Highly Specific ZFN-Based HSC Gene Editing Therapies Identified By In Vivo Barcode Nanoparticle Screens And Rationally Designed Mrna

通过体内条形码纳米粒子筛选和合理设计的 Mrna 鉴定出高度特异性的基于 ZFN 的 HSC 基因编辑疗法

• 批准号: 9810724
• 负责人: James Dahlman
• 金额: $ 71.07万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810724
• 关键词: Address; Affect; Big Data; Biodistribution; Bioinformatics; Biological Assay; Cell Culture Techniques; Cells; Chemicals; Clinical; Clinical Trials; Code; Codon Nucleotides; Custom; DNA; Data; Development; Dose; Drug Delivery Systems; Drug Targeting; Effectiveness; Endothelial Cells; Exhibits; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Diseases; Genomics; Goals; Hematological Disease; Hematopoietic stem cells; Hemoglobin; Hemoglobinopathies; Hepatocyte; Hereditary Disease; High Pressure Liquid Chromatography; Immune; In Vitro; Macaca mulatta; Messenger RNA; Methods; Modification; Mus; Mutation; Names; Nucleotides; Patients; Pharmaceutical Preparations; Phase; Positioning Attribute; RNA; Regimen; Safety; Sickle Cell Anemia; Site; Stem cells; System; Technology; Testing; Therapeutic; Time; Toxic effect; Toxicity due to chemotherapy; Translations; Transplant Recipients; Transplantation; base; beta Globin; beta Thalassemia; cell type; chemical property; clinical translation; conditioning; design; experimental study; gene therapy; immunogenicity; improved; in vivo; interdisciplinary approach; lipid nanoparticle; nanomedicine; nanoparticle; nonhuman primate; novel; physical property; protein expression; response; screening; targeted nucleases; therapeutic target; tool; zinc finger nuclease

Summary. Hemoglobinopathies such as β-thalassemia and sickle cell disease are genetic disorders caused by mutations in the HBB gene that codes for the β-globin component of hemoglobin. Currently, the only gene therapy available for these prevalent hereditary diseases is based on transplantation of genetically corrected hematopoietic stem cells (HSPCs) from fully matched donors. However, the efficacy of this approach is limited by multiple factors. Gene editing is a promising alternative approach for curing hemoglobinopathies. Using this approach, synthetic mRNA-based drugs encoding nucleases that target the HBB gene can be utilized to permanently correct the patient’s DNA. Combining nanoparticle-based drug delivery with zinc- finger nucleases (ZFNs) has the potential to facilitate targeted gene-editing in HSPCs. However, the reliance on in vitro screening of nanoparticles impedes the discovery of safe and efficient in vivo delivery vehicles. Furthermore, current ZFN-mRNA based drugs targeting the HBB gene in HSPCs exhibit immunogenicity and are expressed in off-target cells. The PIs have recently been shown that DNA barcoded nanoparticles can ‘evolve’ nanoparticles to target endothelial cells more efficiently than hepatocytes directly in vivo. The team has also demonstrated that it is possible to (i) design low immune stimulating mRNA via nucleotide modification and HPLC purification, and that (ii) mRNAs can be designed to completely preclude translation in hepatocytes using rationally designed ‘on’ and ‘off’ switches. Based on these supporting data, it is posited that nanoparticles can be evolved to specifically target HSPCs while avoiding hepatocytes, and that ZFN- mRNA based drugs can be rationally optimized to generate safe gene editing therapeutics targeting HSPCs. Thus, the team proposes to create an mRNA-based drug that safely and specifically edits HSPCs in non-human primes in two phases. The development (UG3) phase will address 2 aims: (1) to iteratively evolve nanoparticles that target HSPCs and avoid hepatocytes in vivo, and (2) to reduce mRNA immunogenicity and improve cell type specific delivery to HSPCs. The demonstration (UH3) phase will address the aim (3) to analyze functional gene editing in non-human primates (Rhesus macaques). These will be achieved using a cutting edge multidisciplinary approaches recently developed. Specifically, the team will combine a DNA barcoded nanoparticle technology to screen 4,500 nanoparticles in vivo, synthesize mRNA-based drugs with low immunogenicity and cell type-specific expression, and utilize customized bioinformatics pipeline that facilitates ‘big data’ experiments with a statistical power new to nanomedicine. By creating an mRNA-based drug that safely edits HSPCs, the project is poised to advance gene editing as a viable therapeutic approach for curing genetic blood disorders and pave the way for clinical trials.

概括。血红蛋白病如β-丘脑贫血和镰状细胞疾病是遗传疾病引起的 由HBB基因中的突变编码为血红蛋白的β-珠蛋白成分。目前，唯一的基因 可用于这些流行的雌性疾病的治疗是基于一般校正的移植 完全匹配的供体的造血干细胞（HSPC）。但是，这种方法的效率是 受多种因素的限制。基因编辑是治愈血红蛋白病的承诺替代方法。 使用这种方法，可以编码针对HBB基因的核素的基于MRNA的药物可以是 用于永久纠正患者的DNA。将基于纳米颗粒的药物递送与锌结合 手指核（ZFN）具有促进HSPC中靶向基因编辑的潜力。但是，依赖 纳米颗粒的体外筛查阻碍了在体内递送车辆中的安全有效的发现。 此外，针对HSPC中HBB基因的当前基于ZFN-MRNA的药物暴露了免疫原性和 在脱靶细胞中表达。 PI最近已显示DNA条形码纳米颗粒可以 “进化”纳米颗粒比直接在体内的肝细胞更有效地靶向内皮细胞。团队 还证明了（i）通过核苷酸设计低免疫刺激mRNA 修改和HPLC纯化，并且（ii）mRNA可以设计为完全排除翻译 在肝细胞中使用理性设计的“ On”和“ OFF”开关。基于这些支持数据，列出了 可以将纳米颗粒进化为特异性靶向HSPC，同时避免肝细胞，并ZFN- 基于mRNA的药物可以合理优化以产生安全的基因编辑疗法靶向 HSPC。这是团队提出的，以制作一种基于mRNA的药物，该药物安全，专门编辑HSPC 非人类素数分为两个阶段。开发（UG3）阶段将解决2个目标：（1）迭代发展 靶向HSPC并避免体内肝细胞的纳米颗粒，以及（2）降低mRNA免疫原性 并改善细胞类型的特异性输送到HSPC。演示（UH3）阶段将解决目标（3） 分析非人类素数（Rhesus猕猴）中的功能基因编辑。这些将使用 最新的多学科方法最近开发了。具体来说，团队将结合DNA 在体内筛选4,500纳米颗粒的条形码纳米颗粒技术，将基于mRNA的药物与 免疫原性和细胞类型特异性表达低，并利用自定义的生物信息学管道 具有纳米医学新的统计能力促进“大数据”实验。通过创建基于mRNA的 安全编辑HSPC的药物，该项目被毒化以推进基因编辑作为一种可行的疗法 治愈遗传性血液疾病的方法，为临床试验铺平道路。