(567p) Bioorthogonal Approach to Purification and Functionalization of Semi-Synthetic Retroviruses

Due to their ability to achieve sustained expression of its integrated gene with relatively low immunogenicity, retroviruses have been key vectors in clinical gene therapy and genetically manipulation of mammalian cells (e.g., preparation of induced pluripotent stem (iPS) cells). Nevertheless, there are many shortfalls to working with this type of vector. Producing high-titered retroviral stocks and concentrating/purifying retroviruses are unattainable and highly cumbersome, respectively. Moreover, the retroviral envelope impedes engineering effective and versatile vectors because of the challenges in modifying the envelope without affecting its crucial roles on virus/cell interactions and stability. Therefore, in this study, we have developed a novel and convenient method to molecularly tune the exterior surface of retroviruses such that a variety of functionalities can be incorporated, in order to achieve 1) directed transduction, 2) targeted delivery, and 3) facile production/concentration. The glycoproteins on the surface of viruses and virus-producing cells were activated via glycosylic oxidization and were further bioorthogonally conjugated with various desired functional molecules. It was confirmed that the retroviral envelope was able to be labeled with a fluorescent dye, which can be useful for tracking retroviral vectors in a cell as well as in vivo (molecular imaging). In addition, it was demonstrated that magnetic particle (MP)-conjugated retroviruses can be easily purified and concentrated using magnetic column separation (improved preparation) and also be directed towards target cells using an external magnetic field (enhanced and directed transduction). Lastly, the potential of targeted transduction to specific cell types was explored via the approach of conjugating monoclonal antibodies to the activated retroviruses using the same bioorthagonal conjugation strategy. In conclusion, this facile method of molecularly tuning the surface functionality of retroviruses offers wide potential applicability, addressing important technical obstacles in utilizing retroviruses as emerging therapeutics as well as research tools for genetic modifications.