601 - Efficacy of DNM2 Reduction and Mesenchymal Stem Cell-Derived Extracellular Vesicles in SPEG Deficiency

Publication Number: 4589.601

Sara Ali, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Victtoria O. Ardellini, Holtz Children's Hospital Jackson Memorial Hospital, Sunny Isles Beach, FL, United States; Qifei Li, University of Miami Miller School of Medicine and Holtz Children's Hospital, Miami, FL, United States; Shiyu Luo, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Haoyue sheng, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United States; Aisha Khan, University of Miami, Miami, FL, United States; Yee-Shuan Lee, University of Miami, Miami, FL, United States; Huijun Yuan, University of Miami Leonard M. Miller School of Medicine, miami, FL, United States; Shu Wu, University of Miami, miami, FL, United States; Pankaj Agrawal, Holtz Children's Hospital Jackson Memorial Hospital, Miami, FL, United States

Background: Centronuclear myopathy (CNM) is a congenital myopathy characterized by muscle weakness and mislocalized nuclei within myofibers. Recessive striated preferentially expressed gene (SPEG) variants cause CNM with or without dilated cardiomyopathy. SPEG is a large (~350 kDa) protein that functions as a kinase, making therapeutic approaches challenging. We previously demonstrated that dynamin-2 (DNM2) reduction improved skeletal muscle function in conditional SPEG-knockout (CKO) mice; however, cardiac dysfunction persisted. Mesenchymal stem cells (MSCs) have cardio-reparative properties through reduction of fibrosis, stimulation of angiogenesis, and activation of endogenous cardiac stem cells. We therefore investigated whether MSC-derived extracellular vesicles (MSC-EVs), alone or in combination with DNM2 reduction, could improve cardiac function.

Objective: To evaluate the therapeutic effects of MSC-EVs in SPEG-deficient mice, alone and in combination with DNM2 reduction.

Design/Methods: SPEG-CKO (SPEGfl/fl/MCK-Cre+/DNM2+/+), DNM2-haploinsufficiency in SPEG-CKO (SPEGfl/fl/MCK-Cre+/DNM2+/-), and control (SPEGfl/fl/MCK-Cre-/DNM2+/+ or SPEGfl/+/MCK-Cre+/DNM2+/+) mice were randomized to receive Wharton's Jelly-derived MSC-EVs (5×10⁹ particles) or sham (phosphate-buffered saline) via retro-orbital injections at 6 and 8 weeks, and then every 4 weeks thereafter. To target early cardiac involvement, a subset began EV injections at 3 weeks of age. Survival was the primary outcome measure.

Results: Untreated SPEG-CKO mice showed the poorest survival, with most dying between 6 and 9 weeks. In contrast, EV-treated CKO mice had several long-term survivors, living up to 19 weeks, suggesting a potential trend toward improved survival, although not statistically significant. EV-treated DNM2-haploinsufficiency in SPEG-CKO mice did not show consistent benefit; several died as early as 4 weeks. Potential contributing factors include fragility at the time of early injection, increased isoflurane sensitivity, and variability in EV batches. Echocardiograms of both EV-treated SPEG-CKO and DNM2-haploinsufficiency in SPEG-CKO mice demonstrated decreased left ventricular ejection fraction and ventricular dilation.

Conclusion(s): While Kaplan-Meier analysis suggests a possible survival benefit in EV-treated CKO mice, findings remain inconclusive. There is a need to optimize dosing, timing, and delivery protocols. Ongoing work aims to refine these factors and develop strategies for this rare disorder ( < 100 known patients), in which the large size of SPEG and its allelic heterogeneity complicate precision-based therapeutic development.

Figure 1. Survival of SPEG Mouse Models Treated With MSC-Derived Extracellular Vesicles (EVs)
Figure 1. Kaplan-Meier survival curves showing a trend toward prolonged survival in SPEG-KO mice treated with Wharton's Jelly-derived MSC extracellular vesicles (EVs). Untreated KO mice show early mortality (6-9 weeks).