Mao Yingying, Wang Xuejun, Yan Renhe, Hu Wei, Li Andrew, Wang Shengqi, Li Hongwei
School of Biotechnology, Southern Medical University, 1023 South Shatai Road, Guangzhou, Guangdong, 510515, China.
Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing, China.
BMC Biotechnol. 2016 Jan 5;16:1. doi: 10.1186/s12896-015-0230-0.
Rational design of AAV capsids is a simple method for enhancing AAV transduction efficiency. AAV-DJ is a highly recombinogenic hybrid vector created from DNA shuffling of eight AAV serotypes, which mediates efficient gene expression both in vitro and in vivo. AAV2 and AAV8 are the closest parental vectors of AAV-DJ and it has been reported that mutations on the 137/251/503 ubiquitination or phosphorylation sites of the AAV2 or AAV8 capsid lead to dramatic enhancement of gene delivery. Here, we aimed to find out whether the same point mutations on the AAV-DJ capsid could lead to significant improvement for gene delivery both in vitro and in vivo.
We constructed three single point mutants (K137R/T251A/S503A) of AAV-DJ and the transduction efficiency of these mutants and AAV-DJ were investigated using two reporter gene systems including green fluorescent protein (GFP) and dual-luciferase (Gaussia luciferase and Firefly luciferase). Data indicated that single point mutations T251A/S503A lead to significant improvement of dual-luciferase expression in vivo after tail vein (TV) injection in mice respectively, despite limited enhancement of GFP expression in 293 T, Hela and HepG2 cells in vitro. Moreover, in vivo bioluminescence image and viral genome DNA copy number in tissue analysis showed that these mutants reserved the liver tropism characteristics, consistent with AAV-DJ.
Single point mutations on the 251/503 sites of AAV-DJ capsid can lead to a significant improvement for in vivo gene expression. These enhanced AAV vectors have great potential in gene therapy applications.
合理设计腺相关病毒（AAV）衣壳是提高AAV转导效率的一种简单方法。AAV-DJ是一种通过对8种AAV血清型进行DNA改组而构建的高度重组的杂交载体，它在体外和体内均能介导高效的基因表达。AAV2和AAV8是AAV-DJ最接近的亲本载体，据报道，AAV2或AAV8衣壳的137/251/503泛素化或磷酸化位点发生突变会导致基因传递显著增强。在此，我们旨在探究AAV-DJ衣壳上的相同点突变是否能在体外和体内显著改善基因传递。
我们构建了AAV-DJ的三个单点突变体（K137R/T251A/S503A），并使用包括绿色荧光蛋白（GFP）和双荧光素酶（海肾荧光素酶和萤火虫荧光素酶）在内的两种报告基因系统研究了这些突变体和AAV-DJ的转导效率。数据表明，单点突变T251A/S503A分别在小鼠尾静脉注射后在体内显著提高了双荧光素酶的表达，尽管在体外293T、Hela和HepG2细胞中GFP表达的增强有限。此外，体内生物发光成像和组织分析中的病毒基因组DNA拷贝数表明，这些突变体保留了肝脏嗜性特征，与AAV-DJ一致。
AAV-DJ衣壳251/503位点的单点突变可显著改善体内基因表达。这些增强型AAV载体在基因治疗应用中具有巨大潜力。
