Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as satellite cells, is less studied. Here, we used a Pax7nGFP;Ai9 dual reporter mouse to quantify AAV transduction events in satellite cells. We assessed a panel of AAV serotypes for satellite cell tropism in the mdx mouse model of Duchenne muscular dystrophy and observed the highest satellite cell labeling with AAV9 following local or systemic administration. Subsequently, we used AAV9 to interrogate CRISPR/Cas9-mediated gene editing of satellite cells in the Pax7nGFP;mdx mouse. We quantified the level of gene editing using a Tn5 transposon-based method for unbiased sequencing of editing outcomes at the Dmd locus. We also found that muscle-specific promoters can drive transgene expression and gene editing in satellite cells. Lastly, to demonstrate the functionality of satellite cells edited at the Dmd locus by CRISPR in vivo , we performed a transplantation experiment and observed increased dystrophin-positive fibers in the recipient mouse. Collectively, our results confirm that satellite cells are transduced by AAV and can undergo gene editing to restore the dystrophin reading frame in the mdx mouse.

Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin + myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.

Background. Leiomyosarcomas (LMS) are malignancies with smooth muscle differentiation. Metastasis to the bone is not uncommon. The literature on the clinical course and management of such metastases is limited. Our study describes the clinical course of LMS to the bone, including survival rates, prognostic factors, and surgical management. Methods. We retrospectively reviewed 396 LMS patients presenting at an academic center between 1995 and 2020. We included LMS patients diagnosed with bone metastases and excluded patients with primary LMS of bone. We evaluated survival time with the Kaplan–Meier survival method and used Cox’s proportional hazards regression analysis to determine factors associated with survival. Results. Forty-five patients with LMS (11%) had bone metastases. The most common LMS subtypes with bone metastases were uterine (N = 18, 40%) and retroperitoneal (N = 15, 33%). Bone metastasis was not an independent predictor of mortality by Cox regression analysis (HR 1.0, 95% CI: 0.67–1.5). Patients more frequently metastasized to the axial (N = 29, 64%) than to the appendicular (N = 5, 11%) skeleton. Bone was the first site of metastasis in 13 patients (29%). Patients presented with bone metastases at a median of 32.7 months (IQR: 5.2, 62.6) after initial LMS diagnosis. Twelve patients (27%) sustained a pathologic fracture. Twenty (44%) required surgical management, with 30 surgeries total. Three (15%) had a failure of reconstructive constructs. The median overall survival time was 69.7 months (IQR: 43.2, 124.5). There were no associations between the LMS subtype and survival. Pathologic fracture was an independent predictor of mortality by Cox regression analysis (HR 5.4, 95% CI: 1.8–16). Conclusion. The majority of patients with metastatic LMS to bone survive greater than 5 years and frequently require surgical intervention. Extended survival in this patient population should inform fixation and implant choice. No anatomic subtype was associated with risk for bone metastases. Pathologic fracture was associated with worse survival.