Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells.
Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, alpha(v)beta5- and alpha(v)beta3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was 'trans-complementation' of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.
Fechner
H
H
Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität, Berlin, Germany.
Wang
X
X
Wang
H
H
Jansen
A
A
Pauschinger
M
M
Scherübl
H
H
Bergelson
J M
JM
Schultheiss
H P
HP
Poller
W
W
D000256:Adenoviridae / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005770:Gastrointestinal Neoplasms / Q000628:therapy*; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000235:genetics*; D019030:Receptors, Vitronectin / Q000235:genetics; D019076:Transgenes; D014779:Virus Replication
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D016023:Integrins; D008164:Luminescent Proteins; D011991:Receptors, Virus; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5; D049452:Green Fluorescent Proteins; D008156:Luciferases