Patent Publication Number: US-2011071209-A1

Title: RNAi Modulation of the RHO-A Gene and Uses Thereof

Description:
RELATED APPLICATIONS 
     This application is a divisional application of U.S. application Ser. No. 11/491,367, filed on Jul. 21, 2006, which 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. 
    
    
     SEQUENCE LISTING 
     This application includes a Sequence Listing submitted electronically as a text file named “17189US_sequencelisting.txt,” created on Jul. 30, 2010, with a size of 404,000 bytes. The sequence listing consists of 1165 sequences and is incorporated by reference. 
     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.,  Nature  391:806-811, 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 the Switch 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-6177. 
     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). 
     
    
    
     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.,  Virology  199:491, 1994; Castelli et al.,  J. Exp. Med.  186:967, 1997; Zheng et al.,  RNA  10:1934, 2004; Heidel et al., “Lack of interferon response in animals to naked siRNAs”  Nature Biotechn. advance online publication  doi: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 
                 RNA b   
                 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 
                 gaaagacaugcuugcugauaguc 
                 double 
                 41 
                 aagacaugcuugcucauagTT 
                 42 
                 cuaugagcaagcaugucuuTT 
               
               
                   
               
               
                 6491 
                 327 
                 43 
                 aaagacaugcuugcugauagucu 
                 double 
                 44 
                 agacaugcuugcucauaguTT 
                 45 
                 acuaugagcaagcaugucuTT 
               
               
                   
               
               
                 6492 
                 328 
                 46 
                 aagacaugcuugcucauagucuu 
                 double 
                 47 
                 gacaugcuugcucauagucTT 
                 48 
                 gacuaugagcaagcaugucTT 
               
               
                   
               
               
                 6493 
                 329 
                 49 
                 agacaugcuugcugauagucuuc 
                 double 
                 50 
                 acaugcuugcucauagucuTT 
                 51 
                 agacuaugagcaagcauguTT 
               
               
                   
               
               
                 6494 
                 330 
                 52 
                 gacaugcuugcugauagucuuca 
                 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 
               
               
                   
               
               
                 6529 
                 542 
                 154 
                 cugauaguuuagaaaacauccca 
                 double 
                 155 
                 gauaguuuagaaaacauccTT 
                 156 
                 ggauguuuucuaaacuaucTT 
               
               
                   
               
               
                 6529 
                 543 
                 157 
                 ugauaguuuagaaaacaucccag 
                 double 
                 158 
                 auaguuuagaaaacaucccTT 
                 159 
                 gggauguuuucuaaacuauTT 
               
               
                   
               
               
                 6530 
                 544 
                 160 
                 gauaguuuagaaaacaucccaga 
                 double 
                 161 
                 auguuuagaaaacaucccaTT 
                 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 
                 gaagaaggcagagauauggcaaa 
                 double 
                 218 
                 agaaggcagagauauggcaTT 
                 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 
                 cuccaauccuguuugccauTT 
               
               
                   
               
               
                 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 
                 uugcugaacacuccauguaTT 
               
               
                   
               
               
                 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 
                 cugcucugcaagcuagacgTT 
                 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 
                 uaaucuuaguguaugauuacugg 
                 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 
                 aguguaugauuacuggccuuuuu 
                 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 
                 uaauuuaccuaagauuacaaauc 
                 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 
                 975 
                 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 
                 uguuaguuauaaaguaguuTT 
               
               
                   
               
               
                 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 
                 gacauguuaguuauaaaguTT 
               
               
                   
               
               
                 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 
               
               
                   
               
               
                 6756 
                 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 
                 gauguaguauuuuuuguauaauu 
                 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 
                 aauccaauuauacaaaaaaTT 
               
               
                   
               
               
                 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 
                 1002 
                 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 
                 1015 
                 uauuuggaaauaaagucagaugg 
                 double 
                 1016 
                 uuuggaaauaaagucagauTT 
                 1017 
                 aucugacuuuauuuccaaaTT 
               
               
                   
               
               
                 6816 
                 1542 
                 1018 
                 auuuggaaauaaagucagaugga 
                 double 
                 1019 
                 uuggaaauaaagucagaugTT 
                 1020 
                 caucugacuuuauuuccaaTT 
               
               
                   
               
               
                 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 
               
               
                   
               
               
                 6823 
                 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 
                 uguaccaguuaauuuuuccaacu 
                 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 
               
               
                   
               
               
                   a Single: 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 function of the nucleotide sequence than strand length. For example, Yang, et al.,  PNAS  99:9942-9947 (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 Notes  10(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 as  35 S,  32 P,  33 P, or  3 H; 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., EC 50  or ED 50 ). 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 internucleotide 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 the sequence contexts 5′-ua-3′ and 5′-cc-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 the sequence 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 the sequence 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(CH 2 CH 2 O) n CH 2 CH 2 OR; “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(CH 2 ) n AMINE, (e.g., AMINE=NH 2 ; 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), (OCH 2 CH 2 OCH 3 , 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. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE (AMINE=NH 2 ; 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′-OCH 3 , 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, Nr 2 , or Br 3 . 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 the motif 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—(CH 2 ) n NH 2  may 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&#39;s cytoskeleton, e.g., by disrupting the cell&#39;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. USA  91:3054-3057, 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 Mg 2+ ), 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&#39;s Disease, spongiform encephalopathy, Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), multiple sclerosis, transverse myelitis, motor neuron disease, Guillan Bane, 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 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&#39;s body or cranium, or to an implanted drug pump located in the patient&#39;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 function 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. Nos. 09/872,698 (filed Jun. 1, 2001) and 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&#39;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 raffinose, 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. 
               
            
           
           
               
               
            
               
                 Abbreviation a   
                 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 
               
               
                   
               
               
                   a capital 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 1 
     Selection 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 degrading siRNA 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 2 
     siRNA 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 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 &amp; 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, UnterschleiBheim, 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 in  FIG. 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. The reaction mixture 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 stiffing 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 NaH 2 PO 4 .H 2 O 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 to pH 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/CHCl 3 ) (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 1M 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/CHCl 3 ) (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 funnel 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 in  FIG. 1 . 
     The siRNAs listed Table 3 were synthesized for activity screening. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 siRNAs specific for RhoA 
               
            
           
           
               
               
               
               
               
               
            
               
                 Agent 
                   
                 SEQ 
                   
                 SEQ 
                 C. a. 
               
               
                 number 
                 Sense strand 
                 ID NO. 
                 Antisense strand 
                 ID NO. 
                 # 1   
               
               
                   
               
               
                 AL-DP-5850 
                 gauuaugaccgucugaggcT T   
                 1081 
                 gccucagucggucauaaucT T   
                 1082 
                 n.a. 
               
               
                   
               
               
                 AL-DP-5851 
                 ggaucuucggaaugaugagT T   
                 1083 
                 cucaucauuccgaagauccT T   
                 1084 
                 n.a. 
               
               
                   
               
               
                 AL-DP-5852 
                 agaccaaagacggagugagT T   
                 1085 
                 cucacuccgucuuuggucuT T   
                 1086 
                 n.a. 
               
               
                   
               
               
                 AL-DP-5853 
                 ugaagcaggagccgguaaaT T   
                 1087 
                 uuuaccggcuccugcuucaT T   
                 1088 
                 n.a. 
               
               
                   
               
               
                 AL-DP-5972 
                 gcmumacmcmagumaumumumagaagcmT T   
                 1089 
                 gcuucumaaaumacuggumagcT T   
                 1090 
                 6661 
               
               
                   
               
               
                 AL-DP-5973 
                 cmumacmcmagumaumumumagaagcmcmT T   
                 1091 
                 ggcuucumaaaumacuggumagT T   
                 1092 
                 6662 
               
               
                   
               
               
                 AL-DP-5974 
                 gcmumgumaacmumacmumumumaumaacmT T   
                 1093 
                 guumaumaaagumaguumacmagcT T   
                 0194 
                 6712 
               
               
                   
               
               
                 AL-DP-5975 
                 gumumacmumgumgumaaumumagumgcmT T   
                 1095 
                 gcmacumaauumacmacmagumaacT T   
                 1096 
                 6751 
               
               
                   
               
               
                 AL-DP-5976 
                 cmcmacmumumaaumgumaumgumumacmcmT T   
                 1097 
                 ggumaacmaumacmauumaaguggT T   
                 1098 
                 6769 
               
               
                   
               
               
                 AL-DP-5977 
                 cmagcmcmcmumgaumagumumumagaaT T   
                 1099 
                 uucumaaacumaucmagggcugT T   
                 1100 
                 6521 
               
               
                   
               
               
                 AL-DP-5978 
                 gcmcmcmumgaumagumumumagaaaaT T   
                 1101 
                 uuuucumaaacumaucmagggcT T   
                 1102 
                 6523 
               
               
                   
               
               
                 AL-DP-5979 
                 cmcmcmumgaumagumumumagaaaacmT T   
                 1103 
                 guuuucumaaacumaucmagggT T   
                 1104 
                 6524 
               
               
                   
               
               
                 AL-DP-5980 
                 gaumagumumumagaaaacmaumcmcmT T   
                 1105 
                 ggauguuuucumaaacumaucT T   
                 1106 
                 6528 
               
               
                   
               
               
                 AL-DP-5981 
                 umagumumumagaaaacmaumcmcmcmaT T   
                 1107 
                 ugggauguuuucumaaacumaT T   
                 1108 
                 6530 
               
               
                   
               
               
                 AL-DP-5982 
                 cmagacmumagaumgumagumaumumumT T   
                 1109 
                 aaaumacumacmaucumagucugT T   
                 1110 
                 6790 
               
               
                   
               
               
                 AL-DP-5983 
                 cmcmcmcmagacm8maga8mg8mag8maT T   
                 1111 
                 umacumacmaucumagucuggggT T   
                 1112 
                 6787 
               
               
                   
               
               
                 AL-DP-5984 
                 cmcmagacmumagaumgumagumaumumT T   
                 1113 
                 aaumacumacmaucumagucuggT T   
                 1114 
                 6789 
               
               
                   
               
               
                 AL-DP-5985 
                 cmcmcmagacmumagaumgumagumaumT T   
                 1115 
                 aumacumacmaucumagucugggT T   
                 1116 
                 6788 
               
               
                   
               
               
                 AL-DP-5986 
                 umgcmcmacmumumaaumgumaumgumumaT T   
                 1117 
                 umaacmaumacmauumaaguggcmaT T   
                 1118 
                 6767 
               
               
                   
               
               
                 AL-DP-5987 
                 umgcmumgumumumaumumaaumcmumumagT T   
                 1119 
                 cumaagauumaaumaaacmagcmaT T   
                 1120 
                 6614 
               
               
                   
               
               
                 AL-DP-5988 
                 umcmaumgumumagumumacmcmumumaumaT T   
                 1121 
                 umaumaaggumaacumaacmaugaT T   
                 1122 
                 6732 
               
               
                   
               
               
                 AL-DP-5989 
                 cmcmagumumaaumumumumumcmcmaacmumT T   
                 1123 
                 aguuggaaaauumaacuggT T   
                 1124 
                 6832 
               
               
                   
               
               
                 AL-DP-5990 
                 umacmcmumaagaumumacmaaaumcmaT T   
                 1125 
                 ugauuugumaaucuugammumaT T   
                 1126 
                 6650 
               
               
                   
               
               
                 AL-DP-5991 
                 umcmumumgcmumacmcmagumaumumumagT T   
                 1127 
                 cumaaaumacuggumagcmaagaT T   
                 1128 
                 6657 
               
               
                   
               
               
                 AL-DP-5992 
                 umgumgumaaumumagumgcmcmacmumumT T   
                 1129 
                 aaguggcmacumaauumacmacmaT T   
                 1130 
                 6756 
               
               
                   
               
               
                 AL-DP-5993 
                 aumcmumumgcmumacmcmagumaumumumaT T   
                 1131 
                 umaaaumacuggumagcmaagauT T   
                 1132 
                 6656 
               
               
                   
               
               
                 AL-DP-5994 
                 cmumgumaacmumacmumumumaumaacmumT T   
                 1133 
                 aguumaumaaagumaguumacmagT T   
                 1134 
                 6713 
               
               
                   
               
               
                 AL-DP-5995 
                 gumgaaumumaggcmumgumaacmumaT T   
                 1135 
                 umaguumacmagccumaauucmacT T   
                 1136 
                 6703 
               
               
                   
               
               
                 AL-DP-6176 
                 cmumacmcmagumaumumumagaagcmcmT T-Chol   
                 1137 
                 ggcuucumaaaumacuggumagT T   
                 1138 
                 6662 
               
               
                   
               
               
                 AL-DP-6177 
                 umgcmumgumumumaumumaaumcmumumagT T-Chol   
                 1139 
                 cumaagauumaaumaaacmagcmaT T   
                 1140 
                 6614 
               
               
                   
               
               
                   1 C. 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 3 
     siRNA 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 represented 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 1.5×10 4  cells/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 Lipofectamine-2000 (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 Lipofectamine-2000-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-Lipofectamine-2000-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% CO 2  in 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&#39;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 IC 50  concentrations (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 IC 50  was determined by curve fitting using the computer software X1fit using the following parameters: Dose Response One Site, 4 Parameter Logistic Model, fit=(A+((B−A)/(1+4(10̂C)/x)̂D)))), inv=((10̂C)/(((((B−A)/(y−A))−1)̂(1/D))), res=(y−fit). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Rat RhoA probes 
               
            
           
           
               
               
               
            
               
                 Probe 
                   
                 SEQ 
               
               
                 type 1   
                 Nucleotide sequence 
                 ID NO. 
               
               
                   
               
               
                 CE 
                 CCATTTTTCTGGGATGTTTTCTAAATTTTT 
                 1141 
               
               
                   
                 CTCTTGGAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 ACAGAAATGCTTGACTTCTGGAGTTTTTTC 
                 1142 
               
               
                   
                 TCTTGGAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 CTTCAGGTTTTACCGGCTCCTTTTTCTCTT 
                 1143 
               
               
                   
                 GGAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 CTGTTTGCCATATCTCTGCCTTTTTTTCTC 
                 1144 
               
               
                   
                 TTGGAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 TTGGTCTTTGCTGAACACTCCATTTTTCTC 
                 1145 
               
               
                   
                 TTGGAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 CCCGCGTCTAGCTTGCAGATTTTTCTCTTG 
                 1146 
               
               
                   
                 GAAAGAAAGT 
                   
               
               
                   
               
               
                 LE 
                 AGGATGATGGGCACATTTGGTTTTTAGGCA 
                 1147 
               
               
                   
                 TAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 GCCTTGTGTGCTCATCATTCCTTTTTAGGC 
                 1148 
               
               
                   
                 ATAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 TGCTTCATTTTGGCTAACTCCCTTTTTAGG 
                 1149 
               
               
                   
                 CATAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 TGTACCCAAAAGCGCCAATCTTTTTAGGCA 
                 1150 
               
               
                   
                 TAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 GCAGCTCTCGTGGCCATCTTTTTTAGGCAT 
                 1151 
               
               
                   
                 AGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 AGGCACCCCGACTTTTTCTTTTTTTAGGCA 
                 1152 
               
               
                   
                 TAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 BL 
                 CTATCAGGGCTGTCGATGGAA 
                 1153 
               
               
                   
               
               
                 BL 
                 GAAGATCCTTCTTGTTCCCAACT 
                 1154 
               
               
                   
               
               
                 BL 
                 CAAAAACCTCTCTCACTCCGTCT 
                 1155 
               
               
                   
               
               
                   1 CE = Capture Extender probe; LE = Label Extender probe; BL = blocking probe 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Rat GAPDH probes 
               
            
           
           
               
               
               
            
               
                 Probe 
                   
                 SEQ 
               
               
                 type 1   
                 Nucleotide sequence 
                 ID NO. 
               
               
                   
               
               
                 CE 
                 CCAGCTTCCCATTCTCAGCCTTTTTCTCTTG 
                 1156 
               
               
                   
                 GAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 TCTCGCTCCTGGAAGATGGTTTTTTCTCTTG 
                 1157 
               
               
                   
                 GAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 CCCATTTGATGTTAGCGGGATTTTTCTCTTG 
                 1158 
               
               
                   
                 GAAAGAAAGT 
                   
               
               
                   
               
               
                 CE 
                 CGGAGATGATGACCCTTTTGGTTTTTCTCTT 
                 1159 
               
               
                   
                 GGAAAGAAAGT 
                   
               
               
                   
               
               
                 LE 
                 GATGGGTTTCCCGTTGATGATTTTTAGGCAT 
                 1160 
               
               
                   
                 AGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 GACATACTCAGCACCAGCATCACTTTTTAGG 
                 1161 
               
               
                   
                 CATAGGACCCGTGTCT 
                   
               
               
                   
               
               
                 LE 
                 CCCAGCCTTCTCCATGGTGGTTTTTAGGCAT 
                 1162 
               
               
                   
                 AGGACCCGTGTCT 
                   
               
               
                   
               
               
                 BL 
                 TTGACTGTGCCGTTGAACTTG 
                 1163 
               
               
                   
               
               
                 BL 
                 CCCCACCCTTCAGGTGAGC 
                 1164 
               
               
                   
               
               
                 BL 
                 GGCATCAGCGGAAGGGG 
                 1165 
               
               
                   
               
               
                   1 CE = 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 IC 50  value for selected agents. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Ability of siRNAs specific for RhoA to reduce 
               
               
                 RhoA mRNA levels in cultured cells 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Rem. RhoA 
                 Rem. RhoA 
                   
               
               
                   
                   
                 mRNA at 100 nM 
                 mRNA at 100 nM 
                 IC 50   
               
               
                 Agent 
                 Pos. in 
                 agent, first 
                 agent, second 
                 RhoA 
               
               
                 number 
                 mRNA 1   
                 screen 
                 screen 
                 [nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 AL-DP-5850 
                   
                   
                 73 ± 8% 
                 142 
               
               
                 AL-DP-5852 
                   
                   
                 11 ± 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 
               
               
                   
               
               
                   1 Position 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 4 
     Stability 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 NaC15M, 4.0 ml SDS 10%) was prepared fresh and kept at 50° C. until use to avoid precipitation. 
     A 40 mer of poly(L-dT), (L-dT) 40  was 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 occurring 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) 40  were 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 NaClO 4 , 20 mM TRIS-HCl, 
               
               
                   
                 1 mM EDTA; 10% acetonitrile, 
               
               
                   
                 pH = 8.0 
               
               
                 Eluent B: 
                 800 mM NaClO 4 , 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% −&gt; 35% B 
               
               
                   
                 11-12 min: 
                 35% B −&gt; 100% B 
               
               
                   
                 12-14 min: 
                 100% B −&gt; 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 NaClO 4  by 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) 40  onto the HPLC column, and calculation of the theoretical peak area corresponding to 25 pmole (L-dT) 40  with 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 1/2  may 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 
                 cation 1   
                 # 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 
               
               
                   
               
               
                   1 0 = 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 T T  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 
               
               
                   2 C.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 5 
     Inhibition of RhoA Expression in Rat Primary Dorsal Root Ganglia (DRG) Cells in Culture 
     The inhibition of RhoA expression was assessed in in rat primary dorsal root ganglia (DRG) cells in culture in order to validate results obtained using Neuroscreen 1 cells as described above. 
     DRG cells were isolated from Sprague-Dawley rats at postnatal 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&#39;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% CO 2  in 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&#39;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% CO 2  in a humidified incubator supernatant was removed from the cells, fresh F12-HAM&#39;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% CO 2  in a humidified incubator, and rhoA mRNA was quantified. 
     RhoA mRNA levels were measured using the QuantiGene™ bDNA kit (Genospectra, Fremont, USA) according to manufacturer&#39;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 30 min 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 Victor 2 -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.