title: Recombination-deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution.
abstract: Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination-deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination-deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence.
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Eung Jun
authors.initials: EJ
authors.lastname: Im
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Bais
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Yang
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Qiangzhong
authors.initials: Q
authors.lastname: Ma
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Xiuyang
authors.initials: X
authors.lastname: Guo
authors.affiliation: Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sepe
authors.affiliation: Biotherapeutics Development Lab, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA ; Department of Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, Rhode Island, USA.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Junghans