Genomic modification with purified nuclease proteins for HIV-1 therapy

描述：拟议研究的目标是开发一种潜在的治疗艾滋病的方法。我们假设，新型细胞穿透性锌指核酸酶（ZFN）和转录激活因子样效应核酸酶（TALEN）将允许在人CD 4 + T细胞和CD 34+造血干细胞（HSC）中进行直接基因编辑，从而绕过当代递送系统带来的担忧和困难。直接应用工程核酸酶蛋白将导致CCR 5/CXCR 4基因的双等位基因破坏，并允许HIV-1抗性细胞重新繁殖和保护身体，最终允许受影响的个体停止药物治疗并开始无疾病的生活。成功可能需要将我们的疗法与新兴药物相结合，这些药物可以激活潜伏的病毒库，使它们能够被我们在这里开发的基因编辑免疫细胞消除。推动我们热情的是一名HIV-1阳性患者通过Δ32供体的干细胞移植治愈的报告。“柏林患者”在手术后1年没有检测到HIV水平，尽管停止了逆转录病毒治疗。我们的目标是使用我们的创新方法来重现这一成功，该方法不含核酸和病毒载体。虽然使用ZFN SB-728的腺病毒递送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限制其对Δ32杂合子患者的潜力。需要ZFN核酸酶的显著改进。在初步研究中，我们已经表明，细胞穿透ZFN蛋白可以在其应用于细胞系、T细胞和HSC后敲除CCR 5。我们发现用我们的蛋白质处理的细胞显示出高比率的双等位基因修饰。在一个实验中，58%的修饰细胞在两个等位基因上都显示出破坏。我们的方法的效率类似于DNA载体转染;然而，我们的方法提供了脱靶切割的显著减少。在新的初步数据中，我们已经开发了一种新的细胞可渗透TALEN方法，并证明了在CCR 5处的基因编辑。在这里，我们开发了一个家庭的非常有效的CCR 5/CXCR 4靶向核酸酶的设计和体外进化的方法。ZFN和TALEN将在催化活性、序列特异性、细胞渗透性和双等位基因基因编辑方面进行优化。与核酸酶的电荷工程相结合，应该允许递送到细胞的转导蛋白质的量的急剧增加。因为我们的方法已经允许我们鉴定具有>15倍活性改善和新序列特异性的FokI核酸酶变体，所以我们相信本文开发的核酸酶将以超过病毒递送系统报道的水平编辑T细胞和HSC。这里开发的核酸酶将表现出更高的特异性和改善的安全性。我们将用纯化的蛋白质优化人类T细胞和HSC的治疗，研究它们移植到免疫功能低下的小鼠中，以及它们限制HIV-1感染的能力。ZFN/TALEN蛋白递送的效率和安全性将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会被证明是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 Δ32 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 Δ32 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.