Gene Therapy Basic Research to Treat Inherited Primary Immune Deficiencies

治疗遗传性原发性免疫缺陷的基因治疗基础研究

• 批准号: 9563848
• 负责人: Harry L Malech
• 金额: $ 70.31万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9563848
• 关键词: Affect; Alleles; Allogenic; Animal Model; Annual Reports; Autologous; B-Lymphocytes; Basic Science; Biological Models; Blood Cells; Boston; Busulfan; CD34 gene; Cell Line; Cell model; Cells; Child; Chronic Granulomatous Disease; Clinic; Clinical; Clinical Data; Clinical Protocols; Clinical Trials; Clone Cells; Complementary DNA; Complex; Computer Retrieval of Information on Scientific Projects Database; Defect; Development; Distant; Electroporation; Elements; Evaluation; Exons; Female; Future; Gene Targeting; Gene Transfer; Genes; Genetic; Genetic screening method; Genome; Genomics; Genotype; Goals; HIV; Hematopoietic stem cells; Human; Immune; Immunity; Immunologic Deficiency Syndromes; In Vitro; Infant; Infection; Inherited; Insulator Elements; Introns; Knock-out; Lentivirus Vector; Link; Macaca mulatta; Marrow; Mendelian disorder; Messenger RNA; Methods; Modeling; Multicenter Studies; Mus; Mutagenesis; Mutation; Natural History; Natural Killer Cells; Observational Study; Oncogenic; Outcome; Oxidases; Patients; Pediatric Hospitals; Plasmids; Production; Proto-Oncogenes; Pseudogenes; Publications; Publishing; Reporting; Retroviral Vector; SCID Mice; STAT1 gene; Safety; Severe Combined Immunodeficiency; Site; Somatic Cell; Source; Stem cells; Subfamily lentivirinae; Syndrome; System; T-Lymphocyte; Techniques; Technology; Time; Transplantation; United States National Institutes of Health; Virus; Wiskott-Aldrich Syndrome; Work; Xenograft Model; Xenograft procedure; adenosine deaminase deficiency; base; bone marrow xenograft; clinical application; conditioning; gain of function; gene correction; gene function; gene repair; gene therapy; gene transfer vector; healthy volunteer; immune system function; improved; in vivo; induced pluripotent stem cell; instrument; interest; lentiviral integration; mouse model; mutant; neutrophil; new technology; nonhuman primate; novel; pre-clinical; promoter; repaired; restoration; standard of care; time use; tool; transcription activator-like effector nucleases; vector; young adult; zinc finger nuclease

This project is focused on developing curative gene therapies for primary immune deficiencies (PIDs). We also must understand the basic defects and clinical problems affecting the patient groups for which we are developing gene therapy. To that end we developed and studied integrating and non-integrating lentivectors (Annual report references: 3,18); developed and studied gene editing tools such as CRISP/Cas9 (CRISPR), Zinc Finger Nucleases (ZFNs), and TALENs used to correct PIDs in cellular models of gene correction/mutation repair of primary immune deficiencies (Refs: 1,2,5,15,20,22); developed and studied stem cell models that include both primary CD34+ hematopoietic stem cells (HSCs) obtained from patients and healthy volunteers or iPSC developed from patients and healthy volunteers and from animal models as targets for gene therapy (Refs: 1,2,5,15,16,18,20,21,22); studied immune system function and regulators (Refs: 6,7,8,12,16,19,23,); assessed the outcomes of allogeneic transplants and other cellular therapies given to patients with PIDs that serve as important guides to understand the target goals of gene therapy (Refs: 13,17); conducted studies of carrier females of X-linked PIDs, where such studies may also inform the target goals for gene therapy (Ref: 14); and conducted observational and standard of care studies of patients with PIDs of special interest, which for this project, in particular involves patients with the various genetic forms of chronic granulomatous disease (CGD) (Refs: 4,6,9,24) and the older children and young adults with X-linked severe combined immune deficiency (XSCID) (Ref: 3). We also conducted observational studies with our collaborators of patients with Wiskott-Aldrich Syndrome, patients with adenosine deaminase deficiency who have previously been treated with gene therapy, patients with WHIM syndrome (Warts, hypogammaglobulinemia, infections and myelokathexis) (Ref: 11) and other patients with rare forms of PIDs potentially treatable with gene therapy (Refs: 10,23). We list 24 publications from 2016 and 2017 in the reference section of this Annual Report that relate to aspects of this project outlined above. The importance and impact of the some of the most important of these particularly related to development and clinical application of gene therapy/gene editing are outlined in more detail below: 1. Reference #3: In this publication we report that in 5 children and young adults with XSCID that gene therapy with lentivector transduced autologous CD34 HSC following non-ablative busulfan conditioning can restore T cell, NK cell and humoral (B cell) immunity, leading to significant improvement of clinical status. We have treated 3 additional patients with similar encouraging clinically beneficial outcomes from this gene therapy. 2. Reference #2: We have developed a novel method of gene editing using zinc finger nuclease mRNA targeting the AAVS1 safe harbor genomic site to achieve >15% correction of the X-linked form of CGD in patient CD34+ hematopoietic stem cells by electroporation (MaxCyte instrument) together with delivery of a donor plasmid in an AAV6 vector. This demonstrates important proof of principle for safe harbor approaches to gene editing correction of monogenic disorders. 3. Reference #1: We demonstrate for the first time a CRISPR/Cas9 mutation repair approach for a mutation of the CYBB gene at the start of exon 7 responsible for 6% of cases of X-CGD, achieving unprecedented levels of correction (>20%) of human patient CD34+ HSC engrafting as human zenograft in the NSG mouse model. This leads the way to possible clinical application of this gene editing technology for mutation repair gene therapy of a monogenic disorder. 4. Reference #5: We provided major contribution to studies demonstrating the utility of using iPSC developed from rhesus macaque (non-human primate) as a model system gene editing platform. This system will help advance gene editing technologies by providing a non-human primate model to assess safety and efficacy. 5. Reference #15: We show for the first time using ZFN gene editing of iPSC from patients with the p47phox autosomal recessive form of CGD that correction of the GT deletion at the start of exon 2 of NCF1 (responsible for >90% of mutant alleles causing this form of CGD), can be functionally corrected by repair of not only the NCF1 gene, but by repair of this same mutation found in the two pseudogenes (NCF1B and NCF1C). This is a first demonstration of correction of a monogenic disorder by gene editing resurrection of function in a non-functional pseudogene. 6. Reference #18: We provided major contribution to studies that developed a lentivector capable of efficient correction of ARTEMIS deficient SCID. These studies provide the pre-clinical data needed for our collaborators to prepare an FDA IND and clinical protocol to use this vector to treat infants and children with ARTEMIS deficient SCID. 7. Reference #20: We used CRISPR/Cas9 to knock out the CYBB gene function (X-CGD) in the NSG mouse model (capable of hosting human bone marrow xenograft). This added the X-CGD genotype to this genetically complex model that will allow improved evaluation of gene therapy/gene editing of human X-CGD patient CD34+ HSC in the mouse xenograft model. 8. Reference 22: Using human X-CGD patient iPSCs to assess gene editing cDNA insertion into the CYBB gene to correct CGD, we found that addition of the gp91phox cDNA to the 1st exon worked very poorly with respect to expression of gp91phox, while addition to exon 2 provided excellent corrective production of gp91phox, demonstrating that there are critical control elements in intron 1 necessary to efficient production of gp91phox. This indicates the importance of knowing about intron control elements when planning a gene editing strategy for correction of monogenic disorders. 9. Not yet published is that we have opened here at the NIH a new clinical trial of lentivector gene therapy for X-chronic granulomatous disease as part of a multicenter study, Three patients have now been treated by us and two by our collaborators at Boston Childrens Hospital and at UCLA Childrens Hospital. Early results are very promising showing 20-45% of circulating neutrophils having restoration of normal oxidase activity, and this has been associated with clinical benefit in the control of ongoing infection present at the time of gene therapy.

该项目的重点是开发针对原发性免疫缺陷（PID）的治疗性基因疗法。我们还必须了解影响我们正在开发的基因疗法的患者群体的基本缺陷和临床问题。为此，我们开发并研究了整合型和非整合型慢载体（年度报告参考文献：3,18）；开发和研究了基因编辑工具，如 CRISP/Cas9 (CRISPR)、锌指核酸酶 (ZFN) 和 TALEN，用于纠正原发性免疫缺陷基因校正/突变修复细胞模型中的 PID（参考文献：1,2,5, 15,20,22);开发和研究了干细胞模型，其中包括从患者和健康志愿者获得的原代 CD34+ 造血干细胞 (HSC) 或从患者和健康志愿者以及动物模型中开发的 iPSC，作为基因治疗的靶标（参考文献：1,2,5,15 ,16,18,20,21,22);研究免疫系统功能和调节因子（参考文献：6,7,8,12,16,19,23,）；评估了对 PID 患者进行同种异体移植和其他细胞疗法的结果，作为了解基因治疗目标的重要指南（参考文献：13,17）；对 X 连锁 PID 的女性携带者进行了研究，此类研究也可能为基因治疗的目标提供信息（参考文献：14）；并对特别感兴趣的 PID 患者进行了观察性和护理标准研究，对于该项目，特别涉及患有各种遗传形式的慢性肉芽肿病 (CGD) 的患者（参考文献：4、6、9、24）和患有 X 连锁严重联合免疫缺陷 (XSCID) 的年龄较大的儿童和年轻人（参考文献：3）。我们还与合作者对 Wiskott-Aldrich 综合征患者、先前接受过基因治疗的腺苷脱氨酶缺乏症患者、WHIM 综合征患者（疣、低丙种球蛋白血症、感染和骨髓缺乏症）（参考文献：11）和其他患者进行了观察性研究。患有罕见 PID 的患者可以通过基因疗法进行治疗（参考文献：10,23）。 我们在本年度报告的参考部分列出了 2016 年和 2017 年与上述项目相关的 24 篇出版物。下面更详细地概述了其中一些最重要的问题的重要性和影响，特别是与基因治疗/基因编辑的开发和临床应用相关的问题： 1. 参考文献 #3：在本出版物中，我们报道了 5 名患有 XSCID 的儿童和年轻人，在非消融白消安调理后使用慢载体转导自体 CD34 HSC 进行基因治疗可以恢复 T 细胞、NK 细胞和体液（B 细胞）免疫，导致临床状态的显着改善。我们还治疗了另外 3 名患者，通过这种基因疗法获得了类似的令人鼓舞的临床有益结果。 2. 参考文献#2：我们开发了一种新的基因编辑方法，使用锌指核酸酶 mRNA 靶向 AAVS1 安全港基因组位点，通过电穿孔实现患者 CD34+ 造血干细胞中 X 连锁形式 CGD 的 >15% 校正（ MaxCyte 仪器）以及 AAV6 载体中供体质粒的递送。这证明了单基因疾病基因编辑校正安全港方法的重要原理证明。 3. 参考文献#1：我们首次展示了 CRISPR/Cas9 突变修复方法，用于修复 CYBB 基因在外显子 7 起始处的突变，该突变导致 6% 的 X-CGD 病例，实现了前所未有的校正水平 (> 20%)的人类患者CD34+ HSC作为人类异种移植物移植到NSG小鼠模型中。 这为这种基因编辑技术用于单基因疾病突变修复基因治疗的临床应用开辟了道路。 4. 参考文献 #5：我们为证明使用恒河猴（非人类灵长类动物）开发的 iPSC 作为模型系统基因编辑平台的效用的研究做出了重大贡献。 该系统将通过提供非人类灵长类动物模型来评估安全性和有效性，从而帮助推进基因编辑技术。 5. 参考文献#15：我们首次对 p47phox 常染色体隐性 CGD 患者的 iPSC 进行 ZFN 基因编辑，结果显示，NCF1 外显子 2 起始处的 GT 缺失得到纠正（导致 > 90% 的突变）导致这种形式的 CGD 的等位基因），不仅可以通过修复 NCF1 基因，还可以通过修复两个假基因中发现的相同突变来进行功能纠正（NCF1B 和 NCF1C）。 这是通过基因编辑恢复非功能性假基因功能来纠正单基因疾病的首次演示。 6. 参考文献#18：我们为开发能够有效校正 ARTEMIS 缺陷 SCID 的慢载体的研究做出了重大贡献。 这些研究为我们的合作者提供了准备 FDA IND 和临床方案所需的临床前数据，以使用该载体治疗患有 ARTEMIS 缺陷 SCID 的婴儿和儿童。 7. 参考文献#20：我们使用 CRISPR/Cas9 敲除 NSG 小鼠模型（能够承载人骨髓异种移植物）中的 CYBB 基因功能（X-CGD）。 这将 X-CGD 基因型添加到这个遗传复杂模型中，这将允许在小鼠异种移植模型中改进对人类 X-CGD 患者 CD34+ HSC 的基因治疗/基因编辑的评估。 8. 参考文献 22：使用人类 X-CGD 患者 iPSC 评估基因编辑 cDNA 插入 CYBB 基因以纠正 CGD，我们发现将 gp91phox cDNA 添加到第一个外显子对于 gp91phox 的表达效果非常差，而添加外显子 2 提供了 gp91phox 的出色校正生产，表明内含子 1 中存在有效生产 gp91phox 所必需的关键控制元件gp91phox。 这表明在规划纠正单基因疾病的基因编辑策略时了解内含子控制元件的重要性。 9. 尚未发表的是，我们在 NIH 开展了一项针对 X 慢性肉芽肿病的慢载体基因治疗的新临床试验，作为多中心研究的一部分，现已治疗三名患者，两名患者由我们在波士顿的合作者治疗儿童医院和加州大学洛杉矶分校儿童医院。早期结果非常有希望，显示 20-45% 的循环中性粒细胞恢复了正常氧化酶活性，这与控制基因治疗时存在的持续感染的临床益处相关。