Mitotically Stable Lentiviral Episomes for Stem Cell Gene Therapy

用于干细胞基因治疗的有丝分裂稳定的慢病毒附加体

• 批准号: 9020642
• 负责人: Peter Kurre
• 金额: $ 24.26万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-25 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9020642
• 关键词: Adverse effects; Alleles; Attention; Autologous; CD34 gene; Cancer Etiology; Candidate Disease Gene; Case Study; Cell Line; Cell Nucleus; Cell division; Cells; Chromosomes; Clinical; Clinical Trials; Clonal Evolution; Cost Savings; Development; Disease; Episome; Evaluation; Failure; Fanconi' s Anemia; Funding Opportunities; Gene Delivery; Gene therapy trial; Generations; Genes; Genetic; Genetic Models; Genetic Recombination; Genome; HIV-1; Health; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; Human; Immune system; Immunity; Immunodeficient Mouse; Inborn Genetic Diseases; Inherited; Insertional Activations; Interferons; Lentivirus Vector; Longevity; Maintenance; Matrix Attachment Regions; Mediating; Mitotic; Modeling; Modification; Molecular; Motivation; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nuclear Matrix; Organ; Pancytopenia; Patients; Performance; Phenotype; Physiological; Population; Pre-Clinical Model; Production; Protocols documentation; Replication Origin; Reporting; Research; Research Personnel; Research Project Grants; Residual state; Retroviridae; Risk; Site; Stem cells; Subfamily lentivirinae; System; Testing; Therapeutic; Tissues; Transplantation; Tropism; Vertebral column; Viral; Virus; Work; Xenograft procedure; cost; cytopenia; design; experience; gene delivery system; gene therapy; genotoxicity; in vivo; innovation; integration site; interest; novel; pre-clinical; scaffold; self-renewal; stem; stem cell biology; stem cell population; transgene expression; vector; vector genome

 DESCRIPTION (provided by applicant): Stable genetic modification of autologous stem cells using HIV-1 derived lentivirus vectors confers clinical benefit in patients with inherited disorder of hematopoiesis and immunity. Broad tropism for gene delivery to diverse tissues, stable passage of the proviral integrant to all progeny, and the ability to scale production for clinical use provide strong motivation for using this vector system in stem cell gene therapy. Nevertheless, instances of insertional activation of endogenous genes continue to raise concerns for the use of integrating lentivectors in hematopoietic stem cell gene therapy. Current non- integrating vectors provide long-term expression in postmitotic tissues, but failure to replicate the episomal vector genome leads to rapid loss in dividing stem cell populations. We recently reported the development of a novel, non-integrating episomal lentivector that faithfully replicates through cell division and expresses a gene of interest - without selection. In distinctin to similar vectors, this anchored Non-Integrating LentiVector (aNILV) avoids heterologous viral sequences and relies on human β-interferon nuclear scaffold/matrix attachment region (S/MAR) locus sequences. Our principal hypothesis is that aNILV will optimize biosafety and provide long-term correction of bone marrow failure in a murine model of Fanconi Anemia. We will develop a second-generation episome vector design (aNILV-II) additionally capable of conversion to backbone-free minicircles to further enhance biosafety and long-term transgene expression. This proposal is uniquely responsive to PAS-13-006: New Directions in Hematology Research (SHINE-II). SPECIFIC AIM 1 [A] Demonstrate aNILV-mediated phenotype correction, biosafety and clonal diversity under physiologic and pharmacological selection in a murine model of Fanconi Anemia (FA) associated bone marrow failure. SPECIFIC AIM 1 [B] Track molecular persistence and multilineage expression during serial transplantation of aNILV transduced human CD34 progenitor cells in immunodeficient mice. SPECIFIC AIM 1 [C] Design a minicircle-delivering lentivector (aNILV-II) and systematically determine its performance in clonally-derived cell lines and in vivo. The proposed platform promises to reconcile the efficiency of lentivector delivery with the inherent biosafety of episomally maintained genomes. This vector system will have a strong translational impact on stem cell gene therapy and may allow for significant cost savings, currently associated with long- term integration site surveillance in patients.

 描述（由申请方提供）：使用HIV-1衍生慢病毒载体对自体干细胞进行稳定的遗传修饰，可为遗传性造血和免疫疾病患者带来临床获益。基因递送至不同组织的广泛倾向性、前病毒整合体稳定传递至所有后代以及临床应用规模生产的能力为在干细胞基因治疗中使用该载体系统提供了强大的动力。然而，内源基因的插入激活的情况继续引起对在造血干细胞基因治疗中使用整合慢病毒载体的关注。目前的非整合载体提供在有丝分裂后组织中的长期表达，但未能复制附加型载体基因组导致分裂干细胞群体的快速损失。我们最近报道了一种新的非整合的附加型慢病毒载体的开发，该载体通过细胞分裂忠实地复制并表达感兴趣的基因-而没有选择。与类似载体不同，这种锚定的非整合慢病毒载体（aNILV）避免了异源病毒序列，并依赖于人β-干扰素核支架/基质附着区（S/MAR）基因座序列。我们的主要假设是aNILV将优化生物安全性，并在范可尼贫血小鼠模型中提供骨髓衰竭的长期纠正。我们将开发第二代附加体载体设计（aNILV-II），其另外能够转化为无骨架微环以进一步增强生物安全性和长期转基因表达。本提案是对PAS-13-006：血液学研究新方向（SHINE-II）的唯一响应。具体目的1 [A]证明在范可尼贫血（FA）相关骨髓衰竭的鼠模型中，在生理和药理学选择下aNILV介导的表型校正、生物安全性和克隆多样性。特异性目的1 [B]在免疫缺陷小鼠中连续移植aNILV转导的人CD 34祖细胞期间跟踪分子持久性和多谱系表达。具体目的1 [C]设计微环递送慢病毒载体（aNILV-II）并系统地确定其在克隆衍生的细胞系和体内的性能。所提出的平台有望将慢病毒载体递送的效率与附加型维持的基因组的固有生物安全性相协调。该载体系统将对干细胞基因治疗具有强烈的翻译影响，并且可以允许显著的成本节省，目前与患者中的长期整合位点监测相关。