Gene therapy is emerging as a therapeutic option for patients with Duchenne muscular dystrophy, caused by loss of functional dystrophin protein. The dystrophin coding sequence exceeds the packaging limit of adeno-associated virus (AAV), the preferred delivery vector for muscle gene therapy. Several truncated, AAV-compatible “microdystrophins” have been designed and shown to retain many, though not all, functional features of the full-length protein. Microdystrophins differ from one another according to their combinations of hinges and spectrin-like repeats and the inclusion of dystrophin functional domains. While several microdystrophins have advanced to the clinic, an optimal microdystrophin configuration has not been empirically determined.
The highly conserved dystrophin C-Terminal domain (CT) is absent from all but one clinical-stage microdystrophin (RGX-202, REGENXBIO), despite evidence that this region recruits important signaling proteins to the sarcolemma and prevents force loss following damaging muscle contraction. To test whether the CT contributes to µDys functionality we produced AAV vectors encoding versions of µDys without (“μDysCT48”) or with (“μDysCT194”) an extended piece of the CT. We found that µDysCT194 is maintained at higher levels in transduced muscles, recruits the dystrophin-associated protein complex more effectively to the sarcolemma, and provides improved histopathological benefit in dystrophin-deficient mdx mice. A protocol of 20 consecutive lengthening contractions reduced force to 18.3% of initial value in mdx and 56.6% in WT controls. In mdx mice expressing µDysCT48, a reduction to 34.4% was measured, whereas in µDysCT194 treated animals, force drop was limited to 49.4%. To survey contraction-induced muscle damage, mice were injected with myofiber impermeant Evans blue dye prior to lengthening contraction. In mdx Vehicle mice, 22.5% of fibers were dye-positive following ECC indicating significant cell membrane compromise, which was attenuated more effectively by µDysCT194 than µDysCT48 (4.6% vs. 11.2%, respectively). Our results indicate that incorporation of the CT enhances microdystrophin function.