Methods and compositions for treating neurological disease

Abstract

This invention relates to methods and compositions for treating neurological disease, and more particularly to methods of delivering iRNA agents to neural cells for the treatment of neurological diseases.

1 . A method of downregulating expression of a target gene in a neural cell distal to the site of administration, the method comprising contacting an iRNA agent with the neural cell for a time sufficient to allow uptake of the iRNA agent into the cell, wherein (i) the iRNA agent comprises a sense and an antisense strand that form an RNA duplex, and (ii) the sequence of the antisense strand of the iRNA agent comprises a nucleotide sequence sufficiently complementary to a target sequence of about 18 to 25 nucleotides of an RNA expressed from the target gene.  
     
     
         2 . A method of downregulating expression of a target gene in a neural cell, the method comprising contacting an iRNA agent with the neural cell for a time sufficient to allow uptake of the iRNA agent into the cell, wherein (i) the iRNA agent comprises a sense and an antisense strand that form an RNA duplex, (ii) the iRNA agent comprises a lipophilic moiety, and (iii) the sequence of the antisense strand of the iRNA agent comprises a nucleotide sequence sufficiently complementary to a target sequence of about 18 to 25 nucleotides of an RNA expressed from the target gene.  
     
     
         3 . The method of  claim 1  or  2 , wherein the cells are contacted for a time sufficient to allow axonal transport of said iRNA.  
     
     
         4 . The method of  claim 1 , wherein the iRNA agent comprises a lipophilic moiety.  
     
     
         5 . The method of  claim 2  or  4 , wherein the lipophilic moiety is a cholesterol.  
     
     
         6 . The method of  claim 2  or  4 , wherein the lipophilic moiety is conjugated to the sense strand.  
     
     
         7 . The method of  claim 2  or  4 , wherein the lipophilic moiety is conjugated to the 3′ end of the sense strand.  
     
     
         8 . The method of any of the preceding claims, wherein the antisense sequence differs by no more than four nucleotides from an antisense sequence listed in Table 1.  
     
     
         9 . The method of any of claims  1 - 7 , wherein the antisense strand is selected from an antisense strand listed in Table 1.  
     
     
         10 . The method of any of the preceding claims, wherein the iRNA agent further comprises a phosphorothioate or a 2′-OMe modification.  
     
     
         11 . The method of any of the preceding claims, wherein the iRNA agent is provided in a solution that lacks a transfection reagent.  
     
     
         12 . The method of any of claims  1 - 10 , wherein the iRNA agent is provided in a solution comprising a transfection reagent.  
     
     
         13 . A method of treating a human comprising identifying a human having or at risk for developing a neurological disorder, the method comprising administering to the human an iRNA agent that targets a gene expressed in a neural cell distal to the site of administration, wherein the expression of the gene is associated with symptoms of the neurological disorder, and wherein (i) the iRNA agent comprises a sense and an antisense strand that form an RNA duplex, and (ii) the antisense strand of the iRNA agent comprises a nucleotide sequence sufficiently complementary to a target sequence of about 18 to 25 nucleotides of an RNA expressed from the target gene.  
     
     
         14 . A method of treating a human comprising identifying a human having or at risk for developing a neurological disorder, and administering to the human an iRNA agent that targets a gene expressed in a neural cell, wherein the expression of the gene is associated with symptoms of the neurological disorder, and wherein (i) the iRNA agent comprises a sense and an antisense strand that form an RNA duplex, (ii) the iRNA agent comprises a lipophilic moiety, and (iii) the antisense strand of the iRNA agent comprises a nucleotide sequence sufficiently complementary to a target sequence of about 18 to 25 nucleotides of an RNA expressed from the target gene.  
     
     
         15 . The method of  claim 13 , wherein the iRNA agent comprises a lipophilic moiety.  
     
     
         16 . The method of  claim 14  or  15 , wherein the lipophilic moiety is a cholesterol.  
     
     
         17 . The method of  claim 14  or  15 , wherein the lipophilic moiety is conjugated to the sense strand.  
     
     
         18 . The method of  claim 14  or  15 , wherein the lipophilic moiety is conjugated to the 3′ end of the sense strand.  
     
     
         19 . The method of any of claims  13 - 18 , wherein the iRNA agent further comprises a phosphorothioate or a 2′-OMe modification.  
     
     
         20 . The method of any of claims  13 - 19 , wherein the antisense sequence differs by no more than four nucleotides from an antisense sequence listed in Table 1.  
     
     
         21 . The method of any of claims  13 - 19 , wherein the antisense strand is selected from an antisense strand listed in Table 1.  
     
     
         22 . The method of any of claims  13 - 21 , wherein the antisense strand of the iRNA agent comprises a sequence complementary to a sequence comprising a polymorphism of a huntingtin (htt) RNA.  
     
     
         23 . The method of any of claims  13 - 22 , wherein the human carries a genetic variation in a Parkin gene or a ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene.  
     
     
         24 . The method of any of claims  13 - 23 , wherein the neurological disorder is Huntington's disease.  
     
     
         25 . The method of any of claims  13 - 23 , wherein the neurological disorder is Parkinson's disease.  
     
     
         26 . The method of any of claims  13 - 23 , wherein the neurological disorder is Alzheimer's Disease, multiple system atrophy, or Lewy body dementia.  
     
     
         27 . The method of any of claims  13 - 26 , wherein the iRNA agent comprises a nucleotide overhang having 1 to 4 unpaired nucleotides.  
     
     
         28 . The method of any of claims  13 - 27 , wherein the iRNA agent is provided in a solution that lacks a transfection reagent.  
     
     
         29 . The method of any of claims  13 - 28 , wherein the iRNA agent is provided in a solution comprising a transfection reagent.  
     
     
         30 . The method of any of claims  13 - 29 , wherein the iRNA agent is administered as a sustained dose formulation.  
     
     
         31 . The method of any of claims  13 - 29 , wherein the iRNA agent is administered in multiple doses over a prolonged time period.  
     
     
         32 . The method of any of claims  13 - 29 , wherein the iRNA agent is administered as a single dose.  
     
     
         33 . The method of any of claims  13 - 32 , comprising the administration of a second iRNA agent, wherein (i) the second iRNA agent comprises a sense and an antisense strand that form an RNA duplex, and (ii) the antisense strand of the iRNA agent comprises a nucleotide sequence sufficiently complementary to a second target sequence of about 18 to 25 nucleotides of the RNA expressed from the target gene.  
     
     
         34 . A method of reducing the amount of huntingtin (htt) RNA in a neural cell of a subject, comprising: 
 contacting the neural cell with an iRNA agent, wherein said neural cell is distal to the site of action and the iRNA agent comprises a sense and an antisense strand, wherein the sense and the antisense strands form an RNA duplex, wherein the antisense strand comprises a nucleotide sequence that differs by no more than four nucleotides from an antisense sequence listed in Table 1.    
     
     
         35 . The method of  claim 33 , wherein the iRNA agent further comprises a lipophilic moiety.  
     
     
         36 . The method of  claim 33  or  34 , wherein the cells are contacted for a time sufficient to allow axonal transport of said iRNA.  
     
     
         37 . The method of  claim 36 , wherein the iRNA agent further comprises a phosphorothioate or a 2′-OMe modification.  
     
     
         38 . The method of  claim 36 , wherein the iRNA agent comprises an antisense strand comprising a sequence selected from the antisense strands listed in Table 1.  
     
     
         39 . The method of  claim 36 , wherein the iRNA agent comprises a sense strand selected from the sense strands listed in Table 1.  
     
     
         40 . The method of  claim 36 , wherein the iRNA agent comprises an antisense strand comprising a sequence complementary to sequence comprising a polymorphism of an htt RNA.  
     
     
         41 . The method of  claim 36 , wherein the polymorphism is an A to C at position 171 according to the sequence of GenBank Accession No. NM — 002111.  
     
     
         42 . The method of  claim 36 , wherein the iRNA agent comprises a nucleotide overhang having 1 to 4 unpaired nucleotides.  
     
     
         43 . An isolated iRNA agent comprising a sense and an antisense strand, wherein the sense and the antisense strands form an RNA duplex, wherein the antisense strand comprises a nucleotide sequence that differs by no more than four nucleotides from an antisense sequence listed in Table 1, and wherein the iRNA agent comprises a lipophilic moiety.  
     
     
         44 . The iRNA agent of  claim 43 , wherein the lipophilic moiety is a cholesterol molecule.  
     
     
         45 . The iRNA agent of  claim 43 , wherein the lipophilic moiety is attached to the sense strand.  
     
     
         46 . The iRNA agent of  claim 43 , wherein the lipophilic moiety is attached to the 3′ end of the sense strand.  
     
     
         47 . The iRNA agent of  claim 43 , further comprising a phosphorothioate modification, or a 2′-OMe modification.  
     
     
         48 . The iRNA agent of  claim 43 , wherein the antisense strand comprises a sequence selected from the antisense sequences listed in Table 1.  
     
     
         49 . The iRNA agent of  claim 43 , wherein the sense strand of the iRNA agent comprises a sequence selected from the sense sequences listed in Table 1.  
     
     
         50 . The iRNA agent of  claim 43 , wherein the iRNA agent is at least 21 nucleotides in length, and the duplex region of the iRNA agent is about 18-25 nucleotides in length.  
     
     
         51 . The iRNA agent of  claim 40 , comprising a nucleotide overhang having 1 to 4 unpaired nucleotides.  
     
     
         52 . A pharmaceutical composition, comprising: 
 (i) an iRNA agent comprising a sense and an antisense strand, wherein the sense and the antisense strands form an RNA duplex, wherein the antisense strand comprises a nucleotide sequence that differs by no more than four nucleotides from an antisense sequence listed in Table 1, and wherein the iRNA agent comprises a lipophilic moiety; and    (ii) a pharmaceutically acceptable carrier.    
     
     
         53 . The pharmaceutical composition of  claim 52 , wherein the iRNA agent further comprises a phosphorothioate or a 2′-OMe modification.  
     
     
         54 . The pharmaceutical composition of  claim 52 , wherein the antisense strand of the iRNA agent comprises a sequence selected from the antisense sequences listed in Table 1.  
     
     
         55 . The pharmaceutical composition of  claim 52 , wherein the sense strand of the iRNA agent comprises a sequence from the selected from the sense sequences listed in Table 1.  
     
     
         56 . The pharmaceutical composition of  claim 52 , wherein the iRNA agent comprises a nucleotide overhang having 1 to 4 unpaired nucleotides.  
     
     
         57 . A method of evaluating an iRNA agent for enhanced uptake into neural cells comprising: providing a candidate iRNA agent conjugated to a lipophilic agent, wherein the iRNA agent is in a solution that does not contain a transfection reagent, contacting the iRNA agent with a neural cell for a time sufficient for uptake into the neural cell and determining if the iRNA agent is taken up by the neural cell.  
     
     
         58 . The method of  claim 57 , wherein the method includes a step of evaluating the iRNA agent in a cell culture system; and, if a predetermined level of uptake into the neural cell is observed, evaluating the candidate in an animal.  
     
     
         59 . The method of  claim 57  or  58 , wherein the iRNA agent comprises an antisense strand that is substantially complementary to a target RNA in the neural cell, and the method further comprises determining whether the candidate iRNA agent decreases expression of a target RNA in the neural cell.  
     
     
         60 . The method of any of claims  57 - 59 , wherein the lipophilic agent is a cholesterol.  
     
     
         61 . The method of any of claims  57 - 60 , wherein the target RNA is a huntingtin RNA.  
     
     
         62 . The method of any of claims  57 - 61 , wherein the iRNA agent comprises an antisense strand that is substantially complementary to a target RNA in the neural cell, and the method further comprises determining whether the candidate iRNA agent decreases expression of the target RNA in a neural cell of the animal.  
     
     
         63 . The method of any of claims  57 - 62 , wherein the target RNA is a huntingtin RNA.  
     
     
         64 . The method of any of claims  57 - 63 , wherein the animal is monitored for an effect of the iRNA agent.  
     
     
         65 . The method of any of claims  57 - 64 , wherein brain tissue from the animal is examined for an effect of the iRNA agent on target gene expression.  
     
     
         66 . The method of any of claims  57 - 65 , wherein the determining step comprises performing a method selected from the group consisting of Northern blot, Western blot, RT-PCR, and RNAse protection assay.