Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

Yoshinori Ishikawa, Nobuyuki Tanaka, Kazuhiro Murakami, Toru Uchiyama, Satoru Kumaki, Shigeru Tsuchiya, Hiroyuki Kugoh, Mitsuo Oshimura, Michele P. Calos, Kazuo Sugamura

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33 Citations (Scopus)

Abstract

Background: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor γ chain (γc) gene. Although ex vivo gene therapy, i.e., transduction of the γc gene into autologous CD34+ cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the φC31 integrase-based integration system using human T-cell lines, including the γc-deficient ED40515(-). Methods: A φC31 integrase and a neor gene expression plas mid containing the φC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a γc expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced γc in an ED40515(-)-derived clone. Results: Following co-introduction of t he φC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated γc gene exhibited stable expression of the γc protein, with normal IL-2 signaling, as assessed by STAT5 activation. Conclusions: This study supports the poss ible future use of this φC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

Original languageEnglish
Pages (from-to)646-653
Number of pages8
JournalJournal of Gene Medicine
Volume8
Issue number5
DOIs
Publication statusPublished - 2006 May
Externally publishedYes

Keywords

  • Hematopoietic cells
  • SCID-X1
  • Site-specific integration
  • φC31 integrase

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

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    Ishikawa, Y., Tanaka, N., Murakami, K., Uchiyama, T., Kumaki, S., Tsuchiya, S., Kugoh, H., Oshimura, M., Calos, M. P., & Sugamura, K. (2006). Phage φC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. Journal of Gene Medicine, 8(5), 646-653. https://doi.org/10.1002/jgm.891