In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors.
BACKGROUND
Both viral and non-viral gene transfer vectors transduce vascular endothelial cells (EC) with low efficiency compared with other cell types such as hepatocytes. Generation of EC-selective vectors would enhance the clinical utility of gene therapy for diverse vascular-targeted applications.


METHODS
12mer peptides derived by in vitro phage display with EC binding specificity [MTPFPTSNEANL (MTP) and MSLTTPPAVARP (MSL)] were inserted at position T542 in the exposed HI loop of the adenovirus (Ad) serotype 5 fiber using overlapping oligonucleotides; in combination with a double point mutation (KO1) to ablate virus : cell binding via the coxsackie-adenovirus receptor (CAR). The resulting modified viruses were tested in vitro and in vivo for their ability to direct endothelial-specific gene transfer.


RESULTS
Peptide insertion was not deleterious to fiber trimerisation or virion maturation. In vitro gene transfer studies using a panel of cell types demonstrated that both peptide-targeted Ad vectors mediated efficient CAR-independent gene transfer to vascular EC compared with non-modified Ads. Neither peptide supported gene delivery to non-EC. Upon systemic injection into mice and subsequent evaluation of transgene expression we failed to observe a reduction in hepatic Ad accumulation but observed a significant elevation in beta-galactosidase in blood vessels with the MSLTTPPAVARP-targeted Ad vector.


CONCLUSIONS
We have genetically engineered two novel Ads that transduce human EC selectively in vitro, one of which leads to altered Ad biodistribution in vivo. The successful generation of genetically engineered tropism for EC has broad implications for cardiovascular gene therapy. Further modifications to the Ad capsid will be required to improve in vivo biodistribution profiles.
Nicklin
Stuart A
SA
Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow G11 6NT, UK.
White
Steve J
SJ
Nicol
Campbell G
CG
Von Seggern
Dan J
DJ
Baker
Andrew H
AH
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D042783:Endothelial Cells / Q000378:metabolism* / Q000821:virology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000627:therapeutic use; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000235:genetics / Q000302:isolation & purification; D019151:Peptide Library; D011991:Receptors, Virus / Q000378:metabolism; D014161:Transduction, Genetic; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000906:Antibodies; C568771:CLMP protein, human; C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D009842:Oligopeptides; D019151:Peptide Library; D011991:Receptors, Virus; D015678:Viral Structural Proteins