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

通过体内条码纳米粒子筛选和合理设计的 mRNA 鉴定出基于 ZFN 的高度特异性 HSC 基因编辑疗法

• 批准号: 10809430
• 负责人: James Dahlman
• 金额: $ 62.37万
• 依托单位: EMORY UNIVERSITY
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10809430
• 关键词: Acceleration; Administrative Supplement; Affect; Affinity; Animal Model; Animals; Antibodies; Autopsy; Bar Codes; Big Data; Cell Culture Techniques; Cells; Chemicals; Chemistry; Chronic; Clinical; Clinical Trials; Code; DNA; Data; Development; Disease; Dose; Drug Delivery Systems; Drug Modelings; Drug Screening; Drug Targeting; Effectiveness; Endothelial Cells; Enrollment; Ethics; Exhibits; Future; Gene Transfer; Genes; Genetic; Genetic Diseases; Globin; Goals; Health; Hematological Disease; Hematopoietic stem cells; Hemoglobin; Hemoglobinopathies; Hepatocyte; Hereditary Disease; High Pressure Liquid Chromatography; Histology; Hour; Human; Immunologic Stimulation; In Vitro; Inflammation; Inflammatory; Libraries; Life; Macaca mulatta; Messenger RNA; Methods; Modification; Mus; Mutation; Notification; Nucleotides; Patients; Pharmaceutical Preparations; Phase; Primates; RNA; RNA delivery; Regimen; Research; Schedule; Serum; Sickle Cell Anemia; Specificity; System; Technology; Testing; Thalassemia; Therapeutic; Tissues; Toxic effect; Translations; Transplant Recipients; Transplantation; Veterinarians; Work; bioinformatics pipeline; cell type; chronic inflammatory disease; clinically relevant; conditioning; delivery vehicle; design; disease phenotype; drug discovery; experimental study; gene therapy; immunogenicity; improved; in vivo; interdisciplinary approach; multiple omics; nanomedicine; nanoparticle; nonhuman primate; novel; nuclease; rational design; screening; stem cells; targeted nucleases; therapeutic genome editing; therapeutic target; zinc finger nuclease

Summary. Hemoglobinopathies such as-thalassemia and sickle cell disease are genetic disorders caused by mutations in the HBB gene that codes for the -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.

总结。-地中海贫血和镰状细胞病等血红蛋白疾病是由遗传性疾病引起的 这是由于编码血红蛋白中-珠蛋白成分的HBb基因发生突变。目前，唯一的基因 对这些流行的遗传性疾病的治疗是基于移植经过基因矫正的 来自完全匹配的供者的造血干细胞(HSPC)。然而，这种方法的效果是 受多种因素的制约。基因编辑是治疗血红蛋白疾病的一种很有前途的替代方法。 使用这一方法，编码针对HBB基因的核酸酶的合成基于mRNA的药物可以 用来永久纠正病人的DNA。将基于纳米颗粒的药物输送与锌相结合- 指状核酸酶(ZFN)有可能促进HSPC中的靶向基因编辑。然而，这种依赖 纳米颗粒的体外筛选阻碍了安全有效的体内给药载体的发现。 此外，目前针对HSPC中HBB基因的ZFN-mRNA药物显示出免疫原性和 在非靶标细胞中表达。PI最近被证明DNA条码纳米颗粒可以 在体内，“进化”纳米颗粒比直接针对肝细胞更有效地靶向内皮细胞。该队 也证明了(I)通过核苷酸设计低免疫刺激的mrna是可能的。 修饰和高效液相纯化，以及(Ii)mRNAs可以被设计成完全排除翻译 在肝细胞中使用合理设计的“开”和“关”开关。基于这些支持数据，我们假设 纳米粒子可以进化成专门针对HSPC，同时避免肝细胞，而且ZFN- 合理优化基于信使核糖核酸的药物以产生安全的基因编辑治疗靶向 HSPC。因此，该团队建议创造一种基于信使核糖核酸的药物，这种药物可以安全和特异地编辑HSPC 非人类素数分两个阶段。开发(UG3)阶段将实现两个目标：(1)迭代发展 靶向HSPC并在体内避开肝细胞的纳米颗粒，以及(2)降低mRNA的免疫原性 并改善对HSPC的细胞类型特异性递送。演示(UH3)阶段将针对目标(3) 分析非人类灵长类动物(猕猴)的功能基因编辑。这些将通过使用 前沿的多学科方法是最近发展起来的。具体地说，该团队将结合DNA 条形码纳米颗粒技术在体内筛选4500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， 利用纳米医学的一种新的统计能力促进“大数据”实验。通过创建基于mRNA的 安全编辑HSPC的药物，该项目有望推动基因编辑作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。