Codon Swapping of Zinc Finger Nucleases Confers Expression in Primary Cells and In Vivo from a Single Lentiviral Vector
Eli J. Fine,
Laure Fournier Le Ray,
Thomas J. Cradick,
Tuan Huy Nguyen.
Background: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well
as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are
highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse
transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within
ZFN monomers. Methods: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN
monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codonswapped
ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats
were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to
deliver ZFNs into target cells. Results: We reduced total identity between ZFN monomers from 90.9% to 61.4% and
showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codonswapping,
leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60%
activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Offtarget
analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time
successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery
of a single IDLV encoding two ZFNs. Conclusion: These results demonstrate that a codon-swapping approach allowed
a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFNmediated
genome editing of primary cells, for both ex vivo or in vivo applications.
Keywords: Codon swapping, lentiviral vectors, liver, off-target sites, primary cells, zinc finger nuclease.
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