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Evaluating baculovirus as a vector for human prostate cancer gene therapy.

Swift SL, Rivera GC, Dussupt V, Leadley RM, Hudson LC, Ma de Ridder C, Kraaij R, Burns JE, Maitland NJ, Georgopoulos LJ - PLoS ONE (2013)

Bottom Line: Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects.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.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research Unit, Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
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.

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Reduction in cell viability following transduction with a BV expressing a nitroreductase-IRES-EGFP expression cassette and treatment with the pro-drug CB1954.(A) Percentage of non-viable cells at 48 h post-transduction with BV-[CMV-NTR-EGFP] (MOI = 500) and treatment with CB1954 in a panel of prostate cell lines, shown relative to frequency of transduced cells and adjusted for untreated controls. Malignant cell lines: PC346C (♦), PC3 (▴),LNCaP (▪), P4E6 (×); Non-malignant cell lines: PNT1A (+), PNT2C2 (•). Statistics represent t-test between PC346C/PC-3/LNCaP vs. PNT1A/PNT2C2. (B) Percentage of non-viable cells at 30 h post-transduction with BV-[CMV-NTR-EGFP] and treatment with CB1954 in individual primary prostate epithelial cell samples (n = 3 malignant and n = 2 non-malignant), adjusted for untreated controls.
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pone-0065557-g004: Reduction in cell viability following transduction with a BV expressing a nitroreductase-IRES-EGFP expression cassette and treatment with the pro-drug CB1954.(A) Percentage of non-viable cells at 48 h post-transduction with BV-[CMV-NTR-EGFP] (MOI = 500) and treatment with CB1954 in a panel of prostate cell lines, shown relative to frequency of transduced cells and adjusted for untreated controls. Malignant cell lines: PC346C (♦), PC3 (▴),LNCaP (▪), P4E6 (×); Non-malignant cell lines: PNT1A (+), PNT2C2 (•). Statistics represent t-test between PC346C/PC-3/LNCaP vs. PNT1A/PNT2C2. (B) Percentage of non-viable cells at 30 h post-transduction with BV-[CMV-NTR-EGFP] and treatment with CB1954 in individual primary prostate epithelial cell samples (n = 3 malignant and n = 2 non-malignant), adjusted for untreated controls.

Mentions: In order to test the ability of BV to achieve a cell-lethal outcome, we employed a gene-directed enzyme pro-drug therapy (GDEPT) approach focussed on bacterial nitroreductase (NTR), which converts the pro-drug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a potent DNA cross-linking agent [41]. Therefore, we constructed a BV bearing an expression cassette where the strong mammalian CMV promoter drove expression of mNTR-IRES-EGFP (BV-[CMV-NTR-EGFP]). A panel of malignant and non-malignant prostate cell lines were transduced with BV-[CMV-NTR-EGFP], and cell viability was evaluated following administration of the pro-drug, CB1954. The proportion of non-viable cells, as measured by MTS assay after 48 h, was significantly higher in malignant samples with high metastatic potential (PC346C, PC3 and LNCaP) compared to non-malignant samples (PNT1A and PNT2C2) (P<0.001) (Figure 4A). Early-stage malignant samples (P4E6) fell into the intermediate range of viability. Furthermore, cell viability following pro-drug administration was directly related to the rate of BV transduction (Figure 4A). This analysis was extended into primary patient-derived epithelial cell cultures. Three malignant and two non-malignant samples were transduced with BV-[CMV-NTR-EGFP] and treated with CB1954. At 30 h post-pro-drug administration, cell death in these cultures ranged from 10% to 50%, irrespective of disease status (Figure 4B).


Evaluating baculovirus as a vector for human prostate cancer gene therapy.

Swift SL, Rivera GC, Dussupt V, Leadley RM, Hudson LC, Ma de Ridder C, Kraaij R, Burns JE, Maitland NJ, Georgopoulos LJ - PLoS ONE (2013)

Reduction in cell viability following transduction with a BV expressing a nitroreductase-IRES-EGFP expression cassette and treatment with the pro-drug CB1954.(A) Percentage of non-viable cells at 48 h post-transduction with BV-[CMV-NTR-EGFP] (MOI = 500) and treatment with CB1954 in a panel of prostate cell lines, shown relative to frequency of transduced cells and adjusted for untreated controls. Malignant cell lines: PC346C (♦), PC3 (▴),LNCaP (▪), P4E6 (×); Non-malignant cell lines: PNT1A (+), PNT2C2 (•). Statistics represent t-test between PC346C/PC-3/LNCaP vs. PNT1A/PNT2C2. (B) Percentage of non-viable cells at 30 h post-transduction with BV-[CMV-NTR-EGFP] and treatment with CB1954 in individual primary prostate epithelial cell samples (n = 3 malignant and n = 2 non-malignant), adjusted for untreated controls.
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Related In: Results  -  Collection

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pone-0065557-g004: Reduction in cell viability following transduction with a BV expressing a nitroreductase-IRES-EGFP expression cassette and treatment with the pro-drug CB1954.(A) Percentage of non-viable cells at 48 h post-transduction with BV-[CMV-NTR-EGFP] (MOI = 500) and treatment with CB1954 in a panel of prostate cell lines, shown relative to frequency of transduced cells and adjusted for untreated controls. Malignant cell lines: PC346C (♦), PC3 (▴),LNCaP (▪), P4E6 (×); Non-malignant cell lines: PNT1A (+), PNT2C2 (•). Statistics represent t-test between PC346C/PC-3/LNCaP vs. PNT1A/PNT2C2. (B) Percentage of non-viable cells at 30 h post-transduction with BV-[CMV-NTR-EGFP] and treatment with CB1954 in individual primary prostate epithelial cell samples (n = 3 malignant and n = 2 non-malignant), adjusted for untreated controls.
Mentions: In order to test the ability of BV to achieve a cell-lethal outcome, we employed a gene-directed enzyme pro-drug therapy (GDEPT) approach focussed on bacterial nitroreductase (NTR), which converts the pro-drug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a potent DNA cross-linking agent [41]. Therefore, we constructed a BV bearing an expression cassette where the strong mammalian CMV promoter drove expression of mNTR-IRES-EGFP (BV-[CMV-NTR-EGFP]). A panel of malignant and non-malignant prostate cell lines were transduced with BV-[CMV-NTR-EGFP], and cell viability was evaluated following administration of the pro-drug, CB1954. The proportion of non-viable cells, as measured by MTS assay after 48 h, was significantly higher in malignant samples with high metastatic potential (PC346C, PC3 and LNCaP) compared to non-malignant samples (PNT1A and PNT2C2) (P<0.001) (Figure 4A). Early-stage malignant samples (P4E6) fell into the intermediate range of viability. Furthermore, cell viability following pro-drug administration was directly related to the rate of BV transduction (Figure 4A). This analysis was extended into primary patient-derived epithelial cell cultures. Three malignant and two non-malignant samples were transduced with BV-[CMV-NTR-EGFP] and treated with CB1954. At 30 h post-pro-drug administration, cell death in these cultures ranged from 10% to 50%, irrespective of disease status (Figure 4B).

Bottom Line: Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects.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.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research Unit, Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus