Anti-HIV Gene Therapy: Defend and Attack

 描述（由申请人提供）：本 U19 计划要测试的假设是，将基因修饰试剂与不同的作用方式相结合将对 HIV-1 疾病产生重大影响，并有可能实现治愈。我们将利用我们之前在抗 HIV-1 基因疗法方面的丰富经验，扩大我们的知识并开发新技术，从而开发出基于慢病毒载体的抗 HIV-1 治疗候选药物。该计划是开发 2 种载体，一种用于 HSPC，一种用于 T 细胞移植，并在资助期限结束前提交 I 期临床试验的 IND 申请。已经在临床测试的少数基于基因的 HIV-1 疾病疗法主要通过消除或减少 CCR5 表达来保护分化的后代 T 细胞和巨噬细胞。在“柏林患者”这一著名案例中，CCR532 供体细胞的同种异体移植实现了功能性治愈，且没有残留 HIV-1 的证据。通过移植基因工程细胞来模拟这种 CCR5 消除的努力已经取得了一些成功，但也遇到了一些障碍，我们将在本提案中解决这些障碍。首先，干细胞移植的普遍限制是难以达到足以提供治疗效果的植入水平。我们建议通过测试使用移植细胞的遗传选择来选择性富集基因修饰的造血干/祖细胞（HSPC）再增殖的方法来解决这一基本问题。所有 HIV-1 疗法都面临的第二个主要问题是 HIV-1 产生耐药性。与针对 HIV-1 疾病的小分子疗法的开发一样，基因疗法也需要有效的组合。因此，我们的企业合作伙伴 Calimmune, Inc. 目前正在人体、T 细胞和 HSPC 基因疗法中测试使用 CCR5 敲低 (shRNA1005) 与跨膜融合抑制剂 (C46) 相结合的疗法。在这里，我们建议添加第三种试剂，即识别 HIV-1 感染细胞的嵌合抗原受体 (CAR)，通过过继性 T 细胞免疫疗法传递。早期人类研究已证明，使用肿瘤特异性 CAR 的 T 细胞免疫疗法可有效对抗癌症。虽然 CAR 多年前在人体中针对 HIV-1 疾病进行了测试，并被证明是安全的，但它仍存在许多局限性，现在人们对此有了更深入的了解，并在此予以解决。最后，HSPC 和 T 细胞移植是复杂的生物过程，需要彻底了解数千个功能多样的干细胞、祖细胞或成熟细胞的再增殖。每个项目负责人不仅在 HIV-1 方面拥有丰富的工作经验，而且在一般干细胞生物学及其在 HIV-1 疾病中的应用方面也拥有丰富的经验。专业知识范围广泛，从载体和转基因开发（Chen、An、Kitchen、Symonds）、HSPC 生物学动物模型的开发和使用（Kitchen、An、Chen）、抗 HIV-1 免疫功能（Yang、Kitchen）、对 HSPC 行为的理解（Chen）到 I 期和 II 期临床试验实施（Symonds）。

 DESCRIPTION (provided by applicant): The hypothesis to be tested in this U19 program is that combining gene modifying reagents with different modes of action will have a significant impact on HIV-1 disease with the possibility of achieving a cure. We will build upon our previous extensive experience in anti-HIV-1 genetic therapies to both broaden our knowledge and develop new technologies that will result in lentiviral vector based anti-HIV-1 therapeutic development candidate(s). The plan is to develop 2 vectors, one for HSPC and one for T-cell transplant and file an IND for a Phase I clinical trial by the end of the grant term. The few gene-based therapies for HIV-1 disease that have been tested in the clinic have been focused on protecting the differentiated progeny T-cells and macrophages, principally through ablation or reduction of CCR5 expression. In the single remarkable case of the "Berlin patient", allogeneic transplant of CCR532 donor cells resulted in a functional cure without evidence for remaining HIV-1. Efforts to mimic this CCR5 ablation through transplant of gene-engineered cells has shown some success, but suffers from several roadblocks which we will address in this proposal. First, a universal limitation in stem cell transplant is the difficulty of achieving engraftment levels sufficient to provide therapeutic efficacy. We propose to address this fundamental issue by testing approaches to selectively enrich for repopulation of gene-modified hematopoietic stem/progenitor cells (HSPC) using genetic selection for engrafted cells. A second major issue, one faced by all HIV-1 therapies, is the development of resistance by HIV-1. As with the development of small molecule therapies for HIV-1 disease, gene therapies will also require effective combinations. As such, our corporate partner, Calimmune, Inc., is currently testing in humans, T-cell and HSPC genetic therapy using CCR5 knockdown (shRNA1005) combined with a transmembrane fusion inhibitor (C46). Here, we propose to add a third reagent, a chimeric antigen receptor (CAR) recognizing HIV-1 infected cells, delivered by adoptive T-cell immunotherapy. T-cell immunotherapy with tumor specific CARs has proven to be effective against cancer in early human studies. While a CAR was tested years ago in humans for HIV-1 disease and found to be safe, it suffered from a number of limitations, now better understood, and to be addressed here. Finally, HSPC and T-cell transplants are complex biological processes that require a thorough understanding of repopulation by thousands of functionally diverse stem, progenitor, or mature cells. Each of the project leaders has had extensive experience working not only with HIV-1, but also in general stem cell biology and its applications to HIV-1 disease. The breadth of expertise ranges from vector and transgene development (Chen, An, Kitchen, Symonds), development and use of animal models for HSPC biology (Kitchen, An, Chen), anti-HIV-1 immune function (Yang, Kitchen), understanding of HSPC behavior (Chen) to Phase I and II clinical trial implementation (Symonds).