Chen Yuxuan, Ping Yuan
College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, China.
Acc Chem Res. 2023 Aug 15;56(16):2185-2196. doi: 10.1021/acs.accounts.3c00279. Epub 2023 Aug 1.
ConspectusClustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) is emerging as a powerful genome-editing tool, enabling precise and targeted modifications of virtually any genomic sequence in living cells. These technologies have potential therapeutic applications for cancers, metabolic diseases, and genetic disorders. However, several major challenges hinder the full realization of their potential. Specifically, CRISPR-Cas9 gene editors, whether delivered as plasmid DNA, mRNA/sgRNA, or ribonucleoprotein (RNP), exhibit poor membrane permeability, restricting their access to the intracellular genome, where the editing occurs. Additionally, these editors lack tissue or organ specificity, raising concerns about off-target editing at the tissue level that causes unwanted genotoxicity. Though a range of delivery carriers has been developed to deliver Cas9 editors, their effectiveness is often limited by a number of barriers at both the extracellular and intracellular levels. Moreover, the prolonged activity of Cas9 increases the risk of off-target editing at the genomic level. Therefore, it is crucial to develop efficient delivery vectors, along with molecular switches to safely regulate Cas9 activity.In this Account, we summarize our recent achievements in developing different types of materials that can efficiently deliver the plasmid DNA encoding Cas9 protein and single-guide RNA (sgRNA), or Cas9 RNP into cells to highlight the design considerations of carriers for safe and efficient delivery in vitro and in vivo. After elucidating the chemical and physical factors that are responsible for encapsulating and delivering these biomacromolecules, we further elucidate how we design the biodegradable polymeric carriers using dynamic disulfide chemistry, emphasize their safe and efficient delivery features for genome-editing biomacromolecules, and also introduce the integration of the intracellular delivery of genome-editing biomacromolecules with microneedle-based transdermal delivery to promote therapeutic genome editing for inflammatory skin disorders. Finally, we review how we exploit optical, chemical, and genetic switches to control the Cas9 activity in conjunction with targeted delivery to address the spatiotemporal specificity of gene editing in vivo and demonstrate their precision therapy against cancer and colitis treatment as proof-of-concept examples. In the final part, we will summarize the progress we have made and propose the future directions that may impact the field based on our own research outcomes.
综述
成簇规律间隔短回文重复序列（CRISPR）/相关蛋白9（CRISPR/Cas9）正成为一种强大的基因组编辑工具，能够对活细胞中的几乎任何基因组序列进行精确且有针对性的修饰。这些技术在癌症、代谢疾病和遗传疾病方面具有潜在的治疗应用。然而，几个主要挑战阻碍了它们潜力的充分发挥。具体而言，CRISPR-Cas9基因编辑器，无论是作为质粒DNA、mRNA/单导向RNA（sgRNA）还是核糖核蛋白（RNP）进行递送，都表现出较差的膜通透性，限制了它们进入发生编辑的细胞内基因组。此外，这些编辑器缺乏组织或器官特异性，引发了对组织水平脱靶编辑导致不必要的基因毒性的担忧。尽管已经开发了一系列递送载体来递送Cas9编辑器，但其有效性往往受到细胞外和细胞内水平的多种障碍的限制。而且，Cas9的长时间活性增加了基因组水平脱靶编辑的风险。因此，开发高效递送载体以及用于安全调控Cas9活性的分子开关至关重要。
在本综述中，我们总结了我们在开发不同类型材料方面的最新成果，这些材料能够有效地将编码Cas9蛋白和单导向RNA（sgRNA）的质粒DNA或Cas9核糖核蛋白递送至细胞中，以突出载体在体外和体内进行安全高效递送的设计考量。在阐明负责封装和递送这些生物大分子的化学和物理因素后，我们进一步阐述了如何使用动态二硫键化学设计可生物降解的聚合物载体，强调其对基因组编辑生物大分子的安全高效递送特性，并介绍将基因组编辑生物大分子的细胞内递送与基于微针的透皮递送相结合，以促进对炎症性皮肤病的治疗性基因组编辑。最后，我们回顾了我们如何利用光学、化学和遗传开关来控制Cas9活性并结合靶向递送，以解决体内基因编辑的时空特异性问题，并展示它们针对癌症的精准治疗和作为概念验证示例的结肠炎治疗。在最后一部分，我们将总结我们取得的进展，并根据我们自己的研究成果提出可能影响该领域的未来方向。
