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用于原发性免疫缺陷（PID）的治愈基因疗法。我们还必须了解影响我们正在开发基因治疗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和非整合的慢病毒（年度报告参考：3,18）；开发和研究了基因编辑工具，例如CRISP/CAS9（CRISPR），锌指核酸酶（ZFN）和Talens，用于纠正基因校正/突变修复原发性免疫缺陷的细胞模型中的PID（参考文献：1,2,5,15,15,20,22）;开发和研究了干细胞模型，包括从患者和健康志愿者和健康志愿者开发的患者，健康志愿者或IPSC的主要CD34+造血干细胞（HSC）以及动物模型作为基因治疗的靶标（参考：1,2,5,15,15,15,15,16,18,20,20,21,21,22）;研究的免疫系统功能和调节剂（参考：6,7,8,12,16,19,23，）；评估了对PID患者的同种异体移植和其他细胞疗法的结局，这些患者是了解基因疗法目标目标的重要指南（参考文献：13,17）；对X连锁PID的载体女性进行了研究，其中此类研究还可以为基因治疗的目标目标提供信息（参考：14）；并对具有特殊感兴趣的PID患者进行了观察性和护理研究的观察和标准，特别是涉及患有各种遗传形式的慢性肉芽肿性疾病（CGD）（REFS：4,6,9,24）的患者，以及具有X-Linked严重组合免疫缺陷（XSCID）（XSCID）（XSCID）（XSCID）（XSCID）的年龄较大的儿童和年轻人。我们还与我们的Wiskott-Aldrich综合征患者的合作者进行了观察性研究，患有腺苷脱氨酶缺乏症患者以前曾接受过基因治疗，WHIM综合征患者（WART，低血糖蛋白血症，感染和骨髓触及疗法）以及稀有形式的稀有患者（Ref Sentery of PIDS）（Ref Senters）（疣，低毒素血症，疣状综合症患者）（warts，疣，低震颤血症患者）。 10,23）。 我们在本年度报告的参考部分中列出了与上面概述的该项目相关的本报告的参考部分中的24个出版物。以下更详细地概述了其中最重要的其中一些与基因疗法/基因编辑的发展和临床应用有关的重要性和影响： 1。参考＃3：在本出版物中，我们报告说，在非富有性能的Busulfan调节后，在5名患有XSCID的儿童和年轻人中，用烟叶转导的自体CD34 HSC可以恢复T细胞，NK细胞和体液（B细胞）免疫，从而恢复T细胞，从而恢复临床状况的显着改善。我们已经治疗了3名与该基因疗法相似的临床有益结果的类似临床有益结果的患者。 2。参考＃2：我们已经开发了一种新颖的基因编辑方法，该方法使用锌指核酸酶mRNA靶向AAVS1安全港基因组位点，以通过电型（maxcy仪器）（Maxcy仪器）在患者CD34+ CD34+造血性干细胞中对CGD进行> 15％的校正，并与供体质体在AAV 6 vecordy中的供体校正。这证明了安全港方法的基因编辑方法的重要原理证明。 3。参考＃1：我们首次演示了CRISPR/CAS9突变修复方法，用于在外显子7开始时使用CYBB基因的突变，负责6％的X-CGD病例，在人类NSG小鼠模型中作为人类患者的Zenogroft as Grunder CD34+ HSC的校正水平（> 20％）。 这导致了该基因编辑技术在单基因疾病的突变修复基因治疗中的可能临床应用。 4。参考＃5：我们为研究提供了主要贡献，证明了使用从恒河猴（非人类灵长类动物）作为模型系统基因编辑平台开发的IPSC的实用性。 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功效来帮助推进基因编辑技术。 5。参考＃15：我们首次使用ZFN基因编辑IPSC的p47phox常染色体隐性形式CGD的CGD校正NCF1的外显子2开始时校正GT缺失（负责> 90％的突变等位基因，可以通过cgd的这种形式来维修ncf1的ncf1（CGD），而不仅可以修复CGD），但是ncf ncf1的cGD校正不仅可以修复nCF1的cGD。在两个假基因（NCF1B和NCF1C）中。 这是通过基因编辑非功能伪元中的功能复活来校正单基因障碍的第一个证明。 6。参考＃18：我们为开发出能够有效校正ARTEMIS缺乏SCID的研究的研究提供了重大贡献。 这些研究为我们的合作者提供了临床前数据，以准备FDA IND和临床方案，以使用该矢量来治疗Artemis缺乏SCID的婴儿和儿童。 7。参考＃20：我们使用CRISPR/CAS9在NSG小鼠模型（能够托管人骨髓异种移植物）中淘汰CYBB基因函数（X-CGD）。 这将X-CGD基因型添加到了这种遗传复杂的模型中，该模型将改善对小鼠异种移植模型中人X-CGD患者CD34+ HSC的基因治疗/基因编辑的评估。 8。参考22：使用人X-CGD患者IPSC评估插入CYBB基因以纠正CGD的cDNA插入的基因编辑，我们发现，在第1外显子中添加GP91Phox cDNA非常差，相对于GP91Phox的表达而言，在GP91Phox的表达方面效果非常差，而在Exon 2中则提供了良好的exon extron，该exon的生产量很大，以至于有足够的exton extron，该extor exton的exton extor extor extor extor的构成了GP91Phox的良好元素。 gp91phox。 这表明在计划基因编辑策略以纠正单基因疾病时了解内含子控制元素的重要性。 9。尚未发表的是，我们已经在NIH开放了一项新的针对X-智商颗粒疾病的慢病毒基因治疗的临床试验，这是一项多中心研究的一部分，现在，美国和两名患者在波士顿儿童医院和UCLA Childrens医院的合作者治疗。早期结果非常有前途，表明20-45％的循环中性粒细胞恢复了正常的氧化酶活性，这与临床益处有关，在控制基因治疗时存在的正在进行的感染中。