Suicide of HIV-Infected Cells by TAT-Inducible shRNA

描述（由申请人提供）：拟议项目的目标是生成必要的临床前数据，为使用小发夹RNA（shRNA）靶向“HIV相关生命保护因子”（HALP）和环指蛋白7（RNF 7）（获得性HIV感染中的2种关键抗凋亡基因）的促凋亡慢病毒载体（LV）基因治疗的未来临床研究提供原理验证。艾滋病毒/艾滋病是一种理想的候选人，一种新的基因治疗方法，因为它是一种不治之症和绝症。尽管高效抗逆转录病毒疗法显著提高了艾滋病毒感染者的存活率，但由于耐药病毒的发展、长期毒性和大量潜伏感染的T细胞库，反应的持续时间受到限制。潜伏性静息记忆CD 4 T细胞的长半衰期使得HIV本质上无法用目前可用的治疗方案治愈。因此，迫切需要探索新的分子和遗传策略来减缓或阻止HIV-1复制并防止病毒潜伏。我们假设抗凋亡基因如HALP和RNF 7是HIV感染细胞存活和潜伏感染的主要因素之一。这些细胞对HAART疗法有抗性。我们预测，通过沉默这些基因并诱导细胞凋亡，感染的细胞将被杀死，病毒载量和潜伏感染将减少。在STTR应用的第一阶段，我们将通过3个目标来测试我们的假设。在目标1中，我们将产生LV载体，其表达针对抗凋亡基因产物HALP和RNF 7的可诱导短发夹RNA（shRNA），但仅在HIV-1感染的细胞中可诱导。这些LV载体将易于扩展以用于以后的临床使用。在进行质量控制后，将使用原代T细胞和造血祖细胞在转导方案中测试载体。在目标2中，我们将测试针对HALP和RNF 7抗凋亡基因产物的TAT诱导型shRNA是否抑制HIV-1感染细胞中这些基因的表达，从而导致感染细胞死亡和病毒载量降低以及潜伏感染。在目标3中，我们将用慢病毒构建体转染CD 4 T细胞和脐带血CD 34+细胞，将转导的细胞过继转移到SCID/NOD小鼠，并评估移植和体内载体持久性。所提出的实验的结果将作为这种方法的未来临床研究的基础，作为一种新的治疗方法，重点是杀死感染和潜伏细胞，并在HIV/AIDS感染过程中诱导降低病毒载量。该提案的重点是开发一种新的艾滋病毒/艾滋病免疫治疗策略。全世界约有4 000万人感染艾滋病毒;仅美国就有100万人。这些数字已经达到了流行病的程度。三联鸡尾酒药物疗法（HAART）曾被认为在有效对抗艾滋病病毒方面有很大的希望，但尚未被证明是治愈艾滋病的方法。此外，对这种药物疗法的耐药性正在增加;因此，药物疗法显然不是控制艾滋病流行病的解决办法。因此，最终需要新的疗法。在这里，我们建议开发新的治疗方法，重点是沉默HIV感染细胞中的2个基因，这些基因在过度活跃时会导致HIV感染的延长和药物无法杀死感染细胞。

DESCRIPTION (provided by applicant): The goal of the proposed project is to generate the pre-clinical data necessary to provide proof-of-principle for future clinical studies of pro-apoptotic lentiviral vector (LV) gene therapy using small hairpin RNA (shRNA) to target "HIV-Associated Life Preserver" (HALP) and ring finger protein 7 (RNF7), 2 of the crucial anti-apoptotic genes in acquired HIV infection. HIV/AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease. Although highly active anti-retroviral therapy has significantly improved the survival of HIV-infected individuals, the duration of response is limited by development of drug-resistant viruses, long-term toxicities and a substantial reservoir of latently infected T-cells. The long half-life of latent resting memory CD4 T-cells renders HIV intrinsically incurable with the treatment regimens currently available. Therefore, there is an urgent need to explore novel molecular and genetic strategies to slow or halt HIV-1 replication and to prevent viral latency. We hypothesize that anti-apoptotic genes like HALP and RNF7 are among the major contributors to survival of HIV-infected cells and latent infection. These cells are resistant to HAART therapy. We predict that by silencing these genes and inducing apoptosis, infected cells will be killed, and viral load and latent infection will decrease. During Phase I of this STTR application we will test our hypothesis through 3 aims. In Aim 1, we will generate LV vectors expressing an inducible short hairpin RNA (shRNA), directed against anti-apoptotic gene products HALP and RNF7, but inducible only in HIV-1 infected cells. These LV vectors will be easily scalable for later clinical use. After performing quality control, vectors will be tested in transduction protocols using primary T-cells and hematopoietic progenitor cells. In Aim 2 we will test whether TAT-inducible shRNAs, directed against HALP and RNF7 anti-apoptotic gene products, inhibit expression of these genes in HIV-1 infected cells, leading to death of infected cells and decreased viral load and latent infection. In Aim 3, we will transduce CD4 T-cells and cord blood CD34+ cells with the lentiviral constructs, adoptively transfer the transduced cells to SCID/NOD mice, and assess engraftment and in vivo vector persistence. The results of the proposed experiments will serve as a basis for future clinical investigation of this approach as a novel therapy focused on killing of infected and latent cells, and induction of decreased viral load during HIV/AIDS infection. This proposal focuses upon developing a novel immunotherapy strategy for HIV/AIDS. Approximately 40 million people worldwide are infected with HIV; 1 million people in the United States alone. The numbers have reached epidemic proportions. Triple cocktail drug therapy (HAART), once believed to have great promise in effectively combating the AIDS virus, has not proven to be a cure for AIDS. Moreover, resistance to this drug therapy is increasing; and thus it has become apparent that drug therapy is not the solution for controlling an AIDS epidemic. Therefore, there is an ultimate need for novel therapies. Here we propose to develop novel therapy that is focused on silencing 2 genes in HIV-infected cells which when overactive contribute to prolongation of HIV infection and inability of drugs to kill infected cells.