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.
Highlights
Gene introduction into T cells is a very useful technique for gene therapy of HIV infection and the immunotherapy of fatal diseases including cancer
Because human herpesvirus 6 (HHV-6) has low virulence [33] and infects CD4+ T cells, macrophages, and dendritic cells [14][15][34][35], recombinant HHV-6 vectors have been expected to be useful in gene therapy of HIV infection and cancer
The generation of recombinant HHV-6 vectors has lagged behind that of other herpesviruses. Reasons underlying this delay are that HHV-6 has fewer genes than does the closely related human cytomegalovirus (HCMV), and HHV-6 lacks homologs of the HCMV genes that are dispensable for viral replication and replaced with transgenes during the construction of recombinant viral vectors [29][36][37][38]
Summary
Gene introduction into T cells is a very useful technique for gene therapy of HIV infection and the immunotherapy of fatal diseases including cancer. This method currently relies on vectors derived from members of the lentivirus family of retroviruses to introduce genes into T cells [1]. Oncogenicity is a characteristic of wild-type retroviruses [2]; another risk factor is the potential recombination of retroviral vectors with endogenous retroviruses in the host to yield replication-competent virus [3]. Adeno-associated virus (AAV) vectors have been developed to improve the safety of viral vectors and their transduction into hematologic cells [4]. Because (unlike wild-type AAV) recombinant AAV vectors can integrate randomly into host chromosomes [7][8], recombinant AAV vectors cannot be guaranteed to be free from carcinogenic effects
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