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：制定新型HSPC基因校正策略由于在FA中已经鉴定出多种病因突变，因此，无论下游突变的性质如何，都希望在内源性芬卡遗传基因座上引入治疗性开放式阅读框。由于FA细胞中的DNA修复机制故障，因此在FA HSPC中，基因组编辑方法依赖于CRISPR-Cas9介导的DNA DSB和随后的修复是不切实际的。因此，我们正在开发新的策略，这些策略绕过了目标整合的DNA损伤响应（DDR）。为此，我们将CRISPR-CAS9的可编程性与已知可介导酶促DNA连接反应的DNA重组酶结构域的整合效率结合在一起，而不会形成从头DNA的无端。 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范卡遗传基因座。这些方法的测试正在进行中。目标2：促进基因编辑的HSPC的安全有效植入带有无化疗方案的移植前调节。我们假设CMPL可能是人类HSPC的基于抗体的靶向耗竭的相关抗原，并为移植前更安全的条件方案提供了基础。为了调查这种可能性，我们生产了与残基390（DT390-BISCFV（CMPL））截断的白喉毒素（DT）融合的重组抗CMPL（BI）单链片段变量（SCFV）。该药物在体外和非人类灵长类动物（NHP）模型中启用了HSPC耗竭。在具有人类TPO和CMPL基因序列的敲门小鼠模型中正在进行剂量的进一步优化。我们还在以DT390-BISCFV（CMPL）为条件的NHP中追求基因条形码HSPC的自体移植。长期监测安全性和疗效曲线，并在条形码动物中进行详细的定量纵向随访，以评估植入的HSPC克隆的贡献的稳定性。通过体内扩展基因编辑的HSPC来增加细胞剂量。为了开发一个临床上可行的平台，用于扩展基因经过转基因的长期重塑成人HSPC，我们正在基于最新的进步，以开发一种合成，无细胞因子的扩张培养系统，以解决当前方法的有限效率和批次变化。结合了三种策略，以进一步优化HSPC扩展的培养条件：1）促进HSPC自我更新。关键的转录调节剂（例如HOXB4）已被确定为增强培养物中HSPC自我更新的潜在靶标。由于病毒转导对生长促进转录因子（TF）的本构表达构成安全风险，因此我们使用脂质纳米粒子（LNP）基于基于TF mRNA的转移在培养中暂时表达靶HSPC中的单个或组合自我更新调节剂； 2）抑制HSPC分化。我们最近确定了78C（CD38分化标记物的有效抑制剂），是用于主动抑制培养物中HSPC分化的铅合成候选者。 3）缓解内质网（ER）应力。最近的研究强调了增加的增殖需求如何触发ER应激扰动，从而统一损害了离体培养中HSC功能。为了限制在扩展过程中ER应力途径的激活，我们在低氧条件下评估了HSPC培养物，并补充了HSF1的合成激动剂（例如，17-AAG）最近显示出来通过重新平衡蛋白质量来限制ER应力。通过克服先天免疫反应来增加细胞健身。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遗传操作的疗效中的关键作用。 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 （例如，DNA和mRNA）。这些发现将为克服核酸递送的免疫阻滞的新方法提供信息，并通过促进细胞存活和适应性来提高基因矫正效率。评估移植后G-CSF给药对基因编辑的HSPC的影响。粒细胞落刺激因子（G-CSF）通常用作辅助治疗，以加快化学疗法和自体移植造血茎和祖细胞（HSPC）的自体移植后从中性粒细胞减少症的辅助治疗。但是，尚未对靶向人类HSPC的离体基因治疗程序后G-CSF给药的效用尚未得到彻底评估。我们提供的证据表明，移植后G-CSF的施工阻碍了异种移植模型中的CRISPR-CAS9基因植入人类HSPC。 G-CSF通过加剧p53介导的DNA损伤响应而作用于Cas9介导的DNA DSB触发的DNA损伤响应。培养中的瞬时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G-CSF的施用不会损害未经操纵的人类HSPC或通过慢病毒载体转导进行基因设计的无操纵的人类HSPC或HSPC的重现特性。在离体自体HSPC基因编辑临床试验的设计中，应考虑移植后G-CSF给药加重与CRISPR-Cas9基因编辑相关的HSPC毒性的潜力。 Biorxiv 2023：doi：10.1101/2023.06.29.547089。目标3：制定体内基因治疗策略遗传有效载荷的体内递送可以避免当前体内基因校正方法的缺点，并代表了范科尼贫血基因治疗的明显进步。在可用的体内递送方法中，LNP是临床用途最发达的。正在进行一项三步临床前研究，以提供对新型LNP输送系统的效率（AIM 1），安全性（AIM 2）和治疗适用性（AIM 3）的全面评估。 AIM 1-在试验实验中，我们根据可电离的树枝状聚合物氨基脂质脂质4A3-SC852鉴定并优化了一种新型的LNP公式，并显示出在体外纯化纯化的人类HSPC的核酸货物的功效。基于我们先前的研究，表明CMPL是针对HSPC的相关抗原，优化的LNP配方已在化学上与重组抗CMPL二价单链片段变量（BISCFV（CMPL））化学结合，以促进NHP模型中的NHP模型中的HSPC的选择性递送HSPC。目标2- LNP介导的CRISPR试剂向HSPC的递送的特异性必须限制靶向基因编辑和体内的全身毒性。为了解决这个问题，使用已建立的体内跟踪方法研究了对NHP静脉内施用的LNP制剂的生物分布。简而言之，BISCFV（CMPL）偶联的LNP配方用钠/碘化物分类器（NIS）mRNA封装，注入动物，并在静脉下静脉内被静脉下的时间点上的全身PET/CT扫描成像跟踪，并在18f-tetrafluoroboberation静脉下受压。 AIM 3-将通过CMPL偶联的LNP递送DCAS9指导的，重组酶介导的DDR独立的基因组编辑系统来追求NHP中的内源性基因座的FA供体底物在NHP中的内源性基因座的整合。