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Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells.

Liu S, Mao Q, Zhang W, Zheng X, Bian Y, Wang D, Li H, Chai L, Zhao J, Xia H - Biosci. Rep. (2009)

Bottom Line: The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR.The results provide some new clues as to how PTD.AdeGFP infects target cells.This new vector would be valuable in gene-function analysis and for gene therapy in cancer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, People's Republic of China.

ABSTRACT
The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.AdeGFP (where eGFP is enhanced green fluorescent protein) was constructed by modifying the HI loop of Ad5 (Ad type 5) fibre with the Tat (trans-activating) PTD (protein transduction domain) derived from HIV. The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR. The improvement in gene transfer was not the result of charge-directed binding between the virus and the cell surface. Although PTD.AdeGFP formed aggregates, it infected target cells in a manner different from AdeGFP aggregates precipitated by calcium phosphate. In addition, PTD.AdeGFP was able to transduce target cells in a dynamin-independent pathway. The results provide some new clues as to how PTD.AdeGFP infects target cells. This new vector would be valuable in gene-function analysis and for gene therapy in cancer.

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The effect of PTD.AdeGFP infection on cells by heparinase 1 or mutant dynamin(A) The effect of heparinase 1 on infection of T24 or HUVEC cells by PTD.AdeGFP. GFP-positive cells was detected as described in the Materials and methods section. (B) The effect of mutant dynamin on infection of A549 cells by PTD.AdeGFP or AdeGFP. A549 cells were pre-treated with mutant dynamin virus (5000 particles/cell) or E1-deleted Ad5 (empty vector, 5000 particles/cell). After 48 h, cells were infected with PTD.AdeGFP or AdeGFP and then washed and incubated at 37°C for a further 48 h, after which the GFP-positive cells were detected as described in the Materials and methods section. Results are means±S.D. (n=3). Statistical significance was determined using the Student's t test.
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Figure 4: The effect of PTD.AdeGFP infection on cells by heparinase 1 or mutant dynamin(A) The effect of heparinase 1 on infection of T24 or HUVEC cells by PTD.AdeGFP. GFP-positive cells was detected as described in the Materials and methods section. (B) The effect of mutant dynamin on infection of A549 cells by PTD.AdeGFP or AdeGFP. A549 cells were pre-treated with mutant dynamin virus (5000 particles/cell) or E1-deleted Ad5 (empty vector, 5000 particles/cell). After 48 h, cells were infected with PTD.AdeGFP or AdeGFP and then washed and incubated at 37°C for a further 48 h, after which the GFP-positive cells were detected as described in the Materials and methods section. Results are means±S.D. (n=3). Statistical significance was determined using the Student's t test.

Mentions: Most mammalian cell types express heparin-containing cellular receptors that could bind to charged amino acids [36,37]. Previous studies indicated that adenoviral capsids modified to contain a C-terminal poly-lysine tract allowed for an efficient transduction of multiple cell types via facilitated binding to cell surface heparan sulfate proteoglycans [20]. The Tat PTD is enriched in positively charged amino acids (YGKKKRRQRRR), and, in order to rule out the possibility that the improvement of gene transfer is a result of increased charge-directed binding between viruses and cell surface, the cells were treated with heparinase 1 for 1 h at 25°C prior to incubation with PTD.AdeGFP. The results indicated that heparinase 1 had no effect on the infection of HUVECs or T24 cells by PTD.AdeGFP (Figure 4A). After attaching to the fibre receptor on the cell surface, Ad5 is internalized through the clathrin-coated-pit pathway, and dynamin is required for the pathway [16]. To explore whether PTD.AdeGFP entered into the cells via the clathrin-coated-pit pathway, A549 cells were treated by Ad expressing mutant dynamin or E1-deleted Ad prior to incubation with AdeGFP (control virus) or PTD.AdeGFP. The results demonstrated that mutant dynamin inhibited transduction by control viruses significantly (P<0.01), but had no effects on transduction by PTD.AdeGFP (Figure 4B), indicating that PTD.AdeGFP infected the cells by a dynamin-independent pathway.


Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells.

Liu S, Mao Q, Zhang W, Zheng X, Bian Y, Wang D, Li H, Chai L, Zhao J, Xia H - Biosci. Rep. (2009)

The effect of PTD.AdeGFP infection on cells by heparinase 1 or mutant dynamin(A) The effect of heparinase 1 on infection of T24 or HUVEC cells by PTD.AdeGFP. GFP-positive cells was detected as described in the Materials and methods section. (B) The effect of mutant dynamin on infection of A549 cells by PTD.AdeGFP or AdeGFP. A549 cells were pre-treated with mutant dynamin virus (5000 particles/cell) or E1-deleted Ad5 (empty vector, 5000 particles/cell). After 48 h, cells were infected with PTD.AdeGFP or AdeGFP and then washed and incubated at 37°C for a further 48 h, after which the GFP-positive cells were detected as described in the Materials and methods section. Results are means±S.D. (n=3). Statistical significance was determined using the Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2630516&req=5

Figure 4: The effect of PTD.AdeGFP infection on cells by heparinase 1 or mutant dynamin(A) The effect of heparinase 1 on infection of T24 or HUVEC cells by PTD.AdeGFP. GFP-positive cells was detected as described in the Materials and methods section. (B) The effect of mutant dynamin on infection of A549 cells by PTD.AdeGFP or AdeGFP. A549 cells were pre-treated with mutant dynamin virus (5000 particles/cell) or E1-deleted Ad5 (empty vector, 5000 particles/cell). After 48 h, cells were infected with PTD.AdeGFP or AdeGFP and then washed and incubated at 37°C for a further 48 h, after which the GFP-positive cells were detected as described in the Materials and methods section. Results are means±S.D. (n=3). Statistical significance was determined using the Student's t test.
Mentions: Most mammalian cell types express heparin-containing cellular receptors that could bind to charged amino acids [36,37]. Previous studies indicated that adenoviral capsids modified to contain a C-terminal poly-lysine tract allowed for an efficient transduction of multiple cell types via facilitated binding to cell surface heparan sulfate proteoglycans [20]. The Tat PTD is enriched in positively charged amino acids (YGKKKRRQRRR), and, in order to rule out the possibility that the improvement of gene transfer is a result of increased charge-directed binding between viruses and cell surface, the cells were treated with heparinase 1 for 1 h at 25°C prior to incubation with PTD.AdeGFP. The results indicated that heparinase 1 had no effect on the infection of HUVECs or T24 cells by PTD.AdeGFP (Figure 4A). After attaching to the fibre receptor on the cell surface, Ad5 is internalized through the clathrin-coated-pit pathway, and dynamin is required for the pathway [16]. To explore whether PTD.AdeGFP entered into the cells via the clathrin-coated-pit pathway, A549 cells were treated by Ad expressing mutant dynamin or E1-deleted Ad prior to incubation with AdeGFP (control virus) or PTD.AdeGFP. The results demonstrated that mutant dynamin inhibited transduction by control viruses significantly (P<0.01), but had no effects on transduction by PTD.AdeGFP (Figure 4B), indicating that PTD.AdeGFP infected the cells by a dynamin-independent pathway.

Bottom Line: The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR.The results provide some new clues as to how PTD.AdeGFP infects target cells.This new vector would be valuable in gene-function analysis and for gene therapy in cancer.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, People's Republic of China.

ABSTRACT
The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.AdeGFP (where eGFP is enhanced green fluorescent protein) was constructed by modifying the HI loop of Ad5 (Ad type 5) fibre with the Tat (trans-activating) PTD (protein transduction domain) derived from HIV. The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR. The improvement in gene transfer was not the result of charge-directed binding between the virus and the cell surface. Although PTD.AdeGFP formed aggregates, it infected target cells in a manner different from AdeGFP aggregates precipitated by calcium phosphate. In addition, PTD.AdeGFP was able to transduce target cells in a dynamin-independent pathway. The results provide some new clues as to how PTD.AdeGFP infects target cells. This new vector would be valuable in gene-function analysis and for gene therapy in cancer.

Show MeSH
Related in: MedlinePlus