A soluble CAR-SCF fusion protein improves adenoviral vector-mediated gene transfer to c-Kit-positive hematopoietic cells.
BACKGROUND
Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR).


METHODS
We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF.


RESULTS
Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following Ad5-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells.


CONCLUSIONS
The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies.
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Japan.
Akira
A
Itoh
Takashi
T
Okada
Hiroyuki
H
Mizuguchi
Takao
T
Hayakawa
Hiroaki
H
Mizukami
Akihiro
A
Kume
Masaaki
M
Takatoku
Norio
N
Komatsu
Yutaka
Y
Hanazono
Keiya
K
Ozawa
D000256:Adenoviridae; D045744:Cell Line, Tumor; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism*
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D008164:Luminescent Proteins; D011991:Receptors, Virus; D019089:Stem Cell Factor; D049452:Green Fluorescent Proteins; D019009:Proto-Oncogene Proteins c-kit