Mesenchymal stem cells (MSC) are in clinical development for cardiovascular, neurologic, orthopedic, andother indications. Ossium is developing a novel source of MSC from vertebral bodies (vbMSC). Geneticmodification 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 donot initiate premature senescence or diminish their therapeutic properties. Moreover, an important safety andregulatory barrier to clinical implementation of gene modified vbMSC will be estimating the risk of insertionalmutagenesis. Gammaretroviral vectors used to modify hematopoietic stem cells (HSC) led to leukemia in atleast 4 clinical trials. While lentiviral vectors appear safer, clonal expansion of HSC and mature T cells havenow 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) willefficiently 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 cellimmortalization, 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 greenfluorescent protein (GFP) pseudotyped with VSVG and FV will be used to transduce vbMSC. Cells will beassessed 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 threelineages (bone, adipose and cartilage). Specific Aim 2: Evaluate Insertional Mutagenesis Risk in LVtransduced 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 comparedto published data on HSC integrations. Specific Aim 2B. Evaluating LV-transduced vbMSC forImmortalization. In this sub-aim we will seek to develop an assay for assessing IM risk by evaluating vbMSCafter gene transfer. The findings will have broad applicability given the number of disease states in whichMSC may play a therapeutic role. This proposal also uses a variety of innovative technologies, including anovel LV-FV, a novel stem cell source (vertebral body MSC), and will be the first study aimed at assessingthe 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. Adipose tissue ; Fatty Tissue ; adipose ; white adipose tissue ; yellow adipose tissue ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; bone ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cardiovascular system ; Cardiovascular ; Cardiovascular Body System ; Cardiovascular Organ System ; Heart Vascular ; circulatory system ; Cartilage ; Cartilaginous Tissue ; Cell Survival ; Cell Viability ; Cells ; Cell Body ; Child ; 0-11 years old ; Child Youth ; Children (0-21) ; youngster ; Clinical Trials ; Clone Cells ; Disease ; Disorder ; Canis familiaris ; Canine Species ; Dogs ; Dogs Mammals ; canine ; domestic dog ; Face ; faces ; facial ; Flow Cytometry ; Flow Cytofluorometries ; Flow Cytofluorometry ; Flow Microfluorimetry ; Flow Microfluorometry ; flow cytophotometry ; Genes ; Chronic Granulomatous Disease ; Hematopoietic stem cells ; Blood Precursor Cell ; Hematopoietic Progenitor Cells ; blood stem cell ; hematopoietic progenitor ; hematopoietic stem progenitor cell ; hemopoietic progenitor ; hemopoietic stem cell ; Primary carcinoma of the liver cells ; Hepatocarcinoma ; Hepatocellular Carcinoma ; Hepatocellular cancer ; Hepatoma ; Liver Cells Carcinoma ; liver carcinoma ; HIV-1 ; HIV-I ; HIV1 ; Human Immunodeficiency Virus Type 1 ; Human immunodeficiency virus 1 ; Human ; Modern Man ; leukemia ; Mus ; Mice ; Mice Mammals ; Murine ; Orthopedics ; Orthopedic ; Orthopedic Surgical Profession ; Patients ; Phenotype ; Play ; Publishing ; Research Personnel ; Investigators ; Researchers ; Risk ; Role ; social role ; Safety ; stem cells ; Progenitor Cells ; T-Lymphocyte ; T-Cells ; thymus derived lymphocyte ; Thalassemia ; Transplantation ; transplant ; Vesicular stomatitis Indiana virus ; VSV ; Vesicular Stomatitis Virus ; Wiskott-Aldrich Syndrome ; Aldrich Syndrome ; Wiskott syndrome ; eczema thromocytopenia diarrhea syndrome ; eczema thromocytopenia immunodeficiency syndrome ; eczema thromocytopenia syndrome ; Work ; Insertional Mutagenesis ; GTP-Binding Protein alpha Subunits, Gs ; G(s), alpha Subunit ; G(s), α Subunit ; G(s)alpha ; G(s)α ; GTP-Binding Protein α Subunits, Gs ; Gs alpha Family G-Protein ; Gsα ; Gαs ; Regulatory Ns Protein ; Stimulatory Gs G-Protein ; alpha Subunit Stimulatory GTP-Binding Protein ; alpha-Gs ; α-Gs ; Green Fluorescent Proteins ; base ; improved ; Site ; Surface ; Clinical ; premature ; prematurity ; Phase ; Neurologic ; Neurological ; Gammaretrovirus ; Mammalian Type C Retroviruses ; γ-retrovirus ; Link ; Ensure ; vertebra body ; Vertebral Body ; Hematopoietic ; hemopoietic ; cell mediated therapies ; cell-based therapeutic ; cell-based therapy ; cellular therapy ; Cell Therapy ; Therapeutic ; Genetic ; Frequencies ; Clinic ; Source ; cell type ; Pattern ; Viral ; preference ; innovative technologies ; particle ; genotoxicity ; cell immortalization ; Animal Models and Related Studies ; model of animal ; model organism ; Animal Model ; novel ; Reporting ; Vertebral Bone ; Property ; cell bank ; Mesenchymal Progenitor Cell ; Mesenchymal progenitor ; Mesenchymal Stem Cells ; CD34 ; HPCA1 ; CD34 gene ; Address ; Data ; Mature T-Cell ; Mature T-Lymphocyte ; Clonal Expansion ; Gene-Modified ; gene modification ; Gene Transfer ; Leukemic Cell ; Risk Estimate ; Immunologics ; Immunochemical Immunologic ; Immunologic ; Immunological ; Immunologically ; Modification ; Development ; developmental ; safety study ; pre-clinical ; preclinical ; vector ; cost ; novel strategies ; new approaches ; novel approaches ; novel strategy ; gene therapy clinical trial ; gene transfer vector ; Lentivirus Vector ; Lentiviral Vector ; genetically modified cells ; genetically engineered cells ; therapeutic effectiveness ; lentiviral integration ; lentivirus integration ; lentivirally transduced ; lentiviral-transduced ; lentivirus transduced ; cellular transduction ; cell transduction ; transduced cells ; senescence ; senescent ; chimeric antigen receptor ; chimeric antigen T cell receptor ; High-Throughput Nucleotide Sequencing ; High-Throughput Sequencing ; integration site ; efficacy study ; clinical development ; clinical implementation ; hematopoietic stem cell expansion ; HSC expansion ; blood stem cell expansion ; stem cell model ; stem cell based model ; stem cell derived model ; detection assay ; bone marrow mesenchymal stem cell ; stem cell genes ;
