Use of split intein and myotropic AAV vectors enables effective preclinical gene therapy for dysferlinopathy

Limb-girdle muscular dystrophy type R2 (LGMDR2) is a genetic disorder characterized by progressive skeletal muscle weakness and degeneration. It is caused by mutations in the DYSF gene, which prevent dysferlin expression. Dysferlin protein is essential for muscle cell membrane repair. Gene therapy using adeno-associated viral (AAV) vectors to restore full-length dysferlin expression is a promising therapeutic approach for LGMDR2. However, the large size of the dysferlin cDNA (6.2 kilobases) exceeds the ∼4.7-kilobase packaging capacity of AAV vectors, posing a challenge to effective delivery of the full-length protein. Here, we describe the use of split inteins and myotropic AAV vectors for efficient systemic gene delivery and expression of full-length dysferlin in the muscles of an LGMDR2 mouse model. We generated and validated a potent split-intein version of dysferlin that enables reconstitution of scarless full-length dysferlin protein. In vitro assessment of these constructs demonstrated a high capacity of the reconstituted dysferlin to restore membrane repair defects in dysferlinopathic mouse myoblasts. Systemic administration of split-intein and dysferlin dual AAVMYO1 vectors improved muscle strength, increased contractile force, and reduced histopathological abnormalities in the LGMDR2 mouse model at both the presymptomatic stage and late stage of the disease. Compared with the homologous recombination dual–AAV vector strategy, the split-intein technology demonstrated superior efficiency in restoring the full-length dysferlin protein and resulted in greater functional improvements. These findings demonstrate the curative potential of this split intein–based gene therapy approach for LGMDR2.