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A nanoparticle delivery system for CRISPR/Cas9 based therapeutics

用于基于 CRISPR/Cas9 疗法的纳米颗粒递送系统

基本信息

批准号：
9769918
负责人：
Kenichi Kuroda
金额：
$ 73.3万
依托单位：
ATGC, INC.
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：
2017-06-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9769918
关键词：
Address Affinity Amino Acid Sequence Animal Model Animals Apolipoprotein E Basic Science Biological Assay CCR5 gene CD34 gene CRISPR Therapeutics Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats DNA Double Strand Break DNA delivery Development Disease Elements Event Fluorescence Genes Genetic Hepatocyte Human In Vitro Knockout Mice Length Lipofectamine Liver Long-Term Effects Mediating Methods MicroRNAs Molecular Weight Mouse Protein Mus Mutation Nonhomologous DNA End Joining Nucleic Acid Binding Nucleic Acids Oryctolagus cuniculus Pathogenicity Phase Polyesters Polyethylene Glycols Polymers Proteins Reporter Safety Small Business Technology Transfer Research Specificity Stem cells Structure System Tail Technology Testing Therapeutic Time Tissues Translational Research Veins Virus Weight Work atheroprotective base clinically relevant cytotoxicity cytotoxicity test experimental study gene correction homologous recombination implantation improved in vivo induced pluripotent stem cell mouse model nano nanoparticle nanoparticle delivery nanopolymer novel nuclease nucleic acid-based therapeutics plasmid DNA repaired response scaffold side effect success

项目摘要

Abstract Applications of customizable nucleases such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9) have enabled efficient and precise gene correction in vitro, and hold promises for eventually achieving in vivo gene correction/therapy. However, to apply CRISPR/Cas9 in therapeutic settings, several major challenges remain to be addressed: (i) homologous recombination (HR), even with the help of Cas9, is still of low efficiency; (ii) Cas9 is associated with off-target effects; and (iii) there is a lack of an efficient virus-free system to deliver CRISPR/Cas9 elements in vivo. The present proposal focuses on the challenge of lack of an efficient non-viral in vivo delivery system. We recently developed novel hyperbranched polymers (HPs) with high nucleic acid binding affinity, negligible cytotoxicity, and achieved satisfactory delivery of microRNAs both in vitro and in vivo. Here we propose to develop hyperbranched HP- based system for effective delivery of Cas9 plasmid DNA (pDNA). In Phase I, we will work to formulate and test new HPs for effectively packaging of Cas9 pDNA, and evaluate the safety and efficacy of these HPs in vitro. In Phase II, we will expand the capability of HPs for Cas9 in vivo, and apply HPs for Cas9 therapeutics in animal models. Success of the proposed work will have significant impacts on basic and translational research and accelerate the development of Cas9 therapeutics.

摘要可定制的核酸酶如CRISPR（成簇的规则间隔的短回文序列）的应用重复序列）/Cas9（CRISPR相关蛋白9）已经能够在体外进行有效和精确的基因校正，有望最终实现体内基因校正/治疗。然而，要将CRISPR/Cas9应用于在治疗背景下，几个主要的挑战仍有待解决：（i）同源重组（HR），即使在Cas9的帮助下，效率仍然很低;（ii）Cas9与脱靶效应相关;以及（iii）存在缺乏有效的无病毒系统来体内递送CRISPR/Cas9元件。现时的建议集中于缺乏有效的非病毒体内递送系统的挑战。我们最近开发了一种新的超支化聚合物（HP）具有高核酸结合亲和力、可忽略的细胞毒性，并实现了在体外和体内都有令人满意的microRNA递送。在这里，我们建议开发超支化HP- 该系统用于有效递送Cas9质粒DNA（pDNA）。在第一阶段，我们将努力制定和测试新的HP用于有效包装Cas9 pDNA，并评估这些HP在以下方面的安全性和有效性：体外在第二阶段，我们将扩大HPs在体内对Cas9的能力，并将HPs用于Cas9治疗，动物模型拟议工作的成功将对基础研究和转化研究产生重大影响并加速Cas9疗法的发展。