Modulation of repopulation of anti HIV-1 gene-modified cells to enhance efficacy and safety

项目3：摘要/摘要 项目3的总体目标是通过制定最有效和安全的阳性和阴性选择策略来调节抗HIV-1嵌合抗原受体(CAR)和广谱中和抗体(BNAb)修饰的免疫细胞的水平，以(1)实现治疗水平的再繁殖和(2)加入安全的“杀死开关”，以在发生意想不到的不良反应(如细胞因子风暴、自身免疫反应和恶变)的情况下消除基因工程抗HIV-1免疫效应细胞。基于造血干细胞的基因治疗方法已经显示出实现HIV-1治愈的巨大希望。然而，主要的限制之一是很难达到足够的植入水平来提供治疗效果，特别是对于艾滋病毒-1感染患者，在那里强化清髓条件将是不利的风险收益。因此，一种安全和可滴定的阳性选择策略是非常必要的，以最大限度地提高抗HIV-1基因工程免疫细胞的水平，以治疗HIV-1患者，而不需要危险的密集骨髓清除术。此外，根据从癌症免疫治疗的严重不良反应中吸取的经验教训，采用安全的“杀死开关”程序来消除基因工程抗HIV-1免疫效应细胞，这一点很重要。因此，我们将开发一种负选择策略，作为消除基因工程免疫细胞的安全“杀死开关”。我们将确定最有效、最安全的选择策略，通过(1)通过RNA干扰抑制次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HPRT)的表达，使我们能够通过临床上可获得的前药6-硫代鸟嘌呤或甲氨蝶呤有效地富含或消除抗HIV-1基因修饰的HSPC，(2)共表达截短的无功能的人表皮生长因子受体(HuEGFRt)，一个用于快速体外正选择和体内负选择的细胞表面标志，由FDA批准的抗EGFR单抗西妥昔单抗(Erbitux)和(3)P140K突变形式的人O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMTP140K)进行阳性选择。我们假设，通过严格评估我们为抗HIV-1 CAR和scFv-Fc bNAb联合治疗而提出的选择策略，可以开发出临床上相关、安全和有效的阳性和阴性选择策略，以实现HIV疾病的治愈。

Project 3: Summary/Abstract The overall goal of Project 3 is to modulate the levels of anti-HIV-1 chimeric antigen receptor (CAR) and broadly neutralizing antibodies (bNAb) modified immune cells by developing the most effective and safe positive and negative selection strategy to (1) achieve a therapeutic level of repopulation and (2) incorporate a safety “kill-switch” to eliminate the genetically engineered anti-HIV-1 immune effector cells in cases of unexpected adverse effects, such as cytokine storm, autoimmune reaction and malignant transformation. The hematopoietic stem cell-based gene therapy approach has shown great promise to achieve an HIV-1 cure. However, one of the major limitations has been the difficulty of achieving the engraftment levels sufficient to provide therapeutic efficacy, in particular for HIV-1 infected patients where intensive myeloablative conditionings would be an unfavorable risk-benefit. Thus, a safe and titratable positive selection strategy is highly desirable to maximize the level of anti-HIV-1 gene engineered immune cells to treat patients with HIV-1 without dangerous intensive myeloablation. Furthermore, it is important to incorporate a safety “kill-switch” procedure to eliminate the genetically engineered anti-HIV-1 immune effector cells based on lessons learned from severe adverse effects in cancer immunotherapy. Therefore, we will develop a negative selection strategy as a safety “kill-switch” to eliminate genetically engineered immune cells. We will identify the most effective and safe selection strategy from (1) knocking down hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression using RNA interference that enables us to effectively enrich or eliminate anti-HIV-1 gene-modified HSPC using clinically available prodrug 6-thioguanine or methotrexate, (2) co-expressing truncated non-functional human epidermal growth factor receptor (huEGFRt), a cell surface marker for a rapid ex vivo positive selection and in vivo negative selection by an FDA-approved anti-EGFR monoclonal antibody Cetuximab (Erbitux) and (3) the P140K mutant form of human O6-methylguanine-DNA-methyltransferase (MGMTP140K) for a positive selection. We hypothesize that a clinically relevant, safe and effective positive and negative selection strategy can be developed by rigorously evaluating our proposed selection strategies for our anti-HIV-1 CAR and scFv-Fc bNAb combining therapies to achieve a cure of HIV disease.