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: 8555791
• 负责人: Harry L Malech
• 金额: $ 41.81万
• 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/8555791
• 关键词: Adult; Affect; Animal Model; Appearance; Ascaridil; Autologous; Basic Science; Biological Models; Blood; Blood Circulation; Busulfan; CD34 gene; Canis familiaris; Cathepsin G; Cell Line; Cells; Chickens; Child; Chronic Granulomatous Disease; Clinic; Clinical; Clinical Protocols; Clinical Trials; Codon Nucleotides; Collaborations; Communities; Complementary DNA; Defect; Development; Differentiated Gene; Disease; Distant; Elements; Engineering; European; Future; Gene Activation; Gene Transfer; General Population; Genes; Genome; Globin; Goals; Hematopoietic stem cells; Hospitals; Human; Hybrids; Hypersensitivity; Immune; Immunity; Immunologic Deficiency Syndromes; In Vitro; Indiana; Infant; Infection; Inherited; Institutional Review Boards; Insulator Elements; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Introns; LMO2 gene; Laboratories; Laboratory Study; Lentivirus Vector; Lesion; Link; London; Lymphocyte; Marrow; Medicine; Methods; Molecular; Mus; Mutagenesis; Mutation; Myelogenous; Newly Diagnosed; Oncogenic; Oxidases; Parents; Patients; Phase; Process; Production; Property; Proteins; Proto-Oncogenes; Protocols documentation; Publishing; Recruitment Activity; Research; S-1 Antimetabolite agent; SCID Mice; Safety; Saint Jude Children' s Research Hospital; Site; Solutions; Somatic Cell; Source; Specificity; Stem cells; Subfamily lentivirinae; System; Techniques; Testing; Transplantation; Treatment Protocols; United States National Institutes of Health; Universities; Vertebral column; Virus; Work; Xenograft procedure; Zinc Fingers; base; cell type; clinical lot; conditioning; conventional therapy; design; gene correction; gene repair; gene therapy; gene therapy clinical trial; gene transfer vector; human CYBA protein; human EEF1A1 protein; improved; in vivo; induced pluripotent stem cell; mouse model; murine retroviral vector; neutrophil; neutrophil cytosol factor 40K; neutrophil cytosol factor 67K; new technology; novel; nuclease; peripheral blood; pre-clinical; programs; promoter; repaired; therapeutic transgene; tool; vector

This project involves laboratory studies and studies in animal models of the tools and methods that need to be developed to correct or repair the genetic defects causing the gp91phox deficient X-linked form of chronic granulomatous disease (X-CGD), the p47phox deficient autosomal recessive form of CGD (AR-CGD), and X-linked severe combined immune deficiency (SCID-X-1 or XSCID). This work involves studies of a variety of lentivirus vectors and the critical functional sub-elements that go into the design of safe and effective lentivirus vectors. These function sub-elements include assessment of gene promoters or hybrid promoter constructs, assessment of insulator elements that may protect nearby genes from activation by vector inserts in the genome, assessment of selectable elements that could increase level of gene marking, development of novel pseudotyping envelopes. The work also involves studying vectors in a variety of cell types and in particular optimizing gene transfer into human CD34+ hematopoietic stem cells (HSC). This project also involves the engineering of induced pluripotent stem cells from adult somatic cells of patients with CGD or XSCID for the purpose of achieving gene correction of the functional immune defect in the iPSC, including the differentiation in culture to the mature blood or immune cells affected by the primary immune deficiency under study. In the past fiscal year year we have accomplished, competed and/or published in final form the following results toward the general goals of the project: 1. Together with our collaborators (Dr. B Sorrentino at St. Jude) we have developed a high titer lentivirus vector encoding the common gamma chain of the IL2 receptor for a planned gene therapy trial for XSCID. The CL20 backbone-based lentivector has the following safety elements: self-inactivating lesions in the 3LTR, internal promoter that is the elongation factor 1 alpha short version (EF1a-s), 400 bp version of the Chicken H4 globin insulator, codon-optimized therapeutic transgene cDNA. This vector appears to perform well at transducting human hematopoietic stem cells and correcting the immune defect in both XSCID mice and XSCID dogs. Most important is that this construct does not activate LMO2 when inserted into the first intron of this gene. During the past year clinical lots of vector have been produced and are available for treating patients. Also during the past year, a clinical protocols of gene therapy for XSCID using this vector that have been approved by the IRBs, IBCs, reviewed by the RAC, and the IND process completed at the FDA for protocols at the NIH and at St. Jude Childrens Research Hospital. The clinical trial in our program at the NIH will study treatment of older children with XSCID who had received lymphocyte depleted haploidentical transplants from a parent, but whose immunity had not been adequately restored or was waning. The first patient in this trial is scheduled for treatment in October 2012. The clinical trial of gene transfer at St. Jude will study treatment of infants newly diagnosed with XSCID, and they are open and recruiting. 2. We completed the laboratory assessment of a completed clinical trial of gene therapy for X-CGD patients with severe ongoing infection not responsive to conventional therapy using a murine retrovirus vector and busulfan conditioning. All three patients demonstrated early marking with appearance in the circulation of 24%, 5% and 4% neutrophils that were oxidase normal. However, marking persisted in only two of the patients such that after the first year to the third year marking was 1% and 0.03% , respectively. In the two patients with long term marking their infections cleared. Laboratory assessment of gene insertions sites showed no clonal dominance. We conclude that even when not curative or permanent, gene therapy can provide clinical benefit in the treatment of persistent severe infection in X-CGD. (Kang EM et al, Blood 115:783, 2010). 3. Preclinical work toward the next phase of development of gene therapy for X-CGD has involved the development and study of a new lentivirus vector with features very similar to the CL20 lentivector that we have developed for the clinical trial XSCID noted in section 1. above except that this vector includes the codon optimized cDNA encoding the gp91phox gene product of the CYBB gene. Using our NSG mouse model that can engraft human hematopoietic stem cells we have shown that this vector can achieve full functional oxidase correcion up to 50% of the neutrophils that arise from gene corrected stem cells. We are currently in the process of production of a clinical lot of this vector in collaboration with the Indiana University Vector Production Facility (Dr. Kenneth Cornetta). We have also used the NSG mouse system to test an alternate lentivector developed by our collaborators in London and Frankfurt that uses a hybrid promoter from Fes plus Cathepsin G genes that provides myeloid specificity to expression. This vector also has excellent properties and our European collaborators plan to bring that vector to the clinic (Santilli G et al, Mol Ther 19:122, 2011) in their program. 5. Beginning a few years ago, together with our collaborator (Dr. L Cheng at Johns Hopkins Sch of Medicine) we have developed iPSC from the somatic cells of a patient with X-CGD, demonstrated that neutrophils differentiated from patient iPSC do not have oxidase activity but those from normal iPSC do, recapitulating the disorder. We also demonstrated that gene transfer can correct the oxidase defect in the X-CGD iPSC in that neutrophils differentiated from the gene corrected X-CGD iPSC have restored oxidase activity (Zou J et al, Blood 117:5561, 2011). We have now moved forward in the laboratory with Zinc Finger Nucleases to demonstrate that this general approach of targeting a corrective minigene to the AAVS1 safe harbor site can be applied to correction of iPSC lines derived from patients with each of the four autosomal recessive forms of CGD (p47phox, p40phox, p22phox and p67phox deficient CGD). We have also developed ZNFs and TALENs that target the CYBB gene to achieve insertion of a minigene designed to correct X-linked CGD, and to target the NCF1 gene to achieve gene repair for correction of the p47phox deficient autosomal recessive form of CGD. We have also developed a novel highly efficient method for reprogramming iPSC lines derived from the CD34+ hematopoietic stem cells present in only 10-20ml of peripheral blood and applied this method to generate iPSC lines from many of our patients with CGD, XSCID and some other inherited immune deficiencies. 6. We have published a number of chapters and reviews about gene therapy, thus communicating to the scientific community and to the general public information about progress in the field of gene therapy in general and for gene therapy of CGD and XSCID in particular (Segal et al, Biol Blood Marrow Transplant 17(S-1): S123, 2011; Kang et al, J Allergy Clin Immunol 127:1319, 2011); Kand and Malech, Methods Enzymol 507:125, 2012; Corrigan-Curay, J et al, Mol Ther 20:1084.

该项目涉及在动物模型中进行的实验室研究和研究，这些工具和方法需要开发为纠正或修复导致gp91phox慢性肉芽肿性疾病（X-CGD）的GP91Phox缺乏X连锁形式，P47Phox缺乏的常染色体疗程缺陷疗法（AR-CGD）（AR-CGD）和X-linne nune and comenne and comenne commantine and xsssc-comenne commandect（ac）。这项工作涉及对各种慢病毒载体的研究以及对安全有效的慢病毒载体设计的关键功能子元素。这些功能子元素包括评估基因启动子或混合动力启动子构建体，评估可能保护附近基因的绝缘因子免受基因组中载体插入物激活的激活，评估可能增加基因标记水平的可选元件的评估，对新型的假型信封的发展。这项工作还涉及研究各种细胞类型的载体，特别是优化基因转移到人CD34+造血干细胞（HSC）中。该项目还涉及从CGD患者或XSCID患者的成年体细胞中引起的多能干细胞的工程，以实现IPSC中功能免疫缺陷的基因校正，包括在研究中受主要免疫缺陷影响的成熟血液或免疫细胞的培养分化与成熟的血液或免疫细胞的分化。在过去的财政年度中 1。与我们的合作者（St. Jude的B Sorrentino博士）一起，我们开发了一个高滴度慢病毒矢量，编码IL2受体的常见伽马链，用于XSCID的计划基因治疗试验。基于CL20骨架的烯烃具有以下安全元件：3LTR内部启动子中的自动灭活病变，该病变是延伸因子1 Alpha短版（EF1A-S），400 bp的鸡H4 Globin绝缘子，密码子易于使用的治疗疗法的Transgene Transgene cdna。该载体似乎在转移人造血干细胞并纠正XSCID小鼠和XSCID犬的免疫缺陷方面表现良好。最重要的是，当插入该基因的第一个内含子时，该构建体不会激活LMO2。 在过去的一年中，已经生产了许多载体，可用于治疗患者。 同样在过去的一年中，使用该向量的XSCID基因治疗临床方案，该载体已获得IRB，IBCS批准的RAC审查，而在NIH和St. Jude Childrens Research Hospital在FDA完成的IND过程。我们在NIH计划中的临床试验将研究接受XSCID的老年儿童的治疗，这些儿童接受了淋巴细胞耗尽的单倍性移植，但其免疫力尚未得到充分恢复或正在减弱。 该试验中的第一位患者计划于2012年10月进行治疗。圣裘德基因转移的临床试验将研究新诊断为XSCID的婴儿的治疗，并且它们是开放和招募的。 2。我们完成了针对严重持续感染的X-CGD患者对基因治疗的完整临床试验的实验室评估，但使用鼠逆转录病毒载体和Busulfan条件对常规治疗反应迅速。所有三名患者均表现出早期标记，出现在24％，5％和4％氧化酶正常的嗜中性粒细胞的循环中。但是，仅在两名患者中持续标记，因此在第一年至第三年后，标记分别为1％和0.03％。在两名长期标记感染的患者中，已清除。基因插入部位的实验室评估没有克隆优势。我们得出的结论是，即使没有治愈或永久性，基因疗法也可以在X-CGD中持续的严重感染治疗临床益处。 （Kang Em等人，血液115：783，2010）。 3.临床前的X-CGD基因疗法开发的临床前工作涉及对新的慢病毒载体的开发和研究，其特征与Cl20 lentivector非常相似，我们为临床试验XSCID开发了第1节中提到的XSCID。除了该矢量包括CODON优化的CDNA，该矢量包括编码GP91Phox Gene Gene gene gene的CDNA。使用我们的NSG小鼠模型可以植入人类造血干细胞，我们已经表明，该载体可以实现由基因校正的干细胞产生的完全功能性氧化酶矫正。 目前，我们正在与印第安纳大学媒介生产设施（Kenneth Cornetta博士）合作生产该载体的临床媒介。我们还使用了NSG小鼠系统来测试由我们在伦敦和法兰克福的合作者开发的替代烟虫，该齿轮使用了FES和calter蛋白蛋白酶G基因的混合动力启动子，该基因为表达提供了髓样特异性。该向量还具有出色的特性，我们的欧洲合作者计划将该媒介带到诊所（Santilli G等，Mol Ther 19：122，2011）。 5。从几年前，我们与我们的合作者（Johns Hopkins of Medicine的L Cheng博士）一起从患有X-CGD患者的体细胞中开发了IPSC，这表明与患者IPSC区分开的中性粒细胞没有氧化酶活性，但是来自正常IPS的氧化酶，但从正常的IPS中进行了氧化酶的活性。我们还证明了基因转移可以纠正X-CGD IPSC中的氧化酶缺损，因为中性粒细胞与基因校正后的X-CGD IPSC有所区别已恢复氧化酶活性（Zou J等，血液117：5561，2011）。 现在，我们已经用锌指核酸酶向前迈进，以证明将矫正微基因靶向AAVS1安全港位点的这种一般方法可以应用于从具有四种常染色体隐性形式的CGD中的每种患者中得出的IPSC线（p47phox，p47phox，p40phox，p40phox，p40phox，p22phox，p22phox和p22phox和p67phox）。 我们还开发了针对CYBB基因的ZNF和TALEN，以实现旨在纠正X连锁CGD的微基因的插入，并靶向NCF1基因以实现基因修复，以校正P47PHOX缺乏的无染色体隐性菌群CGD。 我们还开发了一种新型的高效方法，用于重编程源自仅存在10-20mL外周血中的CD34+造血干细胞的IPSC系，并应用了这种方法来生成来自许多CGD，XSCID和其他一些遗传性免疫缺陷的患者的IPSC系。 6。我们发表了许多有关基因疗法的章节和评论，从而与科学界以及有关基因治疗领域的进步以及CGD和XSCID的基因疗法的一般公众信息（Segal等人，Biol Blood Marrow Marrow 17（s-1）：S123，2011年; Kang et Al，Jang Al imn，2011年137：137; Kand and Malech，方法酶507：125，2012; Corrigan-Curay，J等，Mol Ther 20：1084。