Yilmazer Açelya, Tian Bowen, Kostarelos Kostas
Nanomedicine Laboratory, Faculty of Life Sciences, University College London, London, United Kingdom.
School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
PLoS One. 2014 Dec 12;9(12):e114985. doi: 10.1371/journal.pone.0114985. eCollection 2014.
Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.
利用人5型腺病毒（Ad5）进行基因治疗已被广泛探索用于治疗对传统疗法耐药的疾病。静脉内给药会导致其从血液循环中迅速清除并在肝脏中大量蓄积，这限制了基于Ad的载体在涉及全身给药的临床基因治疗方案中的应用。我们之前曾提出，通过在Ad周围构建人工脂质包膜可以改善这些局限性，并围绕病毒衣壳设计了多种人工脂质双分子层包膜。在本研究中，我们试图通过设计一种以前未报道的基因治疗载体来进一步探索人工包膜病毒构建体可能带来的机会，即将Ad和siRNA同时包裹在同一脂质双分子层中。这种双活性载体可以为需要开启和关闭基因的不同遗传疾病提供有效的治疗。利用动态光散射、透射电子显微镜和原子力显微镜对这些载体进行了表征。琼脂糖凝胶电泳、Ribo green和斑点印迹分析表明，siRNA和Ad病毒粒子可以以高包裹效率共同包裹在脂质双分子层内。进行了细胞摄取和体外转染实验，以证明使用这些新设计的双活性载体将siRNA介导的基因沉默与病毒基因转移相结合的可行性。
