Larocca D, Jensen-Pergakes K, Burg M A, Baird A
Selective Genetics, Inc., 11035 Roselle Street, San Diego, California 92121, USA.
Mol Ther. 2001 Apr;3(4):476-84. doi: 10.1006/mthe.2001.0284.
Although growth factor- and antibody-targeted filamentous phage have recently been demonstrated to transduce mammalian cells, there is a significant need to increase transduction efficiency so as to improve the usefulness of targeted phage vectors for gene therapy and ligand discovery. Here, we describe the use of multivalent phagemid vectors that are specifically designed for ligand-targeted mammalian cell transduction. This phagemid system has certain advantages over phage vectors, such as larger insert size and vector stability, and it retains the multivalent display necessary for efficient cell binding and internalization. Immunoblotting revealed that the most efficient multivalent display (exceeding that of a phage vector) was achieved in the phagemid system when epidermal growth factor (EGF) was fused to the C-terminal domain of the pIII coat protein. We compared phagemid particles displaying EGF at high or low valence by rescuing the vector with R408d3 (pIII deleted) or wild-type R408 helper phage, respectively. More efficient display of EGF correlated with increased internalization, vector potency, and transduction efficiency ( approximately 9%). The findings described here support our original hypothesis that phage-based vectors can be modified for more efficient gene transfer and suggest that directed evolution may be applied to increase their potential even further.
尽管最近已证明生长因子靶向和抗体靶向的丝状噬菌体可转导哺乳动物细胞，但仍迫切需要提高转导效率，以增强靶向噬菌体载体在基因治疗和配体发现中的实用性。在此，我们描述了一种多价噬菌粒载体的应用，该载体是专门为配体靶向的哺乳动物细胞转导而设计的。这种噬菌粒系统相对于噬菌体载体具有某些优势，如更大的插入片段大小和载体稳定性，并且它保留了有效细胞结合和内化所需的多价展示。免疫印迹显示，当表皮生长因子（EGF）与pIII外壳蛋白的C末端结构域融合时，在噬菌粒系统中实现了最有效的多价展示（超过噬菌体载体）。我们分别用R408d3（缺失pIII）或野生型R408辅助噬菌体拯救载体，比较了高价或低价展示EGF的噬菌粒颗粒。EGF更有效的展示与内化增加、载体效力和转导效率提高（约9%）相关。此处描述的研究结果支持了我们最初的假设，即基于噬菌体的载体可进行修饰以实现更有效的基因转移，并表明定向进化可能可进一步应用以增加其潜力。
