ABSTRACT
Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized that the administration of VEGF-expressing human MSCs would stimulate neurite outgrowth and proliferation of cell-types involved in neural repair. When cultured with dorsal root ganglion (DRG) explants in vitro, control and VEGF-expressing MSCs (VEGF-MSCs) increased neurite extension and proliferation of Schwann cells (SCs) and endothelial cells, while VEGF-MSCs stimulated significantly greater proliferation of endothelial cells. When embedded within a 3D fibrin matrix, VEGF-MSCs maintained overexpression and expressed detectable levels over 21 days. After transplantation into a murine sciatic nerve injury model, VEGF-MSCs maintained high VEGF levels for 2 weeks. This study provides new insight into the role of VEGF on peripheral nerve injury and the viability of transplanted genetically engineered MSCs. The study aims to provide a framework for future studies with the ultimate goal of developing an improved therapy for nerve repair.
ABSTRACT
OBJECTIVE: Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities. METHODS: Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking. RESULTS: Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied. CONCLUSIONS: These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.
