Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

A new clinical-scale serum-free xeno-free medium efficient in ex vivo amplification of mesenchymal stromal cells does not support mesenchymal stem cells.

BACKGROUND: We evaluated a new serum-free, xeno-free medium (Xuri, GE HealthCare) in ex vivo cultures for amplification of mesenchymal stromal cells (MStroC) in comparison with classical culture supplemented with fetal calf serum and basic fibroblast growth factor. STUDY DESIGN AND METHODS: MStroC and mesenchymal stem cell (MSC) proliferative capacities were studied in bulk cultures and single-cell cultures with assay of secondary replating capacity of individual clones. Flow-cytometric phenotype analysis and proliferative history analysis were also performed. RESULTS: In cultures initiated with previously amplified and cryopreserved MStroC from human marrow, Xuri medium enabled a total cell expansion fold comparable to one obtained in control fetal calf serum (FCS)-supplemented culture. However, both the number and the proliferative capacity of colony-forming unit-fibroblast were greatly reduced in Xuri medium cultures. This is even more evident in single-cell cultures, where, in rare positive wells, only several cells were found in Xuri cultures, compared to abundant cell content in FCS and α-minimal essential medium cultures. Replating these single-cell clones in secondary cultures (FCS in both cases) revealed a total exhaustion of MSC proliferative capacity after Xuri primary culture. CONCLUSION: Since in both conditions after a 7-day bulk culture, similar immunophenotype and proliferative history were found when the standard MSC immunophenotype panel was employed, the loss of proliferative capacity in Xuri medium shows that it cannot maintain functional MSC population. This is a drastic example showing that the real MSC activity can be completely unrelated to the immunophenotype considered as MSC phenotype.