A nanoparticle delivery system for CRISPR/Cas9 based therapeutics

摘要 可定制核酸酶的应用，如CRISPR(簇状规则间隔短回文 Repeats)/Cas9(CRISPR相关蛋白9)在体外实现了高效和精确的基因校正，并且 对最终实现体内基因纠正/治疗抱有希望。但是，要将CRISPR/Cas9应用于 在治疗环境下，仍有几个主要挑战有待解决：(I)同源重组(HR)， 即使在Cas9的帮助下，效率仍然很低；(Ii)Cas9与脱靶效应有关；以及(Iii) 缺乏有效的无病毒系统在体内传递CRISPR/Cas9元件。目前的建议 重点关注缺乏有效的非病毒体内递送系统的挑战。我们最近开发了一本小说 超支化聚合物(HPS)具有很高的核酸结合亲和力，细胞毒性可以忽略不计，并实现了 在体外和体内都能满意地传递microRNAs。在这里，我们建议开发超支化HP- 构建了有效传递Cas9质粒DNA(PDNA)的BASE系统。在第一阶段，我们将致力于制定和 测试用于有效包装Cas9 PDNA的新HPS，并评估这些HPS在 体外培养。在第二阶段，我们将扩大HPS在体内治疗Cas9的能力，并将HPS应用于Cas9治疗 动物模型。拟议工作的成功将对基础研究和翻译研究产生重大影响 加快了Cas9疗法的发展。

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.