Specific and efficient targeting of adenovirus vectors to macrophages: application of a fusion protein between an adenovirus-binding fragment and avidin, linked to a biotinylated oligonucleotide.
BACKGROUND
The application of serotype 5 adenoviruses (Ad5) in macrophages is hampered by the absence of the endogenous coxsackie adenovirus receptor (CAR).


METHODS
To overcome this limitation, we first generated a linker protein consisting of the virus-binding domain of CAR and the C-terminus of avidin. Second, to target macrophages, this linker protein was equipped with the biotinylated (bio) oligonucleotide dA6G10, which was previously shown to display a high affinity for the scavenger receptor A (SR-A).


RESULTS
As compared to nontargeted virus, the linker protein equipped with bio-dA6G10 showed a 500-fold increased reporter gene expression in mouse macrophage RAW264.7 cells. A linker protein equipped with a bio-dA16 control oligonucleotide was inactive. Moreover, the bio-dA6G10-equipped linker showed a 390-fold increased luciferase expression in the macrophage cell line J774 and 276- and 150-fold increased reporter gene expression in primary peritoneal and bone marrow (BM)-derived macrophages, respectively. Using BM-derived macrophages from SR-A knockout mice, it was shown that the dA6G10-mediated uptake is predominantly SR-A-mediated.


CONCLUSIONS
Thus, we have developed a novel tool to link biotinylated ligands to a virus-binding fragment of CAR and have exploited this linker protein to extend the applicability of Ad5 to infect transformed and primary macrophages.
Emile Gras
J C
JC
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Verkuijlen
Paul
P
Frants
Rune R
RR
Havekes
Louis M
LM
van Berkel
Theo J C
TJ
Biessen
Erik A L
EA
van Dijk
K Willems
KW
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001360:Avidin / Q000378:metabolism*; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D005260:Female; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
C568771:CLMP protein, human; C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D008024:Ligands; D009841:Oligonucleotides; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D001360:Avidin