Prime editing therapeutics for LGMD2i

While animal models can provide surrogates for the in vivo evaluation of many aspects of gene editing therapeutics, the efficacy and genotoxicity of editing reagents in the context of human genome cannot be readily modeled in vivo for many organ systems. Our proposal is focused on developing human muscle xenograft models for the in vivo optimization of efficacy and assessment of genotoxicity of genome editing therapeutics for muscular dystrophies. As a proof- of-concept target, we are developing a prime editing therapeutic strategy for the correction of the most common FKRP mutation [L276I (c.826C>A)] associated with Limb Girdle Muscular Dystrophy R9 (LGMD2i or LGMDR9). Mutations in FKRP disrupt skeletal and cardiac muscle function, leading to progressive disability and often premature death. As the FKRP L276I LGMDR9 mutation is autosomal recessive, precise correction of a single L276I allele in muscle and satellite cells will provide an enduring treatment for this disease for which there are no curative therapeutics. Overall project goals are to develop a humanized muscle xenograft model of LGMDR9 for the in vivo assessment of the efficacy and the genotoxicity of prime editing therapeutics that correct the L276I mutation. The project has two Specific Aims. In Aim 1, prime editing correction of the FKRP L276I LGMDR9 mutation will be optimized to therapeutic levels in iPSC derived LGMDR9 myotubes in vitro, and genome-wide off-target analysis will be performed to assess and improve the precision of editing. In Aim 2, prime editing reagents (prime editor mRNA and chemically modified guide RNAs) will be packaged in lipid nanoparticles for delivery to LGMDR9 xenograft muscle and resident regenerative satellite-like cells (iSCs) by direct intramuscular injection or systemic delivery. Dose regimens will be optimized to maximize prime editing correction of FKRP L276I in myotubes and iSCs and minimize off-target editing, as assessed by deep sequencing. Therapeutic efficacy will be assessed using RNA-seq disease biomarker expression and TUNEL apoptosis assays. Our expectation is that development of efficient prime editing systems in the context of a patient muscle xenograft model will have broad impact on the development of safer and more effective editing therapeutics, and that this experimental approach will establish a general pathway for the translation of gene editing therapies to the clinic to treat the diversity of musculoskeletal disorders. Project Number: 1R21HD118293-01A1 | Fiscal Year: 2026 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: SCOT WOLFE (+1 co-PI) | Institution: UNIV OF MASSACHUSETTS MED SCH WORCESTER, WORCESTER, MA | Award Amount: $460,625 | Activity Code: R21 | Study Section: Therapeutic Approaches to Genetic Diseases Study Section[TAG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11310083