P423 AAV-mediated Therapeutic Effect of Linker Protein-Mediated Gene Therapy on Muscle and Nerve Pathology in Mouse Models for LAMA2 MD

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, one chain of the heterotrimeric extracellular matrix protein laminin-211 (α2β1γ1). The large size of the coding sequence for laminin-α2 (∼ 9.4kb) and the heterotrimeric structure of laminin-211 present a challenge for AAV-mediated 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. Prior work 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 the muscular dystrophy (Reinhard et al., 2017) and when expressed ubiquitously also prevents nerve pathology (Reinhard et al., 2023). We now compared the effect of the two linkers on disease progression when delivered by intravenous injection of AAVs into LAMA2 MD mice. We compared the disease ameliorative effects by the use of either muscle-specific or ubiquitous promoters. High expression of the linkers was observed in skeletal muscle with both promoters. Only the ubiquitous promoter also resulted in high linker expression in peripheral nerves. Irrespective of the promoter, AAV treatment led to a tremendous improvement of the dystrophic phenotype in all muscles as shown by the increase in body- and muscle weights, grip strength, myofiber size and the reduction of fibrosis. The use of the ubiquitous promoter, in addition, ameliorated the nerve pathology, which presents in LAMA2 MD mice as a progressive hindlimb paralysis. All in all, these studies firmly establish that systemic delivery of AAVs expressing the two linkers might be a possible strategy to treat LAMA2 MD patients. As the linkers are designed from proteins (agrin, nidogen-1 and laminin-α1) that are all expressed in LAMA2 MD patients, also the ubiquitous expression of the linkers is likely to be well tolerated. LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, one chain of the heterotrimeric extracellular matrix protein laminin-211 (α2β1γ1). The large size of the coding sequence for laminin-α2 (∼ 9.4kb) and the heterotrimeric structure of laminin-211 present a challenge for AAV-mediated 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. Prior work 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 the muscular dystrophy (Reinhard et al., 2017) and when expressed ubiquitously also prevents nerve pathology (Reinhard et al., 2023). We now compared the effect of the two linkers on disease progression when delivered by intravenous injection of AAVs into LAMA2 MD mice. We compared the disease ameliorative effects by the use of either muscle-specific or ubiquitous promoters. High expression of the linkers was observed in skeletal muscle with both promoters. Only the ubiquitous promoter also resulted in high linker expression in peripheral nerves. Irrespective of the promoter, AAV treatment led to a tremendous improvement of the dystrophic phenotype in all muscles as shown by the increase in body- and muscle weights, grip strength, myofiber size and the reduction of fibrosis. The use of the ubiquitous promoter, in addition, ameliorated the nerve pathology, which presents in LAMA2 MD mice as a progressive hindlimb paralysis. All in all, these studies firmly establish that systemic delivery of AAVs expressing the two linkers might be a possible strategy to treat LAMA2 MD patients. As the linkers are designed from proteins (agrin, nidogen-1 and laminin-α1) that are all expressed in LAMA2 MD patients, also the ubiquitous expression of the linkers is likely to be well tolerated.