Site-specific integration and tailoring of cassette design for sustainable gene transfer

• Published in: Nature methods Vol. 8; no. 10; pp. 861 - 869
• Main Authors: Lombardo, Angelo, Cesana, Daniela, Genovese, Pietro, Di Stefano, Bruno, Provasi, Elena, Colombo, Daniele F, Neri, Margherita, Magnani, Zulma, Cantore, Alessio, Lo Riso, Pietro, Damo, Martina, Pello, Oscar M, Holmes, Michael C, Gregory, Philip D, Gritti, Angela, Broccoli, Vania, Bonini, Chiara, Naldini, Luigi
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.10.2011; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/2300; 631/208/199; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedical research; Cells; Dependovirus - genetics; Design; Gene expression; Gene therapy; Gene Transfer Techniques; Genetic aspects; Genetic transformation; Genomics; Health aspects; Humans; Life Sciences; Lymphocytes; Mutagenesis; Mutagenesis, Insertional - genetics; Mutagenesis, Site-Directed; Physiological aspects; Proteomics; Receptors, CCR5 - genetics; Stem cells; Sustainability; Virus Integration - genetics; United States; Italy
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/nmeth.1674

Presented is an experimental analysis of the stability of transgene expression, the perturbation of endogenous expression and the perturbation of epigenetic organization upon site-directed delivery of transgenes to the CCR5 and AAVS1 loci in human cells. It provides guidelines for optimal cassette design for stable and nonperturbative gene transfer. 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.