Bioengineering Laboratory, 908 Furnas Hall, Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY 14260-4200, USA.
Biomaterials. 2011 Apr;32(12):3330-9. doi: 10.1016/j.biomaterials.2011.01.035. Epub 2011 Feb 5.
We recently demonstrated that fibrin hydrogels can be used as vehicles for efficient lentivirus gene delivery. Gene transfer in fibrin gels was strongly dependent on matrix degradation by target cells but a fraction of lentiviral particles diffused out of the gels over time compromising spatial control of gene transfer. To overcome this challenge, we engineered lentiviral particles that bind covalently to fibrin during polymerization. To this end, we fused into the viral envelope glycoprotein (VSV-G) peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. Lentivirus pseudotyped with the modified envelopes bound to fibrinogen in a factor XIII dose dependent manner and was released upon plasmin treatment. The peptide/VSV-G fusion envelope variants did not compromise the transduction efficiency of the resulting virus except when lacking any flexible linkers separating the peptide from the VSV-G envelope. Diffusion of virus from the gels decreased dramatically, especially at high concentrations of FXIII, even for fibrin gels with low fibrinogen concentration that were loaded with high titer virus. Lentivirus arrays prepared with fibrin-conjugated lentivirus yielded highly efficient gene transfer that was confined to virus-containing fibrin spots. As a result, signal/noise ratio increased and cross-contamination between neighboring sites was minimal. Finally, in addition to lentivirus microarrays this strategy may be used to achieve spatially-controlled gene transfer for therapeutic applications.
我们最近证明纤维蛋白水凝胶可用作高效慢病毒基因传递的载体。纤维蛋白凝胶中的基因转移强烈依赖于靶细胞对基质的降解，但随着时间的推移，一部分慢病毒颗粒会从凝胶中扩散出来，从而影响基因转移的空间控制。为了克服这一挑战，我们设计了在聚合过程中与纤维蛋白共价结合的慢病毒颗粒。为此，我们将识别因子 XIII 的肽结构域融合到病毒包膜糖蛋白（VSV-G）中，并将识别纤溶酶的蛋白酶切割位点融合到包膜中。用修饰的包膜假型化的慢病毒以因子 XIII 剂量依赖性方式结合纤维蛋白原，并在纤溶酶处理时释放。除了缺乏将肽与 VSV-G 包膜分离的任何柔性接头的情况外，带有肽/VSV-G 融合包膜变体的病毒不会影响所得病毒的转导效率。病毒从凝胶中的扩散显著减少，尤其是在高浓度的因子 XIII 时，即使对于加载高滴度病毒的纤维蛋白原浓度低的纤维蛋白凝胶也是如此。用纤维蛋白结合的慢病毒制备的慢病毒阵列可实现高效的基因转移，且仅限于含有病毒的纤维蛋白点。因此，信号/噪声比增加，相邻位点之间的交叉污染最小。最后，除了慢病毒微阵列外，这种策略还可用于实现治疗应用中的空间控制基因转移。
