Targeted Non Viral Gene Therapy for Duchenne Muscular Dystrophy
Final Report Abstract
Limb girdle muscular dystrophies types 2B (LGMD 2B) and 2D (LGMD 2D) are atrophic, degenerative muscle diseases affecting the shoulders, pelvic, hip and femoral muscles. After onset, which ranges from early childhood (3-10) for 2D, and from adolescence to adulthood (15-25) for 2B both diseases lead to progressively impaired mobility. So far, no cure exists for both diseases. 2B and 2D are caused by mutations in the “dysferlin” and “alpha-sarcoglycan” genes and are inherited in an autosomal recessive pattern. Using patient-derived human induced pluripotent stem cells (hiPSC) we carried out precise gene addition strategies for correction of the LGMD 2B iPSC by integration of wild-type dysferlin cDNA into the H11 genomic safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We also corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan missense mutation c.229C>T; p.R77C, by single-stranded oligonucleotide (ssODN)-mediated gene editing, using the CRISPR/Cas9 gene editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. We purified corrected iPSC by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.
