Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Gene Ther. 2013 Jan;20(1):35-42. doi: 10.1038/gt.2011.211. Epub 2012 Jan 19.
An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.
一种新兴的单基因疾病治疗策略是利用基因工程在基因组水平上精确纠正突变。该技术的最新进展表明，使用锌指核酸酶 (ZFN) 诱导的 DNA 双链断裂，并结合外源性 DNA 供体底物，可实现治疗水平的基因校正。该策略需要高效的核酸递送来实现，在病毒载体中，重组腺相关病毒 (rAAV) 在没有病理学的情况下表现出了临床成功。然而，rAAV 的一个主要限制是其 DNA 包装容量小，迄今为止，尚未有报道使用 rAAV 进行 ZFN 基因传递。从理论上讲，理想的情况是在单个载体中同时递送 ZFN 和修复底物，以避免低效的基因靶向和不必要的突变，这两种情况都是 rAAV 共转导策略的并发症。因此，设计了一种 rAAV 格式，其中单个多肽通过核糖体跳过 2A 肽和弗林裂解序列连接 ZFN 单体。在此基础上，还在该载体中包含了一个 750 个核苷酸的 DNA 修复底物。实验证明了高效的多肽处理到离散的 ZFN ，以及这种单个载体格式在人细胞系和源自小鼠模型的成纤维细胞中刺激有效基因靶向的能力。此外，我们使用了食品和药物管理局批准的药物组合将 rAAV 介导的基因校正提高了六倍，这些药物作用于 AAV 载体转导水平。总的来说，这些实验证明了通过单个 AAV 载体递送 ZFN 和修复底物的能力，并为使用药物治疗优化 rAAV 介导的基因校正提供了思路。
