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B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

122: Modeling Gene Editing Outcomes in Microphysiological Human Tissue System Models of Duchenne Muscular Dystrophy

Type: Oral Abstract Session

Presentation Details
Session Title: Genome Editing Therapies & Safety II
Location: Concourse Hall 152 & 153
Start Time: 5/18/2023 15:00
End Time: 5/18/2023 15:15

Gene editing has shown potential to treat human diseases, however there remains an unmet need for physiologically relevant preclinical models that can accurately predict the safety and efficacy of gene editors in humans. To address this, we have engineered human skeletal muscle microphysiological tissue systems (myobundles) that model the dystrophic phenotype characteristic of Duchenne muscular dystrophy (DMD). In this study, we report successful editing of the DMD gene using two different myobundle models and three different gene editing strategies delivered via AAV. We show that gene correction leads to significantly improved muscle health and function, demonstrating the potential of myobundles as a preclinical model.
We first determined whether myobundles could accurately model the effects of gene editors delivered via AAV. We completed an AAV serotype screen where hiPSC-derived DMDΔ48-50 myoblasts were transduced at the time of 3D tissue formation with a panel of AAV serotypes encoding GFP. Based on GFP+ cross-sectional area (CSA), AAV6 had the highest transduction efficiency at 40%, followed by AAV2 at 20%, and AAV8 and 9 had little to no transduction.
We then evaluated the editing efficiency of deleting exon 51 by a CRISPR-based editing strategy delivered via two AAV vectors (g51). We observed productive edits in 15% of alleles, which restored the reading frame in 40% of dystrophin transcripts and rescued 12% of wild-type dystrophin protein levels. To further demonstrate the utility of this model, we created an additional myobundle line lacking DMD exon 44 and evaluated two exon skipping strategies: an adenine base editor and SpCas9 targeted to the exon 45 splice acceptor. Treatment with the adenine base editor and SpCas9 led to exon 45 skipping in 30% and 6% of transcripts and restoration of 17% and 5% of wild-type dystrophin protein levels, respectively.
We also compared the function of myobundles treated with g51 vs. a non-targeting gRNA (gCtrl). Interestingly, we found that g51-treated myobundles had a small, but significant decrease in specific force (contractile force/CSA) compared to gCtrl-treated myobundles. However, g51-treated myobundles showed significantly less reduction of force generation compared to gCtrl after injury, suggesting partial amelioration of the dystrophic phenotype. Further histological analysis will characterize the effect of editing on myobundle health and function.
Finally, we treated an immortalized patient myoblast line lacking DMD exons 48-50 with g51 and isolated edited cells to create a monoclonal line of uniformly corrected cells (DMDΔ48-51), which were then used to make myobundles. Histological analysis showed that DMDΔ48-51 had significantly increased myotube diameter and improved sarcomere structure, suggesting improved muscle structure and health, compared to uncorrected controls. DMDΔ48-51 myobundles generated a significantly greater specific force and were protected from injury compared to unedited myobundles. Together, these results suggest that restoration of dystrophin reverses the functional deficits seen in dystrophic human myobundles.
Collectively, this foundational work shows that 3D microphysiological muscle tissue models can be used to perform safety, efficacy, and functional assays in a model that accurately captures human muscle physiology, directly addressing a need in the gene editing field that has been unmet by 2D cell culture and animal models. Additionally, improvements to the model, including the incorporation of immune cells and vasculature, may continue to increase its ability to accurately predict the safety and efficacy of gene therapies in patients.

Madeleine J. Sitton, Alastair Khodabukus, Joel D. Bohning, Nenad Bursac, Charles A. Gersbach

Biomedical Engineering, Duke University, Durham, NC
 M.J. Sitton: None.

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