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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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Proliferation of wild-type (wt) HHV-6 and H6R28LEP.A: Growth curves for wtHHV-6 and H6R28LEP in peripheral blood mononuclear cells (PBMCs). PBMCs were infected with wtHHV-6 virus or H6R28LEP at a multiplicity of infection (MOI) of 1. Culture supernatant was collected every 3 d, and 1×106 fresh PBMCs were added after each collection. Progeny viruses were titered on MT4 cells by using immunofluroescent assays (wtHHV-6) or by counting EGFP-positive cells (H6R28LEP). Virus titers are indicated in focus-forming units per milliliter (FFU/ml). B: Growth curves for H6R28LEP in PBMCs and cord blood mononuclear cells (CBMCs). PBMCs and CBMCs were infected with H6R28LEP, and progeny viruses were titered every 3 d as described for panel A. C: Replication of viral DNA in PBMCs. PBMCs were infected with H6R28LEP as described, and infected cells were harvested at the indicated times and frozen at −80°C. Infected cells then were incubated with lysis buffer containing 1 mg/ml proteinase K, and the copy numbers of H6R28LEP in cell lysates were quantified by real-time PCR using primers specific for EGFP. Results are given as mean ±1 standard deviation (n = 3).
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pone-0056027-g002: Proliferation of wild-type (wt) HHV-6 and H6R28LEP.A: Growth curves for wtHHV-6 and H6R28LEP in peripheral blood mononuclear cells (PBMCs). PBMCs were infected with wtHHV-6 virus or H6R28LEP at a multiplicity of infection (MOI) of 1. Culture supernatant was collected every 3 d, and 1×106 fresh PBMCs were added after each collection. Progeny viruses were titered on MT4 cells by using immunofluroescent assays (wtHHV-6) or by counting EGFP-positive cells (H6R28LEP). Virus titers are indicated in focus-forming units per milliliter (FFU/ml). B: Growth curves for H6R28LEP in PBMCs and cord blood mononuclear cells (CBMCs). PBMCs and CBMCs were infected with H6R28LEP, and progeny viruses were titered every 3 d as described for panel A. C: Replication of viral DNA in PBMCs. PBMCs were infected with H6R28LEP as described, and infected cells were harvested at the indicated times and frozen at −80°C. Infected cells then were incubated with lysis buffer containing 1 mg/ml proteinase K, and the copy numbers of H6R28LEP in cell lysates were quantified by real-time PCR using primers specific for EGFP. Results are given as mean ±1 standard deviation (n = 3).

Mentions: We infected populations of PHA-stimulated PBMCs with wtHHV-6 and H6R28LEP, collected the culture supernatants 3 to 15 d after the infection, and determined the viral titers of these supernatants. This process revealed that the recombinant virus (H6R28LEP) did not grow in the PBMCs in which wtHHV-6 grew robustly (Fig. 2A). Viral proliferation of H6R28LEP in anti-CD3 antibody-stimulated PBMCs was impaired similarly to that in PHA-stimulated PBMCs (data not shown). When PHA-stimulated CBMCs were infected instead of PBMCs, H6R28LEP produced sufficient titers of infectious viral particles and showed productive infection (Fig. 2B). H6R28LEP and wtHHV-6 demonstrated approximately equivalent proliferation in CBMCs (data not shown). These results indicated that H6R28LEP was a proliferation-defective virus in peripheral blood cells although it behaved as a proliferative virus in the PHA-stimulated CBMCs.


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)

Proliferation of wild-type (wt) HHV-6 and H6R28LEP.A: Growth curves for wtHHV-6 and H6R28LEP in peripheral blood mononuclear cells (PBMCs). PBMCs were infected with wtHHV-6 virus or H6R28LEP at a multiplicity of infection (MOI) of 1. Culture supernatant was collected every 3 d, and 1×106 fresh PBMCs were added after each collection. Progeny viruses were titered on MT4 cells by using immunofluroescent assays (wtHHV-6) or by counting EGFP-positive cells (H6R28LEP). Virus titers are indicated in focus-forming units per milliliter (FFU/ml). B: Growth curves for H6R28LEP in PBMCs and cord blood mononuclear cells (CBMCs). PBMCs and CBMCs were infected with H6R28LEP, and progeny viruses were titered every 3 d as described for panel A. C: Replication of viral DNA in PBMCs. PBMCs were infected with H6R28LEP as described, and infected cells were harvested at the indicated times and frozen at −80°C. Infected cells then were incubated with lysis buffer containing 1 mg/ml proteinase K, and the copy numbers of H6R28LEP in cell lysates were quantified by real-time PCR using primers specific for EGFP. Results are given as mean ±1 standard deviation (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569415&req=5

pone-0056027-g002: Proliferation of wild-type (wt) HHV-6 and H6R28LEP.A: Growth curves for wtHHV-6 and H6R28LEP in peripheral blood mononuclear cells (PBMCs). PBMCs were infected with wtHHV-6 virus or H6R28LEP at a multiplicity of infection (MOI) of 1. Culture supernatant was collected every 3 d, and 1×106 fresh PBMCs were added after each collection. Progeny viruses were titered on MT4 cells by using immunofluroescent assays (wtHHV-6) or by counting EGFP-positive cells (H6R28LEP). Virus titers are indicated in focus-forming units per milliliter (FFU/ml). B: Growth curves for H6R28LEP in PBMCs and cord blood mononuclear cells (CBMCs). PBMCs and CBMCs were infected with H6R28LEP, and progeny viruses were titered every 3 d as described for panel A. C: Replication of viral DNA in PBMCs. PBMCs were infected with H6R28LEP as described, and infected cells were harvested at the indicated times and frozen at −80°C. Infected cells then were incubated with lysis buffer containing 1 mg/ml proteinase K, and the copy numbers of H6R28LEP in cell lysates were quantified by real-time PCR using primers specific for EGFP. Results are given as mean ±1 standard deviation (n = 3).
Mentions: We infected populations of PHA-stimulated PBMCs with wtHHV-6 and H6R28LEP, collected the culture supernatants 3 to 15 d after the infection, and determined the viral titers of these supernatants. This process revealed that the recombinant virus (H6R28LEP) did not grow in the PBMCs in which wtHHV-6 grew robustly (Fig. 2A). Viral proliferation of H6R28LEP in anti-CD3 antibody-stimulated PBMCs was impaired similarly to that in PHA-stimulated PBMCs (data not shown). When PHA-stimulated CBMCs were infected instead of PBMCs, H6R28LEP produced sufficient titers of infectious viral particles and showed productive infection (Fig. 2B). H6R28LEP and wtHHV-6 demonstrated approximately equivalent proliferation in CBMCs (data not shown). These results indicated that H6R28LEP was a proliferation-defective virus in peripheral blood cells although it behaved as a proliferative virus in the PHA-stimulated CBMCs.

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