Combination Therapy Using CRISPR/Cas Gene Editing Plus Human Monoclonal Antibodies for a Functional HIV Cure

 描述(由申请人提供) 艾滋病毒感染者体内潜伏蓄水池的持续存在是根除这种疾病的主要障碍。虽然联合抗逆转录病毒疗法(CART)在抑制潜伏库的病毒生产方面非常有效，但CART的退出会导致病毒的快速反弹，这是由潜伏库中的病毒转录重新激活驱动的。在这 在这个项目中，我们将开发一种多靶点疗法，我们预计这种疗法将在没有CART的情况下显著降低甚至潜在地消除HIV-1感染靶细胞的能力。这种联合疗法将针对艾滋病毒感染的不同关键方面，在没有CART的情况下，每一方面都会导致持续性病毒血症。这些成分包括病毒颗粒、病毒产生细胞、靶细胞上表达的CCR5共受体和整合的前病毒。为了清除病毒反弹期间出现的艾滋病毒和产生艾滋病毒的细胞，我们设计了一组抗艾滋病毒的单抗。这些抗体与HIV-1上的各种表位结合，并已被修饰以表达功能增强的Fc结构域。这些改变的Fc结构域促进了髓系细胞上Fc受体的功能，如抗体依赖的细胞毒性(ADCC)、吞噬作用和补体募集。我们已经开发了靶向并切割CCR5基因的CRISPR/Cas结构。CD34+造血干细胞(HSC)群体中CCR5基因的裂解将导致CCR5阴性免疫后代的分化和增殖，从而抵抗R5嗜性HIV-1感染。此外，分化的白细胞中CCR5基因的裂解也会导致对HIV-1的抵抗。我们已经开发了CRISPR/Cas基因编辑结构来切割整合的前病毒序列，从而使转导这些基因的感染细胞无法转录完整的病毒基因组。为了传递这些CRISPR/Cas转基因基因，我们开发了慢病毒载体，我们将用不同的病毒包膜蛋白和抗受体抗体来伪型，将慢病毒导向特定的靶细胞。由LV传递的CRISPR基因稳定地整合到靶细胞基因组中，从而使转导的细胞及其后代永久免受艾滋病毒感染。通常，这些方法中的每一种都将独立提出，并根据没有治疗的情况来衡量疗效。然而，这项应用的一个关键创新是将这些方法结合到一个治疗方案中。在前三个目标中，我们将开发和选择这些方法中的每一种最有效的试剂，使用体外细胞分析和体内评估相结合的方法在感染HIV的人源化小鼠(HU-小鼠)中。在目标4中，我们将结合这些疗法，并在HIV感染的人-鼠模型中测试它们，并确定它们在CART治疗中断后抑制和延迟病毒产生的能力。我们预计，这些研究不仅将开发出可用于临床的试剂，这些试剂将在临床前动物模型中经过严格的测试和验证，而且我们还将证明，与联合抗逆转录病毒疗法一样，联合靶向治疗在减少艾滋病毒患者的病毒负担和发展抗艾滋病毒免疫系统方面比单一疗法更有效。这项工作汇集了Susan Eszterhas博士和George O‘Toole博士在CRISPR基因靶向结构设计方面的专业知识，Bryan Luikart博士在体内主要细胞靶向LV载体开发方面的专业知识，玛格丽特·阿克曼博士在治疗开发抗体优化方面的专业知识，以及Dorothy Wallace博士在人类疾病数学建模方面的专业知识。

 DESCRIPTION (provided by applicant) The persistence of the latent reservoir in HIV-infected patients is a major barrier to the eradication of this disease. Although combination anti-retroviral therapy (cART) is highly effective at suppressing viral production from the latent reservoir, cART withdrawal leads to a rapid viral rebound driven by the reactivation of viral transcription from the latent pool. In this project, we will develop a multi-targeted therapy that we expect will significantly reduce and potentially eliminate the ability of HIV-1 to infect target cells in the absence of cART. This combination therapeutic will target different key aspects of HIV infection, each of which contributes to sustained viremia in the absence of cART. These components include viral particles, virus producing cells, the CCR5 co-receptor expressed on target cells, and the integrated provirus. To clear HIV and HIV-producing cells that emerge during viral rebound, we have engineered a panel of anti-HIV monoclonal antibodies. These antibodies bind to various epitopes on HIV- 1 and have been modified to express functionally enhanced Fc domains. These altered Fc domains promote Fc receptor functions on myeloid cells such as antibody dependent cellular cytotoxicity (ADCC), phagocytosis, and complement recruitment. We have developed CRISPR/Cas constructs that target and cleave the CCR5 gene. CCR5 gene cleavage in the CD34+ hematopoietic stem cell (HSC) population will lead to the differentiation and proliferation of CCR5-negative immune progeny that are resistant to infection with R5-tropic HIV-1. Moreover, CCR5 gene cleavage in differentiated leukocytes will also result in HIV-1 resistance. We have developed CRISPR/Cas gene editing constructs to cleave the integrated proviral sequence, so that infected cells transduced with these genes are unable to transcribe intact viral genomes. To deliver these CRISPR/Cas transgenes, we have developed lentiviral (LV) vectors that we will pseudotype with different viral envelope proteins and anti-receptor antibodies to direct LV to specific target cells. CRISPR genes delivered by LV are stably integrated into the target cell genome so that the transduced cells and their progeny are permanently protected from HIV infection. Typically, each of these approaches would be proposed independently, and efficacy measured against the absence of treatment. However, a key innovation of this application is to combine these approaches into one therapeutic regimen. In the first 3 aims, we will develop and select the most efficacious reagents for each of these approaches using a combination of in vitro cellular assays and in vivo assessments in the HIV-infected humanized mouse (hu-mouse). In Aim 4, we will combine the therapeutics and test them in the HIV-infected hu-mouse model, and determine their ability to suppress and delay viral production following interruption of cART treatment. We expect that these studies will not only develop clinic-ready reagents that will have undergone rigorous testing and validation in a pre-clinical animal model, but we will also demonstrate that combination targeting, like combination anti-retroviral therapy, is more effective than a single therapeutic in reducing the viral burden in HIV patients and in developing an HIV-resistant immune system. This work brings together the combined expertise of Drs. Susan Eszterhas and George O'Toole in the design of CRISPR gene targeting constructs, Dr. Bryan Luikart in the development of LV vectors for primary cell targeting in vivo, Dr. Margaret Ackerman in the optimization of antibodies for therapeutic development, and Dr. Dorothy Wallace in mathematical modeling of human diseases.