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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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Down-regulation of CD4 protein and mRNA levels in PBMCs infected with H6R28LEP shCD4.PBMCs were cultured on plates coated with 1 µg/ml anti-CD3 antibody for 3 d, and then infected (MOI, 2) with H6R28LEP or H6R28LEP shCD4. A: At 4 d after infection, cells underwent FACS analysis for expression of CD4 and EGFP. B: Numbers of CD4-positive cells among those infected with H6R28LEP shCD4 (solid line) or H6R28LEP (dashed line) are shown. Results are representative of at least three independent experiments. C: Cells were collected at different times after infection, total RNA was isolated, and CD4 mRNA transcripts were quantified by real-time RT-PCR. D: Number of CD4-positive cells at 6, 8, and 10 d after infection, as seen in panel B. Data are given as mean ±1 standard deviation (n = 3).
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pone-0056027-g011: Down-regulation of CD4 protein and mRNA levels in PBMCs infected with H6R28LEP shCD4.PBMCs were cultured on plates coated with 1 µg/ml anti-CD3 antibody for 3 d, and then infected (MOI, 2) with H6R28LEP or H6R28LEP shCD4. A: At 4 d after infection, cells underwent FACS analysis for expression of CD4 and EGFP. B: Numbers of CD4-positive cells among those infected with H6R28LEP shCD4 (solid line) or H6R28LEP (dashed line) are shown. Results are representative of at least three independent experiments. C: Cells were collected at different times after infection, total RNA was isolated, and CD4 mRNA transcripts were quantified by real-time RT-PCR. D: Number of CD4-positive cells at 6, 8, and 10 d after infection, as seen in panel B. Data are given as mean ±1 standard deviation (n = 3).

Mentions: Flow cytometry at 4 d after infection showed that, whereas infection rates were similar between the 2 viruses (Fig. 11A), CD4 expression at the cell surface of PMBCs infected with H6R28LEP shCD4 was one tenth that of PBMCs infected with H6R28LEP (Fig. 11B). In addition, CD4 mRNA expression in cells infected with H6R28LEP shCD4 was decreased to less than 10% that of cells infected with H6R28LEP (Fig. 11C). The results confirmed that this effect of H6R28LEP shCD4 continued even at 10 d after infection (Fig. 11D).


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)

Down-regulation of CD4 protein and mRNA levels in PBMCs infected with H6R28LEP shCD4.PBMCs were cultured on plates coated with 1 µg/ml anti-CD3 antibody for 3 d, and then infected (MOI, 2) with H6R28LEP or H6R28LEP shCD4. A: At 4 d after infection, cells underwent FACS analysis for expression of CD4 and EGFP. B: Numbers of CD4-positive cells among those infected with H6R28LEP shCD4 (solid line) or H6R28LEP (dashed line) are shown. Results are representative of at least three independent experiments. C: Cells were collected at different times after infection, total RNA was isolated, and CD4 mRNA transcripts were quantified by real-time RT-PCR. D: Number of CD4-positive cells at 6, 8, and 10 d after infection, as seen in panel B. Data are given as mean ±1 standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056027-g011: Down-regulation of CD4 protein and mRNA levels in PBMCs infected with H6R28LEP shCD4.PBMCs were cultured on plates coated with 1 µg/ml anti-CD3 antibody for 3 d, and then infected (MOI, 2) with H6R28LEP or H6R28LEP shCD4. A: At 4 d after infection, cells underwent FACS analysis for expression of CD4 and EGFP. B: Numbers of CD4-positive cells among those infected with H6R28LEP shCD4 (solid line) or H6R28LEP (dashed line) are shown. Results are representative of at least three independent experiments. C: Cells were collected at different times after infection, total RNA was isolated, and CD4 mRNA transcripts were quantified by real-time RT-PCR. D: Number of CD4-positive cells at 6, 8, and 10 d after infection, as seen in panel B. Data are given as mean ±1 standard deviation (n = 3).
Mentions: Flow cytometry at 4 d after infection showed that, whereas infection rates were similar between the 2 viruses (Fig. 11A), CD4 expression at the cell surface of PMBCs infected with H6R28LEP shCD4 was one tenth that of PBMCs infected with H6R28LEP (Fig. 11B). In addition, CD4 mRNA expression in cells infected with H6R28LEP shCD4 was decreased to less than 10% that of cells infected with H6R28LEP (Fig. 11C). The results confirmed that this effect of H6R28LEP shCD4 continued even at 10 d after infection (Fig. 11D).

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