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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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Related in: MedlinePlus

Schematic diagram of PTD.AdeGFP productionPTD.AdeGFP contains the eGFP gene in the E1 region under the control of the RSV promoter. The pBS shuttle vector containing the Tat PTD motif in the HI loop of Ad5 fibre linearized by BamHI and EcoRI was co-transformed into E. coli. BJ5183 cells along with SwaI linearized pTG360RSVeGFP/SwaI. The positive clones were screened by enzyme digestion and DNA sequencing. Appropriate recombinants were digested by PacI and transfected into HEK-293 cells. Viruses were harvested, amplified and purified using standard methods. Gluc, glucuronidase.
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Figure 1: Schematic diagram of PTD.AdeGFP productionPTD.AdeGFP contains the eGFP gene in the E1 region under the control of the RSV promoter. The pBS shuttle vector containing the Tat PTD motif in the HI loop of Ad5 fibre linearized by BamHI and EcoRI was co-transformed into E. coli. BJ5183 cells along with SwaI linearized pTG360RSVeGFP/SwaI. The positive clones were screened by enzyme digestion and DNA sequencing. Appropriate recombinants were digested by PacI and transfected into HEK-293 cells. Viruses were harvested, amplified and purified using standard methods. Gluc, glucuronidase.

Mentions: Using a homologous strategy in E. coli BJ5185 cells, Ad5 was modified with the Tat PTD in the HI loop of the fibre to obtain a new construct, PTD.AdeGFP. The PTD.AdeGFP virus was then produced by transfecting PacI-linearized PTD.AdeGFP plasmids into HEK-293 cells (Figure 1). PTD.AdeGFP was first tested in A549 (a human lung epithelial cancer cell line), a CAR-positive cell line. The results indicated that PTD.AdeGFP infected A549 cells less efficiently when compared with the unmodified control virus AdeGFP (Figure 2A). Flow cytometry showed that 53% of A549 cells were infected by PTD.AdeGFP (revealed by GFP expression) and 91% of the cells were infected by AdeGFP. However, in contrast with the control virus AdeGFP, PTD.AdeGFP infected A549 cells in a fashion that was not blocked by the presence of the soluble fibre knob (Figure 2A). We next tested if PTD.AdeGFP could transduce CAR-deficient cells by using CHO cells, and the results revealed that 31% of the cells were infected, which was not reversed by pre-treatment with the soluble Ad5 fibre knob (results not shown). In addition, there were no significant differences in transduction efficiency between CHO and CHO–CAR cells infected by PTD.AdeGFP (P>0.05) (Figure 2B). These results suggested that PTD.AdeGFP tranduces cells in a CAR-independent manner. Other CAR-deficient cells, for example, NIH 3T3 (mouse fibroblast cell line), C39 (human fibroblast cell line), T24 (human prostate cancer cell line) and HUVECs all showed efficient transduction (30%–50% GFP expression in cells) by PTD.AdeGFP (Figures 2C and 2D). To test whether the enhanced gene transfer mediated by PTD.AdeGFP in CAR-deficient cells is the result of the insertion of the Tat PTD into the HI loop of the adenoviral vector, PTD.AdeGFP was incubated with CHO cells in the presence or absence of the Tat PTD peptide. The results indicated that the Tat PTD peptide could block the infection of CHO cells by PTD.AdeGFP by 50% (Figure 2E), suggesting that the entry of PTD.AdeGFP into target cells could be the result of an interaction between the Tat PTD and molecules present on the surface of target cells.


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)

Schematic diagram of PTD.AdeGFP productionPTD.AdeGFP contains the eGFP gene in the E1 region under the control of the RSV promoter. The pBS shuttle vector containing the Tat PTD motif in the HI loop of Ad5 fibre linearized by BamHI and EcoRI was co-transformed into E. coli. BJ5183 cells along with SwaI linearized pTG360RSVeGFP/SwaI. The positive clones were screened by enzyme digestion and DNA sequencing. Appropriate recombinants were digested by PacI and transfected into HEK-293 cells. Viruses were harvested, amplified and purified using standard methods. Gluc, glucuronidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic diagram of PTD.AdeGFP productionPTD.AdeGFP contains the eGFP gene in the E1 region under the control of the RSV promoter. The pBS shuttle vector containing the Tat PTD motif in the HI loop of Ad5 fibre linearized by BamHI and EcoRI was co-transformed into E. coli. BJ5183 cells along with SwaI linearized pTG360RSVeGFP/SwaI. The positive clones were screened by enzyme digestion and DNA sequencing. Appropriate recombinants were digested by PacI and transfected into HEK-293 cells. Viruses were harvested, amplified and purified using standard methods. Gluc, glucuronidase.
Mentions: Using a homologous strategy in E. coli BJ5185 cells, Ad5 was modified with the Tat PTD in the HI loop of the fibre to obtain a new construct, PTD.AdeGFP. The PTD.AdeGFP virus was then produced by transfecting PacI-linearized PTD.AdeGFP plasmids into HEK-293 cells (Figure 1). PTD.AdeGFP was first tested in A549 (a human lung epithelial cancer cell line), a CAR-positive cell line. The results indicated that PTD.AdeGFP infected A549 cells less efficiently when compared with the unmodified control virus AdeGFP (Figure 2A). Flow cytometry showed that 53% of A549 cells were infected by PTD.AdeGFP (revealed by GFP expression) and 91% of the cells were infected by AdeGFP. However, in contrast with the control virus AdeGFP, PTD.AdeGFP infected A549 cells in a fashion that was not blocked by the presence of the soluble fibre knob (Figure 2A). We next tested if PTD.AdeGFP could transduce CAR-deficient cells by using CHO cells, and the results revealed that 31% of the cells were infected, which was not reversed by pre-treatment with the soluble Ad5 fibre knob (results not shown). In addition, there were no significant differences in transduction efficiency between CHO and CHO–CAR cells infected by PTD.AdeGFP (P>0.05) (Figure 2B). These results suggested that PTD.AdeGFP tranduces cells in a CAR-independent manner. Other CAR-deficient cells, for example, NIH 3T3 (mouse fibroblast cell line), C39 (human fibroblast cell line), T24 (human prostate cancer cell line) and HUVECs all showed efficient transduction (30%–50% GFP expression in cells) by PTD.AdeGFP (Figures 2C and 2D). To test whether the enhanced gene transfer mediated by PTD.AdeGFP in CAR-deficient cells is the result of the insertion of the Tat PTD into the HI loop of the adenoviral vector, PTD.AdeGFP was incubated with CHO cells in the presence or absence of the Tat PTD peptide. The results indicated that the Tat PTD peptide could block the infection of CHO cells by PTD.AdeGFP by 50% (Figure 2E), suggesting that the entry of PTD.AdeGFP into target cells could be the result of an interaction between the Tat PTD and molecules present on the surface of target cells.

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