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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

Southern blot hybridization to detect H6R28LEP concatemeric region.Total genomic DNA was purified from H6R28LEP-infected CBMCs and PBMCs after 4 d and digested with the restriction enzyme Apa I. A: Possible H6R28LEP forms, Apa I site locations, probe binding sites, and band sizes as predicted by Southern blot hybridization. Given the impossibility of predicting the gene sequence on the chromosome in the case of chromosomally integrated DNA, we have marked this as >4433 bp and >4094 bp. B: In both CBMCs and PBMCs, we detected a 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (where the DNA is cleaved into a single genome and packaged into particles).
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pone-0056027-g005: Southern blot hybridization to detect H6R28LEP concatemeric region.Total genomic DNA was purified from H6R28LEP-infected CBMCs and PBMCs after 4 d and digested with the restriction enzyme Apa I. A: Possible H6R28LEP forms, Apa I site locations, probe binding sites, and band sizes as predicted by Southern blot hybridization. Given the impossibility of predicting the gene sequence on the chromosome in the case of chromosomally integrated DNA, we have marked this as >4433 bp and >4094 bp. B: In both CBMCs and PBMCs, we detected a 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (where the DNA is cleaved into a single genome and packaged into particles).

Mentions: We used Southern blot hybridization to investigate the form of the H6R28LEP genomic DNA in PBMCs. The results above showed that viral particles could be formed in PBMCs even though H6R28LEP was not intact. This suggests that in PBMCs, H6R28LEP is producing concatemeric DNA, which comprises DNA replicates and is the form assumed by viral DNA when the virus is proliferating. We investigated the ratio of concatemeric DNA to virion DNA, which comprises the viral DNA cleaved into a single genome for packaging into a particle, to see whether the suppression of H6R28LEP proliferation in PBMCs occurs at the gene replication level (Fig. 5A). By using Southern blot hybridization after Apa I digestion, we detected an 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (single viral DNA genome cleaved for packaging into a particle) in both CBMCs and PBMCs (Fig. 5B). On the basis of the detection sensitivity of Southern hybridization used in this experiment, it is unlikely that we detected plasmid DNA in relatively low numbers or single copies of chromosomally integrated DNA per cell. Therefore, the 8527 bp and 4120 bp bands probably indicate the concatemer binding site and virion DNA, respectively. H6R28LEP viral DNA in PBMCs was also lower than the total amount in CBMCs, although there was no difference in the ratio of concatemeric DNA to virion DNA between PBMCs and CBMCs (Fig. 5B). This indicates that gene replication does not have a major effect on the suppression of H6R28LEP proliferation in PBMCs.


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)

Southern blot hybridization to detect H6R28LEP concatemeric region.Total genomic DNA was purified from H6R28LEP-infected CBMCs and PBMCs after 4 d and digested with the restriction enzyme Apa I. A: Possible H6R28LEP forms, Apa I site locations, probe binding sites, and band sizes as predicted by Southern blot hybridization. Given the impossibility of predicting the gene sequence on the chromosome in the case of chromosomally integrated DNA, we have marked this as >4433 bp and >4094 bp. B: In both CBMCs and PBMCs, we detected a 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (where the DNA is cleaved into a single genome and packaged into particles).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056027-g005: Southern blot hybridization to detect H6R28LEP concatemeric region.Total genomic DNA was purified from H6R28LEP-infected CBMCs and PBMCs after 4 d and digested with the restriction enzyme Apa I. A: Possible H6R28LEP forms, Apa I site locations, probe binding sites, and band sizes as predicted by Southern blot hybridization. Given the impossibility of predicting the gene sequence on the chromosome in the case of chromosomally integrated DNA, we have marked this as >4433 bp and >4094 bp. B: In both CBMCs and PBMCs, we detected a 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (where the DNA is cleaved into a single genome and packaged into particles).
Mentions: We used Southern blot hybridization to investigate the form of the H6R28LEP genomic DNA in PBMCs. The results above showed that viral particles could be formed in PBMCs even though H6R28LEP was not intact. This suggests that in PBMCs, H6R28LEP is producing concatemeric DNA, which comprises DNA replicates and is the form assumed by viral DNA when the virus is proliferating. We investigated the ratio of concatemeric DNA to virion DNA, which comprises the viral DNA cleaved into a single genome for packaging into a particle, to see whether the suppression of H6R28LEP proliferation in PBMCs occurs at the gene replication level (Fig. 5A). By using Southern blot hybridization after Apa I digestion, we detected an 8527 bp band showing concatemeric DNA (viral DNA replicates formed during viral proliferation) and a 4120 bp band showing virion DNA (single viral DNA genome cleaved for packaging into a particle) in both CBMCs and PBMCs (Fig. 5B). On the basis of the detection sensitivity of Southern hybridization used in this experiment, it is unlikely that we detected plasmid DNA in relatively low numbers or single copies of chromosomally integrated DNA per cell. Therefore, the 8527 bp and 4120 bp bands probably indicate the concatemer binding site and virion DNA, respectively. H6R28LEP viral DNA in PBMCs was also lower than the total amount in CBMCs, although there was no difference in the ratio of concatemeric DNA to virion DNA between PBMCs and CBMCs (Fig. 5B). This indicates that gene replication does not have a major effect on the suppression of H6R28LEP proliferation in PBMCs.

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