METHODS OF TREATING DUCHENNE MUSCULAR DYSTROPHY USING AAV MINI-DYSTROPHIN GENE THERAPY
The disclosure describes methods of treating humans with Duchenne muscular dystrophy by providing doses of an AAV9 vector that expresses a mini-dystrophin protein in transduced muscle cells.
1 . A method for preparing a pharmaceutical composition for treating Duchenne muscular dystrophy (DMD), comprising:
titering a sample of purified recombinant AAV vector by a quantitative PCR (qPCR) assay,
wherein said recombinant AAV vector comprises an AAV capsid and a vector genome comprising a first AAV ITR, a muscle-specific transcriptional regulatory element operably linked to a nucleotide sequence encoding a mini-dystrophin protein consisting of the amino acid sequence of SEQ ID NO:7, a transcription termination sequence, and a second AAV ITR, and
formulating said sample with a pharmaceutically acceptable carrier to contain a predetermined number of vector genomes per unit volume.
2 . The method of claim 1 , wherein each of said AAV ITRs is an AAV2 ITR.
3 . The method of claim 2 , wherein said nucleotide sequence encoding mini-dystrophin protein is codon optimized.
4 . The method of claim 3 , wherein said nucleotide sequence encoding mini-dystrophin protein comprises the nucleotide sequence of SEQ ID NO:1, or a sequence at least 90% identical thereto.
5 . The method of claim 1 , wherein said muscle-specific transcriptional regulatory element is derived from the human or mouse creatine kinase gene.
6 . The method of claim 5 wherein said muscle-specific transcriptional regulatory element comprises an enhancer and a promoter.
7 - 10 . (canceled)
11 . The method of claim 1 , wherein a therapeutically effective dose of said recombinant AAV vector about 2×10 14 vg/kg, or a dose range selected from the group consisting of 1.80×10 14 vg/kg-2.20×10 14 to vg/kg, 1.85×10 14 vg/kg-2.15×10 14 vg/kg, 1.90×10 14 vg/kg-2.10×10 14 vg/kg, and 1.95×10 14 vg/kg-2.05×10 14 vg/kg.
12 - 38 . (canceled)
39 . The method of claim 1 , wherein said qPCR assay is a transgene qPCR assay performed using forward and reverse primer oligonucleotides that specifically anneal to said nucleotide sequence encoding human mini-dystrophin protein, wherein the amplification product is detected using a probe oligonucleotide, and wherein quantification of the amplification product is in reference to a standard curve produced from serial dilutions of a standard comprising the same nucleotide sequence encoding human mini-dystrophin protein.
40 . The method of claim 39 , wherein said nucleotide sequence encoding human mini-dystrophin protein is provided by SEQ ID NO:1.
41 . The method of claim 40 , wherein the forward primer oligonucleotide (F), probe oligonucleotide (P) and reverse primer oligonucleotide (R) used in the assay are selected from the group of primer-probe sets consisting of:
15 F
77 P
132 R,
38 F
82 P
126 R,
42 F
77 P
131 R,
98 F
156 P
275 R,
113 F
156 P
241 R,
178 F
205 P
259 R,
240 F
283 P
356 R,
256 F
384 P
430 R,
333 F
382 P
422 R,
397 F
431 P
488 R,
398 F
431 P
487 R,
400 F
420 P
489 R,
739 F
773 P
836 R,
740 F
773 P
837 R,
805 F
828 P
894 R,
805 F
850 P
903 R,
1009 F
1072 P
1118 R,
1010 F
1072 P
1121 R,
1042 F
1072 P
1127 R,
1102 F
1128 P
1173 R,
1112 F
1221 P
1273 R,
1112 F
1157 P
1201 R,
1143 F
1221 P
1307 R,
1177 F
1221 P
1335 R,
1288 F
1311 P
1370 R,
1316 F
1341 P
1399 R,
1345 F
1387 P
1434 R,
1351 F
1387 P
1475 R,
1370 F
1407 P
1476 R,
1406 F
1437 P
1495 R,
1408 F
1437 P
1497 R,
1585 F
1607 P
1645 R,
1609 F
1688 P
1769 R,
1750 F
1804 P
1929 R,
1838 F
1868 P
1927 R,
1910 F
1934 P
1986 R,
1955 F
1985 P
2028 R,
2116 F
2138 P
2237 R,
2218 F
2285 P
2379 R,
2319 F
2367 P
2408 R,
2349 F
2399 P
2453 R,
2434 F
2504 P
2573 R,
2450 F
2504 P
2552 R,
2470 F
2492 P
2549 R,
2483 F
2492 P
2547 R,
2485 F
2525 P
2574 R,
2533 F
2574 P
2623 R,
2601 F
2623 P
2690 R,
2602 F
2623 P
2691 R,
2604 F
2624 P
2736 R,
2652 F
2673 P
2737 R,
2720 F
2746 P
2809 R,
2721 F
2746 P
2805 R,
2721 F
2746 P
2810 R,
2776 F
2825 P
2865 R,
2786 F
2828 P
2868 R,
2950 F
2970 P
3028 R,
2955 F
2972 P
3027 R,
2970 F
3016 P
3059 R,
2971 F
3017 P
3060 R,
2972 F
3017 P
3061 R,
3041 F
3086 P
3130 R,
3148 F
3178 P
3238 R,
3149 F
3178 P
3237 R,
3166 F
3208 P
3255 R,
3206 F
3237 P
3306 R,
3218 F
3238 P
3304 R,
3218 F
3264 P
3307 R,
3220 F
3264 P
3309 R,
3273 F
3302 P
3362 R,
3285 F
3324 P
3375 R,
3286 F
3324 P
3375 R,
3462 F
3515 P
3557 R,
3538 F
3570 P
3610 R,
3587 F
3658 P
3748 R,
3677 F
3709 P
3765 R,
3729 F
3749 P
3880 R, and
3755 F
3845 P
3915 R.
42 . The method of claim 39 , wherein said probe oligonucleotide comprises a fluorescent reporter dye and a quencher dye.
43 . The method of claim 42 , wherein said fluorescent reporter dye is selected from the group consisting of: 6-FAM™, FAM™, VIC™, NED™, HEX™, TET™, TAMRA™, JOE™, ROX™, Cyanine 3, Cyanine 5, Cyanine 5.5, Cal Fluor® Gold 540, Cal Fluor® Orange 560, Cal Fluor® Red 590, Quasar® 570, Quasar® 670, and TxRd (Sulforhodamine 101-X).
44 . The method of claim 43 , wherein said quencher dye is selected from the group consisting of: TAMRA, DABCYL dT, BHQ®-1, BHQ®-2, BHQ®-3, OQ, MGB NFQ, Iowa Black® FQ, and Iowa Black® RQ.
45 . The method of claim 39 , wherein the amplification reactions of said transgene qPCR assay are performed using forward and reverse primer oligonucleotides at a concentration of 50-1200 nM.
46 . The method of claim 39 , wherein the amplification reactions of said transgene qPCR assay are performed using probe oligonucleotide at a concentration of 50-500 nM.
47 . The method of claim 39 , wherein said transgene qPCR assay is performed using a two-step thermocycler program wherein the denaturation step occurs at 95° C. for 10-30 seconds, and the combined annealing and elongation step occurs at 60° C. for 30-90 seconds.
48 . The method of claim 39 , wherein the standard curve of said transgene qPCR assay has an efficiency value E between 90% and 110%, and an R 2 value of at least 0.98.