Phase I study of IM Injection of VEGF-Producing MSC for the Treatment of Critical Limb Ischemia

Critical limb ischemia (CLI) represents a significant unmet medical need without any approved medical therapies for patients who fail surgical or angioplasty procedures to restore blood flow to the lower leg. CLI affects 2 million people in the U.S. and is associated with an increased risk of leg amputation and death. Amputation rates in patients not suitable for surgery or angioplasty are reported to be up to 30-50% after 1 year. Patients who are not eligible for revascularization procedures are managed with palliative care, but would be candidates for the proposed Phase I clinical trial. In an effort to combat CLI, prior and ongoing clinical trials that our group and others have conducted have evaluated direct injection of purified growth factors into the limb that has low blood flow. Some trials have tested plasmids that would produce the blood vessel growth factors for a short period of time. These therapies did show benefit in early stage clinical trials but were not significantly better than controls in Phase III (late stage) trials, probably due to the short duration of presence of the growth factors and their inability to spread to the areas most needed. Other clinical trials ongoing in our vascular center and others are testing the patient’s own stem cells, moved from the bone marrow to the damaged limb, and those studies are showing some benefit, although the final assessments are not yet completed. Stem cells can have benefit in limb ischemia because they can actively seek out areas of low oxygen and will produce some growth factors to try to encourage blood vessel growth. But in cases where the circulation needs very high levels of rescue, this strategy might not be enough. We have discovered that mesenchymal stem/stromal cells (MSC), a type of adult stem cell, are remarkably effective delivery vehicles, moving robustly through the tissue and infusing therapeutic molecules into damaged cells they contact. In animal models of CLI injections of MSC into the area of decreased blood flow have rapidly restored blood flow to the limbs of rodents who had zero circulation in one leg. As an improved strategy we are combining the stem cell and growth factor approaches to make a more potent therapy. We have engineered human MSCs from normal donor bone marrow to produce high levels of the strong angiogenic agent VEGF for this novel approach (MSC/VEGF). We and others have documented that MSC are capable of sustained expression of growth factors, migrate into the areas of lowest oxygen in the tissues after injection, and wrap around the damaged or tiny blood vessels to secrete their factors where they are needed most, to restore blood flow. We propose to use these MSCs as “nature's own paramedic system”, arming them with VEGF to enhance collateral blood vessel growth. During our first year of funding, we have successfully engineered human MSCs to produce VEGF . We shared with our collaborative funding partners (CFP) at Hospital Reina Sofia in Cordoba, Andalucía, Spain the Standard Operating Procedures developed and product required to enable them to replicate the process. We have also provided our pivotal GMP-grade VEGF lentiviral vector to their group. In addition, personnel from their laboratory came to learn our methods for MSC isolation, expansion, viral transduction, etc. Later that year, our team at UC Davis visited their facilities in Spain with the primary goal of comparing our clinical protocols. We plan to follow almost identical procedures, with the only difference being the route of administration of MSC/VEGF into patients: intramuscular injection in California and intra-arterial in Andalucia. The clinical trials in both, California and Spain will enable us to directly compare our results from the same cellular product, strengthening our conclusions. We had a pre-preIND meeting with the Food and Drug Administration, and have performed preliminary safety and efficacy studies to test our development candidate product, MSC/VEGF. These pilot efficacy and safety studies include the following: measuring secreted VEGF levels, Examining genetic stability, performing rule-out tumorigenicity assays, conducting rule-out hemangioma and edema assays, pericyte study, retention and efficacy studies.

Background: Critical limb ischemia (CLI) represents a significant unmet medical need without any effective medical therapies for patients at high risk of amputation. Currently the only method of treatment for this very severe form of CLI is amputation. Treatment of patients with critical limb ischemia results in a high economic burden to the healthcare system and the individual with the disease, moreover, the mortality (death) of patients undergoing amputation for CLI is very high. Within three months of presentation, 12% of CLI patients will require major amputation, and limb loss rates as high as 30% at 1-2 years have been reported. It is estimated that 160,000 to 180,000 major and minor amputations are performed annually in the United States due to CLI. Fewer than half of all CLI subjects achieve full mobility after an amputation and only one in four above-the-knee amputees will ever wear prosthesis. Prior and ongoing clinical trials have investigated injection of gene therapy agents for patients with CLI. These therapeutic agents were not significantly better than controls in Phase III trials. Other clinical trials using the patient’s own stem cells, from the bone marrow and injected into the damaged limb are showing some benefit, although the final assessments are not yet completed. Stem cells have shown benefit in treating limb ischemia as they can actively seek out areas of low oxygen and produce some growth factors enabling blood vessel growth. In more severe cases, these strategies alone may not be enough. Proposed therapy: We have discovered that mesenchymal stem/stromal cells (MSC), a type of adult stem cell, are effective delivery vehicles, moving robustly through the tissue, infusing therapeutic molecules into damaged cells they contact. In mouse models of CLI, injections of MSC into the ischemic area restored blood flow to the limbs. As an improved strategy, we have combined the stem cell and growth factor approaches to make a more potent therapy. We have engineered human MSC from normal donor bone marrow to produce high levels of the angiogenic agent VEGF (MSC/VEGF). It is well documented that MSC are capable of sustained expression of growth factors, migrate to areas of lowest oxygen in the tissues after injection, and wrap around the damaged or tiny blood vessels to secrete factors where they are needed most to restore blood flow. We propose to use these MSC as “nature's own paramedic system”, arming them with VEGF to enhance collateral blood vessel growth. Progress, Year 2 of grant: Based on feedback from the CIRM Clinical Development Advisory Panel (CDAP), we altered our vector. We have successfully engineered human MSCs to produce VEGF with this new pivotal vector and performed additional efficacy and safety studies. We successfully manufactured the new VEGF vector using Standard Operating Procedures (SOPs) from our UC Davis Good Manufacturing Practices (GMP) Facility and repeated all quality testing. We have developed GMP SOPs to produce MSC/VEGF in large quantities for animal model testing and for the future planned human clinical study. We have shown that MSC/VEGF produces high levels of VEGF with bioactivity, and that a multiplicity of infection of one virus particle per cell generates a single unrearranged integrant per cell, on average. This data is critical to the Recombinant DNA Advisory Committee (RAC), for whom we have prepared and submitted an Appendix M application. RAC approval is needed prior to FDA approval because it is a proposed stem cell gene therapy trial. We shared this information and our pivotal VEGF vector with our collaborative funding partners (CFP) at Hospital Reina Sofia in Cordoba, Andalucía, Spain. Personnel from both groups have visited each other’s facilities and we continue to have regular meetings to ensure we are collectively moving forward. We shared our RAC document with our CFP which includes the proposed clinical protocol. We plan to follow similar procedures, with the exception being the route of administration of MSC/VEGF into patients: intramuscular injection at UCD and intra-arterial in Spain. The clinical trials in both California and Spain will enable us to compare results from the same cellular product, strengthening our conclusions. We have successfully completed four efficacy experiments; the MSC/VEGF cell product caused significantly increased blood flow in the ischemic limb, as compared to the saline controls in each study. In addition we have performed safety studies including the following: measuring secreted VEGF levels, karyotypic stability, examining genetic stability, performing rule-out tumorigenicity assays, conducting rule-out hemangioma and edema assays, pericyte study, and retention. We have fully addressed the suggestions put forth at the CDAP meeting in Year two. We plan to: 1) Complete our pre-clinical studies within the next few months 2) Move toward regulatory approval 3) Initiate the planned clinical trial in 2016

Background: Critical limb ischemia (CLI) represents a significant unmet medical need without any effective medical therapies for patients at high risk of amputation. Currently the only method of treatment for this very severe form of CLI is amputation. Treatment of patients with critical limb ischemia results in a high economic burden to the healthcare system and the individual with the disease, moreover, the mortality (death) of patients undergoing amputation for CLI is very high. Within three months of presentation, 12% of CLI patients will require major amputation, and limb loss rates as high as 30% at 1-2 years have been reported. It is estimated that 160,000 to 180,000 major and minor amputations are performed annually in the United States due to CLI. Fewer than half of all CLI subjects achieve full mobility after an amputation and only one in four above-the-knee amputees will ever wear prosthesis. Prior and ongoing clinical trials have investigated injection of gene therapy agents for patients with CLI. These therapeutic agents were not significantly better than controls in Phase III trials. Other clinical trials using the patient’s own stem cells, from the bone marrow and injected into the damaged limb are showing some benefit, although the final assessments are not yet completed. Stem cells have shown benefit in treating limb ischemia as they can actively seek out areas of low oxygen and produce some growth factors enabling blood vessel growth. In more severe cases, these strategies alone may not be enough. Proposed therapy: We have discovered that mesenchymal stem/stromal cells (MSC), a type of adult stem cell, are effective delivery vehicles, moving robustly through the tissue, infusing therapeutic molecules into damaged cells they contact. In mouse models of CLI, injections of MSC into the ischemic area restored blood flow to the limbs. As an improved strategy, we have combined the stem cell and growth factor approaches to make a more potent therapy. We have engineered human MSC from normal donor bone marrow to produce high levels of the angiogenic agent VEGF (MSC/VEGF). It is well documented that MSC are capable of sustained expression of growth factors, migrate to areas of lowest oxygen in the tissues after injection, and wrap around the damaged or tiny blood vessels to secrete factors where they are needed most to restore blood flow. We propose to use these MSC as “nature's own paramedic system”, arming them with VEGF to enhance collateral blood vessel growth. Progress, Year 3 of grant: We have successfully engineered human MSCs to produce VEGF with the pivotal vector and performed additional efficacy and safety studies. We have shown that MSC/VEGF produces high levels of VEGF with bioactivity, and that a multiplicity of infection of one virus particle per cell generates a single unrearranged integrant per cell, on average. This data is critical to the Recombinant DNA Advisory Committee (RAC), for whom we submitted an Appendix M application and received RAC approval to proceed to the FDA with the preIND submission for the proposed stem cell gene therapy trial. We shared this information and our pivotal VEGF vector with our collaborative funding partners (CFP) at Hospital Reina Sofia in Cordoba, Andalucía, Spain. Personnel from both groups have visited each other’s facilities and we continue to have regular meetings to ensure we are collectively moving forward. We shared our RAC document with our CFP which includes the proposed clinical protocol. We plan to follow similar procedures, with the exception being the route of administration of MSC/VEGF into patients: intramuscular injection at UCD and intra-arterial in Spain. The clinical trials in both California and Spain will enable us to compare results from the same cellular product, strengthening our conclusions. We have successfully completed four efficacy experiments; the MSC/VEGF cell product caused significantly increased blood flow in the ischemic limb, as compared to the saline controls in each study. In addition we have performed safety studies including the following: measuring secreted VEGF levels, karyotypic stability, examining genetic stability, performing rule-out tumorigenicity assays, conducting rule-out hemangioma and edema assays, pericyte study, and retention.