LAMA2-related dystrophies (LAMA2-RD) are a subtype of congenital onset muscular dystrophy caused by mutations in the LAMA2 gene encoding laminin-α2, an extracellular protein essential for skeletal muscle and Schwann cell functions. Individualized correction is hampered by the mutation heterogeneity in patients, for which there are currently over 300 disease-associated mutations. In contrast, the upregulation of the compensatory gene LAMA1 can serve as a mutation-independent approach applicable to all 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 dual-AAVs, which directly contributes to a high dose, toxicity, and cost.
In this project, we aim to generate a mini-CRISPRa (termed 2.0) system suitable for a single AAV9 packaging to improve the clinical relevance of this technology.
First, we swapped the commonly-used CMV promoter with a novel, 7.5x-shorter synthetic promoter termed 4xNRF1 to drive SadCas9 expression. Subsequently, we substituted the previously used 2xVP64 transcriptional activators with a miniaturized tripartite VP64, P65, and RTA activators (miniVPR), and coupled it with a single gRNA targeting the mouse Lama1 promoter.
We observed that the 2.0 system induces Lama1 upregulation in LAMA2-RD mouse myoblasts at a 1.88-fold significantly higher level than the original CRISPRa. Subsequently, we treated a LAMA2-RD mouse model (termed dyw) with an intramuscular injection of AAV9 carrying the 2.0 and observed successful Lama1 upregulation in tibialis anterior muscles. We tested different AAV9 dosages and observed a higher Lama1 upregulation in the 2.0-treated muscles even when the dosage was 15 times less than the original system. In parallel, we have also started studying the effects of systematically administered 2.0 in dyw P1 pups, initial results from 4-week-old post-treatment mice suggest that 2.0 can greatly increase the body weight of diseased mice to a level comparable to wild-type littermates.
Current experiments involve the validation of initial in vivo observations and examination of the AAV biodistribution, neuromuscular functions, and immune responses. Successful completion will lead to new therapeutic avenues based on the upregulation of compensatory genes applicable to other diseases, such as haploinsufficiency-related ones.