Auricchio A, Kobinger G, Anand V, Hildinger M, O'Connor E, Maguire A M, Wilson J M, Bennett J
Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, The Wistar Institute, Philadelphia, PA, USA.
Hum Mol Genet. 2001 Dec 15;10(26):3075-81. doi: 10.1093/hmg/10.26.3075.
Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.
基于腺相关病毒（AAV）或人类免疫缺陷病毒1型（慢病毒）的重组载体是用于长期体内基因递送的有前景的工具。它们的设计允许分别交换衣壳或包膜，理论上提供了转导一系列细胞类型的机会。我们构建了在AAV2、五聚体或单聚体衣壳（分别称为AAV2/2、AAV2/5和AAV2/1）所包含的AAV2血清型基因组内编码增强型绿色荧光蛋白（EGFP）的AAV载体。同样，我们生产了慢病毒载体，其编码与AAV载体中相同的表达盒，并用来自水疱性口炎病毒糖蛋白（VSVG）或莫科拉包膜的蛋白进行假型化。在视网膜下或玻璃体内给药后，在小鼠视网膜中评估了这些载体的转导特性。不同重组体之间转基因表达的起始时间和靶向细胞类型有所不同。慢病毒载体和AAV2/1的转基因表达起始时间为3-4天。相比之下，AAV2/5和AAV2/2的起始时间分别为2-4周。视网膜下注射后，慢病毒-VSVG和AAV2/5均能有效转导视网膜色素上皮（RPE）和光感受器，而使用带有莫科拉包膜的慢病毒或AAV2/1时，转基因表达仅限于RPE细胞。玻璃体内给药后，只有AAV2/2和慢病毒-VSVG转导了视网膜内层。所有载体在视网膜中均检测到载体介导的荧光超过12周。我们得出结论，假型化提供了一种有用的手段来操纵病毒载体的细胞靶向特异性以及含有相同基因组的载体的视网膜转导特性。
