Prime editing therapeutics for LGMD2I

The National Institute of Child Health and Human Development awarded University of Massachusetts Medical School $460,625 on June 1, 2026, under the Child Health and Human Development Extramural Research program (CFDA 93.865) to develop humanized muscle xenograft models for optimizing and assessing genome editing therapeutics targeting limb-girdle muscular dystrophy R9 (LGMDR9).

The project delivers two specific research aims. Aim 1 optimizes prime editing correction of the FKRP L276I mutation in induced pluripotent stem cell-derived LGMDR9 myotubes in vitro and performs genome-wide off-target analysis to assess and improve editing precision. Aim 2 packages prime editing reagents—prime editor mRNA and chemically modified guide RNAs—into lipid nanoparticles for delivery to LGMDR9 xenograft muscle and resident regenerative satellite-like cells via direct intramuscular injection or systemic delivery, with dose regimens optimized to maximize correction of the FKRP L276I mutation in myotubes and satellite-like cells while minimizing off-target editing.

The award targets the most common FKRP mutation (L276I) associated with LGMDR9, an autosomal recessive muscular dystrophy that disrupts skeletal and cardiac muscle function leading to progressive disability and premature death. Precise correction of a single L276I allele in muscle and satellite cells would provide an enduring treatment for a disease with no current curative therapeutics.

Work is performed in Worcester, Massachusetts. The period of performance runs from June 1, 2026, through May 31, 2028.

Abstract

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.