A novel approach to Mesenchymal Stem Cell Transduction

项目摘要 间充质干细胞（MSC）正在临床开发中，用于心血管、神经、骨科和 其他迹象。Ossium正在开发一种新的来自椎体的MSC来源（vmMSC）。遗传 使用慢病毒载体（LV）修饰vbMSC具有改善治疗潜力的潜力。 了解遗传修饰如何改变vbMSC表型对于确保LV在体外发育过程中发挥作用至关重要。 不会引发过早衰老或降低其治疗特性。此外，重要的安全和 基因修饰的vbMSC的临床实施的监管障碍将是估计插入的风险， 诱变用于修饰造血干细胞（HSC）的γ逆转录病毒载体导致白血病， 4临床试验虽然慢病毒载体看起来更安全，但HSC和成熟T细胞的克隆扩增具有更高的安全性。 现在被报道了。Ossium寻求解决I期提案中的重要安全性和有效性问题。 我们假设：（1）一种具有新型泡沫病毒包膜的HIV-1假型LV（LV-FV）， 与用VSV-G假型化的LV相比，有效地转染vbMSC，细胞表型变化较小; (2)LV整合模式、癌症相关基因的分布以及诱导细胞凋亡的能力 永生化，将类似于在其他细胞类型中看到的。为了研究这个问题，我们提出：具体目标1： 评估LV基因转移和载体假型对vbMSC表型的影响。LV表达绿色 用VSVG和FV假型化的荧光蛋白（GFP）将用于转染vbMSC。细胞将被 评估基因转移（载体拷贝数）和表达（GFP，通过流式细胞术），活力， 扩增能力、MSC相关表面标志物、分泌组和分化成三种细胞的能力 谱系（骨、脂肪和软骨）。特定目的2：评价LV中的插入突变风险 转导的vbMSC。该目的旨在研究LV载体在MSC中的遗传毒性。具体目标2A. 评估LV转导的vbMSC的整合模式和癌症相关基因偏好。 将比较LV和γ逆转录病毒载体在低代和高代vbMSC中的作用 发表的HSC整合数据。具体目标2B。评价LV转导的vbMSC用于 不朽。在这个子目标中，我们将寻求开发一种通过评估vbMSC来评估IM风险的测定法。 基因转移后。考虑到疾病状态的数量， MSC可能发挥治疗作用。该提案还使用了各种创新技术，包括 新的LV-FV，一种新的干细胞来源（椎体MSC），将是第一项旨在评估 MSC中永生化的风险。

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.