A novel approach to Mesenchymal Stem Cell Transduction

间充质干细胞转导的新方法

• 批准号: 10325618
• 负责人: KENNETH CORNETTA
• 金额: $ 24.85万
• 依托单位: OSSIUM HEALTH, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-03-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325618
• 关键词: Address; Adipose tissue; Animal Model; Biological Assay; CD34 gene; Canis familiaris; Cardiovascular system; Cartilage; Cell Survival; Cell Therapy; Cells; Child; Chronic Granulomatous Disease; Clinic; Clinical; Clinical Trials; Clonal Expansion; Clone Cells; Data; Development; Disease; Ensure; Face; Flow Cytometry; Frequencies; GTP-Binding Protein alpha Subunits, Gs; Gammaretrovirus; Gene Transfer; Gene-Modified; Genes; Genetic; Green Fluorescent Proteins; HIV-1; Hematopoietic; Hematopoietic stem cells; High-Throughput Nucleotide Sequencing; Human; Immunologics; Insertional Mutagenesis; Lentivirus Vector; Leukemic Cell; Link; Malignant Neoplasms; Mature T-Lymphocyte; Mesenchymal Stem Cells; Modification; Mus; Neurologic; Orthopedics; Patients; Pattern; Phase; Phenotype; Play; Primary carcinoma of the liver cells; Property; Publishing; Reporting; Research Personnel; Risk; Risk Estimate; Role; Safety; Site; Source; Surface; T-Lymphocyte; Thalassemia; Therapeutic; Transplantation; Vertebral Bone; Vesicular stomatitis Indiana virus; Viral; Wiskott-Aldrich Syndrome; Work; base; bone; bone marrow mesenchymal stem cell; cell bank; cell immortalization; cell type; cellular transduction; chimeric antigen receptor; clinical development; clinical implementation; cost; detection assay; efficacy study; gene therapy clinical trial; gene transfer vector; genetically modified cells; genotoxicity; hematopoietic stem cell expansion; improved; innovative technologies; integration site; lentiviral integration; lentivirally transduced; leukemia; novel; novel strategies; particle; pre-clinical; preference; premature; safety study; senescence; stem cell genes; stem cell model; stem cells; therapeutic effectiveness; vector; vertebra body

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.

项目概要 间充质干细胞 (MSC) 正在临床开发中，用于心血管、神经、骨科和 其他指示。 Ossium 正在开发一种新的椎体 MSC 来源 (vbMSC)。遗传 使用慢病毒载体（LV）修饰 vbMSC 有可能提高治疗潜力。 了解基因修饰如何改变 vbMSC 表型对于确保 LV 发挥作用至关重要 不会引发过早衰老或削弱其治疗特性。此外，重要的安全和 基因修饰 vbMSC 临床实施的监管障碍将是估计插入风险 诱变。用于修饰造血干细胞 (HSC) 的伽玛逆转录病毒载体在 至少4次临床试验。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 现在已被报道。 Ossium 寻求在第一阶段提案中解决重要的安全性和有效性问题。 我们假设 (1) 基于 HIV-1 的 LV 假型化具有新型泡沫病毒包膜 (LV-FV) 将 与用 VSV-G 假型化的 LV 相比，有效转导 vbMSC，且细胞表型变化较小； (2)、LV整合模式、癌症相关基因的分布以及诱导细胞的能力 永生化，将与其他细胞类型中看到的相似。为了研究这一点，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。表达绿色的LV 用 VSVG 和 FV 假型化的荧光蛋白 (GFP) 将用于转导 vbMSC。细胞将是 评估基因转移（载体拷贝数）和表达（通过流式细胞术检测 GFP）、活力、 扩增能力、MSC 相关表面标记、分泌组以及分化为三种的能力 谱系（骨、脂肪和软骨）。具体目标 2：评估 LV 中的插入突变风险 转导的vbMSC。该目的旨在研究 MSC 中 LV 载体的遗传毒性。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。 将比较低传代和高传代 vbMSC 中 LV 和 γ 逆转录病毒载体之间的比较 有关 HSC 集成的已发布数据。具体目标 2B。评估 LV 转导的 vbMSC 永生化。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 风险的测定方法 基因转移后。鉴于疾病状态的数量，这些发现将具有广泛的适用性 MSC可能发挥治疗作用。该提案还使用了多种创新技术，包括 新型 LV-FV，一种新型干细胞来源（椎体 MSC），将是第一项旨在评估 MSC 永生化的风险。

项目成果

Gene therapy access: Global challenges, opportunities, and views from Brazil, South Africa, and India.

基因治疗的获取：巴西、南非和印度的全球挑战、机遇和观点。

• 发表时间: 2022-06-01
• 作者: Cornetta, Kenneth;Bonamino, Martín;Mahlangu, Johnny;Mingozzi, Federico;Rangarajan, Savita;Rao, Jayandharan
• 通讯作者: Rao, Jayandharan