Miniaturized CRISPR activation for a single AAV delivery method to treat LAMA2-Related Muscular DystrophiesCheng Zhang, Jia Qi (2023) Miniaturized CRISPR activation for a single AAV delivery method to treat LAMA2-Related Muscular Dystrophies. Doctoral Dissertation, University of Pittsburgh. (Unpublished)
AbstractProgrammable recruitment of transcription factors to activate genes in a targeted and specific manner, commonly known as CRISPR activation (CRISPRa), offers unprecedented opportunities for therapeutic interventions. For example, the upregulation of the compensatory gene LAMA1 using CRISPRa to treat LAMA2-related deficient congenital muscular dystrophies (LAMA2-RD) has emerged as a mutation-independent therapeutic approach for this condition. However, the viral delivery of CRISPRa components has been complicated by their size and the limited packaging capacity of adeno-associated viruses (AAVs), necessitating the use of dual AAV approaches that contribute to a common bottleneck in gene therapy, including high dose, potential toxicity, and production cost. Here, I present a miniaturized CRISPRa system that can be packaged into a single AAV and apply this approach to upregulate a compensatory gene Lama1 in a mouse model representing a severe form of LAMA2-RD. Single-AAV9 carrying S. aureus dCas9, driven by novel mini promoter 4XNRF1, combined with tripartite activators VP64-delp65-delRTA and a single guide RNA targeting the mouse Lama1 promoter resulted in systemic LAMA1 expression, including critical target tissues for LAMA2-RD such as the skeletal muscles and peripheral nerves. Importantly, the single AAV-mediated Lama1 upregulation significantly improved neuromuscular functions and extended the lifespan of the severe LAMA2-RD mouse model. Compared to the current state of CRISPRa therapeutic in LAMA2-RD, here, I successfully rescued the disease phenotype in a much more severe mouse model with only half the viral load. These results are crucial to advance the therapeutic development of upregulating the disease modifier gene for LAMA2-RD. Moreover, the components and engineered cassettes presented are adaptable for AAV-based gene therapy for different genetic diseases, such as other neuromuscular disorders and haploinsufficiency-related diseases. Share
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