Suicide of HIV-Infected Cells by TAT-Inducible shRNA

描述(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相关生命保护器”(HALP)和RNF7(获得性HIV感染中的两个关键抗凋亡基因)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证明。艾滋病毒/艾滋病是一种不治之症，是一种新型基因治疗方法的理想候选者。虽然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，但由于抗药性病毒的发展、长期毒性和大量潜伏感染的T细胞的储存，应答持续时间受到限制。潜伏的静止记忆CD4T细胞的长半衰期使艾滋病毒在现有的治疗方案下本质上是无法治愈的。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的复制，并防止病毒潜伏。我们假设像HALP和RNF7这样的抗凋亡基因是HIV感染细胞存活和潜伏感染的主要贡献者。这些细胞对HAART治疗有抵抗力。我们预测，通过沉默这些基因并诱导细胞凋亡，被感染的细胞将被杀死，病毒载量和潜伏感染将减少。在此STTR应用的第一阶段，我们将通过3个目标来检验我们的假设。在目标1中，我们将构建表达针对抗凋亡基因产物HALP和RNF7的可诱导短发夹状RNA(ShRNA)的LV载体，但仅在HIV-1感染的细胞中诱导。这些LV载体将很容易扩展到以后的临床使用。在进行质量控制后，载体将在使用原始T细胞和造血祖细胞的转导方案中进行测试。在目标2中，我们将测试针对HALP和RNF7抗凋亡基因产物的TAT诱导shRNAs是否抑制HIV-1感染细胞中这些基因的表达，导致感染细胞死亡，降低病毒载量和潜伏感染。在目标3中，我们将用慢病毒载体转导CD4T细胞和脐血CD34+细胞，过继地将转导的细胞转移到SCID/NOD小鼠，并评估植入和体内载体的持久性。拟议的实验结果将作为未来临床研究的基础，该方法将作为一种新的治疗方法，专注于杀死感染细胞和潜伏细胞，并在艾滋病毒/艾滋病感染期间诱导病毒载量下降。这项提议的重点是开发一种新的艾滋病毒/艾滋病免疫治疗战略。全世界约有4000万人感染艾滋病毒；仅美国就有100万人。这一数字已经达到了流行的程度。三重鸡尾酒药物疗法(HAART)曾被认为在有效对抗艾滋病病毒方面有很大的前景，但事实证明它并不是治愈艾滋病的方法。此外，对这种药物疗法的抗药性正在增加；因此，药物疗法显然不是控制艾滋病流行的解决方案。因此，最终需要新的治疗方法。在这里，我们建议开发新的治疗方法，专注于沉默HIV感染细胞中的2个基因，当这些基因过度活跃时，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.