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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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Structure of H6R28LEP.The straight line at the top represents the genome of the HHV-6B HST genome, with the U1 to U10 region expanded below. In the middle, the shaded arrows show the positions from start codon to poly(A) signal of the genes in the U2–U8 gene cluster that was replaced by the EGFP-IRES-LNGFR-Puro cassette. The bottom diagram shows the structure of pU2-U8 LEP. Open boxes represent the low-affinity nerve growth factor receptor (LNGFR), enhanced green fluorescent protein (EGFP), and puromycin-N-acetyl-transferase (Puro) genes; filled boxes represent the internal ribosome entry site (IRES), human cytomegalovirus major immediate early promoter (HCMV MIEP), and simian virus 40 (SV40) promoter. The locations of the PCR primers used in the cloning of the U2 and U8 gene clusters (U2 XbaI, U2 AflII, U8 BamHI, and U8 EcoRI) and the primers for verification of the recombinant and wild-type virus (EGFP F, EGFP R, U5 F, and U5 R) are indicated. DR, direct repeat.
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pone-0056027-g001: Structure of H6R28LEP.The straight line at the top represents the genome of the HHV-6B HST genome, with the U1 to U10 region expanded below. In the middle, the shaded arrows show the positions from start codon to poly(A) signal of the genes in the U2–U8 gene cluster that was replaced by the EGFP-IRES-LNGFR-Puro cassette. The bottom diagram shows the structure of pU2-U8 LEP. Open boxes represent the low-affinity nerve growth factor receptor (LNGFR), enhanced green fluorescent protein (EGFP), and puromycin-N-acetyl-transferase (Puro) genes; filled boxes represent the internal ribosome entry site (IRES), human cytomegalovirus major immediate early promoter (HCMV MIEP), and simian virus 40 (SV40) promoter. The locations of the PCR primers used in the cloning of the U2 and U8 gene clusters (U2 XbaI, U2 AflII, U8 BamHI, and U8 EcoRI) and the primers for verification of the recombinant and wild-type virus (EGFP F, EGFP R, U5 F, and U5 R) are indicated. DR, direct repeat.

Mentions: In our previous report, we generated a recombinant HHV-6 by inserting a selectable marker, the puromycin-resistance gene, into wtHHV-6 by homologous recombination [30]. In the present study, we built on this earlier work by using MACS LNGFR microbeads in addition to puromycin treatment to select for transfected cells. In doing so, we created a recombinant HHV-6 virus (H6R28LEP) in a shorter time than previously and were able to concentrate the infected cells. In H6R28LEP, the Puro and LNGFR genes replace the part of HHV-6 genome from the promoter of the U2 gene to the poly(A) signal of the U8 gene (Fig. 1). Using real-time PCR, we confirmed that H6R28LEP lacks expression of the U2–U8 genes (data not shown).


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)

Structure of H6R28LEP.The straight line at the top represents the genome of the HHV-6B HST genome, with the U1 to U10 region expanded below. In the middle, the shaded arrows show the positions from start codon to poly(A) signal of the genes in the U2–U8 gene cluster that was replaced by the EGFP-IRES-LNGFR-Puro cassette. The bottom diagram shows the structure of pU2-U8 LEP. Open boxes represent the low-affinity nerve growth factor receptor (LNGFR), enhanced green fluorescent protein (EGFP), and puromycin-N-acetyl-transferase (Puro) genes; filled boxes represent the internal ribosome entry site (IRES), human cytomegalovirus major immediate early promoter (HCMV MIEP), and simian virus 40 (SV40) promoter. The locations of the PCR primers used in the cloning of the U2 and U8 gene clusters (U2 XbaI, U2 AflII, U8 BamHI, and U8 EcoRI) and the primers for verification of the recombinant and wild-type virus (EGFP F, EGFP R, U5 F, and U5 R) are indicated. DR, direct repeat.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056027-g001: Structure of H6R28LEP.The straight line at the top represents the genome of the HHV-6B HST genome, with the U1 to U10 region expanded below. In the middle, the shaded arrows show the positions from start codon to poly(A) signal of the genes in the U2–U8 gene cluster that was replaced by the EGFP-IRES-LNGFR-Puro cassette. The bottom diagram shows the structure of pU2-U8 LEP. Open boxes represent the low-affinity nerve growth factor receptor (LNGFR), enhanced green fluorescent protein (EGFP), and puromycin-N-acetyl-transferase (Puro) genes; filled boxes represent the internal ribosome entry site (IRES), human cytomegalovirus major immediate early promoter (HCMV MIEP), and simian virus 40 (SV40) promoter. The locations of the PCR primers used in the cloning of the U2 and U8 gene clusters (U2 XbaI, U2 AflII, U8 BamHI, and U8 EcoRI) and the primers for verification of the recombinant and wild-type virus (EGFP F, EGFP R, U5 F, and U5 R) are indicated. DR, direct repeat.
Mentions: In our previous report, we generated a recombinant HHV-6 by inserting a selectable marker, the puromycin-resistance gene, into wtHHV-6 by homologous recombination [30]. In the present study, we built on this earlier work by using MACS LNGFR microbeads in addition to puromycin treatment to select for transfected cells. In doing so, we created a recombinant HHV-6 virus (H6R28LEP) in a shorter time than previously and were able to concentrate the infected cells. In H6R28LEP, the Puro and LNGFR genes replace the part of HHV-6 genome from the promoter of the U2 gene to the poly(A) signal of the U8 gene (Fig. 1). Using real-time PCR, we confirmed that H6R28LEP lacks expression of the U2–U8 genes (data not shown).

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