Merosin-deficient congenital muscular dystrophy (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-?2, an extracellular protein essential for skeletal muscle and Schwann cell functions. Individualized correction of LAMA2 mutations is hampered by the heterogeneity found in patient populations. In contrast, upregulation of compensatory gene LAMA1 can serve as a mutation-independent approach and benefit larger number of patients. We previously showed that upregulation of Lama1 via AAV-mediated CRISPR activation (CRISPRa) rescues disease phenotypes in mice. However, it required the use of two AAVs, which directly contributes to the high dose, toxicity, and production cost.
In this project, we aim to generate a “mini-and-mighty” CRISPRa suitable for a single AAV9 delivery system.
First, we swapped a commonly used CMV promoter with a novel, 7.5X-shorter synthetic promoter 4XNRF1 to drive dCas9 expression. Subsequently, we substituted previously used 2XVP64 transcriptional activators with miniaturized tripartite VP64, P65, RTA activators (miniVPR). We coupled the 4XNRF1-miniVPR with sgRNA targeting mouse Lama1 promoter. We show that the mini-CRISPRa system induces robust Lama1 upregulation in mouse myoblasts. Importantly, we can reduce the number of sgRNAs from three to 1 and fit all the miniaturized components within the 4.7 kb-packaging capacity of the AAV9. Current experiments involve testing the new mini-CRISPRa in dyW/dyW mouse model, which has low Lama2 and severe dystrophic phenotypes, including limited lifespan and respiratory problems.
Successful completion of this project will lead to new therapeutic avenues
based on the upregulation of compensatory genes that are applicable for many conditions, including neuromuscular disorders and haploinsufficiency-related diseases.