A nanoparticle delivery system for CRISPR/Cas9 based 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，仍然是低效率的;（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。

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.