In vivo analysis of a genetically modified adenoviral vector targeted to human CD40 using a novel transient transgenic model.
BACKGROUND
Retargeting is necessary to overcome the limitations of adenovirus (Ad)-based gene therapy vectors. To this end, we previously constructed an adenovirus with the fiber knob domain replaced by a fibritin trimerization motif fused to the CD40 ligand (Ad5Luc.FF/CD40L). We demonstrated the utility of this fiber replacement strategy for targeting CD40 (hCD40) on human dendritic cells in vitro. The in vivo targeting capacity of this virus, however, is unknown, and there is a limited repertoire of animal models that present hCD40 at an accessible site. Therefore, a new animal model for evaluating CD40-targeted vectors is required.


METHODS
We constructed a recombinant adenovirus that expresses hCD40 under transcriptional control of the flt-1 promoter (AdflthCD40). Expression of hCD40 was validated both in vitro and in transgenic mice expressing the human coxsackie adenovirus receptor (hCAR mice). We then evaluated the targeting efficiency of Ad5Luc.FF/CD40L to hCD40 expressed in the pulmonary vasculature of the hCAR mice.


RESULTS
Infection of flt-1-positive cells with AdflthCD40 resulted in abundant hCD40 expression in vitro, which could subsequently be targeted by Ad5Luc.FF/CD40L. In vivo administration of AdflthCD40 to hCAR mice resulted in hCD40 expression in the pulmonary vasculature, which was successfully targeted with systemically administered Ad5Luc.FF/CD40L.


CONCLUSIONS
This is the first data showing that genetically modified Ad5Luc.FF/CD40L can successfully target hCD40 in vivo. Our data also establishes the utility of transcriptionally targeted, Ad-mediated transient expression of human target molecules in the pulmonary vasculature of hCAR mice as models for in vivo analysis of targeted gene therapy vectors.
Izumi
Miiru
M
Division of Human Gene Therapy, Department of Medicine, Gene Therapy Center, University of Alabama at Birmingham, AL 35294-2172, USA.
Kawakami
Yosuke
Y
Glasgow
Joel N
JN
Belousova
Natalya
N
Everts
Maaike
M
Kim-Park
SangAe
S
Yamamoto
Seiji
S
Wang
Minghui
M
Le
Long P
LP
Reynolds
Paul N
PN
Curiel
David T
DT
D000818:Animals; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism*; D045744:Cell Line, Tumor; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D003713:Dendritic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases; D008168:Lung / Q000098:blood supply; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011991:Receptors, Virus / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics
D019013:CD40 Antigens; C568771:CLMP protein, human; C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D011991:Receptors, Virus; D023201:CD40 Ligand; D008156:Luciferases; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1