Improved Zinc Finger Nuclease Delivery for HIV Gene Therapy

改进的锌指核酸酶递送用于 HIV 基因治疗

• 批准号: 9098594
• 负责人: Guohua Yi
• 金额: $ 7.65万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-26 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9098594
• 关键词: Adenovirus Vector; Antibodies; Binding; CCR5 gene; CD4 Positive T Lymphocytes; Cells; Conserved Sequence; Effectiveness; Engineering; Fc domain; Genes; Glycoproteins; Goals; HIV; HIV therapy; HIV-1; Health; Histone Deacetylase Inhibitor; Immune system; In Vitro; Individual; Infection; Infusion procedures; Lentivirus Vector; Mediating; Methods; Mus; Patients; Procedures; Protein Binding Domain; Proviruses; Reagent; Receptor Gene; Regimen; Reporting; Research; Rest; Sindbis Virus; Source; Subfamily lentivirinae; T-Lymphocyte; Testing; Viral reservoir; Virus; base; gene therapy; improved; in vivo; memory CD4 T lymphocyte; mouse model; protective efficacy; receptor; reconstitution; virus envelope; zinc finger nuclease

 DESCRIPTION (provided by applicant): Zinc finger nucleases (ZFN)-mediated CCR5 disruption has shown to be a promising strategy for HIV gene therapy, but the latent viral reservoir still remains the major obstacle towards sterilizing or even a functional cure for HIV-1 infection. Resting CD4+ memory T cells are thought to be the major source of latency, and these also constitute a particularly stable reservoir of virus. ZFN targeting the conserved sequences in the 3' and the 5' LTR was reported to precisely excise the entire provirus in J-Lat cells, but the difficulty in delivering ZFNs to resting primary CD4+ T cells limited the potential f this approach for latency reduction. Recently, we have developed a method by using HIV env pseudotyping non-integrating lentivirus, successfully delivered CCR5-ZFN to resting CD4+ T cells and showed effectiveness in vivo in Hu-PBL mice. We have also found in preliminary studies that using Sindbis virus glycoprotein engineered to express ZZ-domain (Fc binding domain from protein A) provides another easy way to target resting CD4+ T cells. Here the pseudotyped lentivirus is treated with CD4 antibody (the ZZ domain containing lentivirus can bind any antibody) before infecting T cells. We could demonstrate targeting in >70% of resting CD4+ T cells. Thus we propose to target resting CD4+ T cells with non-integrating lentivirus carrying LTR and/or CCR5 ZFNs via HIV env-pseudotyped or Sindbis virus env-ZZ-domain, by which it would enable easy gene editing. We will use this method to edit CCR5 and/or HIV-LTR. Therefore, CCR5 disruption in bystander CD4+ T cells would protect uninfected cells and LTR/ZFN would excise integrated provirus in infected primary CD4+ T cells. The combination of these two strategies would potentially provide complete HIV protection. The long-term goal of this proposed research is to develop an alternative effective and safe HIV therapy that might offer a functional cure with simplified regimen. We propose to: 1) Generate and optimize reagents for Zinc Finger Nuclease (ZFN)- based gene editing in resting CD4+ T cells; 2) Evaluate whether ex vivo ZFN-modified PBMCs from ART suppressed individuals are protected from reactivation of endogenous virus after reconstitution in Hu/PBL mice

 描述（由申请人提供）：锌指核酸酶（ZFN）介导的CCR 5破坏已被证明是HIV基因治疗的一种有前景的策略，但潜伏的病毒库仍然是HIV-1感染的灭菌甚至功能性治愈的主要障碍。静息的CD 4+记忆T细胞被认为是潜伏期的主要来源，并且这些也构成了特别稳定的病毒库。据报道，靶向3'和5' LTR中的保守序列的ZFN精确地切除了J-Lat细胞中的整个前病毒，但是将ZFN递送至静息的原代CD 4 + T细胞的困难限制了这种方法减少潜伏期的潜力。最近，我们开发了一种方法，通过使用HIV env假型化非整合慢病毒，成功地将CCR 5-ZFN递送到静息的CD 4 + T细胞，并在Hu-PBL小鼠体内显示出有效性。我们还在初步研究中发现，使用经工程改造以表达ZZ-结构域（来自蛋白A的Fc结合结构域）的辛德毕斯病毒糖蛋白提供了另一种靶向静息CD 4 + T细胞的简单方法。在这里，假型化的慢病毒在感染T细胞之前用CD 4抗体处理（含有ZZ结构域的慢病毒可以结合任何抗体）。我们可以证明靶向>70%的静息CD 4 + T细胞。因此，我们提出用携带LTR和/或CCR 5 ZFN的非整合慢病毒通过HIV env-假型或辛德毕斯病毒env-ZZ-结构域靶向静息CD 4 + T细胞，通过这将使基因编辑变得容易。我们将使用此方法编辑CCR 5和/或HIV-LTR。因此，旁观者CD 4 + T细胞中的CCR 5破坏将保护未感染的细胞，并且LTR/ZFN将切除感染的原代CD 4 + T细胞中的整合的前病毒。这两种策略的结合可能会提供全面的艾滋病毒保护。这项拟议研究的长期目标是开发一种替代的有效和安全的艾滋病毒治疗方法，可能提供一个功能性治疗与简化的方案。 我们建议：1）生成并优化用于静息CD 4 + T细胞中基于锌指核酸酶（ZFN）的基因编辑的试剂; 2）评估来自ART抑制个体的离体ZFN修饰的PBMC在Hu/PBL小鼠中重构后是否被保护免于内源性病毒的再活化