RNAi Modulation of the RHO-A Gene and Uses Thereof
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.
1 . An iRNA agent 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 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.
2 . An iRNA agent 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.
3 . An iRNA agent 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.
4 . The iRNA agent of claim 1 , wherein 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
5 . The iRNA agent of claim 1 , wherein the sense and/or antisense strand sequence is chosen from the group consisting of: the sense and antisens 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.
6 . The iRNA agent of claim 1 , wherein the antisense RNA strand is 30 or fewer nucleotides in length, and the duplex region of the iRNA agent is 15-30 nucleotide pairs in length.
7 . The iRNA agent of claim 1 , comprising a modification that causes the iRNA agent to have increased stability in a biological sample.
8 . The iRNA agent of claim 7 , wherein said modification is a phosphorothioate, a 2′-modified nucleotide, a locked nucleotide, an abasic nucleotide, morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.
9 . The iRNA agent of claim 1 , comprising 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.
10 . The iRNA agent of claim 1 ,
wherein every 5′-nucleotide in 5′-ua-3′,5′-uu-3′,5′-ca-3′, and 5′-ug-3′ motifs 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
wherein 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
wherein 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
wherein 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
wherein every pyrimidine nucleotide in the sense strand is 2′-modified, and no nucleotide is 2′-modified in the antisense strand.
11 . The iRNA agent of any one of claim 8 , wherein the 2′-modification is 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).
12 . The iRNA agent of claim 1 , comprising a nucleotide overhang having 1 to 4 unpaired nucleotides.
13 . The iRNA agent of claim 1 , comprising a cholesterol moiety.
14 . The iRNA agent of claim 13 , wherein the cholesterol moiety is conjugated to the 3′-end of the sense strand of the iRNA agent.
15 . The iRNA agent of claim 1 , wherein the iRNA agent is targeted for uptake by nerve cells or nerve sheath cells.
16 . A method of treating a subject having a pathological process mediated in part by RhoA comprising administering to the subject an iRNA agent of claim 1 .
17 . The method of claim 16 , wherein the pathological process is the inhibition of nerve growth or elongation.
18 . The method of claim 17 , wherein the pathological process is the inhibition of nerve growth or elongation as a result of nerve injury or damage.
19 . The method of any one of claim 16 , wherein the iRNA agent is administered in an amount sufficient to reduce the expression of RhoA in a cell or tissue of the subject.
20 . The method of any one of claim 16 , wherein the subject is a human.
21 . A pharmaceutical composition, comprising:
a.) an iRNA agent of any one of claim 1 ; and
b.) a pharmaceutically acceptable carrier.
22 . A cell comprising an iRNA agent of any one of claim 1 .
23 . 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 any one of claim 1 ; 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.