Allele Specific Knockdown for LGMDD1

PROJECT SUMMARY / ABSTRACT While the promise of gene-based therapies for disabling neuromuscular diseases is finally becoming a reality, research efforts thus far have primarily focused on gene replacement strategies for recessive, loss-of-function disorders. Such strategies are not translatable to most dominant muscular dystrophies, hindering the development of new treatment strategies.1,2 Our group recently identified mutations in DNAJB6 that cause limb girdle muscular dystrophy D1 (LGMDD1), a dominantly inherited disabling myopathy with no current treatment options.3 The overarching goal of this proposal is to develop novel therapies for this devastating disease. Addressing this unmet need will advance the field’s understanding of how to treat LGMDD1 and establish the optimal approach to therapeutic development for other dominantly inherited disorders with complex, heterogeneous disease mechanisms.1,2 Several lines of preliminary data indicate that mutations in DNAJB6 exert a dominant effect via a toxic gain-of-function.4,5 The potential for deleterious effects preclude a global DNAJB6 knockdown strategy, as DNAJB6 knockout (KO) mice are embryonic lethal due to aggregation of client proteins.6 Haploinsufficiency appears to be tolerated, as heterozygous KO mice are viable, with no apparent skeletal muscle phenotype, and DNAJB6 frameshift and nonsense mutations are seen in healthy “control” patients in genetic databases. We propose to selectively knockdown mutant DNAJB6 using silencing RNA (siRNA). Our preliminary results indicate that allele specific knockdown (ASKD) is feasible and capable of correcting a proteomic signature of LGMDD1 disease in vitro. Thus, our central hypothesis is that ASKD of mutant DNAJB6 is a viable therapeutic approach to address the toxic gain-of-function mechanism of LGMDD1, while avoiding complete knockdown. In this project we will validate that ASKD of mutant DNAJB6 improves disease phenotypes in a LGMDD1 mouse model (Aim 1) and in vitro human LGMDD1 models (Aim 2). Finally, we will optimize ASKD targeting a common DNAJB6 single nucleotide polymorphism (SNP) to expand the applicability of this therapy to multiple DNAJB6 mutations (Aim 3). We have generated a knock in LGMDD1 mouse with a heterozygous p.F90I mutation, orthologous to human p.F89I, and a FLAG-tagged wild type (WT) allele, enabling size-based distinction of WT vs. mutant RNA and protein. We also generated induced pluripotent stem cells (iPSCs), gene-corrected isogenic controls, and primary myoblast cultures from LGMDD1 patients. Successful completion of the proposed aims will produce essential preclinical data supporting the therapeutic translation of ASKD for LGMDD1.