
273 AA~a~ rnr-AT~r~rr~rmA1~rr-^rr-~A AAUI~UA
SUBSTITUTE SHEET (RULE 26) WO 95123225 ~ 2 1 8 3 9 9 2 P IIIL . 156 1274 Aa~Aa rnr~arr,rrr ~r~rrrr`~ AAAUAA77 1216 ,r,D;~AA7JA rrTr~-Tr~rrrrr~'r-^~rr~ A~a~
1277 ~Aa~7 rrr~T~r~r~r,rrr~ rr,rrr~A aA~7 1278 CG~AA rr,7r~Tr:~r~r.rrt:A~rr.rrraA AAAI~AA
1280 A~:UGUA rnr~T7r~r~r.rrr-~ r~rrrr`~ AUaAAUA
1281 CT~UGU rrTr~-r~rrr~ r~rrrr~ ~a.AAU
1 282 ~UG rrraTTr.~^rrr.AAArrrrr~ a 1294 AAA~ rnr~ r^~r^rrr`~ AC~UC~
1296 cr~Aaua rnr~rr~rrrr~r~r.r~rr~ AD~}1U
1297 CCQAAU rrTr~lrArr,n~rrrrr~ ~U
1298 77CC_~A rr~7r~TTr~ r~ r ~rr~ AAAlIaC~
1300 ~,7Ccr~ rrr~TTr~rr7rr.^7~ ^rrraa ~.AA7la 1301 GZ~7CCC rrr`~Trarrrrr`~r^~ AA~AAU
1315 ccraGGa rrr~TTr~r--~rrr~a~r-~rr-a~ ;~CC~r,CGG
1317 Ccccca,~7 rTTr~TTr~ r~r~-rr~ ~ ~r-rrr~ ~ A~.7Acccc 1334 CA~CUCC rrr-T~TTr~r~~rr''~arrr rr~A ACAliUGG
1345 Cl;~CC rrTr~ Tr~r^~r-,.-rr~ AGGCAGC
1350 CADG~Ja7 rrTr`~` ~ rrrr~ ~ ~r-^~'ma ~ AGCCaAL-7 1359 C~SGGAA rTTr ATTr`r--~ r--~ ~ ~C
1360 ~CGGa rrTr ~rTr~r~r~crr~ r-rrra~ ~GL7 1361 7~13~G rnr~Tr~r~rrrr~r- rrr`~ ACAUG
1362 ~æG rr7t ArTr~rr^rr:AA~r~rrrr a~ AaCA77 1386 AAl aGCC rrTr~Tlr~~G~r~rr.~rr~ A~WC
1393 Ar~JGGG rrP~ArTr-~r^~Crr:~Aar~:A~ ACAGCCU
1394 7-~GG rr7r~ rr7rrr~ aACAGCC
1401 AG~GC rrrArTr.~r,r~r--rrrA~ Aral~GGG
1414 AGGCaCA tlTr`T-r`-~-rr`a~r rrr~A AGGCCa.~7`
1422 ~G cnr.ATTr.Arrrrr.AA~r-~rrr.Aa AGGC~CA
1423 A~LAA cr7r~ --r~r~A~rrrrA~ A~GGCAC
1425 7.~CaA rr~Arlr~-rrrr~r~rrrr.~ AGAaGGC
1426 A~aAS7CA ~-TrArr~ rrfr~' AaGaAGG
1427 rU~AL7C C~ rTr-~--~rr~r~rrrr~Aa AAACAAG
1431 AAAaCA~7 rrJr`~A~---rY-r`''`r---r~r`' A7~1 aAAA
1432 A~AAACA ~rrJr~rTr,Arrrrr~Tr ~ aAI7Ca~
1436 7~AAA r~7r~ArTr~A~rrrr~AA~r,crr-~ Ar~A~A7J
1437 U~A~ rrTrAr7r~-rrrr7~r- maA aA~aUAA
1438 A7~ rrrr~ Tr.A~rrrr~ r,~ rraa AAACaUA
1439 U~7A rrTr-~-Tr~--~r^rr~ 1 ~r-~ AA7~U
1440 AUA7~UrJ7 r~7r-~ r~r~rrr.~aAr rrraP AAAAACA
1441 A~7 rnr~ Tr~~rrrr-~P~7r~r-Aa A~aAAAAC
1446 CA~;U;aA rTTr~T7r~rr~rr~ r-rcrr~ A~A~A
1448 A7~GaU rr~rr~ p~r~rrrr~
1449 AA~CaGa rrTr~ A~r~rr~rr7rrr~
1451 ~AAUCA rn~7r~ - Gr~ ~ ~r.~,rrr~ A~
1456 ACAA~U rrTrATTr,~A~rrrr~rr.rrr~ ~UCAGAU
1457 GAC~7 rrTr~-Tr~r~rrrr~r-~r.~A AAW~
14~1 7~ aGAC rrTr~TTrT--7rrr~r-~rrraA ACU~LAAU
1464 U~JA rrTr-~rTr~arrrrr-aha~rr~rr~Aa A~L7 1466 CAD~7 rT7r.ATTr~r~rrr~rr.~rrP~ ~GACAAC
SUBSrlTUTE SHEET (RULE 26) ~ W0 95123225 249 2 1 8 3 ~ 9 ~ .' 156 1479 G~CaCCA rnr~TT( arrrrr.a~Ar rrr~ AUca.
1480 C~.7CACC rnr~aTTr~arrrrraaar-rrrr~a~ Aa~5;C
14g4 ~aAIJG.~ rnraTTr~-^rrrAAAr--rrT~ Aca~G
1498 CaGt aAlJ rnr~r~ r,rrr~a~--rrrr- ~ AG~ACA
1501 CC~C~C rnr~aTTr~-r~crraaarrrrr~aA AU~G
1517 GG~aGCA rn-~r~ rrr~arrrrr~ Ar,Gcc~C
1517 Ca:~;GG rTTr~ Arr~rrr~ rrrrraA AGCaGAG
1528 cAr~AcAc rTTr~aTTr~-~t;rrr~r~rrrr~a A~CCC~
1533 GAT~ACA rTTr.ATTr.arrrrr.AaArrrrr.aA ACa~ aAc 1537 G~}J rnraTTr~-rrrr~ r~rr~:aa ACA~aCA
1540 GU~G~_C rTT~ aTTr~r r~rrr~ r~rrrr~ ~ AU~AG
1546 Kah.i~.G rnraTTr.arr~-rr-. ` ar.-rrr~ ` ~-CGA
154g CAaJGaA rUr`~:~r--rrr~A~Ar-rrrr~a AG~GC
1551 GCC~;G rT~ r~crr~aAArr-rr~ Al~Ç
1552 CGccAaJ rtT~~T~r~-r-rrr~aa~rrrrr.AA A~A
1566 C~ r~Jr.aTTr.arrrr.2~aAr-~:aA A~C
1572 CC3~AGC rnr~T~.ar~rrrr.AAArr,m~.Aa ACCW~A
1576 .:UU~U rnr~aTT~arrrrr~aar-crr~ AGCaACC
1577 I~ CC rnrT~TTr~Ar-r~rr~7~aar-rrr~AA A~CaAC

SUBSTITUTE SHEET (RULE 26) W0 95/2322~ - P~ 5C

Table 26: ~ouse TNF~ ~H T~rget Sequences 3t. ~ T~ t 5-qu~c~ ~t. ~ ~r~get S~ ~c~ -it r er~ Pos r t r on 66 ;7g&aAAU ~L GcucCcA 324 ~A~r C r~uCCCC
lal GrXaG5U U aJg~rccc 347 GAGAa~7U u cCCAazlU
101 GGC~7U u CuWccC 364 CC~CcCU C rUrcA~a~
02 GC~GG0~ C ~ 7~rccc~ 3 606 IJ~CCC~ c A~uu 102 ~rl~ c ug~7 366 Ircccuc~J C ar~CAGuU
106 wa~glJ c CC~t~UCA 369 C~cAU C AGuuCUa 110 ~rc~rcccu u ~cA 376 CAGuuClJ a ~ICGCCCA
rGCcC~ u czaJcac 390 ~r C AcaC~rcA
111 ~ CCCuU u CACu~ ac 396 ucaCAcU C AG~CaU
112 ~rcCc~r C ACuca~r 401 cUCA~ C A~CCUCU
116 Ut~CP~ C Ac~gcc 404 A5ADC~ C r~CaA
137 r3CCaCAU C uCCc~Cc 406 AWWCU U CUCaAA~
139 caCA~ C CC~/CcAg 406 A~rcA~cU ~ cUcaAaA
177 Ga~r C CrJ5 407 ~wclrGr C ~rc2Aa~u 207 AGGCa~r C CCCcA~A 409 AU~ J C aAA~luuC
228 GG5GCu~:r C cal;AAcGr 409 AucuuCr;r c AaAur~rc 228 GGGGCu~ c C~Ga~c~ 409 ~Ucr~urcu c AAA~u~rc 236 CA~ C C~GCG3 432 Ar~rrGU A GCCCAcG
236 CAG~A~r c cAr~7cr g 249 GGugCC~r a ~r~rcucA
249 GGuGCClJ ~ iJ~clrca 444 Ac5r~rcw A GCAaACC
501 A~r C CUG5CCA
261 ~X:aGCaJ C WCUCaU 560 g~r~ A CC~UC
261 TJC~çrCCU C WC~Jcau 560 r~GguCG&r A Cc~ rc 263 AGCCUC~r ~r C~ aDl;C 564 ~ Cur u g~rurA
263 A~rCCUCU U ccrc u/JC 567 ACCUusrl:J C l~rCCC
264 GCC~JU C ~t;trcc 569 CUug~CtJ A Cl7CCCAG
264 g~CW C Uc urrcc 572 g~CU C CCaGG~u 266 Cr~rcu C a~lC~G 572 G~rCuac~r c CCAGguu ~urcalJ ~r CCUGclJu 572 GuC~cU C CCAsGUu 270 ~CaW C c~rGcuuG 579 CCCA~;W u CUCWCA
276 UCCUGclJ u 5~rGGCAG 580 CCAG~ruU c uCUCrcAa 297 r;~r C ~rGuC 580 CCaGGuU c ~rCUUC~A
299 ACG~W U CU5uCUa 582 AGGWCU C ~UCaagSJ
300 CGC~rCW C ~aC 582 AG51~uCU C UUCAAG5 304 crruCU~7U c uAc~rG~a 584 GUuCUCU U CAAGG5a 306 UcUGUcU A crrç1AAc~r 585 UuC~CW C AaGGGaC
314 CUGaACU U CGGÇGtrG 608 Cr CGaClJ CgugCCrc 3 lS UGaACW c GG6G~A 615 ~ rGcu C CUCAcCC
315 ~GaaCW c GG5guGa 615 AcG~GCU C CUCACCC
324 gGGUGaU c GgtJCCcC 618 UGCIJCCU C ACCCACA
SUBSTITUTE SHEET ~RULE 26) WO 9S/23225 251 2 1 8 3 9 9 2 ~ iSG
630 ~U C AGCCGzu 940 GuCU~CU c ~aGaG
630 ACACCgU C AgCCgaU 943 '~CUccl:J C AGaGcCc 638 rlgcCgAU u uGC~alJc 972 IJCUzaClJ u ~gAAAGg 643 A~GC~r ~ uC5cAuA g72 ucl:raAcu u AGaaAgG
645 ~uGCuzU C ~JCalJACC 973 CUzACuU A G~AAggG
647 GCualJCU C acacc~ 984 ~GsGgA~J ~ auGacuc 663 agAaa~ C AACC~JCC 984 ~GGGga~ IJ alJG~CUc 669 ~ ctJ C C~JC~G g85 GGGGaulJ a uCGcUCa 669 UcaAcclJ c cl~c~Cr~'G 997 ~c~gU c CAAc~cu 672 ACCUCCU C I~Ct;GCCg ' 010 Cu~ c 1~
674 C~JCCCCU C lJGCC~C 1017 cAG~agCU ~ ;7c~CAA
681 c~JGCCgl:J C AagaGcC 018 ~sCt.lJ 1~ cAaCAAC
681 COGCC~ C AAGAGCC 1 0~ 9 GaSCt~ c ~ACu 68~ CUGcC~U C a~cC :0, 3 l:rsGGccu c ucAlJgC~
~34 CCC~ A ~GaGCCC 1096 A~Ac~ C ~gGG
734 CcclJGGU a u5~aGCCc ~ 1 06 a~;GGclJ ~ uccGAAU
744 AG~ a l~acClJGG 1 1 07 UG;;GclW u ccGAAIJu 746 CCCAlJ~J A cCUGGGA ~108 GagCuDU c CGAA~UC
759 GA~ C uuCCaGc 1 1 1 5 Cc~'~aAul7 C AC~GGzG
759 GaGGaGU C ~CCAGC 1133 CG~AusU C CAuuCcU
761 GGaGlJCU U CCAGCUG 1164 g~ ;gU c AgaJlJGc 7QGa~JCU~ C CAGC~;a 1180 UcrJr,UcU c Agaal~
786 ACCaACU C AGCG.~JG 1203 a~ c ~GGCCUtJ
798 CUGaG5U C AAlJCuGC 1210 cAGGCCU ~ CCUacCU
802 Gg~ aUJ C uGCCCaa 1211 A~ C CUacCUu 812 CCCaA~U A culJaGa~c 1214 CCQiCCU a c~CAG
816 AglJAcuU a GaCUWG 1218 CcuACcU u CaGACCu 821 ul/aGAClJ ~ I~GCgGA 218 CC aCC5 U C~GACcu 822 IJaGACûlJ U GC~ = 1213 cCIACcU u cAgACCU
830 GCg~U C cGGGCaG lZ18 CCUacCU u CA AccU
840 GGCaG~J C Ua(:C~ 1219 CuaCCW C AGACcuu 842 CaGGUW A C~GGa 1219 C~cCW c agACcW
842 CAGgucU a CC~G~ 1226 C~ACC~J U uCCagAC
842 cag~uCU ~ CuuugGA 1226 CaG;~ccrJ ~ ~'CCA~AC
845 G~ U lJGGaq~C lZ27 ~CCW u ccaSrAcu 846 ~JCUaCû~J ~ GGagUCA 1227 AG~ccW U CCAGACU
852 WGGagU C AI~JGCuC 1228 G~c_WW C CA~c 855 GagllCa~ J GCuC~W 1238 SA~;CUu c cCUGAGG
887 AUCCaW c uclJACCC lZ6Z ca~cuU C CuCAcaG
891 AuucuCU a CCCaGCC 1283 CCCCccU C ual;WAU
gO5 CCcCaCU C IJgaCCCC 1283 cCcCCCU C ~UA~
905 cCCCacU c IJgACCCC 1285 cCCw~:u A l;~lJaU
905 CcCCACU c uGAccCC 1287 Cc~Ct~U u UauAuW
914 GAcCCclJ U uac~G lZ87 C 'G~C~U ~ ;~aU~U
915 ACCCCuU u aclJCuGA 1288 C~IJAW U ;~IJaDWG
919 CW~lAcU c ugaCCcC 1289 UCUA'uW ~ UaWUGC
928 GACCcCU u l:raUugUC 1293 UUUAUzU U ~c-cacw 928 gAcCCCU U UAlJUguC 1293 uWaUaU u IJGcAcUu 932 CCWUAU U guCUaCU 1294 WA~a~U U GCACWa SU~STITUTE SHEET (RULE 26) 218~992 WO 9SI23225 . . ~ r~ ~ c 1300 l~ca~ U a~Uu 1462 aCCuUGU u GCCuCCU
1303 ca~au u Au~U L470 GccuCcU C ~IU;~GcU
1304 ac~a~u A ~rA 1472 cuCcUCU IJ UO~c~A
1306 Ut~AU U 13a~.DU 1473 uCcUCL~U U ~Gc~
1307 uAl~U ;7 Alit~ 14.74 Ccu~:uW lr Gc~G
1307 UaClJa~7[J iJ AuuAlJuU 1478 ~ UGcU U Al;GUWa 1308 AUUhU~ A ~7A 1~7g V~cW a UG_uuAa 1310 IJauCuAiJ ~ A~U~U 1479 UWG~c~U A I~WUUaa 1310 ~W ~ A~ 14.84 UCAUGUU l:J aaaAcAA
1310 1~ IJ Al~ 1498 AAAuauU U AiJCl7aAc 1311 A~l7 A ~= 1511 ~cccAaU ~ G~AA
1311 ~W A IJ~aD~ 1514 cAaUCTGU C IJuAAuAA
1311 Auul~J A UuUau~J 1516 aStJGU~ u AAuAAcG
1313 ~ 17 ~U~ 1529 Cscu.7AlJ u {~;GuGAC
1313 ~ IJ ~U7CiJA~ 1529 cG~GAU U iJGGUGAC
1313 u71J u IJau~7Au 1530 sCUGA~ u sGiJsacC
1314 ~7 J A~iU 1530 GC~GAl:~tJ U G''UGaCC
1314 ~ ~ A1~7 1563 il~aAcCU c ~c~CCC
1315 Al~ A ~A 1563 uSaaCCtJ C u~uc:CC
1317 ~.7 i7 ~7 1568 C~CIJGCU C CCCAcGG
1318 A~ ~ A~U 1589 UGaC~ A AlJuGcCC
1319 ~ A ~A 1592 CO~rAAU u GcCCUAC
1326 A~ A ~CU 1617 GAGaAAu A AAGa~cG
1328 ~JAI:r U ~W~C 1623 UAAAGaU c G~lAaa 1329 AU~ i7 AWlJgCu 1633 ~UAaaaU a aaAAaCC
1330 ~U A IJtWs;Cuu 25 AsGsaCU a gC"açGA
1332 IJA~aU ~ ~sCuuAU
1333 A~ IJ sCuua~G
1337 auU~ IJ AUGaAuG
1338 u~ A uGaAuGu 1346 UGaA~ A ~U
1348 Aa~JAi7 17 ~aDtn~GG
1349 A~la}lU ~7 Al~.
1350 ~clr A ~;GaA
1352 uaL7u~Au u UGGaAGG
1352 ~ U ~Ga~
1353 A~ ~ GCaAGgC
1369 GCGaJgU C C~GGaGG
1398 gCl~suaJ U cA~;aCAs 13g8 G~GuCJ IJ casaCAG
1412 GACA17GU 17 ~JCuGlJG
1413 ACU~DU U I~CuGCGA
1414 CAI~U IJ CuGCGAA
1415 AUG~ C uGlJGAaA
1415 A~WU~ c l:rsugA~A
1438 saGC17GU c CCCAccU
1451 CU~ C ~cUaCCU ,~
1453 Sgca~ C UaCCu~G
SUBSTITUTE SHEET (RULE 2~

~ wo ss~2322s 253 2 1 8 3 9 9 2 r~ 56 Table 26: ~ouse TNF~ ~rrrnPrh~ Riboz9~e Sequellces - ~t. 160u5-- ~ R~o::ym~ S~lr~ue~
Pos~ t- r~
~ ~CCCGGC rnr~ -rr'~r^rrra~ XCJ
66 ~;GGAGC rrr~ r~,-~-rr~ ~C~
101 ~ rnr~ ~r~ ---rr.~
101 GGG~CaG rn--~ -rrr~---rr~r d~ C~CC
02 ;U~c~ rrTr~ r~ rr~ 5 C~C
102 ~ Ca rnr~rTr~ AA~C
106 ~GG rrr~sr~----rr~_rr~ ~C
110 ~ rnr~rTr~ ~rrr~ ` ` ~,rrr~ ~ AGG;3AC~
~ ~ ~ r~G rr?~~ rr~ ;GGaC

11 ~ r~aG~ rr~ rrr~ GGaC
-7~Ga~ rnr~ ~r,rrr~
~ ~ 5 rGCCaGJ rnr~nr-~--r-rr~ rr~
137 ~r,G~ r~rr~nr:~r--,rrr~ rrrs~ Aa~GC
135 r~GG rrR ~nr~:rrrr.~r,-,rr~, AG;UJWG
177 CG;~GCG rr~`TTr~r--rrr~ rrr~ A~GWG~-207 IJ~GGG rnr`~`--r.^rr~r-,rr.~ A~CaT
228 ~G rnr'~'--~'`~ aA~CCC
228 ~ ~ AGCCCC
236 CCGCCUG rnr~ rr~rrr~
236 CCGCCtJG ~rTr~nr~-----r--rr~ -rr-~ A~WWG
249 I~'Gai~ rrrr~3~rr~rrr~rrrrr~' ACGCaCC
249 I~ rrrrr~ A~CC
261 AI~A rrrr~ Tr~~rrrr~`~rrrrr`` ~;G~7Ga.
261 A~aGi~A rrrr~nr~--rrr~--rrr~ ~G,~a 263 r~A= rnr nr.~r--rrr~---rr~ AGaGGCtjT
263 r~alulGaG r~nr~rrr~r~r~rrr~~rrrr~ ACaGGCJ
264 G~3Ga rnr~sr~------rr~r~r~-, AA(;aGGC
2 64 r,GaA~a rnr~ rTr~--~rr~ ` ` _rrr` ~ Aa~;GC
266 CAGGa~T rrTr~TTr~ rrrrr~
2 69 MCCal3G rrr` ~ ~r--rr~ ~ ~ r rrr~ 71 A~G~.a 270 CA1~GCa~ "~ rrrrr`` AA~A~a.
276 C~C rrrr`~`-rrrr~r-,rrn~ AGC~GGA
297 Ga~G~A rnr~ nr.~ rr-rr~ ~ 7 rrr~r~ ~ AGCGUGG
2g9 ~;hGaCaG rrr~ --r~--rr~
304 ~JGC= rnr~Tlr~--~r~ --rrr~ Aca~
306 ~G rrr~--rrrr~rr-r-~
314 C ~CC rnr~rTr~ rrr~ r~r7rrr~
315 UC~CCCC rrTr~nr~:~r~sorr~ r--~rrr~ ~ AAGGUca.
SUBSTITUTE SHEET (~ULE 26) WO 95123225 - 21 8 3 9 9 2 F_l/ll 5 '~ ~ 156 324 GGGG.~C ~ r--rr~ ~ A3CAK t 324 GGGQACC I IJ' - ' I' '' ~ '----T--~-~ ~ A~CCC
347 AUaUGGC ~`'~ ''~```~~rr`' ACI~
3 64 C~a.DGA (~ ~r~ ~ T r,r~:~ ~ A~GG
366 ~aC~A'3 rr~r.~r~ r~ AGaGG~
366 AA~JGa3 ~ --rrr.~ AGA~ A
369 ~3 "~ r rr~ Al;G~G
376 ~GCCA ~ ' A~ G
390 ~'G'3 rr~ AGG ~U
3g6 A;~3 ~ r----rr~ ~ AC~A
401 ~GaAhA'3 t~ rr--rr~ ~-----r~ r, 404 ~A ~TT~ ~C3 406 ~5 f~r ~-rr~ ;aDGa' 406 ~ r.:~r.rr~r.--rr~A A~,u 407 A~CCA ~ AaG~lGa 409 G~UU~ r~u 40g GaAlltJC'3 ~ A A~3 409 G~ ~ r~ ~ Ar~GA~
432 CWa;Gc ~ ~r rr--~rrrrr~ ACAGGC3 444 GG~TGC rrr~r~rr,rrr~,rrrrr.~, AcGa~
501 ~æcaG ~ Tr~r~rrr~r rrr~ A~GCGU
~60 Ga~aAGG ~ AC~ACCC
560 Ga~G ~ rr~rrr~ A~ACCC
564 ~laGAC (~r. ~rr~r---r~ AGG~ A
567 GG;;aa3A ~TTr~r------rr~T~rrrrr~ A~r-~a~G;r 569 0GGGaC ~ u - ~ ``rr~r`~ AG~U3 r 72 Aa~GG rr~r--rrr~,~r--~rrr~ AG~GAC
572 Aa~G ~ T~rrr~` A~GAC
572 ~ C~ rrTr~rrrrr~ ~ T rrrrr~ ~ AG~ÇAC
579 ~AG~ ~ ~ r--rr~ ~ ~ rrrrr~ ~ ACC~GGG
380 ~13a~GA ~ T r--rrr~ ~ A~;;
~80 ~A~A rrTr~ r~r--rr~rr~

582 Ct ~A rrTr~TTr~rrrt~ rrrrr~ AMj~
582 CCDl~aA rrTr~r~rrr~r-rrr~ A~raa;X~J
584 UCCC~3~; ~ u~ rr~rrr~ ~ AGaG ~Ac 585 Gt~ CC~3 rrTr~TTr~rrrrr~ r~aa A~araGAA
608 Gaa ~ TTr~ rrrrr~ Tl Ar.;JCGGG
61 5 GG.,~ r rrrr~ ~ Al;C;~3 615 GGGuaac ~r-~-r~rrr~rr~r~ A~r,~3 618 ~;GG3 rrTr~TTr~rrrrr~ rrrr~ AGG~;CA
630 AU~GGCU ~ u~ r~rrrr~ ACGGUGU
630 A~GGCU ~ rrrrr~rrrrr~ ACGG~7G3 638 r,~CA ~ r - 'f ~ rrrrr~ A~3 643 UAUGaGA l~ TTr~--r~rrr~ ~ ~r---rrf ~ ;CaAAT3 645 ~r,~ ~A ~ rrrr~ T ~ rrr~ T~ A;~;caA
647 CDGG3AU Cr~ r`-r-rr'~ rrr`` AGa~Gc SUE~STITUTE SHEET (R~ILE 26~

W0 95~3225 255 2 1 8 3 9 9 2 P~ 156 663 r~ ~?~ r~ a ~ r--^^r~ a A~
669 C~ ~r~r~ _rr~ r^,~
669 C~ rrTr~rTr~_rr~rrr~
672 CSGCa~ ~ r~rr~.~ar^,r~
674 Ga~ nr~Af:rr~a~rr^rr~
681 GGCt~7 r~`~'' -r~ r^~` ~ ;~CGGC
681 a~u ~ ranr~rrr~rr~
681 GGu~ ~nr~r~r^~rrr~`7l ,~CaG
734 G~, cnr~r r~ r~rn~~
734 GûGC JCa r~ Tr~rrrrra~rr~m~ aC~
744 c~a ~r~ tr~ rr~rr~ a ~G~
746 ~Ca;;û ~r~ rr~r~r-~
759 GC~ An~ Af~ r~ aar ~mAA A~
759 Gu~GGha ~rTr~ rr~ r~r~
761 C~;G ~r~ ~rrr~rrrr~
7 62 CY~ rrrrr~ ~ ~ rr~r~ ~ AA~;;~C
786 C~7 ~ Drrf-rr~ r--rrr~
7g 8 ra~w , ,,,, f ~ a ~ rrrrr~
802 UlJ~GGCA ~ r`^rrrr~r-rrr`~ ADoeac~
812 G~A~ ~r~rrrr~`r ~:~`` Aa3tæGG
816 C~AU;UC ~tTr~'T~--~rr~rrrrr~
821 Cl~ ~ " ~rr~`~
822 a~JCCGC rTlr:3TTr~ rrrr~r` ` ` rr^rC~ ` .aAG;7C~a, 830 C~ nr~ r rrr~a Aa~5C
a40 C~AA~ ", ",~ ", .~ I I .~rr~rrr~
842 IJCCaAAG ~ rrrrr~
842 ~aA~ rnr~ rrr~r~
842 UCCaAA~ ~`rJr`~`-^rrr`~`~r~
845 Ga~CCA rnr~-~r~rrrrr~r rrra~ Du ac 846 UG~a7CC ( o~ a~r~rrr~
852 G~;CA~ ~ rr~` al D~A
855 Al:Pl~ r7Tr~ ~rrra~r^~rrr~ ~C
887 GGGU~GA rrlr~r~ rrrrr~ ~ ~ rrrrr~ ~ =
891 GG,~0 ~ a~r~rrr~ A0,a;U~
gos GGG~ a rnr~^r7rrr~a~r~
905 r,GGGo( a t~ rr~--rrr~ ~;GG;;
905 GGGGU~ rTl--~r~rr~rrr~ r-rrrr~
914 CA~a~ rnr~r~--r~rr.rrr`` AGGu~C
gl5 ~JCAGaGtJ rTTr~rTr~rrr~r--.rrr~ ~GG~,T
919 ruGG~ rT~r~3nr~Arrrrr`~rrrrr`a AG;~.A~
928 G~ rT~ T1r~Arr~rrrrr,~ ~C
928 Ga~ rr~ a~rrrrr~,3~ AR~C
932 A~iaC Il~.DII. '`' ' ' I I A~rrf--rr~
940 CUC~G ~ r^rrr~r rrr~ ~c 943 GGGaJ~J ~ ar rrr~
972 C~UCIT ~ r-rrr~
g72 CWWW rTT~.7r~_rrrr-~rrr~
g73 CCCtJt~ r7r~ ~rrr` ~ ~ 7rrr~
g84 r~GCCA~ ~ r~rrr~r ,rrr~ ;~CCCW
SUBSTITUTE SHEET (RllLE 26) 218~992 WO 95l23225 P~ l 5. ~S6 984 Ga~CCalJ r~ rar~ JCCCC~
g85 ~;CC.~.. rnrarrr~ ~ r~ ~ aA~cccc 1010 AAGW~ rr~
1017 ~ a~~rrrAA AGC~JC~;
1018 G~;G~ rr-rr~ ~ ~.GC~
1019 ~J, " " ,, .,~rraa AAaGCJC
073 ~Ga. rrT~ r--rr~ GGCCCA
09 6 CCCA~W ~ -, .. . .. I . A ~ ~ ~ ~ ~I lU;~CC~U
1~06 ~;Ga ~ r~ ilc;:cc~J
1107 ~CGG ~ a ;~AG~--cca.
1108 GaA~G ~ ~aGC-C
1115 Ct~, "~ ~ " ,,..... ,~rrrr~ AA~ J
1133 ~
1164 GcPa~ rr~ ~r~ a~C
1180 ~llIJCJ rr~TTr~ar-~a~ QaC;~
1~03 A~GGCCU rrr~ r.^rr~
l Zl 4 Ct~iWG ~ ~r~
1 ~18 AGOE~tJG rrT~~ ~rr~ rrr~ ~ ~.GGCaGG
1218 A~C~7G rnr~ rrr~ rr~ ~ aa;tlaGG
1218 A~ r~ rr~ ~ ~aGG
12i9 .aAG~CC ~ll - .. ,.. aa~''~
1219 AA~;GD~7 C~r~a7~rrrr~
1226 GD~ ~" ~ ~rr~ A~CGG
1226 G~ rrr~ ~rrr~
1227 ~XG rr~ -~r~
1227 aC~aJGG r~ rrr~ ~rr~ ~ U~WCU
1 228 GA~ nr~ A~C
1238 CCUa~ AAGaGCC
1262 C~
1283 AI~AAI~ rr~ ~rrr~
1283 AI~AA~ ~ ~ ~ A~
1285 Al~aAA ~ "'`Grrr`` }U~aGGGG
1287 AAA~ rrr~ rr~l AZ~a~;G
1288 C,A~ ~ ~1~ 1 -, .,, .~.~` A~u~aG.~
1289 GC~M~ rrrr~ rrrr~
1293 ;~ r~rr~Tr~r-rrr~-,rrrrr~ A~AA
1 293 AAG;~C.~ rt~rrrA~ ADh;~AA
1294 ~ C rrrr~r~rrrrr~rrrrr~
1300 A.~ rnr~ rrr~?~rrrrr~
1303 AA~AAIJ rl7r~-7r~--rrr~rr~-rr~
1304 ~.AA rrrr.~rr~rrrrr~ar--rrr~
1306 A~ rnr~r~~--rr~ rrrr~
1307 .a.~ r~ rr~rrr`~rr,rrr`` AA~.
1307 ~Aa~ nrTn~r~ r~~rr.~
SUBSTITUTE SHEET (RULE 26) WO9SI23125 r. l,~,J,ccls6 257 21839g2 1 3 08 ~AI~A ~ r ATlr`--~
1310 ~aA~ rrr~ ~r~
1313 P.~aA~ ~' ----I--A~Ar--~ ~ ~a 13 3 A.~.A1~. ~ .AI _ .1 1 1 .A Z~ I------~.1:~ a~.A
1319 T~aA rTTr~TTr~----rrr~ r~ AAa~3A
1325 AaAlJAaA crTr~TTr~rr~ ~r rrr~ AaAT~UW
1328 GC~TA I l l ~ r-r~rr- ~ AI~AArlA
1329 AGC~ --rr--~ ~ ~aAA~
_330 AaGcAaA rTTr~-Tr~rrrrr ~ r-~-rr~
1332 A~1U3CA t nr~r~ _rr~r rrr~ ATiaAA~JA
_333 ~IJAaGc rrT-~ Tr.~rr~ ---rrr~a aa~.
1337 CAT~W rrTr~-~r~r~^r~
1338 AC~ r l ` K .. . I l ~--`rrr~ AaGCAaA
7346 ~aA~lAaA r ~ r r ~ rr--rr ~ AC~
1348 CCi~ A rTTr`-7r~r----rr~arr~rrr~ ACAT-W
1349 ~1~ rrTr~-Tr~--~rr` ~ ~r-rrr~ ~ Aa~lac uJ
_350 TwccAaA rTTr~r~ çrr r~rr~ JACA
1352 CCWCCA rrTr~rTr`~7rrr```--~zla ~AA.~A
_352 CC~-CCA rrTr~Ar ~ AUhAA~A
~3r3 G~-CWCC ~TTr~ r-rr~ rrrr~ AAU~ T
1353 CC~XaG t nr~TTr`_rrrr~rr~--rr~ ACACCCC
_398 C~ r~-Tr`-~rr~`~--rrr~ AG.~u aGC
_398 CI~GCJCUG rr7r~ Tr~ rrr~ AGAG~GC
14 2 Ca aGaA r I1. .. ~ rrrrr~ ACZU~GUC
1413 ~t aGA rTr`~ T~rr~r rrr~ AAC~U
1414 ~a~ rrTr.~TTr~ r~`~` ~r`~ A~A~3JG
1415 T~U~ ACA rrTr~.TrJ~rr rr~ ~ ~rrrrr~ ~ AA.~alT
14_5 ~A rrTr~lr-`-~~rr~ rrrrr~ AaA~J
143 8 ~G;JG~ rrTr~rTr~ _~rrr~ ~ ~ r~ ~ ACa~C
14Cl ~s~GA rTTr~TTr-~r~rr~~ - rrr~ aGGCCaG
14S3 QAGGC7A rTTr ~ ~r--~rrrrt~ AGAGGCC
1455 A~AGG T ........ ~ rr~:~ AGAGaGG
1462 AGG~GGC rrTr` Tr~r~-rrr~ rrrr~ ACA~G;J
1470 AÇCaAAA ~r~T7r~ - r~ AGGAGGC
1472 ~ aA r~ Tr~Ar~rrrrr~r~r~ AG~
1473 AT~AGCA r~Tr.T~TTr.~r~r,rrr~rr~ A~GGA
1~74 Ca~C rTTr~rTr~r-rrr~r~rr-rr~ A~aGG
l~7a UA,a;~U rr;r ~TTr~r~rr-rr~r^~
SUBSTITUTE SHEET (RULE 26) W0 95/23225 P~

1479 ~a rnr~ r~ AACcaA~
1479 ~AA~ rrr~TT~
1484 ~WCUU ~ I r ~ ~ .. " r 1 . a ar ~rr~ ` AA~:a~.A
1498 Gr~17 ~T~ ~rr~``~`" AA~
1Çll TJtlAACAC ~ ~r~ r-rrr~
lÇ~ r~` ~ rrr` ~ AC~G
1529 GD~LccA rr~T7-~ a~ A~;CG
Ç29G~ a~CG
1530 GG~CACC rr~Tr.ar~r~ MDCaGC
lÇ30 GG~TCaCC C~ r.rrr~a~r.rrr~` M.~.~;C
1563 GG;~ ~`'1r`~``~`~ A
1_63 Gaa~ rn~ r-rrr~ r-,rrr~
1568 CCG~;GGG ~nr.aT~r~ a A~GaG
~Ç89 GGGCAA~ rrT~rlr:ar-~rrr`~'~r~`~ ACaG~
lÇ92 G~ ~r~r~r~ r~` ` ~ aG
1617 CG~lU ~u~ rr~r~a A~C
16Z3 =AGC ~ `~rrr`~ .~WC~7C~
1633 GG~ t~ rTr~rrr~ ar--r~t~ a A~
.

SUBSTITUTE SHEET (RULE 26) L
WO 95123225 259 ~ 1 8 3 9 9 2 P~ 156 f ~ _ . .r f .~ ~ f f f. f f. f _ ~ ~ ~ .' ~: ~ ~ .' .' .r .' f f r d ~ q ~ d '~ ,Z~ q~8~
O ~ o c U~ ~ r O U~ O ~o o c C ~ ~ o ~
-SUBSTITUTE SHEET (RULE 26~

W0 9S/23225 . . - ~ 2 1 8 ~ 9 9 ~ IS6 ~ .. ~ ~ .

SUBSTITUTE SHEET (RULE 26) ~, W0951 ~225 Z61 2183992 P~ M0156 -3a~ 8'3'~ 3'~'33~
r ~r ~ .-- ~ ~r ~rr ~ ~r ~ ~ ~ f ~ ,cr ~ .

5 ~ a o ~ 8 ~ ~ O ,~7 ~;; ~7 ~, rD ~ rn ~ O ~ ~ o o E-SUBSTIT~7TE SHEET (RULE 2 WO g5/2322~ , 21 ~ 3 9 9 ~ r~ 156 . ~ 262 8~1 ~,, ,~, Ul SUBSTITUTE SHEET (RULE 26) WO 95123~25 P~ ~ 15G
~ 263 2183~S2 Ta~le 29: Hu~ bcr/~bl HH Targe~ Se~uenc~
Se~ 8t Soques:c8 'o.
k~
.7~7-7. ~ ;,.
'~. A~ ~ AAt AAt~
2 AP~ ~ IA
b3-~2 ~-~,.~
Z, W.~7 UUC A~AAI~lrr ,~
SUBSTITUTE SHEET (RULE 26) 21~92 Wo 9sl2322s 1 ~l/-L. Is6 Ta~le 30: ~Iu~au bct-abi ~IH Ribozyme Seque~ceS
c- ~ R~o~a- S~;u~c~
ID ~o.
26~ JI l Iul I) (~ rr c~ ~ Al 111~ `' 11~,1 1~ 'A
27~ ~r~` ~ ~I-r~ ~ llJ~
28rr-rTrr~r rr7~ rr~ r-rcrrrJrr~r 30~ ~r~ r ~ r ~ a 31rr~ rrcrrr r~p~ ,.~c~

SUBSTITUTE SHEET ~RULE 26) wo ss/2322s 265 1 ~l~, s. . 156 Table 31: RS~ ~ H Ta3 get Se~uence -t. }~ ~ t S~ C~I ~t. EE~ t S~q~ Lc~
Po-itio:l ~?o~ t~ o~
1~ GGC~aA~ A AiiUCA~ 276 AAaa~7 A C~lGaa~JA
14 A7U~a.A~ C A~G 2a3 AC~JG~U A C~ACaC~
AAIJC ~A./7 IJ C~A 295 ACa;~aA~ A ~iG;3CaCJ
19 AI~J C AGC,CaAC 303 ~GC.~C17 rJ UCCC~
54 C~AK;a/7 A AI~CACC 304 G~UC ~J CCC~'G
57 1~7 A CI~CCACA 305 GC~U~7 C CC;~UGC
77 ~ C AC~Ga( A 309 I~CCU A 2CCA~
94 AG~CCt ;~J IJ G~:U 317 ~:GCCAA~J A ~CA
97 CCWI~J C .aCDt~GaG 319 C~ ai7 ~J C~AI~
101 W~ '~7 Ga~ 320 CAADa/W C al~UUC
0 AGaa ~.7 A AQACalJ 323 I~IU~ C iU~17G
113 CC~A~ A A~ aDCAC 327 CA~CaAl7 C ;I.CG~
118 A~IIA~ C I~ACC 337 G~--l7 ~J CtJ~L
122 CA/JCAa7 A ACC~G~ 338 ~JGGGJ~7 C ~aA;3 134 Ga.~;aCaU C A~ACAC 340 GG;30UCJ IJ A~AI'GC
137 ACa~U7 A ACaCACA 341 GOE7CCOU A CAAUGCA
148 C~M/J ~7 ~UIPDAC 350 ~D(~ U GGCUJJA
149 ACMalJI.7 U A~ 356 ~U U A~GCC~7A
1~0 CaAAl~U A ~SC~J 357 I~;GCaUU A AGC~7AC
152 = A ~A 363 1~W.7 A CAAA~CA
154 ~.7 A CIJC~;P17A 372 AaA~a alJ A CIJCCC~U
157 AI~CU ~ t~AAU 375 Gca~c~7 C CCA~
161 AC~ A A~JCaDG 380 COCCC W A Al~;JACA
16~i G~;7 C A~GaAIJG 383 CC~AU A ~AW
176 AA~ A G;3Ga~iAA 385 A~A~7 A CaAl~U
188 G~AAA~ ~7 Gal~;AM 391 ~CaAG~ A ~GA~C
208 GC~i7 ~J ~JC 3g6 G~liGAU C IJ~ aAlJCC
209 CC~DU IJ ACAOIJCC 398 AI~A~ C M/JCC;W
210 CA~D~J A CA~ 402 UCUC ~UJ C CA~
214 =CA~7 IJ CC~;G;JC 406 A iL7CCAU A M~UIJCA
215 1~ C C~ 410 C~A~U U UCAACAC
221 ~C~IGGl7 C MC~G ~11 A~AlWl7 ~7 CMCACA
226 G13~ 7 A lla;~AA~ 412 UAaa~UU C AU
239 ~ A I~A 421 ACACaAU A ~ACAC
241 AaaCOau U ACacAAA 423 A~7 U CaCACAa 242 AaC!lUD.7 A CaCAAAG 424 CAa~DWU C ACaCAalJ
251 ACAMG;J A GGAAGCA 432 AC~CMU C UMaaCA
261 A~GCaaJ A Aa~M 434 ACAAUCU A MaCAAC
265 AC~A~J A ~aAM 446 MCAACU C UAUG
267 1~AA~17 A Al~AAA17A 448 C lPa7C~J A ~A
274 AaAAlUW A UACI/GM 454 UArS;CAU A AC~aI/AC
SUBSTITUTE SHEET (RULE 26J

458 C~A~ A 1~0CCA
460 UA~7 A C~CCU7A
463 C1~7 C CaI~C
467 ACD~ C~ A GVCC
470 CC~J C CaGP~G
489 ~A~ U ~GI~A
490 G~AAAUl7 A ~AU
492 AaAI~ A GDaAD~
495 ~ P. ~A~
.

SUBSTITUTE StlEET (RULE 26) -WO 95/23225 - 2 1 ~ 3 9 9 2 P~ 15C

Table 3~: RSV (lB) ~I R~bozyme Sequence 3t. ~8 P~bozymi~ g~ nc-P o 2~ ~ t ~
AI~G~U ~-~`--.r~--~A A~U~'GCC
18 ~--~ A~'GA~J
54 ~iW~lU7 ~ 1 A~;
~;7 ~7W = rrK:~n~`~~~`~-~`~ AI~CA
77 U~J ~ AU~WCA
97 CIJC U~7 ~ AC \Aa;G
101 ~JCL7C ( l ~ AG~Q.
122 Ca~J ~`~ AG~UJG
137 IJGO~ `~`rr~rr~rr~rr~ A~G~UI;U
148 G~;a~A l~`'Tr`~r.~r_rrr ~ A~JG
149 AW~ ~Tr~r~r~rr~7 ~rr~` ~ Aa~U
a,ljG~\~, ~ ~r A 11.(~ r--rrr~ ~ A~AU~G
152 a~aGCA ~or~ r~rr-rrr~ Alla.3A~U
154 Ua~ cnn~TTr~ rrr~/ r'rrr~T~ ~.~P~AA
137 AIU;~C .~nr~r~rr,ror~rr.cr~ Ar~W
161 C~J, ", - 11 `'_.~ (T ~rr~r~ A~AG~
163 C~UJ f~ -- rrr~ r-C~, A~JC
176 ~CD~AC ~ rrrrr~ ~l A~
188 ~UO~, " , , I ~rrrrr~ AG~7C
208 GaAI~GCA l ll ~ rrrr`7 A~WGGC
209 GG~DW , ~ rrrrr~ Aa~;U~G
210 Ar~G~WG . (I -, T . ~r_rrr~ AaAD~G
2l4 GaCCAGG , ~ rr~T~` A~AA
213 ~JGacCAG ~_.rrr~r--.~ AU;~A
221 CAII~U rnr,~T~rJ~rrrrr~ ~ T rr,~.rr~ ~ ACCAr, 226 CA~IUUCA I I1 , --_ " ~rr, rr~ AG~aC
239 UW~A ~ --r.r~ ---rr~ A~UCA
241 131~DW ~r:~T~r~rr~rrr~lr--rrO~ AUa~U~
242 C~; rTr~r--rr~ rrr~
251 ~CU~CC ~ " ~ 11 `'r~rrr~` ACI~UUGC
261 ~ ~T~ r~r--rrr~ AGUGCU~
265 ~CI~A I I ~ r~ A~W
267 1~ ~ rr.rrr~' A1~31JA
274 l~lA ~Tr~Tlrpr~rr~ rrrcn~ A~UU~UU
276 ~aa rr~ T~ ~c~r~`` AU~
SUBSTITUTE SHEET (RULE 26) W0 95123225 2 1 8 3 9 9 2 . ~ S~i 283 I~ GI~ C~---~
2g5 Al~;X;CC.lL I
303 A~
304 C~a~GGG

320 GU~
323 C U~IJ ~T~
327 CC~UJ r l l~ . J I . ~ ~ ~ A~.D'G
337 ~C~A~ ACOCai7C
338 ;~O~A r ~
340 GC~ ~ EaaCCC
341 ~GCWtJC ~ AGaACC
3 5 0 I~ZIADGCC ADW~l7 357 G~lila;~ nY~T?~~~~~~~~ AA~
3 63 ~WG r I ~
375~GG ~ AGGWGC
3 8 0 I;G~17 383 ~ r - .
385 Auac~ T .. a~
39} GaG~JCa ( ~ a~
396 G~~ I .. a~ pC
3g8 Al~WA~ r l l~ ~ -- ~ ~ - ``"-~` ~ A~;i~
402 ;~ a AD~.GA
4 0 6 ~7 ( ~ I . ~ ... . . ~ ~ A ~ ~ ~,~ ~ ~ =~
412 uu~ u~uu r 423 ~i ~ a~
424 A~Gl;GtJ ~ a 43 2 ~cca ( ~ a ~~~~
434 G;~Ot;~31J ( ~ .. a~ a 446 A~:G;:a~. ( lll ` = - ~ l ~~ ~Y`-~ U~
448 t=C~ I'I' '''' ~ ll ~~~ ~GaG~GG
454 G~

4 67 I~C I I ll . . I I 1 n ~ ~1~ ~ A~G
4 7 0 C~G ~ I 1 -- 1' I ~ A ~ ~ ~Cy~ ~ ~ A~CIDU3GG
48g ~\~ ' ll~ ~'l~ '~ ~``----~`` AD;;O~
4gO A~ ~ AA~WC
4g2 ~AA~C ~ u~ rrrr~
495 ~AAIJ r~r~ rrrl~a ~ ~r~rrr~ ~ .aa~a.A
SUBSTI~UTE SHEET (RU~E 26) w09s(2322s 2 i 8 ~ ~ g ~ P~./. 0~6 Ta~le 33: RSV (lC) ~I target SequeIlc~
3t. ~ r~l~t S~ 3c- =t. 1!~ t 5-g~-3c-3i'o~ t~ 03 ~o~ t~ 03 10 GGCa.aAU A A~ 165 UACa~'U A AC~aCG
16 ~ i~ 16g ~ A ;I~
17 AA~7 V Ga~ l~S UM~'~ ~GC~.
21 A~ A AalACC?. ~76 A~ 7 GC.~AG
25 ~aAi~ A CC~C~it;A 181 1~'GG~7 A A~
31 l~acca~ u AM11titJA 192 C~G~W A C~lIlaCaA
32 ACC~lJ A A=A 196 G~C~U A C~A17C;~A
36 c~7 Ir ~cc 201 AUaC~17 C AaA~7G~
37 ~7 17 AAC~CCC 206 A~aL7 IJ Ga.~.CGGC
38. ~aA~ A ~ 216 ~ u w~;
42 ~ c ~:uu~u 221 ~7 ~ ~GC:W
46 ACt;lXC~ U GG~aGA 2a 50 C0~GGl7 17 A = G 23i uGcaD~ u AU~aCAA
51 WUG.,~O A G~17GG 232 GC~u~J A UU~
67 ca~Au ~ ~c~ 234 ~;D~7 17 ACA~2UA
68 i~;aAUU C A~GaGU 23S ~A~ A C~G
71 A~AU U GAG~WG 241 ~2~ A Ga~JAU
76 AWGaGL7 A ~AA 247 lIPG;iGAU A ~CCC
81 G'~;AU A ~AA5~A 249 G;;Ga~U ~ JA
87 UAA~AGU U AG;U~l7AC 250 XAUAW IJ G-CC~
88 AaaAG~u A =ACA 256 UGGCCCU A A~A
92 G;~al3AlJ IJ AC~AAa~ 259 CCt 1~17 A ~AU
93 ~Ga~ A CA~AAlll7 262 ~AU A AUAi~GU
100 ACAAMU IJ I~W~WGA 265 ~A~ A IWW~
101 CA;~AA~ U G~aC 267 ~AU U G~A
104 Aa~ U ~G~aAU 270 Al;aD~G~ A G~A;iAU
105 AWOG~ U GA~WG 273 WWi~7 A Aaa~CCA
120 A~aG~J A Gt~=J 278 G;~aAAAU C CA~iUC
125 G;~aGcAU IJ G~hAA 283 AUCCAal7 IT ~ AAC
128 GCAW~ U A~A 284 li~ CaAW 17 CACAaCA
129 caucwu A AaaU~A 285 CCAa~W C ACAaCP~A
135 ~AAaAI~ A ACAIGCIJ 300 I;GCCaG~ A ~ aAA
143 ACAIJGC~J A ll~CIJGAU 303 CaGUACU A CAaaAUG
145 AUGCUAU A ClXal~aA 316 17GG~GGV U ~JG
151 UACUGAU A AAIJ~ 317 GGAGGW A UAl~alJGG
155 GA~AAU U Aal~iW 319 AGG~IJ A WUiGGGA
156 A~ A AI~C1WU 321 G~ 1JAU A ~aAA
59 Aal~aU ~ CAW~A 338 AUGGaAD 17 A~ U
1 63 AAUACAU U UAaC~aA 339 l~ A ACAC~WU
164 A~C~IIJU U AaCt~aAC 346 ~.ca~,' U G~C~
SUBSTITUTE SHEET (RULE 26) W0 95123225 ,, . ,~ 218 ~ 9 9 2 r~ 5G

350 C~J C ~JC~CU
352 l~r C A~A
358 ~CU A A~JCI~
364 I~G~J C lla~laGA
366 A~U A CllaG~;
369 G~ A GA~ GaCA
3?9 T~aA~ ~J G~aAA~7 387 G~aaAai7 ~ AaAllOW
388 UGaA~7 A AU7;1C~7C
392 Alltl~ Ci~AA
393 l~aAA~lU C ~UA
395 ~AhD~ C CAAAAaA
405 A~AAA~ A Aa~U
412 Aa~iG~i7 tJ CA
413 A~;lJ C Aa~ a~G
427 G~CI~ IJ ADaXaA
428 ACCL~ A IDU~
430 CAA~ A ~AD~
436 I~DGM~ C Ahl~C
440 A~ ~J ADCXaA
441 AIJC~ 7 A ~WUJ
443 CAal~ C I~JA
449 ~ ~7 ACt l~
450 Ca~aA~ A CI~W
453 AX;~tJ ~J G~COG
458 C~J ~7 ~V
459 1~1= ~ Ga~lA
463 A~W C ~ADCC
465 I~ CC ~J AADCC~
466 UGaDW~r A A~JA
469 ~ C CA~AA~
473 AA~I C1UJ A Aal~hi7A
477 CAlli~ 7 AI~Ar1UA
478 AI~AA~ A ~aI~A
480 A~A~hIJ A A~JA
483 1~ ~7 AP~A
484 ~ A AllPDt AA
487 AaDl~a~7 A l~ l7A
489 lil~AI~ C A~0hGC
494 AIJC~ A G~aAi7c 501 AGCaAUJ C A~WCA
507 17Ca~ C ACI~ACA
511 ~GU~ A ACACt UJ
519 ACACCAIJ IJ A~aA;7 520 CACC~ A G~AIJA
523 CA~ IJ Aa~A
524 Al~tJ A AI~AA
-SUBSTITUTE SHEET (RULE 26) Wo 95/23225 218 3 9 9 ~ F~l/~ c 156 Table 34: RSV (lC) E~ Ribozyme Sequence 'C . E~ P~CZY=I~ S--r~3c--PO~ t~ 03 io ;~ rr,rrr~r ~ r~ ~CG~r 16 ~r,~ ~11~ r--rrr~
17 ~C rnr~r~A~r--~rr~
~!1 r~CU c'r71-~r~r~l ^.rrr~a~r--.~ D~aaL7 r~A~ --^-rr~r--rr~
32 rJU~U ,rr~r~ rrr~r^~ ~aa~.7 36 GG~ . rnr~rar^~rrr~r^-rr~ AD~G
3'7 GGG~ cnr~- rrr~
38 A.~ ~ rrr.~ rr~t~r~rr~^rr~ AAAD~
42 ~G ~r~r~ rr~ rrrrr~ ~ =
46 ~ " ~ aa .a~
51 U~CUc ,rrr ~ r~ r^r,r_~
67 ~r,~l aAL~ ,rnr~ ~r~-. a ~rr-:rr~ A~;G~--;G
68 A~:~ rnr~ r~r~rr_ 76 I~Ut~C~ rr~ -r~r~ ~ ~r^-rr~
al ~C~ ~r~r~r~TTr~ rrr~a~r~r~rrr~ AD~C
87 ~r,;~ rnr~rlr~--~rr~ aa AC2WUI~
88 ~UJC ,_ ~ rrr~ Ai~['L7 g2 ~,r,U rnr~ -^-~r```r--rrr.~
53 ~.ADtlr~l~G rrlr~rr~--rrr~--rrrr~ .aA~A
100 r~A; A rr~rr~rr_rr~ 7 101 Gt~a~ ~ rrrrr~
104 ;~rJ~ .r~rrrr~
105 r U~ C rrrr~rlr~rrrrr~a~rrrrr~
20 A~, I l' n ~ rrrrr~
~5 ~AC ~ I 11 A ~ r ~ r--rrr~ ~ A~.C
78 ~UU rr~ r~ rrrrr~ ~ ~.AD~;C
~25 ~7;1UW~cr rrr`~`r--~rr~ r--rr~
~ 3 ~ ~r ., ~ rr~-~
143 AD~ ", A ~ rr_~
14i ~-QG rnr~-rrr-r.r~^rr~ rrrr 1~1 ~IJ rnr~ ---rrr~rrrrr~
15~i A~7U ~ 11~ -, ~. 1. ~rrrrr~ A},~C
156 A~ 'rr--rr`~ AA~
159 lir~G rnr7~ r,r~ rrr 163 ~ rnr~anr~r-rr-rr~rr~rrr~ r,~
154 ~r,~rr rnr~rr~---rrr~rr,rrr~ A~r 15~ a;~a~r,u rrr~ rr-~ r-rrr~
SUBSTITUTE SHEET (RULE 26) WO 95/23225 - 218 3 9 ~ 2 r ~ c -156 169 .~ rnr~rrr~r~rrrr~rr-rrr~
175 r~GCC~ r~r~r~r,3r,r~r 176 r~cc . ~ rr~rr~
181 AaJaxu rnr-~rr~r~rrrr~rr-^rr~ ACCC~A~
192 r,~ rnr~rrr~rrt~rr~rrrrr~
~96 ~DW rr7r~rr~ rr~rr.rrr.3~ A~K
201 ~ rrrrr~ ~ WW~7 206 r~CCWUC r~r~rrr;~rrrr~rrrrr~ ~Gar;r 216 c3aAcac r~r~rrr~r~rrrrr~
221 A~ r A ~ r~ ~rr7 ~ 3 AC~AU
222 C.3DG~ r~` ` AA~a.Ca.~
231 ~AU . "- , r ~ Ar~rrr.33 ;~, 232 r~A rrr~rr~rrrr~`rr-~`` .~
234 ~ rr~r~rrr~rrrrr`~'rr-rrr`` A~
235 r,~lJG r~r~rrr~ - rr~rr~rrr~ A~ACa 241 ~iUJCaC ~r~rrr~rrrrr~rrrrr~
247 GGGC~ rrrr~rrr~r-rrr~rrrrr~ cra~;P.
249 ~GGGCA ,,, ~ rrrrr~ A~J~C
250 r,~iGGC . . ~ rr~rr~
256 r,~DDa~ r~` ` .~GGGcaA
259 J~7 . Ir.~ll ' f ~ --rrr~ A~aGGr, 26a ~la~ rr,~r~rr,~rrrrr~~rr.r.~3 ~D~7 265 AC~ "" A.I~ rrrrr~
267 r~C rnr~ rrr~rrr~ rrrrr~
270 ~ac ~ rrl~rr~
273 lX;GU~ r '- ~ r_rrr~
278 r~c~ ''r~ rcrrr~ C
283 r~aWGa rnr~rrr~ 3~----rrr~ aar7 284 ~GDG rnr~rrr~rrr~r-rrr~
285 ~ ;DOW rr~rr~ rrr~-lrrrrr~
300 ~r, ~rr~^r~ ~ ~r~ ~rr~ ~ ACI~Ca 303 CPlJ~C
316 = rnr~rrf~ r~rr~r~r~-rr~
317 rc~ rnr~rr~r~rr~"~rrr~rr~ MCC~
319 ~ rnr~rr~^rrrr~` rr~r~ 7 321 r,~C~ rnr~ r~r~rrrrr ~ AD~C
338 Al~WG~ rr~rrr~^r~rrr~rrrrr~l ~7 339 .~ rrlr:3rrr~rr~rrrrr~ Aa~cca 346 r,=Gr rnr~-rr~_rrr~ r~^rr~ G~cr 350 ~GA rnr~rrr~rrrr~r~rr.~ 3A AGCaA;JG
352 ~;G~7 rn~--rrr~----rr~r~rr~
358 AGaC~J ~ ~r A~ rr,rrr33 AGGD~
3 64 UC~ ~ rr.rrr~ ~
366 CAI~ rrr~rrn~rrrrr~rrrrr~ aC~r 369 ~G~ A17C ~r`rr`rrrrr~ r~~r~rrr.31~ A~aGac 379 AUt~C - I. ~ --^rrr~
387 AG.~U ~rr~rrr~-rrrr```rr~`rr`~ AD~aC
388 0~ ~ "- n ~ 7 ~ rr~` AaL~W~
392 ~ rr7r~7r~ r,rrr`~71r-r~r~` A~7 I~U~E SHEr~ (RU' ~ 26) W0 95123225 ~ 3 rg 9 2 P~ 156 393 W~a r~7r7~r 395 ~ r ~ ~
405 ~;j~ r 11~p~r ...
412 A~ r~ r~r-~ r ~ ~ ~ rr,rr,r~ a ,~U
413 CP~ y ~ _r~r~
427 ~U ~ AD~WC
428 ~ 1~ I~lJ~ r~,r,~
- 430 ~ ~r~r~r~r~r~
436 GU;PaD~
440 ~ r l ~ _~rr~
441 ~A r l l~ rr~' ~ AAG~7 443 ~ ( I ~ rr.
449 ~ r l l. ~ r _r~
450 ;~a.aG ~Jr~r,r-.r~r~
453 C~AuJcc ~rTr~r~r~ rr,~ ~ ~ rryr~
459 ~AG~WC
465 AlhG~ I l ~ .. r. .
466 ~WG~ ( I ~ rr,~ ~
469 a~ r-rlr~_rr~r~r~rrrr~ A~A
473 ~ I ~ rr,~r~ A~
477 ~PA~U I 1 ll - r . ~ rrr~
478 ~ rr~rrr~rr~ A~DI~
480 I;hlJC~Ai7 ~rnr~-~r~_.~rr~rr,r~r~
483 UG11UU~U I 1 l-~ rrrrr~ ~A
484 ~ r~r.rr~,~r~r~ r~ AA~.
487 ~ r ~ r~ ~ A~7 489 G~J r 1 ll. - ~ r ~ r~r~ ~ A~.a 494 Gal~C ~:~nr`~--rr~7~rrcrr-~ GAU
501 I~A aDU ~ `r~rr~ A~iC~,7 507 ;~WC~.7 ~ ~ rrr~-~ ACa~A
511 Al~ ~--~Ir~rr~r--Crr~ AGDG.~CA
5 1 9 A~ ~ r~ rv- A rrr~
~20 I~AC t~r~r1r~rrr~ rrr~ A~
523 = ~ lrrC~rr~ ~ AC ;~
52- 1~7 1 ~ AaCU~A~

SUBSTITUTE SHEET (RULE 26) W095123225 ~ ~ ~ 218399~ P l/~ 156 Table 3~: RSV (N) ~ Target Sequence ~t . ~ ~ t S--qu--nc-- t . ~ 1~ ~J~t S--cu~:~c--~o- I t~ o-~ ~?ol~ ~ t~ o~
9~:aA~J A C IAAGAU 217 GGSaIJG.7 ~.J .a~CQ
21Ga~U C ~A 2'8 G:~ i~. ~a.
231~ ~ ~aAG 220 A~ A
24GGC~C~7 ~ GC~ 229 Ga;al;~;7 C ;
32GCa~u C ~AGDDI~ 231 G~C~ .~. GOE;~GG
37aaG~ IJ Ga~7 235 45GAA~7 ~ CAC~3A 236 C~ A GGaaG
;0~CU C AACaA~ 254 AC~IJ A ~C
60CAAi~GaU C AAa~CU 260 U~IJ A C~
65:UJC~ U ct~a~ 263 ;~AADaC~ C AGaGal~
66~IQ~ C ~ 1JC 277 GCGGGaU A ~JA
70Ctl~W C ~GC 279 Ga;al~ C Ati~A~.
73CDG~JCA~J C CACCaAA 284 AWU7GU ~ A~a~;CaA
82AI~AAU ;~ C~CC;~CC 299 ~ A GaDWAA
89ACAI -~7 C C~ 305 ~aGAriW A Ai aaCAC
108 ACGW A GCAlraGA 315 A~ C G;;~AGA
GA~L7 A ~AC 318 AC~CGU C ~;aCAU
113 A~ IJ G~CJC 326 Aa~aSaIJ ~7 ~;GaA
117 ~GAU A CD~A 327 AGa~JtJ A ~DGGA~A
120 I;G~ C C~A~A 346 ~U~aAaU U i;G~
123 ~7 A AU~DGA 347 ~'G;UA~U U Ga~GU
126 I~AU ~ AI~UJGU 355 G?~GU ~ AaC~
127 CC~ADtJ A UCal~JG 356 AaGa~ A ACAI;~JGG
146 AaCACA~J C A~;J 361 ~U IJ a;C~AGC
150 CA~C~7 A ~ 370 GCa~ U A;UaACU
154 Aa~W IJ AI~WGGC 371 CAaGCl~U ~ ACAA;l',G
155 A~aAG~ A I~IGGCA 383 CDG~ U CA~JCA
166 GG--AI~ 7 AUII~al7C 384 ~;&a~ C A~A;~aA
167 GC~ A l~AIJCA 389 UG~7 C Aa~G
10A~JA~J U A~DCaCA 395 UCaA~ U GAG~G
170 ~ A A~ll a; aG 40~ UtlGa(~AU A G;iAU~TA
173 ~AU C A18aGA~ 406 ~U7 C ua~ A
186 A5a~-t,~ A A~A 408 AGAAI~ A G~C
189 ~AU C A~7 415 AGA~aAU C Cl~AA
192 ~WCAU A Aa~ac 418 AaAUCcU A CA~AAaA
196 CA~AA;J U CA~GG 431 AaAu~J A AAAG~AA
197 Al~aAl~U C AC~GGuU 449 G.~ A GC~CCaG
205 AC~WGU U Aa~GW 453 GG~ C CAGalUJA
206 CtJ5GWU A A~Wt~a 460 CCAG~AU A CA5GCAU
209 G~ A GGW~ 472 CaUGACU C UCC.7GAU
213 AaL~.7 A UGU.7AUA 474 UG~C.7W C CGI~J
SUBSTITUTE SHEET (RULE 26~

WO 9S123225 2 18 3 9 9 2 T ~I/ _ 'I 156 480 IJCC~UJ 17 GIJGGG~ 696 I~U~ûU A UAGCACA
491 GGADGa~ A AU~ 698 I~'IJAIJ ~ GCACaAlJ
494 ~IG~ A I~A 706 GCaG~AU C IJt~lACC
496 A~ ~ A~A 708 ACaAI~ U C~LCCAG
497 ~ A l~aG 709 CAAD~ alJ C ~JACCAGA
501 A~DCrJ A l~C 711 A~JC~J A CCAGAGG
503 ~IJ A GCa~W 726 ~ca~ A Gal;rWGA
511 G~ ~ = 731 GCaGa~`,V U G~GGA
512 C~ A WLu~aA 740 A~;GGUJ U Wt~.G
515 C~DtlaG0 A AI~ACtJA 741 A~l U ~CaGG
518 =UU~ A AC~AAU 742 GG~iDGU U UGC~GGa 522 AaDMI ~ A A~l;C 743 GGalJlraU IJ GCaGGaU
5a6 AC~laAAU ~ A~ 751 G~aGGAU U Gal;l~
5a7 C~Aal ~ A GCAI~G 754 GGa~luGl7 U ~:aAU
544 G~ C u~u~uu 755 G~G~ U ~aA~G
549 A13CDGG~J C ~CaGC 756 A~7~ A UGaADG--551 COGGI~IIJ IJ ACA~XG 766 AAIi~;CCU A ~JGCA
55a ~ A CAGCCGC 787 G;~W U ACGGZGG
563 CCWGi~J TJ A~;; aG1iG 788 T~Ga}~U A CGG;~GGG
564 CW= A GG ~;AGC 800 GGGt~ C li~CaA
573 Ga0~''tJ A A~A~ 8aa GGaG;Jt~ U AGCAAaA
576 A~AIJ A A~ C~ 803 GaG~U A GCAAM~J
581 A~ADW C CDAaAAA 811 GCA~ C AG~7t;1~AA
584 A~C~J A AhAAl}JG 815 AWC~J IJ AaAAhUA
603 GAAaa;lJ U ACAAAGG 816 AI~Ca~ A AaAA~
604 A~ACGDtJ A CaAA~C 8aa UAaAAAU A
613 APAa;C~ IJ ACIJACCC 8a4 AAaA~ U AUG~
614 A~ tW A CtlACCCA 825 AAAliiWU A UG;~GG
617 GC~J A CCC~AGG 829 AUI~aD(;~ U A~
6ag AGGa~ UJ A GCCAlCA 830 l~G~ A G;3AI ~
640 Aa( ~iW IJ C~;aA 840 ACAI~OEU A G~A
641 ACaG~ C I~PI23aAG 866 A~ ~ G~;AGG
643 AGC~IJ A l=G 869 A~G~J IJ GAGG~U
652 GaA~7 U l~aAAA 875 U~Ga~7 I:J ~G~
653 A~aWW TJ GaaA~AC 876 ~ U A~
663 A~AACPIJ C CCCACI~ 877 GaGW~ A ~JGa.W
670 CCCC~ l:J ~a5au 883 UA~Ga11U A UGCCCAA
671 CCCACI~J IJ A~G;iDG 895 CAa~AAU U GGGK;.7 672 CCA~;;J A I~AG~J 913 GC~ U CUAlXaIJ
674 AC13t7t~U A G~WW 914 CA;AW C VACCAUA
680 llaGa~ ~ C 916 GGatW U A CCAVAUA
681 A~AVGW U VC~A 921 C~V A UAUUGAa 682 Gal~GWlJ ~J ~ 923 ACCAUAU A WGAACA
683 AVGGVW U GWC~J 925 CAUAUAU U ~ AAC
686 W~Ct~ IJ CA~!G 943 A~AGCAU C AUUAWA
687 ~UUt7GW C AWOtlGG 946 GCaUCAU ~ AUUAlJCtJ
6go ~ucca~ U ~C~IJ 947 CAUCAW A UUAVC~lU
691 GI~AW U I~;GVAVA 949 UCAWAU l:J AUCW~JG
692 I~W ~7 GGI~G 9S0 CAWAW A UCWUGA
SUBSTITUTE SHEET IRULE 26) ~- 218~992 WO 95/23225 p~ "~. _ ~ ~ ASC

952 1~ C Til~7 954 A~ IJ 1~
955 ~ IJ G~A
960 I~U~ C A~ICC
964 ACtJCaW IJ I~C~C
965 CtlC~UW ~J CCI~CU
966 ~AD~ C CD~ al W
969 Al~ C ACJt7CtiC
973 CC~aJ ~J CO~J
974 Ct~ al GtJ C
976 C~IJ C CAG~A
983 CC~W A G~
986 Gt~a~J A
988 G~A~J IJ AÇGCaA17 989 1~17 A GGCMI~:
1007 C~-Cl7 A GGCU17~A
013 1~ A AII~GGAC
024 G~J A CA~
' 032 CaG~ A CA~aG
1 044 G~GGaA~ C A~G~J
1050 ~aA~ C ~
' 052 ~G~CU A I~U;;:
1054 Gall~ A ~G~CA
072 MGGC~ ~ ~;CC~;aA
085 A~ tJ C A~AGaAA
03 OEIW~' IJ A~A
1104 l~a~ A AC~3 :108 AI~IaA~ A Ca~A
'-5 ACAG~ J A Ct~
8 G~ A Ga~lCG;I
23 CllaG~ IJ Ga~GCA
39 A~ A GaGGC~A
1146 AGAGG"IJ A ~A
1148 A~ C A~A~C
155 C~AACA17 C ACCD~laA
1 1 60 A~ U A~C/~A
'161 u~ A Al~AA
64 G~iA~ C C~AAliGA
173 AA~AGAU A A~G~
' 1 81 ADGA~ A G~CC~J
1~87 II~Qa~C~r IJ UG~WA
1188 AGal~ 7 ;J GaGO~
1 ' 93 ~aG~7 ~ A~DaAAA
1'94 ~ A Al~aAAAA

SUBSTITUTE SHEET (RULE 26) WO 95/23225 218 3 9 9 2 r~ 56 ~ 277 Table 36: RSV (~ I Ribozyme Sequence ~ t . ~EI Rlbozy=~ S~
Po~it ~ o:~
9 ~U7C~CG rrv-~ rrrrr~rrr~
21 urJt~7AA rnr~:arrrr~
23 C= 7 rr~ro~r?-~--~ ^~`~ AGai;CCa 24 A~3GC ~ r 32 TJC~UO rr?~ rr~ -^^rr~ A~UGC
37 Al.7C.U~ rr~ ~J~
~G ~ ` ` ~rr ~ ~ ` a~,c c~Grra rr~ r~rr,~r~
AS~ rr~nr~ -r~
Al~ç rr~ r~ a A~
r~O I l " ~ "~ r-r,rrr~ ~ al3.~AG
13 TJa~G ~ ~^rr~
82 r~7~ rrv-~ r~rr`'`--~r~
89 ~UG rr~ r~rr 108 ~ rrTr~ r~7~r~rr~7 A}JCD~
111 G~\D~ rr~rrr~rrr~ ~CJ
113 Ga~c rr~ --^rr~
~17 ~Q
120 ~WC~G rrr~ rr~
126 ~ rr~
127 r~CP. rr~ ~rr~i~-^rr~ ;~.GG
146 ACCtlUJJ ~ r~-rr~ ~ A;i~
150 C~hA7 rr~ ^rrrr~ --rrr~
15~ rcca~ rrv-~rrrDrr~-~rrrrr~
155 T~CCU:a rr~`~Crr`'l~^rr` ` AAC~
166 ru~A~ ~ ``~rrr~
167 I~lJUaA rr~ ~rr~
170 C~W~W rrrranr~rr~ r7rrr~ A.a;~
173 ~uu~:u~iu rr~ r~rrr~
186 ~U ~ rr~rrr~
189 ~ ^rrrr~ ^rrr~
192 G~GaA~ rrV-~-~r.~rrrrr~rrrrr~
196 rCcaGJG rrrr~ rrrrr~`~~,l~rr~` A/~CGWG
197 A~CCA5J rr~ rrrrr~ rrr~
205 ~X= ~ rr~
206 U~CCG~ rr~rrr~ AA~CCAG
209 AC~CC rnr~ ^r~rr~r~rrrr~ A~ACC
213 1~ rnr~ rr^rr.~`rrrrr-~ ACC~.UU
SUBSTITUTE SHEET (RUL~ 26) WO g5/23225 i 2 18 3 ~ ~ 2 P~,,, r ~ ~156 ~

220 ~ `'`--~`` Al~
22g ~ ~
235 ~ ACC~iGa 236 C~iJ;JCC r 254 G~
260 C~
263 ca~J
j~77 ~a~ I r ~
a79 ~ ~ Il' -'~--' ' ' 1,1 "`1'~` A~CC

zg5 ~~ ~r.a~ AC~alT
318 AR~ll UtJ r l ll "
326 ~U r~
327 ~ r I ~
346 CA1~3~ I,ll. ",, .. ~ ~-~a AWt~AlJ
347 ~WC
35~ CaAD~ ~'~`~
356 CCaADW ~`'~`~'Xrr`a`'~r~`- AAC1~
361 GCU~^C ' l l~ ihA
370 AG~I . ~ AGCDOt;C
383 I~ ``~-~`` A~D~G
384 I~C~ ~`'1'~~`'`~`' ~A11~
389 C~7 ~rr~~~-~`` AD;:t~GaA
395 al~, ", ~ . ,, .... ,, .. ~ ~--rrr.DD A~ D~a.
401 ~,GPDCC ~`~```~``
406 liW~L r u ~
408 GUlC~ '` ~` ~` ` ` ~` ` ~7 415 ~G ~ ~ r 418 I~
431 D~ 7tJ Cn~.a~
44g CWtaGC rll l -- ~l ~''~`` ~C
453 ~3 ~ ur " ~ a A~acc 460 A~ ,.
472 AUC~ ~ ", ~,,, ,.,, ~cn ~D A5 480 ~C ' 491 ~ ~ .D~
494 u~aa ~`-?"`''~```~`` AD~laDl~A
496 ~ ~
497 Cl ~ ~ Aall7U~JA
501 GC~JA
503 A~GC l ll' "' l' ~ aa SUBSIITUTE SI~EET (RULE 2~

WO 95123225 218 3 9 9 2 r~.~ 156 512 ~ ~ . . ~ f, ~
515 r~ J rr~ r~''-~`' A~AUG
518 A~aGU ~ r~
522 GC~WU rn~ r-rrr~
526 r~ 7 ~ bl ~ f . 1 . .` ` ` ~r~
527 CUGC~GC rntarr~;~rrr-~r~_ 5 44 AAaaC~. ~ _r 531 r~ I ", ~ ,-rrr~
552 A~tJG rr~
563 CtD ~J r~^~ ---564 G~CC I ll` ~
573 A~aU ~ ~rm-~ a A~;CD~C
576 AIX~7 r~ a,r~
581 ~U~G r~r~ r~~ rrr~ ~a~.U
584 CU~tJ rr~ r--rr~f^
603 CC~ r 604 G~ rr~ -----rr~ ~ AACG~
613 ;G13~T rTTr~ AGCCl~ll7 614 ~G, "~ r~r~ ~ Aa~XsU
617 CC~GGG ., .~ _f ~ r ~r~ AG~C
629 ~GGC rnr~rT~!~rrr~ r~--rrr.~ aDWCC~r 6~0 ~ rrTr.lTTr~r.rrC~ rrr-~ AGCO~U
641 0CCA~. 1 ~ l l. r 1 ~ n T ~rr-rr~ ~ Aa~;c~;u 643 C~t;C~ rr~rrr~ ~ AGaaGC7 652 U~ ~rTr~ rr~ ~ rrr~ ~ AC~'C
653 G~ WC l~r~r~,_r~r~ ACac5v 663 Aa~GG ,,, ,_,~_rrrr`~ A~W
670 A~L'A ~rTr~rr,rrr~rrrrr~ AG~GGGG
671 CAr~laL7 ~ rT~`-----rr~ r~ AAG~;GGG
672 ACa~ rrTr~ ~7r~ rr~rr~ ~ ~ r-rrr~ ~ Aa~7GC7 67~ C ~Tr~rr~rr7rrr~ Ar~
680 Gaa~ ~rTr~ rrr~~Grrr~ AC~'~'A
681 ~ 7r~ frrr~ r~rr~ Aa~Tcu 682 A~h~ r~ aTTr~r ~T ~r--rrr~ A.~A~U'C
683 A~C ~ rrf~` ` AaAACAU
686 C~ rTr~r~r~rrr~ ACaAAAA
687 CCAAAp~T r~Tr~ -rr~r~r----rr~ AA~MA
690 Ar~ U Cp,A rTTr~ 7 rr~~rr~
691 r~, rnr~rT-A--~rr~ rrr~ Ar~aAC
692 cwu~acc r~ ArTI~~~rrrr~--r~ AA~3GAA
696 ~ ~ r--~ lrrrrr~7l ACCAAAA
698 A~ ;C ~ r~rrr~ ~ A~CAA
706 GGt~aGaA rnr~rlr~rrC~r-rr~ A~ ;C
708 COGG;~G rr~ rTr~-~r~rrr~ rr~ AG~GI.7 709 ~ A ~ .rrrrr~ ~ AAGA~
711 CC~aW ~-rTr7~ r~rrf-rr~ AG;U~U
726 ~iCaA( ~C r~ -rrrr~rrrrr~ ACtJGCCA
731 UCCC~JUC rr~ Tlr~rr~rrr~r~r~rrr~ ~AC
SUBSrITUTE SHEE~ (RULE 26) 7~ CDGC~aA ~`~`~~~`'`'~f`~ A13 741 C~t3GCaA U ~ ~r~ Aa~XC'J
7q.2 VCCDGca ~ AAAI~C--743 Al~: r l P, ~ . ~ . T
751 CA~UC ( ~ ~aa A~GC
754 ~DUCiWA ~ ~~''~` ` ACaAl~:C
755 QD~ " ' r I T A ~ T~ ~--C~ a AA! ILUJC
756 G~aDC~ ~
766 IJGC~XA ~ r ~ a AGGC~J
787 CC~U
788 ~ r - I T ~ a 80a I~UOE~A~'~`--crr`'`-~` ACD~
802 ~CGC~
803 A~IIJGC ~
811 I~J ~ v~ ' r l ~ A~hC
815 I~DCC~ T a`~`~ Aa~
816 At~ ~ A~CaGA~J
8Z2 =a~A I " ~ a~
824 C~ a~rr:~a A~
825 ca~Aca I I r 82g A17~CJ
830 C~WI~
840 ~ A~,J
8 66 cca~ ~ ~ f I I
869 A~ ~ a A~
875 Ai~ ~ ~~~ a A~.A
876 I~W ~ a A~
877 A~IU~ A l ll r ~
883 ~ a ~ aa-~r.a~ ~D~A
895 AC~C ~ A~G
916 U~GG ~ ~ AGWC~
921 ~A Cnr~
923 ~A . ~ a A~GW
925 GI~CC C~~`~`'~ A~
943 ~ crr~
946 AGU~a~ `' ~'` A~DGC
947 Aa~aA ~`"~`'~```~' A
949 C.aAAG~ ~~r~r7~~rr~```~C~
950 IJ~AAGA ~`~`~'~``'~` AalD~G
952 ~ a~A ~ a~ A~,aA
954 UG~, t~ pTTr`--~``~r`a AGa~
955 ~ r ~ Aa~z~A
960 GGii,aAl~ ;DCAAA
964 G~2A5~, ". h~ r . ., I A~rr~_~ AIL~
965 ~G I ~l~ - r n~ APIWG~G
g66 Aa~G~ AAAIJC~A
969 GA~ (~`~'`~'``-~`` AGG~AU
SUBS~ITUT~ SHEET (RULE 26) WO 95/23225 218 3 9 9 2 r~l ~ 156 973 A~:O~;GaG ~-nr~ ~` ~~~r~ ~ ~ ~~~f ~ .3~ a ~7GaGG
976 ll~a~JG r~
983 Cr~C ~`~`~~a~~~`` AC.U:~G
986 1~ r~ r~
988 A~GCr ~r ~rlr:a~ rr~ C
989 ~GCC ~~rJr~rlr~ ,~,~, ~`` ~.A~
1 007 ~arx:cr r-r~ --~r~
1013 cr~ u r~ a ;~ca~
1024 ACC~G r~rb~
1032 Cr~G ~`~`-~f~```~---~`` ;U:C~i:DC
1044 ~ r r~r~ ~r~ ~c 1050 U~ ~-rlr~rlr`~--~`~
1052 car7t~ a. cr~rlr~--,rr~ a ,~r 1054 ~ ", ~ . " . . . _ ~ ar~ r--r ~li~C
1072 U~ ~--- r~ A~r 1085 ~cr r r~rr ~ r 1103 ~W ~ A~C
1104 cr~G;r~
1108 l~hl:DG r~ aa;~r ~- AG~a.G r~r~ a~r 1~18 ~caAG~c c~r~ ~C
1139 ~;CCUC r~ ` ~CW
48 Ga~G~W ~ A~GCCrJ
~135 WhA5C~ r~r~ a iU;W~WG
1160 WGGPW ~~ ~r 1164 UW~ ( ~ ~C
1173 ~r Au r~rr~ a~ cr~w 1188 WAAC~C r~`~`~r~ aAGc~r 1193 wr~W ~rar~ .a~
1194 ~ur~r rn SUBSTITUTE SHE~T (RULE 26) WO 95/23225 . 2 18 3 ~ 9 2 PCTllBgS/00156 B ~ 5 . . .
~^ .
~ ~: ~
E~
SUBSTITUTE SHEET (RULE 26) ~ W095123225 283 2183992 r_l,~ 156 -.
e CO ' ~ 2 U7 U~
UBSTITUTE SHEET (,~ULE 26~

218~9~ p~ - IS6 wo ss/2322s , 284 Table 39: Large-Scaie Synthesis Sequence Activator Am~dite Time~ % Full [AddedlFinall ~AddedlFinal] Length tmin~ (min) Product A5TT [0.50/0.33] [0.1/0.02~ 15 m 85 AgTS [0.25/0.17] [0.1/0.02] 15 m 89 (GGU)3GGT T~0.50/0-331 [0.1/0.02] 15 m 78 (GGV)3GGT S [0.25/0.17~ [0.1/0.02] 15 m 81 CgTT~0.50/0.333 [0.1/0.02I 15 m go CgTS [0.25/0.17] [0.1/0.02] 15 m 97 UgTT [0.50/0.33I [0~l/o~o2l 15 m 80 UgTS [0.25/0-17i [0.1/0.02] 15 m 85 A (36-mer~ T[0.50/0.33] [0.1/0.~2; 15/15m 21 A (36-mer) S [0.25/0.17~ [0.1/0.02] 15/15 m 25 A (36-mer) S [0.5010.24] [0.1/0.03] 15/15 m 25 A ~36-mer) S [0.5010.18] [0.1/0.05-i 15/15 m 38 A (36-mer~ S [0.50/0.18] [0.~/0.05] 10/5 m 42 ~Where two coupiing times are indicated the first refers to FiNA coupling and the second to 2'-0-methyl coupling. S = 5-S-Ethyltetrazole, T =
tetraz~le activator. A is 5' -~Icu ccA UCU GAU GAG GCC GAA AGG CCG
AAA Auc ccu -3' where luvn ~.,dse represents 2'-O-methyln~ otirl~5 SUBSTlTU~E SHEET ~RULE 26) ~183992 wo 9S123~2s 1 ~,~1~. ;. 156 Table 40: Base Depru~ ion Sequence Deprotection Time T C % Full - Reagent (min) Length Product i8u(GGU)4 NH40H/EtOH 16 h 55 62.5 MA10 m 65 62.7 AMA 10 m 65 74.8 MA10 m 55 75.0 AMA 10 m 55 77.2 iPrP(GGU)4 NH40H/EtOH 4 h 65 44.8 MA10m 65 659 AMA 10 m 65 59.8 MA10 m 55 61.3 AMA 10 m 55 60.1 CgUNH40H/EtOH 4 h 65 75.2 MA10 m 65 79.1 AMA 10 m 65 77.1 MA10 m 55 79.8 AMA 10 m 55 75.5 A (36-mer) NH40H/EtOH 4 h 65 22.7 MA10 m 65 28.9 SUB5~U~E SHEr (RU~E

21~9~2 WO95/2322S r~ L 5 156 Table 41: 2'-O-Alkylsilyl Deprotection Sequence Deprotect~on Time T C % Full Reagent (min) Length Product AgTTBAF 24 h 20 84.5 1.4M HF 0.5 h 65 81.0 (GGU)4 TBAF 24 h 20 60.9 1.4 M HF 0.5 h 65 67.8 C.oTBAF 24 h 20 86.2 1.4 M HF 0.5 h 6~ 86.1 U1oTBAF 24 h 20 84.8 1.4M HF 0.5 h 65 84.5 B (36-mer) TBAF 24 h 20 25.2 ~.4 M HF 1.5 h 65 30.6 A (36-mer) TBAF 24 h 20 29.7 1.4M HF 1.5 h 65 30.4 B is 5'- UCU CCA UCU GAU GAG GCC GM AGG CCG AAA AUC CCU
-3.

SUBSTITUTE SHEET ~RULE 26) ~ WO 95123225 - 2 1 8 3 9 9 2 ~ 156 ~ 1~
~Z 0 CD ~-1 ~ ~ ~ o 0 W G 0 ~ ~ ~ O O ~
o ~ ff X o tt ~
O ~ ~ .Q
~ 1:~ 0 ~ C~ ~
L~ L~ IC) ~ L~ L~l C ~ .
'~
SUBSTITUTE SHtET ~RllLE 26) WO 9!il2322~ ~. ' 2 1 8 ~ 9 9 2 ~ SC

.
~ CD ~
r r r ~ !_ r ~ '~
~ N
O
~
X
C~' Et SUBSTIT~TE SH~T (RULE 26) P~~ r 156 ~ woss/2322s 289 2 1 8 3 9 9 2 Table 44. ~i~etic3 of Se f-P. u~ g In Vitro Self~ ` g Constr~cts k (~
EE 1.16 + 0.08 ~DV 0.~6 + 0 l5 lp(GC) 0.36 + 0.06 HP(G7 J) 0.0s4 + 0.003 k, ~ t~e ~ rl~ ~IA rate co~stant for riboz~me self-cleavage rl~i~ ,,; ,r~d from a ron-lillear, le~st-squares fit (RAlr~i~a~rrqrh~ Sy3~ergy Softw re, Reedi~g, P~ to the equat~on (Fraction Uncleaved ~ L,) = kt (1-e~kt~
The equation desc~oes t71e e~te~t of r hozyme l'"~ , g n t~e prese~se of orlgoi~lg~ L; (Long~ n7~eck.1994Proc.~q~7 ~rqr7 Sr- U~:~91, 6917) as a fw~o~ of tirae (t) a~d t~Le "~;,"r,l~ lql- rate co~ ta t for ciea~age(k). Eac7 value of k ~ ~" ~ ~A the average (+ range) of values r~ od irom tvro ,~

SUBSTITUTE SHEET (RULE 26 218 3 9 9 2 F~ s6 WO ss/2322s , .

Tabl ~ 4 5 Fntry Mod~fication tll2 (m) t1~2 (m) ~ = tSISA
A~tlvity S~ability % 10 (tA) (tS~
U4 & U7 = U 1 0.1 2 U4& U7=2'-~M~U 4 260 650 3 U4 = 2'=C1 12-U 6.5 1 20 1 80 4 U7 = 2'=CH2-U 8 280 350 5 U4 & U7=2'~CH2-U 8.5 120 130 6 U4 = 2'=CF2-U 5 320 640 7 U7 = 2'=CFz-U 4 220 550 8 U4 & U7 = 2'=CF2-U 20 320 160 g U4 =2'-F-U 4 320 800 10 U7 = 2'-F-U 8 400 500 11 U4 & U7 = 2'-F-U 4 300 750

12 U4 = Z'-C-Allyl-U 3 ~500 >1700

13 U7 = 2'-C-A~lyl-U 3 220 730

14 U4 & U7 = 2'-GAllyl-U 3 120 400

15 U4 = 2'-araF-U 5 ~500 >1000

16 U7 = 2'-araF-U 4 350 875

17 U4 & U7 = Z-araF-U 15 500 330

18 U4 = 2'-NH2-U 10 500 500

19 U7 = 2'-NH2-U 5 500 1000

20 U4 & U7 =2'-NH2-U 2 300 1500

21 U4 = dU 6 100 170

22 U4 & U7 = dU 4 240 600 SU6STITUTE SHEET (RULE 261

Claims (97)

291 What is claimed is;

1. An enzymatic nucleic acid molecule which cleaves ICAM-1 mRNA, IL-5 mRNA, re? A mRNA, TNF-.alpha. mRNA sites shown in Table 23, 25, 27, or 28, CML associated mRNA selected from those identified as SEQ. ID NOS 1-25, or RSV mRNA or RSV genomic RNA in a region selected from the group consisting of 1C, 1B and N.

2. The enzymatic nucleic acid molecule of claim 1, the binding arms of which contain sequences complementary to any one of the sequences defined in any of those in Tables 2, 3, 6-9, 11, 13, 15-23, 27, 28, 31, 33, 34, 36, and 37.

3. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said nucleic acid molecule is in a hammerhead motif.

4. The enzymatic nucleic acid molecule of claim 1 or 2, wherein said RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA or RNaseP RNA motif.

5. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 12 and 100 bases complementary to said mRNA or genomic RNA.

6. The enzymatic nucleic acid molecule of claim 5 comprising between 14 and 24 bases complementary to said mRNA or genomic RNA.

7. The enzymatic nucleic acid molecule of claim 1 or 2, comprising between 5 and 23 bases complementary to said mRNA or genomic RNA.

8. The enzymatic nucleic acid molecule of claim 7 comprising between 10 and 18 bases complementary to said mRNA or genomic RNA.

9. An enzymatic nucleic acid molecule consisting essentially of a sequence selected from the group of those shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38.

10. A mammalian cell including an enzymatic nucleic acid molecule of claims 1 or 2.

11. The cell of claim 10, wherein said cell is a human cell.

12. An expression vector including nucleic acid encoding an enzymatic nucleic acid molecule or multiple enzymatic molecules of claims 1 or 2 in a manner which allows expression of that enzymatic RNA
molecule(s) within a mammalian cell.

13. A mammalian cell including an expression vector of claim 12.

14. The cell of claim 13, wherein said cell is a human cell.

15. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, rel A, TNF-.alpha., or RSV by administering to a patient an enzymatic nucleic acid molecule of claim 1 or 2.

16. A method for treatment of a pathological condition related to the mRNA level of ICAM-1, IL-5, rel A, TNF-.alpha., or RSV by administering to a patient an expression vector of claim 12.

17. The method of claims 15 or 16, wherein said patient is a human.

18. The method of claim 17 wherein said condition is selected from the group consisting of atherosclerosis, myocardial infraction, stroke, restenosis, heart diseases, cancer, rheumatoid arthritis, asthma, reperfusion injury, inflammatory or autoimmune disorders, transplant rejection, myocardial ischemia, stroke, psoriasis, Kawasaki disease, HIV and AIDS, and septic shock.

19. A nucleoside selected from the group consisting of 5'-C-alkylnucleoside, 2'-deoxy-2'-alkylnucleoside, nucleoside 5'-deoxy-5'-dihalo-methylphosphonate, nucleoside 5'-deoxy-5'-difluoro-methylphosphonate, nucleoside 3'-deoxy-3'-dihalo-methylphosphonate. and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

20. A nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'-deoxy-5'-dihalo-methylnucleotide, 5'-deoxy-5'-difluoro-methylnucleoticle, 3'-deoxy-3'-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

- 293 -

21. A nucleotide triphosphate comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'-deoxy-5'-dihalo-methylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

22. The 5'-C-alkylnueleoside of claim 19, wherein the sugar portion is in a talo configuration.

23. The 5'-C-alkylnueleoside of claim 19, wherein the sugar portion is in an allo configuration.

24. An oligonueleotide comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, 5'-deoxy-5'-dihalo-methylnucleotide, 5'-deoxy-5'-difiuoro-methylnucleotide, 3'-deoxy-3'-dihalo-methylnucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

25. An oligonucleotide comprising a moiety having the formula I:
wherein B is a nucleotide base or hydrogen; R1, R2 and R3 independently is selected from the group consisting of hydrogen, an alkyl group containing between 2 and 10 carbon atoms inclusive, an amine, an amino aeid, and a peptide containing between 2 and 5 amino acids inclusive; and the zigzag lines are independently hydrogen or a bond.

26. An oligonucleotide comprising a 3'-amido or peptido group.

27. An oligonucleotide comprising a 5'-amido or peptido group.

28. The oligonucleotide of claim 24, 25, 26, or 27 having enzymatic activity.

29. Method for producing an enzymatic nucleic acid moleeule having activity to cleave an RNA or single-stranded DNA molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'-position.

30. Method for conversion of a protected allo sugar to a protected talo sugar, comprising the step of contacting said protected allo sugar with triphenyl phosphine, diethylazodicarboxylate, p-nitrobenzoic acid under inversion causing conditions to provide said protected talo sugar.

31. Method for the synthesis of a nucleoside 5' or a 3'-dihalo-methylphosphonate comprising the step of condensing a difluoromethylphosphonate-containing sugar with a pyrimidine or purine under conditions suitable for forming a nucleoside 5'- or 3'-difluoromethylphosphonate.

32. The oligonucleotide of claim 3, wherein the nommal hammerhead U4 and/or U7 positions are substituted with 2'-NH-amino acid.

33. A method for the synthesis of RNA comprising the step of providing 5-S-alkyltetrazole at a delivered 0.1-1.0 M concentration for the activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

34. A method forthe synthesis of RNA comprising the step of providing 5-S-alkyltetrazole at 0.15-0.35 M effective, or final, concentration for the activation of a RNA amidite during a coupling step for less than or equal to 10 minutes.

35. A method for the deprotection of RNA comprising the step of providing alkylamine (MA) or NH4OH/alkylamine (AMA) at between 60°C - 70°C for 5 to 15 minutes to remove any exocyclic amino protecting groups from protected RNA; wherein said alkyl is selected from the group consisting of methyl, ethyl, propyl and butyl.

36. A method for the deprotection of RNA alkylsilyl protecting groups comprising, contacting said groups with anhydrous triethylaminehydrogen fluoride (aHFTEA) trimethylamine or disopropylethylamine at between 60 °C-70 °C for 0.25-24 h.

37. A method for the purification of an RNA molecule by passing said enzymatic RNA molecule over an HPLC column, wherein said HPCC column is an anion exchange chromatography column.

38. Method for one pot deprotection of RNA comprising, contacting a protected base with anhydrous methyl amine at between 60 °C-70 °C for at least 5 min, cooling the resulting mixture and contacting said mixture with TEA-3HF reagents under conditions which remove a protecting group of the 2'-hydroxyl position.

39. Method for synthesizing RNA containing a phosphorothioate linkage comprising the step of contacting 6-10 equivalents of 3H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) with the growing RNA chain for 5 seconds with a reaction time of at least 300 seconds.

40. Method of synthesizing RNA containing a phosphorothioate linkage comprising the step of achieving coupling with 5-S-ethyltetrazole or 5-S-methyltetrazole prior to sulfurization.

41. Method of claims 38, 39 or 40 wherein said RNA is enzymatically active.

42. Method for synthesizing 2'-deoxy-2-amino-nucleoside phosphoramidite, comprising the step of protecting the 2'-amino group with a N-phtaloyl group.

43. The method of claim 42 wherein the said nucleoside lacks a base.

44. Method for synthesis of RNA comprising the step of: protecting the 2'-position of a nucleotide during said synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group.

45. Method for covalently linking a SEM group to the 2'-position of a nucleotide, comprising the step of: contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions.

46. The method of claim 45, wherein said conditions comprise dibutyltin oxide and tetrabutylammonium fluoride and SEM-Cl.

47. Method for removal of an SEM group from a nucleoside molecule or an oligonucleotide, comprising the step of: contacting said molecule or oligonucleotide with boron trifluoride etherate (BF3-OEt2) under SEM removing conditions.

48. The method of claim 57 wherein said (BF3OEt2) is provided in acetonitrile.

49. One or more vectors comprising a first nucleic acid sequence encoding a first ribozyme having intramolecular or intermolecular cleaving activity, said first ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif;
and a second nucleic acid sequence encoding a second ribozyme having intermolecular cleaving activity, said Second ribozyme being selected from the group consisting of a hammerhead, hairpin, hepatitis delta virus, Neurospora VS RNA, Group I, and RNaseP motif and said second nucleic acid being flanked by other nucleic acid sequences encoding RNA which is cleaved by said first ribozyme to release said second ribozyme from RNA encoded by said vector;
wherein said first and second nucleic acid sequences may be on the same or separate nucleic acid molecules, and said vector encodes mRNA or comprises RNA which lacks secondary structure which reduces release of said second ribozyme by more than 20%.

50. Cell comprising the vector of claim 49.

51. A transcribed non-naturally occurring RNA molecule, comprising a desired therapeutic RNA portion, wherein said molecule comprises an intramolecular stem formed by base-pairing interactions between a 3' region and 5' complementary nucleotides in said RNA, wherein said stem comprises at least 8 base pairs.

52. The RNA molecule of claim 51, wherein said molecule is transcribed by a RNA polymerase III based promoter system.

53. The RNA molecule of claim 51, wherein said molecule is transcribed by a type 2 pol III promoter system.

54. The RNA molecule of claim 51, wherein said molecule is a chimeric tRNA.

55. The RNA molecule of claim 53, said RNA having A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases.

56. The RNA molecule of claim 53, wherein said desired RNA molecule is at the 3' end of said B box.

57. The RNA molecule of claim 53, wherein said desired RNA molecule is in between the said A and the B box.

58. The RNA molecule of claim 53, wherein said desired RNA molecule includes said B box.

59. The RNA molecule of claim 51, wherein said desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA.

60. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 12 bases of said 3' region.

61. The RNA molecule of claim 51, wherein said 5' terminus is able to base-pair with at least 15 bases of said 3' region.

62. DNA vector encoding the RNA molecule of claim 51

63. The vector of claim 62, wherein said vector is derived from an AAV
or adeno virus.

64. RNA vector encoding the RNA molecule of claim 51.

65. The vector of claim 64, wherein said vector is derived from an alpha virus or retro virus.

66. The vector of claim 62 wherein the portions of the vector encoding said RNA function as a RNA pol III promoter.

67. Cell comprising the vector of claim 62.

68. Cell comprising the vector of claim 53.

69. Cell comprising the RNA of claim 51.

70. Method to provide a desired RNA molecule in a cell, comprising introducing said molecule into said cell a RNA comprising a desired RNA molecule, having a 5' terminus able to base pair with at least 8 bases of a 3' region of said RNA molecule.

71. The method of claim 70, wherein said introducing comprises providing a vector encoding said RNA molecule.

72. Hammerhead ribozyme having 2 or 3 base pairs in stem II with an interconnecting loop of 4 or more bases between said base pairs.

73. Hairpin ribozyme lacking a substrate moiety, comprising at least six bases in helix 2 and able to base-pair with a separate substrate RNA, wherein the said ribozyme comprises one or more bases 3' of helix 3 able to base-pair with the said substrate RNA to form a helix 5 and wherein the said ribozyme can cleave and/or ligate said separate RNA(s) in trans.

74. The ribozyme of claim 73, wherein said ribozyme comprises six bases in helix 2.

75. The ribozyme of claim 73, having the structure of Fig. 3, wherein each N and N' is independently any base and each dash may represent a hydrogen bond, r is 1-20, q is 2-20, o is 0-20, n is 1 -4, and m is 1 - 20.

76. Method for increasing the activity of a hairpin ribozyme by providing one or more bases 3' of helix 3 able to base-pair with a substrate RNA to form a helix 5.

77. Trans-cleaving Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

78. Trans-ligating Hairpin ribozyme comprising at least 6 base pairs in helix 2 lacking a substrate RNA moiety.

79. The ribozyme of claim 73 having the structure of Fig. 73.

80. The ribozyme of claim 73 having the structure of Fig. 74.

81. A cell including the ribozyme of any of claims 73-80.

82. An expression vector comprising nucleic acid encoding the ribozyme of any of claims 73-80, in a manner which allows expression of that ribozyme within a cell.

83. A cell including an expression vector of claim 82.

84. Method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule, comprising the steps of:
contacting said nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid able to form a duplex or biplex molecule with said nucleic acid molecule, wherein formation of said duplex or triplex molecule directly, or after nucleic acid repair in vivo, causes at least one base in said nucleic acid molecule to be chemically modified to functionally alter the nucleotide base sequence of said nucleic acid sequence.

85. The method of claim 84, wherein said oligonucleotide is of a length sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine in an RNA molecule.

86. The method of claim 84, wherein said oligonucleotide comprises an enzymatic nucleic acid molecule which is active to chemically modify a base.

87. The method claim 84, wherein said nucleic acid molecule is DNA or RNA.

88. The method of claim 84, wherein said oligonucleotide comprises a chemical mutagen.

89. The method of claim 88, wherein said mutagen is nitrous acid.

90. The method of claim 84 wherein said oligonucleotide causes deamination of 5-methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methylation of cytosine to 5-methylcytosine.

91. The method of claim 84, wherein an endogenous mammalian editing system is co-opted to cause said chemical modification.

92. Method for introduction of enzymatic nucleic acid into a cell or tissue, comprising the steps of;
providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions;
and contacting said complex with said cell or tissue under conditions in which said enzymatic nucleic acid molecule is produced in said cell or tissue.

93. Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of;
providing a complex of a first nucleic acid molecule encoding said desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions;
and contacting said complex with said cell or tissue under conditions in which said desired acid molecule is produced in said cell or tissue.

94 Method for introduction of a desired nucleic acid into a cell or tissue, comprising the steps of;
providing a complex of a first nucleic acid molecule encoding said enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule; wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions;
and wherein said second nucleic acid further comprises a localization factor;
and contacting said complex with said cell or tissue under conditions in which said desired nucleic acid molecule is produced in said cell or tissue.

95. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule;
wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

96. Complex of a first nucleic acid molecule encoding a desired nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule;
wherein said first nucleic acid molecule lacks a promoter region and said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions.

97. Complex of a first nucleic acid molecule encoding an enzymatic nucleic acid associated with a second nucleic acid molecule having sufficient complementarity with said first nucleic acid molecule so that it is able to form an R-loop base-paired structure under physiological conditions with said first nucleic acid molecule;
wherein said R-loop is formed in a region of said first nucleic acid molecule at a location which promotes expression of RNA from said first nucleic acid under said conditions, and wherein said second nucleic acid further comprises a localization factor.