Genetically modified mesenchymal stem cells overexpressing CCR2 for cell therapy in critical limb ischemia

Critical Limb Ischemia (CLI) is an advanced stage of Peripheral Arterial Disease (PAD) and a global health problem. Patients with CLI may present with rest pain, gangrene and/or ulceration of the lower limb. Effective treatment options are limited and thus, the incidence of limb amputation is high with associated high morbidity and mortality rates. Mesenchymal Stem Cells (MSCs) are widely used with therapeutic efficacy in various diseases, including Critical Limb Ischemia (CLI). However, the use of MSCs is not without challenges. One of the major challenge in the use of MSCs in CLI therapy is the bio-distribution and retention of the transplanted cells in the target tissue. This reduced retention of locally transplanted cells at the target tissue is due to a variety of factors including the hostile environment where the cells are introduced to; while the reduced bio-distribution of transplanted cells is due to the clearance of these cells into organs like lungs, liver and spleen. Here, MSCs are genetically modified to overexpress a potent chemokine receptor on their surface. The chemokine receptor overexpressed on MSCs in this study is, CC Chemokine Receptor type-2 (CCR2), which is known to bind with high affinity to Monocyte Chemoattractant Protein-1 (MCP-1), a chemokine secreted in high levels by ischemic and injured tissue. CCR2 is normally expressed on immune cells like monocytes that enable their migration towards injured tissue. The hypothesis is; transplantation of CCR2 overexpressing MSCs will assist their increased persistence in ischemic tissue, enabling prolonged secretion of paracrine factors leading to therapeutic angiogenesis. In Chapter 2, a lentiviral vector carrying CCR2 gene was designed and validated for its efficiency and CCR2 integration. The results showed, stable CCR2 integration and efficient transduction of these viruses without affecting the viability of cells. In Chapter 3, Mouse MSCs (mMSCs) were transduced with lentiviral vector carrying CCR2, Transduced cells were selected, culture expanded and the stable overexpression of CCR2 on these mMSCs were confirmed at the transcript level and at the protein level. Characterisation and functional assessment of CCR2 overexpressing mMSCs are detailed in chapter 4. CCR2 overexpressing mMSCs were analysed for their stem cell properties such as expression of mMSC specific surface markers and differentiation potential. The functional properties of these genetically modified cells were assessed by their ability to migrate to MCP-1 in vitro. CCR2 overexpressing mMSCs were found to be positively expressing CD90, CD105 and SCA-1, while negative for the expression of CD11b, CD34 and CD45. CCR2 overexpressing mMSCs were found to be migrating at significantly higher levels towards MCP-1. Therapeutic efficacy of CCR2 overexpressing mMSCs were assessed in a mouse model of hind limb ischemia. Their ability to retain in the ischemic tissue after intramuscular injection were assessed using IVIS fluorescence imaging. The bio-distribution of these cells in various organs in a hind limb ischemia mouse model was also assed after 14 days of cell delivery. CCR2 overexpressing mMSCs were found to improve blood flow in ischemic limbs when compared to animals that received injection of cells expressing a fluorescent protein DsRed and animals that received saline injections. Histological analysis of samples showed there were increase in the number of endothelial cells forming capillaries in animals that received CCR2 overexpressing mMSCs compared to controls. There was improved skeletal muscle regeneration and increased immune cell infiltration in these tissues proving improvement in tissue regeneration. IVIS imaging of tissue samples showed increased retention of transplanted cells in ischemic limb of animals that received CCR2 overexpressing MSCs when compared to animals that received control transduced cells. Moreover, analysis of bio-distribution of transplanted cells showed there were no significant clearing of these transplanted cells into any organs of these animals.

Funder

HEA PRTLI5, SFI

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

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University of Galway Theses (PhD Theses)