Triple AAV Intein-Mediated Gene Therapy Ameliorates Dystrophic Phenotype in MDC1A Mice.

Abstract

Several therapies using adeno-associated virus (AAVs) as gene delivery tool have received marketing approval in the past. However, potential applications of AAVs are limited by their restricted gene packaging capacities (<4,7kb). Many monogenic diseases are caused by large complex genes with hundreds of pathogenic variants that exceed AAV capacity. Merosin-deficient Congenital Muscular dystrophy type 1A is a severe monogenic recessive disease caused by the absence of functional copies of the LAMA2 gene. To date, no treatment options are available, with the most promising approach being gene replacement therapy to provide a functional copy. The LAMA2 coding sequence, spanning 9,3 kb, encodes for the laminin-α2, a subunit of the trimeric protein Laminin-211 found in the basement membrane of skeletal muscle cells and Schwan cells. Efforts have been focused on gene or protein replacement along with basement membrane engineering. Here, we exploit the ability of inteins to reconstitute full-length protein in a scarless manner. Using a combination of three AAVs, each encoding for one fragment of the laminin-α2 protein flanked by short split intein, resulted in the complete reconstitution of the Laminin-α2 and an improvement of the histopathological features of the dy2j dystrophic mouse model.