Bilbao Guadalupe, Contreras Juan L, Dmitriev Igor, Smyth Cheryl A, Jenkins Stacie, Eckhoff Devin, Thomas Francis, Thomas Judith, Curiel David T
Department of Medicine, University of Alabama at Birmingham 35294, USA.
Am J Transplant. 2002 Mar;2(3):237-43. doi: 10.1034/j.1600-6143.2002.20308.x.
The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.
将免疫调节、细胞保护或抗凋亡基因导入胰岛（PI）的能力可能会提高移植后的存活率。现有的基因转移载体在不同细胞类型中感染和表达基因的能力差异很大。与使用病毒载体相关的一个限制与病毒对其主要结合位点的依赖性有关。可以使用重新靶向策略改变病毒载体的嗜性。噬菌体生物淘选结果证明RGD基序具有体内靶向能力。该基序尤其与αvβ3和αvβ5类型的细胞整合素相互作用，这些整合素在胰岛上高度表达。在本报告中，我们探索了一种在纤维钮的HI环中含有RGD基序的重新靶向腺病毒载体（Ad）的效用，以提高对完整分离的非人灵长类动物胰岛的感染效率，并降低基因修饰后的毒性。通过半自动技术分离非人灵长类动物的胰岛。将链脲佐菌素诱导的患有严重联合免疫缺陷疾病（SCID）的糖尿病小鼠用作受体。在我们实验室中构建了一种含有异源RGD肽并表达荧光素酶（AdRGDLuc）或绿色荧光蛋白（AdRGDGFP）的重组腺病毒。使用不含RGD肽的类似腺病毒作为对照（AdLuc和AdGFP）。与AdGFP相比，AdRGDGFP显示出更高的转染效率（使用AdRGDGFP在10个颗粒形成单位（pfu）/细胞时，超过80%的胰岛细胞被感染，而感染AdGFP后为7%）。超过90%的感染细胞是产生胰岛素的细胞。在不同滴度下，与AdLuc相比，AdRGDLuc感染后显示出显著更高的转基因表达。对葡萄糖刺激的胰岛素反应分析表明，与高滴度的AdLuc相比，低滴度（10 pfu/细胞以实现>80%的转染效率）的AdRGDLuc转染的胰岛表现更好。最后，接受AdRGDLuc感染的胰岛的动物中有89%观察到长期血糖正常（>250天），而接受AdLuc感染的胰岛的动物中没有观察到。本研究提供了关于腺病毒载体嗜性修饰可能性的新信息，以提高对分离的胰岛的转染效率和转基因表达。在纤维钮的HI环中掺入RGD序列可实现对非人灵长类动物产生胰岛素细胞的高效转染效率，并使移植后的β细胞具有足够的长期功能。
