Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector.
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.
Shayakhmetov
D M
DM
Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
Papayannopoulou
T
T
Stamatoyannopoulos
G
G
Lieber
A
A
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D018952:Antigens, CD34; D001667:Binding, Competitive; D016466:CHO Cells; D002213:Capsid / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D036022:Capsid Proteins; D002460:Cell Line; D002478:Cells, Cultured; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D006224:Cricetinae; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000276:immunology; D006801:Humans; D039564:Integrin alphaV; D020014:K562 Cells; D011991:Receptors, Virus / Q000096:biosynthesis; D012703:Serotyping; D014779:Virus Replication
D015703:Antigens, CD; D018952:Antigens, CD34; C568771:CLMP protein, human; D036022:Capsid Proteins; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D039564:Integrin alphaV; D011991:Receptors, Virus; C018315:hexon capsid protein, Adenovirus