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Supplementary Figures 1-6. Figure S.I.1 - 1HNMR spectra of the six sunitinib analogues Figure S.I.2 - LC-MS/MS characterization of SAN1 Figure S.I.3 - Molecular models of VEGFR-2 kinase domain complexed with SAN1,2,3 Figure S.I.4 - Synthesis and characterization of SAN1GSC by NMR and LC-MS/MS Figure S.I.5 - Body weights of xenografted mice during the course of treatment Figure S.I.6 - Evaluation of the hematological toxicities following treatment

Supplementary Methods 1 - Overall Synthesis of sunitinib analogues (SAN1-SAN5) Supplementary Methods 2 - Computational analysis Supplementary Methods 3 - Synthesis of the conjugate SAN1GSC Supplementary Methods 4 - Biochemical Tyrosine Kinase Assay Supplementary Methods 5 - Cellular autophosphorylation assay Supplementary Methods 6 - Overall phenotypic health evaluation and monitoring of body weight Supplementary Methods 7 - Cardiotoxicity studies Supplementary Methods 8 - Blood toxicity Table S1 - List of GST- fusion proteins Table S2 - List of Antibodies

Supplementary figure legends for supplementary figures 1-6

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 phosphorylation 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. Cancer Res; 76(5); 1181–92. ©2015 AACR.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

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 phosphorylation 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. Cancer Res; 76(5); 1181–92. ©2015 AACR.

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 in vitro colony forming assay together with in vivo 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.

Breast cancer (BrCa) remains an unmet medical need despite the revolutionary development of antibody treatments and protein kinase inhibitors. In the current study, a series of novel substituted pyridopyrazine derivatives have been rationally designed and evaluated as multi-kinase inhibitors in the PI3K pathway. The target compounds were prepared from 6-amino-2-picoline, which upon nitration and selective reduction was converted to suitably substituted 6-methyl-7-aminopyrido[2,3-b]pyrazines. Suitable manipulation of the former amines provided the designed analogues, which were then assessed in vitro against several BrCa cell lines using the MTT cytotoxicity assay. The most potent compounds underwent evaluation in a broad spectrum of protein kinases, while their pharmacokinetic parameters were measured by LC-MS/MS. In vivo evaluation of a hit compound (14a) was performed in a HER2 amplified BrCa xenograft model (HCC1954) and efficacy was determined using Western blot based phosphokinase assays and immunohistochemistry. This derivative showed low micromolar cytotoxic potency in all BrCa cell lines, a mild inhibition of the PI3Kα wild type and H1047R mutated enzyme and excellent pharmacokinetic parameters following oral and intraperitoneal administration at the designed dose of 10 mg/kg, with absence of in vivo phenotypic toxicity. Interestingly, compound 14a inhibited the growth of xenografted tumors. Analysis of excised tumors from the treated animals showed a significantly reduced population of Ki-67 positive cells, as well as downregulated levels of phosphorylated AKT, ERK1/2 and SRC compared to vehicle treated animals. Finally, the specificity of 14a was assessed in a panel of 31 kinases where a mild, but direct, inhibition of the MET receptor tyrosine kinase was observed.

Objective: A series of novel 2,4-diaminosubstituted pyrrolo[3,2-d]pyrimidines was synthesized together with their corresponding 7-phenyl or 7-isopropyl counterparts. Results: Among the target derivatives, the 7-substituted analogues exhibited interesting cytotoxic activity against a panel of PI3Kα related human breast cancer cell lines, namely MCF7, T47D, MDA-MB-231 and HCC1954. Selected compounds were tested for potential PI3Kα inhibitory activity as well as for their cytotoxic effect in prostate cancer cell lines (DU145 and PC3). Conclusion: Derivatives bearing a specific substitution pattern consisting of 7-phenyl as well as a 2-(4- aminocyclohexylamino) moiety (16c, 16f) display kinase inhibitory activity, elucidated on the basis of molecular simulation studies, which revealed their interaction with the DFG motif of the kinase.

The clinical efficacy of antiangiogenic small molecules (e.g., sunitinib) in breast carcinoma has largely failed with substantial off-target toxicity. We rationally designed and evaluated preclinically a novel sunitinib analogue, SAP, with favourable pharmacological properties and the ability to be readily conjugated to a targeting peptide or antibody for active tumour targeting. SAP was evaluated in silico and in vitro in order to verify target engagement (e.g., VEGFR2). Pharmacokinetic and biodistribution parameters were determined in mice using LC-MS/MS. SAP efficacy was tested in two breast cancer xenograft and two syngeneic animal models and pharmacodynamic evaluation was accomplished using phosphokinase assays and immunohistochemistry. Cardiac and blood toxicity of SAP were also monitored. SAP retained the antiangiogenic and cytotoxic properties of the parental molecule with an increased blood exposure and tumor accumulation compared to sunitinib. SAP proved efficacious in all animal models. Tumors from SAP treated animals had significantly decreased Ki-67 and CD31 markers and reduced levels of phosphorylated AKT, ERK and S6 compared to vehicle treated animals. In mice dosed with SAP there was negligible hematotoxicity, while cardiac function measurements showed a reduction in the percentage left ventricular fractional shortening compared to vehicle treated animals. In conclusion, SAP is a novel rationally designed conjugatable small antiangiogenic molecule, efficacious in preclinical models of breast cancer.

The early potential of gene therapy is slowly becoming realized following the recent treatment of patients with severe combined immunodeficiency and ocular diseases. However at present the field of gene therapy is tempered by the toxicity issues, mainly that of the integrated retroviral vector used in most trials which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors is therefore vital for further progress in this field, in particular vectors which remain episomal and are therefore less genotoxic. One such unique class of vectors are those based on scaffold matrix attachment regions (S/MARs) elements, which are maintained extra-chromosomally and replicate in vitro and in vivo.The overview here describes the most relevant studies utilizing the S/MAR element to episomally modify mammalian cells and tissues with a particular focus on liver tissue, as well as the brain, the muscle, the eye, cancer cells, embryonic cells and neonatal mice. For this purpose, recently published data in these areas (mainly articles published between 2000 and 2010) are reviewed.The utilisation of vectors harbouring an S/MAR element is an efficient, safe and cost-effective way to episomally modify mammalian cells.

The use of nanoparticles in biology, especially in cellular imaging, is extremely promising and offers numerous advantages over existing organic dye systems. There are, however, constraints that need to be addressed before the use of such materials in mainstream clinical applications can be realized. One of the main concerns is the use of metal-containing particles that are potentially toxic or interfere with other diagnostic processes. Here, we present the use of simple conjugated polymer nanoparticles as alternative photostable cellular optical imaging agents.

DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

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.

Establishing structure–activity relationships is vital if the efficacy of non-viral vectors is to match that of their viral counter-parts. Recently, we reported on the ability of a series of small molecule, cyclic polyamine disulfides to condense and cage plasmid DNA (pDNA) by a process of thermodynamically controlled templated polymerization, leading to a series of corresponding pDNA-polyplex nanoparticles able to mediate high levels of transfection with no associated cytotoxicities. The leading cyclic polyamine disulfide was shown to be the spermine tetra-amine disulfide ( TetraN-3,4,3 ). Herein we report on the significantly more challenging syntheses of cyclic disulfides with longer polyamine motifs. Two new cyclic polyamine disulfides, based on hexa- and octa-amine inserts, were prepared and their transfection efficacies and cytotoxicities compared with our previously reported cyclic tri- and tetra-amine disulfides. The new cyclic hexa- and octa-amine disulfides prove more effective at transfection in vitro , especially of lung epithelial A549 cell line. By contrast, our original cyclic tetra-amine disulfide remains the most efficient agent for the transfection of lung epithelial cells in vivo following intra-nasal administration. Hypothetical mechanistic reasons are presented to explain this outcome. Our data in toto support the concept of shorter cyclic polyamine disulfides as preferred agents for polycation-mediated controlled condensation and functional delivery of pDNA to lung epithelial cells in vivo .

Gene therapy vectors based on viruses are the most effective gene delivery systems in use today and although efficient at gene transfer their potential toxicity (Hacein-Bey-Abina et al., Science 302:415-419, 2003) provides impetus for the development of safer non-viral alternatives. An ideal vector for human gene therapy should deliver sustainable therapeutic levels of gene expression without affecting the viability of the host at either the cellular or somatic level. Vectors, which comprise entirely human elements, may provide the most suitable method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production, and great versatility. The development of more efficient, economically prepared, and safer gene delivery vectors is a crucial prerequisite for their successful clinical application and remains a primary strategic task of gene therapy research.

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.

Gene therapy vectors based on viruses are the most effective gene delivery systems in use today and although efficient at gene transfer their potential toxicity (Hacein-Bey-Abina et al., Science 302:415-419, 2003) provides impetus for the development of safer non-viral alternatives. An ideal vector for human gene therapy should deliver sustainable therapeutic levels of gene expression without affecting the viability of the host at either the cellular or somatic level. Vectors, which comprise entirely human elements, may provide the most suitable method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production, and great versatility. The development of more efficient, economically prepared, and safer gene delivery vectors is a crucial prerequisite for their successful clinical application and remains a primary strategic task of gene therapy research.

Nonviral gene therapy continues to require novel synthetic vectors to deliver therapeutic nucleic acids effectively and safely. The majority of synthetic nonviral vectors employed in clinical trials to date have been cationic liposomes; however, cationic polymers are attracting increasing attention. One of the few cationic polymers to enter clinical trials has been polyethylenimine (PEI); however, doubts remain over its cytotoxicity, and in addition it displays lower levels of transfection than viral systems. Herein, we report on the development of a series of small molecule analogues of PEI that are bioresponsive to the presence of pDNA, forming poly(disulfide)s that are capable of efficacious transfection with no associated toxicity. The most effective small molecule developed, a cyclic disulfide based upon a spermine backbone, is shown to form very well-defined polyplexes (100-200 nm in diameter) that mediate murine lung transfection in vivo to within an order of magnitude of in vivo jetPEI, and at the same time display a much improved cytotoxicity profile.

The ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.

Although gemcitabine has been proved to be a valuable ally against several types of cancer, it is characterized by some weaknesses that limit its anticancer potential. These limitations are mainly associated with gemcitabine rapid inactivation as well as the different types of resistance that are related with its use. We have previously shown that conjugation of gemcitabine to a ligand peptide (molecule GSG) enhanced its metabolic stability systemically and locally (within the cancer cell), and lead to improved efficacy in a castration resistant prostate cancer (CRPC) animal model. In order to refine our previous findings, a bioanalytical methodology based on mass spectrometry that allows the simultaneous monitoring of gemcitabine as well as its active (dFdCDP, dFdCTP) and inactive (dFdU) metabolites was developed in the context of this study. The balance of gemcitabine and its active/inactive metabolites was evaluated in in vitro and in vivo systems after treatment with gemcitabine or selected gemcitabine based peptide conjugates (GSG). Furthermore, since GSG's structure contains a potent agonist peptide of the GnRH-R we investigated whether it could have a central effect through the pituitary by performing testosterone measurements in animals dosed with GSG. Interestingly, GSG appears to offer metabolic advantages in comparison to gemcitabine, an important attribute if we had to overcome gemcitabine acquired resistance. In addition, GSG is a potent GnRH-R agonist based on our in vivo biomarker model, suggesting that testosterone ablation might also represent a key attribute of the mechanism of action for our conjugate of interest. These findings along with the targeted delivery nature of this approach could ameliorate the therapeutic status of gemcitabine and expand the use of gemcitabine based conjugates in other types of cancers. Citation Format: Constantin Tamvakopoulos, Theodoros Karampelas, Orestis Argyros. Gemcitabine based peptide conjugates: Overcoming the pitfalls of conventional therapies by targeted approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2015-2618

Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a novel non-viral episomal expression system which fulfils these criteria, in principle, was reported. This vector, pEPI, replicates episomally in CHO, HeLa, K562, as well as in primary human fibroblast-like cells, and is mitotically stable in the absence of selection for more than 100 generations. Here we show the long term expression of pEPI in a range of cells including cystic fibrosis airway (IB3) cells to be stable for at least 10 weeks. We demonstrate, however, that antibiotic selection pressure, is necessary for the first weeks following transfection. FACS sorting of CHO and IB3 cells, show that expression and maintenance in cells without initial antibiotic selection diminished rapidly, whereas constructs under selection pressure for an initial period of 2 weeks were mitotically stable and expressed transgene for at least 10 weeks, even following the removal of selection pressure. However, initial in vivo results showed no persistance of vector and limited expression of the reporter gene when using viral promoters. We describe the developments we have made to overcome these limitations and the results of the application of an S/MAR plasmid carrying a human promoter and chromosomal destabilising elements to drive the expression and episomal maintenance of these vectors in vivo.