Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored Dystrophin reading frame in myofibers, cardiomyocytes and muscle stem cells following local or systemic delivery. AAV-Dmd CRISPR-treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

BackgroundHuman respiratory epithelia function in airway mucociliary clearance and barrier function and have recently been implicated in sensory functions.ObjectiveWe investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying Ca2+ influx into human airway epithelia elicited by diesel exhaust particles (DEP).Methods and ResultsUsing primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates Ca2+-permeable TRPV4, which leads to activation of human respiratory disease–enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-Cβ3 in human airway epithelia and their DEP-enhanced protein–protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)–predisposing TRPV4P19S variant enhances Ca2+ influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease.ConclusionDEP evoked protracted Ca2+ influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism TRPV4P19S. This mechanism reprograms maladaptive inflammatory and extracellular-matrix–remodeling responses in human airways. The novel concept of air pollution–responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route.

Background Epicardial adipose tissue (EAT) volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate EAT’s formation and expansion are unclear. Methods We used a novel modified mRNA (modRNA)-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart. Results Using two independent lineage tracing strategies in murine models, we show that cells originating from the Wt1+ mesothelial lineage, which includes epicardial cells, differentiate into EAT following myocardial infarction (MI). This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation, in the context of exogenous, IGF1 modRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1-lineage cell differentiation into adipocytes after MI. Conclusions Our results establish IGF1R signaling as a key pathway that governs EAT formation in the context of myocardial injury by redirecting the fate of Wt1+ lineage cells. Our study also demonstrates the power of modRNA-based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.

Genome-editing therapeutics are poised to treat human diseases. As we enter clinical trials with the most promising CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) modalities, the risks associated with administering these foreign biomolecules into human patients become increasingly salient. Preclinical discovery with CRISPR-Cas9 and CRISPR-Cas12a systems and foundational gene therapy studies indicate that the host immune system can mount undesired responses against the administered proteins and nucleic acids, the gene-edited cells, and the host itself. These host defenses include inflammation via activation of innate immunity, antibody induction in humoral immunity, and cell death by T-cell-mediated cytotoxicity. If left unchecked, these immunological reactions can curtail therapeutic benefits and potentially lead to mortality. Ways to assay and reduce the immunogenicity of Cas9 and Cas12a proteins are therefore critical for ensuring patient safety and treatment efficacy, and for bringing us closer to realizing the vision of permanent genetic cures.