Gene Therapy for Inherited Blood Disorders

遗传性血液疾病的基因治疗

• 批准号: 10706176
• 负责人: Andre LaRochelle
• 金额: $ 118.4万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/10706176
• 关键词: Address; Adoptive Transfer; Adult; Affect; Animals; Antibodies; Antigens; Apoptotic; Autologous Transplantation; Baculoviruses; CD34 gene; CD34+CD38- cell; CRISPR/Cas technology; CSF3 gene; CXCR4 gene; Cell Line; Cells; Cessation of life; Clinical; Clone Cells; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA Sequence; Data; Diphtheria Toxin; Dose; Electroporation; Engineering; Engraftment; Fanconi' s Anemia; Frequencies; Gene Delivery; Gene Dosage; Genes; Genetic; Genetic Heterogeneity; Genome; Genomic DNA; Genomics; Growth; Guide RNA; Half-Life; Harvest; Hematological Disease; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Human; IL3 Gene; ITGB2 gene; Immune; Immunodeficient Mouse; Impairment; In Vitro; Individual; Infusion procedures; Inherited; Insertional Mutagenesis; Intravenous infusion procedures; K-562; K562 Cells; Lentivirus; Lentivirus Vector; Lesion; Leukocyte adhesion deficiency - Type 1; Macaca mulatta; Marrow; Maximum Tolerated Dose; Measures; Mediating; Mendelian disorder; Methodology; Methods; Modeling; Modification; Molecular; Monoclonal Antibodies; Morbidity - disease rate; Mus; Mutation; Necrosis; Neutropenia; Nonhomologous DNA End Joining; Open Reading Frames; PTPRC gene; Pathway interactions; Patients; Pharmaceutical Preparations; Population; Pre-Clinical Model; Preparation; Procedures; Proteins; Proto-Oncogene Protein c-kit; Publishing; RNA Splicing; Radiation; Recombinants; Recovery; Regimen; Reporter; Reporter Genes; Retroviridae; Safety; Sampling; Sequence Homologs; Serum; Single Nucleotide Polymorphism; Site; Somatic Mutation; Spleen; Statistical Data Interpretation; Stem cell transplant; Stimulator of Interferon Genes; Sum; System; Technology; Testing; Therapeutic; Time; Toxic effect; Toxin; Transgenes; Translations; Transplantation; Transplantation Conditioning; Variant; Vesicular stomatitis Indiana virus; Viral Vector; Virus; Work; alanine aminopeptidase; antibody conjugate; base; causal variant; cellular transduction; chemotherapeutic agent; chemotherapy; clinically relevant; conditioning; cytotoxic; delivery vehicle; density; engineered nucleases; experimental group; experimental study; flexibility; gene therapy; genome editing; genome-wide; genomic locus; genotoxicity; immune function; in vivo; in vivo engraftment; insertion/deletion mutation; mortality; mouse model; novel; nuclease; post-transplant; programs; receptor; reconstitution; repaired; risk minimization; senescence; single cell analysis; small molecule inhibitor; subcutaneous; targeted treatment; therapeutic gene; therapeutic transgene; vector

Objective 1: Develop a targeted preparative regimen for HSPC gene therapy We hypothesized that cMPL might be a relevant antigen for an antibody-based targeted depletion of human HSPCs and provide the basis for a safer conditioning regimen prior to transplant. To investigate this possibility, we produced a recombinant anti-cMPL bivalent (bi) single-chain fragment variable (scFV) fused with diphtheria toxin (DT) truncated at residue 390 (DT390-biscFV(cMPL). We first confirmed the cMPL-dependent cytotoxic effects of the DT390-biscFV(cMPL) conjugate in a HEK293A cell line engineered to express the human cMPL receptor. Next, we assessed DT390-biscFV(cMPL) for its ability to inhibit growth of human CD34+ cells in vitro. Consistent with a cMPL dependent cytotoxic effect, increased cellular death was measured in populations expressing higher densities of cMPL receptors, suggesting preferential targeting of the most primitive hematopoietic compartment. We then assessed whether DT390-biscFV(cMPL) could safely deplete human HSPCs in vivo in a humanized NBSGW mouse model. At 12 weeks post-transplantation, engrafted animals received a single maximum tolerated dose (1.2 mg/kg) of DT390-biscFV(cMPL) or vehicle. We observed a gradual decline in HSPC activity, with a peak 2.6-fold reduction in frequency of human CD45+CD13+ cells at 6 weeks following administration of DT390-biscFV(cMPL) compared to untreated animals (p = 0.003). To further evaluate safety and efficacy in vivo in a more clinically-relevant model, we administered single doses of 0.2, 0.4, 0.6 or 0.8 mg/kg DT390-biscFV(cMPL) by intravenous infusion into three independent rhesus macaques. We selectively depleted >90% cMPL+CD34+ cells at all doses tested. Notably, the drug displayed a short serum half-life (33-163 minutes), thus conferring a distinct advantage for pre-transplant conditioning applications. The overall safety profile was favorable. Objective 2: Evaluate the impact of post-transplant G-CSF administration on gene-edited HSPCs. G-CSF is commonly administered to hasten recovery from chemotherapy-induced neutropenia after autologous transplantation. However, in patients pre-treated with chemotherapeutic agents, G-CSF was shown to exacerbate HSPC toxicity triggered by these drugs by prompting their differentiation or senescence. We reasoned that G-CSF might also negatively impact HSPCs treated with DNA-damaging programmable nucleases and potentially reduce their engraftment in vivo. To address this question, human CD34+ cells were gene-edited by electroporation of sgRNA/Cas9 RNP complexes and subsequently transplanted into NSG mice. We subcutaneously injected G-CSF or a vehicle control solution once daily for the first 14 days after cell infusion and compared hematopoietic reconstitution between experimental groups. From week 10 post-transplantation, administration of G-CSF resulted in a 3- to 4-fold reduction in PB human cell engraftment compared to untreated mice and a marked reduction in human repopulating activity within the spleen and marrow at the endpoint (22 weeks) analysis. In limiting-dilution secondary transplant experiments, the overall frequency of HSPCs with long-term repopulating ability was reduced by 9.7-fold with G-CSF administration post-transplant (p = 0.011). Notably, vehicle- and G-CSF-treated mice revealed no difference in human cell engraftment after transplantation of unmanipulated HSPCs or HSPCs electroporated with Cas9 alone or HSPCs transduced with lentivirus vectors expressing GFP. Collectively, our data suggest that G-CSF use post-transplant significantly impairs long-term engraftment of CRISPR-Cas9 gene-edited HSPCs. Objective 3: Develop safe and efficient HSPC gene editing approaches HDR-based gene editing of human HSPCs. To facilitate HDR-mediated targeted integration of large DNA cargoes for therapeutic applications, we have evaluated the use of vectors based on baculovirus (BV) as alternative gene delivery vehicles in human HSPCs. Baculovirus vectors are capable of packaging inserts of at least 38 kb in size. We constructed VSV-G pseudotyped BV vectors harboring a copGFP reporter flanked by 4kb sequences homologous to the ITGB2 genomic locus implicated in LAD-1. To evaluate vector functionality, we first transduced 293A and K562 cell lines as well as primary human HSPCs. Exogenous expression of copGFP was robust in 293A cells, but approximately 7-fold lower in the hematopoietic cell line K562 and mostly undetectable in CD34+ and CD34+CD38- cell populations, independent of vector MOI or duration of transduction. We uncovered an early innate immune block to BV transduction in K562 cells and primary human HSPCs mediated by the cGAS-STING cytosolic DNA sensing pathway. The transduction blockade could be overcome in part by a brief (45-minute) pre-treatment with H-151, a potent small molecule inhibitor of STING, in combination with zVAD and Nec-1 compounds to limit activation of apoptotic and necrotic pathways. We next evaluated the ability of electroporated sgRNA/Cas9 RNPs to direct the integration of a BV-packaged copGFP reporter gene construct at the ITGB2 locus in K562 cells. The levels of GFP expression observed in cells treated with BV+RNP stabilized by day 21, with an average of 12% GFP+ cells observed during the remaining duration of the time-course. Stable targeted integration at the ITGB2 locus was confirmed by in-out PCR amplification and ddPCR of genomic DNA isolated from edited K562 cells at the end of culture. NHEJ-based gene editing of human HSPCs. Since DNA DSB lesions are predominantly repaired by the NHEJ mechanism in HSPCs, we hypothesized that an NHEJ-based approach to gene addition in HSPCs might facilitate efficient site-specific transgene integration in these cells and provide an alternative to HDR-mediated gene editing methods. In this study, we utilized a homology-independent targeted integration (HITI)-based approach to achieve robust site-specific transgene integration in human adult CD34+ HSPCs. As proof-of-concept, a reporter gene was targeted to a clinically relevant genetic locus (ITGB2) using a rAAV6 vector and sgRNA/Cas9 RNP complexes. We demonstrate high levels of stable HITI-mediated genome editing (21%) in repopulating HSPCs after transplant into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs for the treatment of monogenic diseases affecting the hematopoietic system. This work was published Molecular Therapy 2021, and J. Clin. Med. 2021. Objective 4. Assess genotoxicity related to gene editing of human HSPCs To address the significance of off-target CRISPR/Cas9 activity for the treatment of inherited blood disorders, we performed Cas9 RNP-based gene editing at two genetic loci (CXCR4 and AAVS1) in human mobilized PB CD34+ cells and characterized the genome-wide accumulation of post-editing somatic mutations using high-throughput WGS analysis of single-cell-derived HSPC clones. Total somatic variants, including indels, single nucleotide variants (SNVs), and structural variants (SVs), were compared to Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. The median number of novel SNVs was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. SVs were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. In sum, we found that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity. This work was published Genes 2020.

目标1：制定HSPC基因治疗的靶向准备方案 我们假设 cMPL 可能是基于抗体的人类 HSPC 靶向清除的相关抗原，并为移植前更安全的预处理方案提供了基础。为了研究这种可能性，我们产生了与在残基 390 处截短的白喉毒素 (DT) 融合的重组抗 cMPL 二价 (bi) 单链片段变量 (scFV) (DT390-biscFV(cMPL)。我们首先证实了 cMPL 依赖性DT390-biscFV(cMPL) 缀合物在表达人 cMPL 的 HEK293A 细胞系中的细胞毒性作用接下来，我们评估了 DT390-biscFV(cMPL) 体外抑制人 CD34+ 细胞生长的能力，与 cMPL 依赖性细胞毒性作用一致，在表达较高密度的 cMPL 受体的群体中测量到细胞死亡增加，表明优先靶向。然后我们评估了 DT390-biscFV(cMPL) 是否可以在体内安全地消耗人类 HSPC。移植后 12 周，移植动物接受单次最大耐受剂量 (1.2 mg/kg) DT390-biscFV(cMPL) 或载体，观察到 HSPC 活性逐渐下降，峰值为 2.6-。与未治疗相比，施用 DT390-biscFV(cMPL) 后 6 周时，人 CD45+CD13+ 细胞的频率减少了几倍动物（p = 0.003）。为了在更具临床相关性的模型中进一步评估体内安全性和有效性，我们通过静脉输注给三只独立的恒河猴进行单剂量 0.2、0.4、0.6 或 0.8 mg/kg DT390-biscFV(cMPL)。我们在所有测试剂量下选择性地去除>90%的cMPL+CD34+细胞。值得注意的是，该药物表现出较短的血清半衰期（33-163 分钟），从而为移植前调理应用提供了明显的优势。总体安全状况良好。 目标 2：评估移植后 G-CSF 给药对基因编辑 HSPC 的影响。 G-CSF 通常用于加速自体移植后化疗引起的中性粒细胞减少症的恢复。然而，在接受化疗药物预处理的患者中，G-CSF 通过促进其分化或衰老而加剧了这些药物引发的 HSPC 毒性。我们推断，G-CSF 也可能对用 DNA 损伤性可编程核酸酶处理的 HSPC 产生负面影响，并可能减少它们在体内的植入。为了解决这个问题，通过电穿孔 sgRNA/Cas9 RNP 复合物对人类 CD34+ 细胞进行基因编辑，然后移植到 NSG 小鼠体内。在细胞输注后的前 14 天，我们每天一次皮下注射 G-CSF 或载体对照溶液，并比较实验组之间的造血重建情况。从移植后第 10 周开始，与未治疗的小鼠相比，给予 G-CSF 导致 PB 人类细胞植入减少 3 至 4 倍，并且在终点（22 周）时，脾脏和骨髓内的人类再生活性显着降低） 分析。在有限稀释二次移植实验中，移植后给予 G-CSF 后，具有长期再增殖能力的 HSPC 的总体频率降低了 9.7 倍（p = 0.011）。值得注意的是，在移植未操作的 HSPC 或仅用 Cas9 电穿孔的 HSPC 或用表达 GFP 的慢病毒载体转导的 HSPC 移植后，用媒介物和 G-CSF 处理的小鼠显示人类细胞植入没有差异。总的来说，我们的数据表明，移植后使用 G-CSF 会显着损害 CRISPR-Cas9 基因编辑的 HSPC 的长期植入。 目标3：开发安全高效的HSPC基因编辑方法 基于 HDR 的人类 HSPC 基因编辑。为了促进 HDR 介导的大 DNA 货物的靶向整合用于治疗应用，我们评估了基于杆状病毒 (BV) 的载体作为人类 HSPC 中替代基因递送载体的用途。杆状病毒载体能够包装大小至少为 38 kb 的插入片段。我们构建了 VSV-G 假型 BV 载体，其中含有 copGFP 报告基因，两侧是与 LAD-1 涉及的 ITGB2 基因组位点同源的 4kb 序列。为了评估载体功能，我们首先转导 293A 和 K562 细胞系以及原代人类 HSPC。 copGFP 的外源表达在 293A 细胞中很强，但在造血细胞系 K562 中低约 7 倍，并且在 CD34+ 和 CD34+CD38- 细胞群中几乎检测不到，与载体 MOI 或转导持续时间无关。我们发现了由 cGAS-STING 胞质 DNA 传感途径介导的 K562 细胞和原代人 HSPC 中 BV 转导的早期先天免疫阻断。通过使用 H-151（一种有效的 STING 小分子抑制剂）进行短暂（45 分钟）预处理，结合 zVAD 和 Nec-1 化合物限制细胞凋亡和坏死途径的激活，可以部分克服转导阻断。接下来，我们评估了电穿孔 sgRNA/Cas9 RNP 指导 BV 包装的 copGFP 报告基因构建体在 K562 细胞中 ITGB2 基因座处整合的能力。在用 BV+RNP 处理的细胞中观察到的 GFP 表达水平在第 21 天稳定，在剩余时间过程中观察到平均 12% 的 GFP+ 细胞。在培养结束时对从编辑的 K562 细胞中分离的基因组 DNA 进行 in-out PCR 扩增和 ddPCR 证实了 ITGB2 基因座的稳定靶向整合。 基于 NHEJ 的人类 HSPC 基因编辑。由于 DNA DSB 损伤主要由 HSPC 中的 NHEJ 机制修复，我们假设在 HSPC 中基于 NHEJ 的基因添加方法可能会促进这些细胞中有效的位点特异性转基因整合，并为 HDR 介导的基因编辑方法提供替代方案。在这项研究中，我们利用基于同源性独立靶向整合（HITI）的方法在人类成人 CD34+ HSPC 中实现稳健的位点特异性转基因整合。作为概念验证，使用 rAAV6 载体和 sgRNA/Cas9 RNP 复合物将报告基因靶向临床相关遗传位点 (ITGB2)。我们在移植到免疫缺陷小鼠体内后，在 HSPC 重新增殖过程中展示了高水平的稳定 HITI 介导的基因组编辑 (21%)。我们的研究表明，HITI 介导的基因组编辑为 CD34+ HSPC 中基于 HDR 的转基因整合提供了一种有效的替代方案，用于治疗影响造血系统的单基因疾病。这项工作发表于《分子治疗 2021》和《J. Clin》。医学。 2021 年。 目标 4. 评估与人类 HSPC 基因编辑相关的遗传毒性 为了解决脱靶 CRISPR/Cas9 活性对于治疗遗传性血液疾病的重要性，我们在人类动员的 PB CD34+ 细胞中的两个基因位点（CXCR4 和 AAVS1）进行了基于 Cas9 RNP 的基因编辑，并表征了全基因组积累使用单细胞衍生的 HSPC 克隆的高通量 WGS 分析进行后编辑体细胞突变。将总体细胞变异，包括插入缺失、单核苷酸变异 (SNV) 和结构变异 (SV) 与 Cas9 处理和非 Cas9 处理的对照 HSPC 克隆进行比较。统计分析显示样本中新型非靶向插入缺失的数量没有显着差异。与基线相比，Cas9 RNP 受体样本组中新型 SNV 的中位数略有升高，但未达到统计学显着性。 SV 很罕见，并且与 Cas9 介导的基因编辑程序没有明确的因果关系。总之，我们发现在 Cas9 RNP 编辑的人类 HSPC 克隆中观察到的集体体细胞突变负担与自然发生的背景遗传异质性水平无法区分。该工作发表于Genes 2020。