CCR5-mutant monkey model to facilitate the development of novel stem cell-based therapies for AIDS

 描述(申请人提供)：造血干细胞(HSC)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CCR5突变(CCR5-delta32)造血干细胞治愈HIV证明了基于干细胞的治疗在消除潜伏病毒和控制疾病方面的可行性和有效性 艾滋病。然而，确定抗病毒保护的范围、植入将是至关重要的 保护所需的CCR5突变的HSCs的阈值，以及同种异体HSC移植后通过“同种异体效应”耗尽病毒库的可能性，以促进向人类患者的转换。我们建议在威斯康星州国家灵长类研究中心(WNPRC)开发一种MHC定义的CCR5突变的非人类灵长类(NHP)骨髓移植模型，以解决基于HSC的HIV治疗的关键问题。该模型将使用具有明确MHC的毛里求斯食蟹猴(MCM)来控制异基因骨髓移植中的遗传因素。在目标1中，我们将在MCM诱导的多能干细胞(IPSC)系中使用CRISPR/Cas9方法来优化MCM CCR5基因座的有效靶向，并将脱靶效应降至最低。在目标2中，我们将确认CCR5基因组编辑在体外受精来源的MCM胚胎中的可行性和准确性，并产生CCR5突变的MCM。在目的3中，我们将使用清髓性骨髓移植方案评估WT和CCR5突变的HSCs在SHV感染、MHC和血型相合的MCM受者中的植入情况，并评估这些移植对SHIV感染持久性的影响。这项拟议的研究将建立CCR5突变的MHC定义的NHP模型，并将展示对基于HSC的艾滋病治疗的评价的实用性。这些资源的开发将使包括ORIP、NHLBI、NIAID、NIDKK、NICHD和NINDS在内的多个NIH机构的艾滋病研究人员受益。虽然CCR5突变可以预防艾滋病毒感染，但缺乏CCR5也与西尼罗河病毒感染以及其他感染的易感性增加有关，包括弓形虫、结核分枝杆菌、沙眼衣原体、李斯特菌和疟原虫。因此，例如，获得CCR5突变的NHP将为建立西尼罗河感染的NHP模型提供独特的机会，用于评估疫苗的有效性，并促进NHP用于其他感染的研究，这些感染可能需要CCR5突变的宿主复制在易感人类中观察到的疾病过程。此外，NHP模型中基因编辑技术的进步将促进它们的临床翻译，用于治疗人类的遗传疾病。

 DESCRIPTION (provided by applicant): Hematopoietic stem cell (HSC) transplantation has become a standard of care for the treatment of otherwise incurable blood cancers and genetic diseases. The cure of HIV by transplanting CCR5-mutant (CCR5-delta32) HSCs demonstrated the feasibility and power of stem cell-based therapies for eliminating latent virus and controlling AIDS. However, it will be critical to define the spectrum of anti-viral protection, the engraftment threshold of CCR5-mutant HSCs required for protection, and the potential for depletion of the virus reservoir through "allo-effect" following allogeneic HSC transplantation, to facilitate translation to human patients. We propose to develop an MHC-defined CCR5-mutant nonhuman primate (NHP) bone marrow transplantation model at the Wisconsin National Primate Research Center (WNPRC) to address the critical questions of HSC-based therapies for HIV. This model will employ Mauritian Cynomolgus monkeys (MCMs) with well-defined MHC to control genetic factors in the setting of allogeneic bone marrow transplant. In aim 1, we will use CRISPR/Cas9 methods in MCM induced pluripotent stem cell (iPSC) lines to optimize efficient targeting of the MCM CCR5-locus with minimal off-target effects. In aim 2, we will confirm the feasibility and accuracy of CCR5 genomic editing in IVF-derived MCM embryos and generate CCR5-mutant MCMs. In aim 3, we will evaluate the engraftment of WT and CCR5-mutant HSCs in SHIV-infected, MHC and blood group identical MCM recipients using a myeloablative BMT regimen and assess the effect of these transplants on persistence of SHIV infection. The proposed studies will establish a CCR5-mutant MHC- defined NHP model and will demonstrate utility for evaluation of HSC-based therapies for AIDS. Development of these resources will benefit AIDS researchers at multiple NIH institutions, including ORIP, NHLBI, NIAID, NIDKK, NICHD, and NINDS. Although CCR5-mutant mutation protects from HIV infection, the lack of CCR5 is also associated with increased susceptibility to West Nile virus infection, and other infections including Toxoplasma gondii, Mycobacterium tuberculosis, Chlamydia trachomatis, Listeria, and Plasmodium. Thus, for example, access to CCR5-mutant NHPs will open unique opportunities to establish NHP models for West Nile infection for evaluation of vaccine efficacy and advance the use of NHPs for studies of other infections which may require CCR5-mutant hosts to reproduce a disease course observed in susceptible humans. In addition, advances in gene editing technologies in NHP models will facilitate their clinical translation for treatment of genetic disorders in humans.