iPSC-based blood regenerative therapies for AIDS

描述(申请人提供)：造血干细胞移植已经成为治疗其他无法治愈的血癌和遗传性疾病的标准护理。通过移植具有CCR5-D32突变的艾滋病毒耐药患者的造血干细胞来治愈艾滋病毒和白血病，证明了基于干细胞的艾滋病疗法的力量。然而，在自体造血干细胞的遗传修饰和寻找与人类白细胞抗原相容的CCR5-D32捐赠者方面的困难大大阻碍了基于体细胞的艾滋病治疗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(IPSCs)为治疗血液和免疫系统疾病提供了一个独特的机会，即产生免疫匹配的基因编辑的治疗细胞，因为IPSCs可以在体外无限扩增，使用同源重组进行基因修饰，并分化为造血细胞。然而，将这种方法转移到临床需要改进IPSC来源的血细胞植入，开发强大的兼容cGMP的IPSCs血液生产方案，以及提供抗HIV效果的双等位基因CCR5中断。这些研究利用了我们最近在鉴定人ESC/IPSC培养中HSC前血源性内皮(HE)阶段的进展，以及利用ZNF介导的同源重组在ESC/IPSCs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从人PSCs到HE阶段控制HSCs发育的分子机制，最终目标是开发临床相关的方案，用于体外生产用于艾滋病治疗的CCR5基因敲除的自体HSCs。在目标1中，我们将确定引导从HE形成可植入造血细胞的生物调节因素，以促进从人PSCs产生具有再生能力的血细胞。在目标2中，我们将开发基于同源重组的技术来敲除IPSCs中的双等位CCR5，并测试经过基因校正的IPSC来源的血细胞在NOD/SCD/IL2RG-/-(NSG)小鼠身上移植后的可植入性和安全性。在目标3中，我们将在NSG小鼠中测试IPSC来源的CCR5缺失细胞是否受到HIV-1攻击的保护。这些研究的成功完成将验证一种从IPSCs产生再生血细胞的方法及其在艾滋病毒治疗中的潜在用途。这里提出的方法学的应用也将对基础研究和未来用于修饰IPSCs中任何基因组靶点的临床应用有用。

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.