PRODUCTION OF RECOMBINANT AAV VECTORS FOR TREATING MUSCULAR DYSTROPHY
The present disclosure provides gene therapy vectors, such as recombinant adeno-associated virus (rAAV), produced in mammalian adherent cells cultured in suspension conditions, for expressing a human micro-dystrophin gene. The present disclosure also provides compositions and methods of using these rAAV to treat muscular dystrophy, such as, e.g., Duchenne Muscular Dystrophy.
1 . A method of producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seed process, comprising:
(a) culturing cells with a first growth medium comprising serum in a N-2 container;
(b) removing the cells from the first medium;
(c) inoculating the cells from step (b) into a second medium comprising no serum or serum at a concentration less than the first medium in a N-1 container;
(d) culturing the cells in the N-1 container under suspension conditions; and
(e) inoculating a third medium in a bioreactor with the cells from step (d).
2 . The method of claim 1 , wherein the rAAV comprises the human micro-dystrophin nucleotide sequence of SEQ ID NO:1.
3 . The method of claim 2 , wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7.
4 . The method of claim 1 , wherein the rAAV comprises the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO: 7.
5 . The method of claim 1 , wherein the suspension seed process further comprises:
(f) transfecting the adherent cells with a transgene plasmid comprising a rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid.
6 . The method of claim 5 , wherein the transgene plasmid comprising a rAAVrh74.MHCK7.microdystrophin construct comprises:
the nucleic acid sequence of SEQ ID NO: 9;
nucleotides 55-5021 of SEQ ID NO: 3; or
nucleotides 1-4977 of SEQ ID NO: 8.
7 . The method of claim 5 , wherein the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and an rAAVrh74 cap gene.
8 . The method of claim 5 , wherein the adenovirus helper plasmid comprises an adenovirus 5 E2A, E4ORF6, and a VA RNA gene.
9 . The method of claim 1 , wherein the suspension seed process further comprises:
(g) lysing the adherent cells.
10 . The method of claim 9 , wherein the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high-pressure extrusion, detergent lysis, or combinations thereof.
11 . The method of claim 1 , wherein the suspension seed process further comprises:
(h) purifying the rAAV by at least one column chromatography step.
12 . The method of claim 11 , wherein the at least one column chromatography step comprises an anion exchange chromatography, a size exclusion chromatography, or a combination thereof.
13 .- 14 . (canceled)
15 . The method of claim 1 , wherein the bioreactor is an adherent bioreactor.
16 . (canceled)
17 . The method of claim 1 , wherein the third medium in the bioreactor comprises at least one factor that promotes cell adherence.
18 . The method of claim 17 , wherein the at least one factor that promotes cell adherence is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.
19 . The method of claim 17 , wherein the third medium in the bioreactor comprises DMEM and 10% FBS.
20 . The method of claim 1 , wherein the adherent cells are cultured under suspension conditions for about 48-72 hours.
21 . The method of claim 1 , wherein the N-1 container is a suspension shake flask.
22 . The method of claim 1 , wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells.
23 . The method of claim 22 , wherein the adherent cells are HeLa cells or HEK-293 cells.
24 . The method of claim 23 , wherein the adherent cells are HEK-293 cells.
25 . The method of claim 1 , wherein the adherent cells are not suspension-adapted.
26 . The method of claim 1 , wherein culturing the cells under suspension conditions does not alter the adherent-dependency of the cells.
27 . (canceled)
28 . A composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is made by the method of claim 1 .
29 . The composition of claim 28 , wherein the composition comprises:
a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO: 9;
b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO: 3; and/or
c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO: 8.
30 . A method of treating muscular dystrophy in a human subject in need thereof comprising administering the composition of claim 29 to said human subject.
31 . The method of claim 30 , wherein the rAAV is administered using a systemic route of administration and at a dose of about 5.0×10 12 vg/kg to about 1.0×10 15 vg/kg.
32 . The method of claim 31 , wherein the systemic route of administration is an intravenous route and the dose of the rAAV administered is about 2×10 14 vg/kg.
33 . The method of claim 30 , wherein the dose of rAAV is administered at a concentration of about 10 mL/kg.
34 . The method of claim 30 , wherein the rAAV is administered by injection, infusion, or implantation.
35 . The method of claim 34 , wherein the rAAV is administered by infusion over approximately one hour.
36 . The method of claim 30 , wherein the rAAV is administered by an intravenous route through a peripheral limb vein.
37 . The method of claim 30 , wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker's muscular dystrophy.
38 . The method of claim 37 , wherein the muscular dystrophy is Duchenne muscular dystrophy.
39 . The method of claim 30 , wherein the level of micro-dystrophin gene expression in a cell of the subject is increased after administration of the rAAV as compared to the level of micro-dystrophin gene expression before administration of the rAAV.
40 . The method of claim 39 , wherein expression of the micro-dystrophin gene in the cell is detected by measuring the micro-dystrophin protein level by Western blot in muscle biopsied before and after administration of the rAAV.
41 . The method of claim 40 , wherein the expression is at least 55.4% after administration of the rAAV, as compared to before.
42 . The method of claim 30 , wherein the mean percentage of micro-dystrophin positive fibers in the muscle tissue of the subject is increased after administration of the rAAV, as compared to the number of micro-dystrophin positive fibers before administration of the rAAV.
43 . The method of claim 42 , wherein the mean percentage of micro-dystrophin positive fibers is at least 70.5% and the mean intensity is at least 116.9% as detected by immunofluorescence (IF) in muscle biopsies before and after administration of the rAAV.
44 . The method of claim 30 , wherein micro-dystrophin transduction by vector genome count is at least 3.87 mean vector genome copies per nucleus.
45 . The method of claim 30 , wherein the composition is administered to a genotyped patient.
46 . The method of claim 45 , wherein the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human dystrophin (DMD) gene.
47 . The method of claim 45 , further comprising genotyping the DMD gene of the human subject prior to administering the composition to said human subject.
48 . The method of claim 47 , wherein the genotyping detects at least one mutation in exons 18 to 79 of the DMD gene, wherein the at least one mutation is a frameshift deletion, a frameshift duplication, a premature stop, or other pathogenic variant resulting in the absence of expression of the human dystrophin protein.
49 .- 53 . (canceled)