Publications
Grant Duration
01/23 – 03/25
AAV-based gene replacement therapies have proven to be an effective approach for treating patients with monogenic diseases, or diseases arising from mutations within a single gene. However, this approach cannot be easily translated to treat patients with dysferlinopathy because the Dysferlin (DYSF) gene is too large to be packaged into a single AAV. We have recently developed a novel RNA trans-ligation technology called StitchR, that is able to reconstitute large mRNA sequences to express full-length proteins delivered by two separate AAV vectors.
With generous support from the Jain Foundation, we developed a StitchR dual AAV vector system to deliver and express full-length human Dysferlin (2,080 amino acid, 6,240 ORF isoform). In cell-based assays using plasmid DNA, co-transfection of the StitchR Dysferlin vector pair resulted in 79.9% of the expression of full-length Dysferlin, compared to expression of Dysferlin from a single open reading frame. In a mouse model of dysferlinopathy (A/J, Dysferlin deficient mice), StitchR dual AAV Dysferlin injected at postnatal day 8, under the control of the muscle-specific CK8e promoter and serotyped with AAV9 capsid, resulted in broad expression of human Dysferlin protein throughout skeletal muscle and heart tissues at 1 month of age, with correct localization to myofiber membranes.
Western blot on muscle and heart tissue lysates using primary antibodies specific to Dysferlin showed rescued expression of Dysferlin protein in StitchR-treated A/J (Dysferlin deficient) mice. Human Dysferlin in StitchR treated mice was expressed at levels 150.8% in quadriceps, 114.1% in tibialis anterior (TA) and 510.6% in heart compared to endogenous levels in wild type mice injected with saline.
These studies demonstrate that StitchR is an effective approach for delivery and expression of full-length human Dysferlin to therapeutically relevant tissues in vivo and warrants future studies. Due to the similarities between mouse and man, the same vector sequences and AAV serotypes tested here in mice could be translated to treat patients with dysferlinopathy.
