Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs.
Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs.
复制标题
使用单个 AAV 载体转导和食品和药物管理局批准的药物增强的锌指核酸酶介导的基因校正。
DOI：
10.1038/gt.2011.211
复制
发表时间：
2013-01
期刊：
影响因子：
5.1
通讯作者：
Porteus MH
中科院分区：
文献类型：
--
作者：
Ellis BL;Hirsch ML;Porter SN;Samulski RJ;Porteus MH
单基因疾病治疗的新兴策略使用基因工程来精确纠正基因组水平的突变。该技术的最新进展表明，使用锌指核酸酶（ZFN）诱导的DNA双链与外源性DNA供体底物结合使用的DNA双链断裂水平。该策略需要有效的核酸递送以及在病毒载体中，重组腺相关病毒（RAAV）已证明没有病理学的临床成功。但是，RAAV的一个主要局限性是小型DNA包装能力，迄今为止，RAAV用于ZFN基因递送尚未报告。从理论上讲，理想的情况是在单个矢量中同时提供ZFN和维修底物，以避免效率低下的基因靶向和不良诱变，这是RAAV共同散射策略的并发症。因此，产生了RAAV格式，其中单个多肽编码通过跳过2A肽和脂肪蛋白裂解序列连接的ZFN单体。根据这种布置，该载体还包括750个核苷酸的DNA修复底物。证明了有效的多肽加工到离散的ZFNS，以及该单个矢量格式刺激人类细胞系中有效基因靶向的能力，而小鼠模型得出的成纤维细胞。此外，我们使用食品和药物给药批准的药物的组合将RAAV介导的基因校正提高到六倍，这些药物在AAV载体转导的水平上起作用。总的来说，这些实验证明了通过单个AAV载体提供ZFN和维修底物的能力，并提供了使用药物治疗优化RAAV介导的基因校正的见解。
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.