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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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Lack of cytotoxic effects of H6R28LEP as indicated through mRNA expression of housekeeping genes.At the indicated times, infected and uninfected cells (5×105 each) were harvested. Total RNA was isolated and quantified by real-time RT PCR using the Human Housekeeping Gene Primer Set (Takara Bio). Relative mRNA expression levels were compared by using two-tailed Student's t tests. Data are given as mean ±1 standard deviation (n = 3). GAPDH, glyceraldehydes-30phosphate dehydrogenase; B2M, β2 microglobulin; HPRT1, hypoxanthine guanine phosphoribosyl transferase 1.
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pone-0056027-g008: Lack of cytotoxic effects of H6R28LEP as indicated through mRNA expression of housekeeping genes.At the indicated times, infected and uninfected cells (5×105 each) were harvested. Total RNA was isolated and quantified by real-time RT PCR using the Human Housekeeping Gene Primer Set (Takara Bio). Relative mRNA expression levels were compared by using two-tailed Student's t tests. Data are given as mean ±1 standard deviation (n = 3). GAPDH, glyceraldehydes-30phosphate dehydrogenase; B2M, β2 microglobulin; HPRT1, hypoxanthine guanine phosphoribosyl transferase 1.

Mentions: We determined the mRNA expression levels of several housekeeping genes (GAPDH, B2M, HPRT1) to examine the effect of infection with H6R28LEP on PBMCs. mRNA expression of these housekeeping genes remained consistent for 12 d after infection (Fig. 8), indicating a lack of effect of H6R28LEP on the function of 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)

Lack of cytotoxic effects of H6R28LEP as indicated through mRNA expression of housekeeping genes.At the indicated times, infected and uninfected cells (5×105 each) were harvested. Total RNA was isolated and quantified by real-time RT PCR using the Human Housekeeping Gene Primer Set (Takara Bio). Relative mRNA expression levels were compared by using two-tailed Student's t tests. Data are given as mean ±1 standard deviation (n = 3). GAPDH, glyceraldehydes-30phosphate dehydrogenase; B2M, β2 microglobulin; HPRT1, hypoxanthine guanine phosphoribosyl transferase 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056027-g008: Lack of cytotoxic effects of H6R28LEP as indicated through mRNA expression of housekeeping genes.At the indicated times, infected and uninfected cells (5×105 each) were harvested. Total RNA was isolated and quantified by real-time RT PCR using the Human Housekeeping Gene Primer Set (Takara Bio). Relative mRNA expression levels were compared by using two-tailed Student's t tests. Data are given as mean ±1 standard deviation (n = 3). GAPDH, glyceraldehydes-30phosphate dehydrogenase; B2M, β2 microglobulin; HPRT1, hypoxanthine guanine phosphoribosyl transferase 1.
Mentions: We determined the mRNA expression levels of several housekeeping genes (GAPDH, B2M, HPRT1) to examine the effect of infection with H6R28LEP on PBMCs. mRNA expression of these housekeeping genes remained consistent for 12 d after infection (Fig. 8), indicating a lack of effect of H6R28LEP on the function of 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