Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.
Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.
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基因编辑核酸酶如CRISPR/Cas9已经实现了有效和精确的体外基因编辑，并有望最终实现基于体内基因编辑的治疗。然而，使用它们的主要挑战是缺乏安全有效的无病毒系统来递送基因编辑核酸酶元件。与目前使用的病毒载体相比，聚合物由于其更高的安全性而成为一类有前途的递送载体，但聚合物的转染效率较低。聚合物载体已用于小核苷酸递送，但尚未成功地与质粒DNA（pDNA）一起使用，质粒DNA（pDNA）通常比小核苷酸大几百倍，这提出了工程挑战。为了解决这个问题，我们通过合成几种HP变体：HP-800、HP-1.8K、HP-10 K、HP-25 K，扩展了我们先前报道的用于pDNA递送的超支化聚合物（HP）递送系统。我们证明了所有HP在各种培养细胞中具有低毒性，其中HP-25 K在包装和递送pDNA方面最有效。重要的是，HP-25 K介导的CRISPR/Cas9 pDNA递送在不同细胞类型中的几个临床显著基因座处导致比所有其他HP和Lipofectamine更高的基因编辑率。一致地，与Lipofectamine相比，当将CRISPR碱基编辑器“BE 4-max”pDNA递送至细胞时，HP-25 K也导致更稳健的碱基编辑。目前的工作表明，HP纳米颗粒代表了一类有前途的载体，用于非病毒递送pDNA，以实现基因编辑治疗的临床应用。
Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor “BE4-max” pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.
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