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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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Viral mRNA and protein expression in H6R28LEP-infected PBMCs.A: PBMCs and CMBCs were infected with H6R28LEP, total RNA was purified after 24 h, and mRNA expression of immediate early (U90, U94), early (U41, U69), and late (U24, U39, U48) genes was assayed by real-time PCR after RT. The data are shown as mRNA expression volumes in PBMCs relative to those in CBMCs. Data are given as mean ±1 standard deviation (n = 3). B: At 24 h after infection, H6R28LEP-infected CBMCs or PBMCs were fixed in cold acetone and expression of the viral proteins U90, U39, U41, and U48 was observed using an indirect immunofluorescent assay. C: Proportion of H6R28LEP intact virus particles produced in the culture supernatant. CBMCs or PBMCs were infected with H6R28LEP, and real-time PCR was used to quantify the number of H6R28LEP genome DNA copies present in the supernatant after 3 d. The supernatant from each culture was used to infect PBMCs by using centrifugation (left) or the normal adsorption method (right), and the H6R28LEP intact virus particles in the supernatant were assayed. The graph shows the percentage of intact virus particles relative to H6R28LEP genome DNA copy numbers in the supernatant. Data are given as mean ±1 standard deviation (n = 3).
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pone-0056027-g003: Viral mRNA and protein expression in H6R28LEP-infected PBMCs.A: PBMCs and CMBCs were infected with H6R28LEP, total RNA was purified after 24 h, and mRNA expression of immediate early (U90, U94), early (U41, U69), and late (U24, U39, U48) genes was assayed by real-time PCR after RT. The data are shown as mRNA expression volumes in PBMCs relative to those in CBMCs. Data are given as mean ±1 standard deviation (n = 3). B: At 24 h after infection, H6R28LEP-infected CBMCs or PBMCs were fixed in cold acetone and expression of the viral proteins U90, U39, U41, and U48 was observed using an indirect immunofluorescent assay. C: Proportion of H6R28LEP intact virus particles produced in the culture supernatant. CBMCs or PBMCs were infected with H6R28LEP, and real-time PCR was used to quantify the number of H6R28LEP genome DNA copies present in the supernatant after 3 d. The supernatant from each culture was used to infect PBMCs by using centrifugation (left) or the normal adsorption method (right), and the H6R28LEP intact virus particles in the supernatant were assayed. The graph shows the percentage of intact virus particles relative to H6R28LEP genome DNA copy numbers in the supernatant. Data are given as mean ±1 standard deviation (n = 3).

Mentions: For HHV-6, one-step growth is completed and progeny viruses produced in around 24 h. We therefore infected PBMCs with H6R28LEP and investigated mRNA expression of typical immediate early, early, and late genes after 24 h. The results showed that in PBMCs, H6R28LEP mRNA expression was suppressed in the late stage compared with the immediate early stage (Fig. 3A). The results also showed that the expression of late-gene proteins was markedly lower in PBMCs than in CBMCs (Fig. 3B).


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)

Viral mRNA and protein expression in H6R28LEP-infected PBMCs.A: PBMCs and CMBCs were infected with H6R28LEP, total RNA was purified after 24 h, and mRNA expression of immediate early (U90, U94), early (U41, U69), and late (U24, U39, U48) genes was assayed by real-time PCR after RT. The data are shown as mRNA expression volumes in PBMCs relative to those in CBMCs. Data are given as mean ±1 standard deviation (n = 3). B: At 24 h after infection, H6R28LEP-infected CBMCs or PBMCs were fixed in cold acetone and expression of the viral proteins U90, U39, U41, and U48 was observed using an indirect immunofluorescent assay. C: Proportion of H6R28LEP intact virus particles produced in the culture supernatant. CBMCs or PBMCs were infected with H6R28LEP, and real-time PCR was used to quantify the number of H6R28LEP genome DNA copies present in the supernatant after 3 d. The supernatant from each culture was used to infect PBMCs by using centrifugation (left) or the normal adsorption method (right), and the H6R28LEP intact virus particles in the supernatant were assayed. The graph shows the percentage of intact virus particles relative to H6R28LEP genome DNA copy numbers in the supernatant. Data are given as mean ±1 standard deviation (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569415&req=5

pone-0056027-g003: Viral mRNA and protein expression in H6R28LEP-infected PBMCs.A: PBMCs and CMBCs were infected with H6R28LEP, total RNA was purified after 24 h, and mRNA expression of immediate early (U90, U94), early (U41, U69), and late (U24, U39, U48) genes was assayed by real-time PCR after RT. The data are shown as mRNA expression volumes in PBMCs relative to those in CBMCs. Data are given as mean ±1 standard deviation (n = 3). B: At 24 h after infection, H6R28LEP-infected CBMCs or PBMCs were fixed in cold acetone and expression of the viral proteins U90, U39, U41, and U48 was observed using an indirect immunofluorescent assay. C: Proportion of H6R28LEP intact virus particles produced in the culture supernatant. CBMCs or PBMCs were infected with H6R28LEP, and real-time PCR was used to quantify the number of H6R28LEP genome DNA copies present in the supernatant after 3 d. The supernatant from each culture was used to infect PBMCs by using centrifugation (left) or the normal adsorption method (right), and the H6R28LEP intact virus particles in the supernatant were assayed. The graph shows the percentage of intact virus particles relative to H6R28LEP genome DNA copy numbers in the supernatant. Data are given as mean ±1 standard deviation (n = 3).
Mentions: For HHV-6, one-step growth is completed and progeny viruses produced in around 24 h. We therefore infected PBMCs with H6R28LEP and investigated mRNA expression of typical immediate early, early, and late genes after 24 h. The results showed that in PBMCs, H6R28LEP mRNA expression was suppressed in the late stage compared with the immediate early stage (Fig. 3A). The results also showed that the expression of late-gene proteins was markedly lower in PBMCs than in CBMCs (Fig. 3B).

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