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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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Expression of siRNA determined by real-time RT-PCR.Shown is the relative fold expression of A: siCD4 or B: siGag in PBMCs. siRNA was transduced through infection of an shRNA-containing H6R28LEP vector or by lipofection or electroporation. Total cellular RNA, including siRNA, was extracted from transduced cells. RNA samples were reverse-transcribed and then amplified by using siRNA-specific primers (siCD4 F and siGag F). Expression of siCD4 and siGag was significantly increased in PBMCs infected with the H6R28LEP-shRNA constructs compared with that after lipofection or electroporation. Data are given as mean ±1 standard deviation (n = 2).
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pone-0056027-g010: Expression of siRNA determined by real-time RT-PCR.Shown is the relative fold expression of A: siCD4 or B: siGag in PBMCs. siRNA was transduced through infection of an shRNA-containing H6R28LEP vector or by lipofection or electroporation. Total cellular RNA, including siRNA, was extracted from transduced cells. RNA samples were reverse-transcribed and then amplified by using siRNA-specific primers (siCD4 F and siGag F). Expression of siCD4 and siGag was significantly increased in PBMCs infected with the H6R28LEP-shRNA constructs compared with that after lipofection or electroporation. Data are given as mean ±1 standard deviation (n = 2).

Mentions: To examine the usefulness of siRNA transfected by using H6R28LEP, we generated the virus constructs H6R28LEP shCD4 and H6R28LEP shGag, in which expression of shRNA against the HIV receptor CD4 or the HIV Gag protein, respectively, was driven by using a U6 promoter (Fig. 9). We compared the expression levels of these shRNAs in PBMCs infected by using H6R28LEP shCD4 and H6R28LEP shGag with that from the shRNA expression plasmids transfected into PBMCs by using electroporation or lipofection. The amounts of siCD4 and siGag in the PBMCs infected with the H6R28LEP-based viruses were greater than those in the cells that had been transfected by electroporation or lipofection. Specifically, the amount of siCD4 RNA due to H6R28LEP shCD4 was 4300 times greater than that after lipofection and 30 times greater than that after electroporation. The amount of siGag RNA due to H6R28LEP shGag was 2700 times greater than that after lipofection and 160 times greater than that after electroporation (Fig. 10).


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)

Expression of siRNA determined by real-time RT-PCR.Shown is the relative fold expression of A: siCD4 or B: siGag in PBMCs. siRNA was transduced through infection of an shRNA-containing H6R28LEP vector or by lipofection or electroporation. Total cellular RNA, including siRNA, was extracted from transduced cells. RNA samples were reverse-transcribed and then amplified by using siRNA-specific primers (siCD4 F and siGag F). Expression of siCD4 and siGag was significantly increased in PBMCs infected with the H6R28LEP-shRNA constructs compared with that after lipofection or electroporation. Data are given as mean ±1 standard deviation (n = 2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056027-g010: Expression of siRNA determined by real-time RT-PCR.Shown is the relative fold expression of A: siCD4 or B: siGag in PBMCs. siRNA was transduced through infection of an shRNA-containing H6R28LEP vector or by lipofection or electroporation. Total cellular RNA, including siRNA, was extracted from transduced cells. RNA samples were reverse-transcribed and then amplified by using siRNA-specific primers (siCD4 F and siGag F). Expression of siCD4 and siGag was significantly increased in PBMCs infected with the H6R28LEP-shRNA constructs compared with that after lipofection or electroporation. Data are given as mean ±1 standard deviation (n = 2).
Mentions: To examine the usefulness of siRNA transfected by using H6R28LEP, we generated the virus constructs H6R28LEP shCD4 and H6R28LEP shGag, in which expression of shRNA against the HIV receptor CD4 or the HIV Gag protein, respectively, was driven by using a U6 promoter (Fig. 9). We compared the expression levels of these shRNAs in PBMCs infected by using H6R28LEP shCD4 and H6R28LEP shGag with that from the shRNA expression plasmids transfected into PBMCs by using electroporation or lipofection. The amounts of siCD4 and siGag in the PBMCs infected with the H6R28LEP-based viruses were greater than those in the cells that had been transfected by electroporation or lipofection. Specifically, the amount of siCD4 RNA due to H6R28LEP shCD4 was 4300 times greater than that after lipofection and 30 times greater than that after electroporation. The amount of siGag RNA due to H6R28LEP shGag was 2700 times greater than that after lipofection and 160 times greater than that after electroporation (Fig. 10).

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