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: 9916793
• 负责人: Yizhou Dong
• 金额: $ 39.28万
• 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/9916793
• 关键词: Adult; Affect; Albumins; Animal Model; Bacteria; Blood Coagulation Disorders; Blood Coagulation Factor; Body Weight decreased; Cells; Chemicals; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Complementary DNA; Cytoplasm; DNA; Engineering; Enzymes; Esters; F8 gene; Factor VIII; Formulation; Fumarylacetoacetase; Future; Gene Mutation; Genes; Genetic Diseases; Genome; Goals; Guide RNA; Half-Life; Hemophilia A; Hemophilia B; Hemorrhage; Hepatocyte; 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; nanoparticle delivery; novel; novel strategies; novel therapeutics; plasmid DNA; preclinical study; prevent; programs; repaired; side effect; standard care; therapeutic RNA; 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.

项目摘要 人类患有6,500多种罕见疾病，其中血友病A（HA）影响了估计的 在美国，有20,000名患者和全球40万名患者。这些患者的生命风险很高 - 威胁出血和严重的并发症，包括关节和肌肉疾病。 HA是由基因引起的 VIII因子（FVIII）的突变，导致凝血受损。目前，没有有效的疗法 显示能够治愈HA的表现，因为FVIII蛋白的半衰期（标准处理）或新的FVIII 患者的产品不到20小时。该项目的目的是开发可生物降解 纳米颗粒（BNP），这是一种基因组编辑平台，用于治愈血友病A。以前，我们报道 有效地递送CRISPR/CAS9和维修模板DNA可以诱导基因编辑和校正 通过结合病毒和非病毒递送系统，成人哺乳动物的遗传疾病。这种治疗完全 恢复体重减轻，减轻肝脏损伤并产生的富马乙酸乙酸酯水解酶（FAH） - 阳性 通过纠正FAH突变小鼠的FAH剪接突变，肝细胞。此外，我们开发了类似脂质的 通过正交阵列设计的纳米颗粒，可有效地递送mRNA。我们优化的TT3 LLNS能够 在血友病B小鼠模型中恢复功能蛋白为正常生理值。在此提案中， 我们旨在开发可生物降解的纳米颗粒，用于递送CRISPR/CPF1和HFVIII cDNA。 CRISPR/CPF1 是带有单个指南RNA的新系列CRISPR效应子。我们预计最大化的功能 CRISPR/CPF1并提高了HA治疗的基因组编辑效率。在这项研究中，我们将合成并 表征BNP，研究其白蛋白（MALB）基因座的切割效率，这是一种设计的避风港基因 在体内插入站点，从而将这个新颖的平台推向了未来治疗HA的临床试验。这 与目前用于HA治疗的其他策略相比，方法具有几个优势。 （i）如果成功，BNP 提供可治愈的HA疗法。 （ii）CPF1 mRNA可以翻译以进行短期表达，以诱导 基因切割，避免由于CPF1蛋白的长期表达而导致潜在的脱靶作用和毒性。 （iii） mRNA不整合到宿主细胞的基因中，避免了潜在的遗传毒性。 （iv）BNP是 可生物降解，从而最大程度地减少肝脏中的积累和相关的副作用。以下特定目标 将执行我们的目标：1）。合成并表征新颖的生物降解 纳米颗粒（BNP）。 2）。优化化学修饰的CPF1 mRNA和SGRNA，以用于MALB基因切割 体外和3）。评估BNP的基因组编辑效率和小鼠的安全概况 模型。基于当前的铅材料以及从拟议的研究中鉴定出的新的BNP，这是 研究计划将能够成功发现和开发用于治疗的新药候选者 血友病A和潜在的其他罕见疾病。