Site-Specific Integration by Recruitment of a Complex of Φc31 Integrase and Donor DNA to a Target Site by Using a Tandem, Artificial Zinc-Finger Protein

Tatsuhiko Sumikawa, Serika Ohno, Takeharu Watanabe, Ryo Yamamoto, Miyu Yamano, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

Research output: Contribution to journalArticle

Abstract

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated "Tandem AZP"). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA. Thereafter, an exogenously added integrase was expected to integrate the donor DNA into a specific site in the acceptor DNA (as it might be in the human genome). We demonstrated in vitro that in the presence of Tandem AZP, ΦC31 integrase selectively integrated a donor plasmid into a target acceptor plasmid not only at 30 °C (the optimum temperature of the integrase) but also at 37 °C (for future application in humans). We expect that with further improvement of our current system, a combination of Tandem AZP with integrase/recombinase will enable site-specific integration in mammalian cells and provide safer gene therapy technology.

Original languageEnglish
Pages (from-to)6868-6877
Number of pages10
JournalBiochemistry
Volume57
Issue number50
DOIs
Publication statusPublished - Dec 18 2018

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Integrases
Zinc Fingers
Zinc
DNA
Gene therapy
Proteins
Genes
Genetic Therapy
Plasmids
Recombinases
Human Genome
Gene expression
Fingers
Cells
Technology
Gene Expression
Peptides
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-Specific Integration by Recruitment of a Complex of Φc31 Integrase and Donor DNA to a Target Site by Using a Tandem, Artificial Zinc-Finger Protein. / Sumikawa, Tatsuhiko; Ohno, Serika; Watanabe, Takeharu; Yamamoto, Ryo; Yamano, Miyu; Mori, Tomoaki; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi.

In: Biochemistry, Vol. 57, No. 50, 18.12.2018, p. 6868-6877.

Research output: Contribution to journalArticle

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