Recombination-deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution

• Published in: Molecular therapy. Methods & clinical development 2014-07, Vol.1 (C), p.14022-14022, Article 14022
• Main Authors: Im, Eung Jun, Bais, Anthony J, Yang, Wen, Ma, Qiangzhong, Guo, Xiuyang, Sepe, Steven M, Junghans, Richard P
• Format: Article
• Language: English
• Subjects: Lentivirus; Retroviridae
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1038/mtm.2014.22

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 ( , 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.