Author(s): Dwi U. Kemaladewi, UPMC Children’s Hospital of Pittsburgh; Jia Qi Cheng-Zhang, UPMC Children’s Hospital of Pittsburgh; Annie I. Arockiaraj, UPMC Children’s Hospital of Pittsburgh; Marie Johnson, UPMC Children’s Hospital of Pittsburgh; Anushe Munir, UPMC Children’s Hospital of Pittsburgh; Dwi U. Kemaladewi, UPMC Children’s Hospital of Pittsburgh //

ABSTRACT: LAMA2-deficient congenital muscular dystrophy (LAMA2-CMD) is caused by mutations in the LAMA2 gene encoding laminin-α2, 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 disease modifier gene LAMA1 can serve as a mutation-independent approach and benefit a larger number of patients.     We previously showed that upregulation of Lama1 via AAV-mediated CRISPR activation (CRISPRa) rescues disease phenotypes in mice. Despite the promising outcome, the CRISPRa technology requires the use of dual AAV delivery system, which directly contributes to the high dose, toxicity, and production cost.    In this project, our goal is to develop a miniaturized CRISPRa (mini-CRISPRa) technology 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-dCas9-miniVPR with sgRNA targeting either mouse Lama1 or human LAMA1 promoter. We were able to reduce the number of sgRNAs and fit all the miniaturized components within the 4.7 kb-packaging capacity of the AAV9. Importantly, our data show that the mini-CRISPRa system induces robust Lama1 upregulation in vivo at clinically viable doses. Furthermore, upregulation of human LAMA1 in LAMA2-CMD patient-derived cells rescues several molecular impairments associated with the disease, including mitochondrial bioenergetic and migration defects.    Collectively, our data demonstrate the feasibility and therapeutic benefit of the mini-CRISPRa-mediated upregulation of disease compensatory gene LAMA1, which may enable a mutation-independent treatment for all patients with LAMA2-CMD. This approach has a broad applicability to a variety of modifier genes and could serve as a therapeutic strategy for many conditions, including other neuromuscular disorders and haploinsufficiency-related diseases.

Source of Funding: AFM-Telethon, Cure-CMD, Duchenne Parent Project Netherlands, Muscular Dystrophy Association