Biodegradable nanoparticles, a genome editing platform to treat hemophilia

可生物降解的纳米颗粒，治疗血友病的基因组编辑平台

• 批准号: 9286161
• 负责人: Yizhou Dong
• 金额: $ 37.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9286161
• 关键词: Adult; Adverse effects; Affect; Albumins; Animal Model; Bacteria; Blood Coagulation Disorders; Blood Coagulation Factor; Body Weight decreased; Cells; Chemicals; Clinical Trials; Coagulation Process; Complementary DNA; Cytoplasm; DNA; Engineering; Enzymes; Esters; F8 gene; Factor VIII; Formulation; Fumarylacetoacetase; Future; Gene Mutation; Genes; Genome; Goals; Guide RNA; Half-Life; Hemophilia A; Hemophilia B; Hemorrhage; Hepatocyte; Hereditary Disease; Hour; Human; Impairment; In Vitro; Inherited; Introns; Lead; Life; Lipids; Liver; Mammals; Messenger RNA; Methods; Mus; Mutate; Mutation; Myopathy; Pathway interactions; Patients; Physiological; Process; Production; Proteins; RNA Decay; RNA Splicing; Rare Diseases; Regimen; Reporting; Research; Safety; Series; Site; System; Therapeutic; Time; Toxic effect; Translating; Treatment Efficacy; Viral; Zebrafish; adeno-associated viral vector; arm; arthropathies; base; clinical application; design; drug candidate; effective therapy; endonuclease; enzyme replacement therapy; esterase; genome editing; genotoxicity; high risk; immunogenicity; improved; in vivo; intravenous administration; liver injury; mouse model; nanoparticle; novel; novel strategies; novel therapeutics; plasmid DNA; preclinical study; prevent; programs; repaired; standard care; transcription activator-like effector nucleases

PROJECT SUMMARY Humans suffer from more than 6,500 rare diseases, of which hemophilia A (HA) affects an estimated 20,000 patients in the US and over 400,000 patients worldwide. These patients have a high risk of life- threatening bleeding and serious complications, including joint and muscle diseases. HA is caused by gene mutations of factor VIII (FVIII), resulting in impaired coagulation. Currently, no effective therapy has been shown capable of curing HA, because the half-life of the FVIII protein (the standard treatment) or new FVIII products is less than twenty hours in patients. The goal of this project is to develop biodegradable nanoparticles (BNPs), a genome editing platform to achieve a cure for hemophilia A. Previously, we reported that the efficient delivery of CRISPR/Cas9 and repair template DNA can induce gene-editing and correction of genetic disease in adult mammals by combining viral and non-viral delivery systems. This treatment fully restored weight loss, alleviated liver damage and generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes by correcting a FAH splicing mutation in FAH-mutated mice. Moreover, we developed lipid-like nanoparticles via an orthogonal array design for efficient delivery of mRNA. Our optimized TT3 LLNs was able to restore functional protein to normal physiological values in a hemophilia B mouse model. In this proposal, we aim to develop biodegradable nanoparticles for delivery of CRISPR/Cpf1 and hFVIII cDNA. CRISPR/Cpf1 is a new series of CRISPR effectors with single guide RNA. We anticipate maximizing the function of CRISPR/Cpf1 and enhancing genome editing efficiency for HA therapy. In this study, we will synthesize and characterize BNPs, study their cutting efficiency of the albumin (mAlb) locus, a designed safe-harbor gene- insertion site in vivo, thereby advancing this novel platform toward future clinical trials for treating HA. This approach has several advantages over other strategies currently used for HA treatment. (i) If successful, BNPs offer a curable therapy for HA. (ii) Cpf1 mRNA can be translated for short-term expression in order to induce gene-cutting, avoiding potential off-target effects and toxicity due to long term expression of Cpf1 protein. (iii) mRNA does not integrate into the genes of host cells, avoiding potential genotoxicity. (iv) BNPs are biodegradable, thus minimizing accumulation in the liver and relevant side effects. The following specific aims will be carried out to accomplish our goals: 1). To synthesize and characterize novel biodegradable nanoparticles (BNPs). 2). To optimize chemically modified Cpf1 mRNA and sgRNA for mAlb gene-cutting in vitro; and 3). To evaluate genome editing efficiency and safety profiles of BNPs in a hemophilia A mouse model. Based on current lead material as well as new BNPs to be identified from the proposed study, this research program will be able to successfully discover and develop new drug candidates for treating hemophilia A and potentially other rare diseases.

项目总结 人类患有6500多种罕见疾病，其中血友病A(HA)影响估计 美国有2万名患者，全球有超过40万名患者。这些患者有很高的生命风险- 威胁出血和严重并发症，包括关节和肌肉疾病。HA是由基因引起的 因子VIII(FVIII)突变，导致凝血功能受损。目前，还没有有效的治疗方法 显示能够治愈HA，因为FVIII蛋白的半衰期(标准治疗)或新的FVIII 产品在患者体内使用时间不到20小时。这个项目的目标是开发可生物降解的 纳米粒子(BNPS)，一个实现血友病A治愈的基因组编辑平台。此前，我们报道 CRISPR/Cas9和修复模板DNA的高效传递可以诱导基因编辑和纠正 通过结合病毒和非病毒递送系统在成年哺乳动物中引起的遗传病。这项治疗完全 恢复减肥，减轻肝脏损伤，产生富马酸乙酰乙酸酯水解酶(FAH)阳性 通过纠正FAH突变小鼠中的FAH剪接突变。此外，我们还开发了类脂 通过正交表设计的纳米颗粒可有效传递信使核糖核酸。我们优化的TT3 LLN能够 在血友病B小鼠模型中将功能蛋白恢复到正常生理值。在这份提案中， 我们的目标是开发可生物降解的纳米颗粒，用于运送CRISPR/Cpf1和hFVIII基因。CRISPR/CPF1 是一种新的具有单引导RNA的CRISPR效应器。我们期望最大限度地发挥 CRISPR/Cpf1和提高HA治疗的基因组编辑效率。在这项研究中，我们将综合和 鉴定BNPS，研究其对白蛋白(MAlb)基因座的切割效率，设计的安全港基因-- 体内插入部位，从而将这一新平台推向未来治疗HA的临床试验。这 与目前用于HA治疗的其他策略相比，该方法有几个优点。(I)如成功，英国国家核动力局 为HA提供一种可治愈的治疗方法。(Ii)Cpf1mRNA可以翻译为短期表达，以诱导 基因切割，避免由于Cpf1蛋白的长期表达而可能产生的脱靶效应和毒性。(Iii) MRNA不会整合到宿主细胞的基因中，从而避免了潜在的遗传毒性。(Iv)英国属土核电厂 可生物降解，从而最大限度地减少在肝脏中的蓄积和相关副作用。以下是具体目标 将为实现我们的目标而进行：1)。新型生物可降解材料的合成与表征 纳米粒子(BNPS)。2)。化学修饰Cpf1mRNA和sgRNA用于mAlb基因切割的优化 3)体外培养。评价BNPS在血友病A小鼠体内的基因组编辑效率和安全性 模特。根据目前的铅材料以及从拟议的研究中确定的新的BNPS，这 研究计划将能够成功地发现和开发用于治疗的新药候选 血友病A和其他可能的罕见疾病。