Biology subjects

Pokharel, R.

Publications and source records attributed to Pokharel, R..

3 recordsLinked to original sources

bioRxiv2026

Coupled Transcriptomic and ECM-Mechanical Remodeling Reveal Mechanotransductive Pathways in Spinal Cord Injury

Spinal cord injury (SCI) leads to the formation of a chronic scar composed of glial and fibrotic components that severely restrict neural regeneration and functional recovery. While the scar composition has been widely studied, the spatiotemporal evolution of tissue mechanics and the role it plays in regulating the post-injury responses remain poorly understood. Here we present an integrated mechanobiological and multi-omics analysis of spinal cord remodeling following a severe thoracic contusion injury. Using nanoindentation and viscoelasticity measurements taken via atomic force microscopy (AFM), we demonstrate that SCI induces a dynamic mechanical response characterized by rapid tissue softening during the acute phase reaching a minimum at one-month post-injury, followed by progressive stiffening associated with chronic scar maturation at six months. Bulk RNA sequencing reveals that early mechanical softening coincides with strong activation of inflammatory and matrix-degrading pathways whereas chronic stiffening correlates with upregulation of collagen synthesis, extracellular matrix (ECM) organization and fibrotic remodeling pathways. Concurrently, mechanotransduction regulators exhibit temporally coordinated activation, indicating that cells dynamically sense and respond to evolving mechanical cues. Viscoelastic analysis further shows that chronic scar tissue exhibited increased stiffness and prolonged relaxation dynamics, reflecting dense collagen deposition and proteoglycan accumulation that reinforces a mechanically restrictive microenvironment. Together, these findings establish that the post-injury scar represents a dynamic mechanobiological system in which the evolving tissue mechanics, viscoelasticity and mechanotransduction collectively regulate ECM remodeling, resulting in regenerative failure. This study provides a comprehensive mechanobiological framework for SCI progression and highlights the opportunities for mechanically informed therapeutic strategies aimed at modulating scar mechanics to promote tissue repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/736147v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@543cc4org.highwire.dtl.DTLVardef@6185c6org.highwire.dtl.DTLVardef@95c3ceorg.highwire.dtl.DTLVardef@6a12ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2024

Curative levels of endogenous gene replacement achieved in non-human primate liver using programmable genomic integration

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

bioengineering↗