A novel approach to Mesenchymal Stem Cell Transduction

PROJECT SUMMARY Mesenchymal stem cells (MSC) are in clinical development for cardiovascular, neurologic, orthopedic, and other indications. Ossium is developing a novel source of MSC from vertebral bodies (vbMSC). Genetic modification of vbMSC using lentiviral vector (LV) has the potential to improve the therapeutic potential. Understanding how genetic modification might alter the vbMSC phenotype will be critical to ensuring LV do not initiate premature senescence or diminish their therapeutic properties. Moreover, an important safety and regulatory barrier to clinical implementation of gene modified vbMSC will be estimating the risk of insertional mutagenesis. Gammaretroviral vectors used to modify hematopoietic stem cells (HSC) led to leukemia in at least 4 clinical trials. While lentiviral vectors appear safer, clonal expansion of HSC and mature T cells have now been reported. Ossium seeks to address important safety and efficacy issues in this Phase I proposal. We hypothesize that (1) A HIV-1 based LV pseudotyped with a novel foamy viral envelope (LV-FV) will efficiently transduce vbMSC with less change in cell phenotype compared to LV pseudotyped with VSV-G; (2), the LV integration pattern, distribution among cancer associated genes and ability to induce cell immortalization, will be similar to that seen in other cell types. To study this, we propose: Specific Aim 1: Assess the Effect of LV Gene Transfer and Vector Pseudotype on vbMSC phenotype. LV expressing green fluorescent protein (GFP) pseudotyped with VSVG and FV will be used to transduce vbMSC. Cells will be assessed for gene transfer (vector copy number) and expression (GFP by flow cytometry), viability, expansion capacity, MSC-associated surface markers, secretome, and ability to differentiate into three lineages (bone, adipose and cartilage). Specific Aim 2: Evaluate Insertional Mutagenesis Risk in LV transduced vbMSC. This aim seeks to investigate the genotoxicity of LV vectors in MSC. Specific Aim 2A. Evaluating LV-transduced vbMSC for Integration Pattern and Cancer-Associated Gene Preferences. Comparisons between LV and gammaretroviral vectors in low and high passage vbMSC will be compared to published data on HSC integrations. Specific Aim 2B. Evaluating LV-transduced vbMSC for Immortalization. In this sub-aim we will seek to develop an assay for assessing IM risk by evaluating vbMSC after gene transfer. The findings will have broad applicability given the number of disease states in which MSC may play a therapeutic role. This proposal also uses a variety of innovative technologies, including a novel LV-FV, a novel stem cell source (vertebral body MSC), and will be the first study aimed at assessing the risk of immortalization in MSC.PROJECT NARRATIVE This proposal seeks to address safety and efficacy studies important to clinical development of gene modified vertebral body derived Mesenchymal Stem Cells (vbMSC). Ossium will evaluate vbMSC genetically modified with different lentiviral pseudotypes seeking to minimize changes in the MSC phenotype previously reported after vector transduction. Secondly, given that leukemias and clonal cell outgrowth have been reported in clinical trials of retroviral and lentiviral transduced hematopoietic cells, we will begin to assess the risk of insertional mutagenesis using high throughput sequencing of transduced vbMSC. We also seek to develop a detection assay for vector-associated MSC immortalization.