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

Angelo Lombardo, Daniela Cesana, Pietro Genovese, Bruno Di Stefano, Elena Provasi, Daniele F. Colombo, Margherita Neri, Zulma Magnani, Alessio Cantore, Pietro Lo Riso, Martina Damo, Oscar M. Pello, Michael C. Holmes, Philip D. Gregory, Angela Gritti, Vania Broccoli, Chiara Bonini, Luigi Naldini

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)861-869
Number of pages9
JournalNature Methods
Issue number10
Publication statusPublished - Oct 2011

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Biochemistry
  • Cell Biology


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