Gene Therapy for Inherited Blood Disorders

遗传性血液疾病的基因治疗

基本信息

批准号：
10012688
负责人：
Andre LaRochelle
金额：
$ 46.22万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10012688
关键词：
Address Adverse effects Adverse event Algorithms Antibodies Autologous Transplantation Binding Biological Biological Assay CD18 Antigens CD3 Antigens CD34 gene CMV promoter CRISPR/Cas technology CXCR4 gene Capsid Cell Culture Techniques Cell surface Cells Cleaved cell Clinic Clinical Clinical Trials Clone Cells Collaborations Complementary DNA Complex Cultured Cells Cytotoxic Chemotherapy DNA Repair DNA Repair Pathway DNA cassette Data Data Set Development Diagnosis Diphtheria Toxin Disease Drug Kinetics Ectopic Expression Electroporation Embryo Engraftment Enhancers Episome Evaluation Evaluation Research Event Experimental Designs Flow Cytometry Frequencies G1 Phase Gene Dosage Gene Expression Gene Transduction Agent Gene Transfer Gene therapy trial Genes Genetic Genetic Recombination Genomic DNA Goals Guide RNA Hematological Disease Hematopoietic stem cells High-Throughput Nucleotide Sequencing Hour Human ITGB2 gene Immunologic Deficiency Syndromes Immunotoxins In Vitro Inherited Insertional Mutagenesis Integrins Knock-in Lentivirus Vector Leukocyte Adhesion Deficiency Leukocytes Libraries Life Lymphoid MS4A1 gene Macaca mulatta Mediating Methods Molecular Mus Mutagenesis Mutation Myelogenous Non-Malignant Nonhomologous DNA End Joining Nuclear Nucleotides Oncogene Activation PTPRC gene Patients Pattern Phase Pichia Population Preparation Production Proto-Oncogenes Radiation Recombinant adeno-associated virus (rAAV)Recombinants Regimen Reporter Reporter Genes Reporting Retroviral Vector Retroviridae Ribonucleoproteins Safety Sampling Series Serotyping Site Somatic Mutation Stem cell transplant Stem cells Structure System Techniques Technology Therapeutic Effect Time Toxic effect Toxin Transgenes Transplantation Tumorigenicity Variant Vertebral column Viral Viral Vector Yeasts alanine aminopeptidase antibody conjugate base cellular transduction clinical efficacy clinically relevant conditioning cytotoxicity design dimer experimental study first-in-human gene replacement gene therapy gene therapy clinical trial genetic approach genetic manipulation genome editing genome sequencing genome-wide genotoxicity in vivo induced pluripotent stem cell insertion/deletion mutation integration site interest leukemia meetings member neutrophil next generation sequencing nonhuman primate novel novel strategies peripheral blood post-transplant pre-clinical preclinical study preference prevent promoter regenerative therapy safety testing single cell sequencing success targeted treatment therapeutic gene therapeutic target therapeutic transgene transgene expression treatment group vector whole genome

项目摘要

Objective 1: Develop a FV-based gene therapy approach for the treatment of subjects with LAD-1. 1.1 Pre-clinical studies to evaluate the efficacy of FV in human LAD-1 CD34+ cells. We have conducted extensive pre-clinical studies to investigate the efficacy of clinical grade FV expressing the human CD18 cDNA (FV-hCD18) in HSPCs collected from subjects with a molecularly confirmed diagnosis of LAD-1. Cells were transduced ex vivo with FV-hCD18 for 16 hours. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 39-42% of cells. Genetic correction of HSPCs from LAD-1 patients restored the chemotactic function of neutrophils differentiated from these progenitor cells in vitro. Transplantation of FV-hCD18-transduced LAD-1 HSPCs into immuno-deficient (NSG) mice resulted in high-level, clinically relevant gene marking levels in vivo. The average percentages of human cells expressing CD18 in the murine BM 5 months after transplantation were 36.0 3.9%. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in most of long-term repopulating HSPCs. Flow cytometry-based lineage analysis of BM from mice transplanted with CD34+ cells transduced with FV-hCD18 revealed human CD18+ cells in both CD13+ myeloid and CD20+ lymphoid compartments. Using next-generation sequencing technology, a total of 101 unique integration sites were recovered in repopulating cells and revealed a polyclonal pattern of integration with no evidence of insertional mutagenesis or tumorigenicity five months after transplantation. 1.2 First-in-human clinical trial testing safety/efficacy of FV for gene therapy of patients with LAD-1. Based on pre-clinical evidence of safety and efficacy discussed above, we have designed a first-in-human phase I/II gene therapy clinical trial using FV for the gene therapy of LAD-1. A pre-IND type B meeting was held with members of the Center for Biologics Evaluation and Research (CBER) within the FDA. Regulatory review and accrual are currently on hold due to delays in production of GMP grade FV. Objective 2: Develop and evaluate safety of CRISPR/Cas9-based strategies for permanent site-specific delivery of a therapeutic gene in human HSPCs. 2.1 Evaluate off-target Cas9 activity in human HSPCs We performed CRISPR-Cas9-based genome editing in human HSPCs and assessed the acquisition of de novo somatic mutations in an unbiased, genome-wide manner via high-throughput, whole-genome sequencing (WGS) of single-cell-derived HSPC clones. Importantly, to distinguish between naturally occurring spontaneous somatic mutation, potential cell culture-induced mutagenesis, and bona fide Cas9-mediated genetic alterations, the experimental design included a parallel set of control treatments applied to samples of the same bulk HSPC population. HSPC samples were either i) mock electroporated, ii) electroporated in the absence of effector molecules, iii) electroporated with recombinant Cas9 alone, or iv) electroporated with ribonucleoprotein complexes (RNPs) consisting of Cas9 bound to single guide RNAs (sgRNAs) targeting either the cxcr4 gene or the safe harbor locus AAVS1. Experimentally manipulated cells were cloned by limiting dilution and single-cell isolates were expanded in vitro to obtain sufficient genomic DNA for WGS library preparation and high-throughput sequencing. We employed somatic variant calling algorithms to identify de novo insertions/deletions (indel) mutations, single nucleotide variants (SNVs) and structural variants (SVs) captured within the WGS datasets for each treatment group relative to the un-treated, bulk HSPC reference sequence. Consistent with previous studies investigating CRISPR-Cas9 off-target activity in mouse embryos and human iPSCs, our WGS-based analysis of potential Cas9 RNP-associated off-target mutational events in primary human CD34+ HSPCs suggests that genome-editing utilizing electroporated Cas9 RNP complexes does not result in a significant, Cas9-specific mutational burden within recipient cells. This observation has relevance to current Cas9-based ex vivo genome-editing strategies. 2.2 Homology-independent targeted integration (HITI). We have utilized a novel NHEJ-based approach to Cas9-mediated transgene knock-in, known as HITI, to achieve robust site-specific transgene integration within human CD34+ HSPCs. As proof-of-concept, a copGFP expression cassette was targeted to the genetic locus ITGB2 encoding the beta-2 integrin subunit CD18. First, a HITI donor template bearing a CMV promoter-driven copGFP reporter gene flanked by 20-nt ITGB2-specific sgRNA target sequences (designated ITGB2-ts) was constructed and packaged within recombinant adeno-associated virus serotype 6 (rAAV6) capsids. Mobilized human CD34+ HSPCs isolated from healthy donors were transduced with rAAV6-copGFP and electroporated with pre-formed, ITGB2-targeted Cas9/sgRNA ribonucleoprotein (RNP) complexes. Upon nuclear entry, the ITGB2-ts-flanked reporter cassette and the endogenous ITGB2 target gene are concomitantly cleaved by Cas9, thus promoting NHEJ-mediated transgene insertion at the site of the Cas9-induced chromosomal double-strand break. To determine the optimal time of rAAV transduction, HSPCs were transduced with rAAV6-copGFP at either 48 hr or 36 hr pre- or 0.5 hr post-RNP electroporation. Gene edited cells were cultured for up to 28 days post-electroporation and periodically sampled for flow cytometry and genomic DNA (gDNA) extraction. Transduction of HSPCs prior to electroporation resulted in enhanced cellular viability compared to post-electroporation transduction. Flow cytometry revealed efficient rAAV transduction at 4 days post-transduction (range 18-46% copGFP+ cells). The percentage of cells expressing copGFP slowly decreased over the extended culture period, stabilizing at approximately 5-10 percent of the bulk cell population at >2 weeks post-electroporation. Site-specific transgene integration was confirmed by PCR analysis of bulk cell gDNA using reporter- and flanking gene-specific primer pairs. To estimate the frequency of transgene integration, CD34+ cells were plated in a CFU assay and transplanted into NSG mice. Approximately 12% of colonies and similar percentages of human cells at 5 months after transplantation demonstrated copGFP expression. Integration junction-specific PCR analysis of gDNA derived from GFP+ colonies and post-transplant human CD45+ cells confirmed that reporter gene expression was attributable to integrated donor template sequences, as opposed to rAAV-copGFP episomes or random transgene integration events. In summary, HITI-based transgene knock-in provides an effective alternative to HDR-mediated donor template recombination in human CD34+ HPSCs. Objective 3: Develop a targeted, non-genotoxic conditioning regimen based on anti-c-MPL antibodies conjugated to immunotoxin. In collaboration with Dr. Zhirui Wang and Dr. Diogo Magnani, proof-of-concept experiments have been initiated in non-human primates (NHP). Construction of anti-c-MPL monomeric and dimeric single-chain variable fragment (scFv) fused with the diphtheria toxin fragment 390 (DT390) is underway. This recombinant toxin fragment is safer because it lacks the native toxin-binding domain, therefore, preventing internalization of toxin in off-target cells. The resulting scFv-DT390 (69.55 kDa) and svFv2-DT390 (96.5 KDa) will be produced using the yeast Pichia pastoris expression system, as previously described for anti-CD3-DT390. Evaluation of in vitro and in vivo rhesus macaque HSPC target cytotoxicity and pharmacokinetic studies will be performed in FY20. Pending success of these proof-of-concept experiments, options will be explored to develop this concept clinically.

目标 1：开发一种基于 FV 的基因治疗方法，用于治疗 LAD-1 受试者。 1.1 评估 FV 在人 LAD-1 CD34+ 细胞中功效的临床前研究。我们进行了广泛的临床前研究，以调查表达人 CD18 cDNA (FV-hCD18) 的临床级 FV 在从分子确诊为 LAD-1 的受试者收集的 HSPC 中的功效。用FV-hCD18离体转导细胞16小时。转导后培养 3 天的 CD34+ 细胞的流式细胞术显示 39-42% 的细胞有 CD18+ 细胞表面表达。对 LAD-1 患者的 HSPC 进行基因校正可恢复这些祖细胞在体外分化的中性粒细胞的趋化功能。将 FV-hCD18 转导的 LAD-1 HSPC 移植到免疫缺陷 (NSG) 小鼠体内，可在体内产生高水平的临床相关基因标记水平。移植后5个月，小鼠骨髓中表达CD18的人类细胞的平均百分比为36.0±3.9%。对移植人类细胞内载体整合体的定量 PCR 分析表明，在大多数长期重新增殖的 HSPC 中发生了单一整合事件。对移植有 FV-hCD18 转导的 CD34+ 细胞的小鼠的 BM 进行基于流式细胞术的谱系分析，结果显示在 CD13+ 骨髓细胞和 CD20+ 淋巴细胞中均存在人类 CD18+ 细胞。使用新一代测序技术，在重新增殖的细胞中总共恢复了 101 个独特的整合位点，并揭示了多克隆整合模式，在移植后 5 个月没有插入突变或致瘤性的证据。 1.2 首次人体临床试验测试 FV 对 LAD-1 患者进行基因治疗的安全性/有效性。基于上述安全性和有效性的临床前证据，我们设计了一项使用 FV 进行 LAD-1 基因治疗的首个人体 I/II 期基因治疗临床试验。 FDA 生物制品评估和研究中心 (CBER) 的成员召开了 IND 前 B 类会议。由于 GMP 级 FV 的生产延迟，监管审查和应计费用目前暂停。目标 2：开发并评估基于 CRISPR/Cas9 的策略的安全性，用于在人类 HSPC 中永久定点特异性递送治疗基因。 2.1 评估人类 HSPC 中的脱靶 Cas9 活性我们在人类 HSPC 中进行了基于 CRISPR-Cas9 的基因组编辑，并通过单细胞衍生的 HSPC 克隆的高通量全基因组测序 (WGS)，以无偏倚、全基因组的方式评估了从头体细胞突变的获得。重要的是，为了区分自然发生的自发体细胞突变、潜在的细胞培养诱导的诱变和真正的 Cas9 介导的遗传改变，实验设计包括一组应用于相同批量 HSPC 群体样本的平行对照处理。 HSPC 样品要么 i) 模拟电穿孔，ii) 在没有效应分子的情况下电穿孔，iii) 单独使用重组 Cas9 进行电穿孔，或 iv) 使用核糖核蛋白复合物 (RNP) 进行电穿孔，该复合物由 Cas9 与靶向任一目标的单引导 RNA (sgRNA) 结合组成cxcr4 基因或安全港基因座 AAVS1。通过有限稀释来克隆实验操作的细胞，并在体外扩增单细胞分离物，以获得足够的基因组 DNA，用于 WGS 文库制备和高通量测序。我们采用体细胞变异检出算法来识别每个治疗组的 WGS 数据集中相对于未经处理的批量 HSPC 参考序列捕获的从头插入/缺失 (indel) 突变、单核苷酸变异 (SNV) 和结构变异 (SV) 。与之前调查小鼠胚胎和人类 iPSC 中 CRISPR-Cas9 脱靶活性的研究一致，我们对原代人类 CD34+ HSPC 中潜在的 Cas9 RNP 相关脱靶突变事件进行基于全基因组测序 (WGS) 的分析表明，利用电穿孔 Cas9 RNP 复合物进行基因组编辑不会在受体细胞内产生显着的 Cas9 特异性突变负担。这一观察结果与当前基于 Cas9 的离体基因组编辑策略相关。 2.2 同源无关的靶向整合（HITI）。我们利用了一种基于 NHEJ 的新型 Cas9 介导的转基因敲入方法（称为 HITI），以在人类 CD34+ HSPC 中实现强大的位点特异性转基因整合。作为概念验证，copGFP 表达盒靶向编码 beta-2 整联蛋白亚基 CD18 的遗传位点 ITGB2。首先，构建了一个 HITI 供体模板，该模板带有 CMV 启动子驱动的 copGFP 报告基因，两侧是 20 nt ITGB2 特异性 sgRNA 靶序列（称为 ITGB2-ts），并包装在重组腺相关病毒血清型 6 (rAAV6) 衣壳内。使用 rAAV6-copGFP 转导从健康供体分离的动员的人类 CD34+ HSPC，并使用预先形成的 ITGB2 靶向 Cas9/sgRNA 核糖核蛋白 (RNP) 复合物进行电穿孔。进入核后，ITGB2-ts 侧翼报告盒和内源性 ITGB2 靶基因同时被 Cas9 切割，从而促进 NHEJ 介导的转基因插入到 Cas9 诱导的染色体双链断裂位点。为了确定rAAV转导的最佳时间，在RNP电穿孔前48小时或36小时或后0.5小时用rAAV6-copGFP转导HSPC。基因编辑细胞在电穿孔后培养长达 28 天，并定期取样进行流式细胞术和基因组 DNA (gDNA) 提取。与电穿孔后转导相比，电穿孔前转导 HSPC 导致细胞活力增强。流式细胞术显示转导后 4 天有效的 rAAV 转导（范围为 18-46% copGFP+ 细胞）。表达 copGFP 的细胞百分比在延长的培养期内缓慢下降，在电穿孔后 2 周以上稳定在大细胞群的约 5-10%。使用报告基因和侧翼基因特异性引物对对大量细胞 gDNA 进行 PCR 分析，证实了位点特异性转基因整合。为了估计转基因整合的频率，将 CD34+ 细胞接种到 CFU 测定中并移植到 NSG 小鼠中。移植后 5 个月时，大约 12% 的集落和类似百分比的人类细胞表现出 copGFP 表达。对来自 GFP+ 集落和移植后人 CD45+ 细胞的 gDNA 进行整合连接特异性 PCR 分析，证实报告基因表达可归因于整合的供体模板序列，而不是 rAAV-copGFP 附加体或随机转基因整合事件。总之，基于 HITI 的转基因敲入为人类 CD34+ HPSC 中 HDR 介导的供体模板重组提供了有效的替代方案。目标 3：开发基于与免疫毒素缀合的抗 c-MPL 抗体的靶向、非基因毒性调理方案。与王志瑞博士和 Diogo Magnani 博士合作，在非人类灵长类动物 (NHP) 上启动了概念验证实验。与白喉毒素片段 390 (DT390) 融合的抗 c-MPL 单体和二聚体单链可变片段 (scFv) 的构建正在进行中。这种重组毒素片段更安全，因为它缺乏天然毒素结合结构域，因此可以防止毒素在脱靶细胞中内化。所得的 scFv-DT390 (69.55 kDa) 和 svFv2-DT390 (96.5 KDa) 将使用酵母毕赤酵母表达系统产生，如先前针对抗 CD3-DT390 所描述的。恒河猴 HSPC 靶点细胞毒性和药代动力学研究的体外和体内评估将在 2020 财年进行。在这些概念验证实验取得成功之前，将探索在临床上开发这一概念的选择。