Identifying Satellite Cell-Targeting Ligands for Novel Vectors
Abstract
Duchenne muscular dystrophy (DMD) is a devastating genetic myopathy caused by mutations in the enormous DMD, or dystrophin, gene. Progress over the past 30 years in understanding the structure and function of dystrophin, together with advances in gene delivery technology have enabled the development of gene therapy approaches to treat this disorder. Adeno-associated viral vector (AAV) gene replacement therapies—many using components developed by our group—are in clinical trials, and AAV-CRISPR/Cas9 genome editing treatments are being preclinically and commercially developed by our lab and others. Both methods are capable of efficient targeting of skeletal muscle myofibers and cardiac myocytes. Despite this progress, there remain critical unresolved issues about to the ability of AAV vectors to target skeletal muscle stem cells, known as satellite cells (SC). SCs are the source of skeletal muscle cell regeneration following injury, dystrophic turnover, and during normal muscle maintenance. In the absence of dystrophin, skeletal muscles undergo continuous cycles of breakdown and regeneration, leading eventually to the loss of regenerative capacity and a further worsening of disease. Impaired myofiber regeneration appears to result from both a depletion of the SCs’ ability to continuously divide, known as proliferative senescence, as well as impaired regulation of the stem cells’ ability to undergo asymmetric divisions after being activated from a quiescent, or non-dividing state. This loss of asymmetric division, which is needed to maintain a pool of stem cells for future rounds of regeneration, has been shown by Rudnicki to be directly regulated by dystrophin. In other words, dystrophin loss in DMD results in both fragile myofibers and impaired regeneration/repair of those myofibers from the satellite stem cells. While current gene therapy approaches are showing a remarkable ability to slow or halt ongoing myofiber breakdown and regeneration, it is important to note that even normal, healthy muscle undergoes slow turnover and exchange of muscle nuclei with those from the SC compartment. Thus, restoring dystrophin expression to myofibers, either by gene editing or gene replacement, is unlikely to be permanent, and such therapies are expected to wane over time. A more permanent therapy will require the ability to target AAV vectors, and hence dystrophin restoration, to both satellite cells and myofibers, something that has proven inefficient. The ability to restore dystrophin expression in satellite cells via gene editing would provide a continuous, lifelong supply of gene corrected stem cells, enabling such gene therapy to be permanent. In contrast, AAV-mediated micro-dystrophin gene delivery would not be permanent, since AAV vectors are lost from dividing cells, but it would enable correction of the asymmetric division defect in quiescent satellite cells, leading to enhanced regeneration to support myofiber maintenance following gene therapy. Consequently, AAV targeting of SCs is expected to greatly enhance the longevity of gene therapy whether it be via gene editing or gene replacement. Here we propose a dual approach to developing AAV vectors that can target SCs. First, we will screen a series of novel AAV vectors showing increased muscle targeting and decreased liver uptake for the ability to enter SCs. Secondly, we will use a novel “nucleocapsid” system carrying millions of small surface peptides that allows screening millions of sequences to identify ones capable of latching onto the surface of SCs. Such a screen can identify surface determinants that can subsequently be inserted into AAV capsids to direct them to target, and deliver genes to, SCs. Together, these dual, complementary strategies could lead to a new generation of AAV vectors with a greatly increased ability to treat muscles of patients with DMD.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010427
Entities
People
- Jeffrey S Chamberlain
Organizations
- United States Army
- University of Washington