Biology subjects

Emerson, C. P.

Publications and source records attributed to Emerson, C. P..

4 recordsLinked to original sources

bioRxiv2025

Efficient Cas9 nuclease-based editing in skeletal muscle via lipid nanoparticle delivery

Gene editing holds great promise for muscular dystrophy treatment, but the rapid evaluation of different editing modalities in skeletal muscle in vivo remains challenging due to lack of simple, effective delivery tools. Here we demonstrate that selective organ targeting (SORT) lipid nanoparticles (LNP) encapsulating optimized Cas9 cargo can facilitate efficient, local delivery to skeletal muscles achieving editing rates [≥]35% and restore protein expression for a proof-of-concept muscular dystrophy target. Interestingly, efficient editing in skeletal muscle was observed despite a strong adaptive immune response to repeat dosing of the Cas9 LNPs. High efficiency editing mediated by LNP-based delivery of Cas9 to skeletal muscle permitted detailed analysis of insertion and deletion (InDel) outcomes in vivo for a set of potential therapeutic target sites, which differed substantially from InDel outcomes observed in proliferating cells in one specific instance. Overall, our findings on enhanced LNP delivery of Cas9, platform-specific immune responses, and differential editing patterns observed between in vitro and in vivo models provide valuable insights that should inform the development of gene editing therapeutics for neuromuscular diseases. One Sentence SummarySORT LNPs permitted efficient Cas9-mediated repair of a pathogenic allele in skeletal muscle in a mouse model of LGMDR7.

molecular biology↗

bioRxiv2024

SMCHD1 activates the expression of genes required for the expansion of human myoblasts

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression. Chromatin modifier SMCHD1 regulates the proliferation of human myoblasts SMCHD1 binds to enhancers of cell cycle regulatory genes to activate their expression Lap2 is an SMCHD1 direct target gene that regulates the expansion of human myoblasts.

cell biology↗

bioRxiv2023

A human immune/muscle xenograft model of FSHD muscle pathology

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) disease progression is associated with muscle inflammation, although its role in FSHD muscle pathology is unknown. MethodsWe have developed a novel humanized mouse strain, NSG-SGM3-W41, that supports the co- engraftment of human hematopoietic stem cells (HSCs) and muscle myoblasts as an experimental model to investigate the role of innate immunity in FSHD muscle pathology. ResultsThe NSG-SGM3-W41 mouse supports the selective expansion of human innate immune cell lineages following engraftment of human HSCs and the co-engraftment and differentiation of patient-derived FSHD or control muscle myoblasts. Immunohistological and NanoString RNA expression assays establish that muscle xenografts from three FSHD subjects were immunogenic compared to those from unaffected first-degree relatives. FSHD muscle xenografts preferentially accumulated human macrophages and B cells and expressed early complement genes of the classical and alternative pathways including complement factor C3 protein, which is a mediator of early complement function through opsonization to mark damaged cells for macrophage engulfment. FSHD muscle xenografts also underwent immune donor dependent muscle turnover as assayed by human spectrin {beta}1 immunostaining of muscle fibers and by NanoString RNA expression assays of muscle differentiation genes. ConclusionsThe NSG-SGM3-W41 mouse provides an experimental model to investigate the role of innate immunity and complement in FSHD muscle pathology and to develop FSHD therapeutics targeting DUX4 and the innate immunity inflammatory responses.

immunology↗

bioRxiv2023

Addressing the dNTP bottleneck restricting prime editing activity

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

biochemistry↗