Compositions and methods of treating amyotrophic lateral sclerosis (als)

Abstract

The present invention relates to adeno-associated viral (AAV) particles encoding siRNA molecules and methods for treating amyotrophic lateral sclerosis (ALS).

1 - 26 . (canceled) 
     
     
         27 . A modulatory polynucleotide targeting a SOD1 gene comprising:
 (i) a 5′ flanking region, wherein the nucleotide sequence encoding the 5′ flanking region comprises a nucleotide sequence selected from SEQ ID NOs: 1255-1263;   (ii) an antisense strand comprising a nucleotide sequence at least 80% identical to a nucleotide sequence selected from SEQ ID NOs: 916-1084;   (iii) a loop region, wherein the nucleotide sequence encoding the loop region comprises a nucleotide sequence selected from SEQ ID NOs: 1264-1273;   (iv) a sense strand comprising a nucleotide sequence at least 80% identical to a nucleotide sequence selected from SEQ ID NOs: 1085-1253, wherein the sense strand sequence is complementary to the antisense strand sequence; and   (v) a 3′ flanking region, wherein the nucleotide sequence encoding the 3′ flanking region comprises a nucleotide sequence selected from SEQ ID NOs: 1274-1280.   
     
     
         28 . The modulatory polynucleotide of  claim 27 , which comprises:
 (i) a 5′ flanking region, wherein the nucleotide sequence encoding the 5′ flanking region comprises the nucleotide sequence of SEQ ID NO: 1256;   (ii) an antisense strand sequence comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 1035;   (iii) a loop region, wherein the nucleotide sequence encoding the loop region comprises the nucleotide sequence of SEQ ID NO: 1266;   (iv) a sense strand sequence comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 1204, wherein the sense strand sequence is complementary to the antisense strand sequence; and   (v) a 3′ flanking region, wherein the nucleotide sequence encoding the 3′ flanking region comprises the nucleotide sequence of SEQ ID NO: 1276.   
     
     
         29 . The modulatory polynucleotide of  claim 28 , wherein the nucleotide sequence encoding the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1347 and the nucleotide sequence encoding the sense strand sequence comprises the nucleotide sequence of SEQ ID NO: 1365. 
     
     
         30 . The modulatory polynucleotide of  claim 27 , wherein the nucleotide sequence encoding the modulatory polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1326. 
     
     
         31 . An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence positioned between a first inverted terminal repeat (ITR) and a second ITR, wherein said nucleotide sequence encodes a modulatory polynucleotide, wherein the modulatory polynucleotide comprises:
 (a) a 5′ flanking region, wherein the nucleotide sequence encoding the 5′ flanking region comprises a nucleotide sequence selected from SEQ ID NOs: 1255-1263;   (b) an antisense sequence comprising a nucleotide sequence at least 80% identical to a nucleotide sequence selected from SEQ ID NOs: 916-1084;   (c) a loop region, wherein the nucleotide sequence encoding the loop region comprises a nucleotide sequence selected from SEQ ID NOs: 1264-1273;   (d) a sense strand sequence comprising a nucleotide sequence at least 80% identical to a nucleotide sequence selected from SEQ ID NOs: 1085-1253, wherein the sense strand sequence is complementary to the antisense strand sequence; and   (e) a 3′ flanking region, wherein the nucleotide sequence encoding the 3′ flanking region comprises a nucleotide sequence selected from SEQ ID NOs: 1274-1280.   
     
     
         32 . The AAV viral genome of  claim 31 , wherein:
 (a) the nucleotide sequence encoding the 5′ flanking region comprises SEQ ID NO: 1256;   (b) the nucleotide sequence encoding the loop region comprises SEQ ID NO: 1266;   (c) the nucleotide sequence encoding the 3′ flanking region comprises SEQ ID NO: 1276; and   (d) the antisense strand sequence is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1035, and the sense strand sequence is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1204.   
     
     
         33 . The AAV viral genome of  claim 31 , the nucleotide sequence encoding the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1347; and the nucleotide sequence encoding the sense strand sequence comprises the nucleotide sequence of SEQ ID NO: 1365. 
     
     
         34 . The AAV viral genome of  claim 31 , wherein the nucleotide sequence encoding the modulatory polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1326. 
     
     
         35 . The AAV viral genome of  claim 31 , wherein:
 (a) the sense strand sequence and/or antisense strand sequence are, independently, between 20-22 nucleotides in length and/or   (b) the sense strand sequence and the antisense strand sequence comprise a 3′ overhang of 1-2 nucleotides.   
     
     
         36 . The AAV viral genome of  claim 31 , wherein:
 (i) the first ITR comprises a nucleotide sequence selected from SEQ ID NOs: 1370-1373, and is positioned 5′ relative to the nucleotide sequence encoding the modulatory polynucleotide; and/or   (ii) the second ITR comprises a nucleotide sequence selected from SEQ ID NOs: 1370-1372, and is positioned 3′ relative to the nucleotide sequence encoding the modulatory polynucleotide.   
     
     
         37 . The AAV viral genome of  claim 31 , wherein the first ITR comprises the nucleotide sequence of SEQ ID NO: 1371 and is positioned 5′ relative to the nucleotide sequence encoding the modulatory polynucleotide, and the second ITR comprises the nucleotide sequence of SEQ ID NO: 1373, and is positioned 3′ relative to the nucleotide sequence encoding the modulatory polynucleotide. 
     
     
         38 . An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence positioned between a first inverted terminal repeat (ITR) and a second ITR, wherein said nucleotide sequence encodes the modulatory polynucleotide of  claim 28 . 
     
     
         39 . The AAV viral genome of  claim 38 , further comprising:
 (i) a promoter;   (ii) an enhancer;   (iii) an intron; and/or   (iv) a poly A sequence region.   
     
     
         40 . The AAV viral genome of  claim 39 , wherein:
 (i) the first ITR comprises the nucleotide sequence of SEQ ID NO: 1371 and is positioned 5′ relative to the nucleotide sequence encoding the modulatory polynucleotide, and the second ITR comprises the nucleotide sequence of SEQ ID NO: 1373, and is positioned 3′ relative to the nucleotide sequence encoding the modulatory polynucleotide;   (ii) the promoter is a CMV promoter, a CBA promoter, a EF-1α promoter, a PGK promoter, a synapsin promoter, an H1 promoter, or a truncation thereof; and/or   (ii) the poly A sequence region comprises the nucleotide sequence of SEQ ID NO: 1410.   
     
     
         41 . A recombinant adeno-associated virus (AAV) particle comprising the AAV viral genome of  claim 31 , and an AAV capsid, wherein the AAV capsid comprises an AAV9 capsid or variant thereof, an AAV5 capsid or variant thereof, or an AAVrh10 capsid or variant thereof. 
     
     
         42 . A pharmaceutical composition comprising the recombinant AAV particle of  claim 41 , and a pharmaceutically acceptable excipient. 
     
     
         43 . A method for inhibiting or suppressing the expression of SOD1 gene in a cell, the method comprising administering to the cell a pharmaceutical composition of  claim 42 . 
     
     
         44 . The method of  claim 43 , wherein the cell is:
 (i) a mammalian cell, a motor neuron, or an astrocyte; and/or   (ii) in a subject, wherein the subject has amyotrophic lateral sclerosis (ALS).   
     
     
         45 . The method of  claim 43 , wherein the SOD1 gene comprises a wild type SOD1 gene, a mutated SOD1 gene, or a combination thereof. 
     
     
         46 . A method for treating amyotrophic lateral sclerosis (ALS) in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of  claim 42 . 
     
     
         47 . The method of  claim 46 , wherein the pharmaceutical composition is administered to the subject via intravenous administration, intracisternal magna administration, or combination thereof. 
     
     
         48 . The method of  claim 46 , wherein the ALS is (i) familial ALS; (ii) sporadic ALS; (iii) early stage ALS; (iv) middle stage ALS; and/or (v) late stage ALS.