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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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BV transduction of prostate cell lines.(A) Percentage of EGFP-positive cells following transduction of a panel of high grade malignant (red), low grade malignant (black) or non-malignant (blue) prostate cell lines with BV-[CMV-EGFPCAT] at MOI = 500 for 48 h. Error bars depict −/+ one standard deviation. (B) Relative expression levels of EGFP following transduction with BV-[CMV-EGFPCAT] at a saturating MOI (500 for LNCaP, PC3 and PNT1A, 1000 for PNT2C2). The percentage of EGFP-positive cells was normalised to the levels achieved following 48 h incubation in the presence of virus for each cell type (set to 1). Malignant cell lines: PC3 (▴), LNCaP (▪); Non-Malignant Cell Lines: PNT2C2 (♦), PNT1A (▾). Error bars depict −/+ one standard deviation. (C) Confocal microscopy images (single slice or Z-stack) of BV-transduced LNCaP, PC3 or PNT1A cells at 8 h post-transduction (MOI = 500). Red fluorescence indicates BV capsid (anti-vp39), nuclear staining in blue (DAPI) and BV-driven EGFP expression in green.
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pone-0065557-g001: BV transduction of prostate cell lines.(A) Percentage of EGFP-positive cells following transduction of a panel of high grade malignant (red), low grade malignant (black) or non-malignant (blue) prostate cell lines with BV-[CMV-EGFPCAT] at MOI = 500 for 48 h. Error bars depict −/+ one standard deviation. (B) Relative expression levels of EGFP following transduction with BV-[CMV-EGFPCAT] at a saturating MOI (500 for LNCaP, PC3 and PNT1A, 1000 for PNT2C2). The percentage of EGFP-positive cells was normalised to the levels achieved following 48 h incubation in the presence of virus for each cell type (set to 1). Malignant cell lines: PC3 (▴), LNCaP (▪); Non-Malignant Cell Lines: PNT2C2 (♦), PNT1A (▾). Error bars depict −/+ one standard deviation. (C) Confocal microscopy images (single slice or Z-stack) of BV-transduced LNCaP, PC3 or PNT1A cells at 8 h post-transduction (MOI = 500). Red fluorescence indicates BV capsid (anti-vp39), nuclear staining in blue (DAPI) and BV-driven EGFP expression in green.

Mentions: In order to determine the capacity of BV to differentially transduce malignant vs. non-malignant prostate targets, we evaluated the susceptibility of a panel of established human prostate cell lines to BV transduction. Cells were incubated with a recombinant BV vector modified to carry an EGFPCAT transgene (BV-[CMV-EGFPCAT]) using a fixed MOI of 500 and an incubation time of 48 h. Malignant cell lines with high metastatic potential, including LNCaP, PC3 and PC346C, were highly transduced (∼80% EGFP-positive cells), while non-malignant cell lines PNT1A and PNT2C2 were least transduced (up to 10% EGFP-positive cells) (Figure 1A). P4E6, a cell line derived from an initiated but early stage tumour, was in the intermediate range, with approximately 20% EGFP-positive cells (Figure 1A). Thus, the frequency of cells positively transduced by BV correlated with malignancy and metastatic potential.


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)

BV transduction of prostate cell lines.(A) Percentage of EGFP-positive cells following transduction of a panel of high grade malignant (red), low grade malignant (black) or non-malignant (blue) prostate cell lines with BV-[CMV-EGFPCAT] at MOI = 500 for 48 h. Error bars depict −/+ one standard deviation. (B) Relative expression levels of EGFP following transduction with BV-[CMV-EGFPCAT] at a saturating MOI (500 for LNCaP, PC3 and PNT1A, 1000 for PNT2C2). The percentage of EGFP-positive cells was normalised to the levels achieved following 48 h incubation in the presence of virus for each cell type (set to 1). Malignant cell lines: PC3 (▴), LNCaP (▪); Non-Malignant Cell Lines: PNT2C2 (♦), PNT1A (▾). Error bars depict −/+ one standard deviation. (C) Confocal microscopy images (single slice or Z-stack) of BV-transduced LNCaP, PC3 or PNT1A cells at 8 h post-transduction (MOI = 500). Red fluorescence indicates BV capsid (anti-vp39), nuclear staining in blue (DAPI) and BV-driven EGFP expression in green.
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Related In: Results  -  Collection

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pone-0065557-g001: BV transduction of prostate cell lines.(A) Percentage of EGFP-positive cells following transduction of a panel of high grade malignant (red), low grade malignant (black) or non-malignant (blue) prostate cell lines with BV-[CMV-EGFPCAT] at MOI = 500 for 48 h. Error bars depict −/+ one standard deviation. (B) Relative expression levels of EGFP following transduction with BV-[CMV-EGFPCAT] at a saturating MOI (500 for LNCaP, PC3 and PNT1A, 1000 for PNT2C2). The percentage of EGFP-positive cells was normalised to the levels achieved following 48 h incubation in the presence of virus for each cell type (set to 1). Malignant cell lines: PC3 (▴), LNCaP (▪); Non-Malignant Cell Lines: PNT2C2 (♦), PNT1A (▾). Error bars depict −/+ one standard deviation. (C) Confocal microscopy images (single slice or Z-stack) of BV-transduced LNCaP, PC3 or PNT1A cells at 8 h post-transduction (MOI = 500). Red fluorescence indicates BV capsid (anti-vp39), nuclear staining in blue (DAPI) and BV-driven EGFP expression in green.
Mentions: In order to determine the capacity of BV to differentially transduce malignant vs. non-malignant prostate targets, we evaluated the susceptibility of a panel of established human prostate cell lines to BV transduction. Cells were incubated with a recombinant BV vector modified to carry an EGFPCAT transgene (BV-[CMV-EGFPCAT]) using a fixed MOI of 500 and an incubation time of 48 h. Malignant cell lines with high metastatic potential, including LNCaP, PC3 and PC346C, were highly transduced (∼80% EGFP-positive cells), while non-malignant cell lines PNT1A and PNT2C2 were least transduced (up to 10% EGFP-positive cells) (Figure 1A). P4E6, a cell line derived from an initiated but early stage tumour, was in the intermediate range, with approximately 20% EGFP-positive cells (Figure 1A). Thus, the frequency of cells positively transduced by BV correlated with malignancy and metastatic potential.

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