BCCMA: Vascular Regeneration for PAD: Therapeutic Delivery of Angiogenic Stem Cells and mRNA-Based Gene Therapy

ABSTRACT: Rationale: We propose CLIMB (Cell Therapy Leveraging Innovations in BioMaterials and Biomanufacturing) to leverage the complementary expertise of the investigators as a BLRD and CSRD Collaborative Merit Review Award (BCCMA) by sharing knowledge, resources, and data to jump-start the next generation therapeutic platform for improved vascular and muscle repair in small and large animal models of chronic limb threatening ischemia (CLTI). The central hypothesis of this BCCMA that a comprehensive program addressing macro- to micro-vascular and muscle deficits is needed to advance CLTI treatment and that by sharing and synergizing our complementary expertise, resources, and results, the CLIMB team will be best positioned to address the clinical goal of reducing higher level amputation in CLTI patients. We will develop this comprehensive program by completing the following Specific Aims: (i) increase the quality of therapeutic cell preparations by testing new cell types, and by improving their viability and angiogenic function using instructive biomaterials as cell carriers; (ii) develop new delivery methods to deploy the cell and tissue constructs at the target site; (iii) enable the rational design of new biomaterial-based vascular grafts; and (iv) develop new imaging and microfluidic strategies to enable proper selection of therapeutic interventions for CLTI patients. The objective of this specific project is to test the efficacy of a combinatorial strategy to enhance both vascular and muscle regeneration. The first strategy consists of parallel-aligned nanofibrillar collagen scaffolds for spatiotemporal delivery of chemically modified mRNA (mmRNA) encoding a potent muscle regeneration factor. The second strategy consists of human induced pluripotent stem cell-derived ECs (iPSC-ECs) expanded on mechanically tunable hydrogels to improve endothelial phenotype, expansion, and function yet inhibiting de- differentiation during in vitro manufacturing. The global hypothesis is that delivery of both pro-myogenic and pro- angiogenic therapies will maximize tissue repair in small and large animal models of hindlimb ischemia (HLI) as an experimental model of CLTI. Specific Aim 1 will test the hypothesis that treatment of aligned nanofibrillar collagen scaffolds with spatiotemporally controlled delivery of mmRNA encoding pro-myogenic insulin-like growth factor-1 (IGF-1) will induce muscle regeneration in a mouse HLI model. Specific Aim 2 will test the hypothesis that iPSC-ECs maintain more stable endothelial phenotype, higher angiogenic function, and greater expansion potential in mechanically optimized substrates than on conventional tissue culture dishes. Specific Aim 3 will test the hypothesis that a combinatorial approach involving iPSC-EC-seeded IGF-1-mmRNA releasing scaffolds will maximize muscle and vascular regeneration in murine and porcine models of HLI. If successful, these studies will advance the treatment outcomes for Veterans with CLTI.