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：
10929162
负责人：
Andre LaRochelle
金额：
$ 133.26万
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/10929162
关键词：
Address Adult Agonist Animals Antibodies Antigens Apoptosis Attenuated Autologous Autologous Transplantation Bar Codes Biodistribution Bone marrow failure Bypass CRISPR/Cas technology Cell Differentiation process Cells Chimeric Proteins Clinical Clinical Trials Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Damage DNA Double Strand Break DNA Ligation DNA Repair DNA Repair Disorder Dendrimers Differentiation Antigens Diphtheria Toxin Disease Dose Double Strand Break Repair Electroporation Encapsulated Engineering Engraftment Evaluation Event Exposure to Fanconi Anemia Complementation Group A Protein Fanconi&apos s Anemia Formulation Gammaretrovirus Gene Delivery Gene Modified Genes Genetic Genetic Engineering Genetic Transcription Genome Granulocyte Colony-Stimulating Factor Growth HIV Integrase Hematological Disease Hematopoietic stem cells Human Hypersensitivity Hypoxia IL3 Gene Image Immune Impairment In Situ In Vitro Inflammation Mediators Inflammatory Inherited Innate Immune Response Interferons Iodides Knock-in Mouse Lead Lentivirus Lentivirus Vector Lipids Marrow Mediating Messenger RNA Methods Modeling Molecular Monitor Morbidity - disease rate Mutation Nature Neutropenia Nucleic Acids Open Reading Frames Outcome PET/CT scan Pathway interactions Patients Population Procedures Property Proteomics Protocols documentation Radiation Reaction Reagent Recombinants Recovery Regimen Reserve Cell Residual state Retroviral Vector Risk Role Safety Sleeping Beauty Sodium Specificity System TP53 gene Testing Therapeutic Toxic effect Transplantation Transplantation Conditioning Transposase Viral Xenograft Model bone marrow failure syndrome cancer cell causal variant chemical conjugate chemotherapeutic agent chemotherapy conditioning constitutive expression cytokine cytotoxic design dosage endoplasmic reticulum stress experimental study fitness follow-up gene correction gene delivery system gene therapy gene transfer vector genetic approach genetic manipulation genetic payload genetically modified cells genome editing genomic locus hematopoietic engraftment in vivo in vivo Model inhibitor innate immune pathways intravenous administration intravenous injection lipid nanoparticle mortality mouse model mutant nanoparticle delivery nonhuman primate novel novel strategies nuclease nucleic acid delivery post-transplant pre-clinical precise genome editing preclinical study programs proteostasis radiotracer recombinase repaired response risk minimization self-renewal sensor stem cell function symporter systemic toxicity transcription factor transcriptomics vector

项目摘要

Objective 1: Develop novel HSPC gene correction strategies Because multiple causative mutations have been identified in FA, targeted introduction of a therapeutic open reading frame at the endogenous FANCA genetic locus is desirable to allow single-construct genetic correction regardless of the nature of downstream mutations. Due to faulty DNA repair mechanisms in FA cells, genome editing approaches that rely on CRISPR-Cas9-mediated DNA DSBs and subsequent repair are impractical in FA HSPCs. Accordingly, we are developing novel strategies that bypass the DNA damage response (DDR) for targeted integration. To this end, we have incorporated the programmability of CRISPR-Cas9 with the integration efficiency of DNA recombinase domains known to mediate enzymatic DNA ligation reactions without formation of de novo DNA free ends. Namely, we have constructed chimeric proteins with nuclease-deficient mutant of Cas9 (dCas9) molecularly fused to tandem domains of known eukaryotic transposases (e.g., Tn5, PiggyBac, and Sleeping Beauty) or HIV integrase to enable dCas9-directed, recombinase-mediated, DDR-independent integration of an FA donor substrate at the FANCA genetic locus. Testing of these approaches is underway. Objective 2: Facilitate safe and efficient engraftment of gene-edited HSPCs Pre-transplant conditioning with chemotherapy-free regimens. 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 have produced a recombinant anti-cMPL bivalent (bi) single-chain fragment variable (scFV) fused with diphtheria toxin (DT) truncated at residue 390 (DT390-biscFV(cMPL)). This agent has enabled HSPC depletion in pre-clinical in vitro and non-human primate (NHP) models. Further optimization of dosage is underway in a knock-in mouse model harboring human TPO and cMPL gene sequences. We are also pursuing autologous transplantation of genetically barcoded HSPCs in NHPs conditioned with DT390-biscFV(cMPL). The safety and efficacy profiles is monitored long-term and detailed quantitative longitudinal follow-up is performed in barcoded animals to assess stability of contributions from engrafted HSPC clones. Increase cell dose by ex vivo expansion of gene-edited HSPCs. To develop a clinically feasible platform for the expansion of genetically modified long-term repopulating adult HSPCs, we are building upon recent advances to develop a synthetic, cytokine-free expansion culture system that addresses the limited efficacy and batch-to-batch variability of current approaches. Three strategies are combined to further optimize culture conditions for HSPC expansion: 1) Promoting HSPC self-renewal. Key transcriptional regulators (e.g., HOXB4) have been identified as potential targets to enhance HSPC self-renewal in culture. Because constitutive expression of growth-promoting transcription factors (TFs) by viral transduction poses safety risks, we transiently express single or combined self-renewal regulators within target HSPCs in culture using lipid nanoparticle (LNP)-based transfer of TF mRNA; 2) Suppressing HSPC differentiation. We have recently identified 78c, a potent inhibitor of the CD38 differentiation marker, as a lead synthetic candidate for active suppression of HSPC differentiation in culture. 3) Mitigating endoplasmic reticulum (ER) stress. Recent studies have highlighted how increased proliferative demand triggers ER stress perturbations that collectively impair HSC function in ex vivo culture. To limit activation of ER stress pathways during expansion, we evaluate HSPC cultures under hypoxic conditions and supplemented with synthetic agonists of Hsf1 (e.g., 17-AAG) recently shown to limit ER stress by rebalancing proteostasis. Increase cell fitness by overcoming innate immune responses. A growing body of experimental evidence suggests a pivotal role of host antiviral factors and nucleic acid sensors in limiting the efficacy of HSPC genetic manipulation. To characterize innate immune pathways triggered by reagents used for genetic engineering of HSPCs, we are conducting unbiased proteomic and single-cell transcriptomic screens on human HSC-enriched populations exposed to commonly used gene delivery systems, including vectors based on RV, LV, FV and AAV6, as well electroporation and lipid nanoparticle carriers of nucleic acid constituents (e.g., DNA and mRNA). These findings will inform novel approaches to overcome immune blocks to nucleic acid delivery and enhance gene correction efficiencies by promoting cellular survival and fitness. Evaluate the impact of post-transplant G-CSF administration on gene-edited HSPCs. Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. We provided evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA DSBs. Transient p53 inhibition in culture attenuated the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF did not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. BioRxiv 2023: doi: 10.1101/2023.06.29.547089. Objective 3: Develop in vivo gene therapy strategies In vivo delivery of genetic payloads could circumvent the shortcomings of current ex vivo gene correction approaches and represent a distinct advance for gene therapy of Fanconi anemia. Among available in vivo delivery methods, LNPs are the most developed for clinical use. A 3-step preclinical study is underway to provide a comprehensive evaluation of the efficiency (Aim 1), safety (Aim 2) and therapeutic applicability (Aim 3) of novel LNP delivery systems. Aim 1- In pilot experiments, we have identified and optimized a novel LNP formulation based on the ionizable dendrimer amino lipid 4A3-SC852, and shown efficacy for delivery of nucleic acid cargoes to purified human HSPCs in vitro. Building on our previous studies demonstrating cMPL as a relevant antigen to target HSPCs, optimized LNP formulations have been chemically conjugated to a recombinant anti-cMPL bivalent single-chain fragment variable (biscFV(cMPL)) to facilitate selective delivery of CRISPR reagents to HSPCs in a NHP model in vivo. Aim 2- Specificity of LNP-mediated delivery of CRISPR reagents to HSPCs is imperative to limit off-target gene editing and systemic toxicity in vivo. To address this question, biodistribution of LNP formulations administered intravenously to NHPs are investigated using established in vivo tracking approaches. Briefly, biscFV(cMPL)-conjugated LNP formulations are encapsulated with the sodium/iodide symporter (NIS) mRNA, infused to the animals, and tracked by whole-body PET/CT scan imaging at select timepoints following intravenous injection of an 18F-tetrafluoroborate radiotracer. Aim 3- Integration of an FA donor substrate at the endogenous locus within HSPCs will be pursued in NHPs by cMPL-conjugated LNP delivery of a dCas9-directed, recombinase-mediated, DDR-independent genome editing system.

目标 1：开发新的 HSPC 基因校正策略由于 FA 中已鉴定出多个致病突变，因此需要在内源性 FANCA 基因位点上有针对性地引入治疗性开放阅读框，以允许单构建体遗传校正，而不管下游突变的性质如何。由于 FA 细胞中存在缺陷的 DNA 修复机制，依赖 CRISPR-Cas9 介导的 DNA DSB 和后续修复的基因组编辑方法在 FA HSPC 中是不切实际的。因此，我们正在开发绕过 DNA 损伤反应 (DDR) 进行靶向整合的新策略。为此，我们将 CRISPR-Cas9 的可编程性与已知可介导酶促 DNA 连接反应而不形成从头 DNA 自由端的 DNA 重组酶域的整合效率相结合。也就是说，我们构建了嵌合蛋白，其核酸酶缺陷突变体 Cas9 (dCas9) 与已知真核转座酶（例如 Tn5、PiggyBac 和睡美人）或 HIV 整合酶的串联结构域进行分子融合，以实现 dCas9 定向、重组酶介导、 FA 供体底物在 FANCA 基因位点上的 DDR 独立整合。这些方法的测试正在进行中。目标 2：促进基因编辑 HSPC 的安全高效植入采用无化疗方案的移植前调理。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，我们生产了与在残基 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))。该药物已在临床前体外和非人灵长类动物 (NHP) 模型中实现了 HSPC 消耗。在含有人 TPO 和 cMPL 基因序列的敲入小鼠模型中，剂量的进一步优化正在进行中。我们还致力于在 DT390-biscFV(cMPL) 条件下的 NHP 中进行基因条形码 HSPC 的自体移植。对条形码动物进行长期监测和详细的定量纵向随访，以评估移植的 HSPC 克隆贡献的稳定性。通过离体扩增基因编辑的 HSPC 来增加细胞剂量。为了开发一个临床上可行的平台来扩增转基因长期再增殖的成年 HSPC，我们正在利用最新进展来开发一种合成的、无细胞因子的扩增培养系统，该系统可解决当前的有限功效和批次间差异的问题。接近。结合三种策略，进一步优化 HSPC 扩增的培养条件：1）促进 HSPC 自我更新。关键转录调节因子（例如 HOXB4）已被确定为增强培养中 HSPC 自我更新的潜在靶标。由于病毒转导促进生长转录因子 (TF) 的组成型表达存在安全风险，因此我们使用基于脂质纳米颗粒 (LNP) 的 TF mRNA 转移，在培养物中的目标 HSPC 中瞬时表达单个或组合的自我更新调节因子； 2)抑制HSPC分化。我们最近发现了 78c，一种 CD38 分化标记物的有效抑制剂，可作为主动抑制培养物中 HSPC 分化的主要合成候选物。 3）减轻内质网（ER）应激。最近的研究强调了增殖需求的增加如何引发 ER 应激扰动，从而共同损害离体培养中的 HSC 功能。为了限制扩增过程中 ER 应激途径的激活，我们评估了缺氧条件下的 HSPC 培养物，并补充了最近显示可通过重新平衡蛋白质稳态来限制 ER 应激的 Hsf1 合成激动剂（例如 17-AAG）。通过克服先天免疫反应来增强细胞健康。越来越多的实验证据表明，宿主抗病毒因子和核酸传感器在限制 HSPC 基因操作的功效方面发挥着关键作用。为了表征用于 HSPC 基因工程的试剂触发的先天免疫途径，我们正在对暴露于常用基因传递系统（包括基于 RV、LV、FV 和AAV6，以及核酸成分（例如 DNA 和 mRNA）的电穿孔和脂质纳米颗粒载体。这些发现将为克服核酸传递的免疫障碍并通过促进细胞存活和健康来提高基因校正效率的新方法提供信息。评估移植后 G-CSF 管理对基因编辑 HSPC 的影响。粒细胞集落刺激因子 (G-CSF) 通常用作辅助治疗，以加速化疗和造血干细胞和祖细胞 (HSPC) 恶性疾病自体移植后中性粒细胞减少症的恢复。然而，针对人类 HSPC 的离体基因治疗程序后给予 G-CSF 的效用尚未得到彻底评估。我们提供的证据表明，移植后施用 G-CSF 会阻碍 CRISPR-Cas9 基因编辑的人类 HSPC 在异种移植模型中的植入。 G-CSF 通过加剧由 Cas9 介导的 DNA DSB 触发的 p53 介导的 DNA 损伤反应来发挥作用。培养物中短暂的 p53 抑制减弱了 G-CSF 对基因编辑的 HSPC 功能的负面影响。相比之下，移植后施用 G-CSF 不会损害未经操作的人类 HSPC 或通过慢病毒载体转导进行基因工程改造的 HSPC 的再增殖特性。在设计离体自体 HSPC 基因编辑临床试验时，应考虑移植后给予 G-CSF 可能会加重与 CRISPR-Cas9 基因编辑相关的 HSPC 毒性。 BioRxiv 2023：doi：10.1101/2023.06.29.547089。目标 3：开发体内基因治疗策略基因有效负载的体内传递可以克服当前离体基因校正方法的缺点，代表范可尼贫血基因治疗的显着进步。在可用的体内递送方法中，LNP 是最适合临床使用的。一项三步临床前研究正在进行中，以对新型 LNP 递送系统的效率（目标 1）、安全性（目标 2）和治疗适用性（目标 3）进行综合评估。目标 1 - 在初步实验中，我们鉴定并优化了一种基于可电离树枝状聚合物氨基脂质 4A3-SC852 的新型 LNP 制剂，并显示了在体外将核酸货物递送至纯化的人 HSPC 的功效。基于我们之前的研究证明 cMPL 作为靶向 HSPC 的相关抗原，优化的 LNP 制剂已与重组抗 cMPL 二价单链片段变量 (biscFV(cMPL)) 化学缀合，以促进 CRISPR 试剂选择性递送至 HSPC NHP体内模型。目标 2 - LNP 介导的 CRISPR 试剂向 HSPC 的特异性递送对于限制脱靶基因编辑和体内系统毒性至关重要。为了解决这个问题，使用已建立的体内跟踪方法研究了静脉注射至 NHP 的 LNP 制剂的生物分布。简而言之，biscFV（cMPL）缀合的 LNP 制剂用钠/碘同向转运蛋白（NIS）mRNA 封装，注入动物体内，并在静脉注射 18F-四氟硼酸盐后的选定时间点通过全身 PET/CT 扫描成像进行跟踪放射性示踪剂。目标 3 - 将通过 cMPL 缀合的 LNP 传递 dCas9 定向、重组酶介导、不依赖于 DDR 的基因组编辑系统，在 NHP 中实现 FA 供体底物在 HSPC 内源位点的整合。