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

基本信息

批准号：
9348639
负责人：
Peter Kurre
金额：
$ 13.79万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-25 至 2019-03-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9348639
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&apos s Anemia Funding Opportunities Gene Delivery Gene therapy trial Generations Genes Genetic Genetic Models Genetic Recombination Genome HIV-1 Hematological Disease Hematology Hematopoiesis Hematopoietic Hematopoietic stem cells Hereditary Disease Human Immune System Diseases Immunity Immunodeficient Mouse Inborn Genetic Diseases Inherited Insertional Activations Interferon-beta Lentivirus Vector Longevity Maintenance Matrix Attachment Regions Mediating Mitotic Modification Molecular Motivation National Institute of Diabetes and Digestive and Kidney Diseases Nuclear Matrix Organ Pancytopenia Patients Performance Pharmacology Phenotype Physiological Population Pre-Clinical Model Production Protocols documentation Replication Origin Reporting Research Research Personnel Research Project Grants Residual state Retroviridae Site Stem cells Subfamily lentivirinae System Testing Therapeutic Tissues Transplantation Tropism Vertebral column Viral Virus Xenograft procedure cancer risk cost cytopenia design experience gene delivery system gene therapy genotoxicity in vivo innovation integration site interest mouse model novel pre-clinical public health relevance scaffold self-renewal stem cell biology stem cell population transgene expression translational impact vector vector genome

项目摘要

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（由申请人提供）：使用 HIV-1 衍生的慢病毒载体对自体干细胞进行稳定的遗传修饰可为患有遗传性造血和免疫疾病的患者带来临床益处，将基因传递到不同组织，使前病毒整合体稳定传代。所有后代，以及规模化生产以供临床使用的能力，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，内源基因插入激活的情况继续引起人们的关注。在造血干细胞基因治疗中使用整合慢载体目前的非整合载体可在有丝分裂后组织中提供长期表达，但附加型载体基因组的复制失败会导致分裂干细胞群体的快速损失。新颖的非整合附加型慢病毒载体，通过细胞分裂忠实地复制并表达感兴趣的基因 - 与类似载体不同，这种锚定非整合慢病毒载体。 (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) 位点序列。我们的主要假设是 aNILV 将优化生物安全性并提供对小鼠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），该载体还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。该提案独特地响应了 PAS-13-006：血液学研究新方向 (SHINE-II) [A] 在小鼠模型的生理和药理学选择下展示 aNILV 介导的表型校正、生物安全性和克隆多样性。范可尼贫血 (FA) 相关骨髓衰竭具体目标 1 [B] 追踪 aNILV 转导人类连续移植过程中的分子持久性和多谱系表达。免疫缺陷小鼠中的 CD34 祖细胞 [C] 设计一种微环递送慢病毒载体 (aNILV-II) 并系统地确定其在克隆衍生细胞系和体内的性能该平台有望协调慢病毒载体递送的效率。凭借附加型维持基因组固有的生物安全性，该载体系统将对干细胞基因治疗产生强大的转化影响，并可能显着节省目前与长期相关的成本。患者整合位点监测。