Evaluating Baculovirus as a Vector for Human Prostate Cancer Gene Therapy

<div><p>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. <i>In vitro</i>, 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 <i>in vivo</i> 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 <i>in vitro</i> spheroids and <i>in vivo</i> 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.</p></div

BV transduction of cells growing in three dimensions.

<p>(<b>A</b>) PC346C spheroids transduced with Bv-[CMV-EGFP] (or mock transduced) at 3 days post-infection. (<b>B</b>) PC346C tumours harvested from xenografted mice at 3 days post-infection with BV at 3×10⁷ (one representative image shown) or 1×10⁸ (two representative images shown) pfu per inoculation. Red fluorescence shows cell nuclei (Hoescht), while BV-driven EGFP expression is shown in green. Arrows indicate the site of intratumoural BV injection.</p

BV transduction of prostate cell lines.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

BV transduction of primary prostate epithelial cells.

<p>(<b>A</b>) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (<b>B</b>) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (<b>C</b>) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.</p

Additional file 2: of Meta-analysis of human genome-microbiome association studies: the MiBioGen consortium initiative

Meta-analysis of human genome-microbiome association studies: the MiBioGen consortium initiative Acknowledgement and funding information. (DOCX 37 kb

Results of CHARGE exome chip genotype calls compared to exome sequence data in 530 individuals.

<p>Results of CHARGE exome chip genotype calls compared to exome sequence data in 530 individuals.</p

Exome chip minor allele frequency distribution by race.

<p>The following samples were excluded: all AGES individuals, race unknown or not reported, known replicates, HapMap controls, individuals with p10GC <0.38, and individuals with call rate <0.97. Individuals with race designated as other were included in the overall MAF calculation, but data is not shown separately (n = 26). A total of 238,065 variants were used for calculating minor allele frequencies after excluding those that failed laboratory quality control (n = 8,994) and duplicates (n = 811).</p

Best practices criteria used to identify SNPs for visual inspection and manual reclustering.

<p>AA: allele A homozygote; AB: heterozygote; BB: allele B homozygote; Het: heterozygote; MAF: minor allele frequency; MT: mitochondrial; PC: parent-child; PPC: parent-parent-child; R: normalized intensity; Rep, reproducibility.</p

Exome chip content and CHARGE excluded variants by functional category.

1<p>dbNSFP was used for annotating variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068095#pone.0068095-Liu1" target="_blank">[27]</a> (see Methods).</p

Exome chip SNP exclusion criteria.

<p>AA: allele A homozygote; AB: heterozygote; BB: allele B homozygote; Het: heterozygote; R: normalized intensity.</p