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.

摘要 β-地中海贫血和镰状细胞病等血红蛋白病是由遗传性疾病引起的。 编码血红蛋白β-珠蛋白成分的 HBB 基因发生突变，这是目前唯一的基因。 这些流行的遗传性疾病的可用治疗方法是基于基因校正的移植 然而，这种方法的功效是来自完全匹配的捐赠者的造血干细胞（HSPC）。 受多种因素的限制，基因编辑是治疗血红蛋白病的一种有前途的替代方法。 使用这种方法，编码靶向 HBB 基因的核酸酶的基于 mRNA 的合成药物可以 用于永久纠正患者的 DNA，将纳米颗粒药物输送与锌结合。 指状核酸酶 (ZFN) 有潜力促进 HSPC 中的靶向基因编辑，但人们对其的依赖程度较低。 纳米粒子的体外筛选阻碍了安全有效的体内递送载体的发现。 此外，目前针对 HSPC 中 HBB 基因的基于 ZFN-mRNA 的药物表现出免疫原性和 最近表明，DNA 条形码纳米粒子可以在脱靶细胞中表达。 研究小组在体内“进化”纳米颗粒，比直接靶向肝细胞更有效地靶向内皮细胞。 还证明可以 (i) 通过核苷酸设计低免疫刺激 mRNA 修饰和 HPLC 纯化，并且 (ii) mRNA 可以设计为完全阻止翻译 基于这些支持数据，提出了在肝细胞中使用合理设计的“开”和“关”开关。 纳米颗粒可以进化为专门针对 HSPC，同时避开肝细胞，并且 ZFN- 基于 mRNA 的药物可以进行合理优化，以产生安全的基因编辑治疗靶向 因此，该团队建议开发一种基于 mRNA 的药物，可以安全、特异性地编辑 HSPC。 开发（UG3）阶段将实现两个目标：（1）迭代进化。 靶向 HSPC 并避开体内肝细胞的纳米粒子，以及 (2) 降低 mRNA 免疫原性 并改进向 HSPC 的细胞类型特异性递送。示范 (UH3) 阶段将实现目标 (3)。 分析非人类灵长类动物（恒河猴）的功能性基因编辑。这些将通过使用 具体来说，该团队将结合最近开发的尖端多学科方法。 条形码纳米颗粒技术可在体内筛选 4,500 个纳米颗粒，合成基于 mRNA 的药物 低免疫原性和细胞类型特异性表达，并利用定制的生物信息学管道 通过创建基于 mRNA 的纳米医学新统计能力，促进“大数据”实验。 安全编辑 HSPC 的药物，该项目准备推动基因编辑成为一种可行的治疗方法 治疗遗传性血液疾病的方法并为临床试验铺平道路。