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.
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
J C
JC
Emile Gras
Paul
P
Verkuijlen
Rune R
RR
Frants
Louis M
LM
Havekes
Theo J C
TJ
van Berkel
Erik A L
EA
Biessen
K Willems
KW
van Dijk
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