Körbelin J, Hunger A, Alawi M, Sieber T, Binder M, Trepel M
Department of Oncology and Hematology, University Medical Center Hamburg-Eppendorf, Hubertus Wald Cancer Center, Hamburg, Germany.
University Medical Center Hamburg-Eppendorf, Bioinformatics Core, Hamburg, Germany.
Gene Ther. 2017 Aug;24(8):470-481. doi: 10.1038/gt.2017.51. Epub 2017 Jul 13.
Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.
在腺相关病毒（AAV）表面展示随机肽的文库是生成靶向特异性基因治疗载体的强大工具。然而，由于未知原因，AAV文库筛选的成功率各不相同，且生产过程的影响尚未得到充分评估。在文库筛选过程中，具有最有利嗜性的衣壳变体在几个选择轮次中在选定的靶标上富集，并通过随后对编码带有靶向肽插入的文库衣壳的衣壳化病毒基因组进行测序来鉴定。因此，高衣壳-基因组相关性对于获得有关所选衣壳变体的正确信息至关重要。有人提出通过带有假型化文库转移穿梭载体的两步方案来生产AAV文库，作为确保这种相关性的一种方法。在这里，我们表明通过这种方案经由转移穿梭载体产生的AAV2文库在氨基酸组成上表现出意想不到的额外偏差，这赋予了增加的肝素亲和力，从而与野生型AAV2嗜性相似。这种偏差可能从根本上损害AAV文库的预期用途，阻碍未来使用转移穿梭载体来生产AAV文库。
