Engineering enhanced retroviral vectors through the selection of novel protein libraries

Engineering viral vectors through the genetic insertion of peptides or domains that confer novel functions could readily improve their properties, but the lack of structural information on many viral proteins complicates efforts to identify optimal insertion sites. This study describes the development of a transposon-based approach to generate 'saturated insertion libraries' that contain designed peptides randomly incorporated at numerous sites within a target viral protein. This technique can be combined with a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel, designed functionality. We have applied this system to address two key obstacles in viral gene delivery: (1) the need for highly purified vector preparations and (2) the inability of simple retroviruses to infect nondividing cells.; The production of highly purified gene delivery vectors is imperative to avoid an immune response in a patient. Though the vesicular stomatitis virus glycoprotein (VSV-G) is widely used to pseudotype retroviral and lentiviral vectors, the lack of structural knowledge of the protein has made it difficult to rationally determine where to engineer modifications without disrupting the virus' ability to transduce cells. We generated a comprehensive library of VSV-G mutants that have a His6 tag randomly incorporated at as many or all possible points in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high titer viral vectors and could be purified by immobilized metal affinity chromatography. A dramatic reduction in protein and DNA impurities elicited a lower immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. In addition to addressing a critical need for clinical gene therapy, information gained through this work could be useful in developing other desirable extracellular properties for vectors, such as cell-specific targeting.; A key limitation of the use of simple retroviruses, such as murine leukemia virus (MLV), for gene therapy is their inability to infect quiescent cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We generated saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. (Abstract shortened by UMI.)...