In vivo genomic editing of hematopoietic cells for HIV resistance

产品说明：在过去五年中，艾滋病毒/艾滋病治疗领域取得了标志性成就-至少有三名患者在骨髓移植后治愈了艾滋病毒。不幸的是，移植手术往往风险很大，并且仅限于那些因相关恶性肿瘤而面临巨大死亡风险的患者。直接在体内对造血细胞进行抗HIV基因工程将意味着在HIV-AIDS基因治疗领域向前迈进了一大步。 该提案的目的是开发一种有效的方法，为艾滋病毒感染者提供“功能性治疗”。该方法基于以下观察：（i）缺乏或杂合CCR 5（病毒辅助受体）表达的受试者可以对HIV感染具有高度抗性;（ii）当移植到HIV患者（柏林患者）中时，具有突变形式的CCR 5基因的造血CD 34+干细胞和祖细胞（HPC）提供了HIV的“治愈”。我们已经开发了新的肽核酸（PNA），可以形成一个三重螺旋结构，特别是在CCR 5基因。当同时提供供体DNA时，三链体的形成会诱导天然的细胞修复重组途径，从而能够在CCR 5基因中引入终止密码子。这些途径是无错误的，因此可以用于以极低的脱靶率破坏CCR 5基因，从而表达野生型CCR 5蛋白。我们建议使用由FDA批准的聚合物PLGA制成的生物相容性纳米颗粒，用于封装PNA和供体DNA分子，用于在造血细胞中的CCR 5位点进行体内递送和基因组编辑。本申请中的研究旨在实现针对性和特异性 通过在简单的静脉内注射后使其能够渗透到骨髓中，将新一代的PNA和纳米颗粒递送到人类造血细胞。将在新一代人源化HIV感染小鼠模型中评估该方法在预防和治疗方面的功效。 总体目标是建立一个新的微创和创新的治疗模式的HIV-1感染的可行性：三链体和纳米颗粒技术的应用，在体内造血细胞中的CCR 5基因的定点修饰，通过方便的IV输注。

DESCRIPTION: The last five years have witnessed a hallmark achievement in the field of HIV-AIDS therapy- at least three patients have been cured of HIV after bone marrow transplant. Unfortunately, transplantation procedures tend to be very risky and restricted to those patients who are at a substantial risk for death due to associated malignancies. Direct in vivo genetic engineering of hematopoietic cells for HIV resistance would mean a big step forward in the field of HIV-AIDS gene therapy. The objective of this proposal is to develop an effective method for providing a "functional cure" for HIV infected individuals. The approach is based on the observations that (i) subjects lacking or heterozygous for the expression of CCR5, the viral coreceptor, can be highly resistant to HIV infection (ii) hematopoietic CD34+ stem and progenitor cells (HPCs) with a mutated version of the CCR5 gene when transplanted into a HIV patient (The Berlin patient) afforded a "cure" from HIV. We have developed novel peptide nucleic acids (PNA) that can form a triple helical structure specifically within the CCR5 gene. The triplex formation induces natural cellular repair-recombination pathways enabling introduction of a stop codon in the CCR5 gene when a donor DNA is supplied alongside. These pathways are error-free and can thus be used to disrupt the CCR5 gene with extremely low off-target rates and thereby expression of wild-type CCR5 protein. We propose to use biocompatible nanoparticles made from the FDA-approved polymer PLGA for encapsulating PNA and donor DNA molecules for in vivo delivery and genome editing at the CCR5 locus in hematopoietic cells. The studies in this application are directed at enabling targeted and specific delivery of newer generation PNAs and nanoparticles to human hematopoietic cells by enabling penetrance into the bone marrow following simple intravenous injection. The efficacy of the approach in prophylaxis as well as therapy will be evaluated in a new generation humanized mouse model for HIV infection. The overall goal is to establish feasibility of a new minimally invasive and innovative therapeutic paradigm for HIV-1 infection: application of triplex and nanoparticle technology for the site-directed modification of the CCR5 gene in hematopoietic cells in vivo by facile IV infusion.