In one embodiment of the present invention, target mRNA sequences for the design of the siRNA molecules against thymidylate synthase are selected from the group of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29.

siRNA Targeted to Cell Survival Genes

Appropriate siRNA targeted to cell survival genes can be selected based on criteria known in the art and outlined above for the TS siRNA. Examples of suitable cell survival genes include, but are not limited to Bcl-2, BcI-XL, Bcl-w, McI-I, Al and protein kinase B (also known as Akt). The sequences of these genes and/or the mRNA transcribed from the gene are known in the art and are available from GenBank™. For example, the mRNA sequence for Bcl-2 can be obtained from GenBank™ under Accession No. NM 000657 (also shown in Figure 10 (SEQ ID NO:12)). Suitable siRNA target regions within Bcl-2, and methods of selecting same, are known in the art (see, for example, U.S. Patent Application No. 11/083,784 (Publication No. 2005/0245475)). Examplary target regions within the Bcl-2 mRNA are provided in Table 3.

Table 3: Exemplary mRNA Target Sequences from Bcl-2 for the Design of siRNA Molecules

* US 2005/0245475

In one embodiment of the present invention, target mRNA sequences for the design of the siRNA molecules against Bcl-2 are selected from the group of SEQ ID NOs: 23, 24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39. In another embodiment, the target mRNA sequences for the design of the siRNA molecules against Bcl-2 to be used in combination with TS siRNA are selected from the group of SEQ ID NOs: 23 and 24. In another embodiment, the target mRNA sequence for the design of the siRNA molecules against Bcl-2 to be used in combination with TS siRNA is SEQ ID NO: 23.

Design of siRNA Molecules

Following selection of an appropriate target mRNA sequence, as described above, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e. an antisense sequence, can be designed and prepared. As indicated above, the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single-stranded (i.e. a ssRNA molecule comprising just an antisense strand). The siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense strands.

Double-stranded siRNA may comprise RNA strands that are the same length or different lengths. In one embodiment, the siRNA is a double-stranded siRNA. In another embodiment, the siRNA is a double-stranded siRNA wherein both RNA strands are the same length.

Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

Small hairpin RNA (shRNA) molecules thus are also contemplated by the present invention. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands). The spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands). The spacer sequence is typically an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA (see, for example, Brummelkamp et al., 2002 Science 296:550; Paddison et al., 2002 Genes Develop. 16:948; Paul et al., Nat. Biotechnol. 20:505-508 (2002); Grabarek et al., BioTechniques 34:734-44 (2003)). The spacer sequence generally comprises between about 3 and about 100 nucleotides.

Single-stranded siRNA molecules are generally single-stranded RNA molecules with little or no secondary structure.

The overall length of the siRNA molecules can vary from about 14 to about 200 nucleotides depending on the type of siRNA molecule being designed. Between about 14 and about 50 of these nucleotides are complementary to the mRNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 200 nucleotides.

In one embodiment of the present invention, the siRNA molecule is a double- or single-stranded siRNA between about 15 and about 40 nucleotides in length. In another embodiment, the siRNA molecule is a double- or single-stranded siRNA between about 15 and about 35 nucleotides in length. In another embodiment, the siRNA molecule is a double- or single-stranded siRNA between about 17 and about 30 nucleotides in length. In another embodiment, the siRNA molecule is a double- or single-stranded siRNA between about 19 and about 25 nucleotides in length. In another embodiment, the siRNA molecule is a double- or single-stranded siRNA between about 21 to about 23 nucleotides in length.

In an alternative embodiment, the siRNA molecule is a shRNA molecule or circular siRNA molecule between about 50 and about 100 nucleotides in length. In a further embodiment, the siRNA molecule is a shRNA molecule between about 50 to about 60 nucleotides in length.

As indicated above, the siRNA molecule comprises an antisense strand that includes a specific antisense sequence complementary to all or a portion of a target mRNA sequence. One skilled in the art will appreciate that the entire length of the antisense strand comprised by the siRNA molecule does not need to be complementary to the target sequence. Thus, the antisense strand of the siRNA molecules may comprise a specific antisense sequence together with nucleotide sequences at the 5' and/or 3' termini that are not complementary to the target sequence. Such non-complementary nucleotides may provide additional functionality to the siRNA molecule. For example, they may provide a restriction enzyme recognition sequence or a "tag" that facilitates detection, isolation or purification. Alternatively, the additional nucleotides may provide a self-complementary sequence that allows the siRNA to adopt a hairpin configuration. Such configurations are useful when the siRNA molecule is a shRNA molecule, as described above.

Accordingly, within its overall length of about 14 to about 200 nucleotides, the siRNA molecules of the present invention comprise a specific antisense sequence of between about 14 to about 50 nucleotides in length that is complementary to all or a portion of a selected target mRNA sequence. In one embodiment, the length of the specific antisense sequence is from about 14 to about 40 nucleotides. In another embodiment, the length of the specific antisense sequence is from about 14 to about 35 nucleotides. In another embodiment, the length of the specific antisense sequence is from about 14 to about 30 nucleotides. In another embodiment, the length of the specific antisense sequence is from about 15 to about 30 nucleotides. In a further embodiment, the length of the specific antisense sequence is from about 17 to about 30 nucleotides. In other embodiments, the length of the specific antisense sequence is from about 19 to about 25, from about 19 to about 23, and from about 21 to about 23 nucleotides.

In one embodiment of the invention, the siRNA is a TS siRNA that comprises a specific antisense sequence that is complementary to at least 14 consecutive nucleotides of any one of the sequences as set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29. In another embodiment, the TS siRNA comprises a specific antisense sequence that comprises at least 14 consecutive nucleotides of the complementary sequence of any one of SEQ ID NOs: 1, 2, 3. 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29.

In another embodiment of the invention, the siRNA is a siRNA targeted to Bcl-2 that comprises a specific antisense sequence that is complementary to at least 14 consecutive nucleotides of any one of the sequences as set forth in SEQ ID NOs: 23,

24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39. In another embodiment, the siRNA is a siRNA targeted to Bcl-2 comprising a specific antisense sequence that comprises at least 14 consecutive nucleotides of the complementary sequence of any one of SEQ ID NOs: 23, 24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

The specific antisense sequence comprised by the siRNA molecule can be identical or substantially identical to the complement of the target sequence. In one embodiment of the present invention, the specific antisense sequence comprised by the siRNA molecule is at least about 75% identical to the complement of the target mRNA sequence. In another embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 90% identical to the complement of the target mRNA sequence. In a further embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 95% identical to the complement of the target mRNA sequence. In another embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 98% identical to the complement of the target mRNA sequence. Methods of determining sequence identity are known in the art and can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software or provided on the NCBI website.

The specific antisense sequence of the siRNA molecules of the present invention may exhibit variability by differing (e.g. by nucleotide substitution, including transition or transversion) at one, two, three, four or more nucleotides from the sequence of the target mRNA. When such nucleotide substitutions are present in the antisense strand of a dsRNA molecule, the complementary nucleotide in the sense strand with which the substitute nucleotide would typically form hydrogen bond base-pairing may or may not be correspondingly substituted. dsRNA molecules in which one or more nucleotide substitution occurs in the sense sequence, but not in the antisense strand, are also contemplated by the present invention. When the antisense sequence of a siRNA molecule comprises one or more mismatches between the nucleotide sequence of the siRNA and the target nucleotide sequence, as described above, the mismatches may be found at the 3' terminus, the 5' terminus or in the central portion of the antisense sequence.

In one embodiment of the invention, the siRNA molecules comprise a specific antisense sequence that is capable of selectively hybridizing under stringent conditions to a portion of a naturally occurring target gene or target mRNA. Suitable stringent conditions include, for example, hybridization according to conventional hybridization procedures and washing conditions of 1-3 x SSC, 0.1-1% SDS, 50-70⁰C with a change of wash solution after about 5-30 minutes. As known to those of ordinary skill in the art, variations in stringency of hybridization conditions may be achieved by altering the time, temperature, and/or concentration of the solutions used for the hybridization and wash steps. Suitable conditions can also depend in part on the particular nucleotide sequences used, for example the sequence of the target mRNA or gene.

siRNA molecules having a duplex or double-stranded structure, for example dsRNA or shRNA, can have blunt ends, or can have 3' and/or 5' overhangs. As used herein, "overhang" refers to the unpaired nucleotide or nucleotides that protrude from a duplex structure when a 3 '-terminus of one RNA strand extends beyond the 5'- terminus of the other strand (3' overhang), or vice versa (5' overhang). The nucleotides comprising the overhang can be ribonucleotides, deoxyribonucleotides or modified versions thereof. For example, double-stranded siRNA molecules can comprise poly-T or poly-U overhangs at each end to minimise RNase-mediated degradation of the molecules.

In one embodiment of the present invention, the siRNA molecules comprise overhangs at the 3' and 5' ends which consist of two thymidine or two uridine residues.

In another embodiment, at least one strand of the siRNA molecule has a 3' overhang from about 1 to about 6 nucleotides in length. In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.

When the siRNA molecule comprises a 3' overhang at one end of the molecule, the other end can be blunt-ended or have also an overhang (5' or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present invention comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule. In a further embodiment, the siRNA molecule is a dsRNA having a 3' overhang of 2 nucleotides at both ends of the molecule. In yet another embodiment, the nucleotides comprising the overhang of the siRNA are TT dinucleotides or UU dinucleotides.

When determining the percentage identity of the siRNA molecule comprising one or more overhangs to the target mRNA sequence, the overhang(s) may or may not be taken into account. For example, the nucleotides from a 3' overhang and up to 2 nucleotides from the 5'- and/or 3 '-terminus of the double strand may be modified without significant loss of activity of the siRNA molecule.

In the context of this invention, the term "RNA" refers to a nucleic acid molecule of one or more ribonucleotides in length, wherein the ribonucleotide(s) are naturally- occurring ribonucleotides or modified ribonucleotides. In general RNA refers to an oligomer or polymer of ribonucleotides. siRNA molecules, therefore, can be composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages or can comprise one or more non-naturally-occurring nucleotides or linkages, which function similarly. Such modified siRNA molecules are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake and/or increased bioavailability, enhanced affinity for nucleic acid target, and increased stability in the presence of nucleases. For example, certain chemical modifications can improve the bioavailability of the siRNA molecules by targeting particular cells or tissues and/or improving cellular uptake of the molecule. Typically, siRNA molecules comprising modified nucleotides and/or linkages retain substantially the same activity as the unmodified molecule. However, it is also contemplated that the activity of a modified siRNA molecule may be reduced as compared to an unmodified siRNA molecule, but the overall activity of the modified siRNA molecule can be greater than that of the unmodified siRNA molecule due to improved stability and/or delivery of the molecule. Modified siRNA molecules can also minimise the possibility of activating interferon activity.

A modified siRNA molecule can comprise one or more modified nucleotides, for example, a siRNA molecule comprising modified ribonucleotide(s) can comprise about 5% to about 100% modified nucleotides (for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siRN A molecule will depend on the total number of nucleotides present in the siRNA. If the siRNA molecule is a ssRNA molecule, the percent modification will be based upon the total number of nucleotides present in the ssRNA molecule. When the siRNA molecule is a dsRNA molecule, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands of the molecule. In accordance with the present invention, a siRNA molecule that comprises one or more modified nucleotides or linkages maintains the ability to inhibit expression of the target gene.

As is known in the art, a nucleoside is a base-sugar combination and a nucleotide is a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. In forming RNA molecules, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound, with the normal linkage or backbone of RNA being a 3' to 5' phosphodiester linkage. Specific examples of siRNA molecules useful in this invention include siRNA molecules containing modified backbones or non-natural internucleoside linkages. siRNA molecules having modified backbones include both those that retain a phosphorus atom in the backbone and those that lack a phosphorus atom in the backbone.

Exemplary siRNA molecules having modified backbones include, for example, those with one or more modified internucleotide linkages that are phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. The modified linkages can link one or more of the nucleotides comprised by the siRNA, for example, the modified linkages can link the four, five or six 3'- terminal nucleotides of the siRNA molecule. Alternatively, in a further embodiment, the modified linkages can link all the nucleotides of the siRNA molecule.

Exemplary modified RNA backbones that do not include a phosphorus atom are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. Such backbones include morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulphone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulphamate backbones; methyleneimino and methylenehydrazino backbones; sulphonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

The siRNA molecules can also comprise one or more universal base. The term "universal base," as used herein, refers to a nucleotide analogue that forms base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6- nitroindole (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The siRNA molecules can comprise one or more 5' and/or 3'-cap structure. The siRNA molecule can comprise a cap structure at the 3 '-end of the sense strand, the antisense strand, or both the sense and antisense strands; or at the 5 '-end of the sense strand, the antisense strand, or both the sense and antisense strands of the siRNA molecule. Alternatively, the siRNA molecule can comprise a cap structure at both the 3 '-end and 5'-end of the siRNA molecule. The term "cap structure" refers to a chemical modification incorporated at either terminus of an oligonucleotide (see, for example, U.S. Patent No. 5,998,203), which protects the molecule from exonuclease degradation, and may also facilitate delivery and/or localisation within a cell.

Examples of suitable 5 '-cap structures include, but are not limited to, glyceryl, inverted deoxy abasic residue; 4', 5 '-methylene nucleotide; 1 -(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'- inverted abasic moiety; 1 ,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; and bridging or non-bridging methylphosphonate moieties.

Examples of suitable 3 '-cap structures include, but are not limited to, glyceryl, inverted deoxy abasic residue; 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3- diamino-2 -propyl phosphate; 3 -aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L- nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo- pentoruranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'- inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1 ,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (see, for example, Beaucage and Iyer,

1993, Tetrahedron 49, 1925).

The term "abasic residue" refers to a nucleotide comprising a sugar moiety lacking a base or having another chemical group in place of a base at the 1' position (see, for example, U.S. Patent No. 5,998,203).

The present invention also contemplates siRNA comprising ribonucleotide mimetics in which both the sugar and the internucleoside linkage of the nucleotide units are replaced with novel groups. The base units are maintained for hybridisation with an appropriate nucleic acid target compound. An example of such a mimetic, which has been shown to have excellent hybridisation properties, is a peptide nucleic acid (PNA) [Nielsen et al., Science, 254:1497-1500 (1991)]. In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza-nitrogen atoms of the amide portion of the backbone.

The present invention also contemplates siRNA molecules comprising "locked nucleic acids" (LNAs), which are novel conformational^ restricted nucleic acid analogues containing a methylene bridge that connects the 2'-0 of ribose with the 4'-C (see, Singh et al., Chem. Commun., 1998, 4:455-456). LNA and LNA analogues display very high duplex thermal stabilities with complementary DNA and RNA, stability towards 3'-exonuclease degradation, and good solubility properties. Synthesis of the LNA analogues of adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil, their oligomerization, and nucleic acid recognition properties have been described (see Koshkin et al., Tetrahedron, 1998, 54:3607-3630). Studies of mismatched sequences show that LNA obey the Watson-Crick base-pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.

LNAs form duplexes with complementary DNA or RNA or with complementary LNA, with high thermal affinities. The universality of LNA-mediated hybridization has been emphasized by the formation of exceedingly stable LNA: LNA duplexes (Koshkin et al., J. Am. Chem. Soc, 1998, 120:13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of three LNA monomers (T or A) resulted in significantly increased melting points toward DNA complements.

Synthesis of 2'-amino-LNA (Singh et al., J. Org. Chem., 1998, 63, 10035-10039) and 2'-methylamino-LNA has been described and thermal stability of their duplexes with complementary RNA and DNA strands reported. Preparation of phosphorothioate- LNA and 2'-thio-LNA have also been described (Kumar et al., Bioorg. Med. Chem. Lett, 1998, 8:2219-2222).

Modified siRNA molecules may also contain one or more substituted sugar moieties. For example, siRNA molecules may comprise sugars with one of the following substituents at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to Ci₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Examples of such groups are: 0[(CH₂)_n O]_n, CH₃, 0(CH₂)_n OCH₃, 0(CH₂)_n NH₂, 0(CH₂)_n CH₃, 0(CH₂)_n ONH₂, and 0(CH₂)_n 0N[(CH₂)_n CH₃)J₂, where n and m are from 1 to about 10. Alternatively, the siRNA molecules may comprise one of the following substituents at the 2' position: Ci to Ci₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a siRNA molecule, or a group for improving the pharmacodynamic properties of a siRNA molecule, and other substituents having similar properties. Specific examples include 2'-methoxyethoxy (2'-0-CH₂ CH₂ OCH₃, also known as 2'-O-(2- methoxyethyl) or 2'-MOE) [Martin et al., HeIv. Chim. Acta, 78:486-504(1995)], 2'- dimethylaminooxyethoxy (O(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE), 2'- methoxy (2'-0-CH₃), 2'-aminopropoxy (2'-OCH₂ CH₂ CH₂NH₂) and 2'-fluoro (2'-F).

Similar modifications may also be made at other positions on the siRNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked siRNA molecules and the 5' position of 5' terminal nucleotide. siRNA molecules may also comprise sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

siRNA molecules may also include modifications or substitutions to the nucleobase. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those described in U.S. Patent No. 3,687,808; The Concise Encyclopedia Of Polymer Science And Engineering, (1990) pp 858-859, Kroschwitz, J. I., ed. John Wiley & Sons; Englisch et al., Angewandte Chemie, Int. Ed., 30:613 (1991); and Sanghvi, Y. S., (1993) Antisense Research and Applications, pp 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2⁰C [Sanghvi, Y. S., (1993) Antisense Research and Applications, pp 276- 278, Crooke, S. T. and Lebleu, B., ed., CRC Press, Boca Raton].

Another modification applicable to the siRNA molecules is the chemical linkage to the siRNA molecule of one or more moieties or conjugates which enhance the activity, cellular distribution, cellular uptake, bioavailability, pharmacokinetic properties and/or stability of the siRNA molecule. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety [Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556 (1989)], cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4:1053-1060 (1994)], a thioether, e.g. hexyl-S-tritylthiol [Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309 (1992); Manoharan et al., Bioorg. Med. Chem. Lett., 3:2765-2770 (1993)], a thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20:533-538 (1992)], an aliphatic chain, e.g. dodecandiol or undecyl residues [Saison- Behmoaras et al., EMBO J., 10:1111-1118 (1991); Kabanov et al., FEBS Lett., 259:327-330 (1990); Svinarchuk et al., Biochimie, 75:49-54 (1993)], a phospholipid, e.g. di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac-glycero- 3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995); Shea et al., Nucl. Acids Res., 18:3777-3783 (1990)], a polyamine or a polyethylene glycol chain [Manoharan et al., Nucleosides & Nucleotides, 14:969-973 (1995)], or adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)], a palmityl moiety [Mishra et al., Biochim. Biophys. Acta, 1264:229-237 (1995)], or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937 (1996)]. The conjugate molecule can be linked to the siRNA molecule by way of a linker, for example, via a biodegradable linker. The conjugate molecule can be attached at the 3'-end of the sense strand, the antisense strand, or both the sense and antisense strands of the siRNA molecule. Alternatively, the conjugate molecule can be attached at the 5'-end of the sense strand, the antisense strand, or both the sense and antisense strands of the siRNA molecule. It is also contemplated that a conjugate molecule can be attached at both the 3'-end and 5'-end of the siRNA molecule. When more than one conjugate molecule is attached to the siRNA molecule, the conjugate molecules can be the same or different.

One skilled in the art will recognise that it is not necessary for all positions in a given siRNA molecule to be uniformly modified. The present invention, therefore, contemplates the incorporation of more than one of the aforementioned modifications into a single siRNA molecule, or even at a single nucleoside within the siRNA molecule.

In the context of the present invention, a siRNA molecule is "nuclease resistant" when it has either been modified such that it is not susceptible to degradation by nucleases or alternatively has been placed in a delivery vehicle which in itself protects the siRNA molecule from nucleases. Nuclease resistant siRNA molecules include, for example, methyl phosphonates, phosphorothioates, phosphorodithioates, phosphotriesters, and morpholino oligomers. Suitable delivery vehicles for conferring nuclease resistance include, for example, liposomes. In one embodiment of the present invention, the siRNA molecules are nuclease resistant.

PREPARATION OF siRNA MOLECULES

The siRNA molecules can be prepared using several methods known in the art, such as chemical synthesis, in vitro transcription and the use of siRNA expression vectors.

Chemical synthesis of the siRNA molecules can be carried out by conventional techniques well-known to those skilled in the art. In general, RNA synthetic chemistry is based on standard solid-phase synthesis technology using commercially available equipment and the selective incorporation of various protecting groups into the RNA molecule at pre-determined points in the synthetic pathway. General methods of RNA synthesis and use of appropriate protecting groups is well known in the art (see, for example, Scaringe, S. A., et al., J. Am. Chem. Soc, 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc, 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862: Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677- 2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331). As is also well known in the art, modified siRNA molecules, such as phosphorothioated and alkylated derivatives, can also be readily prepared by similar methods.

RNA molecules are typically synthesized as single-stranded RNA oligonucleotides. Once synthesized, complementary RNA oligonucleotides can be annealed by methods known in the art to form double-stranded siRNA molecules, if desired. For example, dsRNA molecules can be formed by combining appropriate amounts of two complementary RNA oligonucleotides in a suitable annealing buffer, heating the solution, for example to about 9O⁰C, then allowing the solution to cool gradually to between 37⁰C and room temperature.

siRNA molecules can also be synthesized by standard in vitro transcription methods by placing a DNA sequence encoding the siRNA molecule downstream of a promoter sequence of a DNA-dependent RNA polymerase, for example, T3, T7 or SP6 RNA polymerase. U.S. Patent No. 5,795,715, for example, teaches a process for the simultaneous transcription of the two complementary strands of a DNA sequence, carried out under pre-determined conditions and in the same reaction compartment. The two resulting transcripts hybridize immediately after transcription giving rise to a dsRNA molecule. In addition, kits providing a rapid and efficient means of constructing siRNA molecules by in vitro transcription are commercially available, for example, from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) and are suitable for constructing the siRNA molecules of the present invention. Standard recombinant techniques in which a sequence encoding the siRNA molecule is inserted into a recombinant expression vector can also be used. Suitable expression vectors for expressing siRNA include chromosomal, nonchromosomal and synthetic DNA sequences (for example, derivatives of SV40); bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; and viral vectors, such as vaccinia virus, adenovirus, fowl pox virus, and pseudorabies virus vectors. Retroviral plasmid vectors are also suitable for use as expression vectors, including for example, those derived from Moloney Murine Leukemia Virus, spleen necrosis virus, Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumour virus.

The appropriate DNA sequence(s) encoding the siRNA molecule can be inserted into the vector by a variety of procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques can be employed, such as those described, for example, in Ausubel et al. (Current Protocols in Molecular Biology, 1993 & updates, John Wiley & Sons, Inc., Boston, Mass.) and Sambrook et al. (Molecular Cloning, Third Ed., 2001, Cold Spring Harbor Laboratory, Plainview, N. Y.).

The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequence, such as a promoter or a regulated promoter, to direct RNA synthesis. Representative examples of such expression control sequences include bacterial promoters such as the lac, lacZ, T3, T7, gpt, lambda P_R, lambda PL and tip; eukaryotic promoters including the human U6 snRNA promoter (see, for example, Miyagishi et al, Nat. Biotechnol. 20:497-500 (2002); Lee et al., Nat. Biotechnol. 20:500-505 (2002); Paul et al., Nat. Biotechnol. 20:505-508 (2002); Grabarek et al., BioTechniques 34:73544 (2003); Sui et al., Proc. Natl. Acad. Sci. USA 99:5515-20 (2002)), the Hl RNA promoter (see, for example, Brummelkamp et al., Science 296:550-53 (2002)), human cytomegalovirus (CMV) immediate early (Miller, et al., Biotechniques 7:980-990 (1989)), HSV thymidine kinase, early and late SV40, LTRs from retrovirus, mouse metallothionein-I and eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters. Other regulatory regions that may be included in the expression vector as necessary include, but are not limited to, enhancers, ribosome binding sites, polyadenylation sites, splice donor and/or acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. Selection of the appropriate vector and regulatory regions is well within the level of ordinary skill in the art.

Transcription of one or more copies of the encoded siRNA molecule can be achieved by an endogenous RNA polymerase of the cell transformed with the expression vector or by a cloned RNA polymerase (for example, T3, T7 or SP6 RNA polymerase), which may be encoded by the same or a different expression vector. When the encoded siRNA molecule is a dsRNA molecule, each strand of the dsRNA can be transcribed separately, each under the direction of a separate promoter, and then can hybridize within the cell to form the dsRNA duplex or each strand can be transcribed from a separate vector (see Lee et al., supra). Alternatively, when the siRNA molecule is a shRNA, then the sense and antisense sequences can be transcribed as a single sequence under the control of a single promoter such that the transcribed RNA molecule forms a hairpin (Paul et al., supra). DNA vectors useful for insertion of sequences for transcription of an siRNA polynucleotide include pSUPER vector (see, for example, Brummelkamp et al., supra); pAV vectors derived from pCWRSVN (see, for example, Paul et al., supra); and pIND (see, for example, Lee et al., supra), or the like.

The expression vector can be introduced into a suitable host cell for propagation of the vector and/or expression of the encoded siRNA molecule by one of a number of standard techniques. In general the host cell is transduced, transformed or transfected by electroporation, the use of liposomes including cationic liposomes, calcium phosphate precipitation, DEAE-dextran mediated transfection, other suitable technique. Suitable host cells include prokaryotic cells, such as a bacterial cells; lower eukaryotic cells, such as a yeast cells or higher eukaryotic cells, such as mammalian cells, plant cells or insect cells.

Suitable prokaryotic host cells for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces and Staphylococcus. Suitable lower eukaryotic host cells include the yeasts, Saccharomyces and Pichia. Representative examples of appropriate higher eukaryotic host cells include insect cells, such as Drosophila S2 and Spodoptera Sf9; mammalian cells, such as CHO, COS, 293, C127, 3T3, HeLa, HEK, and BHK cell lines; and plant cells.

When a retroviral expression vector is employed, the vector can be used to transduce a packaging cell line to form a producer cell line. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi CRE, psi CRIP, GP+E-86, GP+envAml2, and DAN cell lines (see, for example, Miller, Human Gene Therapy, 1 :5-14 (1990)). Transduction can be achieved through various techniques known in the art, including those listed above. The producer cell line generates infectious retroviral vector particles that include the nucleic acid sequence(s) encoding the siRNA molecule of the invention. Such retroviral vector particles can subsequently be employed to transduce eukaryotic cells, either in vitro or in vivo. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, bronchial epithelial cells and various other culture- adapted cell lines.

The engineered host cells into which the expression vector has been introduced can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, amplifying particular sequences, etc. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.

siRNA may also be prepared using the Drosophila in vitro system described in U.S. Patent Application No. 2003/0108923, which entails combining dsRNA with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA of about 21 to about 23 nucleotides.

The siRNAs may be derived from dsRNAs longer than 50 nucleotides, or from microRNAs (miRNAs) according to methods known in the art, including the Drosophila in vitro system described above.

The siRNA molecules can be provided in crude mixtures or as purified or partially purified molecules. For example, recombinant siRNA molecules can be provided in the form of intact host cells, intact organelles (such as cell membranes, intracellular vesicles and the like), disrupted cell preparations (such as cell homogenates, cell lysates, microsomes, uni- and multilamellar membrane vesicles and other preparations). Alternatively, recombinant siRNA molecules can be recovered and purified from host cell cultures by standard purification techniques, including ammonium sulphate precipitation, ethanol precipitation, acid extraction, and various chromatographic techniques (including anion exchange, cation exchange, phosphocellulose, hydrophobic interaction, affinity, hydroxylapatite and lectin chromatography). High performance liquid chromatography (HPLC) is also suitable for final purification steps. Similarly, when synthesized chemically or by in vitro transcription, the siRNA molecules can be purified by standard techniques, such as extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, chemically synthesized and in vitro transcribed siRNA molecules can be used without purification or with a minimum of purification steps in order to minimise losses due to sample processing. The siRNA molecules can be dried for storage or dissolved in an aqueous solution, which may contain buffers or salts to promote annealing, and/or stabilization of duplex strands.

In addition to the above methods, a number of commercial packages and services are available that are suitable for use for the preparation of siRNA molecules. These include the in vitro transcription kits available from Ambion (Austin, TX) and New England Biolabs (Beverly, MA) as described above; viral siRNA construction kits commercially available from Invitrogen (Carlsbad, CA) and Ambion (Austin, TX), and custom siRNA construction services provided by Ambion (Austin, TX), Qiagen (Valencia, CA), Dharmacon (Lafayette, CO) and Sequitur, Inc (Natick, MA).

EFFICACY OF THE siRNA MOLECULES

Various methods of testing the efficacy of the siRNA molecules are known in the art and may be employed to test the efficacy of the TS siRNA and siRNA targeted to a cell survival gene. Representative methods are described below using TS siRNA as an example. One skilled in the art will recognize that analogous methods can be employed to test the efficacy of siRNA targeted to a cell survival gene, such as Bcl-2.

In accordance with the present invention, the siRNA molecules are able to inhibit the expression of their target gene. The ability of TS siRNA to inhibit the expression of thymidylate synthase mRNA or protein can be tested by one or more of a number of standard in vitro or in vivo techniques. In one embodiment of the present invention,

TS siRNA sensitizes cancer cells to the action of one or more chemotherapeutics. In another embodiment, TS siRNA inhibits the proliferation of neoplastic cells. In another embodiment, TS siRNA does not substantially inhibit proliferation of neoplastic cells, but is capable of sensitizing the cells to the action of one or more chemotherapeutics. The chemosensitizing activity and/or the ability of the TS siRNA to inhibit cancer cell proliferation can be tested using standard techniques, as can the effects of the TS siRNA in combination with various anti-cancer therapeutics including other siRNAs. Exemplary, non-limiting tests are provided below and in the

Examples.

In vitro Testing

The ability of TS siRNA to inhibit the expression of a mammalian thymidylate synthase gene can be determined by culturing cells of a selected cell line in a suitable medium. After an appropriate incubation time, the cells are transfected with the siRNA molecule, for example in the presence of a commercial lipid carrier such as lipofectamine or Oligfectamine,™ and are incubated for a further period of time. The expression of the thymidylate synthase can be measured by determining the amount of mRNA transcribed from the gene and/or by determining the amount of protein expressed according to standard methods known in the art. For example, mRNA levels can be measured using Northern blot analysis or quantitative RT-PCR procedures and protein levels can be measured using Western blot analysis. The levels of mRNA and protein corresponding to thymidylate synthase can be compared to an appropriate control. Suitable controls include, for example, untreated cells and/or cells treated with a compound known to inhibit thymidylate synthase expression.

In accordance with the present invention, the TS siRNA inhibits expression of the thymidylate synthase gene in a test cell population by at least 10% as compared to an untreated control cell population. In one embodiment, TS siRNA inhibits expression of the thymidylate synthase gene in a test cell population by at least 33%. In another embodiment, TS siRNA inhibits expression of the thymidylate synthase gene in a test cell population by at least 50%. In other embodiments, TS siRNA inhibits expression of the thymidylate synthase gene in a test cell population by at least 75%, 80%, 90%, 95% and 98%. Alternatively, the extent of inhibition by the TS siRNA can be expressed in terms of the number of cells within a population in which inhibition of expression of the target thymidylate synthase gene is observed. This could be assessed, for example, by FACS analysis. As would be apparent to a worker skilled in the art, lower doses of administered siRNA molecules and longer times after administration of siRNA molecules may result in inhibition being observed in only a small fraction of cells. Thus, the TS siRNA demonstrates inhibition of expression of the target thymidylate synthase gene in at least 10% of targeted cells. In various embodiments, TS siRNA demonstrates inhibition of expression of the target thymidylate synthase gene in at least 20%, 50%, 75%, 90%, and 95% of targeted cells. Quantitation of expression in a cell or cell population may show similar amounts of inhibition at the level of accumulation of target mRNA and translation of target protein, or the levels of inhibition may be different.

The ability of TS siRNA to attenuate the growth or proliferation of neoplastic cells can be tested by a number of standard techniques. For example, the cytotoxicity of the siRNA molecule can be assayed in vitro using a suitable cancer cell line. In general, cells of the selected test cell line are grown to an appropriate density and the candidate siRNA molecule is added. After an appropriate incubation time (for example, about 48 to 72 hours), cell survival is assessed. Methods of determining cell survival are well known in the art and include, but are not limited to, the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11 :48-50; O'Brien et al., (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No. 5,501,959), the sulforhodamine assay (Rubinstein et al., (1990) J. Natl. Cancer Inst. 82:113-118), the neutral red dye test (Kitano et al., (1991) Euro. J. Clin. Investg. 21 :53-58; West et al., (1992) J. Investigative Derm. 99:95-100) or the XTT assay. Cytotoxicity is determined by comparison of cell survival in the treated culture with cell survival in one or more control cultures, for example, untreated cultures and/or cultures pre- treated with a control compound (typically a known therapeutic).

Alternatively, the ability of the TS siRNA to inhibit proliferation of neoplastic cells can be assessed by culturing cells of a cancer cell line of interest in a suitable medium. After an appropriate incubation time, the cells can be transfected with the siRNA molecule, as described above, and incubated for a further period of time. Cells are then counted and compared to an appropriate control, as described above.

In accordance with one embodiment, the TS siRNA is capable of producing a decrease of about 10% or more in the proliferation of neoplastic cells when compared to untreated cells or to cells treated with an appropriate control siRNA, such as a scrambled control siRNA molecule. In another embodiment, the TS siRNA is capable of producing a decrease of about 15% or more in the proliferation of neoplastic cells when compared to untreated cells or to cells treated with an appropriate control siRNA. In another embodiment, the TS siRNA is capable of producing a decrease of about 20% or more in the proliferation of neoplastic cells when compared to untreated cells or to cells treated with an appropriate control siRNA. In a further embodiment, the TS siRNA is capable of producing a decrease of about 25% or more in the proliferation of neoplastic cells when compared to untreated cells or to cells treated with an appropriate control siRNA.

The TS siRNA can also be tested in vitro to determine if it can induce tumour cell accumulation in the Gl phase of the cell cycle. Tumour cells can be cultured in the presence or absence of the siRNA and after a suitable length of time, the number cells in each phase of the cell cycle can be measured using, for example, flow cytometry. The TS siRNA can also be tested in vitro by determining its ability to inhibit anchorage-independent growth of tumour cells. Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage- independent growth is assessed by plating cells from a selected cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the siRNA molecule can then be compared with that of control cells (as described above).

A variety of cancer cell-lines suitable for testing the candidate TS siRNA are known in the art and many are commercially available (for example, from the American Type Culture Collection, Manassas, VA). In one embodiment of the present invention, in vitro testing of the TS siRNA is conducted in a human cancer cell-line. Examples of suitable cancer cell-lines for in vitro testing include, but are not limited to, breast cancer cell-lines MDA-MB-231 and MCF-7, renal carcinoma cell-line A-498, mesothelial cell lines MSTO-211H, NCI-H2052 and NCI-H28, ovarian cancer cell- lines OV90 and SK-OV-3, colon cancer cell-lines CaCo, HCTl 16 and HT29, cervical cancer cell-line HeLa, non-small cell lung carcinoma cell-lines A549 and H 1299, pancreatic cancer cell-lines MIA-PaCa-2 and AsPC-I, prostatic cancer-cell line PC-3, bladder cancer cell-line T24, liver cancer cell-lineHepG2, brain cancer cell-line U-87 MG, melanoma cell-line A2058, lung cancer cell-line NCI-H460. Other examples of suitable cell-lines are known in the art.

If desired, the TS siRNA can be tested to determine whether it activates the interferon pathway. Methods of determining the ability of the siRNA molecules to activate the interferon response pathway are known in the art, and are described in, for example, Sledz, et al. (2003) Nature Cell Biology 5:834-839, and Bridge et al., (2003) Nature Genetics 34:263-264.

In one embodiment of the present invention, the TS siRNA is used in combination with one or more standard chemotherapeutics, for example to enhance sensitivity of cancer cells to the chemotherapeutic. The efficacy of the combinations of siRNA molecules and one or more chemotherapeutic can be tested using standard techniques including those outlined above for the siRNA molecules. Assessment of the efficacy of the TS siRNA in combination with a siRNA targeted to a cell survival gene can also be tested using these methods. Additional controls may be included in such assays, such as cells treated with the siRNA molecule(s) alone and/or the chemotherapeutic(s) alone in order to determine whether the effect of the combination is greater than the effect of each siRNA molecule and/or the chemotherapeutic(s) alone.

Therapeutic efficacy of TS siRNA can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50.

The toxicity of the TS siRNA can also be assessed in vitro, if necessary, using standard techniques. For example, human primary fibroblasts can be transfected in vitro with the siRNA molecule and then tested at different time points following treatment for their viability using a standard viability assay, such as those described above. Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS).

In vivo Testing

The ability of the TS siRNA alone, or in combination with one or more anti-cancer agents, including other siRNA, to inhibit tumour growth or proliferation in vivo can be determined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al., Current Protocols in Pharmacology, J. Wiley & Sons, Inc., New York, NY).

In general, current animal models for screening anti-tumour compounds are xenograft models, in which a human tumour has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumour xenografts, implanted by sub-cutaneous injection or implantation and used in tumour growth assays; human solid tumour isografts, implanted by fat pad injection and used in tumour growth assays; human solid tumour orthotopic xenografts, implanted directly into the relevant tissue and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.

For example, TS siRNA can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0. The animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Animals not bearing tumours may also be subjected to the same treatments as the tumour-bearing animals in order to be able to dissociate any toxic effect of the test siRNA molecule(s) from the specific effect on the tumour. Chemotherapy generally begins from 3 to 22 days after grafting, depending on the type of tumour, and the animals are observed every day. The siRNA molecules of the present invention can be administered to the animals, for example, by i.p. injection or bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.

The tumours are measured after a pre-determined time period, or they can be monitored continuously by measuring about 2 or 3 times a week until the tumour reaches a pre-determined size and/or weight, or until the animal dies if this occurs before the tumour reaches the pre-determined size/weight. The animals are then sacrificed and the tissue histology, size and/or proliferation of the tumour assessed.

For the study of the effect of the siRNA molecules on leukaemias, the animals are grafted with a particular number of cells, and the anti-tumour activity is determined by the increase in the survival time of the treated mice relative to the controls.

To study the effect of the TS siRNA on tumour metastasis, tumour cells are typically treated with the siRNA molecule(s) ex vivo and then injected into a suitable test animal. The spread of the tumour cells from the site of injection is then monitored over a suitable period of time.

In vivo toxic effects of the TS siRNA can be evaluated by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.

Table 3: Examples of Xenograft Models of Human Cancer

PHARMACEUTICAL COMPOSITIONS

The present invention provides for pharmaceutical compositions comprising one or more siRNA molecules in admixture with a conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. The pharmaceutical compositions can be formulated for oral, topical, parenteral or rectal administration or for administration by inhalation or spray. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The siRNA molecules can be present in the compositions in association with one or more other active ingredients, such as conventional chemotherapeutics, if desired.

Compositions containing only "naked" siRNA and a physiologically acceptable solvent or carrier have been shown to be taken up by cells in vivo. The pharmaceutical compositions of the present invention, therefore, may comprise one or more siRNA molecule in aqueous suspension. Suitable solvents and carriers for such compositions include those described below. Alternatively, the siRNA molecules may be formulated for administration by associating the siRNA molecule with a biodegradable polymer or may be encapsulated to protect the siRNA against rapid elimination from the body, for example, in the form of a controlled release formulation, such as an implant or microencapsulated delivery system. Biodegradable, biocompatible polymers that can be used for such formulations include, for example, polypeptides, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, poly(d, 1-lactic-co-glycolic acid) (PLGA), polylysin, polylysin conjugates, polylysine- graft-imidazole acetic acid and poly(beta-amino ester). Microparticles, such as microspheres, nanoparticles or nanospheres can also be employed. Alternatively, the siRNA molecules may be covalently coupled to the polymer or microparticle, wherein the covalent coupling particularly is effected via the 3 '-terminus of the siRNA. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075. Similarly, the pharmaceutical compositions of the present invention can comprise one or more siRNA associated with or encapsulated by liposomes or other artificial membrane vesicles known in the art [for example, see "Liposomes as Drug Carriers" G. Gregoriadis, Wiley & Sons, New- York (1988); Gregoriadis, G., "Liposome preparation and related techniques," in: G. Gregoriadis (Ed.) "Liposome Technology" Vol. 1, 2^nd Edition, CRC Press, Baton Rouge, FL, (1993), pp.1-63].

The pharmaceutical compositions may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with suitable nontoxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Pharmaceutical compositions for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active siRNA in admixture with suitable excipients including, for example, suspending agents, such as sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, hepta- decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in- water emulsions. The oil phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixtures of these oils. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. Other examples are, sterile, fixed oils which are conventionally employed as a solvent or suspending medium, and a variety of bland fixed oils including, for example, synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000).

USE OF TS siRNA

In its most general aspect, the present invention provides for the use of TS siRNA in various cancer treatment regimens based on the properties of the TS siRNA and its interaction with other anti-cancer agents, including siRNA targeted to cell survival genes. The present invention, therefore, provides for the use of TS siRNA alone, or in combination with a siRNA targeting a cell survival gene, and/or in combination with one or more chemotherapeutic in the treatment, stabilization or prevention of various cancers.

In this context, the treatment of cancer can refer to a cytotoxic or cytostatic effect resulting in a reduction in the size of a tumour, the slowing or prevention of an increase in the size of a tumour, an increase in the disease-free survival time between the disappearance or removal of a tumour and its reappearance, prevention of an initial or subsequent occurrence of a tumour (e.g. metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumour, or an increase in the overall survival time of a subject having cancer. In one embodiment, the invention provides for the use of TS siRNA to sensitise cancer cells to the action of a TS-targeting chemotherapeutic. In another embodiment, the invention provides for the use of TS siRNA in combination with siRNA targeted to a cell survival gene, such as Bcl-2, to inhibit cancer cell proliferation in a subject having a cancer. In another embodiment, the invention provides for the use of a combination of TS siRNA, a siRNA targeting a cell survival gene and a non-TS targeting chemotherapeutic in the treatment of cancer.

In one embodiment of the present invention, TS siRNA is used in the treatment of cancer in combination with an anti-cancer agent, such as a chemotherapeutic, immunotherapeutic or other siRNA, to produce a therapeutic effect that is greater than the effect of either the TS siRNA alone. Such an increased therapeutic effect can be, for example, less-than-additive, additive or synergistic.

Examples of cancers which may be treated or stabilized in accordance with the present invention include, but are not limited to haematologic neoplasms, including leukaemias and lymphomas; carcinomas, including adenocarcinomas; melanomas and sarcomas. Carcinomas, adenocarcinomas and sarcomas are also frequently referred to as "solid tumours," examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus, non-small cell lung cancer and colorectal cancer. Various forms of lymphoma also may result in the formation of a solid tumour and, therefore, are often considered to be solid tumours. In one embodiment of the present invention, TS siRNA is used to treat or stabilize a solid tumour. In another embodiment, TS siRNA in combination with a siRNA targeted to a cell surivial gene is used to treat or stabilise a solid tumour. In another embodiment, TS siRNA in combination with a siRNA targeted to a cell surivial gene is used to treat or stabilise breast cancer or small cell lung cancer.

The term "leukaemia" refers broadly to progressive, malignant diseases of the blood- forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1) the duration and character of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic). Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T- cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia, monocytic leukaemia, myeloblasts leukaemia, myelocytic leukaemia, myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia.

The term "lymphoma" generally refers to a malignant neoplasm of the lymphatic system, including cancer of the lymphatic system. The two main types of lymphoma are Hodgkin's disease (HD or HL) and non-Hodgkin's lymphoma (NHL). Abnormal cells appear as congregations which enlarge the lymph nodes, form solid tumours in the body, or more rarely, like leukemia, circulate in the blood. Hodgkin's disease lymphomas, include nodular lymphocyte predominance Hodgkin's lymphoma; classical Hodgkin's lymphoma; nodular sclerosis Hodgkin's lymphoma; lymphocyte- rich classical Hodgkin's lymphoma; mixed cellularity Hodgkin's lymphoma; lymphocyte depletion Hodgkin's lymphoma. Non-Hodgkin's lymphomas include small lymphocytic NHL, follicular NHL; mantle cell NHL; mucosa-associated lymphoid tissue (MALT) NHL; diffuse large cell B-cell NHL; mediastinal large B-cell NHL; precursor T lymphoblastic NHL; cutaneous T-cell NHL; T-cell and natural killer cell NHL; mature (peripheral) T-cell NHL; Burkitt's lymphoma; mycosis fungoides; Sezary Syndrome; precursor B-lymophoblastic lymphoma; B-cell small lymphocytic lymphoma; lymphoplasmacytic lymphoma; spenic marginal zome B-cell lymphoma; nodal marginal zome lymphoma; plasma cell myeloma/plasmacytoma; intravascular large B-cell NHL; primary effusion lymphoma; blastic natural killer cell lymphoma; enteropathy-type T-cell lymphoma; hepatosplenic gamma-delta T-cell lymphoma; subcutaneous panniculitis-like T-cell lymphoma; angioimmunoblastic T- cell lymphoma; and primary systemic anaplastic large T/null cell lymphoma.

The term "sarcoma" generally refers to a tumour which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma. The term "melanoma" is taken to mean a tumour arising from the melanocyte system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large- cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term "carcinoma" also encompasses adenocarcinomas. Adenocarcinomas are carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate.

Additional cancers encompassed by the present invention include, for example, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumours, primary brain tumours, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.

The cancer to be treated may be indolent or it may be aggressive. The present invention contemplates the use of the siRNA molecules in the treatment of refractory cancers, advanced cancers, recurrent cancers and metastatic cancers. One skilled in the art will appreciate that many of these categories may overlap, for example, aggressive cancers are typically also metastatic.

"Aggressive cancer," as used herein, refers to a rapidly growing cancer. One skilled in the art will appreciate that for some cancers, such as breast cancer or prostate cancer the term "aggressive cancer" will refer to an advanced cancer that has relapsed within approximately the earlier two-thirds of the spectrum of relapse times for a given cancer, whereas for other types of cancer, such as small cell lung carcinoma (SCLC) nearly all cases present rapidly growing cancers which are considered to be aggressive. The term can thus cover a subsection of a certain cancer type or it may encompass all of other cancer types. A "refractory" cancer or tumour refers to a cancer or tumour that has not responded to treatment. "Advanced cancer," refers to overt disease in a patient, wherein such overt disease is not amenable to cure by local modalities of treatment, such as surgery or radiotherapy. Advanced disease may refer to a locally advanced cancer or it may refer to metastatic cancer. The term "metastatic cancer" refers to cancer that has spread from one part of the body to another. Advanced cancers may also be unresectable, that is, they have spread to surrounding tissue and cannot be surgically removed. In one embodiment, the TS siRNA may also be used to treat drug resistant cancers, including multidrug resistant tumours. As is known in the art, the resistance of cancer cells to chemotherapy is one of the central problems in the management of cancer.

Certain cancers, such as prostate and breast cancer, can be treated by hormone therapy, i.e. with hormones or anti-hormone drugs that slow or stop the growth of certain cancers by blocking the body's natural hormones. Such cancers may develop resistance, or be intrinsically resistant, to hormone therapy. In one embodiment, the present invention contemplates the use of the TS siRNA in the treatment of such "hormone-resistant" or "hormone-refractory" cancers.

Administration of the siRNA Molecules

Typically in the treatment of cancer, therapeutic compounds are administered systemically to patients. Thus, the TS siRNA, and when used the siRNA targeted to a cell survival gene, can be administered to a subject, for example, orally, by bolus injection or by infusion into the subject's bloodstream. The siRNA molecules can also be administered using a hydrodynamic protocol, such as that described in International patent application PCT/US02/22869 (WO 03/10180). Alternatively, the siRNA molecules can be administered through the use of viral vectors or liposome formulations as is known in the art, or by microparticle bombardment (for example, through use of a "gene gun"; Biolistic, Dupont).

The TS siRNA may be used as part of a neo-adjuvant therapy (to primary therapy), or as part of an adjuvant therapy regimen. "Primary therapy" refers to a first line of treatment upon the initial diagnosis of cancer in a subject. Exemplary primary therapies may involve surgery, a wide range of chemotherapies and radiotherapy. "Adjuvant therapy" refers to a therapy that follows a primary therapy and that is administered to subjects at risk of relapsing. Adjuvant systemic therapy is begun soon after primary therapy to delay recurrence, prolong survival or cure a subject.

In one embodiment, the present invention provides for the use of TS siRNA in combination with one or more anti-cancer therapeutics, such as chemotherapeutic agents, immunotherapeutic agents or siRNA targeted to a cell survival gene, as part of an adjuvant therapy. When employed in combination therapy, the present invention contemplates that the TS siRNA may act as a "sensitizing agent" or "chemosensitizer." In this case, the TS siRNA alone does not have a cytotoxic effect on the cancer cells, but provides a means of weakening the cells, thereby facilitating the benefit from the other anti-cancer therapeutic(s) employed in the combination.

When used in conjunction with one or more other anti-cancer agents, the TS siRNA can be administered prior to, or after, administration of the other agent(s), or can be administered concurrently. In one embodiment of the invention, in a combination treatment regimen comprising a TS siRNA and a siRNA targeted to a cell survival gene, such as Bcl-2, the TS siRNA and the Bcl-2 siRNA are administered sequentially. In another embodiment, in a combination treatment regimen comprising a TS siRNA and a siRNA targeted to a cell survival gene, such as Bcl-2, the TS siRNA and the Bcl-2 siRNA are administered sequentially with an interval of more than 72 hours, for example, between about 72 hours and about 7 days, between administration of each individual agent. In another embodiment, in a combination treatment regimen comprising a TS siRNA and a siRNA targeted to a cell survival gene, such as Bcl-2, the TS siRNA and the Bcl-2 siRNA are administered sequentially with an interval of between 4 days and 7 days, between administration of each individual agent.

The dosage of TS siRNA to be administered is not subject to defined limits, but it will usually be an effective amount. The TS siRNA may be formulated in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of the TS siRNA calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

Daily dosages of TS siRNA will typically fall within the range of about 0.01 to about 100 mg/kg of body weight, in a single dose, a divided dose, or by administration over a pre-determined time period. However, it will be understood that the actual amount of the siRNA molecule(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual siRNA molecule administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. The above dosage range is given by way of example only and is not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day.

Chemother -apeutic Agents

When the TS siRNA is used in combination with one or more chemotherapeutic agents, the chemotherapeutic agent can be selected from a wide range of cancer chemotherapeutic agents known in the art. Known chemotherapeutic agents include those that are specific for the treatment of a particular type of cancer as well as those that are applicable to a range of cancers, such as doxorubicin, docetaxel, 5- fluorouracil, capecitabine, mitoxantrone, irinotecan (CPT-I l), cisplatin and gemcitabine. Etoposide is generally applicable in the treatment of leukaemias (including acute lymphocytic leukaemia and acute myeloid leukaemia), germ cell tumours, Hodgkin's disease and various sarcomas. Cytarabine (Ara-C) is also applicable in the treatment of various leukaemias, including acute myeloid leukaemia, meningeal leukaemia, acute lymphocytic leukaemia, chronic myeloid leukaemia, erythroleukaemia, as well as non-Hodgkin's lymphoma.

The present invention contemplates the use of both cancer-specific and broad- spectrum chemotherapeutic agent in conjunction with the TS siRNA. In one embodiment of the present invention, the TS siRNA is used in combination with a broad-spectrum chemotherapeutic. Exemplary chemotherapeutics that can be used alone or in various combinations for the treatment of specific cancers are provided in Table 4 and are suitable for use in combination with the TS siRNA. One skilled in the art will appreciate that many other chemotherapeutics are available and that the following list is representative only.

In one embodiment of the present invention, TS siRNA is used in conjunction with one or more chemotherapeutic agents that target TS. Examples of suitable TS inhibiting chemotherapeutics include, but are not limited to, the fluoropyrimidine drugs 5-FU, 5-FUdR, capecitabine (an oral form of a pro-drug of 5-FU) and a topical 5-FU cream (Effudex®), as well as the non-fluoropyrimidine drugs raltitrexed, methotrexate and pemetrexed (Alimta®). These chemotherapeutic agents are used alone and in combination in a variety of treatment regimens against various tumours including colorectal, breast, lung and mesothelioma. In one embodiment of the present invention, TS siRNA is used in combination with one or more of 5-FU, 5- FUdR, capecitabine, pemetrexed, methotrexate or raltitrexed. In another embodiment, TS siRNA is used in combination with 5-FU, 5-FUdR, or pemetrexed.

In another embodiment of the invention, TS siRNA is used in combination with a siRNA targeting a cell survival gene, and optionally a conventional chemotherapeutic.

In a further embodiment, TS siRNA is used in combination with a siRNA targeting

Bcl-2 and a non-TS targeting chemotherapeutic, such as those described in Table 4. In another embodiment, TS siRNA is used in combination with a siRNA targeting Bcl-2 and a broad-spectrum non-TS targeting chemotherapeutic such as, doxorubicin, docetaxel, 5-fluorouracil, capecitabine, mitoxantrone, irinotecan (CPT-I l), cisplatin or gemcitabine.

Table 4: Exemplary Chemotherapeutics used in the Treatment of Some Common Cancers

CANCER CHEMOTHERAPEUTIC

Acute Pegaspargase (e.g. Oncaspar®) L-asparaginase lymphocytic Cytarabine leukaemia (ALL)

Acute myeloid Cytarabine Idarubicin leukaemia (AML)

Brain cancer Procarbazine (e.g. Matulane®) Nitrosoureas

Platinum analogues Temozolomide

Breast cancer Capecitabine (e.g. Xeloda®) Cyclophosphamide

5-fluorouracil (5-FU) Carboplatin

Paclitaxel (e.g. Taxol®) Cisplatin

Docetaxel (e.g. Taxotere®) Ifosfamide

Epi-doxorubicin (epirubicin) Tamoxifen

Doxorubicin (e.g. Adriamycin®)

Chronic myeloid Cytarabine

CANCER CHEMOTHERAPEUTIC

Mitoxantrone (e.g. Novantrone®)

Prednisone (e.g. Deltasone®) Liarozole

Nilutamide (e.g. Nilandron®) Flutamide (e.g. Eulexin®)

Finasteride (e.g. Proscar®) Terazosin (e.g. Hytrin®)

Doxazosin (e.g. Cardura®) Cyclophosphamide

Docetaxel (e.g. Taxotere®) Estramustine

Luteinizing hormone releasing hormone agonist

Renal cancer Capecitabine (e.g. Xeloda®) Gemcitabine (e.g. Gemzar®)

Small cell lung Cyclophosphamide Vincristine cancer

Doxorubicin Etoposide

Solid tumours Gemicitabine (e.g. Gemzar®) Cyclophosphamide

Capecitabine (e.g. Xeloda®) Ifosfamide

Paclitaxel (e.g. Taxol®) Cisplatin

Docetaxel (e.g. Taxotere®) Carboplatin

Epi-doxorubicin (epirubicin) 5-fluorouracil (5-FU)

Doxorubicin (e.g. Adriamycin®)

As indicated above, combinations of chemotherapeutics may be employed. Combination therapies using standard cancer chemotherapeutics are well known in the art and such combinations also can be used in conjunction with TS siRNA.

Immunotherapeutic Agents

Immunotherapeutic agents suitable for use in combination with TS siRNA include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non- cytokine adjuvants. "Immunotherapeutic agents" in general refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or augments the body's immune response against cancer cells and/or that lessens the side effects of other anticancer therapies.

Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that it becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves. Immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. The present invention contemplates the use of the siRNA molecules with either non-specific or specific immunotherapeutic agents, or with combinations thereof. In one embodiment, TS siRNA is used in combination therapies with one or more non-specific immunotherapeutic agents.

Non-specific immunotherapeutic agents are substances that stimulate or indirectly augment the immune system. Some of these agents can be used alone as the main therapy for the treatment of cancer. Alternatively, non-specific immunotherapeutic agents may be given in addition to a main therapy and thus function as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines) or reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.

A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines for use in the combination therapies of the present invention include interferons, interleukins and colony- stimulating factors.

Interferons (IFNs) contemplated by the present invention for use in combination with TS siRNA include the common types of IFNs, IFN-alpha (IFN-α), IFN-beta (IFN-β) and IFN-gamma (IFN-γ). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.

Recombinant IFN-α is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). The use of IFN-α, alone or in combination with other immunofherapeutics or with chemotherapeutics, has shown efficacy in the treatment of various cancers including melanoma (including metastatic melanoma), renal cancer (including metastatic renal cancer), breast cancer, prostate cancer, cervical cancer (including metastatic cervical cancer), Kaposi's sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), multiple myeloma, follicular non- Hodgkin's lymphoma and cutaneous T cell lymphoma.

Interleukins contemplated by the present invention for use in combination with TS siRNA include IL-2 (or aldesleukin), IL-4, IL-Il and IL- 12 (or oprelvekin). Examples of commercially available recombinant interleukins include Proleukin (IL-2; Chiron Corporation) and Neumega (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, WA) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Interleukins, alone or in combination with other immunotherapeutics or with chemotherapeutics, have shown efficacy in the treatment of various cancers including renal cancer (including metastatic renal cancer), melanoma (including metastatic melanoma), ovarian cancer (including recurrent ovarian cancer), cervical cancer (including metastatic cervical cancer), breast cancer, colorectal cancer, lung cancer, brain cancer, prostate cancer, leukemias and lymphomas.

Interleukins have also shown good activity in combination with IFN-α in the treatment of various cancers and the present invention contemplates the use of one or more interleukins and IFN-α in combination therapies with one or more siRNA molecules. An interleukin-immunotoxin conjugate known as denileukin diftitox (or Ontak; Seragen, Inc), which comprises IL-2 conjugated to diptheria toxin, has been approved by the FDA for the treatment of cutaneous T cell lymphoma.

Colony-stimulating factors (CSFs) include granulocyte colony stimulating factor (G- CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in patients undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. One embodiment of the present invention provides for the use of higher than standard doses of a chemotherapeutic agent in combination therapies with a siRNA molecule and one or more CSFs.

Various recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erythropoietin). Colony stimulating factors have shown efficacy in the treatment of cancer, including melanoma, colorectal cancer (including metastatic colorectal cancer), lung cancer and leukemia.

Non-cytokine adjuvants include, but are not limited to, levamisole, alum hydroxide (alum), bacillus Calmette-Guerin (BCG), incomplete Freund's Adjuvant (IFA), QS- 21, DETOX, Keyhole limpet hemocyanin (KLH) and dinitrophenyl (DNP). Non- cytokine adjuvants in combination with other immuno- and/or chemotherapeutics have demonstrated efficacy against various cancers including, for example, colon cancer and colorectal cancer (Levimasole); melanoma (BCG and QS-21); renal cancer and bladder cancer (BCG).

In addition to having specific or non-specific targets, immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e. comprise immune system components that were generated external to the body. In one embodiment, TS siRNA is used in combination therapies with one or more active immunotherapeutic agents.

Passive immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor. Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked to such agents. The present invention contemplates the use of one or more monoclonal antibodies in combination with TS siRNA for the treatment of cancer. Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C -225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg), alemtuzumab (Campath) and ibritumomab tiuxetan (Zevalin).

Monoclonal antibodies are used in the treatment of a wide range of cancers including lymphomas (such as non-Hodgkin's lymphoma, B cell chronic lymphocytic leukemia

(B-CLL)), myelomas (such as multiple myeloma), leukemias (such as B cell leukemia), breast cancer (including advanced metastatic breast cancer), colorectal cancer (including advanced and/or metastatic colorectal cancer), ovarian cancer, lung cancer, prostate cancer, cervical cancer, melanoma and brain tumours. Monoclonal antibodies can be used alone or in combination with other immunotherapeutic agents or chemotherapeutic agents.

Active specific immunotherapy typically involves the use of cancer vaccines. Cancer vaccines have been developed that comprise whole cancer cells, parts of cancer cells or one or more antigens derived from cancer cells. Cancer vaccines, alone or in combination with one or more immuno- or chemotherapeutic agents are being investigated in the treatment of several types of cancer including melanoma, renal cancer, ovarian cancer, breast cancer, colorectal cancer, lung cancer and leukemia. Non-specific immunotherapeutics are useful in combination with cancer vaccines in order to enhance the body's immune response.

CLINICAL TRIALS IN CANCER PATIENTS

One skilled in the art will appreciate that, following the demonstrated effectiveness of a TS siRNA molecule or combination in vitro and in animal models, the siRNA or combination should be submitted to standard GLP animal toxicology and pharmacokinetic studies and then be entered into Clinical Trials in order to further evaluate the efficacy in the treatment of cancer and to obtain regulatory approval for therapeutic use. As is known in the art, clinical trials progress through phases of testing, which are identified as Phases I, II, III, and IV.

Initially, the selected treatment regimen will be evaluated in a Phase I trial, which is usually an open-label trial. Typically Phase I trials are used to determine the best mode of administration (for example, by pill or by injection), the frequency of administration, and the toxicity for the components of the regimen. Phase I studies frequently include laboratory tests, such as blood tests and biopsies, to evaluate the effects of the components in the body of the patient. For a Phase I trial, a small group of cancer patients will be treated with a specific dose of the siRNA molecule. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the siRNA molecule. This process determines an appropriate dose to use in a subsequent Phase II trial.

A Phase II trial can be conducted to further evaluate the effectiveness and safety of the selected treatment regimen. Phase II trials are usually open-label, but may also be blinded. In Phase II trials, groups of patients with either one specific type of cancer or with related cancers are treated using the dosage(s) found to be effective in Phase I trials.

Phase III trials focus on determining how the treatment regimen compares to the standard, or most widely accepted, treatment. Phase III trials are generally blinded. In Phase III trials, patients are randomly assigned to one of two or more "arms". In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive siRNA-based treatment (investigational group).

Phase IV trials are used to further evaluate the long-term safety and effectiveness of the treatment regimen. Phase IV trials are less common than Phase I, II and III trials and will take place after the treatment regimen has been approved for standard use.

Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example, age, sex, type of cancer) to specific (for example, type and number of prior treatments, tumour characteristics, blood cell counts, organ function). Eligibility criteria may also vary with trial phase. For example, in Phase I and II trials, the criteria often exclude patients who may be at risk from the investigational treatment because of abnormal organ function or other factors. In Phase II and III trials additional criteria are often included regarding disease type and stage, and number and type of prior treatments.

Phase I cancer trials usually comprise 15 to 30 participants for whom other treatment options have not been effective. Phase II trials typically comprise up to 100 participants who have already received chemotherapy, surgery, or radiation treatment, but for whom the treatment has not been effective. Participation in Phase II trials is often restricted based on the previous treatment received. Phase III trials usually comprise hundreds to thousands of participants. This large number of participants is necessary in order to determine whether there are true differences between the effectiveness of the siRNA-based treatment and the standard treatment. Phase III may comprise patients ranging from those newly diagnosed with cancer to those with extensive disease in order to cover the disease continuum.

One skilled in the art will appreciate that clinical trials should be designed to be as inclusive as possible without making the study population too diverse to determine whether the treatment might be as effective on a more narrowly defined population. The more diverse the population included in the trial, the more applicable the results could be to the general population, particularly in Phase III trials. Selection of appropriate participants in each phase of clinical trial is considered to be within the ordinary skills of a worker in the art.

Assessment of patients prior to treatment

Prior to commencement of the study, several measures known in the art can be used to first classify the patients. Patients can first be assessed, for example, using the Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) scale. ECOG PS is a widely accepted standard for the assessment of the progression of a patient's disease as measured by functional impairment in the patient, with ECOG PS 0 indicating no functional impairment, ECOG PS 1 and 2 indicating that the patients have progressively greater functional impairment but are still ambulatory and ECOG PS 3 and 4 indicating progressive disablement and lack of mobility.

Patients' overall quality of life can be assessed, for example, using the McGiIl Quality of Life Questionnaire (MQOL) (Cohen et al (1995) Palliative Medicine 9: 207-219). The MQOL measures physical symptoms; physical, psychological and existential well-being; support; and overall quality of life. To assess symptoms such as nausea, mood, appetite, insomnia, mobility and fatigue the Symptom Distress Scale (SDS) developed by McCorkle and Young ((1978) Cancer Nursing 1: 373-378) can be used.

Patients can also be classified according to the type and/or stage of their disease and/or by tumour size.

Administration of the siRNA Molecule in Clinical Trials

The selected siRNA molecule is typically administered to the trial participants parenterally, for example, by intravenous infusion. Methods of administering drugs by intravenous infusion are known in the art. Usually intravenous infusion takes place over a certain time period, for example, over the course of 60 minutes to several days. A range of doses of the siRNA molecule can be tested.

Pharmacokinetic monitoring

To fulfil Phase I criteria, distribution of the siRNA is monitored, for example, by chemical analysis of samples, such as blood or urine, collected at regular intervals. For example, samples can be taken at regular intervals up until about 72 hours after the start of infusion. In one embodiment, samples are taken at 0, 0.33, 0.67, 1, 1.25, 1.5, 2, 4, 6, 8, 12, 24, 48 and 72 hours after the start of each infusion of the siRNA.

If analysis is not conducted immediately, the samples can be placed on dry ice after collection and subsequently transported to a freezer to be stored at -70⁰C until analysis can be conducted. Samples can be prepared for analysis using standard techniques known in the art and the amount of the siRNA molecule present can be determined, for example, by high-performance liquid chromatography (HPLC). Pharmacokinetic data can be generated and analyzed in collaboration with an expert clinical pharmacologist and used to determine, for example, clearance, half-life and maximum plasma concentration.

Monitoring of Patient Outcome The endpoint of a clinical trial is a measurable outcome that indicates the effectiveness of the treatment regimen under evaluation. The endpoint is established prior to the commencement of the trial and will vary depending on the type and phase of the clinical trial. Examples of endpoints include, for example, tumour response rate - the proportion of trial participants whose tumour was reduced in size by a specific amount, usually described as a percentage; disease-free survival - the amount of time a participant survives without cancer occurring or recurring, usually measured in months; overall survival - the amount of time a participant lives, typically measured from the beginning of the clinical trial until the time of death. For advanced and/or metastatic cancers, disease stabilization - the proportion of trial participants whose disease has stabilized, for example, whose tumour(s) has ceased to grow and/or metastasize, can be used as an endpoint. Other endpoints include toxicity and quality of life.

Tumour response rate is a typical endpoint in Phase II trials. However, even if a treatment reduces the size of a participant's tumour and lengthens the period of disease-free survival, it may not lengthen overall survival. In such a case, side effects and failure to extend overall survival might outweigh the benefit of longer disease- free survival. Alternatively, the participant's improved quality of life during the tumour-free interval might outweigh other factors. Thus, because tumour response rates are often temporary and may not translate into long-term survival benefits for the participant, response rate is a reasonable measure of a treatment's effectiveness in a Phase II trial, whereas participant survival and quality of life are typically used as endpoints in a Phase III trial.

PHARMACEUTICAL KITS The present invention additionally provides for therapeutic kits comprising a TS siRNA for use in the treatment of cancer. The kit may optionally further comprise a siRNA targeted to a cell survival gene and/or one or more other chemotherapeutic or immunotherapeutic agents for administration in conjunction with the TS siRNA.

Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. In this case the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to a patient or applied to and mixed with the other components of the kit.

The components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components. Irrespective of the number or type of containers, the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.

The kit may further comprise appropriate reagents for formulating the siRNA molecule(s) for delivery, for example, for encapsulation of the siRNA molecule, association with a biodegradable polymer or for the preparation of a liposomal solution.

To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way. EXAMPLES

EXAMPLE 1: siRNAs AGAINST THYMIDYL ATE SYNTHASE (TS) AND BCL-2

The following siRNAs were obtained from Dharmacon RNA Technologies (Lafayette, CO). All siRNAs were obtained in annealed and desalted form, then dissolved in siRNA buffer (supplied by Dharmacon) to obtain a 10 μM working solution.

a) siRNA duplex targeting TS (targeting bases 868-886 of SEQ ID NO:1): antisense (5'-GAUUCAGGUAAAUAUGUGCUU, SEQ ID NO: 10) sense (5'- GCACAUAUUUACCUGAAUCUU, SEQ ID NO: 11);

b) siRNA duplex no. 1 targeting Bcl-2 (targeting bases 927-945 of SEQ ID NO:12, and as shown in Figure 12): antisense (5'-AAGAAGGCCACAAUCCUCCUU, SEQ ID NO: 13), sense (5'-GGAGGAUUGUGGCCUUCUUU, SEQ ID NO: 14);

c) siRNA duplex no. 2 targeting Bcl-2 (targeting bases 528-546 of SEQ ID NO: 12): antisense (5'-UACUUCAUCACUAUCUCCCUU, SEQ ID NO:15), sense (5'- GGGAGAUAGUGAUGAAGUAUU, SEQ ID NO: 16); and

d) Control (nontargeting) siRNA duplex (SC): designed by Dharmacon to have >4 mismatches with all known human mRNAs.

EXAMPLE 2: EFFECT OF THYMIDYLATE SYNTHASE AND BCL-2 siRNAS ON THYMIDYLATE SYNTHASE AND BCL-2 mRNA AND PROTEIN LEVELS IN HELA AND MCF-7 CELLS

The ability of siRNAs against thymidylate synthase or Bcl-2 to decrease their respective mRNA and protein levels in HeLa cells and MCF-7 cells was determined as follows.

Cell lines and cultures Human cervical carcinoma (HeLa) and human breast tumor MCF-7 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)(Invitrogen, Burlington, ON) with 10% fetal bovine serum (InVitrogen, Burlington, ON) in a humidified incubator at 37° C with 5% CO₂ in air. Cells were transfected using Oligofectamine™ Reagent (Invitrogen, Burlington, ON) according to the manufacturer's instructions. Briefly, siRNA: Oligofectamine™ Reagent complexes were formed in DMEM without FBS and then serially diluted to generate desired transfection concentrations. siRNA complexes in DMEM were added to HeLa and MCF-7 cells grown to 50-60% confluence. Transfected siRNA concentrations were either 50 nM (in experiments to measure the effects of siRNA on specific TS and BcI- 2 mRNA and protein) or 100 nM (for experiments to measure siRNA effects on cell cycle, proliferation, and drug sensitivity). Regardless of the concentration of antisense Bcl-2 and/or TS siRNA applied to cells, the total concentration of siRNA applied in every case was kept constant at 50 or 100 nM by addition of SC siRNA duplex. For treatments with combined TS and Bcl-2 siRNA, the ratio of TS siRNA to Bcl-2 siRNA was kept at 1 :1. When TS siRNA or Bcl-2 siRNA was used alone, SC siRNA was added (where necessary) so that the total siRNA concentration added to every aliquot of cultured cells was kept constant.

RNA Isolation, Reverse Transcription, and Quantitative Real- Time PCR

Cells were plated and then transfected with siRNA(s) 24 hours later. The concentrations of siRNA and incubation times are described in the results following.

Cells were lysed in TRI^® Reagent (Sigma, St. Louis, MO) and RNA was extracted from cells and quantified by UV spectrophotometry. Complementary DNA (cDNA) was prepared from 1 μg of total cellular RNA using Moloney murine leukemia virus

(MMLV) reverse transcriptase (Invitrogen, Canada) and random primers according to the manufacturer's recommendations. Relative gene expression ([TS or Bcl-2 mRNA]:[18S rRNA]) was determined by real-time RT-PCR amplification of TS, BcI-

2, and 18S rRNA cDNA. TS primers were: forward (5'-GGCCTCGGTGTGCCTTT-

3', SEQ ID NO: 17), reverse (5'-GATGTGCGCAATCATGTACGT-S', SEQ ID

NO: 18), and the TS TaqMan probe was (5'-6FAM- AACATCGCCAGCTACGCCCTGC-MGBNFQ-3', SEQ ID NO: 19). Bcl-2 primers were: forward (5'-TTGGCCCCCGTTGCTT-S', SEQ ID NO:20), reverse (5'- CGGTTGTCGTACCCCGTTCTC-3', SEQ ID NO:21), and the Bcl-2 TaqMan probe was (5'-6FAM-AGCGTGCGCCATCCTTCCCAG-MGBNFQ-S', SEQ ID NO:22). Standard curves for Bcl-2, TS mRNA, and 18S rRNA, were generated using serial dilutions of cDNA derived from the HeLa and MCF-7 cell lines. Bcl-2 and TS mRNA levels (relative to 18S rRNA levels) were normalized to the 18S rRNA control and then the untreated control.

Immunoblot

Cells were plated, transfected 24 hours later, and lysed to obtain total soluble cellular protein. Total protein (10 -30 μg) was separated using 12% SDS PAGE, followed by transfer onto nitrocellulose membranes (Amersham Biosciences, Baie-d'Urfe, Que). Membranes were blocked (16 h, 4°C) with 5% skim milk powder in Tris-buffered saline with 0.2% Tween-20 (TBS-T) and then probed at 25° C using a rabbit anti- human TS polyclonal antibody (Flynn et al., 2006, MoI Cancer Ther 5:1423-1433) (0.0625 μg/ml) or a monoclonal mouse anti-human Bcl-2 oncoprotein antibody (clone 24, 1 :800, DakoCytomation, Glostrup, Denmark), and an anti-actin antibody ( 1 :1000, Sigma, St. Louis, MO). Following incubation with primary antibodies, membranes were washed with TBS-T and incubated for 1 h with horseradish peroxidase (HRP)- conjugated anti-rabbit or anti-mouse antibodies (1 :2000, 1 :3000 respectively )( Amersham). HRP activity was detected using ECL Plus (Amersham) and X-ray film (Eastman Kodak). Band intensities were quantified by densitometry using a FluorChem 8800 digital image system and AlphaEase FC software (Alpha Innotech, San Leandro, CA). TS and Bcl-2 densitometric band intensities were divided by the intensity of actin bands in the same lanes to yield relative TS and Bcl-2 levels in cell lysates. Relative TS and Bcl-2 protein levels were normalized to relative TS and Bcl-2 protein levels in control cells. Protein from control cells (untreated with siRNAs) were routinely analyzed along with protein from siRNA treated cells. Comparison of control and siRNA-treated cell protein revealed that actin levels were unchanged by siRNA treatment in all cases. Actin was therefore adapted as a suitable loading control for analysis for siRNA-induced changes to TS and/or Bcl-2.

Statistical analysis Data are presented as means ± SE. To determine the significance of differences between means, a Student's t-test was performed. The level of significance for all statistical analyses was chosen a priori to be P<0.05.

Results: To reduce the potential for concentration-dependent, non-specific toxicities to confound observations, both cell lines were treated with siRNA concentrations that yielded significant biological effects at the lowest practical concentrations.

Figure 1 indicates the downregulation of TS and Bcl-2 mRNA by siRNA in HeIa and MCF-7 cells. Figure l(A) shows the results for HeLa or MCF-7 cells treated with TS siRNA (25 nM) or a scrambled control (SC) siRNA, while Figure l(B) shows the result of treating HeLa or MCF-7 cells with Bcl-2 siRNA no. 1 or Bcl-2 siRNA no. 2 (25 nM) or a scrambled control (SC) siRNA. Relative TS and Bcl-2 mRNA levels were assessed 24 h thereafter, as described above. Bars indicate mean ± SE (n=3 independent treatments). [* indicates different from cells treated with SC siRNA (p < 0.05, Student's t-test).]

TS siRNA (50 nM overall duplex RNA concentration, 25 nM TS siRNA + 25 nM SC siRNA) decreased TS mRNA levels by 70-80% in HeLa and MCF-7 cells, compared to the effect of SC siRNA (50 nM), 24 hours following transfection (Fig. IA). Both Bcl-2 siRNA no. 1 and Bcl-2 siRNA no. 2 (50 nM overall duplex RNA concentration, 25 nM of either Bcl-2 siRNA + 25 nM SC siRNA) decreased Bcl-2 mRNA levels by 60-70% in HeLa and MCF-7 cells compared to the effect of SC siRNA alone, 24 hours post transfection (Fig. IB). Bars indicate mean ± SE (n=3 independent treatments). *Different from cells treated with SC siRNA (p < 0.05, Student's t-test).

Figure 2(A) shows the results of treating HeLa cells with TS siRNA, Bcl-2 siRNA no.l or no. 2, a combination of TS siRNA and Bcl-2 siRNA no. 1, SC siRNA, or left untreated. Relative levels of TS mRNA and Bcl-2 mRNA were measured 24 h thereafter, as described above. Bars indicate mean ± SE (n > 4 independent treatments) Figure 2(B) shows a representative immunoblot of TS, Bcl-2 and actin proteins from cells treated as described in (A). Figure 2(C) shows the relative levels of TS and Bcl-2 proteins in HeLa cells treated with TS siRNA, Bcl-2 siRNA no. 1, a combination of TS siRNA and Bcl-2 siRNA no. 1, SC siRNA, or left untreated. Relative levels of TS protein and Bcl-2 protein were measured 24 h thereafter, as described above. Bars indicate mean ± SE (n > 3 independent treatments). *Different from cells treated with TS siRNA (p < 0.05, Student's t-test). **Different from cells treated with SC siRNA (p < 0.05, Student's t-test). Figure 3(A) shows the relative levels of TS mRNA and protein in MCF-7 cells were treated with TS siRNA, Bcl-2 siRNA no. 1, a combination of TS siRNA and Bcl-2 siRNA no. 1, SC siRNA, or left untreated. Relative levels of TS mRNA and Bcl-2 mRNA were measured 24 h thereafter, as described above. Bars indicate mean ± SE (n > 4 independent treatments). Figure 3(B) shows a representative immunoblot of TS, Bcl-2 and actin proteins in cells treated as described in A. Figure 3(C) shows the levels of TS and BcI- 2 proteins in MCF-7 cells treated with TS siRNA, Bcl-2 siRNA no. 1 , a combination of TS siRNA and Bcl-2 siRNA no. 1, SC siRNA, or left untreated. Relative levels of TS and Bcl-2 proteins were measured 24 h thereafter, as described above. Bars indicate mean ± SE (n > 3 independent treatments).* Different from cells treated with TS siRNA (p < 0.05, Student's t-test).

Changes in Bcl-2 protein levels 24 h after treatment of HeLa and MCF-7 cells with either of the two Bcl-2 siRNAs corresponded well, both quantitatively and qualitatively, with siRNA-induced reduction in Bcl-2 mRNA levels (Figures 2 and 3). Similarly, TS siRNA treatment of HeLa and MCF-7 cells reduced TS protein, in accord with siRNA-mediated reduction in TS mRNA (Fig. 2).

Figure 4(A) shows the time course of TS protein level changes in HeLa cells following treatment with TS and/or Bcl-2 siRNA. HeLa cells were treated with TS and Bcl-2 siRNAs as described for Figure 2, and TS protein levels measured by immunoblot as described above. Bars indicate mean ± SE (n > 3 independent treatments). Figure 4(B) depicts a time course of Bcl-2 protein level changes in HeLa cells treated as described in A. *Different from cells treated with SC siRNA (p < 0.05, Student's t-test). f Different from cells treated with TS siRNA (p < 0.05, Student's t- test). Reduction in TS protein levels mediated by TS siRNA in HeLa cells reached approximately 95% by 48 h and 80% at 72 h (Fig. 4A) post-TS siRNA treatment. BcI- 2 siRNA-mediated reduction in Bcl-2 protein was similarly dowregulated by approximately 80% at 48-72 h.

In addition to normalizing relative TS and Bcl-2 mRNA and protein values to untreated controls, the effects of SC treatment alone were assessed. There were no significant differences in either Bcl-2 and TS mRNA or protein in SC-treated HeLa and MCF-7 cells compared to untreated control cells.

EXAMPLE 3: EFFECT OF BCL-2 siRNA ON THYMIDYLATE SYNTHASE (TS) siRNA-MEDIATED REDUCTION OF THYMIDYLATE SYNTHASE (TS) mRNA AND PROTEIN LEVELS IN HELA CELLS AND MCF-7 CELLS

The ability of Bcl-2 siRNA to antagonize the TS siRNA-mediated reduction of TS mRNA and protein was also measured. HeLa cells and MCF-7 cells were cultured and treated as described in Example 2, with the specific treatments and concentrations of siRNAs as indicated in Figures 2 to 5. TS mRNA and protein levels were also measured as described in Example 2. Results:

TS siRNA, administered concurrently with Bcl-2 siRNA no. 1 to HeLa cells (Fig. 2) or MCF-7 cells (Fig. 3), reduced TS mRNA and TS protein less effectively after 24 h of treatment than when administered in combination with equal amounts of SC siRNA.

TS siRNA alone reduced HeLa TS mRNA by 80% and TS protein by 50% at 24 h after administration. However, when delivered in combination with Bcl-2 siRNA no. 1, TS siRNA reduced TS mRNA by only 50% (leaving three times the TS mRNA in cells than after TS siRNA treatment alone) and protein by 30% (leaving about 30% more TS protein in cells than after TS siRNA treatment alone). Thus, co- administration of siRNA targeting a non-TS mRNA (Bcl-2) reduced the effectiveness of TS siRNA after 1 day of treatment. A similar effect was observed in MCF-7 cells, where co-administration of Bcl-2 siRNA no. 1 impaired TS siRNA-mediated reduction of both TS mRNA and protein by leaving 50% more TS mRNA and protein in cells than after TS siRNA treatment alone (Fig. 3). Co-administration of Bcl-2 siRNA no. 2 with TS siRNA did not reduce the effectiveness of TS siRNA to decrease mRNA or protein in either cell line (Fig. 2 and 3).

No evidence of a converse inhibition of the antisense effect of TS siRNA on Bcl-2 siRNA no. 1 was evident in either HeLa cells (Fig. 2) or MCF-7 cells (Fig. 3). In both cell lines, Bcl-2 siRNA no. 1 (25 nM) reduced Bcl-2 mRNA by approximately 60% and Bcl-2 protein by 50-60%, regardless of the presence or absence of TS siRNA.

Interestingly, treatment with Bcl-2 siRNA no. 1 alone for 24 h (in the absence of TS siRNA) led to a small but significant increase (approximately 15%) in both TS mRNA and TS protein in HeLa (Fig. 2) but not MCF-7 cells (Fig. 3), compared to cells treated with SC siRNA.

It was possible that the observed antagonism was due to delay or acceleration of TS siRNA activity such that changes apparent at 24 h of treatment would not be evident at other times. To test this, relative TS and Bcl-2 protein levels were measured in HeLa cells treated with TS siRNA or Bcl-2 siRNA no. 1, alone or in combination, at 12, 24, 48, or 72 h post-treatment. The capacity of Bcl-2 siRNA no. 1 to antagonize TS siRNA-mediated reduction of TS protein persisted to at least 48 and 72 h post- transfection (Fig. 4A). No converse effects of TS siRNA on Bcl-2 protein levels were evident at any time from 24 to 72 h (Fig. 4B).

Figure 5(A) shows the TS mRNA levels in HeLa cells treated with TS siRNA and Bcl-2 siRNA no. 1 as indicated. Relative levels of TS mRNA were measured 24 h later, as described in Example 2. Bars indicate mean ± SE (n = 3 independent treatments). * Different from cells treated with TS siRNA (p < 0.05, Student's t-test). f Different from cells treated with a combination of TS siRNA and Bcl-2 siRNA no. 1 (5 nM) (p < 0.05, Student's t-test). Relative TS mRNA levels in all siRNA-treated cells were lower than in cells treated with SC (p < 0.05, Student's t-test). The antagonism of TS siRNA effectiveness by Bcl-2 siRNA was not restricted to 1:1 ratios of administered siRNAs (Fig. 5A). Other TS siRNA:Bcl-2 siRNA no. 1 ratios (2:1, 5:1 and 1 :2.5) also antagonized TS siRNA effectiveness in HeLa cells in a dose- dependent fashion. The antagonism depended on Bcl-2 siRNA no. 1 dose (Fig 5A). Figure 5(B) shows the TS mRNA levels in HeLa cells first (1) treated with one siRNA (either TS siRNA or Bcl-2 siRNA no. 1) followed by (2) transfection with the other siRNA 24 h later. A control treatment group was transfected twice with 25 nM SC siRNA (1,2). Relative TS mRNA levels were measured 24 h following the last transfection, as described in Example 2. Bars indicate mean ± SE (n = 4 independent treatments). *Different from cells treated with TS siRNA followed by SC siRNA (p < 0.05, Student's t-test). ** Different from cells treated with SC siRNA followed by TS siRNA (p < 0.05, Student's t-test). As observed after concurrent treatment with Bcl-2 siRNA no.l and TS siRNA, sequential treatment of HeLa cells, first with Bcl-2 siRNA no. 1 and then with TS siRNA 24 h later, antagonized TS siRNA effectiveness (Fig. 5B). Bcl-2 siRNA no. 1 treatment in the reverse order (TS siRNA followed by Bcl-2 siRNA no. 1 24 h later) resulted in much less antagonism of TS downregulation, although a small but significant antagosnism was evident.

To assess this possibility that targeting multiple mRNAs mediating more than one mechanism of resistance chemotherapeutic drugs could enhance cancer therapy, the consequences of combined treatment of human tumor cell lines with siRNAs targeting

TS and Bcl-2 was studied. TS and Bcl-2 mRNA and proteins were downregulated by treatment with either TS siRNA or Bcl-2 siRNAs, in both HeLa and MCF-7 cells, with no detectable non-specific toxicity (Figs. 1-4). The degree of siRNA-mediated reduction was consistent with other reports where antisense reagents of several types were employed to target Bcl-2 (Chawla-Sarkar et al., 2004, Cell Death Differ 11 :915-

923; Lima et al., 2004, Cancer Gene Ther 11 :309-316; Basma et al., 2005, J Biomed

Sci 12:999-1011; Ocker et al., 2005, Gut 54:1298-1308) or TS (Ferguson et al., 1999,

Br J Pharmacol 127:1777-1786; Schmitz et al., 2004, ibid.) protein in several human tumor cell lines.

Although both Bcl-2 siRNAs were equally effective at reducing Bcl-2 mRNA and protein, only Bcl-2 siRNA no. 1 antagonized the capacity of TS siRNA to downregulate TS mRNA and protein in HeLa (Figs. 2, 4, 5) and MCF-7 cells (Fig. 3). Direct interaction between the two siRNAs, if it occurred, might mediate such antagonism: such interaction, however, was unlikely. First, no complementary sequences capable of mediating direct hybridization between the TS and Bcl-2 siRNAs exist. Second, TS siRNA did not impede the ability of Bcl-2 siRNA no. 1 to downregulate Bcl-2 mRNA and protein in HeLa or MCF-7 cells (a converse antagonism expected if direct interaction occurred)(Figs. 2, 3, 4). Therefore, it appears that antagonism was a downstream consequence of events mediated by downregulation of Bcl-2 mRNA and/or protein.

Bcl-2 siRNA no. 1, when administered alone, increased TS mRNA and protein in HeLa cells (Fig. 2, 4A). Such an increase might conceivably act counter to the downregulatory activity of TS siRNA and thus contribute to antagonism of antisense effect. This, however, may not be a major contributor since it was evident only in HeLa (Figs. 2,4) and not MCF-7 cells (Fig. 3). Antagonism of TS siRNA by Bcl-2 siRNA no. 1, and increased TS expression induced by Bcl-2 siRNA no. 1 as a single agent, were sustained phenomena evident at all times from 24-72 h post-treatment (Fig. 4A) suggesting that they are unlikely to be easily overcome simply by prolonged treatment with TS siRNA (although a lower the dose of Bcl-2 siRNA no. 1 did diminish antagonism [Fig. 5A]). Accordingly, a combination treatment with both the TS siRNA and the Bcl-2 siRNA would need to employ separate administration, with a break of greater than 72 hours between administration of each individual agent.

Sequential treatment with Bcl-2 siRNA no. 1 followed by TS siRNA also resulted in antagonism, suggesting that a direct interaction of the siRNAs was not responsible for the effect.

EXAMPLE 4: EFFECT OF BCL-2 siRNA ON TUMOR CELL ACCUMULATION IN Gl PHASE OF CELL CYCLE

Cells were plated in T25 flasks and transfected with siRNAs 24 hours later as described in Example 2. Five hours later, they were trypsinized and re-plated in T75 flasks at lower densitities (30% confluency). At the appropriate time-point, cells were trypsinized, diluted in PBS and collected by centrifugation (5 min, 1000Xg, RT). The cell pellet was washed in PBS, cells collected by centrifugation (5 min, 1000Xg, RT), suspended in 300 μL of PBS, and fixed in 95 % ethanol for a minimum of 24 h at 4° C. The cells were precipitated by centrifugation (5 min, 1600Xg, RT) and then treated with ribonuclease and stained with PI to reveal cellular DNA content (0.01 mg/ml PI, 0.25 mg/ml RNase A, 0.25 niM Tris.HCl, 0.38 mM NaCl, pH 7.5) for 30 min at 37° C. Cells were filtered and flow cytometry performed using a Beckman Coulter Epics XL-MCL Flow Cytometer. The distribution of cells in G0/G1, S and G2/M cell cycle phases was calculated using Multi Cycle Software (Version 3.0, Phoenix Flow Systems, San Diego, CA).

Figure 6 shows the flow cytometric analysis of cell cycle distribution in HeLa (A,B) or MCF-7 (C,D) cells treated with Bcl-2 siRNA no. 1, Bcl-2 siRNA no. 2, SC siRNA or left untreated, and collected 24 (A,C) or 48 (B,D) h later. Bars indicate mean ± SE (2 independent experiments, n=3 for each experiment). *Different from cells treated with SC siRNA (p < 0.05, Student's t-test). Treatment with antagonistic Bcl-2 siRNA no. 1 increased the fraction of cells in Gl at 24 and 48 h post -treatment in HeLa cells: a 10% increase [Fig. 6A] and a 17% increase [Fig. 6B], respectively. Qualitatively similar increases were evident in MCF-7 cells: a 7% increase [Fig. 6C] and an 8% increase [Fig. 6D], respectively.

Treatment with non-antagonistic Bcl-2 siRNA no. 2 had a similar effect in both cell lines at 48 h (Fig. 6B,D), although the trend toward increased cells in Gl in MCF-7 cells at 24 h was not significant. As expected for Gl accumulation, there was a concomitant decrease in the number of cells in S phase after treatment with either BcI- 2 siRNA no. 1 or no. 2 at 48 h (Fig. 6B,D), and at 24 h in all cases except in HeLa cells treated with Bcl-2 siRNA no. 2 (where the trend to decrease did not reach significance). As expected, both Bcl-2 siRNAs reduced the number of cells in G2 at 24 h and 48 h (Fig. 6A,B), although only Bcl-2 siRNA no. 2 induced such a decrease in MCF-7 cells, and only at 48 h (Fig. 6D).

Both Bcl-2 siRNAs induced cell cycle changes in HeLa and MCF-7 cells by increasing the fraction of cells in Gl, (Fig. 6). TS is regulated post-transcriptionally, and in a cell cycle-dependent fashion, such that TS protein levels increase in S phase

(Chu and Allegra, 1996). The observed decrease in numbers of cells in S, with increased numbers in Gl in response to Bcl-2 siRNAs is not consistent with an explanation of antagonism based on differential accumulation of cells in specific cell cycle compartments. Therefore, cell cycle effects are unlikely to be responsible for either antagonism of TS siRNA activity or upregulation of TS levels by Bcl-2 siRNA no. 1.

EXAMPLE 5: EFFECT OF THYMIDYLATE SYNTHASE siRNA ON CELL PROLIFERATION IN HELA AND MCF-7 CELLS

To assess the capacity of siRNA treatment to alter the ability of cells to proliferate in the presence or absence of drugs, cells were plated in T25 flasks and transfected 24 hours later as described in Example 2. For all determinations of drug sensitivity (other than the sensitivity of HeLa cells to 5-fluorouracil deoxyribonucleotide [5-FUdR] or raltitrexed 2-[5-[methyl-[(2-methyl-4-oxo- 1 H-quinazolin-6-yl)methyl] amino] thiophen-2-yl] carbonylaminopentanedioic acid [raltitrexed]), cells were replated in 96 well plates (VWR, Mississauga) at 1700 (MCF-7) and 1200 (HeLa) cells per well in a volume of 100 μl DMEM + 10% FBS. Cells were allowed to adhere to tissue culture plastic for 18 h prior to addition of drug. Drug was added as a 100 μL solution in DMEM + 10% FBS. Cells were then grown for 4 days and cell numbers were assessed by the Alamar Blue Fluorescence assay using a Wallac Victor² multilabel counter.

Because of relatively high background levels of Alamar Blue signals in HeLa cells exposed to 5-FUDR and raltitrexed, the number of those cells after growth in the presence and absence of 5-FUdR or raltitrexed was determined by direct cell counting. The number of HeLa cells after growth in the presence and absence of 5-FUdR or raltitrexed, was determined by replating cells in T25's following (9 x 10⁴ cells/flasks) transfection and direct cell counting after a 4 day drug exposure using an electronic particle counter (Beckman Coulter, Hialeah, FL).

Treatment of HeLa and MCF-7 with TS siRNA induced a small reduction in proliferation (less than 5%) as shown in Table 6.

Table 6: Proliferation of HeLa and MCF-7 cells is inhibited by Bcl-2 and TS siRNA treatments

Proliferation a

Treatment⁵ HeLa cells MCF-7 cells A) SC siRNA 100 ± 1.1 100 : t l.O

B) TS siRNA 95 ± 1.3* 94 ± 2.2*

C) Bcl-2 siRNA no. 1 77 ± 2.8* 76 ± 1.6*

D) Bcl-2 siRNA no. 2 85 ± 2.5* 91 ± 2.2*

E) TS siRNA +Bcl-2 siRNA no. 1 68 ± 0.8* 66 ± 1.2

F) TS siRNA + Bcl-2 siRNA no. 2 71 ± 1.8* 78 ± 1.7

G) SC siRNA 98.5 ± 6.0 95.3 ± 1.2*

H) No siRNA (control) 100 ± 2.5 100 - H .6

^aFor treatments A-F, "proliferation" was the number of cells 5 days after treatment with siRNAs targeting TS and/or Bcl-2 (% of the number of cells treated with SC siRNA). For treatment G, "proliferation" was the number of cells 5 days after treatment with SC siRNA is shown (% of the number of control cells, untreated with siRNA). Numbers are means ± SE (2 independent experiments, n=3 for each experiment).^bTotal siRNA concentration was 100 nM for all treatments (A-G). *Different from untreated cells (p<0.05, Student's t-test)

On the other hand, treatment with Bcl-2 siRNA no. 1 or Bcl-2 siRNA no. 2 reduced proliferation of HeLa cells by 23% and 15% respectively, and MCF-7 cells by 24% and 9%. When used in combination with TS siRNA, the inhibition of proliferation was at least additive, and significantly greater than after treatment with Bcl-2 siRNA alone (Table 1). In HeLa cells and MCF-7 cells, treatment with TS siRNA plus Bcl-2 siRNA no. 1 reduced cell proliferation by 32% and 34%, respectively. Treatment with TS siRNA plus Bcl-2 siRNA no. 2 reduced proliferation of HeLa and MCF-7 cells by 29% and 22%, respectively.

Targeting different regions of a particular message using anti sense reagents can have different physiological consequences, regardless of message or protein reduction (Berg et al., 2003, Cancer Gene Ther 10:278-286). Targeting the 3'-UTR of TS mRNA in HeLa cells with antisense has been shown to reduce proliferation and induce G₂M arrest (Berg et al., 2001, J Pharmacol Exp Ther 298:477-484). In the current investigation, targeting the coding region of the TS mRNA had little or no effect on proliferation or cell cycle in HeLa and MCF-7 cells despite both agents being comparable in their ability to decrease mRNA and protein levels. In view of this, differential antagonistic effects of the Bcl-2 siRNAs raises the possibility that Bcl-2 mRNA and possibly protein mediate non-canonical functions additional to its well-characterized regulatory roles in pro- and antiapoptotic signaling pathways (Shore and Viallet, 2005, Hematology (Am Soc Hematol Educ Program):226-230).

Accumulation in Gl can contribute to inhibition of proliferation. Both Bcl-2 siRNA no. 1 and no. 2 reduced the capacity of HeLa and MCF-7 cells to increase in number (Table 6). Combined treatment with siRNAs targeting both TS and Bcl-2 mRNA had at least additive effects on inhibition of human tumor cell proliferation, with no evidence of antagonism. Given the above, potential changes in sensitivity to TS- targeting drugs were investigated as another physiological consequence of the antagonism, as described in Example 6.

EXAMPLE 6: EFFECT OF THYMIDYLATE SYNTHASE AND BCL-2 siRNAs ON CYTOTOXICITY OF RALTITREXED, 5-FU dR AND DOCETAXEL

To assess the capacity of siRNA treatment to alter the ability of cells to proliferate in the presence or absence of drugs, cells were plated in T25 flasks and transfected 24 hours later as described in Example 2. For all determinations of drug sensitivity (other than the sensitivity of HeLa cells to 5-fluorouracil deoxyribonucleotide [5-FUdR] or raltitrexed, cells were replated in 96 well plates (VWR, Mississauga) at 1700 (MCF-7) and 1200 (HeLa) cells per well in a volume of 100 μl DMEM + 10% FBS. Cells were allowed to adhere to tissue culture plastic for 18 h prior to addition of drug. Drug was added as a 100 μL solution in DMEM + 10% FBS. Cells were then grown for 4 days and cell numbers were assessed by the Alamar Blue Fluorescence assay using a Wallac Victor multilabel counter.

Because of relatively high background levels of Alamar Blue signals in HeLa cells exposed to 5 -FUDR and raltitrexed, the number of those cells after growth in the presence and absence of 5-FUdR or raltitrexed was determined by direct cell counting. The number of HeLa cells after growth in the presence and absence of 5-FUdR or raltitrexed, was determined by replating cells in T25's following (9 x 10⁴ cells/flasks) transfection and direct cell counting after a 4 day drug exposure using an electronic particle counter (Beckman Coulter, Hialeah, FL).

TS siRNA sensitized HeLa and MCF-7 cells to the TS-targeting drugs raltitrexed and 5-FUdR (Fig. 7). The shown proliferation rates, following a continuous 4-day drug exposure 24 hours post-transfection reflect the effect of the drug alone: for each siRNA treatment, proliferation was assumed to be 100% in the absence of chemotherapeutic drug and data for each drug treatment are relative to that 100% value. Figure 7 shows the proliferation rates for HeLa (A,C,E) or MCF-7 (B,D,F) cells pre-treated with TS siRNA, SC siRNA or combinations of the above siRNA followed by the indicated doses of raltitrexed (A₅B), 5-FUDR (C,D) or Docetaxel (E,F). Cell numbers were determined after 4 d of exposure to drug, as described above. Bars indicate mean ± SE (n > 3 independent treatments). * indicates different from cells treated with SC siRNA (p < 0.05, Student's t-test).

TS siRNA sensitization to raltitrexed and 5-FUdR was almost completely abrogated by concurrent treatment with Bcl-2 siRNA no. 1 in both HeLa and MCF-7 cells (Fig.

7A,B,C,D). In HeLa cells, at low raltitrexed concentrations (1-4 nM) and at all tested

5-FUdR concentrations, some abrogation of TS siRNA sensitization was also observed following concurrent treatment with Bcl-2 siRNA no. 2, although not to the same degree as concurrent treatment with Bcl-2 siRNA no. 1. Bcl-2 siRNA no. 1 and no. 2, regardless of whether or not they were co-administered with TS siRNA, sensitized MCF-7 (but not HeLa) cells to docetaxel (taxotere)(Figures 7F and 7E).

Figure 8 shows the proliferation rates of HeLa (A,C) or MCF-7 (B,D) cells pre-treated with Bcl-2 siRNA no. 1, Bcl-2 siRNA no. 2 or SC siRNA followed by the indicated doses of raltitrexed (A,B) or 5-FUDR (C,D). MCF-7 cells were pre-treated with Bcl-2 siRNA no. 1, Bcl-2 siRNA no. 2 and SC siRNA followed by the indicated doses of docetaxel (E). Cell numbers were determined after 4 d of continuous exposure to drug and are presented as described for Figure 7 and in Example 6. Bars indicate mean ± SE (n > 3 independent treatments). (* indicates different from cells treated with SC siRNA (p < 0.05, Student's t-test)). Treatment of HeLa cells with Bcl-2 siRNA no. 1 alone reduced sensitivity to both 5-FUdR and raltitrexed, compared to SC siRNA (Fig. 8A,C). Bcl-2 siRNA no. 2 also reduced sensitivity to raltitrexed (Fig. 8A). In MCF-7 cells, there was no clear change in sensitivity to raltitrexed induced by either of the two Bcl-2 siRNAs, compared to the SC siRNA (Fig. 8B). However, each of the Bcl-2 siRNAs reduced sensitivity to 5-FUdR (compared to SC siRNA), to a small degree.

As previously reported using different antisense oligonucleotides or siRNAs (Schmitz et al., 2004, ibid.) (Ferguson et al., 2001, ibid), treatment with TS siRNA sensitized HeLa and MCF-7 cells to 5-FUdR and raltitrexed (Fig. 7). The nearly complete abrogation of this sensitization upon concurrent treatment with the antagonistic Bcl-2 siRNA no.l, but not Bcl-2 siRNA no. 2, demonstrates that antagonism, regardless of its cause, has consequences for a physiological event of potential therapeutic benefit.

Treatment with a combination of TS siRNA and Bcl-2 siRNA no. 2 was less effective in sensitizing HeLa cells to 5-FUdR and raltitrexed than TS siRNA alone. Furthermore, Bcl-2 siRNA no. 1 treatment, as a single agent, decreased HeLa cell sensitivity to raltitrexed and 5-FUdR (Fig. 8A and C); Bcl-2 siRNA no. 2 induced a minor decrease in sensitivity, and only to raltitrexed. Treatment of MCF-7 cells with either Bcl-2 siRNA did not increase TS protein levels and, as a result, had a lesser effect on sensitivity to 5-FUdR and no clear effect on raltitrexed sensitivity (Fig. 8B and D). Since both 5-FUdR and raltitrexed cause cell cycle arrest at Gl, it is possible that agents that induce pre-arrest in Gl (in this case, both Bcl-2 siRNAs) will reduce subsequent sensitization (Johnson et al., 1999, Clin Cancer Res 5:2559-2565).

The mechanism underlying abrogation of sensitization following combination treatment, or treatment with Bcl-2 siRNAs alone, does not appear to be induction of general cellular resistance to cytotoxic agents since addition of Bcl-2 siRNA in combination with TS siRNA enhanced, rather than antagonized, the capacity of the tubule depolymerization-inhibiting drug docetaxel to inhibit MCF-7 proliferation (Fig. 7F). Furthermore, the combination of TS and Bcl-2 no. 2 siRNA sensitized rather than antagonized MCF-7 cells to 5-FUdR and raltitrexed, compared to pre-treatment with TS siRNA alone. It is therefore likely that the mechanism of antagonism of TS sensitization to TS -targeting drugs involves both cell cycle effects and the antagonism/upregulation of TS protein after concurrent or single agent use of Bcl-2 siRNAs.

Previous reports have shown that over-expression of Bcl-2 plays an important role in the survival of cells undergoing thymineless stress induced by TS-targeting drugs such as 5-FudR (Fisher et al., 1993, Cancer Res 53:3321-3326; Houghton et al., 1997, Proc

Natl Acad Sci U S A 94:8144-8149). Therefore, it might be predicted that treatment with Bcl-2 siRNAs would lead to increased sensitivity to TS-targeting drugs.

However, Bcl-2 siRNA no. 1 exerted unexpected, non-reciprocal antagonism of downregulation of TS niRNA and protein by TS siRNA, in two human tumor cell lines, concomitantly reducing potentially therapeutic sensitization to TS-targeting drugs.

The embodiments of the invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Use of a first siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a thymidylate synthase gene in combination with a second siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a cell survival gene for the treatment of cancer in a subject in need thereof.

2. The use according to claim 1, wherein said cell survival gene is Bcl-2.

3. The use according to claim 1 or 2, wherein said first siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29.

4. The use according to claim 2, wherein said second siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 12, 23, 24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

5. The use according to any one of claims 1, 2, 3 or 4, wherein said first siRNA molecule and said second siRNA molecule comprise one or more modified ribonucleotides.

6. The use according to any one of claims 1, 2, 3, 4 or 5, further comprising the use of a non-thymidylate synthase targeting chemotherapeutic.

7. A method of treating cancer comprising administering to a subject having a cancer a first siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a thymidylate synthase gene in combination with a second siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a cell survival gene.

8. The method according to claim 7, wherein said cell survival gene is Bcl-2.

9. The method according to claim 7 or 8, wherein said first siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29.

10. The method according to claim 8, wherein said second siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 12, 23, 24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

11. The method according to any one of claims 6, 7, 8, 9 or 10, wherein said first siRNA molecule and said second siRNA molecule comprise one or more modified ribonucleotides.

12. The method according to any one of claims 6, 7, 8, 9, 10 or 11, further comprising administering to said subject a non-thymidylate synthase targeting chemotherapeutic .

13. An isolated siRNA molecule of between about 14 and about 200 nucleotides in length comprising a nucleotide sequence complementary to a region of a human thymidylate synthase mRNA, wherein said nucleotide sequence comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26 or 27, and wherein said isolated siRNA molecule inhibits expression of thymidylate synthase.

14. The isolated siRNA molecule according to claim 13, wherein said isolated siRNA molecule is between about 14 and about 50 nucleotides in length.

15. The isolated siRNA molecule according to claim 13 or 14, wherein said isolated siRNA molecule is a double-stranded RNA molecule.

16. The isolated siRNA molecule according to any one of claims 13, 14 or 15, wherein said isolated siRNA molecule comprises one or more modified ribonucleotides.

17. A DNA sequence encoding the siRNA molecule according to any one of claims 13, 14 or 15 operatively linked to one or more regulatory control regions.

18. A vector comprising the DNA sequence according to claim 17.

19. A pharmaceutical composition comprising the siRNA molecule according to any one of claims 13, 14, 15 or 16, and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the DNA sequence according to claim 17 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition comprising the vector according to claim 18 and a pharmaceutically acceptable carrier.

22. Use of the isolated siRNA molecule according to any one of claims 13, 14, 15 or 16 for sensitizing cancer cells to a chemotherapeutic agent that targets thymidylate synthase.

23. The use according to claim 22, wherein said chemotherapeutic agent is 5-FU, 5-FUdR, capecitabine, raltitrexed, methotrexate or pemetrexed.

24. A method of sensitizing cancer cells in a subject to the effect of a chemotherapeutic agent that target thymidylate synthase, said method comprising administering to said subject the isolated siRNA molecule according to any one of claims 13, 14 or 15.

25. The method according to claim 24, wherein said chemotherapeutic agent is 5- FU, 5-FUdR, capecitabine, raltitrexed, methotrexate or pemetrexed.

26. A combination product for the treatment of cancer comprising a first siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a thymidylate synthase gene in combination with a second siRNA molecule of between about 14 and about 200 nucleotides in length targeted to a cell survival gene.

27. The combination product according to claim 26, wherein said cell survival gene is Bcl-2.

28. The combination product according to claim 26 or 27, wherein said first siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 25, 26, 27, 28 or 29.

29. The combination product according to claim 27, wherein said second siRNA molecule comprises at least 14 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs: 12, 23, 24, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

30. The combination product according to any one of claims 26, 27, 28 or 29, wherein said first siRNA molecule and said second siRNA molecule comprise one or more modified ribonucleotides.