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.