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J. Biol. Chem., Vol. 278, Issue 35, 32578-32586, August 29, 2003

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Nanoparticles of Compacted DNA Transfect Postmitotic Cells^*

Ge Liu  , DeShan Li ¶, Murali K. Pasumarthy ¶, Tomasz H. Kowalczyk ¶, Christopher R. Gedeon ¶, Susannah L. Hyatt ¶, Jennifer M. Payne ¶, Timothy J. Miller ¶ ||, Peter Brunovskis ¶, Tamara L. Fink ¶, Osman Muhammad ¶, Robert C. Moen ¶, Richard W. Hanson  and Mark J. Cooper ¶ **

From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and ^¶Copernicus Therapeutics, Inc., Cleveland, Ohio 44106-3052

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900–360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.

Received for publication, June 2, 2003

^* This work was supported by Copernicus Therapeutics, Inc. and National Institutes of Health Grant DK-25541 (to R. W. H.). R. W. H. has a significant equity interest in Copernicus Therapeutics, Inc. 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.

Present address: Dept. of Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106.

^|| Present address: Dept. of Pharmacology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106.

^** To whom correspondence should be addressed: Copernicus Therapeutics, Inc., 11000 Cedar Ave., Suite 145, Cleveland, OH 44106-3052. Tel.: 216-231-0227; Fax: 216-231-9477; E-mail: mcooper{at}cgsys.com.

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