Abstract
Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.
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Acknowledgements
We thank B. Celona, A. Anselmo and F. Ungaro for help with some experiments, A. Agresti, M. Bianchi and D. Gabellini for critical discussion, C. Di Serio and A. Nonis for statistical counseling, K. Ponder (Washington University, St. Louis), C. Miao (University of Washington, Seattle) and A. Recchia (University of Modena and Reggio Emilia) for providing reagents. Research was supported by Telethon (Telethon Institute for Gene Therapy grant), 7th EU Framework Programme (grant agreement 222878, PERSIST), European Research Council Advanced grant (249845 Targeting Gene Therapy), Fondazione Cariplo (Nobel Project) to L.N., Italian Ministry of Health (Giovani Ricercatori) to V.B.; Telethon (TGT06B02) to A.G.; Italian Ministry of Research and University (Ideas), Italian Ministry of Health (Giovani Ricercatori), Associazione Italiana per la Ricerca sul Cancro and Fondazione Cariplo to C.B.
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A.L., D.C., P.G., B.D.S., E.P. and D.F.C. designed and performed experiments, and interpreted data. M.N., Z.M., A.C., P.L.R., M.D. and O.M.P. performed experiments and interpreted data. M.C.H. and P.D.G. provided ZFNs. A.G., V.B. and C.B. coordinated NSC, iPSC and T-cell work, respectively. A.L. and L.N. conceived the project, coordinated all work and wrote the paper.
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M.C.H. and P.D.G. are employees of Sangamo BioSciences Inc.
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Lombardo, A., Cesana, D., Genovese, P. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869 (2011). https://doi.org/10.1038/nmeth.1674
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DOI: https://doi.org/10.1038/nmeth.1674
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