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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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Related in: MedlinePlus

Real-time PCR analysis of OAS1 and STAT1 mRNA levels.A, B: PBMCs treated with 100 ng/ml interferon (IFN) γ for 4 d were used as a positive control (PC), and PBMCs treated with buffer alone was used as a negative control (NC). PBMCs were infected (MOI, 0.5) with wtHHV-6 or recombinant HHV-6s, total RNA from transduced PBMCs was isolated at 5 d after infection, and mRNA levels of OAS1 and STAT1 (interferon response genes) were quantified by real-time RT-PCR using IFN Response Watcher (Takara Bio). A: OAS1. B: STAT1. Data are given as mean ±1 standard deviation (n = 3).
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pone-0056027-g013: Real-time PCR analysis of OAS1 and STAT1 mRNA levels.A, B: PBMCs treated with 100 ng/ml interferon (IFN) γ for 4 d were used as a positive control (PC), and PBMCs treated with buffer alone was used as a negative control (NC). PBMCs were infected (MOI, 0.5) with wtHHV-6 or recombinant HHV-6s, total RNA from transduced PBMCs was isolated at 5 d after infection, and mRNA levels of OAS1 and STAT1 (interferon response genes) were quantified by real-time RT-PCR using IFN Response Watcher (Takara Bio). A: OAS1. B: STAT1. Data are given as mean ±1 standard deviation (n = 3).

Mentions: It is well known that double-stranded RNA induces an interferon response in mammalian cells, leading to nonspecific inhibition of transcription; in addition, siRNA and shRNA reportedly can induce this interferon response [24][32]. We examined the mRNA expression of 2 host genes (Oas1 and Stat1) known to be induced through the interferon response [24][25] to assess whether this response led to the suppression of CD4 synthesis and the anti-HIV effect shown above. The expression of Oas1 and Stat1 did not differ between PBMCs infected with each recombinant virus and uninfected PBMCs (Fig. 13). Therefore, the observed inhibitory effect on CD4 and HIV Gag gene expression likely was due to RNA interference and not to the interferon response.


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)

Real-time PCR analysis of OAS1 and STAT1 mRNA levels.A, B: PBMCs treated with 100 ng/ml interferon (IFN) γ for 4 d were used as a positive control (PC), and PBMCs treated with buffer alone was used as a negative control (NC). PBMCs were infected (MOI, 0.5) with wtHHV-6 or recombinant HHV-6s, total RNA from transduced PBMCs was isolated at 5 d after infection, and mRNA levels of OAS1 and STAT1 (interferon response genes) were quantified by real-time RT-PCR using IFN Response Watcher (Takara Bio). A: OAS1. B: STAT1. 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-g013: Real-time PCR analysis of OAS1 and STAT1 mRNA levels.A, B: PBMCs treated with 100 ng/ml interferon (IFN) γ for 4 d were used as a positive control (PC), and PBMCs treated with buffer alone was used as a negative control (NC). PBMCs were infected (MOI, 0.5) with wtHHV-6 or recombinant HHV-6s, total RNA from transduced PBMCs was isolated at 5 d after infection, and mRNA levels of OAS1 and STAT1 (interferon response genes) were quantified by real-time RT-PCR using IFN Response Watcher (Takara Bio). A: OAS1. B: STAT1. Data are given as mean ±1 standard deviation (n = 3).
Mentions: It is well known that double-stranded RNA induces an interferon response in mammalian cells, leading to nonspecific inhibition of transcription; in addition, siRNA and shRNA reportedly can induce this interferon response [24][32]. We examined the mRNA expression of 2 host genes (Oas1 and Stat1) known to be induced through the interferon response [24][25] to assess whether this response led to the suppression of CD4 synthesis and the anti-HIV effect shown above. The expression of Oas1 and Stat1 did not differ between PBMCs infected with each recombinant virus and uninfected PBMCs (Fig. 13). Therefore, the observed inhibitory effect on CD4 and HIV Gag gene expression likely was due to RNA interference and not to the interferon response.

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