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Novel gene therapy viral vector using non-oncogenic lymphotropic herpesvirus.

Shimizu A, Kobayashi N, Shimada K, Oura K, Tanaka T, Okamoto A, Kondo K - PLoS ONE (2013)

Bottom Line: In the present study, we have altered the cell specificity of the resulting recombinant HHV-6 by knocking out the U2-U8 genes.Furthermore, HHV-6 vectors containing short hairpin RNAs against CD4 and HIV Gag remarkably inhibited the production of these proteins and HIV particles.Here we demonstrate the utility of HHV-6 as a new non-carcinogenic viral vector for immunologic diseases and immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, The Jikei University School of Medicine, Tokyo, Japan.

ABSTRACT
Despite the use of retroviral vectors, efficiently introducing target genes into immunocytes such as T cells is difficult. In addition, retroviral vectors carry risks associated with the oncogenicity of the native virus and the potential for introducing malignancy in recipients due to genetic carryover from immortalized cells used during vector production. To address these issues, we have established a new virus vector that is based on human herpesvirus 6 (HHV-6), a non-oncogenic lymphotropic herpesvirus that infects CD4(+) T cells, macrophages, and dendritic cells. In the present study, we have altered the cell specificity of the resulting recombinant HHV-6 by knocking out the U2-U8 genes. The resulting virus proliferated only in activated cord blood cells and not in peripheral blood cells. Umbilical cord blood cells produced replication-defective recombinant virus in sufficiently high titer to omit the use of immortalized cells during vector production. HHV-6 vectors led to high rates (>90%) of gene transduction in both CD4(+) and CD8(+) T cells. These viruses showed low-level replication of viral DNA that supported greater expression of the induced genes than that of other methods but that was insufficient to support the production of replication-competent virus. Furthermore, HHV-6 vectors containing short hairpin RNAs against CD4 and HIV Gag remarkably inhibited the production of these proteins and HIV particles. Here we demonstrate the utility of HHV-6 as a new non-carcinogenic viral vector for immunologic diseases and immunotherapy.

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Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP.PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.
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pone-0056027-g007: Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP.PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.

Mentions: According to fluorescence microscopy, PBMCs infected with H6R28LEP remained positive for expression of EGFP for approximately 2 weeks without demonstrating any cytopathic effects. The intensity of the EGFP signal increased daily and was strongest by day 4 after infection (Fig. 6). Flow cytometry revealed that approximately 85% of PBMCs became infected with H6R28LEP (data not shown). Flow cytometry to determine the types of H6R28LEP-infected PBMCs showed that 91.3% of CD4+ T cells and 91.9% of CD8+ T cells were EGFP-positive, but only 1.6% of CD19+ B cells were EGFP-positive (Fig. 7A, B, C). These results show that in PBMCs, H6R28LEP can infect CD4+ and CD8+ T cells at extremely high rates. We also tested infection of CD34+ blood stem cells in umbilical cord blood. The results showed a low infection rate of 2.0%, demonstrating that hardly any infections occurred (Fig. 7D).


Novel gene therapy viral vector using non-oncogenic lymphotropic herpesvirus.

Shimizu A, Kobayashi N, Shimada K, Oura K, Tanaka T, Okamoto A, Kondo K - PLoS ONE (2013)

Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP.PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569415&req=5

pone-0056027-g007: Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP.PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.
Mentions: According to fluorescence microscopy, PBMCs infected with H6R28LEP remained positive for expression of EGFP for approximately 2 weeks without demonstrating any cytopathic effects. The intensity of the EGFP signal increased daily and was strongest by day 4 after infection (Fig. 6). Flow cytometry revealed that approximately 85% of PBMCs became infected with H6R28LEP (data not shown). Flow cytometry to determine the types of H6R28LEP-infected PBMCs showed that 91.3% of CD4+ T cells and 91.9% of CD8+ T cells were EGFP-positive, but only 1.6% of CD19+ B cells were EGFP-positive (Fig. 7A, B, C). These results show that in PBMCs, H6R28LEP can infect CD4+ and CD8+ T cells at extremely high rates. We also tested infection of CD34+ blood stem cells in umbilical cord blood. The results showed a low infection rate of 2.0%, demonstrating that hardly any infections occurred (Fig. 7D).

Bottom Line: In the present study, we have altered the cell specificity of the resulting recombinant HHV-6 by knocking out the U2-U8 genes.Furthermore, HHV-6 vectors containing short hairpin RNAs against CD4 and HIV Gag remarkably inhibited the production of these proteins and HIV particles.Here we demonstrate the utility of HHV-6 as a new non-carcinogenic viral vector for immunologic diseases and immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, The Jikei University School of Medicine, Tokyo, Japan.

ABSTRACT
Despite the use of retroviral vectors, efficiently introducing target genes into immunocytes such as T cells is difficult. In addition, retroviral vectors carry risks associated with the oncogenicity of the native virus and the potential for introducing malignancy in recipients due to genetic carryover from immortalized cells used during vector production. To address these issues, we have established a new virus vector that is based on human herpesvirus 6 (HHV-6), a non-oncogenic lymphotropic herpesvirus that infects CD4(+) T cells, macrophages, and dendritic cells. In the present study, we have altered the cell specificity of the resulting recombinant HHV-6 by knocking out the U2-U8 genes. The resulting virus proliferated only in activated cord blood cells and not in peripheral blood cells. Umbilical cord blood cells produced replication-defective recombinant virus in sufficiently high titer to omit the use of immortalized cells during vector production. HHV-6 vectors led to high rates (>90%) of gene transduction in both CD4(+) and CD8(+) T cells. These viruses showed low-level replication of viral DNA that supported greater expression of the induced genes than that of other methods but that was insufficient to support the production of replication-competent virus. Furthermore, HHV-6 vectors containing short hairpin RNAs against CD4 and HIV Gag remarkably inhibited the production of these proteins and HIV particles. Here we demonstrate the utility of HHV-6 as a new non-carcinogenic viral vector for immunologic diseases and immunotherapy.

Show MeSH
Related in: MedlinePlus