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

<jats:title>ABSTRACT</jats:title> <jats:p> Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and α <jats:sub>v</jats:sub> 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 <jats:sup>+</jats:sup> 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 <jats:sup>+</jats:sup> 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 α <jats:sub>v</jats:sub> integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34 <jats:sup>+</jats:sup> cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34 <jats:sup>+</jats:sup> cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34 <jats:sup>+</jats:sup> cells expressing α <jats:sub>v</jats:sub> integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34 <jats:sup>+</jats:sup> 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 <jats:sup>+</jats:sup> cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34 <jats:sup>+</jats:sup> c-Kit <jats:sup>+</jats:sup> cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34 <jats:sup>+</jats:sup> c-Kit <jats:sup>+</jats:sup> cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells. </jats:p>