US20070044161A1 - RNAi modulation of the Rho-A gene in research models - Google Patents

Abstract

The invention relates to compositions and methods for modulating the expression of the RhoA gene, and more particularly to the downregulation of RhoA by chemically modified oligonucleotides.

Description

RELATED APPLICATIONS
• This application claims the benefit of U.S. Provisional Application No. 60/701,470, filed Jul. 21, 2005, U.S. Provisional Application No. 60/726,838, filed Oct. 14, 2005, and U.S. Provisional Application No. 60/748,316, filed Dec. 7, 2005. The contents of each of these priority applications are incorporated herein by reference in their entirety.
TECHNICAL FIELD
• The invention relates to compositions and methods for modulating the expression of RhoA, and more particularly to the downregulation of RhoA mRNA and RhoA protein levels by oligonucleotides via RNA interference, e.g., chemically modified oligonucleotides.
BACKGROUND
• RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al.,Nature391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function.
• Numerous myelin-derived axon growth inhibitors have been characterized (see, for review, David et al., WO995394547, 1999; Bandman et al. U.S. Pat. No. 5,858,708, 1999; Schwab,Neurochem. Res.21:755-761, 1996). Several components of CNS white matter, NI35, NI250 (Nogo) and Myelin-associated glycoprotein (MAG), which have inhibitory activity for axonal extension, have been described as well (Schwab et al., WO9005191, 1990; Schwab et al., U.S. Pat. No. 5,684,133, 1997). In particular, RhoA is a member of the large family of Rho (Ras homologue) GTPases, itself belonging to the superfamily of Ras GTPases. All eukaryotes contain at least one Rho GTPase. During the process of evolution the number of Rho GTPases increased from 5 to 6 per organism (yeast) to over 20 (mammals) (Karnoub, A. E., et al., Breast Cancer Res. Treat. 2004, 84:61). Like other GTPases, RhoA has intrinsic GTPase activity and shuttles between an inactive GDP-bound state and an active GTP-bound state. In vitro, the exchange of GDP to GTP occurs very slowly, and is catalyzed by guanine nucleotide exchange factors (GEFs), which exchange GDP for GTP. GTPase activating proteins (GAPs) catalyze hydrolysis of the γ-phosphate of GTP. (Wheeler, A. P., Ridley, A. J., Exp. Cell Res. 2004, 301:43). A third set of regulatory proteins, the guanine nucleotide-dissociation inhibitors (GDIs), sequester GTPAses in the cytosol in the inactive, GDP-bound state.
• The N-terminal half of Rho GTPases contains the majority of the amino acids involved in GTP binding and hydrolysis, together with theSwitch 1 and 2 regions that change conformation between the GTP-bound and GDP-bound states (Bishop, A. L., Hall, A., Biochem. J. 2000, 348 (Pt. 2):241). The C-terminus of Rho family GTPases is essential for correct localization of the proteins. It is post-translationally modified by prenylation of a conserved C-terminal cysteine followed by methylation and proteolytic removal of the last three amino acids (Shao, F., Dixon, J. E., Adv. Exp. Med. Biol. 2003, 529:79). The prenyl group anchors the GTPases into membranes and this modification is essential for cell growth, transformation, and cytoskeleton organization (Allal, C., et al., J. Biol. Chem. 2000, 275:31001). Prenylation of Rho proteins appears to be important for their stability, inhibitors of enzymes that synthesize prenyl groups induce a decrease in Rho protein levels and their function (Stamatakis, K., et al., J. Biol. Chem 2002, 277:49389). In the case of RhoA, prenylation adds a geranylgeranyl group. RhoA is mainly found in the cytoplasm or at the plasma membrane (Adamson, P., et al., J. Cell Biol. 1992, 119:617).
• RhoA may bind to the intracellular portion of p75NTR and is activated by Nogo-R in a p75NTR-dependent manner (Wang, K. C., et al., Nature 2002, 420:74), which is how MAG, Nogo-66, and oligodendrocyte-myelin glycoprotein achieve RhoA activation. The central inhibitory domain of Nogo-A, NiG, distinct from Nogo-66, and Versican V2, a chondroitin-sulfate proteoglycan and another component of myelin, are able to activate RhoA in the absence of p75NTR, by an alternative pathway of RhoA activation remaining to be elucidated (Schweigreiter, R., et al., Mol. Cell Neurosci. 2004, 27:163). Further pathways of activation may exist.
• RhoA is part of the growth inhibitory machinery present in the central nervous system (CNS), but not in peripheral nerves, which prevents the regeneration of CNS tissue after injury. Both the expression and the activation of RhoA is induced in brain and spinal cord injury (Mueller, K., et al., Nature Reviews 2005, 4:387). Activation of RhoA leads to neuronal growth cone collapse, retraction bulb formation and neurite withdrawal. Inactivation of RhoA leads to neurite outgrowth in primary neurons on otherwise inhibitory substrates in vitro, and promotes axon regeneration and functional recovery after spinal cord injury in rats and mice in vivo (Lehmann, M. A., et al., J. Neurosci. 1999, 19:7537; Hara, M, et al., J. Neurosurg. 2000, 93:94; Dergham, P., et al., J. Neurosci. 2002, 22:6570). Furthermore, inactivation of Rho has been shown to protect endogenous cells of the spinal cord from apoptosis induced by spinal cord injury (Dubreuil, C. I., et al, J. Cell Biol. 2003, 162:233). These findings have clinical relevance because neuroprotective treatments after spinal cord injury lead to improved functional recovery (Liu, X. Z., et al., J. Neurosci. 1997, 17:5395).
• Evidently, RhoA is a potential target for therapeutic intervention strategies aimed at diseases and conditions involving, e.g., the destruction and/or impaired regeneration of cells of the CNS. The present invention advances the art by providing methods and medicaments encompassing short dsRNAs leading to the downregulation of RhoA mRNA and protein levels in cells expressing the RhoA gene. These methods and medicaments may be used in the treatment of disorders or pathological processes mediated, at least in part, by RhoA, e.g., by preventing the RhoA inhibition of axonal elongation and regeneration, and consequently stimulating nerve growth and proliferation.
SUMMARY
• The present invention is based, at least in part, on an investigation of the RhoA gene using iRNA agents and further testing of the iRNA agents that target the RhoA site. The present invention provides compositions and methods that are useful in reducing RhoA mRNA levels, RhoA protein levels and the treatment of pathological process mediated, at least in part, by RhoA, e.g. preventing RhoA inhibition of axonal elongation and regeneration, in a subject, e.g., a mammal, such as a human.
• In one aspect, the invention provides iRNA agents comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836 as given in Table 1 below, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836.
• In a further aspect, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and an antisense strand wherein the antisense strand comprises at least 15 contiguous nucleotides of the antisense sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, and wherein the iRNA agent reduces the amount of RhoA MRNA present in cultured human cells after incubation with these agents by 40% or more compared to cells which have not been incubated with the agent.
• In a further aspect, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell comprising a sense strand and an antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical to one of the sequences of any one agent selected from the group consisting of: agents number 6477 to 6836, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit RhoA expression. Preferably, for such agents the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisense strand sequences of agent numbers 6523, 6524, 6530, 6614, 6650, 6656, 6657, 6661, 6662, 6703, 6712, 6713, 6732, 6751, 6756, 6767, 6769, 6787, 6789, 6790, 6832.
• Evidently, in the above embodiments, the sense strands and/or antisense strands of the iRNA agents of the invention can also be identical to the sense strands and antisense strands of the agents, agent numbers 6477 to 6836.
• The iRNA agents of the invention may comprise a modification, e.g a modification that causes the iRNA agent to have increased stability in a biological sample. For example, they may comprise a phosphorothioate, a 2′-modified nucleotide, a locked nucleotide, an abasic nucleotide, morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide. For purposes of the above embodiments, an iRNA agent is considered to comprise one of the sequences of the agents, agent numbers 6477 to 6836, irrespective of the potential presence of nucleotide modifications, i.e. a 2′-O-methyl guanosine would be considered a guanosine for such comparison. However, certain patterns of modifications are particularly preferred embodiments of the present invention. Consequently, in another embodiment, the invention provides iRNA agents for inhibiting the expression of a rhoA gene in a cell wherein the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisense strand sequences of agent numbers AL-DP-5972, AL-DP-5973, AL-DP-5974, AL-DP-5975, AL-DP-5976, AL-DP-5978, AL-DP-5979, AL-DP-5981, AL-DP-5982, AL-DP-5983, AL-DP-5984, AL-DP-5986, AL-DP-5987, AL-DP-5988, AL-DP-5989, AL-DP-5990, AL-DP-5991, AL-DP-5992, AL-DP-5993, AL-DP-5994, AL-DP-5995, AL-DP-6176, AL-DP-6 177.
• In the iRNA agents of the present invention, the antisense RNA strand may be 30 or fewer nucleotides in length, and the duplex region of the iRNA agent may be 15-30 nucleotide pairs in length.
• A 2′-modified nucleotide according to the instant invention may comprise at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide.
• The iRNA agents of the invention may be designed such that
• every 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is a 2′-modified in sense strand, and every 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in antisense strand, or
• every 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in the sense and antisense strand, or
• every pyrimidine nucleotide is 2′-modified in the sense strand, and every 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in the antisense strand, or
• every pyrimidine nucleotide is 2′-modified in sense strand, and every 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in the antisense strand, or
• every pyrimidine nucleotide in the sense strand is 2′-modified, and no nucleotide is 2′-modified in the antisense strand.
• The 2′-modification in the iRNA agents of the invention may be selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O-N-methylacetamido (2′-O-NMA).
• The iRNA agents of the invention may comprise a nucleotide overhang having 1 to 4 unpaired nucleotides, preferably 2 or 3 unpaired nucleotides. The nucleotide overhang may be at the 3′-end of the antisense strand of the iRNA agent. The iRNA agents may comprise a cholesterol moiety, which is preferably conjugated to the 3′-end of the sense strand of the iRNA agent. In a preferred embodiment, the iRNA agent is targeted for uptake by nerve cells or nerve sheath cells.
• The present invention further provides methods for reducing the level of RhoA mRNA in a cell. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade RhoA mRNA in a cell and are comprised of the step of contacting a cell with one of the iRNA agents of the present invention. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the iRNA agents of the present invention. Reduction of RhoA mRNA in a cell results in a reduction in the amount of RhoA protein produced, and in an organism, may result in a decrease in RhoA specific pathological/disease effects, e.g. preventing RhoA inhibition of axonal elongation and regeneration.
• In another aspect of the invention, a method of treating a human subject having a pathological process mediated in part by RhoA is provided, comprising administering an iRNA agent of the invention, e.g. wherein the iRNA agent comprises a sense strand wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences any one of the agents, agent numbers 6477 to 6836, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense strand sequences of any one of the agents, agent numbers 6477 to 6836.
• In one embodiment of the above methods of the invention, the pathological process is the inhibition of nerve growth or elongation, preferably as a result of nerve injury or damage. In another preferred embodiment, the iRNA agent is administered in an amount sufficient to reduce the expression of RhoA in a cell or tissue of the subject. Preferably, the subject is a human.
• In another aspect, the instant invention provides pharmaceutical compositions, comprising:
• a.) an iRNA agent of the invention; and
• b.) a pharmaceutically acceptable carrier
• In another embodiment, the invention provides a cell comprising an iRNA agent of the invention.
• In another embodiment, the invention provides a method for inhibiting the expression of a RhoA gene in a cell, the method comprising:
• • (a) introducing into the cell an iRNA agent of the invention; and
  • (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the RhoA gene, thereby inhibiting expression of the RhoA gene in the cell.
• In another embodiment, the invention provides a vector for inhibiting the expression of a RhoA gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an an iRNA agent of the invention.
• In another embodiment, the invention provides a cell comprising the above vector.
• The methods and compositions of the invention, e.g., the methods and iRNA compositions can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.
• The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from this description and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.
BRIEF DESCRIPTION OF DRAWINGS
• FIG. 1 is a schematic illustrating the synthesis and structure of cholesterol conjugated RNA strands. The sphere represents the solid phase (controlled pore glass, CPG).
• FIG. 2 shows the effect of administration of iRNA agents of the invention, and of a control agent, respectively, to the site of injury in rats that have undergone partial dissection of the spinal cord; shown is the improvement in BBB locomotor score fromday 10 after spinal cord injury, taking the BBB locomotor score of individual rats onday 10 as 0.
DETAILED DESCRIPTION
• For ease of exposition the term “nucleotide” or “ribonucleotide” is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood that the usage of the term “ribonucleotide” or “nucleotide” herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety, as further described below, at one or more positions.
• An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, each of which is described herein or is well known in the RNA synthetic art. While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those that have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′-modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
• An “iRNA agent” (abbreviation for “interfering RNA agent”) as used herein, is an RNA agent, which can downregulate the expression of a target gene, e.g., RhoA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can be a double stranded iRNA agent.
• A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interstrand hybridization can form a region of duplex structure. A “strand” herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g., by a linker, e.g., a polyethyleneglycol linker, to form one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the “antisense strand.” A second strand of the dsRNA agent, which comprises a region complementary to the antisense strand, is termed the “sense strand.” However, a ds iRNA agent can also be formed from a single RNA molecule which is at least partly self-complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term “strand” refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule.
• Although, in mammalian cells, long ds iRNA agents can induce the interferon response which is frequently deleterious, short ds iRNA agents do not trigger the interferon response, at least not to an extent that is deleterious to the cell and/or host (Manche et al.,Mol. Cell. Biol.12:5238, 1992; Lee et al.,Virology199:491, 1994; Castelli et al.,J. Exp. Med.186:967, 1997; Zheng et al.,RNA10:1934, 2004; Heidel et al., “Lack of interferon response in animals to naked siRNAs”Nature Biotechn. advance online publicationdoi: 10.1038/nbt1038, Nov. 21, 2004). The iRNA agents of the present invention include molecules which are sufficiently short that they do not trigger a deleterious non-specific interferon response in normal mammalian cells. Thus, the administration of a composition including an iRNA agent (e.g., formulated as described herein) to a subject can be used to decreased expression of the RhoA genes in RhoA expressing cells in the subject, while circumventing an interferon response. Molecules that are short enough that they do not trigger a deleterious interferon response are termed siRNA agents or siRNAs herein. “siRNA agent” or “siRNA” as used herein, refers to an iRNA agent, e.g., a ds iRNA agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs.
• The isolated iRNA agents described herein, including ds iRNA agents and siRNA agents, can mediate the decreased expression of a RhoA nucleic acid, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a nucleic acid is also referred to as a target gene. Preferably, the RNA to be silenced is a gene product of an endogenous RhoA gene.
• As used herein, the phrase “mediates RNAi” refers to the ability of an agent to silence, in a sequence specific manner, a target gene. “Silencing a target gene” means the process whereby a cell containing and/or expressing a certain product of the target gene when not in contact with the agent, will contain and/or express at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.
• As used herein, the term “complementary” is used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule, e.g., a RhoA mRNA. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ from the target sequences by at least 4 nucleotides.
• As used herein, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA (e.g., a target RhoA mRNA) if the iRNA agent reduces the production of a protein encoded by the target RNA in a cell. The iRNA agent may also be “exactly complementary” to the target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” iRNA agent can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RhoA RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementarity is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference. Preferred iRNA agents will be based on or consist of or comprise the sense and antisense sequences provided in Table 1.
• As used herein, “essentially identical” when used referring to a first nucleotide sequence in comparison to a second nucleotide sequence means that the first nucleotide sequence is identical to the second nucleotide sequence except for up to one, two or three nucleotide substitutions (e.g., adenosine replaced by uracil). “Essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells,” as used herein referring to an iRNA agent not identical to but derived from one of the iRNA agents of Table 1 by deletion, addition or substitution of nucleotides, means that the derived iRNA agent possesses an inhibitory activity not more than 20% (in terms of remaining target mRNA) different from the inhibitory activity of the iRNA agent of Table 1 from which it was derived. For example, an iRNA agent derived from an iRNA agent of Table 1 which lowers the amount of RhoA mRNA present in cultured human Rho-A expressing cells by 70% may itself lower the amount of RhoA mRNA present in cultured human RhoA expressing cells by at least 50% in order to be considered as essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells. Optionally, an iRNA agent of the invention may lower the amount of RhoA mRNA present in cultured human RhoA expressing cells by at least 50%, or at least 40%.
• As used herein, a “subject” refers to a mammalian organism undergoing treatment for a disorder mediated by RhoA protein expression. The subject can be any mammal, such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. In the preferred embodiment, the subject is a human.
• Design and Selection of iRNA Agents
• As used herein, “disorders associated with RhoA expression” refers to any biological or pathological state that (1) is mediated in part by the presence of RhoA mRNA and/or protein and (2) whose outcome can be affected by reducing the level of RhoA mRNA and/or protein present. Specific disorders associated with RhoA expression are noted below and are primarily based on the responsibility of RhoA action in inhibiting axonal elongation and regeneration.
• The present invention is based on the design, synthesis and generation of iRNA agents that target RhoA and the demonstration of silencing of a RhoA gene in vitro in cultured cells after incubation with an iRNA agent, and the resulting RhoA-specific effect.
• An iRNA agent can be rationally designed based on sequence information and desired characteristics. For example, an iRNA agent can be designed according to the relative melting temperature of the candidate duplex. Generally, the duplex should have a lower melting temperature at the 5′ end of the antisense strand than at the 3′ end of the antisense strand.
• Candidate iRNA agents can also be designed by performing, for example, a gene walk analysis of the genes that will serve as the target gene. Overlapping, adjacent, or closely spaced candidate agents corresponding to all or some of the transcribed region can be generated and tested. Each of the iRNA agents can be tested and evaluated for the ability to down regulate the target gene expression (see below, “Evaluation of Candidate iRNA agents”).
• Herein, potential iRNA agents targeting RhoA were designed using the known sequences of RhoA for human, rat and mouse and other known RhoA sequences. The target sequences shown in Table 1 hereinabove were selected from those regions of the human RhoA mRNA sequences that show complete homology with the corresponding sequences in rat and mouse. Therefore, the siRNA agents, agent numbers 6477-6836 should show cross reactivity between these three species. Based on the results provided, the present invention provides iRNA agents that silence RhoA in cultured human RhoA expressing cells and in a subject.
• Table 1 provides exemplary iRNA agents targeting RhoA

  TABLE 1
  Exemplary iRNA agents for targeting RhoA mRNA

  Start

  duplex

  sense strand

  antisense strand

  pos.

  SEQ

  design

  SEQ

  SEQ

  Agent

  in

  ID

  (over-

  ID

  ID

  number	RNAb	NO.	target sequence (5′-3′)	hang)a	NO.	sequence (5′-3′)	NO.	sequence (5′-3′)

  6477	288	1	ccggaagaaacuggugauuguug	double	2	ggaagaaacuggugauuguTT	3	acaaucaccaguuucuuccTT
  6478	289	4	cggaagaaacuggugauuguugg	double	5	gaagaaacuggugauuguuTT	6	aacaaucaccaguuucuucTT
  6479	290	7	ggaagaaacuggugauuguuggu	double	8	aagaaacuggugauuguugTT	9	caacaaucaccaguuucuuTT
  6480	291	10	gaagaaacuggugauuguuggug	double	11	agaaacuggugauuguuggTT	12	ccaacaaucaccaguuucuTT
  6481	292	13	aagaaacuggugauuguugguga	double	14	gaaacuggugauuguugguTT	15	accaacaaucaccaguuucTT
  6482	293	16	agaaacuggugauuguuggugau	double	17	aaacuggugauuguuggugTT	18	caccaacaaucaccaguuuTT
  6483	294	19	gaaacuggugauuguuggugaug	double	20	aacuggugauuguuggugaTT	21	ucaccaacaaucaccaguuTT
  6484	295	22	aaacuggugauuguuggugaugg	double	23	acuggugauuguuggugauTT	24	aucaccaacaaucaccaguTT
  6485	296	25	aacuggugauuguuggugaugga	double	26	cuggugauuguuggugaugTT	27	caucaccaacaaucaccagTT
  6486	297	28	acuggugauuguuggugauggag	double	29	uggugauuguuggugauggTT	30	ccaucaccaacaaucaccaTT
  6487	298	31	cuggugauuguuggugauggagc	double	32	ggugauuguuggugauggaTT	33	uccaucaccaacaaucaccTT
  6488	299	34	uggugauuguuggugauggagcc	double	35	gugauuguuggugauggagTT	36	cuccaucaccaacaaucacTT
  6489	300	37	ggugauuguuggugauggagccu	double	38	ugauuguuggugauggagcTT	39	gcuccaucaccaacaaucaTT
  6490	326	40	gaaagacaugcuugcucauaguc	double	41	aagacaugcuugcucauagTT	42	cuaugagcaagcaugucuuTT
  6491	327	43	aaagacaugcuugcucauagucu	double	44	agacaugcuugcucauaguTT	45	acuaugagcaagcaugucuTT
  6492	328	46	aagacaugcuugcucauagucuu	double	47	gacaugcuugcucauagucTT	48	gacuaugagcaagcaugucTT
  6493	329	49	agacaugcuugcucauagucuuc	double	50	acaugcuugcucauagucuTT	SI	agacuaugagcaagcauguTT
  6494	330	52	gacaugcuugcucauagucuuca	double	53	caugcuugcucauagucuuTT	54	aagacuaugagcaagcaugTT
  6495	331	55	acaugcuugcucauagucuucag	double	56	augcuugcucauagucuucTT	57	gaagacuaugagcaagcauTT
  6496	332	58	caugcuugcucauagucuucagc	double	59	ugcuugcucauagucuucaTT	60	ugaagacuaugagcaagcaTT
  6497	333	61	augcuugcucauagucuucagca	double	62	gcuugcucauagucuucagTT	63	cugaagacuaugagcaagcTT
  6498	334	64	ugcuugcucauagucuucagcaa	double	65	cuugcucauagucuucagcTT	66	gcugaagacuaugagcaagTT
  6499	335	67	gcuugcucauagucuucagcaag	double	68	uugcucauagucuucagcaTT	69	ugcugaagacuaugagcaaTT
  6500	336	70	cuugcucauagucuucagcaagg	double	71	ugcucauagucuucagcaaTT	72	uugcugaagacuaugagcaTT
  6501	337	73	uugcucauagucuucagcaagga	double	74	gcucauagucuucagcaagTT	75	cuugcugaagacuaugagcTT
  6502	338	76	ugcucauagucuucagcaaggac	double	77	cucauagucuucagcaaggTT	78	ccuugcugaagacuaugagTT
  6503	339	79	gcucauagucuucagcaaggacc	double	80	ucauagucuucagcaaggaTT	81	uccuugcugaagacuaugaTT
  6504	340	82	cucauagucuucagcaaggacca	double	83	cauagucuucagcaaggacTT	84	guccuugcugaagacuaugTT
  6505	341	85	ucauagucuucagcaaggaccag	double	86	auagucuucagcaaggaccTT	87	gguccuugcugaagacuauTT
  6506	342	88	cauagucuucagcaaggaccagu	double	89	uagucuucagcaaggaccaTT	90	ugguccuugcugaagacuaTT
  6507	343	91	auagucuucagcaaggaccaguu	double	92	agucuucagcaaggaccagTT	93	cugguccuugcugaagacuTT
  6508	344	94	uagucuucagcaaggaccaguuc	double	95	gucuucagcaaggaccaguTT	96	acugguccuugcugaagacTT
  6509	345	97	agucuucagcaaggaccaguucc	double	98	ucuucagcaaggaccaguuTT	99	aacugguccuugcugaagaTT
  6510	346	100	gucuucagcaaggaccaguuccc	double	101	cuucagcaaggaccaguucTT	102	gaacugguccuugcugaagTT
  6511	347	103	ucuucagcaaggaccaguuccca	double	104	uucagcaaggaccaguuccTT	105	ggaacugguccuugcugaaTT
  6512	348	106	cuucagcaaggaccaguucccag	double	107	ucagcaaggaccaguucccTT	108	gggaacugguccuugcugaTT
  6513	349	109	uucagcaaggaccaguucccaga	double	110	cagcaaggaccaguucccaTT	111	ugggaacugguccuugcugTT
  6514	350	112	ucagcaaggaccaguucccagag	double	113	agcaaggaccaguucccagTT	114	cugggaacugguccuugcuTT
  6515	351	115	cagcaaggaccaguucccagagg	double	116	gcaaggaccaguucccagaTT	117	ucugggaacugguccuugcTT
  6516	352	118	agcaaggaccaguucccagaggu	double	119	caaggaccaguucccagagTT	120	cucugggaacugguccuugTT
  6517	353	121	gcaaggaccaguucccagaggug	double	122	aaggaccaguucccagaggTT	123	ccucugggaacugguccuuTT
  6518	354	124	caaggaccaguucccagaggugu	double	125	aggaccaguucccagagguTT	126	accucugggaacugguccuTT
  6519	425	127	gaaagcagguagaguuggcuuug	double	128	aagcagguagaguuggcuuTT	129	aagccaacucuaccugcuuTT
  6520	426	130	aaagcagguagaguuggcuuugu	double	131	agcagguagaguuggcuuuTT	132	aaagccaacucuaccugcuTT
  6521	535	133	gacagcccugauaguuuagaaaa	double	134	cagcccugauaguuuagaaTT	135	uucuaaacuaucagggcugTT
  6522	536	136	acagcccugauaguuuagaaaac	double	137	agcccugauaguuuagaaaTT	138	uuucuaaacuaucagggcuTT
  6523	537	139	cagcccugauaguuuagaaaaca	double	140	gcccugauaguuuagaaaaTT	141	uuuucuaaacuaucagggcTT
  6524	538	142	agcccugauaguuuagaaaacau	double	143	cccugauaguuuagaaaacTT	144	guuuucuaaacuaucagggTT
  6525	539	145	gcccugauaguuuagaaaacauc	double	146	ccugauaguuuagaaaacaTT	147	uguuuucuaaacuaucaggTT
  6526	540	148	cccugauaguuuagaaaacaucc	double	149	cugauaguuuagaaaacauTT	150	auguuuucuaaacuaucagTT
  6527	541	151	ccugauaguuuagaaaacauccc	double	152	ugauaguuuagaaaacaucTT	153	gauguuuucuaaacuaucaTT
  6528	542	154	cugauaguuuagaaaacauccca	double	155	gauaguuuagaaaacauccTT	156	ggauguuuucuaaacuaucTT
  6529	543	157	ugauaguuuagaaaacaucccag	double	158	auaguuuagaaaacaucccTT	159	gggauguuuucuaaacuauTT
  6530	544	160	gauaguuuagaaaacaucccaga	double	161	uaguuuagaaaacaucccaTT	162	ugggauguuuucuaaacuaTT
  6531	545	163	auaguuuagaaaacaucccagaa	double	164	aguuuagaaaacaucccagTT	165	cugggauguuuucuaaacuTT
  6532	546	166	uaguuuagaaaacaucccagaaa	double	167	guuuagaaaacaucccagaTT	168	ucugggauguuuucuaaacTT
  6533	547	169	aguuuagaaaacaucccagaaaa	double	170	uuuagaaaacaucccagaaTT	171	uucugggauguuuucuaaaTT
  6534	548	172	guuuagaaaacaucccagaaaag	double	173	uuagaaaacaucccagaaaTT	174	uuucugggauguuuucuaaTT
  6535	549	175	uuuagaaaacaucccagaaaagu	double	176	uagaaaacaucccagaaaaTT	177	uuuucugggauguuuucuaTT
  6536	575	178	ccccagaagucaagcauuucugu	double	179	ccagaagucaagcauuucuTT	180	agaaaugcuugacuucuggTT
  6537	576	181	cccagaagucaagcauuucuguc	double	182	cagaagucaagcauuucugTT	183	cagaaaugcuugacuucugTT
  6538	577	184	ccagaagucaagcauuucugucc	double	185	agaagucaagcauuucuguTT	186	acagaaaugcuugacuucuTT
  6539	578	187	cagaagucaagcauuucuguccc	double	188	gaagucaagcauuucugucTT	189	gacagaaaugcuugacuucTT
  6540	579	190	agaagucaagcauuucuguccca	double	191	aagucaagcauuucuguccTT	192	ggacagaaaugcuugacuuTT
  6541	692	193	ugaaaccugaagaaggcagagau	double	194	aaaccugaagaaggcagagTT	195	cucugccuucuucagguuuTT
  6542	693	196	gaaaccugaagaaggcagagaua	double	197	aaccugaagaaggcagagaTT	198	ucucugccuucuucagguuTT
  6543	694	199	aaaccugaagaaggcagagauau	double	200	accugaagaaggcagagauTT	201	aucucugccuucuucagguTT
  6544	695	202	aaccugaagaaggcagagauaug	double	203	ccugaagaaggcagagauaTT	204	uaucucugccuucuucaggTT
  6545	696	205	accugaagaaggcagagauaugg	double	206	cugaagaaggcagagauauTT	207	auaucucugccuucuucagTT
  6546	697	208	ccugaagaaggcagagauauggc	double	209	ugaagaaggcagagauaugTT	210	cauaucucugccuucuucaTT
  6547	698	211	cugaagaaggcagagauauggca	double	212	gaagaaggcagagauauggTT	213	ccauaucucugccuucuucTT
  6548	699	214	ugaagaaggcagagauauggcaa	double	215	aagaaggcagagauauggcTT	216	gccauaucucugccuucuuTT
  6549	700	217	gaagaaggcagagauaugycaaa	double	218	agaaggcagagauaug9caTT	219	ugccauaucucugccuucuTT
  6550	701	220	aagaaggcagagauauggcaaac	double	221	gaaggcagagauauggcaaTT	222	uugccauaucucugccuucTT
  6551	702	223	agaaggcagagauauggcaaaca	double	224	aaggcagagauauggcaaaTT	225	uuugccauaucucugccuuTT
  6552	703	226	gaaggcagagauauggcaaacag	double	227	aggcagagauauggcaaacTT	228	guuugccauaucucugccuTT
  6553	704	229	aaggcagagauauggcaaacagg	double	230	ggcagagauauggcaaacaTT	231	uguuugccauaucucugccTT
  6554	705	232	aggcagagauauggcaaacagga	double	233	gcagagauauggcaaacagTT	234	cuguuugccauaucucugcTT
  6555	706	235	ggcagagauauggcaaacaggau	double	236	cagagauauggcaaacaggTT	237	ccuguuugccauaucucugTT
  6556	707	238	gcagagauauggcaaacaggauu	double	239	agagauauggcaaacaggaTT	240	uccuguuugccauaucucuTT
  6557	708	241	cagagauauggcaaacaggauug	double	242	gagauauggcaaacaggauTT	243	auccuguuugccauaucucTT
  6558	709	244	agagauauggcaaacaggauugg	double	245	agauauggcaaacaggauuTT	246	aauccuguuugccauaucuTT
  6559	710	247	gagauauggcaaacaggauuggc	double	248	gauauggcaaacaggauugTT	249	caauccuguuugccauaucTT
  6560	711	250	agauauggcaaacaggauuggcg	double	251	auauggcaaacaggauuggTT	252	ccaauccuguuugccauauTT
  6561	712	253	gauauggcaaacaggauuggcgc	double	254	uauggcaaacaggauuggcTT	255	gccaauccuguuugccauaTT
  6562	713	256	auauggcaaacaggauuggcgcu	double	257	auggcaaacaggauuggcgTT	258	cgccaauccuguuugccauTT
  6563	714	259	uauggcaaacaggauuggcgcuu	double	260	uggcaaacaggauuggcgcTT	261	gcgccaauccuguuugccaTT
  6564	715	262	auggcaaacaggauuggcgcuuu	double	263	ggcaaacaggauuggcgcuTT	264	agcgccaauccuguuugccTT
  6565	716	265	uggcaaacaggauuggcgcuuuu	double	266	gcaaacaggauuggcgcuuTT	267	aagcgccaauccuguuugcTT
  6566	717	268	ggcaaacaggauuggcgcuuuug	double	269	caaacaggauuggcgcuuuTT	270	aaagcgccaauccuguuugTT
  6567	718	271	gcaaacaggauuggcgcuuuugg	double	272	aaacaggauuggcgcuuuuTT	273	aaaagcgccaauccuguuuTT
  6568	719	274	caaacaggauuggcgcuuuuggg	double	275	aacaggauuggcgcuuuugTT	276	caaaagcgccaauccuguuTT
  6569	720	277	aaacaggauuggcgcuuuugggu	double	278	acaggauuggcgcuuuuggTT	279	ccaaaagcgccaauccuguTT
  6570	721	280	aacaggauuggcgcuuuugggua	double	281	caggauuggcgcuuuugggTT	282	cccaaaagcgccaauccugTT
  6571	722	283	acaggauuggcgcuuuuggguac	double	284	aggauuggcgcuuuuggguTT	285	acccaaaagcgccaauccuTT
  6572	723	286	caggauuggcgcuuuuggguaca	double	287	ggauuggcgcuuuuggguaTT	288	uacccaaaagcgccaauccTT
  6573	724	289	aggauuggcgcuuuuggguacau	double	290	gauuggcgcuuuuggguacTT	291	guacccaaaagcgccaaucTT
  6574	725	292	ggauuggcgcuuuuggguacaug	double	293	auuggcgcuuuuggguacaTT	294	uguacccaaaagcgccaauTT
  6575	726	295	gauuggcgcuuuuggguacaugg	double	296	uuggcgcuuuuggguacauTT	297	auguacccaaaagcgccaaTT
  6576	727	298	auuggcgcuuuuggguacaugga	double	299	uggcgcuuuuggguacaugTT	300	cauguacccaaaagcgccaTT
  6577	728	301	uuggcgcuuuuggguacauggag	double	302	ggcgcuuuuggguacauggTT	303	ccauguacccaaaagcgccTT
  6578	729	304	uggcgcuuuuggguacauggagu	double	305	gcgcuuuuggguacauggaTT	306	uccauguacccaaaagcgcTT
  6579	730	307	ggcgcuuuuggguacauggagug	double	308	cgcuuuuggguacauggagTT	309	cuccauguacccaaaagcgTT
  6580	731	310	gcgcuuuuggguacauggagugu	double	311	gcuuuuggguacauggaguTT	312	acuccauguacccaaaagcTT
  6581	732	313	cgcuuuuggguacauggaguguu	double	314	cuuuuggguacauggagugTT	315	cacuccauguacccaaaagTT
  6582	733	316	gcuuuuggguacauggaguguuc	double	317	uuuuggguacauggaguguTT	318	acacuccauguacccaaaaTT
  6583	734	319	cuuuuggguacauggaguguuca	double	320	uuuggguacauggaguguuTT	321	aacacuccauguacccaaaTT
  6584	735	322	uuuuggguacauggaguguucag	double	323	uuggguacauggaguguucTT	324	gaacacuccauguacccaaTT
  6585	736	325	uuuggguacauggaguguucagc	double	326	uggguacauggaguguucaTT	327	ugaacacuccauguacccaTT
  6586	737	328	uuggguacauggaguguucagca	double	329	ggguacauggaguguucagTT	330	cugaacacuccauguacccTT
  6587	738	331	uggguacauggaguguucagcaa	double	332	gguacauggaguguucagcTT	333	gcugaacacuccauguaccTT
  6588	739	334	ggguacauggaguguucagcaaa	double	335	guacauggaguguucagcaTT	336	ugcugaacacuccauguacTT
  6589	740	337	gguacauggaguguucagcaaag	double	338	uacauggaguguucagcaaTT	339	uugcugaacacuccauguaTT
  6590	741	340	guacauggaguguucagcaaaga	double	341	acauggaguguucagcaaaTT	342	uuugcugaacacuccauguTT
  6591	742	343	uacauggaguguucagcaaagac	double	344	cauggaguguucagcaaagTT	345	cuuugcugaacacuccaugTT
  6592	743	346	acauggaguguucagcaaagacc	double	347	auggaguguucagcaaagaTT	348	ucuuugcugaacacuccauTT
  6593	744	349	cauggaguguucagcaaagacca	double	350	uggaguguucagcaaagacTT	351	gucuuugcugaacacuccaTT
  6594	745	352	auggaguguucagcaaagaccaa	double	353	ggaguguucagcaaagaccTT	354	ggucuuugcugaacacuccTT
  6595	746	355	uggaguguucagcaaagaccaaa	double	356	gaguguucagcaaagaccaTT	357	uggucuuugcugaacacucTT
  6596	747	358	ggaguguucagcaaagaccaaag	double	359	aguguucagcaaagaccaaTT	360	uuggucuuugcugaacacuTT
  6597	748	361	gaguguucagcaaagaccaaaga	double	362	guguucagcaaagaccaaaTT	363	uuuggucuuugcugaacacTT
  6598	749	364	aguguucagcaaagaccaaagau	double	365	uguucagcaaagaccaaagTT	366	cuuuggucuuugcugaacaTT
  6599	750	367	guguucagcaaagaccaaagaug	double	368	guucagcaaagaccaaagaTT	369	ucuuuggucuuugcugaacTT
  6600	770	370	auggagugagagagguuuuugaa	double	371	ggagugagagagguuuuugTT	372	caaaaaccucucucacuccTT
  6601	771	373	uggagugagagagguuuuugaaa	double	374	gagugagagagguuuuugaTT	375	ucaaaaaccucucucacucTT
  6602	797	376	cuacgagagcugcucugcaagcu	double	377	acgagagcugcucugcaagTT	378	cuugcagagcagcucucguTT
  6603	798	379	uacgagagcugcucugcaagcua	double	380	cgagagcugcucugcaagcTT	381	gcuugcagagcagcucucgTT
  6604	799	382	acgagagcugcucugcaagcuag	double	383	gagagcugcucugcaagcuTT	384	agcuugcagagcagcucucTT
  6605	800	385	cgagagcugcucugcaagcuaga	double	386	agagcugcucugcaagcuaTT	387	uagcuugcagagcagcucuTT
  6606	801	388	gagagcugcucugcaagcuagac	double	389	gagcugcucugcaagcuagTT	390	cuagcuugcagagcagcucTT
  6607	802	391	agagcugcucugcaagcuagacg	double	392	agcugcucugcaagcuagaTT	393	ucuagcuugcagagcagcuTT
  6608	803	394	gagcugcucugcaagcuagacgu	double	395	gcugcucugcaagcuagacTT	396	gucuagcuugcagagcagcTT
  6609	804	397	agcugcucugcaagcuagacgug	double	398	cuyoucugcaagcuagacgTT	399	cgucuagcuugcagagcagTT
  6610	895	400	uugaagugcuguuuauuaaucuu	double	401	gaagugcuguuuauuaaucTT	402	gauuaauaaacagcacuucTT
  6611	896	403	ugaagugcuguuuauuaaucuua	double	404	aagugcuguuuauuaaucuTT	405	agauuaauaaacagcacuuTT
  6612	897	406	gaagugcuguuuauuaaucuuag	double	407	agugcuguuuauuaaucuuTT	408	aagauuaauaaacagcacuTT
  6613	898	409	aagugcuguuuauuaaucuuagu	double	410	gugcuguuuauuaaucuuaTT	411	uaagauuaauaaacagcacTT
  6614	899	412	agugcuguuuauuaaucuuagug	double	413	ugcuguuuauuaaucuuagTT	414	cuaagauuaauaaacagcaTT
  6615	900	415	gugcuguuuauuaaucuuagugu	double	416	gcuguuuauuaaucuuaguTT	417	acuaagauuaauaaacagcTT
  6616	901	418	ugcuguuuauuaaucuuagugua	double	419	cuguuuauuaaucuuagugTT	420	cacuaagauuaauaaacagTT
  6617	902	421	gcuguuuauuaaucuuaguguau	double	422	uguuuauuaaucuuaguguTT	423	acacuaagauuaauaaacaTT
  6618	903	424	cuguuuauuaaucuuaguguaug	double	425	guuuauuaaucuuaguguaTT	426	uacacuaagauuaauaaacTT
  6619	904	427	uguuuauuaaucuuaguguauga	double	428	uuuauuaaucuuaguguauTT	429	auacacuaagauuaauaaaTT
  6620	905	430	guuuauuaaucuuaguguaugau	double	431	uuauuaaucuuaguguaugTT	432	cauacacuaagauuaauaaTT
  6621	906	433	uuuauuaaucuuaguguaugauu	double	434	uauuaaucuuaguguaugaTT	435	ucauacacuaagauuaauaTT
  6622	907	436	uuauuaaucuuaguguaugauua	double	437	auuaaucuuaguguaugauTT	438	aucauacacuaagauuaauTT
  6623	908	439	uauuaaucuuaguguaugauuac	double	440	uuaaucuuaguguaugauuTT	441	aaucauacacuaagauuaaTT
  6624	909	442	auuaaucuuaguguaugauuacu	double	443	uaaucuuaguguaugauuaTT	444	uaaucauacacuaagauuaTT
  6625	910	445	uuaaucuuaguguaugauuacug	double	446	aaucuuaguguaugauuacTT	447	guaaucauacacuaagauuTT
  6626	911	448	uaaucuuaguguauyauuacugg	double	449	aucuuaguguaugauuacuTT	450	aguaaucauacacuaagauTT
  6627	912	451	aaucuuaguguaugauuacuggc	double	452	ucuuaguguaugauuacugTT	453	caguaaucauacacuaagaTT
  6628	913	454	aucuuaguguaugauuacuggcc	double	455	cuuaguguaugauuacuggTT	456	ccaguaaucauacacuaagTT
  6629	914	457	ucuuaguguaugauuacuggccu	double	458	uuaguguaugauuacuggcTT	459	gccaguaaucauacacuaaTT
  6630	915	460	cuuaguguaugauuacuggccuu	double	461	uaguguaugauuacuggccTT	462	ggccaguaaucauacacuaTT
  6631	916	463	uuaguguaugauuacuggccuuu	double	464	aguguaugauuacuggccuTT	465	aggccaguaaucauacacuTT
  6632	917	466	uaguguaugauuacuggccuuuu	double	467	guguaugauuacuggccuuTT	468	aaggccaguaaucauacacTT
  6633	918	469	aguguaugauuacugyccuuuuu	double	470	uguaugauuacuggccuuuTT	471	aaaggccaguaaucauacaTT
  6634	919	472	guguaugauuacuggccuuuuuc	double	473	guaugauuacuggccuuuuTT	474	aaaaggccaguaaucauacTT
  6635	939	475	uucauuuaucuauaauuuaccua	double	476	cauuuaucuauaauuuaccTT	477	gguaaauuauagauaaaugTT
  6636	940	478	ucauuuaucuauaauuuaccuaa	double	479	auuuaucuauaauuuaccuTT	480	agguaaauuauagauaaauTT
  6637	941	481	cauuuaucuauaauuuaccuaag	double	482	uuuaucuauaauuuaccuaTT	483	uagguaaauuauagauaaaTT
  6638	942	484	auuuaucuauaauuuaccuaaga	double	485	uuaucuauaauuuaccuaaTT	486	uuagguaaauuauagauaaTT
  6639	943	487	uuuaucuauaauuuaccuaagau	double	488	uaucuauaauuuaccuaagTT	489	cuuagguaaauuauagauaTT
  6640	944	490	uuaucuauaauuuaccuaagauu	double	491	aucuauaauuuaccuaagaTT	492	ucuuagguaaauuauagauTT
  6641	945	493	uaucuauaauuuaccuaagauua	double	494	ucuauaauuuaccuaagauTT	495	aucuuagguaaauuauagaTT
  6642	946	496	aucuauaauuuaccuaagauuac	double	497	cuauaauuuaccuaagauuTT	498	aaucuuagguaaauuauagTT
  6643	947	499	ucuauaauuuaccuaagauuaca	double	500	uauaauuuaccuaagauuaTT	501	uaaucuuagguaaauuauaTT
  6644	948	502	cuauaauuuaccuaagauuacaa	double	503	auaauuuaccuaagauuacTT	504	guaaucuuagguaaauuauTT
  6645	949	505	uauaauuuaccuaagauuacaaa	double	506	uaauuuaccuaagauuacaTT	507	uguaaucuuagguaaauuaTT
  6646	950	508	auaauuuaccuaagauuacaaau	double	509	aauuuaccuaagauuacaaTT	510	uuguaaucuuagguaaauuTT
  6647	951	511	uaauuuacauaagauuacaaauc	double	512	auuuaccuaagauuacaaaTT	513	uuuguaaucuuagguaaauTT
  6648	952	514	aauuuaccuaagauuacaaauca	double	515	uuuaccuaagauuacaaauTT	516	auuuguaaucuuagguaaaTT
  6649	953	517	auuuaccuaagauuacaaaucag	double	518	uuaccuaagauuacaaaucTT	519	gauuuguaaucuuagguaaTT
  6650	954	520	uuuaccuaagauuacaaaucaga	double	521	uaccuaagauuacaaaucaTT	522	ugauuuguaaucuuagguaTT
  6651	974	523	agaagucaucuugcuaccaguau	double	524	aagucaucuugcuaccaguTT	525	acugguagcaagaugacuuTT
  6652	975	526	gaagucaucuugcuaccaguauu	double	527	agucaucuugcuaccaguaTT	528	uacugguagcaagaugacuTT
  6653	976	529	aagucaucuugcuaccaguauuu	double	530	gucaucuugcuaccaguauTT	531	auacugguagcaagaugacTT
  6654	977	532	agucaucuugcuaccaguauuua	double	533	ucaucuugcuaccaguauuTT	534	aauacugguagcaagaugaTT
  6655	978	535	gucaucuugcuaccaguauuuag	double	536	caucuugcuaccaguauuuTT	537	aaauacugguagcaagaugTT
  6656	979	538	ucaucuugcuaccaguauuuaga	double	539	aucuugcuaccaguauuuaTT	540	uaaauacugguagcaagauTT
  6657	980	541	caucuugcuaccaguauuuagaa	double	542	ucuugcuaccaguauuuagTT	543	cuaaauacugguagcaagaTT
  6658	981	544	aucuugcuaccaguauuuagaag	double	545	cuugcuaccaguauuuagaTT	546	ucuaaauacugguagcaagTT
  6659	982	547	ucuugcuaccaguauuuagaagc	double	548	uugcuaccaguauuuagaaTT	549	uucuaaauacugguagcaaTT
  6660	983	550	cuugcuaccaguauuuagaagcc	double	551	ugcuaccaguauuuagaagTT	552	cuucuaaauacugguagcaTT
  6661	984	553	uugcuaccaguauuuagaagcca	double	554	gcuaccaguauuuagaagcTT	555	gcuucuaaauacugguagcTT
  6662	985	556	ugcuaccaguauuuagaagccaa	double	557	cuaccaguauuuagaagccTT	558	ggcuucuaaauacugguagTT
  6663	986	559	gcuaccaguauuuagaagccaac	double	560	uaccaguauuuagaagccaTT	561	uggcuucuaaauacugguaTT
  6664	987	562	cuaccaguauuuagaagccaacu	double	563	accaguauuuagaagccaaTT	564	uuggcuucuaaauacugguTT
  6665	988	565	uaccaguauuuagaagccaacua	double	566	ccaguauuuagaagccaacTT	567	guuggcuucuaaauacuggTT
  6666	1131	568	cuugcuucuuucuagaaagagaa	double	569	ugcuucuuucuagaaagagTT	570	cucuuucuagaaagaagcaTT
  6667	1132	571	uugcuucuuucuagaaagagaaa	double	572	gcuucuuucuagaaagagaTT	573	ucucuuucuagaaagaagcTT
  6668	1133	574	ugcuucuuucuagaaagagaaac	double	575	cuucuuucuagaaagagaaTT	576	uucucuuucuagaaagaagTT
  6669	1134	577	gcuucuuucuagaaagagaaaca	double	578	uucuuucuagaaagagaaaTT	579	uuucucuuucuagaaagaaTT
  6670	1135	580	cuucuuucuagaaagagaaacag	double	581	ucuuucuagaaagagaaacTT	582	guuucucuuucuagaaagaTT
  6671	1136	583	uucuuucuagaaagagaaacagu	double	584	cuuucuagaaagagaaacaTT	585	uguuucucuuucuagaaagTT
  6672	1137	586	ucuuucuagaaagagaaacaguu	double	587	uuucuagaaagagaaacagTT	588	cuguuucucuuucuagaaaTT
  6673	1138	589	cuuucuagaaagagaaacaguug	double	590	uucuagaaagagaaacaguTT	591	acuguuucucuuucuagaaTT
  6674	1139	592	uuucuagaaagagaaacaguugg	double	593	ucuagaaagagaaacaguuTT	594	aacuguuucucuuucuagaTT
  6675	1140	595	uucuagaaagagaaacaguuggu	double	596	cuagaaagagaaacaguugTT	597	caacuguuucucuuucuagTT
  6676	1141	598	ucuagaaagagaaacaguuggua	double	599	uagaaagagaaacaguuggTT	600	ccaacuguuucucuuucuaTT
  6677	1142	601	cuagaaagagaaacaguugguaa	double	602	agaaagagaaacaguugguTT	603	accaacuguuucucuuucuTT
  6678	1143	604	uagaaagagaaacaguugguaac	double	605	gaaagagaaacaguugguaTT	606	uaccaacuguuucucuuucTT
  6679	1144	607	agaaagagaaacaguugguaacu	double	608	aaagagaaacaguugguaaTT	609	uuaccaacuguuucucuuuTT
  6680	1145	610	gaaagagaaacaguugguaacuu	double	611	aagagaaacaguugguaacTT	612	guuaccaacuguuucucuuTT
  6681	1146	613	aaagagaaacaguugguaacuuu	double	614	agagaaacaguugguaacuTT	615	aguuaccaacuguuucucuTT
  6682	1147	616	aagagaaacaguugguaacuuuu	double	617	gagaaacaguugguaacuuTT	618	aaguuaccaacuguuucucTT
  6683	1148	619	agagaaacaguugguaacuuuug	double	620	agaaacaguugguaacuuuTT	621	aaaguuaccaacuguuucuTT
  6684	1149	622	gagaaacaguugguaacuuuugu	double	623	gaaacaguugguaacuuuuTT	624	aaaaguuaccaacuguuucTT
  6685	1150	625	agaaacaguugguaacuuuugug	double	626	aaacaguugguaacuuuugTT	627	caaaaguuaccaacuguuuTT
  6686	1151	628	gaaacaguugguaacuuuuguga	double	629	aacaguugguaacuuuuguTT	630	acaaaaguuaccaacuguuTT
  6687	1152	631	aaacaguugguaacuuuugugaa	double	632	acaguugguaacuuuugugTT	633	cacaaaaguuaccaacuguTT
  6688	1153	634	aacaguugguaacuuuugugaau	double	635	caguugguaacuuuugugaTT	636	ucacaaaaguuaccaacugTT
  6689	1154	637	acaguugguaacuuuugugaauu	double	638	aguugguaacuuuugugaaTT	639	uucacaaaaguuaccaacuTT
  6690	1155	640	caguugguaacuuuugugaauua	double	641	guugguaacuuuugugaauTT	642	auucacaaaaguuaccaacTT
  6691	1156	643	aguugguaacuuuugugaauuag	double	644	uugguaacuuuugugaauuTT	645	aauucacaaaaguuaccaaTT
  6692	1157	646	guugguaacuuuugugaauuagg	double	647	ugguaacuuuugugaauuaTT	648	uaauucacaaaaguuaccaTT
  6693	1158	649	uugguaacuuuugugaauuaggc	double	650	gguaacuuuugugaauuagTT	651	cuaauucacaaaaguuaccTT
  6694	1159	652	ugguaacuuuugugaauuaggcu	double	653	guaacuuuugugaauuaggTT	654	ccuaauucacaaaaguuacTT
  6695	1160	655	gguaacuuuugugaauuaggcug	double	656	uaacuuuugugaauuaggcTT	657	gccuaauucacaaaaguuaTT
  6696	1161	658	guaacuuuugugaauuaggcugu	double	659	aacuuuugugaauuaggcuTT	660	agccuaauucacaaaaguuTT
  6697	1162	661	uaacuuuugugaauuaggcugua	double	662	acuuuugugaauuaggcugTT	663	cagccuaauucacaaaaguTT
  6698	1163	664	aacuuuugugaauuaggcuguaa	double	665	cuuuugugaauuaggcuguTT	666	acagccuaauucacaaaagTT
  6699	1164	667	acuuuugugaauuaggcuguaac	double	668	uuuugugaauuaggcuguaTT	669	uacagccuaauucacaaaaTT
  6700	1165	670	cuuuugugaauuaggcuguaacu	double	671	uuugugaauuaggcuguaaTT	672	uuacagccuaauucacaaaTT
  6701	1166	673	uuuugugaauuaggcuguaacua	double	674	uugugaauuaggcuguaacTT	675	guuacagccuaauucacaaTT
  6702	1167	676	uuugugaauuaggcuguaacuac	double	677	ugugaauuaggcuguaacuTT	678	aguuacagccuaauucacaTT
  6703	1168	679	uugugaauuaggcuguaacuacu	double	680	gugaauuaggcuguaacuaTT	681	uaguuacagccuaauucacTT
  6704	1169	682	ugugaauuaggcuguaacuacuu	double	683	ugaauuaggcuguaacuacTT	684	guaguuacagccuaauucaTT
  6705	1170	685	gugaauuaggcuguaacuacuuu	double	686	gaauuaggcuguaacuacuTT	687	aguaguuacagccuaauucTT
  6706	1171	688	ugaauuaggcuguaacuacuuua	double	689	aauuaggcuguaacuacuuTT	690	aaguaguuacagccuaauuTT
  6707	1172	691	gaauuaggcuguaacuacuuuau	double	692	auuaggcuguaacuacuuuTT	693	aaaguaguuacagccuaauTT
  6708	1173	694	aauuaggcuguaacuacuuuaua	double	695	uuaggcuguaacuacuuuaTT	696	uaaaguaguuacagccuaaTT
  6709	1174	697	auuaggcuguaacuacuuuauaa	double	698	uaggcuguaacuacuuuauTT	699	auaaaguaguuacagccuaTT
  6710	1175	700	uuaggcuguaacuacuuuauaac	double	701	aggcuguaacuacuuuauaTT	702	uauaaaguaguuacagccuTT
  6711	1176	703	uaggcuguaacuacuuuauaacu	double	704	ggcuguaacuacuuuauaaTT	705	uuauaaaguaguuacagccTT
  6712	1177	706	aggcuguaacuacuuuauaacua	double	707	gcuguaacuacuuuauaacTT	708	guuauaaaguaguuacagcTT
  6713	1178	709	ggcuguaacuacuuuauaacuaa	double	710	cuguaacuacuuuauaacuTT	711	aguuauaaaguaguuacagTT
  6714	1179	712	gcuguaacuacuuuauaacuaac	double	713	uguaacuacuuuauaacuaTT	714	uaguuauaaaguaguuacaTT
  6715	1180	715	cuguaacuacuuuauaacuaaca	double	716	guaacuacuuuauaacuaaTT	717	uuaguuauaaaguaguuacTT
  6716	1181	718	uguaacuacuuuauaacuaacau	double	719	uaacuacuuuauaacuaacTT	720	guuaguuauaaaguaguuaTT
  6717	1182	721	guaacuacuuuauaacuaacaug	double	722	aacuacuuuauaacuaacaTT	723	uguuaguuauaaaguaguuIT
  6718	1183	724	uaacuacuuuauaacuaacaugu	double	725	acuacuuuauaacuaacauTT	726	auguuaguuauaaaguaguTT
  6719	1184	727	aacuacuuuauaacuaacauguc	double	728	cuacuuuauaacuaacaugTT	729	cauguuaguuauaaaguagTT
  6720	1185	730	acuacuuuauaacuaacaugucc	double	731	uacuuuauaacuaacauguTT	732	acauguuaguuauaaaguaTT
  6721	1186	733	cuacuuuauaacuaacauguccu	double	734	acuuuauaacuaacaugucTT	735	gacauguuaguuauaaaguTT
  6722	1187	736	uacuuuauaacuaacauguccug	double	737	cuuuauaacuaacauguccTT	738	ggacauguuaguuauaaagTT
  6723	1188	739	acuuuauaacuaacauguccugc	double	740	uuuauaacuaacauguccuTT	741	aggacauguuaguuauaaaTT
  6724	1189	742	cuuuauaacuaacauguccugcc	double	743	uuauaacuaacauguccugTT	744	caggacauguuaguuauaaTT
  6725	1190	745	uuuauaacuaacauguccugccu	double	746	uauaacuaacauguccugcTT	747	gcaggacauguuaguuauaTT
  6726	1191	748	uuauaacuaacauguccugccua	double	749	auaacuaacauguccugccTT	750	ggcaggacauguuaguuauTT
  6727	1192	751	uauaacuaacauguccugccuau	double	752	uaacuaacauguccugccuTT	753	aggcaggacauguuaguuaTT
  6728	1193	754	auaacuaacauguccugccuauu	double	755	aacuaacauguccugccuaTT	756	uaggcaggacauguuaguuTT
  6729	1352	757	uggcagaguuacaguucuguggu	double	758	gcagaguuacaguucugugTT	759	cacagaacuguaacucugcTT
  6730	1353	760	ggcagaguuacaguucugugguu	double	761	cagaguuacaguucuguggTT	762	ccacagaacuguaacucugTT
  6731	1354	763	gcagaguuacaguucugugguuu	double	764	agaguuacaguucugugguTT	765	accacagaacuguaacucuTT
  6732	1374	766	uuucauguuaguuaccuuauagu	double	767	ucauguuaguuaccuuauaTT	768	uauaagguaacuaacaugaTT
  6733	1375	769	uucauguuaguuaccuuauaguu	double	770	cauguuaguuaccuuauagTT	771	cuauaagguaacuaacaugTT
  6734	1376	772	ucauguuaguuaccuuauaguua	double	773	auguuaguuaccuuauaguTT	774	acuauaagguaacuaacauTT
  6735	1377	775	cauguuaguuaccuuauaguuac	double	776	uguuaguuaccuuauaguuTT	777	aacuauaagguaacuaacaTT
  6736	1378	778	auguuaguuaccuuauaguuacu	double	779	guuaguuaccuuauaguuaTT	780	uaacuauaagguaacuaacTT
  6737	1379	781	uguuaguuaccuuauaguuacug	double	782	uuaguuaccuuauaguuacTT	783	guaacuauaagguaacuaaTT
  6738	1380	784	guuaguuaccuuauaguuacugu	double	785	uaguuaccuuauaguuacuTT	786	aguaacuauaagguaacuaTT
  6739	1381	787	uuaguuaccuuauaguuacugug	double	788	aguuaccuuauaguuacugTT	789	caguaacuauaagguaacuTT
  6740	1382	790	uaguuaccuuauaguuacugugu	double	791	guuaccuuauaguuacuguTT	792	acaguaacuauaagguaacTT
  6741	1383	793	aguuaccuuauaguuacugugua	double	794	uuaccuuauaguuacugugTT	795	cacaguaacuauaagguaaTT
  6742	1384	796	guuaccuuauaguuacuguguaa	double	797	uaccuuauaguuacuguguTT	798	acacaguaacuauaagguaTT
  6743	1385	799	uuaccuuauaguuacuguguaau	double	800	accuuauaguuacuguguaTT	801	uacacaguaacuauaagguTT
  6744	1386	802	uaccuuauaguuacuguguaauu	double	803	ccuuauaguuacuguguaaTT	804	uuacacaguaacuauaaggTT
  6745	1387	805	accuuauaguuacuguguaauua	double	806	cuuauaguuacuguguaauTT	807	auuacacaguaacuauaagTT
  6746	1388	808	ccuuauaguuacuguguaauuag	double	809	uuauaguuacuguguaauuTT	810	aauuacacaguaacuauaaTT
  6747	1389	811	cuuauaguuacuguguaauuagu	double	812	uauaguuacuguguaauuaTT	813	uaauuacacaguaacuauaTT
  6748	1390	814	uuauaguuacuguguaauuagug	double	815	auaguuacuguguaauuagTT	816	cuaauuacacaguaacuauTT
  6749	1391	817	uauaguuacuguguaauuagugc	double	818	uaguuacuguguaauuaguTT	819	acuaauuacacaguaacuaTT
  6750	1392	820	auaguuacuguguaauuagugcc	double	821	aguuacuguguaauuagugTT	822	cacuaauuacacaguaacuTT
  6751	1393	823	uaguuacuguguaauuagugcca	double	824	guuacuguguaauuagugcTT	825	gcacuaauuacacaguaacTT
  6752	1394	826	aguuacuguguaauuagugccac	double	827	uuacuguguaauuagugccTT	828	ggcacuaauuacacaguaaTT
  6753	1395	829	guuacuguguaauuagugccacu	double	830	uacuguguaauuagugccaTT	831	uggcacuaauuacacaguaTT
  6754	1396	832	uuacuguguaauuagugccacuu	double	833	acuguguaauuagugccacTT	834	guggcacuaauuacacaguTT
  6755	1397	835	uacuguguaauuagugccacuua	double	836	cuguguaauuagugccacuTT	837	aguggcacuaauuacacagTT
  6756	1398	838	acuguguaauuagugccacuuaa	double	839	uguguaauuagugccacuuTT	840	aaguggcacuaauuacacaTT
  6757	1399	841	cuguguaauuagugccacuuaau	double	842	guguaauuagugccacuuaTT	843	uaaguggcacuaauuacacTT
  6758	1400	844	uguguaauuagugccacuuaaug	double	845	uguaauuagugccacuuaaTT	846	uuaaguggcacuaauuacaTT
  6759	1401	847	guguaauuagugccacuuaaugu	double	848	guaauuagugccacuuaauTT	849	auuaaguggcacuaauuacTT
  6760	1402	850	uguaauuagugccacuuaaugua	double	851	uaauuagugccacuuaaugTT	852	cauuaaguggcacuaauuaTT
  6761	1403	853	guaauuagugccacuuaauguau	double	854	aauuagugccacuuaauguTT	855	acauuaaguggcacuaauuTT
  6762	1404	856	uaauuagugccacuuaauguaug	double	857	auuagugccacuuaauguaTT	858	uacauuaaguggcacuaauTT
  6763	1405	859	aauuagugccacuuaauguaugu	double	860	uuagugccacuuaauguauTT	861	auacauuaaguggcacuaaTT
  6764	1406	862	auuagugccacuuaauguauguu	double	863	uagugccacuuaauguaugTT	864	cauacauuaaguggcacuaTT
  6765	1407	865	uuagugccacuuaauguauguua	double	866	agugccacuuaauguauguTT	867	acauacauuaaguggcacuTT
  6766	1408	868	uagugccacuuaauguauguuac	double	869	gugccacuuaauguauguuTT	870	aacauacauuaaguggcacTT
  6767	1409	871	agugccacuuaauguauguuacc	double	872	ugccacuuaauguauguuaTT	873	uaacauacauuaaguggcaTT
  6768	1410	874	gugccacuuaauguauguuacca	double	875	gccacuuaauguauguuacTT	876	guaacauacauuaaguggcTT
  6769	1411	877	ugccacuuaauguauguuaccaa	double	878	ccacuuaauguauguuaccTT	879	gguaacauacauuaaguggTT
  6770	1412	880	gccacuuaauguauguuaccaaa	double	881	cacuuaauguauguuaccaTT	882	ugguaacauacauuaagugTT
  6771	1413	883	ccacuuaauguauguuaccaaaa	double	884	acuuaauguauguuaccaaTT	885	uugguaacauacauuaaguTT
  6772	1414	886	cacuuaauguauguuaccaaaaa	double	887	cuuaauguauguuaccaaaTT	888	uuugguaacauacauuaagTT
  6773	1415	889	acuuaauguauguuaccaaaaau	double	890	uuaauguauguuaccaaaaTT	891	uuuugguaacauacauuaaTT
  6774	1416	892	cuuaauguauguuaccaaaaaua	double	893	uaauguauguuaccaaaaaTT	894	uuuuugguaacauacauuaTT
  6775	1435	895	aauaaauauaucuaccccagacu	double	896	uaaauauaucuaccccagaTT	897	ucugggguagauauauuuaTT
  6776	1436	898	auaaauauaucuaccccagacua	double	899	aaauauaucuaccccagacTT	900	gucugggguagauauauuuTT
  6777	1437	901	uaaauauaucuaccccagacuag	double	902	aauauaucuaccccagacuTT	903	agucugggguagauauauuTT
  6778	1438	904	aaauauaucuaccccagacuaga	double	905	auauaucuaccccagacuaTT	906	uagucugggguagauauauTT
  6779	1439	907	aauauaucuaccccagacuagau	double	908	uauaucuaccccagacuagTT	909	cuagucugggguagauauaTT
  6780	1440	910	auauaucuaccccagacuagaug	double	911	auaucuaccccagacuagaTT	912	ucuagucugggguagauauTT
  6781	1441	913	uauaucuaccccagacuagaugu	double	914	uaucuaccccagacuagauTT	915	aucuagucugggguagauaTT
  6782	1442	916	auaucuaccccagacuagaugua	double	917	aucuaccccagacuagaugTT	918	caucuagucugggguagauTT
  6783	1443	919	uaucuaccccagacuagauguag	double	920	ucuaccccagacuagauguTT	921	acaucuagucugggguagaTT
  6784	1444	922	aucuaccccagacuagauguagu	double	923	cuaccccagacuagauguaTT	924	uacaucuagucugggguagTT
  6785	1445	925	ucuaccccagacuagauguagua	double	926	uaccccagacuagauguagTT	927	cuacaucuagucugggguaTT
  6786	1446	928	cuaccccagacuagauguaguau	double	929	accccagacuagauguaguTT	930	acuacaucuagucugggguTT
  6787	1447	931	uaccccagacuagauguaguauu	double	932	ccccagacuagauguaguaTT	933	uacuacaucuagucuggggTT
  6788	1448	934	accccagacuagauguaguauuu	double	935	cccagacuagauguaguauTT	936	auacuacaucuagucugggTT
  6789	1449	937	ccccagacuagauguaguauuuu	double	938	ccagacuagauguaguauuTT	939	aauacuacaucuagucuggTT
  6790	1450	940	cccagacuagauguaguauuuuu	double	941	cagacuagauguaguauuuTT	942	aaauacuacaucuagucugTT
  6791	1451	943	ccagacuagauguaguauuuuuu	double	944	agacuagauguaguauuuuTT	945	aaaauacuacaucuagucuTT
  6792	1452	946	cagacuagauguaguauuuuuug	double	947	gacuagauguaguauuuuuTT	948	aaaaauacuacaucuagucTT
  6793	1453	949	agacuagauguaguauuuuuugu	double	950	acuagauguaguauuuuuuTT	951	aaaaaauacuacaucuaguTT
  6794	1454	952	gacuagauguaguauuuuuugua	double	953	cuagauguaguauuuuuugTT	954	caaaaaauacuacaucuagTT
  6795	1455	955	acuagauguaguauuuuuuguau	double	956	uagauguaguauuuuuuguTT	957	acaaaaaauacuacaucuaTT
  6796	1456	958	cuagauguaguauuuuuuguaua	double	959	agauguaguauuuuuuguaTT	960	uacaaaaaauacuacaucuTT
  6797	1457	961	uagauguaguauuuuuuguauaa	double	962	gauguaguauuuuuuguauTT	963	auacaaaaaauacuacaucTT
  6798	1458	964	agauguaguauuuuuuguauaau	double	965	auguaguauuuuuuguauaTT	966	uauacaaaaaauacuacauTT
  6799	1459	967	gauguagauuuuuuuguauaauu	double	968	uguaguauuuuuuguauaaTT	969	uuauacaaaaaauacuacaTT
  6800	1460	970	auguaguauuuuuuguauaauug	double	971	guaguauuuuuuguauaauTT	972	auuauacaaaaaauacuacTT
  6801	1461	973	uguaguauuuuuuguauaauugg	double	974	uaguauuuuuuguauaauuTT	975	aauuauacaaaaaauacuaTT
  6802	1462	976	guaguauuuuuuguauaauugga	double	977	aguauuuuuuguauaauugTT	978	caauuauacaaaaaauacuTT
  6803	1463	979	uaguauuuuuuguauaauuggau	double	980	guauuuuuuguauaauuggTT	981	ccaauuauacaaaaaauacTT
  6804	1464	982	aguauuuuuuguauaauuggauu	double	983	uauuuuuuguauaauuggaTT	984	uccaauuauacaaaaaauaTT
  6805	1465	985	guauuuuuuguauaauuggauuu	double	986	auuuuuuguauaauuggauTT	987	auccaauuauacaaaaaauTT
  6806	1466	988	uauuuuuuguauaauuggauuuc	double	989	uuuuuuguauaauuggauuTT	990	aauccaauuauacaaaaaaTl
  6807	1467	991	auuuuuuguauaauuggauuucc	double	992	uuuuuguauaauuggauuuTT	993	aaauccaauuauacaaaaaTT
  6808	1468	994	uuuuuuguauaauuggauuuccu	double	995	uuuuguauaauuggauuucTT	996	gaaauccaauuauacaaaaTT
  6809	1469	997	uuuuuguauaauuggauuuccua	double	998	uuuguauaauuggauuuccTT	999	ggaaauccaauuauacaaaTT
  6810	1470	1000	uuuuguauaauuggauuuccuaa	double	1001	uuguauaauuggauuuccuTT	1602	aggaaauccaauuauacaaTT
  6811	1471	1003	uuuguauaauuggauuuccuaau	double	1004	uguauaauuggauuuccuaTT	1005	uaggaaauccaauuauacaTT
  6812	1472	1006	uuguauaauuggauuuccuaaua	double	1007	guauaauuggauuuccuaaTT	1008	uuaggaaauccaauuauacTT
  6813	1473	1009	uguauaauuggauuuccuaauac	double	1010	uauaauuggauuuccuaauTT	1011	auuaggaaauccaauuauaTT
  6814	1540	1012	guauuuggaaauaaagucagaug	double	1013	auuuggaaauaaagucagaTT	1014	ucugacuuuauuuccaaauTT
  6815	1541	lOIS	uauuuggaaauaaagucagaugg	double	1016	uuuggaaauaaagucagauTT	1017	aucugacuuuauuuccaaaTT
  6816	1542	1018	auuuggaaauaaagucagaugga	double	1019	uuggaaauaaagucagaugTT	1020	aaucugacuuuauuuccaaTT
  6817	1543	1021	uuuggaaauaaagucagauggaa	double	1022	uggaaauaaagucagauggTT	1023	ccaucugacuuuauuuccaTT
  6818	1544	1024	uuggaaauaaagucagauggaaa	double	1025	ggaaauaaagucagauggaTT	1026	uccaucugacuuuauuuccTT
  6819	1545	1027	uggaaauaaagucagauggaaaa	double	1028	gaaauaaagucagauggaaTT	1029	uuccaucugacuuuauuucTT
  6820	1796	1030	ucccucccagaggagccaccagu	double	1031	ccucccagaggagccaccaTT	1032	ugguggcuccucugggaggTT
  6821	1797	1033	cccucccagaggagccaccaguu	double	1034	cucccagaggagccaccagTT	1035	cugguggcuccucugggagTT
  6822	1798	1036	ccucccagaggagccaccaguuc	double	1037	ucccagaggagccaccaguTT	1038	acugguggcuccucugggaTT
  6823	1799	1039	cucccagaggagccaccaguucu	double	1040	cccagaggagccaccaguuTT	1041	aacugguggcuccucugggTT
  6824	1800	1042	ucccagaggagccaccaguucuc	double	1043	ccagaggagccaccaguucTT	1044	gaacugguggcuccucuggTT
  6825	1801	1045	cccagaggagccaccaguucuca	double	1046	cagaggagccaccaguucuTT	1047	agaacugguggcuccucugTT
  6826	1843	1048	cuucucuccagcugacuaaacuu	double	1049	ucucuccagcugacuaaacTT	1050	guuuagucagcuggagagaTT
  6827	1869	1051	uucuguaccaguuaauuuuucca	double	1052	cuguaccaguuaauuuuucTT	1053	gaaaaauuaacugguacagTT
  6828	1870	1054	ucuguaccaguuaauuuuuccaa	double	1055	uguaccaguuaauuuuuccTT	1056	ggaaaaauuaacugguacaTT
  6829	1871	1057	cuguaccaguuaauuuuuccaac	double	1058	guaccaguuaauuuuuccaTT	1059	uggaaaaauuaacugguacTT
  6830	1872	1060	uguaacaguuaauuuuuccaacu	double	1061	uaccaguuaauuuuuccaaTT	1062	uuggaaaaauuaacugguaTT
  6831	1873	1063	guaccaguuaauuuuuccaacua	double	1064	accaguuaauuuuuccaacTT	1065	guuggaaaaauuaacugguTT
  6832	1874	1066	uaccaguuaauuuuuccaacuac	double	1067	ccaguuaauuuuuccaacuTT	1068	aguuggaaaaauuaacuggTT
  6833	1875	1069	accaguuaauuuuuccaacuacu	double	1070	caguuaauuuuuccaacuaTT	1071	uaguuggaaaaauuaacugTT
  6834	1897	1072	uaauagaauaaaggcaguuuucu	double	1073	auagaauaaaggcaguuuuTT	1074	aaaacugccuuuauucuauTT
  6835	1898	1075	aauagaauaaaggcaguuuucua	double	1076	uagaauaaaggcaguuuucTT	1077	gaaaacugccuuuauucuaTT
  6836	1899	1078	auagaauaaaggcaguuuucuaa	double	1079	agaauaaaggcaguuuucuTT	1080	agaaaacugccuuuauucuTT
  aSingle: Single overhang design 21 mer sense, corresponding 23 mer antisense with 2 nucleotides overhang at 3′ end; Double: double overhang design corresponding 19 mer sense and antisense strand each with 2 dT nucleotide overhang at 3′ end
  b“Start position” corresponds to the position within the sequence of human RhoA (Genbank accession no. NM_001664) mRNA to which the 5′-most nucleotide of the sense strand corresponds for single overhang designs; for double overhang designs, the 5′-most ribonucleotide of the sense strand corresponds to (Start position + 2)

• Based on these results, the invention specifically provides an iRNA agent that includes a sense strand having at least 15 contiguous nucleotides of the sense strand sequences of the agents provided in Table 1 under agent numbers 6477-6836, and an antisense strand having at least 15 contiguous nucleotides of the antisense sequences of the agents provided in Table 1 under agent numbers 6477 to 6836.
• The iRNA agents shown in Table 1 are composed of two strands of 19 nucleotides in length which are complementary or identical to the target sequence, plus a 3′-TT overhang. The present invention provides agents that comprise 15 contiguous nucleotides from these agents. However, while these lengths may potentially be optimal, the iRNA agents are not meant to be limited to these lengths. The skilled person is well aware that shorter or longer iRNA agents may be similarly effective, since, within certain length ranges, the efficacy is rather a finction of the nucleotide sequence than strand length. For example, Yang, et al.,PNAS99:9942-9947 (2002), demonstrated similar efficacies for iRNA agents of lengths between 21 and 30 base pairs. Others have shown effective silencing of genes by iRNA agents down to a length of approx. 15 base pairs (Byrom, et al., “Inducing RNAi with siRNA Cocktails Generated by RNase III”Tech Notes10(1), Ambion, Inc., Austin, Tex.).
• Therefore, it is possible and contemplated by the instant invention to select from the sequences provided in Table 1 under agent numbers 6477 to 6836 a partial sequence of between 15 to 22 nucleotides for the generation of an iRNA agent derived from one of the sequences provided in Table 1 under agent numbers 6477 to 6836. Alternatively, one may add one or several nucleotides to one of the sequences provided in Table 1 under agent numbers 6477 to 6836, or an agent comprising 15 contiguous nucleotides from one of these agents, preferably, but not necessarily, in such a fashion that the added nucleotides are complementary to the respective sequence of the target gene, e.g., RhoA. For example, the first 15 nucleotides from one of the agents can be combined with the 8 nucleotides found 5′ to these sequence in the RhoA mRNA to obtain an agent with 23 nucleotides in the sense and antisense strands. All such derived iRNA agents are included in the iRNA agents of the present invention, provided they essentially retain the ability to inhibit RhoA expression in cultured human RhoA expressing cells.
• The antisense strand of an iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30, nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
• The sense strand of an iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
• The double stranded portion of an iRNA agent should be equal to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs in length. It should be equal to or less than 60, 50, 40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.
• Generally, the iRNA agents of the instant invention include a region of sufficient complementarity to the respective RhoA gene, and are of sufficient length in terms of nucleotides, that the iRNA agent, or a fragment thereof, can mediate down regulation of the RhoA gene. The ribonucleotide portions of the antisense strands of the iRNA agents of Table 1 under agent numbers 6477 to 6836 are fully complementary to the mRNA sequences of the RhoA gene, respectively, and ribonucleotide portion of their sense strands are fully complementary to the ribonucleotide portions of the respective antisense strands, except for the two 3′-terminal nucleotides on the antisense strand in single overhang design iRNA agents. However, it is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of a RhoA mRNA.
• Therefore, the iRNA agents of the instant invention include agents comprising a sense strand and antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical, as defined below, to one of the sequences of Table 1 under agent numbers 6477 to 6836, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit RhoA expression in cultured human RhoA expressing cells, respectively. These agents will therefore possess at least 15 nucleotides identical to one of the sequences of Table 1 under agent numbers 6477 to 6836, but 1, 2 or 3 base mismatches with respect to either the target RhoA mRNA sequence or between the sense and antisense strand are introduced. Mismatches to the target RhoA mRNA sequence, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, most preferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus of the sense strand or the 3′-terminus of the antisense strand. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character of the molecule.
• It is preferred that the sense and antisense strands be chosen such that the iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, an iRNA agent contains sense and antisense strands, preferably paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred siRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 to 4, or preferably 2 or 3 nucleotides, in length, at one or both ends of the iRNA agent. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The unpaired nucleotides forming the overhang can be ribonucleotides, or they can be deoxyribonucleotides, preferably thymidine. 5′-ends are preferably phosphorylated.
• Preferred lengths for the duplexed region are between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA agent range discussed above. siRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the siRNA agent are linked, e.g., covalently linked, are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.
• Evaluation of Candidate iRNA Agents
• A candidate iRNA agent can be evaluated for its ability to downregulate target gene expression. For example, a candidate iRNA agent can be provided, and contacted with a cell, that expresses the target gene, e.g., the RhoA gene, either endogenously or because it has been transfected with a construct from which a RhoA protein can be expressed. The level of target gene expression prior to and following contact with the candidate iRNA agent can be compared, e.g., on an MRNA or protein level. If it is determined that the amount of RNA or protein expressed from the target gene is lower following contact with the iRNA agent, then it can be concluded that the iRNA agent downregulates target gene expression. The level of target RhoA RNA or RhoA protein in the cell can be determined by any method desired. For example, the level of target RNA can be determined by Northern blot analysis, reverse transcription coupled with polymerase chain reaction (RT-PCR), or RNAse protection assay. The level of protein can be determined, for example, by Western blot analysis or immuno-fluorescence. Preferably, the assay also tests the ability of the iRNA agent to inhibit RhoA expression on a functional level, e.g. by assessing the ability of the iRNA agent to facilitate neuronal growth, e.g. the restoration of neurite outgrowth on an otherwise inhibitory substrate, e.g a substrate comprising myelin.
• Stability Testing, Modification, and Retesting of iRNA Agents
• A candidate iRNA agent can be evaluated with respect to stability, e.g., its susceptibility to cleavage by an endonuclease or exonuclease, such as when the iRNA agent is introduced into the body of a subject. Methods can be employed to identify sites that are susceptible to modification, particularly cleavage, e.g., cleavage by a component found in the body of a subject.
• When sites susceptible to cleavage are identified, a further iRNA agent can be designed and/or synthesized wherein the potential cleavage site is made resistant to cleavage, e.g. by introduction of a 2′-modification on the site of cleavage, e.g. a 2′-O-methyl group. This further iRNA agent can be retested for stability, and this process may be iterated until an iRNA agent is found exhibiting the desired stability.
• In Vivo Testing
• An iRNA agent identified as being capable of inhibiting RhoA gene expression can be tested for functionality in vivo in an animal model (e.g., in a mammal, such as in mouse or rat). For example, the iRNA agent can be administered to an animal, and the iRNA agent evaluated with respect to its biodistribution, stability, and its ability to inhibit RhoA gene expression or reduce a biological or pathological process mediated at least in part by RhoA.
• The iRNA agent can be administered directly to the target tissue, e.g. the spinal cord, and, in the case of a spinal cord injury model, to the site of spinal cord injury, such as by injection. Preferably, the iRNA agent is administered to the animal model in the same manner that it would be administered to a human.
• The iRNA agent can also be evaluated for its intracellular distribution. The evaluation can include determining whether the iRNA agent was taken up into the cell. The evaluation can also include determining the stability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNA agent in vivo can be facilitated by use of an iRNA agent conjugated to a traceable marker (e.g., a fluorescent marker such as fluorescein; a radioactive label, such as³⁵S,³²P,³³P, or³H; gold particles; or antigen particles for immunohistochemistry).
• The iRNA agent can be evaluated with respect to its ability to down regulate RhoA gene expression. Levels of RhoA gene expression in vivo can be measured, for example, by in situ hybridization, or by the isolation of RNA from tissue prior to and following exposure to the iRNA agent. Where the animal needs to be sacrificed in order to harvest the tissue, an untreated control animal will serve for comparison. RhoA mRNA can be detected by any desired method, including but not limited to RT-PCR, Northern blot, branched-DNA assay, or RNAase protection assay. Alternatively, or additionally, RhoA gene expression can be monitored by performing Western blot analysis on tissue extracts treated with the iRNA agent.
• Animal models may be used to establish the concentration necessary to achieve a certain desired effect (e.g., EC₅₀or ED₅₀). Such animal models may include transgenic animals that express a human gene, e.g., a gene that produces a target human RhoA RNA. In another embodiment, the composition for testing includes an iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RhoA RNA in the animal model and the target RhoA RNA in a human.
• iRNA Chemistry
• Described herein are isolated iRNA agents, e.g., ds RNA agents that mediate RNAi to inhibit expression of a RhoA gene.
• RNA agents discussed herein include otherwise unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al.Nucleic Acids Res.22: 2183-2196, 1994. Such rare or unusual RNAs, often termed modified RNAs (apparently because they are typically the result of a post-transcriptional modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of the above are discussed herein.
• Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. It may be desirable to modify one or both of the antisense and sense strands of an iRNA agent. As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most, cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. Similarly, a modification may occur on the sense strand, antisense strand, or both. In some cases, the sense and antisense strand will have the same modifications or the same class of modifications, but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it may be desirable to modify only one strand, e.g. the sense strand.
• Two prime objectives for the introduction of modifications into iRNA agents is their stabilization towards degradation in biological environments and the improvement of pharmacological properties, e.g. pharmacodynamic properties, which are further discussed below. Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described in co-owned PCT Application No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can include a non-naturally occurring base, such as the bases described in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule. Exemplary features of non-naturally occurring sugars for use in iRNA agents are described in co-owned PCT Application No. PCT/US2004/11829, filed Apr. 16, 2003.
• An iRNA agent can include an internucleotide linkage (e.g., the chiral phosphorothioate linkage) useful for increasing nuclease resistance. In addition, or in the alternative, an iRNA agent can include a ribose mimic for increased nuclease resistance. Exemplary intemucleotide linkages and ribose mimics for increased nuclease resistance are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.
• An iRNA agent can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10, 2004.
• An iRNA agent can have a ZXY structure, such as is described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.
• An iRNA agent can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with iRNA agents are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.
• In another embodiment, the iRNA agent can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type. iRNA agents complexed to a delivery agent are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.
• An iRNA agent can have non-canonical pairings, such as between the sense and antisense sequences of the iRNA duplex. Exemplary features of non-canonical iRNA agents are described in co-owned PCT Application No. PCT/US2004/07070, filed on Mar. 8, 2004.
• Enhanced Nuclease Resistance
• An iRNA agent, e.g., an iRNA agent that targets RhoA, can have enhanced resistance to nucleases. Naked RNA is often an easy prey for nucleolytic enzymes, such as exonucleases and endonucleases, which are omnipresent in biological media, such as the cellular cytoplasm, blood, or cerebrospinal fluid (CSF). Quick degradation can severly hamper the ability of an siRNA to inhibit the expression of a target gene. The vulnerability towards nucleolytic degradation can be greatly reduced by chemically modifying certain nucleotides of an siRNA. However, adding modifications in order to stabilize an siRNA sometimes represents a trade-off with its activity, and stabilizing modifications may even introduce toxic effects. It is therefore desirable to introduce the minimum number of modifications that still imparts the desired level of stability. Modifications in the sense strand usually have less impact on the activity of an siRNA.
• In order to increase the stability of an siRNA towards nucleolytic degradation by endonucleases, it is therefore advantageous to modify only a limited number of nucleotides in particularly degradation prone positions, as described in co-owned U.S. Application No. 60/559,917, filed on May 4, 2004, co-owned U.S. Application No. 60/574,744, filed on May 27, 2004, and co-owned international application PCT/US2005/018931, filed May 27, 2005. We have determined that pyrimidine nucleotides, and specifically the 5′ nucleotide in a 5′-ua-3′ sequence context, a 5′-ug-3′ sequence context, a 5′-ca-3′ sequence context, a 5′-uu-3′ sequence context, or a 5′-cc-3′ sequence context are particularly prone to degradative attack, in that approximate order. Sufficiently stable and highly active siRNAs have been obtained by our laboratory when the 5′-most pyrimidines in all occurrences of thesequence contexts 5′-ua-3′ and 5′-ca-3′, or in all occurrences of 5′-ua-3′, 5′-ca-3′, and 5′-uu-3′, or in all occurrences of 5′-ua-3′, 5′-ca-3′, 5′-uu-3′, and 5′-ug-3′ were replaced by 2′-modified nucleotides, such as 2′-O-methyl nucleotides, in both strands. Alternatively, 2′-modifying all pyrimidine nucleotides in the sense strand and the 5′-most pyrimidines in all occurrences of thesequence contexts 5′-ua-3′ and 5′-ca-3′ in the antisense strand has given good results in terms of activity and stability. Sometimes, it has been necessary to 2′-modify all pyrimidine nucleotides in the sense strand and the 5′-most pyrimidines in all occurrences of thesequence contexts 5′-ua-3′, 5′-ca-3′, 5′-uu-3′, and 5′-ug-3′ in the antisense strand. The iRNA agent can include at least 2, at least 3, at least 4 or at least 5 of such dinucleotides.
• Preferably, the 2′-modified nucleotides include, for example, a 2′-modified ribose unit, e.g., the 2′-hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
• Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE and aminoalkoxy, O(CH₂)_nAMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
• “Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.
• Preferred substitutents are 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.
• The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An iRNA agent can be further modified by including a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus can be blocked with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′ exonucleolytic cleavage. While not being bound by theory, a 3′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.
• Nucleolytic cleavage can also be inhibited by the introduction of phosphate linker modifications, e.g., phosphorothioate linkages. Thus, preferred iRNA agents include nucleotide dimers enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at a nonbridging position normally occupied by oxygen. The heteroatom can be S, Se, Nr₂, or Br₃. When the heteroatom is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Modified phosphate linkages are particularly efficient in inhibiting exonucleolytic cleavage when introduced near the 5′- or 3′-terminal positions, and preferably the 5′-terminal positions, of an iRNA agent.
• 5′ conjugates can also inhibit 5′-3′ exonucleolytic cleavage. While not being bound by theory, a 5′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.
• An iRNA agent can have increased resistance to nucleases when a duplexed iRNA agent includes a single-stranded nucleotide overhang on at least one end. In preferred embodiments, the nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment, the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide pair contains a purine base, and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs. In further embodiments, the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in an exemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferred embodiments, the nucleotide overhang is on the 3′-end of the antisense strand. In one embodiment, the iRNA agent includes themotif 5′-CGC-3′ on the 3′-end of the antisense strand, such that a 2-nt overhang 5′-GC-3′ is formed.
• Thus, an iRNA agent can include modifications so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or Nuclease Resistance promoting Monomers, the corresponding modifications as NRM modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC, or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.
• One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent.
• NRM modifications include some which can be placed only at the terminus and others which can go at any position. Some NRM modifications can inhibit hybridization so it is preferable to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of a sequence which targets a subject sequence or gene, particularly on the antisense strand. They can be used anywhere in a sense strand, provided that sufficient hybridization between the two strands of the ds iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sense strand, as it can minimize off-target silencing.
• In most cases, NRM modifications will be distributed differently depending on whether they are comprised on a sense or antisense strand. If on an antisense strand, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference). Cleavage of the target occurs about in the middle of a 20 or 21 nt antisense strand, or about 10 or 11 nucleotides upstream of the first nucleotide on the target mRNA which is complementary to the antisense strand. As used herein cleavage site refers to the nucleotides on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means the nucleotides within 1, 2, or 3 nucleotides of the cleavagee site, in either direction.
• Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sense or antisense strand.
• Tethered Ligands
• The properties of an iRNA agent, including its pharmacological properties, can be influenced and tailored, for example, by the introduction of ligands, e.g. tethered ligands.
• A wide variety of entities, e.g., ligands, can be tethered to an iRNA agent, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but that is only preferred, entities can be coupled at other points to an iRNA agent.
• Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. The ligand or tethered ligand may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., TAP-(CH₂)_nNH₂may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.
• In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
• Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
• Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophilic molecules, lipids, lectins, steroids (e.g.,uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.
• The ligand may be a naturally occurring or recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
• Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a thyrotropin, melanotropin, surfactant protein A, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGD peptide mimetic.
• Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. low density lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA), or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
• The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
• In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
• A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
• In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
• In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include the B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
• In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
• 5′-Phosphate Modifications
• In preferred embodiments, iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications of the antisense strand include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).
• The sense strand can be modified in order to inactivate the sense strand and prevent formation of an active RISC, thereby potentially reducing off-target effects. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage.
• Transport of iRNA Agents into Cells
• Not wishing to be bound by any theory, the chemical similarity between cholesterol-conjugated iRNA agents and certain constituents of lipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) may lead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL) in blood and/or the interaction of the iRNA agent with cellular components having an affinity for cholesterol, e.g. components of the cholesterol transport pathway. Lipoproteins as well as their constituents are taken up and processed by cells by various active and passive transport mechanisms, for example, without limitation, endocytosis of LDL-receptor bound LDL, endocytosis of oxidized or otherwise modified LDLs through interaction with Scavenger receptor A, Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver, pinocytosis, or transport of cholesterol across membranes by ABC (ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 or ABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoy facilitated uptake by cells possessing such transport mechanisms, e.g. cells of the liver. As such, the present invention provides evidence and general methods for targeting iRNA agents to cells expressing certain cell surface components, e.g. receptors, by conjugating a natural ligand for such component (e.g. cholesterol) to the iRNA agent, or by conjugating a chemical moiety (e.g. cholesterol) to the iRNA agent which associates with or binds to a natural ligand for the component (e.g. LDL, HDL).
• Other Embodiments
• An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al.Proc. Natl. Acad. Sci. USA91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.
• Formulation
• The iRNA agents described herein can be formulated for administration to a subject.
• For ease of exposition, the formulations, compositions, and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions, and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.
• A formulated iRNA agent composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA agent is in an aqueous phase, e.g., in a solution that includes water.
• The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA agent composition is formulated in a manner that is compatible with the intended method of administration.
• An iRNA agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an iRNA agent, e.g., a protein that complexes with the iRNA agent to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
• In one embodiment, the iRNA agent preparation includes two or more iRNA agent(s), e.g., two or more iRNA agents that can mediate RNAi with respect to the same gene, or different alleles of the gene, or with respect to different genes. Such preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different iRNA agent species. Such iRNA agents can mediate RNAi with respect to a similar number of different genes.
• Where the two or more iRNA agents in such preparation target the same gene, they can have target sequences that are non-overlapping and non-adjacent, or the target sequences may be overlapping or adjacent.
• Disorders Associated with RhoA Expression
• An iRNA agent that targets RhoA, e.g., an iRNA agent described herein, can be used to treat a subject, e.g., a human having or at risk for developing a disease or disorder associated with RhoA gene expression or treating a subject where a biological process mediated by RhoA is unwanted. Since Nogo-L, RhoA, and Nogo-R participate in inhibiting axonal growth and elongations, the iRNA agents of the present invention are used to reverse this inhibition leading to nerve/axonal growth and elongation. Such a treatment is useful in treating injuries to the nervous system such as spinal cord injury or peripheral nerve death (caused by, e.g., Metastatic cancers of the CNS, e.g., gliomas (such as glioblastomas, astrocytomas, oligodendrogliomas, ependymomas), meningiomas, medulloblastomas, neuroblastomas, choroid plexus papillomas, sarcomas can also be treated by the iRNA agents described herein. Other indications include diseases of the central nervous system, including but not limited to encephalomyelitis, ischemic stroke, Alzheimer's Disease, spongiform encephalopathy, Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), multiple sclerosis, transverse myelitis, motor neuron disease, Guillan Barre, Anterior Spinal Artery Syndrome, and schizophrenia.
• For example, an iRNA agent that targets RhoA mRNA can be used to treat a subject with a spinal cord injury or a subject having another pathological state which can be ameliorated, at least in part, by nerve growth and elongation. In such a use, an iRNA agent of the present invention is administered preferably locally at the site of nerve damage or the site at which the inhibitory effects of RhoA is desired to be reversed. Administration of the iRNA agent leads to decrease in RhoA protein resulting in reversing Nogo mediated inhibition of axonal elongation and growth.
• Treatment Methods and Routes of Delivery
• A composition that includes an iRNA agent, e.g., an iRNA agent that targets RhoA, can be delivered to a subject by a variety of routes to achieve either local delivery to the site of action of systemic delivery to the subject. Exemplary routes include direct injection to the site of treatment, intrathecal, parenchymal, intravenous, nasal, oral, and ocular delivery. The preferred means of administering the iRNA agents of the present invention is through direct injection or infusion to the site of treatment.
• An iRNA agent can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more species of an iRNA agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
• The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
• The route of delivery can be dependent on the disorder of the patient. In general, the delivery of the iRNA agents of the present invention is done to achieve systemic delivery into the subject. One preferred means of achieving this is through parenteral administration. In a particularly preferred embodiment, the application is achieved by direct application of the pharmaceutical composition to the site of nerve injury, such as the the site of spinal cord injury. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
• Using the small interfering RNA vectors previously described, the invention also provides devices, systems, and methods for delivery of small interfering RNA to target locations in the nervous system and or/the brain. The envisioned route of delivery is through the use of implanted, indwelling, intrathecal or intraparenchymal catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into local nerves or local brain tissue. The proximal end of these catheters may be connected to an implanted, intrathecal or intracerebral access port surgically affixed to the patient's body or cranium, or to an implanted drug pump located in the patient's torso.
• Alternatively, implantable delivery devices, such as an implantable pump may be employed. Examples of the delivery devices within the scope of the invention include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously in the body or on the cranium, and provides an access port through which therapeutic agents may be delivered to the nerves or brain. Delivery occurs through a stereotactically implanted polyurethane catheter. Two models of catheters that can finction with the Model 8506 access port include the Model 8770 ventricular catheter by Medtronic, Inc., for delivery to the intracerebral ventricles, which is disclosed in U.S. Pat. No. 6,093,180, incorporated herein by reference, and the IPA1 catheter by Medtronic, Inc., for delivery to the brain tissue itself (i.e., intraparenchymal delivery), disclosed in U.S. Ser. Nos. 09/540,444 and 09/625,751, which are incorporated herein by reference. The latter catheter has multiple outlets on its distal end to deliver the therapeutic agent to multiple sites along the catheter path. In addition to the aforementioned device, the delivery of the small interfering RNA vectors in accordance with the invention can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference. Using the teachings of the invention and those of skill in the art will recognize that these and other devices and systems may be suitable for delivery of small interfering RNA vectors for the treatment of pain in accordance with the invention.
• In one such embodiment, the method further comprises the steps of implanting a pump outside the body or brain, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of the at least one small interfering RNA or small interfering RNA vector through the discharge portion of the catheter. A further embodiment comprises the further step of periodically refreshing a supply of the at least one small interfering RNA or small interfering RNA vector to the pump outside said body or brain.
• Thus, the invention includes the delivery of small interfering RNA vectors using an implantable pump and catheter, like that taught in U.S. Pat. Nos. 5,735,814 and 6,042,579, and further using a sensor as part of the infusion system to regulate the amount of small interfering RNA vectors delivered to the nerves or brain, like that taught in U.S. Pat. No. 5,814,014. Other devices and systems can be used in accordance with the method of the invention, for example, the devices and systems disclosed in U.S. Ser. No. 09/872,698 (filed Jun. 1, 2001) and Ser. No. 09/864,646 (filed May 23, 2001), which are incorporated herein by reference.
• Preferably, the outlet of the pump or catheter is placed in close proximity of the desired site of action of the pharmaceutical composition, such as near the site of spinal cord, or other nerve, injury.
• Administration can be provided by the subject or by another person, e.g., a caregiver. A caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.
• The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.
• The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.
• The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.
• The term “co-administration” refers to administering to a subject two or more agents, and in particular two or more iRNA agents. The agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulation and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be co-administered. In one embodiment, both Nogo-L, RhoA, and Nogo-R iRNA agents are co-administered.
• The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
• Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffmose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.
• Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
• Dosage. An iRNA agent can be administered at a unit dose less than about 75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of bodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of iRNA agent per kg of bodyweight. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular, intrathecally, or directly into an organ), an inhaled dose, or a topical application.
• Delivery of an iRNA agent directly to an organ (e.g., directly to the liver) can be at a dosage on the order of about 0.00001 mg to about 3 mg per organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.
• The dosage can be an amount effective to treat or prevent a disease or disorder.
• In one embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. Because iRNA agent mediated silencing can persist for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen.
• In one embodiment, a subject is administered an initial dose, and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into an siRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 to 75 mg/kg of body weight per day, e.g., 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of body weight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
• The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
• Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.001 g to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).
• The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary, or topical, such as intrathecal or at the site of nerve injury. For example, topical formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning.
• Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of an iRNA agent such as an siRNA used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays. For example, the subject can be monitored after administering an iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.
• Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient, or of drug accumulation at the site of application when delivering locally, e.g. at the site of nerve injusry, e.g. at the site of spinal cord injury. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
• Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models as described above.
• The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
• Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 2.

  TABLE 2
  Abbreviations of nucleotide monomers used in nucleic acid
  sequence representation. It will be understood that these
  monomers, when present in an oligonucleotide, are mutually
  linked by 5′-3′-phosphodiester bonds.

  	Abbreviationa	Nucleotide(s)
  	A, a	2′-deoxy-adenosine-5′-phosphate,
  		adenosine-5′-phosphate
  	C, c	2′-deoxy-cytidine-5′-phosphate,
  		cytidine-5′-phosphate
  	G, g	2′-deoxy-guanosine-5′-phosphate,
  		guanosine-5′-phosphate
  	T, t	2′-deoxy-thymidine-5′-phosphate,
  		thymidine-5′-phosphate
  	U, u	2′-deoxy-uridine-5′-phosphate,
  		uridine-5′-phosphate
  	N, n	any 2′-deoxy-nucleotide/nucleotide
  		(G, A, C, or T, g, a, c or u)
  	am	2′-O-methyladenosine-5′-phosphate
  	cm	2′-O-methylcytidine-5′-phosphate
  	gm	2′-O-methylguanosine-5′-phosphate
  	tm	2′-O-methyl-thymidine-5′-phosphate
  	um	2′-O-methyluridine-5′-phosphate
  	A,C,G,T,U,a,	underlined: nucleoside-5′-phosphorothioate
  	c,g,t,u
  	-Chol	1-{6-[cholester-3-yloxycarbonylamino]-
  		hexanoyl}-4-hydroxy-pyrrolidin-3-
  		phosphorothioate diester
  	acapital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)

  
  Source of Reagents
• Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Example 1Selection of Sequences
• Sequence alignment was performed to identify regions within the sequence of human RhoA mRNA with full homology to the respective sequences in both mouse and rat RhoA mRNA (human RhoA mRNA: Genbank accession no. NM_—001664; mouse RhoA MRNA: Genbank accession no. NM_—016802; rat RhoA mRNA: Genbank accession no. NM_—057132). Within the regions of homology thus identified, all possible contiguous sequences of 19 nucleotides were examined by further BLAST comparison for potential cross-reactivity of an siRNA comprising such sequence to other mRNA sequences present in humans. Only sequences with 3 or more mismatches to any other human mRNA or genomic sequence were chosen. The resulting set of 19 nt sequences is represented in the sense strand ribonucleotide sequences of the double-overhang iRNA agents given in Table 1.
• In order to maximise the stability of the siRNAs for testing in biological media, particularly towards nucleolytic attack by endo- and exonucleases, the siRNAs were synthesized such that in the sense strands, all cytidine and uridine nucleotides comprise a 2′-O-methyl group, and in the antisense strand, all cytidines and uridines appearing in a sequence context of 5′-ca-3′ or 5′-ua-3′ comprise a 2′-O-methyl group.
• To the same end, phosphorothioate linkages were introduced between 3′-terminal 5′-TT-3′-group thymidines. It has been our experience that the most active exonucleases in serum and other biological media relevant for the in vivo activity of siRNAs act by degradingsiRNA strands 3′-5′. It has proven advantageous, and often sufficient, to replace the 2 penultimate nucleotides in the antisense strand by 2′-O-methyl-5′-phosphorothioate-modified nucleotides (e.g. the nucleotides in positions 21 and 22, counting 5′ to 3′, of a 23-nucleotide antisense strand); sometimes it is sufficient to modify only the penultimate nucleotide, or to use only 5′-phosphorothioate-modified nucleotides, or both. The sense strand may be protected in a similar fashion, and/or it may be 3′-conjugated to a tethered ligand via a phosphodiester or a phosphorothioate diester.
• In addition to the sequences selected as described above, four siRNAs were synthesized which corresponded to four of those utilized by the authors of Ahmed, Z.,et al, Mol Cell Neurosci. 2005, 28:509-23. AL-DP-5850 corresponds to RHO-A1 of Ahmed et al., supra, AL-DP-5851 to RHO-A2, AL-DP-5852 to RHO-A5 and AL-DP-5853 to RHO-A4 of Ahmed et al., supra.
Example 2siRNA Synthesis
• Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Glen Research, Sterling Va.) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were 30 generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
• Deprotection and purification by anion exchange HPLC of the crude oligoribonucleotides were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The purified RNA solution was stored at −20° C. until use.
• As a result of the synthesis strategy described above, all oligonucleotides synthesized as described above do not comprise a phosphate group on their 5′-most nucleotide.
• Cholesterol was 3′-conjugated to siRNA as illustrated inFIG. 1. For the synthesis of these 3′-cholesterol-conjugated siRNAs, an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
  Diethyl-2-azabutane-1,4-dicarboxylate AA

  
• A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until the completion of reaction was ascertained by TLC (19 h). After 19 h which it was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
  3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB

  
• Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. the completion of the reaction was ascertained by TLC. Thereaction mixture 1 5 was concentrated in vacuum and to the ethylacetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
  3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC

  
• 3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated in vacuum and the residue water was added and the product was extracted with ethyl acetate. The crude. product was purified by converting into hydrochloride salt.
  3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

  
• The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
  1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

  
• Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted topH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to a residue. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
  [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF

  
• Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated in vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl₃) (89%).
  (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG

  
• Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated in vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with IM aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g, 95%).
  Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH

  
• Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
  Cholesterol Derivatised CPG AI

  
• Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mm/g) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered finnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement (37 mM/g).
• The synthesis and structure of cholesterol conjugated RNA strands is illustrated inFIG. 1.
• The siRNAs listed Table 3 were synthesized for activity screening.

  TABLE 3
  siRNAs specific for RhoA

  SEQ

  SEQ

  Agent

  ID

  ID

  C. a.

  number	Sense strand	NO.	Antisense strand	NO.	#1

  AL-DP-5850	gauuaugaccgucugaggcTT	1081	gccucagucggucauaaucTT	1082	n.a.
  AL-DP-5851	ggaucuucggaaugaugagTT	1083	cucaucauuccgaagauccTT	1084	n.a.
  AL-DP-5852	agaccaaagacggagugagTT	1085	cucacuccgucuuuggucuTT	1086	na.
  AL-DP-5853	ugaagcaggagccgguaaaTT	1087	uuuaccggcuccugcuucaTT	1088	na.
  AL-DP-5972	gcmumacmcmagumaumumumagaagcmTT	1089	gcuucumaaaumacuggumagcTT	1090	6661
  AL-DP-5973	cmumacmcmagumaumumumagaagcmcmTT	1091	ggcuucumaaaumacuggumagTT	1092	6662
  AL-DP-5974	gcmumgumaacmumacmumumumaumaacmTT	1093	guumaumaaagumaguumacmagcTT	1094	6712
  AL-DP-5975	gumumacmumgumgumaaumumagumgcmTT	1095	gcmacumaauumacmacmagumaacTT	1096	6751
  AL-DP-5976	cmcmacmumumaaumgumaumgumumacmcmTT	1097	ggumaacmaumacmauumaaguggTT	1098	6769
  AL-DP-5977	cmagcmcmcmumgaumagumumumagaaTT	1099	uucumaaacumaucmagggcugTT	1100	6521
  AL-DP-5978	gcmcmcmumgaumagumumumagaaaaTT	1101	uuuucumaaacumaucmagggcTT	1102	6523
  AL-DP-5979	cmcmcmumgaumagumumumagaaaacmTT	1103	guuuucumaaacumaucmagggTT	1104	6524
  AL-DP-5980	gaumagumumumagaaaacmaumcmcmTT	1105	ggauguuuucumaaacumaucTT	1106	6528
  AL-DP-5981	umagumumumagaaaacmaumcmcmcmaTT	1107	ugggauguuuucumaaacumaTT	1108	6530
  AL-DP-5982	cmagacmumagaumgumagumaumumumTT	1109	aaaumacumacmaucumagucugTT	1110	6790
  AL-DP-5983	cmcmcmcmagacmumagaumgumagumaTT	1111	umacumacmaucumagucuggggTT	1112	6787
  AL-DP-5984	cmcmagacmumagaumgumagumaumumTT	1113	aaumacumacmaucumagucuggTT	1114	6789
  AL-DP-5985	cmcmcmagacmumagaumgumagumaumTT	1115	aumacumacmaucumagucugggTT	1116	6788
  AL-DP-5986	umgcmacmumumaaumgumaumgumumaTT	1117	umaacmaumacmauumaaguggcmaTT	1118	6767
  AL-DP-5987	umgcmumgumumumaumumaaumcmumumagTT	1119	cumaagauumaaumaaacmagcmaTT	1120	6614
  AL-DP-5988	umcmaumgumumagumumacmcmumumaumaTT	1121	umaumaaggumaacumaacmaugaTT	1122	6732
  AL-DP-5989	cmcmagumumaaumumumumumcmcmaacmumTT	1123	aguuggaaaaauumaacuggTT	1124	6832
  AL-DP-5990	umacmcmumaagaumumacmaaaumcmaTT	1125	ugauuugumaaucuumaggumaTT	1126	6650
  AL-DP-5991	umcmumumgcmumacmcmagumaumumumagTT	1127	cumaaaumacuggumagcmaagaTT	1128	6657
  AL-DP-5992	umgumgumaaumumagumgcmcmacmumumTT	1129	aaguggcmacumaauumacmacmaTT	1130	6756
  AL-DP-5993	aumcmumumgcmumacmcmagumaumumumaTT	1131	umaaaumacuggumagcmaagauTT	1132	6656
  AL-DP-5994	cmumgumaacmumacmumumumaumaacmumTT	1133	aguumaumaaagumaguumacmagTT	1134	6713
  AL-DP-5995	gumgaaumumaggcmumgumaacmumaTT	1135	umaguumacmagccumaauucmacTT	1136	6703
  AL-DP-6176	cmumacmcmagumaumumumagaagcmcmTT-Chol	1137	ggcuucumaaaumacuggumagTT	1138	6662
  AL-DP-6177	umgcmumgumumumaumumaaumcmumumagTT-Chol	1139	cumaagauumaaumaaacmagcmaTT	1140	6614
  1C. a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 3 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2′-O-methyl modifications and phosphorothioate linkages, are disregarded

Example 3siRNA Activity Testing
• The ability of the iRNA agents represented in Table 3 to inhibit the expression of human RhoA was tested in human cell lines expressing the respective gene product from an expression construct, or in cell lines constitutively expressing the respective gene product. The iRNA agent is transfected into the cells, e.g., by transfection or electroporation, allowed to act on the cells for a certain time, e.g., 24 hours, and levels of RhoA expression were determined by measurement of RhoA mRNA concentrations in cell lysates. These expression levels were then compared to RhoA expression levels in cells treated equivalently but without addition of the iRNA agent, or to expression levels of housekeeping genes (e.g. GAPDH), and the ability of the iRNA agents representend in Table 3 to inhibit the expression of human RhoA thereby assessed.
• Screening for Inhibition of RhoA Expression
• One day before transfection, Neuroscreen-1 cells (Cellomics Inc., Pittsburgh, USA) were seeded at 1.5×10⁴cells/well on 96-well collagen-coated plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl of growth medium (RPMI 1640, 10% horse serum, 5% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, 2 mM L-glutamine, Biochrom AG, Berlin, Germany). Transfections were performed in triplicates. For each well 0.5 μl Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. 2 μl of a 5 μM solution of siRNA in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride) were mixed with 10.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. During this incubation, growth medium was removed from cells and replaced by 75 μl/well of fresh medium. The 25 μl solution of siRNA-Lipofectamine2000-complex were added, resulting in an overall 100 nM siRNA concentration in the 100 μl incubation volume, and the cells were incubated for 24 h at 37° C. and 5% CO₂in a humidified incubator (Heraeus GmbH, Hanau).
• mRNA levels in cell lysates were quantitated by a commercially available branched DNA hybridization assay (QuantiGene bDNA-kit, Genospectra, Fremont, USA). Cells were harvested by applying 50 μl additional growth medium and 75 μl of Lysis Mixture (from QuantiGene bDNA-kit) to each well and were lysed at 53° C. for 30 min. 50 μl of the lysates were incubated with probes specific to rat RhoA and rGAPDH (sequence of probes given in Table 4 and Table 5) according to the manufacturer's protocol for the QuantiGene bDNA kit assay. Finally, chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with RhoA probes were normalized to the respective GAPDH values for each well. Mock transfected cells (following the same protocol except that no siRNA was added) served as controls and forcomparison of mRNA levels.
• Effective siRNAs from the screen were further characterized by establishment of dose response curves and calculation of IC₅₀concentrations (the concentration at which 50% inhibition of gene expression would be observed). For dose response assessment, transfections were performed at the following concentrations: 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, 137 pM, 46 pM, 15 pM, 5 pM and mock (no siRNA) by serially diluting the 5 μM siRNA stock solution with annealing buffer and using 2 μl of the diluted stock according to the above protocol. The IC₅₀was determined by curve fitting using the computer software Xlfit using the following parameters: Dose Response One Site, 4 Parameter Logistic Model, fit=(A+((B−A)/(1+(((10ˆAC)/x)ˆD)))), inv=((10ˆC)/((((B−A)/(y−A))−1)ˆ(1/D))), res=(y−fit).

  TABLE 4
  Rat RhoA probes

  SEQ

  Probe

  ID

  type1	Nucleotide sequence	NO.

  CE	CCATTTTTCTGGGATGTTTTCTAAATTTTTCTCTTGGAA	1141
  	AGAAAGT
  CE	ACAGAAATGCTTGACTTCTGGAGTTTTTTCTCTTGGAAA	1142
  	GAAAGT
  CE	CTTCAGGTTTTACCGGCTCCTTTTTCTCTTGGAAAGAAA	1143
  	GT
  CE	CTGTTTGCCATATCTCTGCCTTTTTTTCTCTTGGAAAGA	1144
  	AAGT
  CE	TTGGTCTTTGCTGAACACTCCATTTTTCTCTTGGAAAGA	1145
  	AAGT
  CE	CCCGCGTCTAGCTTGCAGATTTTTCTCTTGGAAAGAAAG	1146
  	T
  LE	AGGATGATGGGCACATTTGGTTTTTAGGCATAGGACCCG	1147
  	TGTCT
  LE	GCCTTGTGTGCTCATCATTCCTTTTTAGGCATAGGACCC	1148
  	GTGTCT
  LE	TGCTTCATTTTGGCTAACTCCCTTTTTAGGCATAGGACC	1149
  	CGTGTCT
  LE	TGTACCCAAAAGCGCCAATCTTTTTAGGCATAGGACCCG	1150
  	TGTCT
  LE	GCAGCTCTCGTGGCCATCTTTTTTAGGCATAGGACCCGT	1151
  	GTCT
  LE	AGGCACCCCGACTTTTTCTTTTTTTAGGCATAGGACCCG	1152
  	TGTCT
  BL	CTATCAGGGCTGTCGATGGAA	1153
  BL	GAAGATCCTTCTTGTTCCCAACT	1154
  BL	CAAAAACCTCTCTCACTCCGTCT	1155
  1CE = Capture Extender probe; LE = Label Extender probe; BL = blocking probe

• TABLE 5
  Rat GAPDH probes

  SEQ

  Probe

  ID

  type1	Nucleotide sequence	NO.

  CE	CCAGCTTCCCATTCTCAGCCTTTTTCTCTTGGAAAGAAA	1156
  	GT
  CE	TCTCGCTCCTGGAAGATGGTTTTTTCTCTTGGAAAGAAA	1157
  	GT
  CE	CCCATTTGATGTTAGCGGGATTTTTCTCTTGGAAAGAAA	1158
  	GT
  CE	CGGAGATGATGACCCTTTTGGTTTTTCTCTTGGAAAGAA	1159
  	AGT
  LE	GATGGGTTTCCCGTTGATGATTTTTAGGCATAGGACCCG	1160
  	TGTCT
  LE	GACATACTCAGCACCAGCATCACTTTTTAGGCATAGGAC	1161
  	CCGTGTCT
  LE	CCCAGCCTTCTCCATGGTGGTTTTTAGGCATAGGACCCG	1162
  	TGTCT
  BL	TTGACTGTGCCGTTGAACTTG	1163
  BL	CCCCACCCTTCAGGTGAGC	1164
  BL	GGCATCAGCGGAAGGGG	1165
  1CE = Capture Extender probe; LE = Label Extender probe; BL = blocking probe

• Table 6 lists the agent number, the position of the nucleotide within the human RhoA mRNA sequence (Genbank accession number NM_—001664) corresponding to the 5′-most nucleotide of the sense strand of the agent, the amount of total RhoA mRNA remaining in cells treated with the agent at 100 nM concentration in % of controls, and the IC₅₀value for selected agents.

  TABLE 6
  Ability of siRNAs specific for RhoA to reduce
  RhoA mRNA levels in cultured cells

  		Rem. RhoA
  		mRNA at	Rem. RhoA
  		100 nM	mRNA at 100 nM
  Agent	Pos. in	agent,	agent, second	IC50RhoA
  number	mRNA1	first screen	screen	[nM]

  AL-DP-5850			73 ± 8%	142
  AL-DP-5852			17 ± 4%	3.1
  AL-DP-5853			18 ± 3%	2.8
  AL-DP-5854			17 ± 1%	4.2
  AL-DP-5972	986	30 ± 9%	17 ± 2%
  AL-DP-5973	987	21 ± 2%	15 ± 1%	0.003
  AL-DP-5974	1179	44 ± 12%	48 ± 2%
  AL-DP-5975	1395	33 ± 4%	27 ± 10%
  AL-DP-5976	1413	26 ± 3%	17 ± 2%
  AL-DP-5977	537	n.d.	30 ± 1%
  AL-DP-5978	539	58 ± 4%	51 ± 1%
  AL-DP-5979	540	12 ± 2%	15 ± 2%	0.06
  AL-DP-5980	544	75 ± 3%	95 ± 3%
  AL-DP-5981	546	17 ± 2%	16 ± 1%	0.13
  AL-DP-5982	1452	18 ± 2%	22 ± 2%	0.13
  AL-DP-5983	1449	37 ± 4%	29 ± 3%
  AL-DP-5984	1451	26 ± 1%	33 ± 4%
  AL-DP-5985	1450	n.d.	33 ± 1%	0.37
  AL-DP-5986	1411	18 ± 1%	22 ± 1%	0.4
  AL-DP-5987	901	22 ± 5%	10 ± 0%	0.01
  AL-DP-5988	1376	17 ± 1%	16 ± 1%	0.34
  AL-DP-5989	1876	20 ± 1%	25 ± 3%	3.1
  AL-DP-5990	956	16 ± 2%	17 ± 1%	0.36
  AL-DP-5991	982	55 ± 5%	33 ± 5%
  AL-DP-5992	1400	55 ± 6%	55 ± 6%
  AL-DP-5993	981	32 ± 2%	33 ± 3%
  AL-DP-5994	1180	23 ± 2%	20 ± 1%	0.24
  AL-DP-5995	1170	25 ± 2%	26 ± 2%	6.0
  AL-DP-6176	987		14 ± 2%	1.17
  AL-DP-6177	901		19 ± 5%	0.005
  1Position of nucleotide within human Nogo-R mRNA corresponding to the 5′-most nucleotide of the sense strand of the agent

• In summary, agents AL-DP-5979, AL-DP-5990, AL-DP-5988, AL-DP-5981, AL-DP-5982, AL-DP-5986, AL-DP-5989 AL-DP-6176, and AL-DP-6177 were able to reduce the expression of RhoA mRNA by 80% or more, AL-DP-5973, AL-DP-5987, AL-DP-5994, AL-DP-5995, AL-DP-5976, AL-DP-5984, and AL-DP-5972 were able to reduce the expression of RhoA mRNA by 70% or more, AL-DP-5993, AL-DP-5975, and AL-DP-5983 were able to reduce the expression of RhoA mRNA by 60% or more, AL-DP-5974 was able to reduce the expression of RhoA mRNA by 50% or more, and AL-DP-5991, AL-DP-5992, and AL-DP-5978 were able to reduce the expression of RhoA mRNA by 40% or more. The high activity of AL-DP-6176 and AL-DP-6177 shwos that a cholesteryl moiety may be conjugated to the 3′-end of the sense strand of an siRNA without significant loss of activity. AL-DP-6176 and AL-DP-6177 are identical to AL-DP-5973 and AL-DP-5987, respectively, except for the 3′-conjugated cholesteryl moiety on the sense strand.
Example 4Stability Testing
• In order to verify the stability of siRNAs in the biological matrix most relevant to their intended physiological application, cerebrospinal fluid (CSF), we established a method for determining the degradation half life of siRNAs in this medium. This method comprises the incubation of siRNAs with CSF followed by Proteinase K treatment of the CSF sample and the separation of CSF sample constituents on an HPLC.
• The example below shows the analyses of CSF samples which were contacted with siRNAs in vitro. However, this method can equally be applied to biological samples ex vivo, i.e. obtained from a subject which was contacted with an siRNA in vivo.
• Bovine CSF was obtained from a calf (Bos bovis),age 6 months (Prof. Dr. J. Rehage, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany). Porcine CSF was pooled from 3 healthy weaner pigs (Sus scrofa domesticus), age 3-4 months (Prof. Dr. M. Wendt, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany). Rat CSF was pooled from 20 male Sprague Dawley rats (Rattus norvegicus), 175-200 g in weight (Charles River Laboratories, L'Arbresle Cedex, France). Proteinase K (20 mg/ml) was obtained from peQLab (Erlangen, Germany; Cat.-No. 04-1075) and diluted 1:1 with deionized water (18.2 mΩ) to a final concentration of 10 mg/ml Proteinase K. Proteinase K Buffer (4.0 ml TRIS-HCl 1M pH 7.5, 1.0 ml EDTA 0.5M, 1.2 ml NaCl 5M, 4.0ml SDS 10%) was prepared fresh and kept at 50° C. until use to avoid precipitation.
• A 40 mer of poly(L-dT), (L-dT)₄₀was obtained from Noxxon Pharma AG (Berlin, Germany) and used as an internal standard. Polymers of the L-enantiomers of nucleic acids show an extraordinary stability towards nucleolytic degradation (Klussman S, et al., Nature Biotechn. 1996, 14:1112) but otherwise very similar properties when compared to naturally occuring nucleic acids consisting of R-enantiomers.
• Proteinase K Treatment of siRNA Incubation Samples
• 6 μl of a 50 μM solution of the respective siRNA in phosphate buffered saline (PBS, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was incubated with 54 μl CSF at 37° C. for 30 min, 1, 2, 4, 8, 16, 24 or 48 hours. To terminate the siRNA-degradation, 25 μl of Proteinase K buffer were added to incubation samples immediately after expiry of the respective incubation period, the mixture vortexed at highest speed for 5 s (Vortex Genie 2, Scientific Industries, Inc., Bohemia, N.Y., USA, cat. no. SI 0256), 8 μl Proteinase K (10 mg/ml) were added followed by vortexing for 5 s, and finally the mixture was incubated for 20 min in a thermomixer at 42° C. and 1050 rpm.
• 5 μl of a 50 μM solution (250 pmole) of (L-dT)₄₀were added as an internal standard to each well, the solution was vortexed for 5 s, and the tube centrifuged for 1 min in a tabletop centrifuge to collect all droplets clinging to the inner surfaces of the wells at the bottom. The solution was transferred to a 96 well Captiva 0.2 μm filter plate (Varian, Germany, Cat. No. A5960002) and filtered by centrifugation at 21900 rcf for 45 min.
• The incubation wells were washed with 47.5 μl deionized water (18.2 mΩ), the wash filtered through the Captiva Filter Unit at 21900 rcf for 15 min, and the wash step repeated. Approximately 180 μl of the theoretical total volume of 200 μl are on average recovered after the second washing step.
• Ion exchange chromatographic separation of siRNA single strands from each other and from degradation products:
• A Dionex BioLC HPLC-system equipped with inline-degasser, autosampler, column oven and fixed wavelength UV-detector (Dionex GmbH, Idstein, Germany) was used under denaturing conditions. Standard run parameters were:

  Column:	Dionex DNA-Pac100; 4 × 250 mm
  Temperature:	75° C.
  Eluent A:	10 mM NaClO4, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile,
  	pH = 8.0
  Eluent B:	800 mM NaClO4, 20 mM TRIS-HCl, 1 mM EDTA; 10%
  	acetonitrile, pH = 8.0
  Detection:	@ 260 nm

  Gradient:	0-1 min:	10% B
  	1-11 min:	10% −> 35% B
  	11-12 min:	35% B −> 100% B
  	12-14 min:	100% B −> 10% B
  	14-16 min:	10% B for column reequilibration

  Injection volume:	20 μl

• Where separation between the two strands of an siRNA was not satisfactory or a degradation fragment co-eluted with one strand, the chromatographic parameters were adjusted by changing temperature, pH, replacement of NaClO₄by NaBr, the concentration of acetonitrile, and/or adjusting the slope of the eluent gradient until separation was achieved which allowed separate quantitation of the peaks from sense and antisense strand.
• Peak areas for full length strands were obtained by integration of the UV detector signal using software supplied by the manufacturer of the instrument (Chromeleon 6.6; Dionex GmbH, Idstein, Germany).
• Data Analysis:
• Integrated sense strand, antisense strand, and internal standard peak areas were obtained for all samples and the normalization control.
• A correction factor CF, accounting for liquid losses in the filtration and washing steps, was determined for every sample by calculating the ratio of experimental to theoretical internal standard peak area. The theoretical internal standard peak area is obtained, e.g. from a calibration curve of the internal standard obtained by injecting 50 μl each of a serial dilution of the 50 μM solution of (L-dT)₄₀onto the HPLC column, and calculation of the theoretical peak area corresponding to 25 pmole (L-dT)₄₀with the equation obtained by linear least square fit to the peak areas from the dilution series. The correction factor CF to be applied to the peak areas of the sense and antisense strand is the obtained as:
  CF=PeakArea_intStd(theoretical)/PeakArea_intStd(Sample)
• This treatment assumes that, by virtue of washing the filter twice, virtually complete recovery is achieved in the combined filtrates, and corrects for the variable volume of wash water retained in the filter, such that peak areas from different samples can be compared.
• The peak areas obtained for the sense and antisense strand peaks for each time point are then multiplied with the correction factor CF to obtain Normalized Peak Areas (NPA_sense,t, NPA_antisense,t):
  NPA_{sense or antisense,t}=(Peak Area_{sense or antisense,t})×CF
• To obtain the relative amount of remaining Full Length Product (% FLP) for the sense and antisense strands at time t, the Normalized Peak Area for each strand at time t=0 min (NPA_sense,t=0, NPA_{antisense,t=0}) is set as 100%, and the NPAs from other time points are divided by these values.
  %FLP_{t=1, 2, 3 . . . n}=(NPA_{t=1, 2, 3 . . . n}/NPA_t=0)*100
• The value obtained from the control sample, where the siRNA was incubated with annealing buffer only, may serve as a control of the accuracy of the method. The % FLP for both strands should lie near 100%, within error margins, regardless of time of incubation.
• The degradation half life t_1/2may then be calculated for each strand, assuming first order kinetics, from the slope of a linear least square fit to a plot of ln(% FLP) versus time as:
  t_1/2=ln(0,5)/slope
  Stability of siRNAs Specific for NogoL and RhoA in Rat, Bovine and Porcine CSF
• Table 7 shows the results for select siRNAs of the determination of the relative amount of full length dsRNA present in porcine, rat, and bovine CSF, and PBS, after 48 h of incubation in the respective medium. In addition, the degradation half life was determined for the sense and antisense strands separately for some siRNAs.

  TABLE 7
  Stability of various siRNAs specific for NogoL
  and RhoA in rat, bovine and porcine CSF

  	% full length duplex
  	present after 48 h in

  Agent	Porcine	Rat	Bovine		Specific	Modifi-	C.a.
  number	CSF	CSF	CSF	PBS	for	cation1	#2

  AL-DP-5973	95	3	95	100	RhoA	3/TTs	6662
  AL-DP-5979	99			108	RhoA	3/TTs	6524
  AL-DP-5981	96			103	RhoA	3/TTs	6530
  AL-DP-5982	56			98	RhoA	3/TTs	6790
  AL-DP-5986	100			105	RhoA	3/TTs	6767
  AL-DP-5987	87			97	RhoA	3/TTs	6614
  AL-DP-5988	41			99	RhoA	3/TTs	6732
  AL-DP-5989	87			101	RhoA	3/TTs	6832
  AL-DP-5990	76			92	RhoA	3/TTs	6650
  10 = no 2′-modifications; 1 = 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in sense strand, 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in antisense strand; 2 = 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is
  # 2′-modified in sense and antisense strand, 3 = all pyrmidine nucleotides are 2′-modified in sense strand, 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in antisense strand; 4 = all pyrimidine nucleotides are 2′-modified in sense strand,
  # 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in antisense strand; 5 = all pyrimidine nucleotides are 2′-modified in sense strand, no 2′-modifications in antisense strand; TT = 21 nucleotides and 3′-terminal TT single strand overhangs in sense and antisense strands; TTs = 21 nucleotides and 3′-terminal
  # TT single strand overhangs in sense and antisense strands; 23 = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3′-end of antisense strand; 23s = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3′-end of antisense strand, nucleotides comprise 5′-phosphorothioate groups in positions 21 and 22 of antisense strand
  2C.a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 2 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2′-O-methyl modifications and phosphorothioate linkages, are disregarded

• As is evident from Table 7, the modification of siRNAs in select sites vulnerable to degradation can lead to agents with excellent properties in terms of activity and stability. For example, AL-DP-5871, AL-DP-5938, AL-DP-5963, AL-DP-5973, AL-DP-5979, AL-DP-5981, AL-DP-5986, AL-DP-5987, AL-DP-5989, and AL-DP-5990 all inhibit their respective target gene by more than 70% in the in vitro assays described above, and more than 70% full length duplex remain after incubation with porcine CSF for 48 h. However, there is some indication that rat CSF is more aggressive towards siRNAs than porcine or bovine CSF.
Example 5Inhibition of RhoA Expression in Rat Primary Dorsal Root Ganglia (DRG) Cells in Culture
• The inhibition of RhoA expression was assessed in rat primary dorsal root ganglia (DRG) cells in culture in order to validate results obtained usingNeuroscreen 1 cells as described above.
• DRG cells were isolated from Sprague-Dawley rats atpostnatal day 3 to 6. Rats were dissected and cells dissociated into single cells by addition of 1.3 ml (0.28 Wunsch units/ml) Liberase Blendzyme (Roche) in S-MEM (Invitrogen Gibco, Carlsbad Calif., USA) and incubated for 35 min at 37° C. The cell suspension was pre-plated on tissue-culture plates to remove non-neuronal cells. Neurons were then plated onto tissue-culture Biocoat™ PDL Poly-D-Lysine/Laminin 96 well plates (BD Biosciences, Bedford Mass., USA) in F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5% fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heat inactivated) (both Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp., Madison Wis., USA) and kept at 37° C., 5% CO₂in a humidified incubator until transfection.
• A rhoA-specific siRNA, agent number AL-DP-5987, was tested in DRG cultures at 200 nM concentration using TransMessenger™ Transfection reagent (Qiagen GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid formulation, specific RNA-condensing reagent (Enhancer R™) and an RNA-condensing buffer (Buffer EC-R™) keeping siRNA:Enhancer R™ ratio (μg:μl) constant at 1:2, and siRNA:TransMessenger™ ratio (μg:μl) constant at 1:12.
• DRG neurons were transfected 24 h post-plating. For each well 0.52 μl Enhancer R™ were first mixed with 13.68 μl Buffer EC-R™. 0.8 μl of a 25 μM solution of AL-DP-5987 (0.26 μg) in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), or 0.8 μl of annealing buffer (siRNA-free control) were added and the mixture incubated for 5 min at RT. 3.12 μl TransMesssenger™ Transfection Reagent were diluted with 6.88 μl Buffer EC-R™, added to the mixture, and the mixture incubated for another 10 min at room temperature to allow transfection-complex formation. 75 μl serum free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad Calif., USA) were added to the transfection complexes and complete mixing achieved by gently pipetting up and down. The growth medium was removed from the DRG cells, and 90 μl of the above transfection complex mixture were added onto the cells. After 8 h of incubation at 37° C., 5% CO₂in a humidified incubator supernatant was removed from the cells, fresh F12-HAM's medium containing glutamine supplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA) was added, the cells were incubated for another 16 h at 37° C., 5% CO₂in a humidified incubator, and rhoA mRNA was quantified.
• RhoA mRNA levels were measured using the QuantiGene™ bDNA kit (Genospectra, Fremont, USA) according to manufacturer's protocol. Briefly, the supernatant was removed from the DRG cells, and the cells were lysed by addition of 150 μl of Lysis Working Reagent (1 volume of Lysis Mixture plus 2 volumes of medium) and incubation at 52° C. for 30min. 40 μl of the lysates were incubated at 52° C. for 40 min with the probe sets specific to rat RhoA and rat GAPDH given above in Table 4 and Table 5. Chemoluminescence was read on a Victor²-Light™ (PerkinElmer Life And Analytical Sciences, Inc., Boston Mass., USA) as Relative Light Units (RLU). RLU for RhoA were normalized to GAPDH RLU for each well. Normalized RhoA/GAPDH ratios were then compared to the siRNA-free control, which was set as 100%.
• In several independent experiments, rhoA mRNA was reduced in primary DRG cells treated with AL-DP-5987 in culture consistently to 20-25% of rhoA mRNA levels found in the siRNA free controls.
Example 6Neurite Outgrowth Assessment in Primary DRG Transfected with Selected iRNA Agents of the Invention
• Neuron Dissection
• Dorsal root ganglia (DRG) were isolated from Sprague-Dawley rats at postnatal day 7-12. DRG were dissected into the culture medium (Neurobasal-A supplemented with 1:50 B27, Glutamine, and Pen/Strep; Invitrogen Gibco, Carlsbad Calif., USA), blood was rinsed off with PBS and neurons were dissociated by incubation with 1 mL DMEM/collagenase (0.5%) for 90 minutes at 37° C., inverting the tube every 30 minutes. The collagenase was washed off by serial dilution and the isolated DRG were resuspended in 500 μl of culture medium. The suspension was then triturated 15-20 times with a fire-polished pipette until a homogeneous cell suspension was achieved. Cells were spun down three times for 3 minutes at 1500 RPM, and the cell debris aspirated off. Cells were then resuspended in 500 μl of culture media and passed through a 70 μm strainer (Falcon, 352350). The cell suspension was pre-plated on a 24 well tissue-culture plate in 300 μl of culture media for 24 hours at 37° C., 5% CO2 in a humidified incubator. Neurons were then transfected with appropriate siRNA as follows:
• Transfection of siRNA
• For each well, Lipofectamine™ 2000 was used and transfections were performed according to the manufacturer's protocol. The appropriate amount of siRNA was diluted into Opti-MEM I Reduced Serum Medium and mixed gently. The Lipofectamine™ 2000 was vortexed before use. For each well of a 24 well plate, 1.5 μl was diluted in 25 μl of Opti-MEM I Reduced Serum Medium, mixed gently and incubated for 5 minutes at room temperature. After the 5 minute incubation, 3 μl of the diluted siRNA was combined with the diluted Lipofectamine™ 2000 (total volume is 29.5 μl). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: Lipofectamine™ 2000 complexes to form. 300 μl of Media were added to the transfection complexes and complete mixing was achieved by gently pipetting up and down (total volume: 600 μl). The cells were incubated for another 20-24 h at 37° C., 5% CO2 in a humidified incubator
• Outgrowth Assay Plate Preparation
• Chondroitin sulfate proteoglycan (CSPG) or myelin was dried down overnight in 50 μl in individual wells of a biocoat Poly-D-Lysine plate (BD Biosciences, Bedford Mass., USA). The plate was rinsed once with water to remove salt deposits. laminin (Invitrogen, CA; 1 μg/ml) was then coated for 1 h at room temperature.
• Replating of the Neurons
• Neurons were resuspended from the 24 well plate by gently pipetting up and down, spun down at 1500 RPM for 3 minutes and resuspended in the appropriate volume. Neurons were replated at a concentration of 3000 neurons/well onto the prepared Poly-D-Lysine/Laminin 96-well plates in culture medium. The neurons were allowed to grow for 16-24 hours at 37° C., 5% CO2 in a humidified incubator and subjected to neurite quantification as follows.
• Neurite Quantification
• The cells were fixed in 4% formaldehyde, 20% sucrose in PBS for 15 minutes. The cells were rinsed once with PBS, permeabilized and blocked for an hour at room temperature using 0.1% Triton, 10% Goat serum in PBS. The cells were stained with a beta-III tubulin, polyclonal rabbit antibody (Covance, 1:500) diluted in PBS and incubated overnight at 4° C. After three 5 minutes rinses in PBS, the secondary Alexa-Fluor 488 goat anti-rabbit IgG antibody (Molecular Probes, 1:500 filtered through 0.22 um) was incubated overnight at 4° C. Following 3 rinses in PBS, neurite outgrowth was quantified using an automated cellular imaging and analysis system (Axon Instrument, Union City, Calif.).
• Results
• Two iRNA agents specific for the green fluorescent protein gene (AL-DP 5549, AL-DP 5266; nucleic acid sequences in Table 8) were used as controls.

  TABLE 8
  Nucleic acid sequences of control iRNA agents specific for
  the green fluorescent protein gene

  SEQ

  SEQ

  Agent

  ID

  ID

  number	Sense strand	NO.	Antisense strand	NO.

  AL-DP-5549	ccacaugaagcagcacgacuu-Chol	1166	aagucgugcugcuucauguggmumc	1167
  AL-DP-5266	ccacaugaagcagcacgacuu	1168	aagucgugcugcuucauguggmumc	1169

• The iRNA agent of the invention AL-DP-6176 was tested at a concentration of 250 nM at transfection for its effect on neurite outgrowth on growth substrate supplemented with 0, 40, or 200 ng myelin, and compared to the effect of AL-DP-5266. In Table 9, neurite outgrowth is expressed as percent of neurite outgrowth seen without myelin. Evidently, the green fluorescent protein-specific iRNA agent has no effect on neunte outgrowth of DRG on the inhibitory substrate, while the RhoA-specific iRNA agent strongly counters the growth inhibitory effect of myelin.

  TABLE 9
  Inhibition of neurite outgrowth by myelin in DRG
  in the absence or presence of iRNA agents specific
  for green fluorescent protein (GFP) or RhoA

  Myelin (ng)	no siRNA	AL-DP 5266 (GFP)	AL-DP 6176 (RhoA)

  0	100%	100%	100%
  40	82%	75%	183%
  200	3%	6%	19%

• Furthermore, the iRNA agents of the invention AL-DP 5973, AL-DP 5987, AL-DP 6176, and AL-DP 6177 were tested at a concentration of 250 nM at transfection for its effect on neurite outgrowth on growth substrate supplemented with 30 ng CSPG, and compared to the effect of AL-DP-5549 and AL-DP-5266. In Table 10, neurite outgrowth is expressed as percent of neurite outgrowth seen without CSPG. Evidently, the green fluorescent protein-specific iRNA agent has no effect on neurite outgrowth of DRG on the inhibitory substrate, while the RhoA-specific iRNA agents counter the growth inhibitory effect of CSPG.

  TABLE 10
  Inhibition of neurite outgrowth by CSPG in DRG
  in the absence or presence of iRNA agents specific
  for green fluorescent protein (GFP) or RhoA

  	Relative outgrowth

  	AL-DP-5549	3 ± 2%
  	AL-DP-5266	2 ± 1%
  	AL-DP 5266	6 ± 2%
  	AL-DP 5973	5 ± 2%
  	AL-DP 6176	8 ± 4%
  	AL-DP 5987	10 ± 5%

Example 7Assessing Behavioural Improvement by Intrathecal Administration of iRNA Agents of the Invention Following Hemisection Injury
• Animals and iRNA Agent Adminstration
• Thirty adult female Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass., USA; weight 200-230 grams) were randomly divided into 3 groups. One day before the spinal cord lesion surgery, all the animals were trained for locomotor BBB score in an open field. 3 iRNA agents were administered in blinded fashion to the three different groups. Group A being administered a first rhoA-specific iRNA agent (AL-DP-6177), group B receiving a second rhoA-specific iRNA agent (AL-DP-6176), group C as a control group getting the unrelated control iRNA agent, directed against Luciferase (AL-DP-1956, for nucleic acid sequence see Table 11). The iRNA agents were delivered through an osmotic minipump (Alzet 2004, 0.25 μl/hr, 28 day delivery), which was filled with appropriate drug for the groups A, B, C, and pre-incubated overnight at 37° C. before implantation to the animals (described below). The siRNA was released at a dose of 0.4 mg per day for 28 days.

  TABLE 11
  Nucleic acid sequences of control iRNA agent specific
  for luciferase

  SEQ

  SEQ

  Agent

  ID

  ID

  number	Sense strand	NO.	Antisense strand	NO.

  AL-DP-5549	cmumumacmgcmumgagumacmumumcmgaTT-Chol	1170	ucgaagumacucmagcgumaagTT	1171

  
  Microsurgery
• For the microsurgery procedure generating the hemisection injury the rats were deeply anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg). A complete laminectomy was conducted at spinal level T6-7 under a surgical microscope. The dorsal part of spinal cord was transected with the sharp tip of a 30 gauge needle and a pair of microscissors. The depth of transection (1.85 mm) was confirmed by passing the sharp part of number 11 blade across the dorsal spinal cord for several times with the aid of a stereotaxic (David Kopf Instruments, Tujunga, Calif.). Routinely, the spinal cord was severed past the central canal area (GrandPre, T., et al., Nature 2002, 417:547-551; Li, S., et al., J Neurosci 2004, 24:10511-10520). Rats with an incomplete lesion documented by uninjured dorsal or dorsolateral corticospinal fibers in caudal spinal cord or a less degree of disability (BBB score ≧5) at two days after injury were excluded.
• For intrathecal administration of the siRNA solutions with the osmotic pumps the dura over the dorsal spinal cord was gently lifted with a pair of microforceps and a small hole was made in the midline with a pair of microscissors at T7 level. The minipump was sutured to the muscles under the skin on the back of the animals. The outlet of the minipump was connected to one end of an intrathecal catheter (PE-60, MS-0040, Marsil Scientific). The other end of the catheter with a small diameter of PE-5 was inserted into the subdura space of spinal cord at T7 level through the small opening made for drug infusion. After pump and catheter implantation, muscle and connective tissue layers were sutured with 4.0 silk. The skin incision was closed with sterile animal clips. The animals received a subcutaneous injection of lactated Ringer's solutions (5% of body weight) immediately after surgery and two times per day for the first two days after injury. Postoperative antibiotics and analgesics were given during the first two days post injury. Bladder expression was performed manually three times per day during the survival period of the animal.
• Behavioral Tests
• To monitor the recovery of locomotor functions during and after siRNA treatment after spinal cord hemisection, we used a standard behavioral test, the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale (GrandPre, T., et al., Nature 2002, 417:547-551; Li, S., et al., J Neurosci 2004, 24:10511-10520; Basso, D. M., et al., J Neurotrauma 1995, 12:1-21) to analyze individual components of limp movements, weight support, plantar and dorsal stepping, forelimb-hindlimp coordination, paw rotation, toe clearance, trunk stability, and tail placement. Scores from 0-21 were given based on these observations. The BBB score was evaluated by two individuals unaware of experimental treatments at several time points (Li, S., et al., J Neurosci 2004, 24:10511-10520). In particular, the locomotion was analyzed onday 3, 10, 17, 24, 31 and 45 post hemisection of the spinal cord.
• Results
• The obtained BBB score data were plotted by normalizing all the single day score data of the animals to the score atday 10. The group averages of those normalized scores were charted including the error representing the calculated standard error. By this procedure improvements in locomotion fromday 10 onwards are shown inFIG. 2. As evident, a significant improvement was obtained by virtue of the treatment with the inventive RhoA-specific iRNA agents over the unrelatred control iRNA agent.

Claims (2)

1. A method of stimulating neuronal cell outgrowth in a neural cell culture comprising contacting the neural cell culture with an iRNA agent, wherein the iRNA agent comprises (i) a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of agents number 5850 to 6177, and (ii) an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one agent selected from the group consisting of agents number 5850 to 6177.

2. A method of performing an animal study comprising:

(a) providing an animal having a neural disfunction;

(b) administering an iRNA agent wherein the iRNA agent comprises (i) a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one agent selected from the group consisting of agents number 5850 to 6177, and (ii) an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one agent selected from the group consisting of agents number 5850 to 6177; and

(c) monitoring the animal for an improvement in neural function.

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