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J. Biol. Chem., Vol. 282, Issue 29, 20790-20793, July 20, 2007

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Intranuclear Protein Transduction through a Nucleoside Salvage Pathway^*

James E. Hansen, Chung-Ming Tse^¶, Grace Chan, Emil R. Heinze, Robert N. Nishimura^||1, and Richard H. Weisbart,²

From the Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS), Sepulveda, California 91343, the Department of Medicine, UCLA-San Fernando Valley Program, Sylmar, California 91342, the ^¶Department of Medicine, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^||Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095

Regulation of gene expression by intranuclear transduction of macromolecules such as transcription factors is an alternative to gene therapy for the treatment of numerous diseases. The identification of an effective intranuclear delivery vehicle and pathway for the transport of therapeutic macromolecules across plasma and nuclear membranes, however, has posed a significant challenge. The anti-DNA antibody fragment 3E10 Fv has received attention as a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Elucidation of the pathway that allows 3E10 Fv to cross cell membranes is critical to the development of new molecular therapies. Here we show that 3E10 Fv penetrates cells through a nucleoside salvage transporter. 3E10 Fv is unable to penetrate into cells deficient in the equilibrative nucleoside transporter ENT2, and reconstitution of ENT2 into ENT2-deficient cells restores 3E10 Fv transport into cell nuclei. Our results represent the first demonstration of protein transport through a nucleoside salvage pathway. We expect that our finding will facilitate a variety of methods of gene regulation in the treatment of human diseases, open up new avenues of research in nucleoside salvage pathways, and enhance our understanding of the pathophysiology of autoimmune diseases.

Received for publication, May 10, 2007

^* This work was supported by a Veterans Affairs grant (to R. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors are senior authors.

² To whom correspondence should be addressed: Veterans Affairs Medical Center (111S), 16111 Plummer St., Sepulveda, CA 91343. Fax: 818-895-9423; E-mail: rweisbar{at}ucla.edu.

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