Yan Ziying, Zak Roman, Zhang Yulong, Engelhardt John F
Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, Iowa 52242-1109, USA.
J Virol. 2005 Jan;79(1):364-79. doi: 10.1128/JVI.79.1.364-379.2005.
The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.
随着双载体异源二聚化方法的发展，腺相关病毒（AAV）载体相对较小的包装容量（小于5 kb）已有效翻倍。然而，这种双载体系统的效率不仅受到两个独立载体基因组之间分子间重组程度的限制，还受到串联后成功进行转基因重构所需的方向偏倚的限制。在本研究中，我们试图评估反向末端重复序列（ITR）介导AAV基因组分子间重组的机制，目标是构建更高效的双载体转剪接方法载体。为此，我们构建了一种新型的AAV杂交ITR载体，其特征是在病毒基因组的两端分别有一个AAV-2和一个AAV-5 ITR。这种杂交基因组被有效地包装到AAV-2或AAV-5衣壳中以产生感染性病毒粒子。尽管杂交AV2:5 ITR病毒与同源AV2:2和AV5:5 ITR载体表达编码的增强型绿色荧光蛋白（EGFP）转基因的能力相似，但它们在感染细胞中形成环状中间体的能力明显较低。为了研究杂交AV2:5 ITR载体中包含的不同ITR序列是否能以头对头的方式指导分子间重组，我们构建了两种杂交ITR转剪接载体（AV5:2LacZ供体和AV2:5LacZ受体）。每种载体都递送一个由供体或受体剪接序列侧翼的β-半乳糖苷酶小基因的一个外显子。将这些杂交转剪接载体与同源AV5:5和AV2:2转剪接载体组进行比较，以评估它们重构β-半乳糖苷酶基因表达的能力。比较结果表明，与同源ITR载体相比，杂交ITR双载体组的转剪接效率显著提高（提高6至10倍，具体取决于衣壳血清型）。对病毒基因组结构的分子研究表明，由于线性形式基因组丰度的增加，杂交ITR载体提供了更有效的定向重组。这些研究为ITR序列在指导AAV基因组分子间和分子内同源重组中的重要性提供了直接证据。使用杂交ITR AAV载体基因组为操纵病毒基因组转化产物和指导高效双AAV载体转基因重构所需的分子间重组事件提供了新策略。
