Modified friedreich ataxia genes and vectors for gene therapy

Abstract

The present invention relates to a modified FXN gene providing for increased expression of the encoded protein frataxin that can be used for treatment of Friedreich ataxia.

1 - 33 . (canceled) 
     
     
         34 . A modified nucleic acid encoding frataxin (FXN), wherein the FXN comprises the amino acid sequence of SEQ ID NO:1, wherein the modified nucleic acid is expressed at a greater level compared with the level of expression of the wild type FXN nucleic acid sequence of SEQ ID NO:2 in an otherwise identical cell, and wherein the modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO:5. 
     
     
         35 . The modified nucleic acid of  claim 34 , wherein the nucleic acid comprises a GC content of at least 61%, a number of CpG dinucleotides not greater than 117, and a codon adaptation index (CAI) of at least 0.95. 
     
     
         36 . A recombinant expression vector comprising the modified nucleic acid encoding FXN of  claim 34 . 
     
     
         37 . The recombinant expression vector of  claim 36 , wherein the vector is a recombinant adeno-associated virus (rAAV) vector and the modified nucleic acid encoding FXN is self-complementary. 
     
     
         38 . The recombinant expression vector of  claim 36 , wherein the vector is a recombinant adeno-associated virus (rAAV) vector. 
     
     
         39 . A recombinant adeno-associated virus (rAAV) vector comprising the modified nucleic acid of  claim 34 , and a capsid selected from the group consisting of a capsid of AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, AAVrh74, RHM4-1, RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAVHu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, and AAV-LK03. 
     
     
         40 . The rAAV vector of  claim 39 , wherein the capsid is selected from the group consisting of a capsid of AAV serotype AAV2i8, AAV9, AAV-LK03 and AAV2-TT-S312N. 
     
     
         41 . The rAAV vector of  claim 39 , wherein the modified nucleic acid further comprises at least one element selected from the group consisting of at least one adeno-associated virus (AAV) terminal repeat sequence, an enhancer, a promoter, a collagen stabilizing sequence (CSS), a stop codon, and a poly-adenylation (polyA) signal sequence. 
     
     
         42 . The rAAV vector of  claim 41 , wherein the rAAV vector comprises two AAV terminal repeat sequences, a cytomegalovirus enhancer/chicken beta actin (CBh) promoter, a CSS, and a bovine growth hormone poly-adenylation signal sequence (bGHpolyA). 
     
     
         43 . An rAAV vector comprising a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) a modified nucleic acid encoding frataxin (FXN) comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO:25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat. 
     
     
         44 . The rAAV vector of  claim 43 , further comprising an AAV2i8 capsid wherein the VP1 comprises the amino acid sequence of SEQ ID NO:29, and wherein the vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) a modified nucleic acid encoding FXN comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO: 25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat. 
     
     
         45 . The rAAV vector of  claim 43 , further comprising an AAV2-TT-S312N capsid wherein the VP1 comprises the amino acid sequence of SEQ ID NO:33, and wherein the vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) a modified nucleic acid encoding FXN comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO:25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat. 
     
     
         46 . An rAAV vector for treating Friedreich ataxia (FRDA) in a patient in need thereof, wherein the vector comprises a modified nucleic acid encoding frataxin (FXN) comprising the nucleic acid sequence of SEQ ID NO:5. 
     
     
         47 . A pharmaceutical composition comprising the rAAV vector of  claim 46 , and a pharmaceutically acceptable carrier. 
     
     
         48 . A method of treating FRDA in a mammal, the method comprising administering a therapeutically effective amount of the rAAV vector of  claim 46 . 
     
     
         49 . The method of  claim 48 , wherein the rAAV vector is administered systemically, by direct cardiac administration or by intracranial administration. 
     
     
         50 . The method of  claim 49 , wherein the systemic administration is intravenous administration. 
     
     
         51 . The method of  claim 49 , wherein the rAAV vector is administered directly into the heart. 
     
     
         52 . The method of  claim 49 , wherein the rAAV vector is administered intracranially. 
     
     
         53 . A method of treating a disease, disorder or condition mediated by a decreased level of FXN in a mammal, the method comprising administering a therapeutically effective amount of the rAAV vector of  claim 46 . 
     
     
         54 . An isolated host cell comprising a modified nucleic acid encoding frataxin (FXN) comprising the nucleic acid sequence of SEQ ID NO:5. 
     
     
         55 . The host cell of  claim 54 , wherein the cell is selected from the group consisting of VERO, WI38, MRC5, A549, HEK293, B-50 HeLa, HepG2, Saos-2, HuH7, and HT1080. 
     
     
         56 . The host cell of  claim 55 , wherein the cell is a HEK293 cell adapted to growth in suspension culture. 
     
     
         57 . The host cell of  claim 55 , wherein the cell is a HEK293 cell having American Type Culture Collection (ATCC) No. PTA 13274. 
     
     
         58 . The host cell of  claim 54 , wherein the cell comprises at least one nucleic acid encoding a protein selected from the group consisting of a replication (Rep) protein, a capsid (Cap) protein, an adenovirus early region 1a (E1a) protein, a E1b protein, an E2a protein, an E4 protein and a viral associated (VA) RNA and a combination thereof. 
     
     
         59 . A method for increasing the level of frataxin in a cell, the method comprising transducing the cell with an rAAV vector comprising a modified nucleic acid encoding frataxin (FXN) comprising the nucleic acid sequence of SEQ ID NO:5. 
     
     
         60 . An rAAV vector comprising a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) the modified nucleic acid encoding FXN of  claim 34 ; (d) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (e) an AAV2 terminal repeat. 
     
     
         61 . The rAAV vector of  claim 60 , further comprising a capsid selected from the group consisting of a capsid of AAV serotype AAV2i8, AAV9, AAV-LK03, and AAV2-TT-S312N. 
     
     
         62 . The rAAV vector of  claim 61 , further comprising a CSS comprising the nucleic acid sequence of SEQ ID NO:25 immediately following the sequence encoding FXN. 
     
     
         63 . The rAAV vector of  claim 62 , further comprising a capsid of AAV2i8, and wherein the vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) the modified nucleic acid encoding FXN comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO:25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat. 
     
     
         64 . The rAAV vector of  claim 62 , further comprising a capsid of AAV9, and wherein the vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) the modified nucleic acid encoding FXN comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO:25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat. 
     
     
         65 . The rAAV vector of  claim 62 , further comprising a capsid of AAV-LK03, and wherein the vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat; (b) a CBh promoter comprising the nucleic acid sequence of SEQ ID NO:26; (c) the modified nucleic acid encoding FXN comprising the nucleic acid sequence of SEQ ID NO:5; (d) a CSS comprising the nucleic acid sequence of SEQ ID NO:25; (e) a bGHpolyA signal sequence comprising the nucleic acid sequence of SEQ ID NO:27; and (f) an AAV2 terminal repeat.