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的总体目标是通过开发最有效和最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）和广泛中和抗体（bNAb）修饰的免疫细胞的水平，以（1）实现治疗水平的再增殖和（2）并入安全的“开关”以消除基因工程抗HIV-1嵌合抗原受体（CAR）和广泛中和抗体（bNAb）修饰的免疫细胞。1免疫效应细胞的情况下，意外的不良反应，如细胞因子风暴，自身免疫反应和恶性转化。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，主要的限制之一是难以达到足以提供治疗效果的植入水平，特别是对于HIV-1感染的患者，其中强化清髓性条件治疗将是不利的风险-获益。因此，非常需要一种安全且可滴定的阳性选择策略来最大化抗HIV-1基因工程化免疫细胞的水平，以治疗HIV-1患者而不进行危险的强化骨髓切除。此外，根据从癌症免疫治疗中的严重不良反应中吸取的经验教训，纳入安全的“自动开关”程序以消除基因工程抗HIV-1免疫效应细胞是重要的。因此，我们将开发一种阴性选择策略作为消除基因工程免疫细胞的安全“开关”。我们将从以下方面确定最有效和安全的选择策略：（1）使用RNA干扰敲低次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）表达，使我们能够使用临床可用的前药6-硫鸟嘌呤或甲氨蝶呤有效富集或消除抗HIV-1基因修饰的HSPC，（2）共表达截短的非功能性人表皮生长因子受体（huEGFRt），用于通过FDA批准的抗EGFR单克隆抗体西妥昔单抗（爱必妥）进行快速离体阳性选择和体内阴性选择的细胞表面标记物，和（3）用于阳性选择的人O 6-甲基鸟嘌呤-DNA-甲基转移酶（MGMTP 140 K）的P140 K突变形式。我们假设，通过严格评估我们提出的抗HIV-1 CAR和scFv-Fc bNAb联合疗法的选择策略，可以开发出临床相关、安全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.