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Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells.

Sengupta S, Ulasov IV, Thaci B, Ahmed AU, Lesniak MS - PLoS ONE (2011)

Bottom Line: Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells.In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

View Article: PubMed Central - PubMed

Affiliation: The Brain Tumor Center, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT

Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD).

Methodology/principal finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.

Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

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Related in: MedlinePlus

T cells transduced with oncolytic Ad-RGD undergo rapid cell cycle.(A) Flow cytometric profile of propidium iodide (PI) dilution in virus-transduced T cells at different time-points after treatment. Gates drawn on different cell-cycle phases are M1 sub-G0, M2 G0/G1, M3 S, M4 G2/M and M5 quiescent. Left panel shows cells treated with adenovirus with wild-type fiber (Ad-WT). Right panel shows cells with RGD-modified oncolytic virus (Ad-RGD). (B). Bar diagram representation of the cell-cycle. Top panel represents cells treated with Ad-WT. Bottom panel represents cells treated with Ad-RGD. White bars are cells after 24 h, while patterned and black bars are cells after 48 and 72 h respectively after virus transduction. Error bars represent mean ± SD.
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pone-0018091-g006: T cells transduced with oncolytic Ad-RGD undergo rapid cell cycle.(A) Flow cytometric profile of propidium iodide (PI) dilution in virus-transduced T cells at different time-points after treatment. Gates drawn on different cell-cycle phases are M1 sub-G0, M2 G0/G1, M3 S, M4 G2/M and M5 quiescent. Left panel shows cells treated with adenovirus with wild-type fiber (Ad-WT). Right panel shows cells with RGD-modified oncolytic virus (Ad-RGD). (B). Bar diagram representation of the cell-cycle. Top panel represents cells treated with Ad-WT. Bottom panel represents cells treated with Ad-RGD. White bars are cells after 24 h, while patterned and black bars are cells after 48 and 72 h respectively after virus transduction. Error bars represent mean ± SD.

Mentions: We performed a cell cycle analysis of T cells that were treated with oncolytic Ad-WT, Ad-RGD viruses to test the replication efficiency in the transduced cells. Virus treated cells were stained with propidium iodide to evaluate their cell cycle phases by flow cytometry. Propidium iodide binds to the cellular DNA and the intensity of propidium iodide indicates the levels of DNA in a particular phase of the cell cycle. The results showed that 25–30% of the Ad-RGD treated cells were in G0/G1 and S-phase respectively at 24 h and 48 h after virus treatment while 60–70% of the Ad-WT treated T cells were quiescent during the entire study period. At 72 h, most of the Ad-RGD treated cells were out of G0/G1 and S phase and accumulated in the quiescent phase, which corroborates with viability results where more than 67% of these cells were dead or dying (Fig. 6). Since virus utilizes the cell's cycling machinery to replicate, this rapid cycling of Ad-RGD transduced T cells supports the notion that the virus replicates in the cells.


Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells.

Sengupta S, Ulasov IV, Thaci B, Ahmed AU, Lesniak MS - PLoS ONE (2011)

T cells transduced with oncolytic Ad-RGD undergo rapid cell cycle.(A) Flow cytometric profile of propidium iodide (PI) dilution in virus-transduced T cells at different time-points after treatment. Gates drawn on different cell-cycle phases are M1 sub-G0, M2 G0/G1, M3 S, M4 G2/M and M5 quiescent. Left panel shows cells treated with adenovirus with wild-type fiber (Ad-WT). Right panel shows cells with RGD-modified oncolytic virus (Ad-RGD). (B). Bar diagram representation of the cell-cycle. Top panel represents cells treated with Ad-WT. Bottom panel represents cells treated with Ad-RGD. White bars are cells after 24 h, while patterned and black bars are cells after 48 and 72 h respectively after virus transduction. Error bars represent mean ± SD.
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Related In: Results  -  Collection

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pone-0018091-g006: T cells transduced with oncolytic Ad-RGD undergo rapid cell cycle.(A) Flow cytometric profile of propidium iodide (PI) dilution in virus-transduced T cells at different time-points after treatment. Gates drawn on different cell-cycle phases are M1 sub-G0, M2 G0/G1, M3 S, M4 G2/M and M5 quiescent. Left panel shows cells treated with adenovirus with wild-type fiber (Ad-WT). Right panel shows cells with RGD-modified oncolytic virus (Ad-RGD). (B). Bar diagram representation of the cell-cycle. Top panel represents cells treated with Ad-WT. Bottom panel represents cells treated with Ad-RGD. White bars are cells after 24 h, while patterned and black bars are cells after 48 and 72 h respectively after virus transduction. Error bars represent mean ± SD.
Mentions: We performed a cell cycle analysis of T cells that were treated with oncolytic Ad-WT, Ad-RGD viruses to test the replication efficiency in the transduced cells. Virus treated cells were stained with propidium iodide to evaluate their cell cycle phases by flow cytometry. Propidium iodide binds to the cellular DNA and the intensity of propidium iodide indicates the levels of DNA in a particular phase of the cell cycle. The results showed that 25–30% of the Ad-RGD treated cells were in G0/G1 and S-phase respectively at 24 h and 48 h after virus treatment while 60–70% of the Ad-WT treated T cells were quiescent during the entire study period. At 72 h, most of the Ad-RGD treated cells were out of G0/G1 and S phase and accumulated in the quiescent phase, which corroborates with viability results where more than 67% of these cells were dead or dying (Fig. 6). Since virus utilizes the cell's cycling machinery to replicate, this rapid cycling of Ad-RGD transduced T cells supports the notion that the virus replicates in the cells.

Bottom Line: Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells.In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

View Article: PubMed Central - PubMed

Affiliation: The Brain Tumor Center, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT

Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD).

Methodology/principal finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.

Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

Show MeSH
Related in: MedlinePlus