iPSC-based blood regenerative therapies for AIDS

基于 iPSC 的艾滋病血液再生疗法

• 批准号: 8708198
• 负责人: Igor I. Slukvin
• 金额: $ 62.78万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708198
• 关键词: AIDS therapy; Acquired Immunodeficiency Syndrome; Adult; Alleles; Antiviral Agents; Autologous; B-Lymphocytes; Basic Science; Benefits and Risks; Biological; Blood; Blood Cells; Bone Marrow Purging; CCR5 gene; CD34 gene; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Therapy; Cells; Cellular biology; Clinic; Clinical; Collaborations; Cyclic GMP; Cytoprotection; Development; Endothelium; Engraftment; Evaluation; Frequencies; Future; Generations; Genes; Genetic; Genomics; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hereditary Disease; Human; IL2RA gene; Immune System Diseases; Immune system; In Vitro; Individual; Knock-out; Latent Virus; Liver; Mediating; Methodology; Modification; Molecular; Molecular Profiling; Mus; Mutation; Myeloid Cells; National Heart, Lung, and Blood Institute; Null Lymphocytes; Patients; Population; Production; Protocols documentation; Regulatory Pathway; Research; Resistance; Safety; Somatic Cell; Source; Staging; Stem cells; Stromal Cells; T-Cell Development; T-Lymphocyte; Technology; Testing; Therapeutic; Thymus Gland; Time; Translations; Transplantation; Viral; base; cancer genetics; clinical application; clinically relevant; fetal; hematopoietic repopulating cell; homologous recombination; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; leukemia; mouse model; mutant; pluripotency; public health relevance; reconstitution; regenerative; regenerative therapy; standard of care

DESCRIPTION (provided by applicant): HSC transplantations have become a standard of care for treating otherwise incurable blood cancers and genetic diseases. The curing of HIV and leukemia by transplanting HSCs from HIV-resistant patients with a CCR5-D32 mutation has demonstrated the power of stem cell-based therapies for AIDS. However, difficulties in the genetic modifications of autologous HSCs and in finding HLA-compatible CCR5-D32 donors significantly hamper the widespread use of somatic HSC-based AIDS therapies in the clinical setting. Converting adult human cells to induced pluripotent stem cells (iPSCs) provides a unique opportunity to produce immunologically matched gene-edited therapeutic cells for diseases of the blood and immune system as iPSCs can be expanded indefinitely ex vivo, genetically modified using homologous recombination and differentiated into hematopoietic cells. However, transferring this approach to the clinic requires the improvement of iPSC- derived blood cell engraftment, development of robust cGMP-compatible protocols for blood production from iPSCs, and the bi-allelic CCR5 disruption to provide an anti-HIV effect. The proposed studies capitalize on our recent advances in identification of pre-HSC hemogenic endothelium (HE) stage in human ESC/iPSC cultures and progress in locus-specific gene editing in ESC/iPSCs using ZNF-mediated homologous recombination. The three related specific aims are directed at understanding the molecular mechanisms controlling development of HSCs from human PSCs through the HE stage, with the ultimate goal to develop clinically- relevant protocols for ex vivo production of CCR5-knockout autologous HSCs for AIDS therapies. In aim 1, we will identify the biological regulators guiding the formation of engraftabl hematopoietic cells from HE with a goal to improve production of blood cells with regenerative potential from human PSCs. In aim 2, we will develop homologous recombination-based technology for the bi-allelic CCR5 knockout in iPSCs and test the engraftability and safety of genetically corrected iPSC-derived blood cells following transplantation in NOD/SCD/IL2Rg-/- (NSG) mice. In aim 3, we will test whether iPSC-derived CCR5-null cells are protected from HIV-1 challenge in NSG mice. Successful completion of the studies will validate a methodology for generation of regenerative blood cells from iPSCs and their potential use for HIV therapies. The applications of the methodology proposed here will be also useful for basic research and for future clinical applications for modification of any genomic target in iPSCs.

描述（由申请人提供）：HSC 移植已成为治疗无法治愈的血癌和遗传性疾病的标准治疗方法。通过移植具有 CCR5-D32 突变的 HIV 耐药患者的 HSC 来治愈 HIV 和白血病，证明了干细胞疗法治疗艾滋病的功效。然而，自体 HSC 的基因修饰和寻找 HLA 相容的 CCR5-D32 供体方面的困难极大地阻碍了基于体细胞 HSC 的艾滋病疗法在临床环境中的广泛使用。将成人细胞转化为诱导多能干细胞 (iPSC) 为生产用于血液和免疫系统疾病的免疫匹配基因编辑治疗细胞提供了独特的机会，因为 iPSC 可以在离体无限期扩增、使用同源重组进行基因修饰并分化为造血细胞。然而，将该方法转移到临床需要改进 iPSC 衍生的血细胞植入，开发用于 iPSC 血液生产的强大的 cGMP 兼容方案，以及破坏双等位基因 CCR5 以提供抗 HIV 效果。拟议的研究利用了我们在人类 ESC/iPSC 培养物中鉴定 HSC 前造血内皮 (HE) 阶段的最新进展，以及使用 ZNF 介导的同源重组在 ESC/iPSC 中进行位点特异性基因编辑的进展。这三个相关的具体目标旨在了解控制人类 PSC 至 HE 阶段 HSC 发育的分子机制，最终目标是开发用于离体生产 CCR5 敲除自体 HSC 的临床相关方案，用于艾滋病治疗。在目标 1 中，我们将确定引导 HE 形成可移植造血细胞的生物调节因子，目的是提高人类 PSC 产生具有再生潜力的血细胞。在目标 2 中，我们将开发基于同源重组的 iPSC 双等位基因 CCR5 敲除技术，并测试基因校正的 iPSC 衍生血细胞移植到 NOD/SCD/IL2Rg-/- (NSG) 小鼠后的可移植性和安全性。在目标 3 中，我们将测试 NSG 小鼠中 iPSC 衍生的 CCR5 缺失细胞是否能够免受 HIV-1 攻击。研究的成功完成将验证从 iPSC 生成再生血细胞的方法及其在 HIV 治疗中的潜在用途。这里提出的方法的应用也将适用于基础研究和未来修饰 iPSC 中任何基因组靶标的临床应用。