CAR-dependent and CAR-independent pathways of adenovirus vector–mediated gene transfer and expression in human fibroblasts
Chisa Hidaka, … , Peter Roelvink, Ronald G. Crystal
Chisa Hidaka, … , Peter Roelvink, Ronald G. Crystal
Published February 15, 1999
Citation Information: J Clin Invest. 1999;103(4):579-587. https://doi.org/10.1172/JCI5309.
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Article

CAR-dependent and CAR-independent pathways of adenovirus vector–mediated gene transfer and expression in human fibroblasts

Abstract

Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdΔRGDβgal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) αV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)βgal (bearing seven lysines on the end of fiber) (b) AdF(RGD)βgal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK βgal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.

Authors

Chisa Hidaka, Eric Milano, Philip L. Leopold, Jeffrey M. Bergelson, Neil R. Hackett, Robert W. Finberg, Thomas J. Wickham, Imre Kovesdi, Peter Roelvink, Ronald G. Crystal

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Expression of the transgene in Ad vectors with altered fibers in CAR-suf...
Expression of the transgene in Ad vectors with altered fibers in CAR-sufficient vs. CAR-deficient primary human fibroblasts. Mock-, AdNull-, or AdCAR-infected fibroblasts were infected with 300 pu/cell of Adβgal (first-generation Ad vector expressing the βgal gene) or AdF(pK7) βgal [F(pK7), similar to Adβgal, but seven lysine residues at the end of each fiber protein], AdβgalF(RGD) [F(RGD), similar to Adβgal, but bearing a high-affinity RGD sequence on the end of each fiber protein], or AdF9sK (F9sK, similar to Adβgal, but with a short fiber including the tail and first eight shaft repeats of Ad5 fiber attached to the last shaft repeat and knob of Ad9). βgal activity of cell lysates was assayed 24 h after the second infection. The data are presented as mean ± SE of triplicate measurements.