Pharmacoidea Ltd., H-6726 Szeged, Hungary.
Department of Neuroscience, Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield S10 2HQ, UK.
Int J Mol Sci. 2023 Feb 5;24(4):3141. doi: 10.3390/ijms24043141.
Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9's cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9's cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9's cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS.
由于其低致病性、免疫原性和长期基因表达，腺相关病毒 (AAV) 载体成为安全有效的基因传递工具，克服了早期基因治疗试验中其他病毒基因传递系统遇到的挫折。在 AAV 中，AAV9 可以穿过血脑屏障 (BBB)，使其成为通过系统给药转导中枢神经系统 (CNS) 的有前途的基因传递工具。最近关于 AAV9 介导的基因传递到 CNS 的缺点的报告需要审查 AAV9 细胞生物学的分子基础。更详细地了解 AAV9 的细胞进入将消除当前的障碍，并使更有效的基于 AAV9 的基因治疗方法成为可能。连接蛋白是硫酸乙酰肝素蛋白聚糖的跨膜家族，促进各种病毒和药物传递系统的细胞摄取。利用人细胞系和连接蛋白特异性细胞测定法，我们评估了连接蛋白在 AAV9 的细胞进入中的参与。普遍表达的同种型，连接蛋白-4 证明了其在促进 AAV9 内化方面优于连接蛋白。将连接蛋白-4 引入难以转导的细胞系中可实现强大的 AAV9 依赖性基因转导，而其敲低则降低了 AAV9 的细胞进入。AAV9 与连接蛋白-4 的附着不仅由多阴离子硫酸乙酰肝素链介导，还由细胞结合域介导。共免疫沉淀测定和亲和蛋白质组学也证实了连接蛋白-4 在 AAV9 的细胞进入中的作用。总的来说，我们的发现强调了普遍表达的连接蛋白-4 是 AAV9 细胞内化的重要贡献者，并为 AAV9 进入 CNS 的低基因传递潜力提供了基于分子的合理解释。
