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，用于用LAD-1治疗受试者。 1.1临床前研究评估FV在人LAD-1 CD34+细胞中的功效。我们已经进行了广泛的临床前研究，以研究表达人CD18 cDNA（FV-HCD18）的临床级FV的功效，这些HSPC是从具有分子确定的LAD-1诊断的受试者收集的HSPC中。用FV-HCD18转导细胞16小时。转导后3天培养的CD34+细胞的流式细胞仪显示CD18+细胞表面表达在39-42％的细胞中。来自LAD-1患者的HSPC的遗传校正恢复了与这些祖细胞在体外不同的中性粒细胞的趋化功能。将FV-HCD18转导的LAD-1 HSPC移植到免疫缺陷型（NSG）小鼠中导致体内高水平，临床相关的基因标记水平。移植后5个月5个月，在鼠BM中表达CD18的人类细胞的平均百分比为36.0 3.9％。对植入的人类细胞中载体整合物的定量PCR分析表明，大多数长期重塑HSPC发生了单个整合事件。基于流式细胞仪的基于FV-HCD18转导的CD34+细胞移植的小鼠的BM基于流式细胞仪的谱图分析显示，CD13+髓样和CD20+淋巴室中的人CD18+细胞均显示出人CD18+细胞。使用下一代的测序技术，在重脑细胞中总共回收了101个独特的整合位点，并揭示了整合的多克隆模式，而没有插入诱变或肿瘤性五个月后五个月后插入五个月的迹象。 1.2人类临床试验测试FV对LAD-1患者基因治疗的安全性/功效。基于上面讨论的安全性和功效的临床前证据，我们设计了使用FV使用FV进行LAD-1基因治疗的第一阶段I/II期基因治疗临床试验。在FDA中，与生物制剂评估与研究中心（CBER）的成员举行了预先开发的B型会议。由于GMP级FV的生产延迟，目前监管审查和应计。目标2：开发和评估基于CRISPR/CAS9的策略的安全性，以在人类HSPC中的治疗基因的永久性特定地点传递。 2.1评估人HSPC中的靶向cas9活动我们在人类HSPC中进行了基于CRISPR-CAS9的基因组编辑，并通过高通量，全基因组测序（WGS）以单细胞衍生的HSPC克隆的高通量，全基因组测序（WGS）评估了从头体细胞突变的获取。重要的是，为了区分自然发生的自发性体细胞突变，潜在的细胞培养诱导的诱变和真正的CAS9介导的遗传改变，实验设计包括一组适用于同一体积HSPC种群样品的控制处理。 HSPC样品是i）对电穿孔进行模拟的，ii）在没有效应分子的情况下进行电穿孔，iii）单独用重组Cas9电穿孔，或者用核糖核蛋白复合物（RNP）（RNP）电穿孔，由CAS9与单个指导RNAS（SGRNAS）（SGRNAS）（SGRNAS）（SGRNAS）靶向CXCR 4 gene或CASCR 4 Genee或CASCR 4 Genee kene或CASCR。通过限制稀释来克隆实验操纵的细胞，并在体外扩展单细胞分离株，以获得足够的基因组DNA，以进行WGS文库制备和高通量测序。我们采用了召集算法的体差异来识别从头插入/缺失（INDEL）突变，单核苷酸变体（SNV）和结构变体（SVS）（SVS）在WGS数据集中捕获的每个处理组中相对于未经治疗的散装，散装HSPC参考序列。与先前研究小鼠胚胎和人IPSC中CRISPR-CAS9非靶向活性的研究一致，我们基于WGS对原代人CD34+ HSPC中潜在的CAS9 RNP相关的脱靶突变事件的分析表明，使用电型Cas9 RNP复合物的基因组编辑的CAS9 RNP复合物在Cas9 pec9 prenden of cas9 pectectient bunden中，基因组编辑并不是在内部。该观察结果与当前基于CAS9的离体基因组编辑策略相关。 2.2独立于同源性靶向整合（HITI）。我们已经利用了一种新型的基于NHEJ的方法，用于CAS9介导的转基因敲入（称为HITI），以实现人CD34+ HSPC中强大的位点特异性转基因整合。作为概念验证，COPGFP表达盒针对编码Beta-2整合素亚基CD18的遗传基因座ITGB2。首先，在重组与腺相关的病毒血清型6（RAAV6）Capsids中构建并包装了一个带有CMV启动子驱动的COPGFP报告基因基因的基因（指定的ITGB2-TS）。用RAAV6-COPGFP转导从健康供体中分离的人类CD34+ HSPC，并用预先形成的ITGB2靶向CAS9/SGRNA核糖核蛋白（RNP）配合物进行电穿孔。进入核进入后，ITGB2-TS-FANKERETER盒和内源性ITGB2靶基因也会被CAS9裂解，从而促进了Cas9诱导的染色体染色体染色体双链破裂的NHEJ介导的转基因插入。为了确定RAAV转导的最佳时间，在48小时或36 hr前或0.5小时RNP电穿孔后，用RAAV6-COPGFP转导HSPC。将基因编辑的细胞培养长达28天，并定期采样用于流式细胞仪和基因组DNA（GDNA）提取。与电动倍倍期转导相比，电穿孔之前HSPC的转导导致细胞活力增强。流式细胞仪显示在转变后4天（范围为18-46％的COPGFP+细胞）有效的RAAV转导。在扩展的培养期间，表达COPGFP的细胞百分比慢慢降低，在电动载物后> 2周时，大约5-10％的散装细胞群体稳定。使用报告基因和侧翼基因特异性引物对通过PCR分析Bulk细胞GDNA来证实位点特异性的转基因整合。为了估计转基因整合的频率，将CD34+细胞放在CFU测定中并移植到NSG小鼠中。移植后5个月，大约12％的菌落和类似百分比的人类细胞表现出COPGFP的表达。源自GFP+菌落和移植后人CD45+细胞的GDNA的整合连接特异性PCR分析证实，报告基因表达表达可归因于集成的供体模板序列，而不是RAAV-COPGFP或随机转基因整合事件。总而言之，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替代方法。目标3：基于与免疫毒素结合的抗C-MPL抗体，开发一种靶向的非生物毒性调节方案。与Zhirui Wang博士和Diogo Magnani博士合作，已在非人类灵长类动物（NHP）中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，因为它缺乏天然的毒素结合结构域，因此可以防止靶靶细胞中毒素的内在化。所得的SCFV-DT390（69.55 kDa）和SVFV2-DT390（96.5 kDa）将使用酵母pichia Pastoris表达系统产生，如先前针对抗CD3-DT390所述。体外和体内恒河猕猴HSPC靶标细胞毒性和药代动力学研究将在FY20中进行。在这些概念验证实验的成功之前，将探讨选项以在临床上发展这一概念。