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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 HIV p24 Gag protein and mRNA levels in PBMCs infected with H6R28LEP shGag.PBMCs infected with H6R28LEP or H6R28LEP shGag were transduced with a self-inactivating HIV vector plasmid by using electroporation. A: The p24 level in culture supernatants was determined by ELISA. B: Total RNA from transduced PBMCs was isolated, and Gag mRNA expression was quantified by real-time RT PCR. Firefly luciferase expression was used to normalize transfection efficiency, and the expression level of the Gag transcript was normalized against the transfection efficiency. Data are given as mean ±1 standard deviation (n = 3).
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pone-0056027-g012: Down-regulation of HIV p24 Gag protein and mRNA levels in PBMCs infected with H6R28LEP shGag.PBMCs infected with H6R28LEP or H6R28LEP shGag were transduced with a self-inactivating HIV vector plasmid by using electroporation. A: The p24 level in culture supernatants was determined by ELISA. B: Total RNA from transduced PBMCs was isolated, and Gag mRNA expression was quantified by real-time RT PCR. Firefly luciferase expression was used to normalize transfection efficiency, and the expression level of the Gag transcript was normalized against the transfection efficiency. Data are given as mean ±1 standard deviation (n = 3).

Mentions: Furthermore, we examined protein and mRNA levels of HIV p24 Gag in PBMCs infected with H6R28LEP shGag. In this examination, we infected PBMCs with H6R28LEP and H6R28LEP shGag and then used electroporation to transduce the HIV vector plasmid at 3 d after infection. We used ELISA of culture supernatant at 3 d after electroporation to measure levels of p24 protein as a marker for HIV particle production. Cells infected with H6R28LEP shGag yielded less than one fifth of the amount of HIV virus particles produced by H6R28LEP-infected cells (Fig. 12A). In addition, Gag mRNA expression by PBMCs infected with H6R28LEP shGag was less than one tenth of that in the H6R28LEP-infected PBMCs (Fig. 12B). These data correlate with the inhibition of HIV particle production. Therefore, the recombinant HHV-6 vectors produced siRNAs effectively and strongly induced siRNA-associated silencing effects.


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 HIV p24 Gag protein and mRNA levels in PBMCs infected with H6R28LEP shGag.PBMCs infected with H6R28LEP or H6R28LEP shGag were transduced with a self-inactivating HIV vector plasmid by using electroporation. A: The p24 level in culture supernatants was determined by ELISA. B: Total RNA from transduced PBMCs was isolated, and Gag mRNA expression was quantified by real-time RT PCR. Firefly luciferase expression was used to normalize transfection efficiency, and the expression level of the Gag transcript was normalized against the transfection efficiency. 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-g012: Down-regulation of HIV p24 Gag protein and mRNA levels in PBMCs infected with H6R28LEP shGag.PBMCs infected with H6R28LEP or H6R28LEP shGag were transduced with a self-inactivating HIV vector plasmid by using electroporation. A: The p24 level in culture supernatants was determined by ELISA. B: Total RNA from transduced PBMCs was isolated, and Gag mRNA expression was quantified by real-time RT PCR. Firefly luciferase expression was used to normalize transfection efficiency, and the expression level of the Gag transcript was normalized against the transfection efficiency. Data are given as mean ±1 standard deviation (n = 3).
Mentions: Furthermore, we examined protein and mRNA levels of HIV p24 Gag in PBMCs infected with H6R28LEP shGag. In this examination, we infected PBMCs with H6R28LEP and H6R28LEP shGag and then used electroporation to transduce the HIV vector plasmid at 3 d after infection. We used ELISA of culture supernatant at 3 d after electroporation to measure levels of p24 protein as a marker for HIV particle production. Cells infected with H6R28LEP shGag yielded less than one fifth of the amount of HIV virus particles produced by H6R28LEP-infected cells (Fig. 12A). In addition, Gag mRNA expression by PBMCs infected with H6R28LEP shGag was less than one tenth of that in the H6R28LEP-infected PBMCs (Fig. 12B). These data correlate with the inhibition of HIV particle production. Therefore, the recombinant HHV-6 vectors produced siRNAs effectively and strongly induced siRNA-associated silencing effects.

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