736. Engineering Enhanced Retroviral and Lentiviral Vectors through the Selection of Peptide Insertion Libraries

Abstract

Engineering viral vectors through the genetic insertion of peptides or domains to confer novel functions can readily improve their properties as gene therapy vehicles; however, the lack of structural information for many viral proteins complicates efforts to identify optimal insertion sites. We have developed 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. This library generation and selection approach has enabled successful modifications to both extracellular and intracellular properties of retroviral and lentiviral vectors and can be readily extended to the development of other viral vectors and, more broadly, to other challenges in mammalian protein engineering.The ability to produce highly purified gene delivery vectors is imperative to avoid an immune response in a patient. Though VSV- G is widely used to pseudotype retroviral and lentiviral vectors, the lack of structural knowledge has made it difficult to rationally determine where to engineer modifications without disrupting native protein function. 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. Column purification of vectors reduced protein and DNA impurities over 5,000-fold and 14,000- fold, respectively, from the viral supernatant. This substantially improved purity elicited a lower immune response in the brain, without altering the infectivity or tropism from wild-type VSVG 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 retroviral vectors for gene therapy is their inability to infect quiescent cells. We applied the transposon-based approach to generate libraries of MLV gag-pol variants that had nuclear localization sequences randomly incorporated throughout the overlapping genes. These libraries were selected in the context of retroviral infections of nondividing cells and led to the identification of a novel MLV variant that can successfully transduce growth-arrested cells as well as primary neurons. The ability to engineer specific aspects of intracellular viral infection mechanisms has potential implications for the design and control of other post-entry events, such as site-specific integration.We have applied for a provisional patent on the VSV-G-His6 variants. Engineering viral vectors through the genetic insertion of peptides or domains to confer novel functions can readily improve their properties as gene therapy vehicles; however, the lack of structural information for many viral proteins complicates efforts to identify optimal insertion sites. We have developed 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. This library generation and selection approach has enabled successful modifications to both extracellular and intracellular properties of retroviral and lentiviral vectors and can be readily extended to the development of other viral vectors and, more broadly, to other challenges in mammalian protein engineering. The ability to produce highly purified gene delivery vectors is imperative to avoid an immune response in a patient. Though VSV- G is widely used to pseudotype retroviral and lentiviral vectors, the lack of structural knowledge has made it difficult to rationally determine where to engineer modifications without disrupting native protein function. 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. Column purification of vectors reduced protein and DNA impurities over 5,000-fold and 14,000- fold, respectively, from the viral supernatant. This substantially improved purity elicited a lower immune response in the brain, without altering the infectivity or tropism from wild-type VSVG 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 retroviral vectors for gene therapy is their inability to infect quiescent cells. We applied the transposon-based approach to generate libraries of MLV gag-pol variants that had nuclear localization sequences randomly incorporated throughout the overlapping genes. These libraries were selected in the context of retroviral infections of nondividing cells and led to the identification of a novel MLV variant that can successfully transduce growth-arrested cells as well as primary neurons. The ability to engineer specific aspects of intracellular viral infection mechanisms has potential implications for the design and control of other post-entry events, such as site-specific integration. We have applied for a provisional patent on the VSV-G-His6 variants.