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 人类患有超过6，500种罕见疾病，其中血友病A（HA）影响估计 美国有20，000名患者，全球有400，000多名患者。这些病人有很高的生命危险- 有出血和严重并发症的危险，包括关节和肌肉疾病。HA由基因引起 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（标准治疗）或新FVIII的半衰期 产品在患者体内的使用时间少于20小时。该项目的目标是开发可生物降解 纳米颗粒（BNPs），一种基因组编辑平台，用于治愈血友病A。此前，我们曾报道 CRISPR/Cas9和修复模板DNA的有效递送可以诱导基因编辑和修复。 通过组合病毒和非病毒递送系统，在成年哺乳动物中治疗遗传疾病。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 通过校正FAH突变小鼠中的FAH剪接突变来诱导肝细胞的凋亡。此外，我们开发了脂质样 通过正交阵列设计，使用纳米颗粒来有效递送mRNA。我们优化的TT 3 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。在这一提议中， 我们的目标是开发用于递送CRISPR/Cpf 1和hFVIII cDNA的可生物降解的纳米颗粒。CRISPR/Cpf1 是一个新的系列CRISPR效应与单向导RNA。我们期望最大限度地发挥 CRISPR/Cpf 1和增强HA治疗的基因组编辑效率。在这项研究中，我们将合成和 表征BNP，研究它们对白蛋白（mAlb）位点的切割效率，这是一种设计的安全港基因， 插入位点，从而将这种新型平台推向未来治疗HA的临床试验。这 该方法与目前用于HA治疗的其他策略相比具有几个优点。(i)如果成功， 为HA提供了一种可治愈的治疗方法。(ii)Cpf 1 mRNA可以翻译为短期表达，以诱导 基因切割，避免由于Cpf 1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 生物可降解，从而最大限度地减少在肝脏中的积累和相关的副作用。以下具体目标 我们的目标是：（1）。合成并表征了一种新型的可生物降解的 纳米颗粒（BNPs）。2）。为了优化化学修饰的Cpf 1 mRNA和sgRNA用于mAlb基因切割， 体外;和3）。评价BNP在血友病A小鼠中的基因组编辑效率和安全性特征 模型根据当前的铅材料以及拟从拟定研究中识别的新BNP， 研究计划将能够成功地发现和开发新的候选药物，用于治疗 血友病A和潜在的其他罕见疾病。