FP.38 Linker protein-mediated gene therapy ameliorates muscle and nerve pathology in mouse models for LAMA2-related congenital muscular dystrophy

Abstract

<i>LAMA2</i>-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of congenital muscular dystrophies. It is caused by mutations in <i>LAMA2</i>, the gene encoding laminin-α2, one chain of the heterotrimeric extracellular matrix protein laminin-211 (α2β1γ1). The large size of the cDNA encoding laminin-α2 and the heterotrimeric structure of laminin-211 present a challenge for gene replacement or gene editing strategies. Here, we describe the development of an AAV-based gene therapy to functionally replace laminin-211 by two small linker proteins. The two linker proteins, mini-agrin (mag) and αLNNd, are assembled from selected domains of agrin, nidogen-1 and laminin-α1. Previous work from our lab in transgenic mice has demonstrated that this Simultaneous Expression of Artificial Linkers (SEAL) in skeletal muscle of LAMA2 MD mice has a tremendous ameliorative effect on muscle pathology and survival. However, muscle-specific treatment makes the peripheral neuropathy phenotype in aged LAMA2 MD mice very apparent, as they develop a severe hindlimb paralysis. Here, we show in novel transgenic models that ubiquitous expression of linker proteins additionally overcomes this LAMA2-related peripheral neuropathy. We also show that the linker proteins are efficiently expressed upon systemic delivery by AAV9. Expression by a muscle-specific promoter significantly ameliorates the muscle disease phenotype in LAMA2 MD mice, as shown on the level of body- and muscle mass, fiber size, inflammation and muscle function (grip strength and locomotion). These experiments show that AAV-mediated expression of the two linker proteins is possible and they suggest that this functional replacement strategy by the design of small proteins assembled from protein domains present in patients may overcome some of the current challenges of gene therapy trials. Additionally, the linker proteins are secreted and are deposited in the extracellular matrix, which allows their accumulation upon expression by AAV. In summary, this study serves as a proof-of-concept in mice and establishes systemic delivery of AAVs that express the two linkers (SEAL technology) as a promising gene therapy for treating LAMA2 MD. Our current efforts focus on the use of next-generation AAVs with increased tropisms to skeletal muscle but low infection of liver (AAVMYO) and the use of ubiquitous promoters to evaluate the possibility of ameliorating the peripheral neuropathy.