Enhance Cell Therapies for Peripheral Arterial Disease Using Human-Compatible Protease-Based Controls

Over 8 million people in the US suffer from peripheral artery disease (PAD), which is characterized by narrowing of the arteries in the arms or legs that lead to insufficient blood flow and limb ischemia. A therapeutic strategy to treat PAD is to boost the formation of new vessels through a process known as angiogenesis. We previously demonstrated that the delivery of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) improve blood perfusion and angiogenesis in preclinical models of PAD. However, a major bottleneck is poor post-transplantation iPSC-EC survival and the limited capability to reshape the local immune response. To overcome these limitations, we propose to develop novel synthetic biology strategies that would enable the quantitative and dynamic regulation of growth factors and cytokines produced from iPSC-ECs after transplantation. Specifically, the strategies leverage a novel control knob we developed from a human protease and its FDA-approved inhibitor, which would facilitate its eventual deployment in patients because of its reduced immunogenic risk compared to existing control tools and the availability of off-the-shelf external control. Specific Aim 1 is to engineer versions of a pro-survival growth factor (bFGF) and two immunomodulatory cytokines (IL-10 and IL-19) that can be controlled by the protease. We will engineer variants for protease-dependent activation and inactivation, achieved by fusing the target proteins to “caging” domains that are removable by the protease and inserting protease cleavage sites into internal loops, respectively. Specific Aim 2 is to test the pro-survival effect of the protease-controlled growth factor on iPSC-ECs in vivo. We will achieve compact encoding of the entire system on a single vector and validate its performance in vitro. We will then transplant the modified cells in a mouse model of PAD. Output measures include bioluminescence to track cell survival and laser Doppler spectroscopy to quantify vascular perfusion recovery. Specific Aim 3 is to examine the immunomodulatory effects of the protease-controlled cytokines in vivo. After compact encoding and in vitro testing similar to Aim 2, we will transplant the engineered cells in vivo and perform immunohistochemistry to quantify how they affect the immune response at the transplantation site. These results will not only pave the way for more effective stem cell therapies and immunomodulation of the ischemic limb environment for the treatment of PAD, but also provide the first proof of principle for a control strategy that could be generalized to other cell therapies. Project Number: 1R21HL177570-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Xiaojing Gao (+1 co-PI) | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $410,449 | Activity Code: R21 | Study Section: Cellular and Molecular Technologies Study Section[CMT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21HL17757001