ARTICLES NATURE METHODS | VOL.8 NO.10 | OCTOBER 2011 | 861 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. Integrative gene transfer is widely used to study gene function, confer new properties on cells or organisms and, in the clinical setting, to correct disease. However, the use of conventional vectors that integrate semirandomly throughout the genome has several limitations. Because transgene expression is influenced by the integration site, expression varies and silencing occurs among transduced cells 1 . Some insertions may disrupt genes or perturb their transcription, altering the biological properties of the transduced cell. This may occur when insertion activates a proto-oncogene and triggers cell transformation, as has been reported in some gene-therapy applications 2 . Site-specific inte- gration may overcome these hurdles. We and others have previously described the use of engineered zinc- finger nucleases (ZFNs) to target gene transfer 3–6 . A ZFN-induced Site-specific integration and tailoring of cassette design for sustainable gene transfer Angelo Lombardo 1,2 , Daniela Cesana 1,2,7 , Pietro Genovese 1,2,7 , Bruno Di Stefano 3,6,7 , Elena Provasi 2,4,7 , Daniele F Colombo 1,2,7 , Margherita Neri 1,2 , Zulma Magnani 4 , Alessio Cantore 1,2 , Pietro Lo Riso 1,2 , Martina Damo 1,2 , Oscar M Pello 1 , Michael C Holmes 5 , Philip D Gregory 5 , Angela Gritti 1 , Vania Broccoli 3 , Chiara Bonini 4 & Luigi Naldini 1,2 DNA double-strand break at a predetermined site of the genome can trigger homology-directed repair, a pathway exploited to insert a transgene into the ZFN target site from an exogenous template. With this approach, exogenous DNA sequences can be delivered into specific loci for comparative or subtractive gene function studies without the confounding influences of insertion sites and to replace malfunctioning genes 7,8 . However, the choice of a suitable genomic acceptor site for wide application in gene transfer and the optimal design of the transgene cassette to ensure robust expres- sion without perturbing nearby endogenous transcription remain to be investigated. An ideal site for transgene insertion should allow (i) robust and stable transgene expression across different cell types; (ii) no transcriptional perturbation owing to the transgene cassette and (iii) no disruption of essential regulatory or coding sequences due to the transgene cassette. Such sites may be identified in model organisms such as the mouse from data on established transgenic lines 9 , although these data might not apply to the human system. In humans, such sites may be sought by combining emerging knowledge on sequence variation among genomes 10 and on clinically silent homozygous gene deficiencies with gene expression atlases 11 , and possibly from the few identified func- tional vector insertions associated with a benign outcome in gene therapy clinical trials 12,13 . We and others have previously investigated the potential suit- ability for transgene insertion at two human genomic locations: (i) a common integration site of the human non-pathogenic adeno-associated virus (AAV), found between exon 1 and intron 1 of protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene, known as AAV site 1 (AAVS1) 14 and (ii) the HIV co-receptor chemokine (C-C motif) receptor 5 (CCR5) gene, for which a homozygous deletion is found in apparently healthy individuals 15 . We previously described efficient integration into CCR5 mediated by ZFNs targeting exon 3 of the gene, resulting in stable transgene 1 San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Gene Therapy and Stem Cells, San Raffaele Institute, Milan, Italy. 2 Vita-Salute San Raffaele University, Milan, Italy. 3 Stem Cell and Neurogenesis Unit, Division of Neurosciences, San Raffaele Institute, Milan, Italy. 4 Experimental Hematology Unit, Division of Regenerative Medicine, Gene Therapy and Stem Cells, Program in Immunology and Bio-immunotherapy of Cancer, San Raffaele Scientific Institute, Milan, Italy. 5 Sangamo BioSciences Inc., Richmond, California, USA. 6 Present address: Hematopoietic Stem Cell Biology and Differentiation Group, Department of Differentiation and Cancer Centre for Genomic Regulation, Barcelona, Spain. 7 These authors contributed equally to this work. Correspondence should be addressed to L.N. (naldini.luigi@hsr.it). RECEIVED 8 FEBRUARY; ACCEPTED 29 JULY; PUBLISHED ONLINE 21 AUGUST 2011; DOI:10.1038/NMETH.1674 © 2011 Nature America, Inc. All rights reserved. © 2011 Nature America, Inc. All rights reserved.