Genomic modification with purified nuclease proteins for HIV-1 therapy

描述：拟议研究的目标是开发一种可能治愈艾滋病的治疗方法。我们假设新的细胞穿透锌指核酸酶（ZFNs）和转录激活因子样效应核酸酶（TALENs）将允许在人类CD4+ t细胞和CD34+造血干细胞（hsc）中直接进行基因编辑，从而绕过当代传递系统带来的担忧和困难。直接应用工程核酸酶蛋白将导致CCR5/CXCR4基因的双等位基因破坏，并允许HIV-1抗性细胞重新繁殖并保护身体，最终允许受感染的个体停止药物治疗，过上没有疾病的生活。成功可能需要将我们的疗法与新兴药物结合起来，这些药物可以激活潜伏的病毒库，让它们被我们在这里开发的基因编辑免疫细胞消除。一名HIV-1阳性患者通过干细胞移植治愈的报道激发了我们的热情。32个捐献者。尽管停止抗逆转录病毒治疗，“柏林病人”在手术1年后仍未检测到hiv水平。我们的目标是利用我们的创新方法来重现这一成功，这种方法不含核酸和病毒运载工具。虽然使用腺病毒递送ZFN SB-728的临床试验目前正在进行中，但最近的数据表明，在I期研究中观察到的双等位基因修饰的低效率可能会限制其对？32个杂合的。需要对ZFN核酸酶进行重大改进。在初步研究中，我们已经证明细胞穿透性ZFN蛋白在应用于细胞系、t细胞和造血干细胞后可以敲除CCR5。我们发现用我们的蛋白质处理的细胞显示出高的双等位基因修饰率。在一项实验中，58%的修饰细胞在两个等位基因上都出现了破坏。我们的方法的效率类似于DNA载体转染；然而，我们的方法提供了显著减少脱靶乳沟。在新的初步数据中，我们开发了一种新的细胞渗透性TALENs方法，并在CCR5上展示了基因编辑。在这里，我们利用设计和体外进化方法开发了一个非常有效的CCR5/CXCR4靶向核酸酶家族。ZFNs和TALENs将在催化活性、序列特异性、细胞渗透性和双等位基因编辑方面进行优化。与核酸酶的电荷工程相结合，应允许转导蛋白递送到细胞的数量急剧增加。由于我们的方法使我们能够鉴定出FokI核酸酶变体，其活性和新的序列特异性提高了150倍，我们相信本文开发的核酸酶将以超过病毒传递系统报道的水平编辑T细胞和造血干细胞。这里开发的核酸酶将显示出更高的特异性和更高的安全性。我们将用纯化蛋白优化人类t细胞和造血干细胞的治疗，研究它们移植到免疫功能低下的小鼠体内，以及它们限制HIV-1感染的能力。将ZFN/TALEN蛋白递送的效率和安全性与常规递送方法进行比较。我们相信，这里提出的可渗透核酸酶的发展可能被证明是HIV-1治疗和许多其他遗传疾病的突破。

DESCRIPTION: The goal of the proposed research is to develop a potentially curative AIDS treatment. We hypothesize that novel cell-penetrating zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) will allow for direct gene editing in human CD4+ T-cells and CD34+ hematopoietic stem cells (HSCs), thereby bypassing concerns and difficulties posed by contemporary delivery systems. Direct application of engineered nuclease proteins will lead to biallelic disruption of CCR5/CXCR4 genes and allow for HIV-1 resistant cells to repopulate and protect the body, eventually allowing affected individuals to go off drug therapy and on to a life free of disease. Success may require combining our therapy with emerging drugs that activate latent viral reservoirs allowing them to be eliminated by the gene edited immune cells we develop here. Driving our enthusiasm is the report of an HIV-1 positive patient cured by stem cell transplant from a ?32 donor. The "Berlin patient" had no detectable levels of HIV-1 years after the procedure despite discontinuing retroviral therapy. We aim to recapitulate this success using our innovative approach that is free of nucleic acids and viral delivery vehicles. While clinical trials using adenoviral delivery of ZFN SB-728 are currentl underway, recent data indicates that low efficiency of biallelic modification observed in phase I studies may limit its potential to patients whom are ?32 heterozygous. Significant improvements in ZFN nucleases are needed. In preliminary studies we have shown that cell-penetrating ZFN proteins can knock-out CCR5 after their application to cell lines, T-cells, and HSCs. We found that cells treated with our proteins showed a high rate of biallelic modification. In one experiment, 58% of modified cells showed disruption at both alleles. The efficiency of our approach is similar to DNA vector transfection; however, our approach provides dramatic reductions in off-target cleavage. In new preliminary data, we have developed a novel approach to cell- permeable TALENs and demonstrated gene editing at CCR5. Here we develop a family of very efficient CCR5/CXCR4 targeting nucleases using design and in vitro evolution approaches. ZFNs and TALENs will be optimized with respect to catalytic activity, sequence specificity, cell-permeability, and biallelic gene editing. Coupled with charge engineering of nucleases should allow for dramatic increases in the quantity of transduced protein delivered to cells. Because our approaches have allowed us to identify FokI nuclease variants with >15 fold improvement in activity and novel sequence specificity, we are confident that the nucleases developed herein will edit T cells and HSCs at levels exceeding that reported by viral delivery systems. The nucleases developed here will demonstrate higher specificity and improved safety profiles. We will optimize treatment of human T-cells and HSCs with purified proteins, study their transplant into immunocompromised mice, and their ability to limit HIV-1 infection. The efficiency and safety of ZFN/TALEN protein delivery will be compared to conventional delivery methods. We believe that the development of the permeable nucleases proposed here could prove to be a breakthrough in HIV-1 therapy and many other genetic diseases.