PROJECT SUMMARY/ABSTRACT Duchenne muscular dystrophy (DMD) is a rare (1 in 3500 boys) but devastating disease affecting skeletal and cardiac muscles with onset in early childhood and premature death in early adulthood. DMD is caused by mutations in the dystrophin gene, which result in its poor or incomplete production by muscle cells. Dystrophin anchors cells in the skeletal and heart muscle to their surrounding tissue and loss-of-function mutations cause progressive muscle degeneration. Multiple therapeutic approaches have been evaluated for treating DMD, but there is no cure. To date, dystrophin-replacement approaches have proven very challenging, due the limited ability to efficiently provide dystrophin replacement or editing indefinitely to the resident stem cells in muscle tissues. The proposed work will develop much-needed new methods to establish clinically amenable stem-cell therapies for DMD. Multiple cell therapy candidates have been evaluated for the treatment of DMD, the potency of the defined muscle stem cell population offers an ideal cell therapy profile but this defined population remains challenging to genome-edit in vivo and ex vivo. The use of genome-editing strategies to permanently correct the dystrophin mutations causing the disease in rare muscle stem cells (also called satellite cells) in vivo is highly inefficient and ex vivo has not been demonstrated in purified, expandable stem cells. Our approach aims to engineer combinatorial biomimetic culture platform to enable the screening and optimization of CRISPR/Cas9-mediated Dystrophin gene editing in self-renewing MuSCs for long-term stem cell-expansion cultures. We aim to overcome critical challenges in the ex vivo expansion of dystrophin-deficient MuSCs without loss of their transplantation potential and achieve efficient and selectable and dystrophin editing through both non-homologous end-joining (NHEJ) and template-mediated homology-directed repair (HDR) in expanding MuSC clones. In Aim 1, we will extend recently developed MuSC expansion methods, reliant on tunable rigidity hydrogel biomaterials, to expand young adult MuSCs from dystrophic mdx mice in long-term cultures and test their sufficiency engrafting and generating serial repopulating satellite cells in vivo. In Aim 2, we will establish a high-throughput single-cell MuSC screening platform to optimize dystrophin gene editing in expanding mdx MuSCs, and resolve transfection, Cas9, guide RNA, and dsDNA repair template conditions for efficient, targeted Mdx editing. This MuSC expansion and screening system should prove useful for optimizing gene editing ex vivo for a variety of application, and could serve as a biotechnological starting point for muscle stem cell therapies for a broad set of congential muscle diseases.
NIH ExPorter
NIH ExPorter