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Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector
D M Shayakhmetov1, T Papayannopoulou, G Stamatoyannopoulos
1Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
Abstract:
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.
Insights
Adenovirus serotype 35 (Ad35) vectors show improved gene transfer into hematopoietic stem cells. A chimeric Ad5 vector with an Ad35 fiber enhances transduction efficiency and broadens cell targeting.
Area of Science:
- Gene Therapy
- Virology
- Hematopoietic Stem Cell Biology
Background:
- Adenovirus serotype 5 (Ad5) vectors require coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins for efficient gene transfer.
- Limited expression of these receptors on hematopoietic stem cells restricts Ad5-mediated gene transfer.
- Adenovirus serotype 35 (Ad35) shows higher tropism for CD34(+) cells.
Purpose of the Study:
- To screen adenovirus serotypes for efficient interaction with human CD34(+) cells.
- To develop a chimeric adenovirus vector with enhanced tropism for hematopoietic stem cells.
- To evaluate the gene transfer efficiency of the chimeric vector compared to standard Ad5 vectors.
Main Methods:
- Screening of different Ad serotypes for attachment, internalization, and replication in CD34(+) and K562 cells.
- Generation of a chimeric Ad5 vector (Ad5GFP/F35) with an Ad35 fiber.
- Transduction of CD34(+) cells with Ad5GFP/F35 and Ad5GFP vectors, followed by assessment of GFP expression and viral genome presence.
Main Results:
- Adenovirus serotype 35 demonstrated the highest tropism for CD34(+) cells.
- The chimeric Ad5GFP/F35 vector efficiently transduced CD34(+) cells via a CAR- and alpha(v) integrin-independent pathway.
- Ad5GFP/F35 achieved 54% GFP expression in CD34(+) cells, compared to 25% for Ad5GFP.
- Transduction efficiency was higher than reported due to unactivated CMV promoter in some cells.
- Ad5GFP/F35 transduced a broader spectrum of CD34(+) cells, including CD34(+) c-Kit(+) cells (55% vs. 13%).
Conclusions:
- A chimeric Ad5 vector incorporating the Ad35 fiber significantly enhances gene transfer into hematopoietic stem cells.
- This retargeted vector overcomes limitations associated with CAR and alpha(v) integrin dependency.
- The findings support the potential of Ad5GFP/F35 for stable gene transfer into hematopoietic stem cells.