# Patent

## Title
iRNA agents targeted to the Rho-A gene

## Abstract

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

## Background
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/701,470, filed Jul. 21, 2005, U.S. Provisional Application No. 60/726,838, filed Oct. 14, 2005, and U.S. Provisional Application No. 60/748,316, filed Dec. 7, 2005. The contents of each of these priority applications are incorporated herein by reference in their entirety.


SEQUENCE LISTING
This application incorporates by reference the sequence listing saved as an ASCII text file on CD-ROM. The sequence listing saved on CD-ROM was created on Oct. 5, 2006, and is identified as “14174-139001.txt.” The file contains 280 KB of data. Three identical copies of the sequence listing have been submitted, including one “Computer-Readable Format” (CRF) and two “Official Copies” (Copy 1 and Copy 2).
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 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















Agent
pos.
SEQ

design
SEQ

SEQ



num-
in
ID

(over-
ID

ID


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


















6477
288
1
ccggaagaaacuggugauuguug
double
2
ggaagaaacuggugauuguTT
3
acaaucaccaguuucuuccTT


 


6478
289
4
cggaagaaacuggugauuguugg
double
5
gaagaaacuggugauuguuTT
6
aacaaucaccaguuucuucTT


 


6479
290
7
ggaagaaacuggugauuguuggu
double
8
aagaaacuggugauuguugTT
9
caacaaucaccaguuucuuTT


 


6480
291
10
gaagaaacuggugauuguuggug
double
11
agaaacuggugauuguuggTT
12
ccaacaaucaccaguuucuTT


 


6481
292
13
aagaaacuggugauuguugguga
double
14
gaaacuggugauuguugguTT
15
accaacaaucaccaguuucTT


 


6482
293
16
agaaacuggugauuguuggugau
double
17
aaacuggugauuguuggugTT
18
caccaacaaucaccaguuuTT


 


6483
294
19
gaaacuggugauuguuggugaug
double
20
aacuggugauuguuggugaTT
21
ucaccaacaaucaccaguuTT


 


6484
295
22
aaacuggugauuguuggugaugg
double
23
acuggugauuguuggugauTT
24
aucaccaacaaucaccaguTT


 


6485
296
25
aacuggugauuguuggugaugga
double
26
cuggugauuguuggugaugTT
27
caucaccaacaaucaccagTT


 


6486
297
28
acuggugauuguuggugauggag
double
29
uggugauuguuggugauggTT
30
ccaucaccaacaaucaccaTT


 


6487
298
31
cuggugauuguuggugauggagc
double
32
ggugauuguuggugauggaTT
33
uccaucaccaacaaucaccTT


 


6488
299
34
uggugauuguuggugauggagcc
double
35
gugauuguuggugauggagTT
36
cuccaucaccaacaaucacTT


 


6489
300
37
ggugauuguuggugauggagccu
double
38
ugauuguuggugauggagcTT
39
gcuccaucaccaacaaucaTT


 


6490
326
40
gaaagacaugcuugcucauaguc
double
41
aagacaugcuugcucauagTT
42
cuaugagcaagcaugucuuTT


 


6491
327
43
aaagacaugcuugcucauagucu
double
44
agacaugcuugcucauaguTT
45
acuaugagcaagcaugucuTT


 


6492
328
46
aagacaugcuugcucauagucuu
double
47
gacaugcuugcucauagucTT
48
gacuaugagcaagcaugucTT


 


6493
329
49
agacaugcuugcucauagucuuc
double
50
acaugcuugcucauagucuTT
51
agacuaugagcaagcauguTT


 


6494
330
52
gacaugcuugcucauagucuuca
double
53
caugcuugcucauagucuuTT
54
aagacuaugagcaagcaugTT


 


6495
331
55
acaugcuugcucauagucuucag
double
56
augcuugcucauagucuucTT
57
gaagacuaugagcaagcauTT


 


6496
332
58
caugcuugcucauagucuucagc
double
59
ugcuugcucauagucuucaTT
60
ugaagacuaugagcaagcaTT


 


6497
333
61
augcuugcucauagucuucagca
double
62
gcuugcucauagucuucagTT
63
cugaagacuaugagcaagcTT


 


6498
334
64
ugcuugcucauagucuucagcaa
double
65
cuugcucauagucuucagcTT
66
gcugaagacuaugagcaagTT


 


6499
335
67
gcuugcucauagucuucagcaag
double
68
uugcucauagucuucagcaTT
69
ugcugaagacuaugagcaaTT


 


6500
336
70
cuugcucauagucuucagcaagg
double
71
ugcucauagucuucagcaaTT
72
uugcugaagacuaugagcaTT


 


6501
337
73
uugcucauagucuucagcaagga
double
74
gcucauagucuucagcaagTT
75
cuugcugaagacuaugagcTT


 


6502
338
76
ugcucauagucuucagcaaggac
double
77
cucauagucuucagcaaggTT
78
ccuugcugaagacuaugagTT


 


6503
339
79
gcucauagucuucagcaaggacc
double
80
ucauagucuucagcaaggaTT
81
uccuugcugaagacuaugaTT


 


6504
340
82
cucauagucuucagcaaggacca
double
83
cauagucuucagcaaggacTT
84
guccuugcugaagacuaugTT


 


6505
341
85
ucauagucuucagcaaggaccag
double
86
auagucuucagcaaggaccTT
87
gguccuugcugaagacuauTT


 


6506
342
88
cauagucuucagcaaggaccagu
double
89
uagucuucagcaaggaccaTT
90
ugguccuugcugaagacuaTT


 


6507
343
91
auagucuucagcaaggaccaguu
double
92
agucuucagcaaggaccagTT
93
cugguccuugcugaagacuTT


 


6508
344
94
uagucuucagcaaggaccaguuc
double
95
gucuucagcaaggaccaguTT
96
acugguccuugcugaagacTT


 


6509
345
97
agucuucagcaaggaccaguucc
double
98
ucuucagcaaggaccaguuTT
99
aacugguccuugcugaagaTT


 


6510
346
100
gucuucagcaaggaccaguuccc
double
101
cuucagcaaggaccaguucTT
102
gaacugguccuugcugaagTT


 


6511
347
103
ucuucagcaaggaccaguuccca
double
104
uucagcaaggaccaguuccTT
105
ggaacugguccuugcugaaTT


 


6512
348
106
cuucagcaaggaccaguucccag
double
107
ucagcaaggaccaguucccTT
108
gggaacugguccuugcugaTT


 


6513
349
109
uucagcaaggaccaguucccaga
double
110
cagcaaggaccaguucccaTT
111
ugggaacugguccuugcugTT


 


6514
350
112
ucagcaaggaccaguucccagag
double
113
agcaaggaccaguucccagTT
114
cugggaacugguccuugcuTT


 


6515
351
115
cagcaaggaccaguucccagagg
double
116
gcaaggaccaguucccagaTT
117
ucugggaacugguccuugcTT


 


6516
352
118
agcaaggaccaguucccagaggu
double
119
caaggaccaguucccagagTT
120
cucugggaacugguccuugTT


 


6517
353
121
gcaaggaccaguucccagaggug
double
122
aaggaccaguucccagaggTT
123
ccucugggaacugguccuuTT


 


6518
354
124
caaggaccaguucccagaggugu
double
125
aggaccaguucccagagguTT
126
accucugggaacugguccuTT


 


6519
425
127
gaaagcagguagaguuggcuuug
double
128
aagcagguagaguuggcuuTT
129
aagccaacucuaccugcuuTT


 


6520
426
130
aaagcagguagaguuggcuuugu
double
131
agcagguagaguuggcuuuTT
132
aaagccaacucuaccugcuTT


 


6521
535
133
gacagcccugauaguuuagaaaa
double
134
cagcccugauaguuuagaaTT
135
uucuaaacuaucagggcugTT


 


6522
536
136
acagcccugauaguuuagaaaac
double
137
agcccugauaguuuagaaaTT
138
uuucuaaacuaucagggcuTT


 


6523
537
139
cagcccugauaguuuagaaaaca
double
140
gcccugauaguuuagaaaaTT
141
uuuucuaaacuaucagggcTT


 


6524
538
142
agcccugauaguuuagaaaacau
double
143
cccugauaguuuagaaaacTT
144
guuuucuaaacuaucagggTT


 


6525
539
145
gcccugauaguuuagaaaacauc
double
146
ccugauaguuuagaaaacaTT
147
uguuuucuaaacuaucaggTT


 


6526
540
148
cccugauaguuuagaaaacaucc
double
149
cugauaguuuagaaaacauTT
150
auguuuucuaaacuaucagTT


 


6527
541
151
ccugauaguuuagaaaacauccc
double
152
ugauaguuuagaaaacaucTT
153
gauguuuucuaaacuaucaTT


 


6528
542
154
cugauaguuuagaaaacauccca
double
155
gauaguuuagaaaacauccTT
156
ggauguuuucuaaacuaucTT


 


6529
543
157
ugauaguuuagaaaacaucccag
double
158
auaguuuagaaaacaucccTT
159
gggauguuuucuaaacuauTT


 


6530
544
160
gauaguuuagaaaacaucccaga
double
161
uaguuuagaaaacaucccaTT
162
ugggauguuuucuaaacuaTT


 


6531
545
163
auaguuuagaaaacaucccagaa
double
164
aguuuagaaaacaucccagTT
165
cugggauguuuucuaaacuTT


 


6532
546
166
uaguuuagaaaacaucccagaaa
double
167
guuuagaaaacaucccagaTT
168
ucugggauguuuucuaaacTT


 


6533
547
169
aguuuagaaaacaucccagaaaa
double
170
uuuagaaaacaucccagaaTT
171
uucugggauguuuucuaaaTT


 


6534
548
172
guuuagaaaacaucccagaaaag
double
173
uuagaaaacaucccagaaaTT
174
uuucugggauguuuucuaaTT


 


6535
549
175
uuuagaaaacaucccagaaaagu
double
176
uagaaaacaucccagaaaaTT
177
uuuucugggauguuuucuaTT


 


6536
575
178
ccccagaagucaagcauuucugu
double
179
ccagaagucaagcauuucuTT
180
agaaaugcuugacuucuggTT


 


6537
576
181
cccagaagucaagcauuucuguc
double
182
cagaagucaagcauuucugTT
183
cagaaaugcuugacuucugTT


 


6538
577
184
ccagaagucaagcauuucugucc
double
185
agaagucaagcauuucuguTT
186
acagaaaugcuugacuucuTT


 


6539
578
187
cagaagucaagcauuucuguccc
double
188
gaagucaagcauuucugucTT
189
gacagaaaugcuugacuucTT


 


6540
579
190
agaagucaagcauuucuguccca
double
191
aagucaagcauuucuguccTT
192
ggacagaaaugcuugacuuTT


 


6541
692
193
ugaaaccugaagaaggcagagau
double
194
aaaccugaagaaggcagagTT
195
cucugccuucuucagguuuTT


 


6542
693
196
gaaaccugaagaaggcagagaua
double
197
aaccugaagaaggcagagaTT
198
ucucugccuucuucagguuTT


 


6543
694
199
aaaccugaagaaggcagagauau
double
200
accugaagaaggcagagauTT
201
aucucugccuucuucagguTT


 


6544
695
202
aaccugaagaaggcagagauaug
double
203
ccugaagaaggcagagauaTT
204
uaucucugccuucuucaggTT


 


6545
696
205
accugaagaaggcagagauaugg
double
206
cugaagaaggcagagauauTT
207
auaucucugccuucuucagTT


 


6546
697
208
ccugaagaaggcagagauauggc
double
209
ugaagaaggcagagauaugTT
210
cauaucucugccuucuucaTT


 


6547
698
211
cugaagaaggcagagauauggca
double
212
gaagaaggcagagauauggTT
213
ccauaucucugccuucuucTT


 


6548
699
214
ugaagaaggcagagauauggcaa
double
215
aagaaggcagagauauggcTT
216
gccauaucucugccuucuuTT


 


6549
700
217
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
cgccaauccuguuugccauTT


 


6563
714
259
uauggcaaacaggauuggcgcuu
double
260
uggcaaacaggauuggcgcTT
261
gcgccaauccuguuugccaTT


 


6564
715
262
auggcaaacaggauuggcgcuuu
double
263
ggcaaacaggauuggcgcuTT
264
agcgccaauccuguuugccTT


 


6565
716
265
uggcaaacaggauuggcgcuuuu
double
266
gcaaacaggauuggcgcuuTT
267
aagcgccaauccuguuugcTT


 


6566
717
268
ggcaaacaggauuggcgcuuuug
double
269
caaacaggauuggcgcuuuTT
270
aaagcgccaauccuguuugTT


 


6567
718
271
gcaaacaggauuggcgcuuuugg
double
272
aaacaggauuggcgcuuuuTT
273
aaaagcgccaauccuguuuTT


 


6568
719
274
caaacaggauuggcgcuuuuggg
double
275
aacaggauuggcgcuuuugTT
276
caaaagcgccaauccuguuTT


 


6569
720
277
aaacaggauuggcgcuuuugggu
double
278
acaggauuggcgcuuuuggTT
279
ccaaaagcgccaauccuguTT


 


6570
721
280
aacaggauuggcgcuuuugggua
double
281
caggauuggcgcuuuugggTT
282
cccaaaagcgccaauccugTT


 


6571
722
283
acaggauuggcgcuuuuggguac
double
284
aggauuggcgcuuuuggguTT
285
acccaaaagcgccaauccuTT


 


6572
723
286
caggauuggcgcuuuuggguaca
double
287
ggauuggcgcuuuuggguaTT
288
uacccaaaagcgccaauccTT


 


6573
724
289
aggauuggcgcuuuuggguacau
double
290
gauuggcgcuuuuggguacTT
291
guacccaaaagcgccaaucTT


 


6574
725
292
ggauuggcgcuuuuggguacaug
double
293
auuggcgcuuuuggguacaTT
294
uguacccaaaagcgccaauTT


 


6575
726
295
gauuggcgcuuuuggguacaugg
double
296
uuggcgcuuuuggguacauTT
297
auguacccaaaagcgccaaTT


 


6576
727
298
auuggcgcuuuuggguacaugga
double
299
uggcgcuuuuggguacaugTT
300
cauguacccaaaagcgccaTT


 


6577
728
301
uuggcgcuuuuggguacauggag
double
302
ggcgcuuuuggguacauggTT
303
ccauguacccaaaagcgccTT


 


6578
729
304
uggcgcuuuuggguacauggagu
double
305
gcgcuuuuggguacauggaTT
306
uccauguacccaaaagcgcTT


 


6579
730
307
ggcgcuuuuggguacauggagug
double
308
cgcuuuuggguacauggagTT
309
cuccauguacccaaaagcgTT


 


6580
731
310
gcgcuuuuggguacauggagugu
double
311
gcuuuuggguacauggaguTT
312
acuccauguacccaaaagcTT


 


6581
732
313
cgcuuuuggguacauggaguguu
double
314
cuuuuggguacauggagugTT
315
cacuccauguacccaaaagTT


 


6582
733
316
gcuuuuggguacauggaguguuc
double
317
uuuuggguacauggaguguTT
318
acacuccauguacccaaaaTT


 


6583
734
319
cuuuuggguacauggaguguuca
double
320
uuuggguacauggaguguuTT
321
aacacuccauguacccaaaTT


 


6584
735
322
uuuuggguacauggaguguucag
double
323
uuggguacauggaguguucTT
324
gaacacuccauguacccaaTT


 


6585
736
325
uuuggguacauggaguguucagc
double
326
uggguacauggaguguucaTT
327
ugaacacuccauguacccaTT


 


6586
737
328
uuggguacauggaguguucagca
double
329
ggguacauggaguguucagTT
330
cugaacacuccauguacccTT


 


6587
738
331
uggguacauggaguguucagcaa
double
332
gguacauggaguguucagcTT
333
gcugaacacuccauguaccTT


 


6588
739
334
ggguacauggaguguucagcaaa
double
335
guacauggaguguucagcaTT
336
ugcugaacacuccauguacTT


 


6589
740
337
gguacauggaguguucagcaaag
double
338
uacauggaguguucagcaaTT
339
uugcugaacacuccauguaTT


 


6590
741
340
guacauggaguguucagcaaaga
double
341
acauggaguguucagcaaaTT
342
uuugcugaacacuccauguTT


 


6591
742
343
uacauggaguguucagcaaagac
double
344
cauggaguguucagcaaagTT
345
cuuugcugaacacuccaugTT


 


6592
743
346
acauggaguguucagcaaagacc
double
347
auggaguguucagcaaagaTT
348
ucuuugcugaacacuccauTT


 


6593
744
349
cauggaguguucagcaaagacca
double
350
uggaguguucagcaaagacTT
351
gucuuugcugaacacuccaTT


 


6594
745
352
auggaguguucagcaaagaccaa
double
353
ggaguguucagcaaagaccTT
354
ggucuuugcugaacacuccTT


 


6595
746
355
uggaguguucagcaaagaccaaa
double
356
gaguguucagcaaagaccaTT
357
uggucuuugcugaacacucTT


 


6596
747
358
ggaguguucagcaaagaccaaag
double
359
aguguucagcaaagaccaaTT
360
uuggucuuugcugaacacuTT


 


6597
748
361
gaquguucagcaaagaccaaaga
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
974
523
agaagucaucuugcuaccaguau
double
524
aagucaucuugcuaccaguTT
525
acugguagcaagaugacuuTT


 


6652
975
526
gaagucaucuugcuaccaguauu
double
527
agucaucuugcuaccaguaTT
528
uacugguagcaagaugacuTT


 


6653
976
529
aagucaucuugcuaccaguauuu
double
530
gucaucuugcuaccaguauTT
531
auacugguagcaagaugacTT


 


6654
977
532
agucaucuugcuaccaguauuua
double
533
ucaucuugcuaccaguauuTT
534
aauacugguagcaagaugaTT


 


6655
978
535
gucaucuugcuaccaguauuuag
double
536
caucuugcuaccaguauuuTT
537
aaauacugguagcaagaugTT


 


6656
979
538
ucaucuugcuaccaguauuuaga
double
539
aucuugcuaccaguauuuaTT
540
uaaauacugguagcaagauTT


 


6657
980
541
caucuugcuaccaguauuuagaa
double
542
ucuugcuaccaguauuuagTT
543
cuaaauacugguagcaagaTT


 


6658
981
544
aucuugcuaccaguauuuagaag
double
545
cuugcuaccaguauuuagaTT
546
ucuaaauacugguagcaagTT


 


6659
982
547
ucuugcuaccaguauuuagaagc
double
548
uugcuaccaguauuuagaaTT
549
uucuaaauacugguagcaaTT


 


6660
983
550
cuugcuaccaguauuuagaagcc
double
551
ugcuaccaguauuuagaagTT
552
cuucuaaauacugguagcaTT


 


6661
984
553
uugcuaccaguauuuagaagcca
double
554
gcuaccaguauuuagaagcTT
555
gcuucuaaauacugguagcTT


 


6662
985
556
ugcuaccaguauuuagaagccaa
double
557
cuaccaguauuuagaagccTT
558
ggcuucuaaauacugguagTT


 


6663
986
559
gcuaccaguauuuagaagccaac
double
560
uaccaguauuuagaagccaTT
561
uggcuucuaaauacugguaTT


 


6664
987
562
cuaccaguauuuagaagccaacu
double
563
accaguauuuagaagccaaTT
564
uuggcuucuaaauacugguTT


 


6665
988
565
uaccaguauuuagaagccaacua
double
566
ccaguauuuagaagccaacTT
567
guuggcuucuaaauacuggTT


 


6666
1131
568
cuugcuucuuucuagaaagagaa
double
569
ugcuucuuucuagaaagagTT
570
cucuuucuagaaagaagcaTT


 


6667
1132
571
uugcuucuuucuagaaagagaaa
double
572
gcuucuuucuagaaagagaTT
573
ucucuuucuagaaagaagcTT


 


6668
1133
574
ugcuucuuucuagaaagagaaac
double
575
cuucuuucuagaaagagaaTT
576
uucucuuucuagaaagaagTT


 


6669
1134
577
gcuucuuucuagaaagagaaaca
double
578
uucuuucuagaaagagaaaTT
579
uuucucuuucuagaaagaaTT


 


6670
1135
580
cuucuuucuagaaagagaaacag
double
581
ucuuucuagaaagagaaacTT
582
guuucucuuucuagaaagaTT


 


6671
1136
583
uucuuucuagaaagagaaacagu
double
584
cuuucuagaaagagaaacaTT
585
uguuucucuuucuagaaagTT


 


6672
1137
586
ucuuucuagaaagagaaacaguu
double
587
uuucuagaaagagaaacagTT
588
cuguuucucuuucuagaaaTT


 


6673
1138
589
cuuucuagaaagagaaacaguug
double
590
uucuagaaagagaaacaguTT
591
acuguuucucuuucuagaaTT


 


6674
1139
592
uuucuagaaagagaaacaguugg
double
593
ucuagaaagagaaacaguuTT
594
aacuguuucucuuucuagaTT


 


6675
1140
595
uucuagaaagagaaacaguuggu
double
596
cuagaaagagaaacaguugTT
597
caacuguuucucuuucuagTT


 


6676
1141
598
ucuagaaagagaaacaguuggua
double
599
uagaaagagaaacaguuggTT
600
ccaacuguuucucuuucuaTT


 


6677
1142
601
cuagaaagagaaacaguugguaa
double
602
agaaagagaaacaguugguTT
603
accaacuguuucucuuucuTT


 


6678
1143
604
uagaaagagaaacaguugguaac
double
605
gaaagagaaacaguugguaTT
606
uaccaacuguuucucuuucTT


 


6679
1144
607
agaaagagaaacaguugguaacu
double
608
aaagagaaacaguugguaaTT
609
uuaccaacuguuucucuuuTT


 


6680
1145
610
gaaagagaaacaguugguaacuu
double
611
aagagaaacaguugguaacTT
612
guuaccaacuguuucucuuTT


 


6681
1146
613
aaagagaaacaguugguaacuuu
double
614
agagaaacaguugguaacuTT
615
aguuaccaacuguuucucuTT


 


6682
1147
616
aagagaaacaguugguaacuuuu
double
617
gagaaacaguugguaacuuTT
618
aaguuaccaacuguuucucTT


 


6683
1148
619
agagaaacaguugguaacuuuug
double
620
agaaacaguugguaacuuuTT
621
aaaguuaccaacuguuucuTT


 


6684
1149
622
gagaaacaguugguaacuuuugu
double
623
gaaacaguugguaacuuuuTT
624
aaaaguuaccaacuguuucTT


 


6685
1150
625
agaaacaguugguaacuuuugug
double
626
aaacaguugguaacuuuugTT
627
caaaaguuaccaacuguuuTT


 


6686
1151
628
gaaacaguugguaacuuuuguga
double
629
aacaguugguaacuuuuguTT
630
acaaaaguuaccaacuguuTT


 


6687
1152
631
aaacaguugguaacuuuugugaa
double
632
acaguugguaacuuuugugTT
633
cacaaaaguuaccaacuguTT


 


6688
1153
634
aacaguugguaacuuuugugaau
double
635
caguugguaacuuuugugaTT
636
ucacaaaaguuaccaacugTT


 


6689
1154
637
acaguugguaacuuuugugaauu
double
638
aguugguaacuuuugugaaTT
639
uucacaaaaguuaccaacuTT


 


6690
1155
640
caguugguaacuuuugugaauua
double
641
guugguaacuuuugugaauTT
642
auucacaaaaguuaccaacTT


 


6691
1156
643
aguugguaacuuuugugaauuag
double
644
uugguaacuuuugugaauuTT
645
aauucacaaaaguuaccaaTT


 


6692
1157
646
guugguaacuuuugugaauuagg
double
647
ugguaacuuuugugaauuaTT
648
uaauucacaaaaguuaccaTT


 


6693
1158
649
uugguaacuuuugugaauuaggc
double
650
gguaacuuuugugaauuagTT
651
cuaauucacaaaaguuaccTT


 


6694
1159
652
ugguaacuuuugugaauuaggcu
double
653
guaacuuuugugaauuaggTT
654
ccuaauucacaaaaguuacTT


 


6695
1160
655
gguaacuuuugugaauuaggcug
double
656
uaacuuuugugaauuaggcTT
657
gccuaauucacaaaaguuaTT


 


6696
1161
658
guaacuuuugugaauuaggcugu
double
659
aacuuuugugaauuaggcuTT
660
agccuaauucacaaaaguuTT


 


6697
1162
661
uaacuuuugugaauuaggcugua
double
662
acuuuugugaauuaggcugTT
663
cagccuaauucacaaaaguTT


 


6698
1163
664
aacuuuugugaauuaggcuguaa
double
665
cuuuugugaauuaggcuguTT
666
acagccuaauucacaaaagTT


 


6699
1164
667
acuuuugugaauuaggcuguaac
double
668
uuuugugaauuaggcuguaTT
669
uacagccuaauucacaaaaTT


 


6700
1165
670
cuuuugugaauuaggcuguaacu
double
671
uuugugaauuaggcuguaaTT
672
uuacagccuaauucacaaaTT


 


6701
1166
673
uuuugugaauuaggcuguaacua
double
674
uugugaauuaggcuguaacTT
675
guuacagccuaauucacaaTT


 


6702
1167
676
uuugugaauuaggcuguaacuac
double
677
ugugaauuaggcuguaacuTT
678
aguuacagccuaauucacaTT


 


6703
1168
679
uugugaauuaggcuguaacuacu
double
680
gugaauuaggcuguaacuaTT
681
uaguuacagccuaauucacTT


 


6704
1169
682
ugugaauuaggcuguaacuacuu
double
683
ugaauuaggcuguaacuacTT
684
guaguuacagccuaauucaTT


 


6705
1170
685
gugaauuaggcuguaacuacuuu
double
686
gaauuaggcuguaacuacuTT
687
aguaguuacagccuaauucTT


 


6706
1171
688
ugaauuaggcuguaacuacuuua
double
689
aauuaggcuguaacuacuuTT
690
aaguaguuacagccuaauuTT


 


6707
1172
691
gaauuaggcuguaacuacuuuau
double
692
auuaggcuguaacuacuuuTT
693
aaaguaguuacagccuaauTT


 


6708
1173
694
aauuaggcuguaacuacuuuaua
double
695
uuaggcuguaacuacuuuaTT
696
uaaaguaguuacagccuaaTT


 


6709
1174
697
auuaggcuguaacuacuuuauaa
double
698
uaggcuguaacuacuuuauTT
699
auaaaguaguuacagccuaTT


 


6710
1175
700
uuaggcuguaacuacuuuauaac
double
701
aggcuguaacuacuuuauaTT
702
uauaaaguaguuacagccuTT


 


6711
1176
703
uaggcuguaacuacuuuauaacu
double
704
ggcuguaacuacuuuauaaTT
705
uucuaaaguaguuacagccTT


 


67I2
1177
706
aggcuguaacuacuuuauaacua
double
707
gcuguaacuacuuuauaacTT
708
guuauaaaguaguuccagcTT


 


6713
1178
709
ggcuguaacuacuuuauaacuaa
double
710
cuguaacuacuuuauaacuTT
711
aguuauacaguaguuacagTT


 


6714
1179
712
gcuguaacuacuuuauaacuaac
double
713
uguaacuacuuuauaacuaTT
714
uaguuauaaaguaguuacaTT


 


6715
1180
715
cuguaacuacuuuauaacuaaca
double
716
guaacuacuuuauaacuaaTT
7I7
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
ggacauguuaguuauaaagTT


 


6723
1188
739
acuuuauaacuaacauguccugc
double
740
uuuauaacuaacauguccuTT
741
aggacauguuaguuauaaaTT


 


6724
1189
742
cuuuauaacuaacauguccugcc
double
743
uuauaacuaacauguccugTT
744
caggacauguuaguuauaaTT


 


6725
1190
745
uuuauaacuaacauguccugccu
double
746
uauaacuaacauguccugcTT
747
gcaggacauguuaguuauaTT


 


6726
1191
748
uuauaacuaacauguccugccua
double
749
auaacuaacauguccugccTT
750
ggcaggacauguuaguuauTT


 


6727
1192
751
uauaacuaacauguccugccuau
double
752
uaacuaacauguccugccuTT
753
aggcaggacauguuaguuaTT


 


6728
1193
754
auaacuaacauguccugccuauu
double
755
aacuaacauguccugccuaTT
756
uaggcaggacauguuaguuTT


 


6729
1352
757
uggcagaguuacaguucuguggu
double
758
gcagaguuacaguucugugTT
759
cacagaacuguaacucugcTT


 


6730
1353
760
ggcagaguuacaguucugugguu
double
761
cagaguuacaguucuguggTT
762
ccacagaacuguaacucugTT


 


6731
1354
763
gcagaguuacaguucugugguuu
double
764
agaguuacaguucugugguTT
765
accacagaacuguaacucuTT


 


6732
1374
766
uuucauguuaguuaccuuauagu
double
767
ucauguuaguuaccuuauaTT
768
uauaagguaacuaacaugaTT


 


6733
1375
769
uucauguuaguuaccuuauaguu
double
770
cauguuaguuaccuuauagTT
771
cuauaagguaacuaacaugTT


 


6734
1376
772
ucauguuaguuaccuuauaguua
double
773
auguuaguuaccuuauaguTT
774
acuauaagguaacuaacauTT


 


6735
1377
775
cauguuaguuaccuuauaguuac
double
776
uguuaguuaccuuauaguuTT
777
aacuauaagguaacuaacaTT


 


6736
1378
778
auguuaguuaccuuauaguuacu
double
779
guuaguuaccuuauaguuaTT
780
uaacuauaagguaacuaacTT


 


6737
1379
781
uguuaguuaccuuauaguuacug
double
782
uuaguuaccuuauaguuacTT
783
guaacuauaagguaacuaaTT


 


6738
1380
784
guuaguuaccuuauaguuacugu
double
785
uaguuaccuuauaguuacuTT
786
aguaacuauaagguaacuaTT


 


6739
1381
787
uuaguuaccuuauaguuacugug
double
788
aguuaccuuauaguuacugTT
789
caguaacuauaagguaacuTT


 


6740
1382
790
uaguuaccuuauaguuacugugu
double
791
guuaccuuauaguuacuguTT
792
acaguaacuauaagguaacTT


 


6741
1383
793
aguuaccuuauaguuacugugua
double
794
uuaccuuauaguuacugugTT
795
cacaguaacuauaagguaaTT


 


6742
1384
796
guuaccuuauaguuacuguguaa
double
797
uaccuuauaguuacuguguTT
798
acacaguaacuauaagguaTT


 


6743
1385
799
uuaccuuauaguuacuguguaau
double
800
accuuauaguuacuguguaTT
801
uacacaguaacuauaagguTT


 


6744
1386
802
uaccuuauaguuacuguguaauu
double
803
ccuuauaguuacuguguaaTT
804
uuacacaguaacuauaaggTT


 


6745
1387
805
accuuauaguuacuguguaauua
double
806
cuuauaguuacuguguaauTT
807
auuacacaguaacuauaagTT


 


6746
1388
808
ccuuauaguuacuguguaauuag
double
809
uuauaguuacuguguaauuTT
810
aauuacacaguaacuauaaTT


 


6747
1389
811
cuuauaguuacuguguaauuagu
double
812
uauaguuacuguguaauuaTT
813
uaauuacacaguaacuauaTT


 


6748
1390
814
uuauaguuacuguguaauuagug
double
815
auaguuacuguguaauuagTT
816
cuaauuacacaguaacuauTT


 


6749
1391
817
uauaguuacuguguaauuagugc
double
818
uaguuacuguguaauuaguTT
819
acuaauuacacaguaacuaTT


 


6750
1392
820
auaguuacuguguaauuagugcc
double
821
aguuacuguguaauuagugTT
822
cacuaauuacacaguaacuTT


 


6751
1393
823
uaguuacuguguaauuagugcca
double
824
guuacuguguaauuagugcTT
825
gcacuaauuacacaguaacTT


 


6752
1394
826
aguuacuguguaauuagugccac
double
827
uuacuguguaauuagugccTT
828
ggcacuaauuacacaguaaTT


 


6753
1395
829
guuacuguguaauuagugccacu
double
830
uacuguguaauuagugccaTT
831
uggcacuaauuacacaguaTT


 


6754
1396
832
uuacuguguaauuagugccacuu
double
833
acuguguaauuagugccacTT
834
guggcacuaauuacacaguTT


 


6755
1397
835
uacuguguaauuagugccacuua
double
836
cuguguaauuagugccacuTT
837
aguggcacuaauuacacagTT


 


6756
1398
838
acuguguaauuagugccacuuaa
double
839
uguguaauuagugccacuuTT
840
aaguggcacuaauuacacaTT


 


6757
1399
841
cuguguaauuagugccacuuaau
double
842
guguaauuagugccacuuaTT
843
uaaguggcacuaauuacacTT


 


6758
1400
844
uguguaauuagugccacuuaaug
double
845
uguaauuagugccacuuaaTT
846
uuaaguggcacuaauuacaTT


 


6759
1401
847
guguaauuagugccacuuaaugu
double
848
guaauuagugccacuuaauTT
849
auuaaguggcacuaauuacTT


 


6760
1402
850
uguaauuagugccacuuaaugua
double
851
uaauuagugccacuuaaugTT
852
cauuaaguggcacuaauuaTT


 


6761
1403
853
guaauuagugccacuuaauguau
double
854
aauuagugccacuuaauguTT
855
acauuaaguggcacuaauuTT


 


6762
1404
856
uaauuagugccacuuaauguaug
double
857
auuagugccacuuaauguaTT
858
uacauuaaguggcacuaauTT


 


6763
1405
859
aauuagugccacuuaauguaugu
double
860
uuagugccacuuaauguauTT
861
auacauuaaguggcacuaaTT


 


6764
1406
862
auuagugccacuuaauguauguu
double
863
uagugccacuuaauguaugTT
864
cauacauuaaguggcacuaTT


 


6765
1407
865
uuagugccacuuaauguauguua
double
866
agugccacuuaauguauguTT
867
acauacauuaaguggcacuTT


 


6766
1408
868
uagugccacuuaauguauguuac
double
869
gugccacuuaauguauguuTT
870
aacauacauuaaguggcacTT


 


6767
1409
871
agugccacuuaauguauguuacc
double
872
ugccacuuaauguauguuaTT
873
uaacauacauuaaguggcaTT


 


6768
1410
874
gugccacuuaauguauguuacca
double
875
gccacuuaauguauguuacTT
876
guaacauacauuaaguggcTT


 


6769
1411
877
ugccacuuaauguauguuaccaa
double
878
ccacuuaauguauguuaccTT
879
gguaacauacauuaaguggTT


 


6770
1412
880
gccacuuaauguauguuaccaaa
double
881
cacuuaauguauguuaccaTT
882
ugguaacauacauuaagugTT


 


6771
1413
883
ccacuuaauguauguuaccaaaa
double
884
acuuaauguauguuaccaaTT
885
uugguaacauacauuaaguTT


 


6772
1414
886
cacuuaauguauguuaccaaaaa
double
887
cuuaauguauguuaccaaaTT
888
uuugguaacauacauuaagTT


 


6773
1415
889
acuuaauguauguuaccaaaaau
double
890
uuaauguauguuaccaaaaTT
891
uuuugguaacauacauuaaTT


 


6774
1416
892
cuuaauguauguuaccaaaaaua
double
893
uaauguauguuaccaaaaaTT
894
uuuuugguaacauacauuaTT


 


6775
1435
895
aauaaauauaucuaccccagacu
double
896
uaaauauaucuaccccagaTT
897
ucugggguagauauauuuaTT


 


6776
1436
898
auaaauauaucuaccccagacua
double
899
aaauauaucuaccccagacTT
900
gucugggguagauauauuuTT


 


6777
1437
901
uaaauauaucuaccccagacuag
double
902
aauauaucuaccccagacuTT
903
agucugggguagauauauuTT


 


6778
1438
904
aaauauaucuaccccagacuaga
double
905
auauaucuaccccagacuaTT
906
uagucugggguagauauauTT


 


6779
1439
907
aauauaucuaccccagacuagau
double
908
uauaucuaccccagacuagTT
909
cuagucugggguagauauaTT


 


6780
1440
910
auauaucuaccccagacuagaug
double
911
auaucuaccccagacuagaTT
912
ucuagucugggguagauauTT


 


6781
1441
913
uauaucuaccccagacuagaugu
double
914
uaucuaccccagacuagauTT
915
aucuagucugggguagauaTT


 


6782
1442
916
auaucuaccccagacuagaugua
double
917
aucuaccccagacuagaugTT
918
caucuagucugggguagauTT


 


6783
1443
919
uaucuaccccagacuagauguag
double
920
ucuaccccagacuagauguTT
921
acaucuagucugggguagaTT


 


6784
1444
922
aucuaccccagacuagauguagu
double
923
cuaccccagacuagauguaTT
924
uacaucuagucugggguagTT


 


6785
1445
925
ucuaccccagacuagauguagua
double
926
uaccccagacuagauguagTT
927
cuacaucuagucugggguaTT


 


6786
1446
928
cuaccccagacuagauguaguau
double
929
accccagacuagauguaguTT
930
acuacaucuagucugggguTT


 


6787
1447
931
uaccccagacuagauguaguauu
double
932
ccccagacuagauguaguaTT
933
uacuacaucuagucuggggTT


 


6788
1448
934
accccagacuagauguaguauuu
double
935
cccagacuagauguaguauTT
936
auacuacaucuagucugggTT


 


6789
1449
937
ccccagacuagauguaguauuuu
double
938
ccagacuagauguaguauuTT
939
aauacuacaucuagucuggTT


 


6790
1450
940
cccagacuagauguaguauuuuu
double
941
cagacuagauguaguauuuTT
942
aaauacuacaucuagucugTT


 


6791
1451
943
ccagacuagauguaguauuuuuu
double
944
agacuagauguaguauuuuTT
945
aaaauacuacaucuagucuTT


 


6792
1452
946
cagacuagauguaguauuuuuug
double
947
gacuagauguaguauuuuuTT
948
aaaaauacuacaucuagucTT


 


6793
1453
949
agacuagauguaguauuuuuugu
double
950
acuagauguaguauuuuuuTT
951
aaaaaauacuacaucuaguTT


 


6794
1454
952
gacuagauguaguauuuuuugua
double
953
cuagauguaguauuuuuugTT
954
caaaaaauacuacaucuagTT


 


6795
1455
955
acuagauguaguauuuuuuguau
double
956
uagauguaguauuuuuuguTT
957
acaaaaaauacuacaucuaTT


 


6796
1456
958
cuagauguaguauuuuuuguaua
double
959
agauguaguauuuuuuguaTT
960
uacaaaaaauacuacaucuTT


 


6797
1457
961
uagauguaguauuuuuuguauaa
double
962
gauguaguauuuuuuguauTT
963
auacaaaaaauacuacaucTT


 


6798
1458
964
agauguaguauuuuuuguauaau
double
965
auguaguauuuuuuguauaTT
966
uauacaaaaaauacuacauTT


 


6799
1459
967
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


 


6824
1800
1042
ucccagaggagccaccaguucuc
double
1043
ccagaggagccaccaguucTT
1044
gaacugguggcuccucuggTT


 


6825
1801
1045
cccagaggagccaccaguucuca
double
1046
cagaggagccaccaguucuTT
1047
agaacugguggcuccucugTT


 


6826
1843
1048
cuucucuccagcugacuaaacuu
double
1049
ucucuccagcugacuaaacTT
1050
guuuagucagcuggagagaTT


 


6827
1869
1051
uucuguaccaguuaauuuuucca
double
1052
cuguaccaguuaauuuuucTT
1053
gaaaaauuaacugguacagTT


 


6828
1870
1054
ucuguaccaguuaauuuuuccaa
double
1055
uguaccaguuaauuuuuccTT
1056
ggaaaaauuaacugguacaTT


 


6829
1871
1057
cuguaccaguuaauuuuuccaac
double
1058
guaccaguuaauuuuuccaTT
1059
uggaaaaauuaacugguacTT


 


6830
1872
1060
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





aSingle: Single overhang design 21 mer sense, corresponding 23 mer antisense with 2 nucleotides overhang at 3′ end;


Double: double overhang design corresponding 19 mer sense and antisense strand each with 2 dT nucleotide overhang at 3′ end


b“Start position” corresponds to the position within the sequence of human RhoA (Genbank accession no. NM_001664) mRNA to which the 5′-most nucleotide of the sense strand corresponds for single overhang designs; for double overhang designs, the 5′-most ribonucleotide of the sense strand corresponds to (Start position +2)






Based on these results, the invention specifically provides an iRNA agent that includes a sense strand having at least 15 contiguous nucleotides of the sense strand sequences of the agents provided in Table 1 under agent numbers 6477-6836, and an antisense strand having at least 15 contiguous nucleotides of the antisense sequences of the agents provided in Table 1 under agent numbers 6477 to 6836.
The iRNA agents shown in Table 1 are composed of two strands of 19 nucleotides in length which are complementary or identical to the target sequence, plus a 3′-TT overhang. The present invention provides agents that comprise 15 contiguous nucleotides from these agents. However, while these lengths may potentially be optimal, the iRNA agents are not meant to be limited to these lengths. The skilled person is well aware that shorter or longer iRNA agents may be similarly effective, since, within certain length ranges, the efficacy is rather a 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 35S, 32P, 33P, or 3H; 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., EC50 or ED50). Such animal models may include transgenic animals that express a human gene, e.g., a gene that produces a target human RhoA RNA. In another embodiment, the composition for testing includes an iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RhoA RNA in the animal model and the target RhoA RNA in a human.
iRNA Chemistry
Described herein are isolated iRNA agents, e.g., ds RNA agents that mediate RNAi to inhibit expression of a RhoA gene.
RNA agents discussed herein include otherwise unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. Nucleic Acids Res. 22: 2183-2196, 1994. Such rare or unusual RNAs, often termed modified RNAs (apparently because they are typically the result of a post-transcriptional modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of the above are discussed herein.
Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. It may be desirable to modify one or both of the antisense and sense strands of an iRNA agent. As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most, cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. Similarly, a modification may occur on the sense strand, antisense strand, or both. In some cases, the sense and antisense strand will have the same modifications or the same class of modifications, but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it may be desirable to modify only one strand, e.g. the sense strand.
Two prime objectives for the introduction of modifications into iRNA agents is their stabilization towards degradation in biological environments and the improvement of pharmacological properties, e.g. pharmacodynamic properties, which are further discussed below. Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described in co-owned PCT Application No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can include a non-naturally occurring base, such as the bases described in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule. Exemplary features of non-naturally occurring sugars for use in iRNA agents are described in co-owned PCT Application No. PCT/US2004/11829, filed Apr. 16, 2003.
An iRNA agent can include an internucleotide linkage (e.g., the chiral phosphorothioate linkage) useful for increasing nuclease resistance. In addition, or in the alternative, an iRNA agent can include a ribose mimic for increased nuclease resistance. Exemplary 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 severely 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′-ca-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′-ca-3′, or in all occurrences of 5′-ua-3′,5′-ca-3′, and 5′-uu-3′, or in all occurrences of 5′-ua-3′,5′-ca-3′,5′-uu-3′, and 5′-ug-3′ were replaced by 2′-modified nucleotides, such as 2′-O-methyl nucleotides, in both strands. Alternatively, 2′-modifying all pyrimidine nucleotides in the sense strand and the 5′-most pyrimidines in all occurrences of 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(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH2CH2OCH3, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.
Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.
The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An iRNA agent can be further modified by including a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus can be blocked with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′ exonucleolytic cleavage. While not being bound by theory, a 3′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.
Nucleolytic cleavage can also be inhibited by the introduction of phosphate linker modifications, e.g., phosphorothioate linkages. Thus, preferred iRNA agents include nucleotide dimers enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at a nonbridging position normally occupied by oxygen. The heteroatom can be S, Se, Nr2, or Br3. When the heteroatom is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Modified phosphate linkages are particularly efficient in inhibiting exonucleolytic cleavage when introduced near the 5′- or 3′-terminal positions, and preferably the 5′-terminal positions, of an iRNA agent.
5′ conjugates can also inhibit 5′-3′ exonucleolytic cleavage. While not being bound by theory, a 5′ conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5′-end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3′-5′-exonucleases.
An iRNA agent can have increased resistance to nucleases when a duplexed iRNA agent includes a single-stranded nucleotide overhang on at least one end. In preferred embodiments, the nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment, the unpaired nucleotide of the single-stranded overhang that is directly adjacent to the terminal nucleotide pair contains a purine base, and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs. In further embodiments, the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in an exemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferred embodiments, the nucleotide overhang is on the 3′-end of the antisense strand. In one embodiment, the iRNA agent includes 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-(CH2)nNH2 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's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include the B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
5′-Phosphate Modifications
In preferred embodiments, iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications of the antisense strand include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether-methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).
The sense strand can be modified in order to inactivate the sense strand and prevent formation of an active RISC, thereby potentially reducing off-target effects. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage.
Transport of iRNA Agents into Cells
Not wishing to be bound by any theory, the chemical similarity between cholesterol-conjugated iRNA agents and certain constituents of lipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) may lead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL) in blood and/or the interaction of the iRNA agent with cellular components having an affinity for cholesterol, e.g. components of the cholesterol transport pathway. Lipoproteins as well as their constituents are taken up and processed by cells by various active and passive transport mechanisms, for example, without limitation, endocytosis of LDL-receptor bound LDL, endocytosis of oxidized or otherwise modified LDLs through interaction with Scavenger receptor A, Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver, pinocytosis, or transport of cholesterol across membranes by ABC (ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 or ABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoy facilitated uptake by cells possessing such transport mechanisms, e.g. cells of the liver. As such, the present invention provides evidence and general methods for targeting iRNA agents to cells expressing certain cell surface components, e.g. receptors, by conjugating a natural ligand for such component (e.g. cholesterol) to the iRNA agent, or by conjugating a chemical moiety (e.g. cholesterol) to the iRNA agent which associates with or binds to a natural ligand for the component (e.g. LDL, HDL).
Other Embodiments
An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. Proc. Natl. Acad. Sci. 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 Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the iRNA agent preparation includes two or more iRNA agent(s), e.g., two or more iRNA agents that can mediate RNAi with respect to the same gene, or different alleles of the gene, or with respect to different genes. Such preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different iRNA agent species. Such iRNA agents can mediate RNAi with respect to a similar number of different genes.
Where the two or more iRNA agents in such preparation target the same gene, they can have target sequences that are non-overlapping and non-adjacent, or the target sequences may be overlapping or adjacent.
Disorders Associated with RhoA Expression
An iRNA agent that targets RhoA, e.g., an iRNA agent described herein, can be used to treat a subject, e.g., a human having or at risk for developing a disease or disorder associated with RhoA gene expression or treating a subject where a biological process mediated by RhoA is unwanted. Since Nogo-L, RhoA, and Nogo-R participate in inhibiting axonal growth and elongations, the iRNA agents of the present invention are used to reverse this inhibition leading to nerve/axonal growth and elongation. Such a treatment is useful in treating injuries to the nervous system such as spinal cord injury or peripheral nerve death (caused by, e.g., Metastatic cancers of the CNS, e.g., gliomas (such as glioblastomas, astrocytomas, oligodendrogliomas, ependymomas), meningiomas, medulloblastomas, neuroblastomas, choroid plexus papillomas, sarcomas can also be treated by the iRNA agents described herein. Other indications include diseases of the central nervous system, including but not limited to encephalomyelitis, ischemic stroke, Alzheimer's Disease, spongiform encephalopathy, Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), multiple sclerosis, transverse myelitis, motor neuron disease, Guillan Barre, Anterior Spinal Artery Syndrome, and schizophrenia.
For example, an iRNA agent that targets RhoA mRNA can be used to treat a subject with a spinal cord injury or a subject having another pathological state which can be ameliorated, at least in part, by nerve growth and elongation. In such a use, an iRNA agent of the present invention is administered preferably locally at the site of nerve damage or the site at which the inhibitory effects of RhoA is desired to be reversed. Administration of the iRNA agent leads to decrease in RhoA protein resulting in reversing Nogo mediated inhibition of axonal elongation and growth.
Treatment Methods and Routes of Delivery
A composition that includes an iRNA agent, e.g., an iRNA agent that targets RhoA, can be delivered to a subject by a variety of routes to achieve either local delivery to the site of action of systemic delivery to the subject. Exemplary routes include direct injection to the site of treatment, intrathecal, parenchymal, intravenous, nasal, oral, and ocular delivery. The preferred means of administering the iRNA agents of the present invention is through direct injection or infusion to the site of treatment.
An iRNA agent can be incorporated into pharmaceutical compositions suitable for administration. For example, compositions can include one or more species of an iRNA agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
The route of delivery can be dependent on the disorder of the patient. In general, the delivery of the iRNA agents of the present invention is done to achieve systemic delivery into the subject. One preferred means of achieving this is through parenteral administration. In a particularly preferred embodiment, the application is achieved by direct application of the pharmaceutical composition to the site of nerve injury, such as the site of spinal cord injury. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Using the small interfering RNA vectors previously described, the invention also provides devices, systems, and methods for delivery of small interfering RNA to target locations in the nervous system and or/the brain. The envisioned route of delivery is through the use of implanted, indwelling, intrathecal or intraparenchymal catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into local nerves or local brain tissue. The proximal end of these catheters may be connected to an implanted, intrathecal or intracerebral access port surgically affixed to the patient's body or cranium, or to an implanted drug pump located in the patient's torso.
Alternatively, implantable delivery devices, such as an implantable pump may be employed. Examples of the delivery devices within the scope of the invention include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously in the body or on the cranium, and provides an access port through which therapeutic agents may be delivered to the nerves or brain. Delivery occurs through a stereotactically implanted polyurethane catheter. Two models of catheters that can 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. No. 09/872,698 (filed Jun. 1, 2001) and U.S. Ser. No. 09/864,646 (filed May 23, 2001), which are incorporated herein by reference.
Preferably, the outlet of the pump or catheter is placed in close proximity of the desired site of action of the pharmaceutical composition, such as near the site of spinal cord, or other nerve, injury.
Administration can be provided by the subject or by another person, e.g., a caregiver. A caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.
The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.
The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.
The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.
The term “co-administration” refers to administering to a subject two or more agents, and in particular two or more iRNA agents. The agents can be contained in a single pharmaceutical composition and be administered at the same time, or the agents can be contained in separate formulation and administered serially to a subject. So long as the two agents can be detected in the subject at the same time, the two agents are said to be co-administered. In one embodiment, both Nogo-L, RhoA, and Nogo-R iRNA agents are co-administered.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as 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 injury, e.g. at the site of spinal cord injury. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models as described above.
The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 2.







TABLE 2







Abbreviations of nucleotide monomers used in nucleic acid


sequence representation. It will be understood that these


monomers, when present in an oligonucleotide, are mutually


linked by 5′-3′-phosphodiester bonds.










Abbreviationa
Nucleotide(s)







A, a
2′-deoxy-adenosine-5′-phosphate,




adenosine-5′-phosphate



C, c
2′-deoxy-cytidine-5′-phosphate,




cytidine-5′-phosphate



G, g
2′-deoxy-guanosine-5′-phosphate,




guanosine-5′-phosphate



T, t
2′-deoxy-thymidine-5′-phosphate,




thymidine-5′-phosphate



U, u
2′-deoxy-uridine-5′-phosphate,




uridine-5′-phosphate



N, n
any 2′-deoxy-nucleotide/nucleotide




(G, A, C, or T, g, a, c or u)



am
2′-O-methyladenosine-5′-phosphate



cm
2′-O-methylcytidine-5′-phosphate



gm
2′-O-methylguanosine-5′-phosphate



tm
2′-O-methyl-thymidine-5′-phosphate



um
2′-O-methyluridine-5′-phosphate



A, C, G, T, U, a,
underlined: nucleoside-5′-phosphorothioate



c, g, t, u



-Chol
1-{6-[cholester-3-yloxycarbonylamino]-




hexanoyl}-4-hydroxy-pyrrolidin-3-




phosphorothioate diester







acapital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)






Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Example 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 & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification by anion exchange HPLC of the crude oligoribonucleotides were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The purified RNA solution was stored at −20° C. until use.
As a result of the synthesis strategy described above, all oligonucleotides synthesized as described above do not comprise a phosphate group on their 5′-most nucleotide.
Cholesterol was 3′-conjugated to siRNA as illustrated 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. Diisopropylcarbodiimide (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 stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted 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/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




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




Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Cholesterol Derivatised CPG A1




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
















SEQ



Agent

SEQ ID

ID
C. a.


number
Sense strand
NO.
Antisense strand
NO.
#1





AL-DP-5850
gauuaugaccgucugaggcTT
1081
gccucagucggucauaaucTT
1082
n. a.


 


AL-DP-5851
ggaucuucggaaugaugagTT
1083
cucaucauuccgaagauccTT
1084
n. a.


 


AL-DP-5852
agaccaaagacggagugagTT
1085
cucacuccgucuuuggucuTT
1086
n. a.


 


AL-DP-5853
ugaagcaggagccgguaaaTT
1087
uuuaccggcuccugcuucaTT
1088
n. a.


 


AL-DP-5972
gcmumacmcmagumaumumumagaagcmTT
1089
gcuucumaaaumacuggumagcTT
1090
6661


 


AL-DP-5973
cmumacmcmagumaumumumagaagcmcmTT
1091
ggcuucumaaaumacuggumagTT
1092
6662


 


AL-DP-5974
gcmumgumaacmumacmumumumaumaacmTT
1093
guumaumaaagumaguumacmagcTT
1094
6712


 


AL-DP-5975
gumumacmumgumgumaaumumagumgcmTT
1095
gcmacumaauumacmacmagumaacTT
1096
6751


 


AL-DP-5976
cmcmacmumumaaumgumaumgumumacmcmTT
1097
ggumaacmaumacmauumaaguggTT
1098
6769


 


AL-DP-5977
cmagcmcmcmumgaumagumumumagaaTT
1099
uucumaaacumaucmagggcugTT
1100
6521


 


AL-DP-5978
gcmcmcmumgaumagumumumagaaaaTT
1101
uuuucumaaacumaucmagggcTT
1102
6523


 


AL-DP-5979
cmcmcmumgaumagumumumagaaaacmTT
1103
guuuucumaaacumaucmagggTT
1104
6524


 


AL-DP-5980
gaumagumumumagaaaacmaumcmcmTT
1105
ggauguuuucumaaacumaucTT
1106
6528


 


AL-DP-5981
umagumumumagaaaacmaumcmcmcmaTT
1107
ugggauguuuucumaaacumaTT
1108
6530


 


AL-DP-5982
cmagacmumagaumgumagumaumumumTT
1109
aaaumacumacmaucumagucugTT
1110
6790


 


AL-DP-5983
cmcmcmcmagacmumagaumgumagumaTT
1111
umacumacmaucumagucuggggTT
1112
6787


 


AL-DP-5984
cmcmagacmumagaumgumagumaumumTT
1113
aaumacumacmaucumagucuggTT
1114
6789


 


AL-DP-5985
cmcmcmagacmumagaumgumagumaumTT
1115
aumacumacmaucumagucugggTT
1116
6788


 


AL-DP-5986
umgcmcmacmumumaaumgumaumgumumaTT
1117
umaacmaumacmauumaaguggcmaTT
1118
6767


 


AL-DP-5987
umgcmumgumumumaumumaaumcmumumagTT
1119
cumaagauumaaumaaacmagcmaTT
1120
6614


 


AL-DP-5988
umcmaumgumumagumumacmcmumumaumaTT
1121
umaumaaggumaacumaacmaugaTT
1122
6732


 


AL-DP-5989
cmcmagumumaaumumumumumcmcmaacmumTT
1123
aguuggaaaaauumaacuggTT
1124
6832


 


AL-DP-5990
umacmcmumaagaumumacmaaaumcmaTT
1125
ugauuugumaaucuumaggumaTT
1126
6650


 


AL-DP-5991
umcmumumgcmumacmcmagumaumumumagTT
1127
cumaaaumacuggumagcmaagaTT
1128
6657


 


AL-DP-5992
umgumgumaaumumagumgcmcmacmumumTT
1129
aaguggcmacumaauumacmacmaTT
1130
6756


 


AL-DP-5993
aumcmumumgcmumacmcmagumaumumumaTT
1131
umaaaumacuggumagcmaagauTT
1132
6656


 


AL-DP-5994
cmumgumaacmumacmumumumaumaacmumTT
1133
aguumaumaaagumaguumacmagTT
1134
6713


 


AL-DP-5995
gumgaaumumaggcmumgumaacmumaTT
1135
umaguumacmagccumaauucmacTT
1136
6703


 


AL-DP-6176
cmumacmcmagumaumumumagaagcmcmTT-Chol
1137
ggcuucumaaaumacuggumagTT
1138
6662


 


AL-DP-6177
umgcmumgumumumaumumaaumcmumumagTT-Chol
1139
cumaagauumaaumaaacmagcmaTT
1140
6614





1C. a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 3 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2′-O-methyl modifications and phosphorothioate linkages, are disregarded






Example 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 1.5×104 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 Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. 2 μl of a 5 μM solution of siRNA in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride) were mixed with 10.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. During this incubation, growth medium was removed from cells and replaced by 75 μl/well of fresh medium. The 25 μl solution of siRNA-Lipofectamine2000-complex were added, resulting in an overall 100 nM siRNA concentration in the 100 μl incubation volume, and the cells were incubated for 24 h at 37° C. and 5% CO2 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'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 for comparison of mRNA levels.
Effective siRNAs from the screen were further characterized by establishment of dose response curves and calculation of IC50 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 IC50 was determined by curve fitting using the computer software Xlfit using the following parameters: Dose Response One Site, 4 Parameter Logistic Model, fit=(A+((B−A)/(1+(((10^C)/x)^D)))), inv=((10^C)/((((B−A)/(y−A))−1)^(1/D))), res=(y-fit).







TABLE 4







Rat RhoA probes









Probe type1
Nucleotide sequence
SEQ ID NO.





CE
CCATTTTTCTGGGATGTTTTCTAAATTTTTCTCTTGGAAAGAAAGT
1141


 


CE
ACAGAAATGCTTGACTTCTGGAGTTTTTTCTCTTGGAAAGAAAGT
1142


 


CE
CTTCAGGTTTTACCGGCTCCTTTTTCTCTTGGAAAGAAAGT
1143


 


CE
CTGTTTGCCATATCTCTGCCTTTTTTTCTCTTGGAAAGAAAGT
1144


 


CE
TTGGTCTTTGCTGAACACTCCATTTTTCTCTTGGAAAGAAAGT
1145


 


CE
CCCGCGTCTAGCTTGCAGATTTTTCTCTTGGAAAGAAAGT
1146


 


LE
AGGATGATGGGCACATTTGGTTTTTAGGCATAGGACCCGTGTCT
1147


 


LE
GCCTTGTGTGCTCATCATTCCTTTTTAGGCATAGGACCCGTGTCT
1148


 


LE
TGCTTCATTTTGGCTAACTCCCTTTTTAGGCATAGGACCCGTGTCT
1149


 


LE
TGTACCCAAAAGCGCCAATCTTTTTAGGCATAGGACCCGTGTCT
1150


 


LE
GCAGCTCTCGTGGCCATCTTTTTTAGGCATAGGACCCGTGTCT
1151


 


LE
AGGCACCCCGACTTTTTCTTTTTTTAGGCATAGGACCCGTGTCT
1152


 


BL
CTATCAGGGCTGTCGATGGAA
1153


 


BL
GAAGATCCTTCTTGTTCCCAACT
1154


 


BL
CAAAAACCTCTCTCACTCCGTCT
1155





1CE = Capture Extender probe;


LE = Label Extender probe;


BL = blocking probe













TABLE 5







Rat GAPDH probes









Probe type1
Nucleotide sequence
SEQ ID NO.





CE
CCAGCTTCCCATTCTCAGCCTTTTTCTCTTGGAAAGAAAGT
1156


 


CE
TCTCGCTCCTGGAAGATGGTTTTTTCTCTTGGAAAGAAAGT
1157


 


CE
CCCATTTGATGTTAGCGGGATTTTTCTCTTGGAAAGAAAGT
1158


 


CE
CGGAGATGATGACCCTTTTGGTTTTTCTCTTGGAAAGAAAGT
1159


 


LE
GATGGGTTTCCCGTTGATGATTTTTAGGCATAGGACCCGTGTCT
1160


 


LE
GACATACTCAGCACCAGCATCACTTTTTAGGCATAGGACCCGTGTCT
1161


 


LE
CCCAGCCTTCTCCATGGTGGTTTTTAGGCATAGGACCCGTGTCT
1162


 


BL
TTGACTGTGCCGTTGAACTTG
1163


 


BL
CCCCACCCTTCAGGTGAGC
1164


 


BL
GGCATCAGCGGAAGGGG
1165





1CE = Capture Extender probe;


LE = Label Extender probe;


BL = blocking probe






Table 6 lists the agent number, the position of the nucleotide within the human RhoA mRNA sequence (Genbank accession number NM—001664) corresponding to the 5′-most nucleotide of the sense strand of the agent, the amount of total RhoA mRNA remaining in cells treated with the agent at 100 nM concentration in % of controls, and the IC50 value for selected agents.







TABLE 6







Ability of siRNAs specific for RhoA to reduce


RhoA mRNA levels in cultured cells













Rem. RhoA






mRNA at
Rem. RhoA




100 nM
mRNA at 100 nM


Agent
Pos. in
agent,
agent, second
IC50 RhoA


number
mRNA1
first screen
screen
[nM]














AL-DP-5850


73 ± 8%
142


AL-DP-5852


17 ± 4%
3.1


AL-DP-5853


18 ± 3%
2.8


AL-DP-5854


17 ± 1%
4.2


AL-DP-5972
986
30 ± 9%
17 ± 2%


AL-DP-5973
987
21 ± 2%
15 ± 1%
0.003


AL-DP-5974
1179
 44 ± 12%
48 ± 2%


AL-DP-5975
1395
33 ± 4%
 27 ± 10%


AL-DP-5976
1413
26 ± 3%
17 ± 2%


AL-DP-5977
537
n.d.
30 ± 1%


AL-DP-5978
539
58 ± 4%
51 ± 1%


AL-DP-5979
540
12 ± 2%
15 ± 2%
0.06


AL-DP-5980
544
75 ± 3%
95 ± 3%


AL-DP-5981
546
17 ± 2%
16 ± 1%
0.13


AL-DP-5982
1452
18 ± 2%
22 ± 2%
0.13


AL-DP-5983
1449
37 ± 4%
29 ± 3%


AL-DP-5984
1451
26 ± 1%
33 ± 4%


AL-DP-5985
1450
n.d.
33 ± 1%
0.37


AL-DP-5986
1411
18 ± 1%
22 ± 1%
0.4


AL-DP-5987
901
22 ± 5%
10 ± 0%
0.01


AL-DP-5988
1376
17 ± 1%
16 ± 1%
0.34


AL-DP-5989
1876
20 ± 1%
25 ± 3%
3.1


AL-DP-5990
956
16 ± 2%
17 ± 1%
0.36


AL-DP-5991
982
55 ± 5%
33 ± 5%


AL-DP-5992
1400
55 ± 6%
55 ± 6%


AL-DP-5993
981
32 ± 2%
33 ± 3%


AL-DP-5994
1180
23 ± 2%
20 ± 1%
0.24


AL-DP-5995
1170
25 ± 2%
26 ± 2%
6.0


AL-DP-6176
987

14 ± 2%
1.17


AL-DP-6177
901

19 ± 5%
0.005





1Position of nucleotide within human Nogo-R mRNA corresponding to the 5′-most nucleotide of the sense strand of the agent






In summary, agents AL-DP-5979, AL-DP-5990, AL-DP-5988, AL-DP-5981, AL-DP-5982, AL-DP-5986, AL-DP-5989 AL-DP-6176, and AL-DP-6177 were able to reduce the expression of RhoA mRNA by 80% or more, AL-DP-5973, AL-DP-5987, AL-DP-5994, AL-DP-5995, AL-DP-5976, AL-DP-5984, and AL-DP-5972 were able to reduce the expression of RhoA mRNA by 70% or more, AL-DP-5993, AL-DP-5975, and AL-DP-5983 were able to reduce the expression of RhoA mRNA by 60% or more, AL-DP-5974 was able to reduce the expression of RhoA mRNA by 50% or more, and AL-DP-5991, AL-DP-5992, and AL-DP-5978 were able to reduce the expression of RhoA mRNA by 40% or more. The high activity of AL-DP-6176 and AL-DP-6177 shows 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 NaCl 5M, 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 occuring nucleic acids consisting of R-enantiomers.
Proteinase K Treatment of siRNA Incubation Samples
6 μl of a 50 μM solution of the respective siRNA in phosphate buffered saline (PBS, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was incubated with 54 μl CSF at 37° C. for 30 min, 1, 2, 4, 8, 16, 24 or 48 hours. To terminate the siRNA-degradation, 25 μl of Proteinase K buffer were added to incubation samples immediately after expiry of the respective incubation period, the mixture vortexed at highest speed for 5 s (Vortex Genie 2, Scientific Industries, Inc., Bohemia, N.Y., USA, cat. no. SI 0256), 8 μl Proteinase K (10 mg/ml) were added followed by vortexing for 5 s, and finally the mixture was incubated for 20 min in a thermomixer at 42° C. and 1050 rpm.
5 μl of a 50 μM solution (250 pmole) of (L-dT)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 NaClO4, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile,



pH = 8.0


Eluent B:
800 mM NaClO4, 20 mM TRIS-HCl, 1 mM EDTA; 10%



acetonitrile, pH = 8.0


Detection:
@ 260 nm









Gradient:
 0-1 min:
 10% B



 1-11 min:
 10% −> 35% B



11-12 min:
 35% B −> 100% B



12-14 min:
100% B −> 10% B



14-16 min:
 10% B for column reequilibration








Injection volume:
20 μl









Where separation between the two strands of an siRNA was not satisfactory or a degradation fragment co-eluted with one strand, the chromatographic parameters were adjusted by changing temperature, pH, replacement of NaClO4 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=PeakAreaintStd(theoretical)/PeakAreaintStd(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 (NPAsense,t, NPAantisense,t):

NPAsense or antisense,t=(Peak Areasense 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 (NPAsense,t=0, NPAantisense,t=0) is set as 100%, and the NPAs from other time points are divided by these values.

% FLPt=1,2,3 . . . n=(NPAt=1,2,3 . . . n/NPAt=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 t1/2 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:

t1/2=ln(0,5)/slope

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

Table 7 shows the results for select siRNAs of the determination of the relative amount of full length dsRNA present in porcine, rat, and bovine CSF, and PBS, after 48 h of incubation in the respective medium. In addition, the degradation half life was determined for the sense and antisense strands separately for some siRNAs.







TABLE 7







Stability of various siRNAs specific for NogoL


and RhoA in rat, bovine and porcine CSF










% full length duplex




present after 48 h in














Agent
Porcine
Rat
Bovine

Specific
Modifi-
C. a.


number
CSF
CSF
CSF
PBS
for
cation1
#2

















AL-DP-5973
95
3
95
100
RhoA
3/TTs
6662


AL-DP-5979
99


108
RhoA
3/TTs
6524


AL-DP-5981
96


103
RhoA
3/TTs
6530


AL-DP-5982
56


98
RhoA
3/TTs
6790


AL-DP-5986
100


105
RhoA
3/TTs
6767


AL-DP-5987
87


97
RhoA
3/TTs
6614


AL-DP-5988
41


99
RhoA
3/TTs
6732


AL-DP-5989
87


101
RhoA
3/TTs
6832


AL-DP-5990
76


92
RhoA
3/TTs
6650





10 = no 2′-modifications; 1 = 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in sense strand, 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in antisense strand; 2 = 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in sense and antisense strand, 3 = all pyrmidine nucleotides are 2′-modified in sense strand, 5′-nucleotide in 5′-ua-3′ and 5′-ca-3′ motifs is 2′-modified in antisense strand; 4 = all pyrimidine nucleotides are 2′-modified in sense strand, 5′-nucleotide in 5′-ua-3′, 5′-uu-3′, 5′-ca-3′, and 5′-ug-3′ motifs is 2′-modified in antisense strand; 5 = all pyrimidine nucleotides are 2′-modified in sense strand, no 2′-modifications in antisense strand; TT = 21 nucleotides and 3′-terminal TT single strand overhangs in sense and antisense strands; TTs = 21 nucleotides and 3′-terminal TT single strand overhangs in sense and antisense strands; 23 = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3′-end of antisense strand; 23s = 21 nucleotide sense, 23 nucleotide antisense strand, 2 nucleotide single strand overhang on 3′-end of antisense strand, nucleotides comprise 5′-phosphorothioate groups in positions 21 and 22 of antisense strand


2C. a. # = corresponding agent # in Table 2. The agent given under this agent number in Table 2 possesses the same core nucleotide sequence when nucleotide modifications, e.g. 2′-O-methyl modifications and phosphorothioate linkages, are disregarded






As is evident from Table 7, the modification of siRNAs in select sites vulnerable to degradation can lead to agents with excellent properties in terms of activity and stability. For example, AL-DP-5871, AL-DP-5938, AL-DP-5963, AL-DP-5973, AL-DP-5979, AL-DP-5981, AL-DP-5986, AL-DP-5987, AL-DP-5989, and AL-DP-5990 all inhibit their respective target gene by more than 70% in the in vitro assays described above, and more than 70% full length duplex remain after incubation with porcine CSF for 48 h. However, there is some indication that rat CSF is more aggressive towards siRNAs than porcine or bovine CSF.
Example 5
Inhibition of RhoA Expression in Rat Primary Dorsal Root Ganglia (DRG) Cells in Culture
The inhibition of RhoA expression was assessed in rat primary dorsal root ganglia (DRG) cells in culture in order to validate results obtained 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 BiocoatTM PDL Poly-D-Lysine/Laminin 96 well plates (BD Biosciences, Bedford Mass., USA) in F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5% fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heat inactivated) (both Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp., Madison Wis., USA) and kept at 37° C., 5% CO2 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 TransMessengerTM Transfection reagent (Qiagen GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid formulation, specific RNA-condensing reagent (Enhancer RTM) and an RNA-condensing buffer (Buffer EC-RTM) keeping siRNA:Enhancer RTM ratio (μg:μl) constant at 1:2, and siRNA:TransMessengerTM ratio (μg:μl) constant at 1:12.
DRG neurons were transfected 24 h post-plating. For each well 0.52 μl Enhancer RTM were first mixed with 13.68 μl Buffer EC-RTM. 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 TransMesssengerTM Transfection Reagent were diluted with 6.88 μl Buffer EC-RTM, added to the mixture, and the mixture incubated for another 10 min at room temperature to allow transfection-complex formation. 75 μl serum free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad Calif., USA) were added to the transfection complexes and complete mixing achieved by gently pipetting up and down. The growth medium was removed from the DRG cells, and 90 μl of the above transfection complex mixture were added onto the cells. After 8 h of incubation at 37° C., 5% CO2 in a humidified incubator supernatant was removed from the cells, fresh F12-HAM's medium containing glutamine supplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA) was added, the cells were incubated for another 16 h at 37° C., 5% CO2 in a humidified incubator, and rhoA mRNA was quantified.
RhoA mRNA levels were measured using the QuantiGeneTM bDNA kit (Genospectra, Fremont, USA) according to manufacturer's protocol. Briefly, the supernatant was removed from the DRG cells, and the cells were lysed by addition of 150 μl of Lysis Working Reagent (1 volume of Lysis Mixture plus 2 volumes of medium) and incubation at 52° C. for 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 Victor2-LightTM (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.

## Claims

1. An iRNA agent comprising a sense strand and an antisense strand, wherein each strand is 21 nucleotides in length, and wherein the sequence of the sense strand is identical to the sense strand sequence of agent number 6662 (SEQ ID NO:557), and wherein the sequence of the antisense strand is identical to the antisense strand sequence of agent number 6662 (SEQ ID NO:558), and wherein every pyrimidine in the sense strand except for deoxythimidine is 2′-O-methyl modified, and wherein every 5′-nucleotide in 5′-ua-3′ motifs in the antisense strand is 2′-O-methyl modified, and wherein the 21 nucleotides include two deoxythymidine nucleotides at the 3′ end of each strand.

2. The iRNA agent of claim 1, wherein said deoxythymidine nucleotides at the 3′ end of each strand comprise a phosphorothioate linkage.

3. The iRNA agent of claim 2, further comprising a cholesterol moiety conjugated to the deoxythymidine nucleotide at the 3′-end of the sense strand of the iRNA agent.

4. A composition, comprising:
a.) an iRNA agent of claim 1, 2 or 3; and
b.) a pharmaceutically acceptable carrier.

5. An isolated cell comprising an iRNA agent of claim 1, 2 or 3.