Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Innovative Genomics Institute, 2151 Berkeley Way, Berkeley, CA 94704, USA.
Department of Bioengineering, University of California, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Adv Drug Deliv Rev. 2021 Jan;168:99-117. doi: 10.1016/j.addr.2020.09.004. Epub 2020 Sep 12.
Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.
基因编辑工具，如 Cre 重组酶 (Cre)、锌指核酸酶 (ZFNs)、转录激活因子样效应核酸酶 (TALENs)，以及最近的成簇规律间隔短回文重复 (CRISPR) 相关蛋白系统，已经彻底改变了生物医学研究、农业、微生物工程和治疗开发。与相应的 DNA 和 mRNA 前体相比，直接递送基因组编辑酶具有优势，因为它们不需要转录和/或翻译。此外，当递送病毒载体或质粒 DNA 时，长时间过表达是一个问题，而递送完整蛋白质时则可以避免该问题。这降低了插入突变的风险，并且相对更容易制造。然而，在体内利用基因组编辑蛋白的一个主要限制是其递送效率低，目前最成功的策略是使用潜在的免疫原性病毒载体。缺乏安全有效的非病毒递送系统仍然是此类酶临床转化的一大障碍。本文讨论了广泛使用的基因组编辑酶的非病毒递送策略的挑战，包括 Cre 重组酶、ZFNs 和 TALENs、CRISPR/Cas9 和 Cas12a (Cpf1) 的蛋白形式，并强调了非病毒递送策略的最新创新，这些创新有可能克服当前的递送限制并推进基因组编辑的临床转化。
