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

 描述（由申请人提供）：造血干细胞（HSC）移植已成为治疗无法治愈的血癌和遗传性疾病的护理标准。通过移植 CCR5 突变 (CCR5-delta32) HSC 来治愈 HIV 证明了基于干细胞的疗法消除潜伏病毒和控制病毒的可行性和功效 艾滋病。然而，定义抗病毒保护的范围、植入病毒的范围至关重要。 保护所需的 CCR5 突变 HSC 的阈值，以及同种异体 HSC 移植后通过“同种异体效应”耗尽病毒库的潜力，以促进转化为人类患者。我们建议在威斯康星国家灵长类动物研究中心 (WNPRC) 开发 MHC 定义的 CCR5 突变非人灵长类动物 (NHP) 骨髓移植模型，以解决基于 HSC 的 HIV 疗法的关键问题。该模型将采用具有明确 MHC 的毛里求斯食蟹猴 (MCM) 来控制同种异体骨髓移植中的遗传因素。在目标 1 中，我们将在 MCM 诱导多能干细胞 (iPSC) 系中使用 CRISPR/Cas9 方法，以优化 MCM CCR5 基因座的有效靶向，同时将脱靶效应降至最低。在目标 2 中，我们将确认在 IVF 衍生的 MCM 胚胎中进行 CCR5 基因组编辑的可行性和准确性，并生成 CCR5 突变 MCM。在目标 3 中，我们将使用清髓性 BMT 方案评估 WT 和 CCR5 突变型 HSC 在 SHIV 感染、MHC 和血型相同的 MCM 受者中的植入情况，并评估这些移植对 SHIV 感染持续存在的影响。拟议的研究将建立一个 CCR5 突变型 MHC 定义的 NHP 模型，并将展示用于评估基于 HSC 的艾滋病疗法的实用性。这些资源的开发将使多个 NIH 机构的艾滋病研究人员受益，包括 ORIP、NHLBI、NIAID、NIDKK、NICHD 和 NINDS。尽管 CCR5 突变可以防止 HIV 感染，但 CCR5 的缺乏也与西尼罗河病毒感染以及其他感染（包括弓形虫、结核分枝杆菌、沙眼衣原体、李斯特菌和疟原虫）的易感性增加有关。因此，例如，获得 CCR5 突变 NHP 将为建立西尼罗河感染 NHP 模型以评估疫苗功效提供独特的机会，并促进 NHP 用于其他感染的研究，这些感染可能需要 CCR5 突变宿主重现在易感人群中观察到的疾病过程。此外，NHP模型基因编辑技术的进步将促进其临床转化，用于治疗人类遗传性疾病。

 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.