Manufacture scale up of human pericyte progenitor cells for regenerative medicine

Regenerative stem cell therapy offers new hope for treating cardiovascular disease. The potential of this approach is immense but not completely developed. Clinical trials of cell therapy in patients with myocardial infarction (MI) have so far investigated a relatively small number of options, focusing mainly on the use of blood and bone marrow-derived cells. A host of other cells merit attention as they may offer a specialised means for myocardial and vascular reconstitution. Among these, vascular mural cells and pericytes are gaining attention. The rationale of pericyte-based therapy is to stabilise peri-infarct blood vessels, which would remain otherwise prone to leak and break. We have developed a method for easy isolation and expansion of pericytes from human saphenous veins of patients with coronary artery disease. The vein is obtained without harm to the patient using the leftover from coronary artery bypass surgery. Likewise, a small piece of vein could be obtained through minimal invasive surgery to isolate and expand pericytes in culture and obtain million cells ready for clinical use. We demonstrated that transplantation of human pericytes exerts long-term therapeutic benefit in models of limb ischaemia and MI. Noteworthy, pericytes engraft in peri-infarct vascularisation and exert a spectrum of reparative actions, including attraction of resident cardiac stem cells and circulating angiogenic cells. Furthermore, pericytes improve the survival of cardiac cells thus leading to reduction of the infarct size. No safety concerns were reported. The current isolation/expansion method was developed in a common tissue culture laboratory. Hence, we now need to upgrade it to the standards required for clinical use in patients. The present study aims to develop a clinical grade cell product through a series of regulated procedures and quality controls in preparation of a first-in-man clinical study with autologous pericytes in coronary artery disease patients unresponsive to medications and not amenable to revascularization. The term autologous means that pericytes are extracted from the patient vein, expanded and reintroduced into the patient's heart. Use of patient's own cells avoids their rejection and transmission of infectious disease, which are common problems when using cells from a different donor. The project comprises 3 work-packages (WP), 6 milestones (M) and a contingency plan for prevention, troubleshooting and alternative solutions. The goal of WP1 is to scale up current protocol to improve the yield of cells available for clinical use. The goal of WP2 is to transfer the protocol to the facilities of the National Health Service-Blood and Transplant (NHSBT) for clinical-grade manufacture of at least 6 cell lines. This will be followed by quality control of cell stability. The goal of WP3 is to test the performance of the cell product using in vitro functional assays and models of MI. The project will lead to the clinical manufacture of a novel regenerative medicine cell product through the liaison of the University of Bristol and NHSBT, which is the recommended channel for clinical application of autologous cell therapy in UK. Hence, successful completion of proposed work will make the cell product available immediately to the healthcare system for patient benefit.

This proposal aims to develop a novel cell product for regenerative vascular medicine. The prototype of the cell product, consisting of saphenous vein-derived pericyte progenitor cells (SVPs), proved to be therapeutically effective in preclinical models of peripheral or myocardial ischaemia. Supplementation of pericytes stabilises the peri-infarct neovascularisation which is otherwise prone to leak and break. Furthermore, SVPs exert a spectrum of reparative actions including recruitment of pro-angiogenic monocytes and cardiac progenitor cells and inhibition of apoptosis and fibrosis. No safety concerns were reported. We propose now to validate the capability of a defined system of technical procedures to increase total throughput of cells and obtain a reliable, reproducible and quality-assured cell product in view of performing a first-in-man clinical trial with autologous SVPs in coronary artery disease patients unresponsive to medications and not amenable to revascularization. The project comprises 3 work-packages (WP), 6 milestones (M) and a contingency plan for prevention, troubleshooting and alternative solutions. The goal of WP1 is to scale up current protocol to improve the yield of extraction/expansion (M1) and introduce clinical-grade and xeno-free reagents (M2). The goal of WP2 is to transfer the protocol to the GMP facilities of the National Health Service-Blood and Transplant (NHSBT) for clinical-grade manufacture of at least 6 SVP lines (M3), followed by quality control of antigenic and genomic/epigenetic stability (M4). The goal of WP3 is to test the performance of individual SVP lines (WP5) and assess the therapeutic efficacy in a mouse model of myocardial infarction using a randomised factorial protocol (WP6).

Social Benefit
Cardiovascular disease remains the most serious medical challenge we face. Regenerative medicine could potentially improve health and save public money by reducing the need for long-term care, with benefit for the UK economy as a whole. Our main ambition is to achieve medical benefit through generation of a novel cell product, i.e. human pericytes, which can be used to aid the neovascularisation of ischaemic tissues. The study will bring a new approach to generate blood vessels that are not susceptible to break or regress, as instead often occurs during spontaneous angiogenesis in ischaemic tissues as well as during pathologic angiogenesis, e.g. in diabetes. The end result of this project, three years after initiation, will be the provision of a clinical grade cell product to be tested in a first-in-man clinical trial in patients with coronary artery disease unresponsive to medications and not amenable to revascularization. Moreover, the cell product could be used in association with cardiac stem cells, proangiogenic cells and gene therapy to promote full repair of ischaemic organs. Patients with critical limb ischaemia as well as diabetic patients with microangiopathy could also benefit from the use of pericyte-based therapies. If successful this could benefit millions of UK citizens with cardiovascular disease.

Healthcare Benefit
We have been able to build a team of basic and clinical scientists, bioinformatics/geneticists and specialists from the National Health Service that will collaborate to reach a common objective. Working together will be cross-fertilising and instrumental for the next stage in the development of our overarching strategy. Furthermore, the project will involve the transfer of methodologies from the academia to the National Health Service, where those methodologies will be upgraded to clinical manufacture. Hence, the project will have a profound impact on transferability, medical standards and quality controls of cell manufacture.

Advancement of knowledge
This project is likely to produce results that will advance the knowledge on stem cell behaviour during expansion and following transplantation. For instance, we will assess the effect of expansion and transplantation on genomic/epigenetic stability, which will inform about the safety of this specific cell product and also shed new light on the safety of cell therapy in general. Correlation of therapeutic efficacy in a preclinical model with functional and epigenetic signatures of different cell lines will have an impact on reverse translation, i.e. understanding how the cell product works and if variability of the cell product can predictably influence therapeutic activity. However, perhaps the most important impact will be providing a successful example of translation of basic science discoveries into a medically relevant product. To the best of our knowledge, this will be the first example of a stem cell type isolated, expanded and pre-clinically tested by a UK academic group and further developed by the same investigators into a clinically-grade cell product.

Industrial exploitation
Innovative aspects of our study might be patentable. While this is especially applicable to conventional medical products and in some case allogeneic cell therapy, autologous cell therapies offer less scope for intellectual coverage (since a patient's own cells can't be patented). Hence, we are following the suggestion of the recent document of the UK Ministry of health to embed exploitation plans in an NHS-based model. The University of Bristol and NHSBT will discuss any relevant issue arising from this collaboration that might merit intellectual protection. Finally, the project introduces a series of new commercial reagents for cell manufacture upgrading. Testing these reagents may have an impact on the quality of culture media, matrices etc, thus helping further refinement and improvements by industrial providers.