Genomic modification with purified nuclease proteins for HIV-1 therapy

使用纯化核酸酶蛋白进行基因组修饰用于 HIV-1 治疗

• 批准号: 9267454
• 负责人: Dennis R. Burton
• 金额: $ 77.96万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-25 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267454
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alleles; Architecture; Automobile Driving; Berlin; Bypass; CCR5 gene; CD34 gene; CD4 Positive T Lymphocytes; CXCR4 Receptors; CXCR4 gene; Cell Line; Cells; Charge; Clinical Trials; Coupled; DNA; Data; Development; Directed Molecular Evolution; Disease; Engineering; Evaluation; Evolution; Family; Genes; Genetic Vectors; Genomics; Goals; HIV-1; Hematopoietic stem cells; Hereditary Disease; Human; IL2RA gene; Immune; Immunocompromised Host; In Vitro; Individual; Infection; Knock-out; Life; Messenger RNA; Methods; Modeling; Modification; Mus; Names; Nucleic Acids; Other Genetics; Patients; Performance; Permeability; Pharmaceutical Preparations; Pharmacotherapy; Population; Procedures; Property; Proteins; Reporting; Research; Resistance; Role; Safety; Specificity; Stem cell transplant; Surface; System; T-Lymphocyte; Therapeutic; Time; Toxic effect; Transplantation; Treatment Protocols; Variant; Viral; Viral reservoir; Virus Latency; Zinc Fingers; base; design; direct application; experimental study; gene function; humanized mouse; improved; in vivo; innovation; knockout gene; novel; novel strategies; nuclease; phase 1 study; portability; preference; public health relevance; success; tool; transcription activator-like effector nucleases; zinc finger nuclease

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.

描述：这项拟议研究的目标是开发一种潜在的治愈艾滋病的方法。我们假设，新型细胞穿透性锌指核酸酶(ZFN)和转录激活物样效应核酸酶(TALENS)将允许在人类CD4+T细胞和CD34+造血干细胞(HSCs)中直接进行基因编辑，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CCR5/CXCR4基因的双等位基因破坏，使抗HIV-1的细胞重新繁殖并保护身体，最终使受影响的人停止药物治疗，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经过基因编辑的免疫细胞消除。一位来自Δ32捐赠者的干细胞移植治愈了一名HIV-1阳性患者的报道，这一报道推动了我们的热情。尽管停止了逆转录病毒治疗，但这位“柏林患者”在手术后几年内没有检测到艾滋病毒-1的水平。我们的目标是使用我们的创新方法来重现这一成功，这种方法不需要核酸和病毒载体。虽然用腺病毒传递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效率低可能限制了其在Δ32杂合子患者中的潜力。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，穿透细胞的ZFN蛋白在应用于细胞系、T细胞和HSCs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很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。我们的方法的效率与DNA载体转染法相似，但是，我们的方法显著减少了非靶标切割。在新的初步数据中，我们开发了一种新的细胞渗透性TALEN方法，并在CCR5上演示了基因编辑。在这里，我们利用设计和体外进化的方法开发了一系列非常有效的针对核酸酶的CCR5/CXCR4。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编辑方面进行优化。再加上核酸酶的电荷工程，应该可以显著增加传递给细胞的转导蛋白的数量。由于我们的方法使我们能够识别活性提高15倍的FokI核酸酶变体和新的序列特异性，我们相信这里开发的核酸酶将编辑T细胞和HSCs的水平超过病毒递送系统报告的水平。这里开发的核酸酶将表现出更高的特异性和更好的安全性。我们将用纯化的蛋白质优化人类T细胞和造血干细胞的治疗，研究它们移植到免疫缺陷小鼠体内的情况，以及它们限制HIV-1感染的能力。ZFN/TALEN蛋白质递送的效率和安全性将与传统递送方法进行比较。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性疾病的突破。