Anti-HIV Gene Therapy: Defend and Attack

 描述（由申请人提供）：本U19项目中待检验的假设是，将具有不同作用模式的基因修饰试剂组合使用，将对HIV-1疾病产生显著影响，并有可能实现治愈。我们将在抗HIV-1基因疗法的丰富经验的基础上，扩大我们的知识和开发新技术，这将导致基于慢病毒载体的抗HIV-1治疗开发候选药物。计划开发2种载体，一种用于HSPC，一种用于T细胞移植，并在资助期结束前提交I期临床试验IND。已经在临床上测试的用于HIV-1疾病的少数基于基因的疗法主要通过消融或减少CCR 5表达来保护分化的子代T细胞和巨噬细胞。在“柏林患者”的单一显著病例中，CCR 5 β 32供体细胞的同种异体移植导致了功能性治愈，而没有残留HIV-1的证据。通过移植基因工程细胞来模拟这种CCR 5消融的努力已经取得了一些成功，但存在一些障碍，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功效的植入水平。我们建议通过测试方法来解决这个基本问题，以选择性地富集基因修饰的造血干/祖细胞（HSPC）的再增殖，使用移植细胞的遗传选择。第二个主要问题，也是所有HIV-1疗法都面临的问题，是HIV-1产生耐药性。与HIV-1疾病的小分子疗法的发展一样，基因疗法也需要有效的组合。因此，我们的合作伙伴，Calimmune公司，目前正在人类中测试使用CCR 5敲低（shRNA 1005）与跨膜融合抑制剂（C46）组合的T细胞和HSPC基因疗法。在这里，我们建议添加第三种试剂，一种识别HIV-1感染细胞的嵌合抗原受体（CAR），通过过继性T细胞免疫疗法递送。在早期人类研究中，已经证明使用肿瘤特异性汽车的T细胞免疫疗法对癌症有效。虽然CAR几年前在人类中进行了HIV-1疾病的测试，并发现是安全的，但它受到了一些限制，现在更好地理解，并在这里得到解决。最后，HSPC和T细胞移植是复杂的生物学过程，需要彻底了解数千种功能多样的干细胞、祖细胞或成熟细胞的再增殖。每个项目负责人都有丰富的经验，不仅与艾滋病毒-1，而且在一般干细胞生物学及其应用于艾滋病毒-1疾病。专业知识的范围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），HSPC行为的理解（Chen）到I期和II期临床试验的实施（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).