Biology subjects

Harper, S. Q.

Publications and source records attributed to Harper, S. Q..

4 recordsLinked to original sources

bioRxiv2026

Allele-selective knockdown of mutant DNAJB6 improves molecular features in a primary mouse myotube model of LGMDD1

Dominant variants in DNAJB6 cause limb-girdle muscular dystrophy D1 (LGMDD1), a progressive myopathy associated with disrupted protein homeostasis. Because complete loss of DNAJB6 is embryonic lethal in mice, we evaluated allele-selective knockdown as a strategy to reduce mutant DNAJB6 while preserving wild-type protein. We generated a knock-in mouse carrying an N-terminal 3xFLAG tag on the wild-type Dnajb6 allele, allowing endogenous wild-type and F90I DNAJB6, the murine equivalent of human F89I, to be measured within the same sample. Flag-LGMDD1 mice recapitulated the functional, myopathological, and biochemical features seen in other LGMDD1 models and in human disease. F90I DNAJB6 was more abundant and degraded more slowly than wild-type DNAJB6, and LGMDD1 muscle and primary myotubes showed altered protein handling at baseline and following proteostatic stress. Comparative proteomic analyses identified DNAJA4 as a candidate marker responsive to pathogenic DNAJB6 activity. A two-stage mismatch screen identified a lead candidate siRNA with 264-fold selectivity for F90I DNAJB6. In primary Flag-LGMDD1 myotubes, this siRNA selectively reduced F90I DNAJB6 and shifted several baseline and stress-dependent molecular abnormalities involving p62, HSP70, DNAJA4, and markers associated with unfolded protein response signaling toward control values. These findings establish cellular proof of concept for allele-selective knockdown in LGMDD1.

neuroscience↗

bioRxiv2026

Single Particle Adsorption and Response Quantification for Functional AAVx Titer and Dose Analysis

Accurate quantification of functional adeno-associated virus (AAV) dose remains a critical limitation in gene therapy, where titers are defined using ensemble measurements of viral genomes or capsid concentration that average across heterogeneous populations and weakly predict transduction. We present single-particle adsorption and response quantification (SPARQ), a diffraction-limited imaging platform for quantifying AAVx vectors, regardless of serotype, at single-nanoparticle and single-cell resolution. SPARQ immobilizes and resolves individual AAVx, enabling concurrent measurement of capsid-associated and genome-associated signals before cell exposure. Across multiple serotypes, including AAV2, AAV5, and AAVrh74, SPARQ identifies full-capsid fractions, consistent with bulk measurements obtained by ddPCR/ELISA, DLS/UV-Vis, charge detection, and mass photometry, while reporting narrower distributions than ensemble-derived values and revealing systematic discrepancies in bulk-derived titers arising from population averaging. Surface immobilization is achieved by charge-dependent AAVx-surface interactions, as supported by multiphysics simulations, which capture serotype-dependent adsorption kinetics governed by capsid electrostatics for AAV2, AAV5, AAV6, and AAV9. Furthermore, SPARQ directly measures transduction as a function of particle number and reveals a threshold response. Transduction efficiency increased from [~]29% (S.D {+/-} 3.31%) at [~]18 (S.D {+/-} 7.55) AAVs per cell to [~]66% (S.D {+/-} 1.66%) at [~]42 (S.D {+/-} 9.69) AAVs per cell, which is comparable with conventional in vitro assay requiring 105 AAVs/cell. Cumulative fluorescence measurements across large micro-patterned arrays of single cells recapitulate bulk-like high-throughput scaling, while SPARQs single-nanoparticle resolution reveals functional heterogeneity that is masked by population averaged assays. SPARQ provides a particle-resolved framework for functional AAVx titration and quantitative characterization, enabling the development of quality control and dosing strategies in gene therapy manufacturing. TeaserSPARQ combines light-activated surface viral adsorption with diffraction-limited imaging to directly quantify AAV integrity, heterogeneity, and functional transduction at the single-particle level.

bioengineering↗

bioRxiv2025

Sustained efficacy of CRISPR-Cas13b gene therapy for FSHD is challenged by immune response to Cas13b

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating muscle disease caused by de-repression of the toxic DUX4 gene in skeletal muscle. FSHD patients may benefit from DUX4 inhibition therapies, and although several experimental strategies to reduce DUX4 levels in skeletal muscle are being developed, no approved disease modifying therapies currently exist. We developed a CRISPR-Cas13b system that cleaves DUX4 mRNA and reduces DUX4 protein level, protects cells from DUX4-mediated death, and reduces FSHD-associated biomarkers in vitro. In vivo delivery of the CRISPR-Cas13b system with adeno-associated viral vectors reduced acute damage caused by high DUX4 levels in a mouse model of severe FSHD. However, protection was not sustained over time, with decreases in Cas13b and guide RNA levels between 8 weeks and 6 months after injection. In addition, wild-type mice injected with AAV6.Cas13b showed muscle inflammation with infiltrates containing Cas13b-responsive CD8+ cytotoxic T cells. Our RNA-seq data confirmed that several immune response pathways were significantly increased in human FSHD myoblasts transfected with Cas13b. Overall, our findings suggest that CRISPR-Cas13b is highly effective for DUX4 silencing but successful implementation of CRISPR/Cas13-based gene therapies may require strategies to mitigate immune responses.

molecular biology↗

bioRxiv2022

Posttranslational modifications of the DUX4 protein impact toxic function

ObjectiveFacioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the transcription factor DUX4, which is toxic to muscle in vitro and in vivo. While the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 posttranslational modifications (PTMs) and their impact on the toxic function of DUX4. MethodsDUX4 protein was immunoprecipitated and mass spectrometry performed to identify PTMs. We then extensively characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics and biochemical assays in human cell lines and human myoblast cell lines. ResultsOur in vitro screen of DUX4 PTM mutants identified arginine methyl-null and serine/threonine phosphomimetic mutants that protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate downstream gene targets, including FSHD biomarkers. Using additional proteomics and biochemical approaches, we identified protein kinase A (PKA) and a protein arginine methyltransferase (PRMT1) as components of the DUX4 complex. Importantly, over-expression of PRKACA, a catalytic subunit of the PKA holoenzyme, mitigated DUX4 toxicity, while pharmacologic inhibition of PRMT1 protected human myoblasts from DUX4-mediated apoptosis. InterpretationThese results demonstrate that DUX4 is regulated by PTMs and that DUX4 PTMs, or associated modifying enzymes, may be druggable targets for FSHD therapy.

neuroscience↗