Protein transduction domains fused to virus receptors improve cellular virus uptake and enhance oncolysis by tumor-specific replicating vectors
Protein transduction domains fused to virus receptors improve cellular virus uptake and enhance oncolysis by tumor-specific replicating vectors
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DOI：
10.1128/jvi.78.24.13743-13754.2004
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发表时间：
2004-12-01
影响因子：
5.4
通讯作者：
Kubicka, S
中科院分区：
文献类型：
--
作者：
Kühnel, F;Schulte, B;Kubicka, S
细胞受体的表达决定了病毒的趋向性并限制了病毒载体的基因传递。蛋白质转导结构域（PTDs）已被证明可以将蛋白质、反义寡核苷酸、脂质体或质粒DNA传递到细胞中。在我们的研究中，我们研究了几个PTD基序在腺病毒感染中的作用。当生理表达时，来自人类免疫缺陷病毒转录反激活因子（Tat）的PTD并没有改善腺病毒感染。因此，我们将PTDs融合到柯萨奇病毒-腺病毒受体（CAR(ex)）的外域，将PTDs附着在腺病毒纤维旋钮上。CAR(ex)-Tat和CAR(ex)-VP22可在非受纳细胞中有效感染腺病毒，并显著提高受纳细胞的病毒摄取率。使用CAR（ex）介导的CAR(ex)-PTD介导感染的剂量依赖性竞争和肝素抑制实验表明，CAR(ex)-PTD与腺病毒纤维和细胞糖胺聚糖的结合对于改善感染是必不可少的。CAR(ex)-PTD处理的腺病毒在密度梯度超离心后仍保持其特性，表明CAR(ex)-PTD与腺病毒颗粒的稳定结合。因此，CAR(ex)-PTD介导的感染机制涉及由CAR(ex)-PTD包裹病毒纤维旋孔，而不是将CAR（ex）结构域放置在细胞表面。CAR(ex)-PTDs的表达通过复制腺病毒导致容许性和非容许性肿瘤细胞的裂解增强，这表明CAR(ex)-PTDs是提高溶瘤治疗疗效的有价值的工具。总之，我们的研究表明，CAR(ex)-PTDs促进非受纳细胞中的基因转移，并在降低滴度和感染时间的情况下改善病毒摄取。这些数据表明，与病毒结合受体融合的PTDs可能是克服载体自然趋向性的一种有价值的工具，并且可能对基因治疗方法有很大的兴趣。
Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CAR(ex)) to attach PTDs to adenoviral fiber knobs. CAR(ex)-Tat and CAR(ex)-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CAR(ex)-PTD-mediated infection using CAR(ex) and inhibition experiments with heparin showed that binding of CAR(ex)-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CAR(ex)-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CAR(ex)-PTD to adenoviral particles. Consequently, the mechanism of CAR(ex)-PTD-mediated infection involves coating of the viral fiber knobs by CAR(ex)-PTD, rather than placement of CAR(ex) domains on cell surfaces. Expression of CAR(ex)-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CAR(ex)-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CAR(ex)-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.