Vectors encoding a glucose-6-phosphatase (G6PASE-A) for gene therapy
The invention relates to an adeno-associated virus (AAV) vector comprising a nucleic acid construct for the expression of a glucose-6-phosphatase-a (G6Pase-a) in a cell, the construct comprising a nucleic acid sequence encoding the G6Pase-a, wherein the nucleic acid sequence encoding the G6Pase-a is operably linked to a human alpha-1 antitrypsin (hAAT) promoter, a cell transformed with the vector of the invention, a composition comprising the vector or the cell of the invention, and the use thereof.
1 . An adeno-associated virus (AAV) vector comprising a nucleic acid construct for the expression of a glucose-6-phosphatase-a (G6Pase-a) in a cell, the construct comprising a nucleic acid sequence encoding the G6Pase-a, wherein the nucleic acid sequence encoding the G6Pase-a is operably linked to a human alpha-1 antitrypsin (hAAT) promoter, wherein the nucleic acid sequence encoding the G6Pase-a is codon optimized by decreasing the content of GC and decreasing GC dimers in said nucleic acid sequence encoding the G6Pase-a.
2 . The AAV vector according to claim 1 , wherein the G6Pase-a has an amino acid sequence at least 90% identical to SEQ ID NO: 1.
3 . The AAV vector according to claim 1 , wherein the nucleic acid sequence encoding the G6Pase-a comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 2.
4 . The AAV vector according to claim 1 , wherein the hAAT promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 8.
5 . The AAV vector according to claim 1 , wherein the hAAT promoter is preceded by an enhancer.
6 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 11, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, or SEQ ID NO: 56.
7 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises, in the 5′ to 3′ orientation:
(i) the hAAT promoter preceded by an enhancer;
(ii) optionally an intron;
(iii) the nucleic acid sequence encoding the G6Pase-a; and
(iv) a polyadenylation signal.
8 . The AAV vector according to claim 1 , wherein the cell is a liver cell, a kidney cell, or an intestine cell.
9 . The AAV vector of claim 1 , which is an AAV serotype 8 (AAV8) vector, an AAV9 vector, an AAVrh74 vector, an AAV218 vector, or an AAVmut5 vector.
10 . A cell transformed with the AAV vector of claim 1 .
11 . The cell according to claim 10 , which is a liver cell, an intestinal cell, or a kidney cell.
12 . The AAV vector of claim 1 , a cell transformed with said AAV vector, or a composition comprising said AAV vector or said cell, for use as a medicament.
13 . The AAV vector according to claim 1 , wherein the hAAT promoter is preceded by an ApoE enhancer or cis-regulatory modules (CRMs).
14 . The AAV vector according to claim 1 , wherein the hAAT promoter is preceded by an ApoE enhancer of SEQ ID NO: 9.
15 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises SEQ ID NO: 7.
16 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 48.
17 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises, in the 5′ to 3′ orientation:
(i) the hAAT promoter preceded by an ApoE enhancer or cis-regulatory modules (CRMs);
(ii) optionally an intron of a human β globin gene;
(iii) the nucleic acid sequence encoding the G6Pase-a; and
(iv) a bovine growth hormone polyadenylation signal, a HBB2 polyadenylation signal, a SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal.
18 . The AAV vector according to claim 1 , wherein the nucleic acid construct comprises, in the 5′ to 3′ orientation:
(i) the hAAT promoter preceded by an ApoE enhancer of SEQ ID NO: 9;
(ii) optionally an intron of a human β globin gene of SEQ ID NO: 47;
(iii) the nucleic acid sequence encoding the G6Pase-a; and
(iv) a bovine growth hormone polyadenylation signal, a HBB2 polyadenylation signal, a SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal.
19 . The AAV vector according to claim 1 , which is an AAV8 vector.
20 . A composition comprising the AAV vector of claim 1 , or a cell transformed with said AAV vector.
21 . A method for treating glycogen storage disease (GSD), comprising administering to a subject the AAV vector of claim 1 , a cell transformed with said AAV vector, or a composition comprising said AAV vector or said cell.
22 . The method of claim 21 , wherein the GSD is GSD-Ia.