iPSC-based blood regenerative therapies for AIDS

描述（由申请人提供）：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疾病的标准护理。通过移植来自CCR5-D32突变的HIV耐药患者的造血干细胞来治愈HIV和白血病，已经证明了干细胞治疗艾滋病的力量。然而，在自体造血干细胞的遗传修饰和寻找hla兼容的CCR5-D32供体方面的困难极大地阻碍了基于体细胞造血干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）提供了一个独特的机会，可以产生免疫匹配的基因编辑治疗细胞，用于血液和免疫系统疾病，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衍生的血细胞植入，开发强大的cgmp兼容的iPSC血液生产方案，以及双等位基因CCR5破坏以提供抗hiv效果。拟建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，以及利用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。这三个相关的特定目的是为了了解通过HE阶段控制人类PSCs发育成造血干细胞的分子机制，最终目标是开发出用于艾滋病治疗的体外生产ccr5敲除的自体造血干细胞的临床相关方案。在目标1中，我们将确定引导HE可移植造血细胞形成的生物调节剂，目的是提高人类psc具有再生潜力的血细胞的产生。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，并在NOD/SCD/IL2Rg-/- (NSG)小鼠移植后测试遗传校正的ipsc来源的血细胞的可移植性和安全性。在目的3中，我们将测试ipsc衍生的无ccr5细胞是否能保护NSG小鼠免受HIV-1攻击。这些研究的成功完成将验证从多能干细胞中产生再生血细胞的方法及其在艾滋病治疗中的潜在应用。本文提出的方法的应用也将有助于基础研究和未来的临床应用，以修饰iPSCs中的任何基因组靶点。

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.