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

摘要血红蛋白病如β-地中海贫血和镰状细胞病是由遗传性疾病引起的 编码血红蛋白中β-珠蛋白成分的HBB基因发生突变。目前，唯一的基因 可用于这些流行的遗传性疾病的治疗是基于遗传校正的移植。 造血干细胞（HSPC）来自完全匹配的供体。然而，这种方法的有效性是 受到多种因素的限制。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，可以合成编码靶向HBB基因的核酸酶的基于mRNA的药物。 用来永久性地修正病人的DNA将纳米颗粒药物输送与锌- 指状核酸酶（ZFN）具有促进HSPC中的靶向基因编辑的潜力。然而，依赖 对纳米颗粒的体外筛选阻碍了安全有效的体内递送载体的发现。 此外，目前靶向HSPC中HBB基因的基于ZFN-mRNA的药物表现出免疫原性， 在脱靶细胞中表达。PI最近已经表明，DNA条形码纳米颗粒可以 “进化”纳米颗粒，以靶向内皮细胞比肝细胞更有效地直接在体内。球队 还证明了有可能（i）通过核苷酸设计低免疫刺激性mRNA， 修饰和HPLC纯化，以及（ii）mRNA可以被设计为完全排除翻译 使用合理设计的“开”和“关”开关。根据这些支持数据， 纳米颗粒可以进化为特异性靶向HSPC，同时避免肝细胞，并且ZFN- 基于mRNA的药物可以合理优化，以产生安全的基因编辑治疗靶向 HSPC。因此，该团队建议创建一种基于mRNA的药物，可以安全地特异性编辑HSPC， 两个阶段的非人类启动开发（UG 3）阶段将致力于2个目标：（1）迭代进化 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 并改善向HSPC的细胞类型特异性递送。示范（UH 3）阶段将解决目标（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 最近开发的尖端多学科方法。具体来说，研究小组将联合收割机 条形码纳米颗粒技术，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， 利用纳米医学新的统计能力促进“大数据”实验。通过创建一个基于mRNA的 安全编辑HSPC的药物，该项目准备将基因编辑作为一种可行的治疗方法 这是治疗遗传性血液疾病的方法，并为临床试验铺平了道路。

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.