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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要人类患有 6,500 多种罕见疾病，其中甲型血友病 (HA) 影响估计美国有 20,000 名患者，全球有超过 400,000 名患者。这些患者的生命危险很高—— 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。目前尚无有效的治疗方法显示能够治愈 HA，因为 FVIII 蛋白（标准治疗）或新 FVIII 的半衰期产品在患者体内使用时间不足二十小时。该项目的目标是开发可生物降解的纳米颗粒（BNP），一种基因组编辑平台，可治愈甲型血友病。此前，我们报道过 CRISPR/Cas9 的有效传递和修复模板 DNA 可以诱导基因编辑和校正通过结合病毒和非病毒传递系统来治疗成年哺乳动物的遗传病。这种治疗完全恢复体重减轻，减轻肝损伤并产生富马酰乙酰乙酸水解酶（FAH）阳性通过纠正 FAH 突变小鼠的 FAH 剪接突变来改变肝细胞。此外，我们还开发了类脂质通过正交阵列设计的纳米颗粒可有效递送 mRNA。我们优化的 TT3 LLN 能够将 B 型血友病小鼠模型中的功能蛋白恢复至正常生理值。在这个提案中，我们的目标是开发可生物降解的纳米粒子，用于递送 CRISPR/Cpf1 和 hFVIII cDNA。 CRISPR/Cpf1 是具有单指导RNA的新系列CRISPR效应器。我们预计将功能最大化 CRISPR/Cpf1 和增强 HA 治疗的基因组编辑效率。在本研究中，我们将综合并表征 BNP，研究它们对白蛋白 (mAlb) 位点（一种设计的安全港基因）的切割效率体内插入位点，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) 如果成功，BNP 为HA提供可治愈的疗法。 (ii) Cpf1 mRNA 可以翻译为短期表达，以诱导基因切割，避免由于 Cpf1 蛋白长期表达而导致的潜在脱靶效应和毒性。 (三) mRNA不会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，从而最大限度地减少肝脏中的积累和相关副作用。具体目标如下我们将采取以下措施来实现我们的目标： 1).新型可生物降解材料的合成和表征纳米颗粒（BNP）。 2）。优化化学修饰的 Cpf1 mRNA 和 sgRNA，用于 mAlb 基因切割体外；和3）。评估 A 型血友病小鼠中 BNP 的基因组编辑效率和安全性模型。根据当前的先导材料以及从拟议研究中确定的新 BNP，研究计划将能够成功发现和开发用于治疗的新候选药物 A 型血友病和其他潜在的罕见疾病。