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Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

ABSTRACT: We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.

SUBMITTER: Mahmood K

PROVIDER: S-EPMC548291 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature

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Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

Mahmood Kutubuddin K Prichard Mark N MN Duke Gregory M GM Kemble George W GW Spaete Richard R RR

Genetic vaccines and therapy 20050126 1

We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packa ...[more]

PMID: 15673469

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Project description:Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. In vitro, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV in vivo in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.

Project description:The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions.IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

Dataset Information

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

Publications

Human cytomegalovirus plasmid-based amplicon vector system for gene therapy.

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