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Journal of Virology, March 2000, p. 2567-2583, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector

Dmitry M. Shayakhmetov,1 Thalia Papayannopoulou,2 George Stamatoyannopoulos,1 and André Lieber1,*

Division of Medical Genetics1 and Division of Hematology,2 Department of Medicine, University of Washington, Seattle, Washington 98195

Received 3 September 1999/Accepted 5 December 1999

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha v integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34+ cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34+ cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha v integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34+ cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34+ cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34+ cells expressing alpha v integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34+ cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34+ cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34+ c-Kit+ cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34+ c-Kit+ cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

* Corresponding author. Mailing address: Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195. Phone: (206) 221-3973. Fax: (206) 685-8675. E-mail: lieber00{at}u.washington.edu.

Journal of Virology, March 2000, p. 2567-2583, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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