Inhibitors of cacna1a/alpha1a subunit internal ribosomal entry site (ires) and methods of treating spinocerebellar ataxia type 6

Abstract

The invention provides methods of treating polyglutamine diseases, e.g., spinocerebellar ataxia Type 6, in a subject, comprising administering to the subject an IRES inhibitor in an amount effective for treating the SCA6 in the subject. Also provided herein are the IRES inhibitors, and pharmaceutical compositions comprising the same.

What is claimed is: 
     
         1 . A method of treating spinocerebellar ataxia Type 6 (SCA6) in a subject in need thereof, comprising the step of administering to the subject (i) an antisense molecule, (ii) a vector encoding the antisense molecule, (iii) a cell comprising the vector or antisense molecule, (iv) a extracellular vesicle comprising the antisense molecule, or (v) a combination thereof, wherein the antisense molecule binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4), optionally, wherein the vector is a recombinant expression vector of any one of  claims 18  to  28 , wherein the cell is a cell of  claim 30 , or the extracellular vesicle is an extracellular vesicle of  claim 29 . 
     
     
         2 . The method of  claim 1 , wherein the antisense molecule is an microRNA (miRNA), a pri-miRNA, or a pre-miRNA. 
     
     
         3 . The method of  claim 1  or  2 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the vector comprises a nucleotide sequence encoding a miRNA, optionally, wherein the miRNA comprises the sequence of SEQ ID NO: 179. 
     
     
         5 . The method of  claim 4 , wherein the nucleotide sequence comprises of SEQ ID NO: 181. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the antisense molecule is an antisense nucleic acid analog comprising the base sequence of SEQ ID NO: 179. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein the cell or extracellular vesicle is autologous to the subject. 
     
     
         8 . A method of treating a subject with a predisposition to spinocerebellar ataxia Type 6 (SCA6), comprising the step of administering to the subject (i) an antisense molecule, (ii) a vector encoding the antisense molecule, (iii) a cell comprising the vector or antisense molecule, (iv) a extracellular vesicle comprising the antisense molecule, or (v) a combination thereof, wherein the antisense molecule binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4), optionally, wherein the vector is a recombinant expression vector of any one of  claims 18  to  28 , wherein the cell is a cell of  claim 30 , or the extracellular vesicle is an extracellular vesicle of  claim 29 . 
     
     
         9 . The method of  claim 8 , wherein the antisense molecule is an microRNA (miRNA), a pri-miRNA, or a pre-miRNA. 
     
     
         10 . The method of  claim 8  or  9 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         11 . The method of any one of  claims 8  to  10 , wherein the vector comprises a nucleotide sequence encoding a miRNA, optionally, wherein the miRNA comprises the sequence of SEQ ID NO: 179. 
     
     
         12 . The method of  claim 11 , wherein the nucleotide sequence comprises of SEQ ID NO: 181. 
     
     
         13 . The method of any one of  claims 8  to  12 , wherein the antisense molecule is an antisense nucleic acid analog comprising the base sequence of SEQ ID NO: 179. 
     
     
         14 . The method of any one of  claims 8  to  13 , wherein the cell or extracellular vesicle is autologous to the subject. 
     
     
         15 . A synthetic antisense molecule which specifically binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180 comprising at least one non-naturally occurring nucleotide or at least one non-naturally occurring internucleotide linkage, optionally, wherein the synthetic antisense molecule binds to Argonaute 4 (Ago4). 
     
     
         16 . The synthetic antisense molecule of  claim 15 , which is a synthetic microRNA (miRNA), a synthetic pri-miRNA, or a synthetic pre-miRNA. 
     
     
         17 . The synthetic antisense molecule of  claim 15  or  16 , wherein the synthetic antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         18 . A recombinant expression vector comprising a nucleotide sequence encoding an antisense molecule which specifically binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4). 
     
     
         19 . The recombinant expression vector of  claim 18 , an microRNA (miRNA), a pri-miRNA, or a pre-miRNA. 
     
     
         20 . The recombinant expression vector of  claim 18  or  19 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         21 . The recombinant expression vector of any one of  claims 18  to  20 , comprising a nucleotide sequence encoding a miRNA, optionally, wherein the miRNA comprises the sequence of SEQ ID NO: 179. 
     
     
         22 . The recombinant expression vector of any one of  claims 18  to  21 , wherein the nucleotide sequence comprises of SEQ ID NO: 181. 
     
     
         23 . The recombinant expression vector of any one of  claims 18  to  22 , which is an recombinant adeno-associated viral (AAV) vector. 
     
     
         24 . The recombinant expression vector of  claim 23 , wherein the recombinant AAV vector is an AAV serotype 9 vector. 
     
     
         25 . The recombinant expression vector of  claim 23  or  24 , wherein the recombinant AAV vector comprises one or more of a promoter, a pair of inverted terminal repeats (ITRs), and a polyadenylation signal sequence. 
     
     
         26 . The recombinant expression vector of  claim 25 , wherein (i) the promoter is a human cytomegalovirus (CMV) immediate early promoter, (ii) the ITRs are AAV ITRs, (iii) the polyadenylation signal sequence is an simian virus 40 (SV40) polyadenylation signal sequence, or (iv) a combination thereof. 
     
     
         27 . The recombinant expression vector of any one of  claims 23  to  26 , comprising a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 
     
     
         28 . The recombinant expression vector of  claim 27 , comprising an ITR upstream of the human CMV immediate early promoter, which is located upstream of the nucleotide sequence encoding the antisense molecule, which is located upstream of the WPRE which is located upstream of the SV40 polyadenylation signal sequence which is located upstream of the other ITR. 
     
     
         29 . An extracellular vesicle comprising (i) an antisense molecule which specifically binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, a synthetic antisense molecule of any one of  claims 15  to  17 , a recombinant expression vector of any one of  claims 18  to  28 , or a combination thereof, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4). 
     
     
         30 . A cell comprising (i) an antisense molecule which specifically binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, (ii) the recombinant expression vector of any one of  claims 18 - 28 , (iii) an extracellular vesicle of  claim 29 , or (iv) a combination thereof, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4). 
     
     
         31 . A population of cells comprising at least one cell of  claim 30 . 
     
     
         32 . The extracellular vesicle of  claim 29 , cell of  claim 30 , or population of  claim 31 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         33 . A pharmaceutical composition comprising (i) an antisense molecule, (ii) a vector encoding the antisense molecule, (iii) a cell comprising the vector or antisense molecule, (iv) a extracellular vesicle comprising the antisense molecule, or (v) a combination thereof, wherein the antisense molecule binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, and a pharmaceutically acceptable carrier, diluent, or excipient, wherein the pharmaceutically acceptable carrier, diluent, or excipient is synthetic, when the antisense molecule, cell, or extracellular vesicle is naturally-occurring, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4), optionally, wherein the vector is a recombinant expression vector of any one of  claims 18  to  28 , wherein the cell is a cell of  claim 30 , or the extracellular vesicle is an extracellular vesicle of  claim 29 . 
     
     
         34 . The pharmaceutical composition of  claim 33 , wherein the antisense molecule is an microRNA (miRNA), a pri-miRNA, or a pre-miRNA. 
     
     
         35 . The pharmaceutical composition of  claim 33  or  34 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         36 . The pharmaceutical composition of any one of  claims 33  to  35 , wherein the vector comprises a nucleotide sequence encoding a miRNA, optionally, wherein the miRNA comprises the sequence of SEQ ID NO: 179. 
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein the nucleotide sequence comprises of SEQ ID NO: 181. 
     
     
         38 . The pharmaceutical composition of any one of  claims 33  to  37 , wherein the antisense molecule is an antisense nucleic acid analog comprising the base sequence of SEQ ID NO: 179. 
     
     
         39 . The pharmaceutical composition of any one of  claims 33  to  38 , wherein the cell or extracellular vesicle is obtained from a subject. 
     
     
         40 . A kit comprising (i) an antisense molecule, (ii) a vector encoding the antisense molecule, (iii) a cell comprising the vector or antisense molecule, (iv) a extracellular vesicle comprising the antisense molecule, or (v) a combination thereof, wherein the antisense molecule binds to a portion of an IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180, (ii) and a device for administration to a subject, optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4), optionally, wherein the vector is a recombinant expression vector of any one of  claims 18  to  28 , wherein the cell is a cell of  claim 30 , or the extracellular vesicle is an extracellular vesicle of  claim 29 . 
     
     
         41 . The kit of  claim 40 , wherein the antisense molecule is an microRNA (miRNA), a pri-miRNA, or a pre-miRNA. 
     
     
         42 . The kit of  claim 40  or  41 , wherein the antisense molecule comprises the sequence of SEQ ID NO: 179. 
     
     
         43 . The kit of any one of  claims 40  to  42 , wherein the vector comprises a nucleotide sequence encoding a miRNA, optionally, wherein the miRNA comprises the sequence of SEQ ID NO: 179. 
     
     
         44 . The kit of  claim 43 , wherein the nucleotide sequence comprises of SEQ ID NO: 181. 
     
     
         45 . The kit of any one of  claims 40  to  44 , wherein the antisense molecule is an antisense nucleic acid analog comprising the base sequence of SEQ ID NO: 179. 
     
     
         46 . The kit of any one of  claims 40  to  45 , wherein the cell or extracellular vesicle is obtained from a subject.