AAV1.tMCK.NT-3 gene therapy improves phenotype in Sh3tc2−/− mouse model of Charcot–Marie–Tooth Type 4C

AUTHORS

Burcak Ozes , Lingying Tong , Kyle Moss , Morgan Myers , Lilye Morrison , Zayed Attia , Zarife Sahenk

ABSTRACT

Charcot–Marie–Tooth Type 4C (CMT4C) is associated with mutations in the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene, primarily expressed in Schwann cells (SCs). Neurotrophin-3 (NT-3) is an important autocrine factor for SC survival and differentiation, and it stimulates neurite outgrowth and myelination. In this study, scAAV1.tMCK.NT-3 was delivered intramuscularly to 4-week-old Sh3tc2−/− mice, a model for CMT4C, and treatment efficacy was assessed at 6-month post-gene delivery. Efficient transgene production was verified with the detection of NT-3 in serum from the treated cohort. NT-3 gene therapy improved functional and electrophysiological outcomes including rotarod, grip strength and nerve conduction velocity. Qualitative and quantitative histopathological studies showed that hypomyelination of peripheral nerves and denervated status of neuromuscular junctions at lumbrical muscles were also improved in the NT-3-treated mice. Morphometric analysis in mid-sciatic and tibial nerves showed treatment-induced distally prominent regenerative activity in the nerve and an increase in the estimated SC density. This indicates that SC proliferation and differentiation, including the promyelination stage, are normal in the Sh3tc2−/− mice, consistent with the previous findings that Sh3tc2 is not involved in the early stages of myelination. Moreover, in size distribution histograms, the number of myelinated axons within the 3- to 6-µm diameter range increased, suggesting that treatment resulted in continuous radial growth of regenerating axons over time. In conclusion, this study demonstrates the efficacy of AAV1.NT-3 gene therapy in the Sh3tc2−/− mouse model of CMT4C, the most common recessively inherited demyelinating CMT subtype.

In vivo effects of cell seeding technique in an ex vivo regional gene therapy model for bone regeneration

AUTHORS

Jennifer A. Bell, Cory K. Mayfield, Kevin Collon, Stephanie Chang, Matthew C. Gallo, Elizabeth Lechtholz-Zey, Mina Ayad, Osamu Sugiyam, Amy H. Tang, Sang-Hyun Park, Jay R. Lieberman

ABSTRACT

When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells.