pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

<p>Abstract</p> <p>Background</p> <p>The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed <it>Cytomegalovirus </it>immediate early promoter (CMV-IEP) and directed into a 2000 bp long <it>matrix attachment region sequence </it>(MARS) derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.</p> <p>Results</p> <p>Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both <it>in vitro </it>and <it>in vivo</it>. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies <it>in vitro</it>, as well as more persistent transgene expression profiles <it>in vivo</it>. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the <it>human CMV enhancer/human elongation factor 1 alpha promoter </it>(hCMV/EF1P) element that is known to be less affected by epigenetic silencing events.</p> <p>Conclusions</p> <p>The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications <it>in vitro </it>and for non-viral gene delivery <it>in vivo</it>.</p

GnRH-gemcitabine conjugates for the treatment of androgen-independent prostate cancer : pharmacokinetic enhancements combined with targeted drug delivery

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone- Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG, showed a significant advantage in tumor growth inhibition when compared to gemcitabine.A.G.Leventis foundation and the General Secretariat for Research & Technology of the Greek Ministry of Education (LS7- 1682/17156/6.12.10).MRC and National Research Foundation of South Africa, and the Universities of Pretoria and Cape Townhttp://pubs.acs.org/bc2015-02-28hb201

Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modelling.

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome

Molecular analysis of DNA isolated from tumour tissues at day 35 post delivery, from Huh7 and MIA-PaCa2 injected NOD/SCID mice.

<p>(A) Southern blot analysis of pDNA isolated from two different regions of tumour tissue from NOD/SCID mice, 35days post-delivery of Huh7 and MIA-PaCa2 stable cell lines, performed as described in materials and methods. A representative hybridization pattern of pDNA isolated from one animal of each tumour is shown. Detection of indicator plasmid by M: 1-kbp ladder (Hyperladder I, Bioline); lane 1: pUbC-S/MAR isolated from the tumour tissue formed after Huh7 injection of NOD/SCID mice at 35 days post-injection; lane 2: pUbC-S/MAR isolated from a different region of the tumour tissue formed after Huh7 delivery into NOD/SCID mice at 35 days post-injection; lane 3 pUbC-S/MAR isolated from the tumour tissue formed after MIA-PaCa2 injection of NOD/SCID mice at 35 days post-injection; lane 4: pUbC-S/MAR isolated from a different region of the tumour tissue formed after MIA-PaCa2 delivery into NOD/SCID mice at 35 days post-injection; (+) positive control: 25 ng of linearized pUbC-S/MAR plasmid. (B) Replication-dependent assay of pUbC-S/MAR plasmid DNA isolated from the tumours of mice at 35 days post-administration. lanes 1–3: Southern blot of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with Huh7 stable cell line and double digested with <i>Spe</i>I–<i>Mbo</i>I (lane 1), <i>Spe</i>I–<i>Dpn</i>I (lane 2) or <i>Spe</i>I–<i>Bfu</i>CI (lane 3) enzymes; lanes 7–9: Southern of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with MIA-PaCa2 stable cell line and double digested with <i>Spe</i>I–<i>Mbo</i>I (lane 4), <i>Spe</i>I–<i>Dpn</i>I (lane 5) or <i>Spe</i>I–<i>Bfu</i>CI (lane 6) enzymes; M: 1-kbp ladder (Hyperladder I, Bioline UK Ltd., London, UK). (C) Quantitative PCR performed on tumour DNA obtained at day 35 after injection of Huh7 and MIA-PaCa2 cell lines. DNA was extracted from two different sites of each tumour at the end of the experiment and the number of pUbC-S/MAR vector genomes per diploid genome is shown, after normalisation with GAPDH gene, as described in materials and methods. (D) PCR analysis of DNA isolated <i>in vitro</i> from the Huh7 (lane 1) and MIA-PaCa2 (lane 4) cells before injection into NOD/SCID mice, and <i>in vivo</i> from two different regions of the tumour for each cell line (lanes 2,3 for Huh7 and lanes 5,6 for the MIA-PaCa2 cell lines). Expected PCR product size: 1091 bp. 100 bp DNA ladder (lane M), (+) positive control: pUbC-S/MAR; (-) negative control: PCR mix without DNA. (E) Plasmid rescue experiments of four <i>E.Coli</i> colonies for Huh7 (lanes 1–4) and three colonies for MIA-PaCa2 cell lines (lanes 5–7), showing identical restriction pattern with pure pUbC-S/MAR plasmid (+), following restriction digest with <i>Spe</i>I enzyme. M: 1-kbp ladder (Hyperladder I, Bioline).</p

Histochemistry and Immunohistochemistry of tumour sections at day 35 post delivery, showing the formation of a hepatocellular carcinoma-like tumour and a pancreatic carcinoma tumour, to which luciferase expression localises.

<p>Sections from different parts of the two tumours were cut and stained with haematoxylin and eosin for histological analysis of the tumours. A–D) Sections from Huh7 injected mice. Sections have an amorphous structure and were identified as hepatocellular carcinoma (HCC) of varying degrees of differentiation: (A) Moderately differentiated HCC, magnification×10 (B–C) Sections were analysed by immunohistochemistry to show distribution of luciferase expression. Brown staining indicates luciferase positive cells. (B) Positively stained, Magnification×40 (C) Positively stained, Magnification×10 (D) Negative control: no primary antibody added, magnification×10 E–H) Sections from MIA-PaCa2 injected mice. Sections have an amorphous structure and were identified as Pancreatic carcinoma (PaCa) of varying degrees of differentiation. (E) Moderately differentiated PaCa, magnification×10 (F–G) Sections were analysed by immunohistochemistry to show distribution of luciferase expression. Brown staining indicates luciferase positive cells. (F) Positively stained, Magnification×40 (G) Positively stained, Magnification×10 (H) Negative control: no primary antibody added, magnification×10.</p

Analysis of luciferase expression from pUbC-S/MAR plasmid in stably transfected Huh7 and MIA-PaCa2 tumour cells.

<p>A) The pUbC-S/MAR plasmid used in this study, in which luciferase expression is driven by the human UbC promoter. B) Huh7, and MIA-PaCa2 cells were transfected with pUbC-S/MAR and grown under selection with G418 for about two weeks. Three single colonies were isolated and expanded out of selection with regular imaging using a Xenogen bioimager. C) Southern blot of total DNA isolated from three individual colonies for each cell line at 45 days post transfection. Lanes 1–3: Huh7 isolated colonies; Lanes 4–6 MIA-PaCa2 isolated colonies; (+): Positive control, 10 ng of bacterial pUbC-S/MAR plasmid. D) luciferase bioluminescence assay (in duplicate) on increasing amounts of Huh7 and MIA-PaCa2 cells, showing limits of signal (luciferase) detection, <i>in vitro</i>. E–F) Plasmid rescue experiments of three <i>E.Coli</i> colonies for Huh7 (lanes 1–3) and four colonies for MIA-PaCa2 cell lines (lanes 4–7), showing identical restriction pattern with pure pUbC-S/MAR plasmid (+), following restriction digest with <i>Spe</i>I enzyme. (−) negative control (no DNA); M: 1-kbp ladder (Hyperladder I, Bioline).</p

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy

Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC–MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an <i>in vitro</i> colony forming assay together with <i>in vivo</i> whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine’s bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution, and safety while also agonistically ablating testosterone levels

Peptide-Drug conjugate gnrh-sunitinib targets angiogenesis selectively at the site of action to inhibit tumor growth

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [D-Lys6]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphor-ylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.</p