Successful large gene augmentation of USH2A with non-viral episomal vectors.

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Birt-Hogg-Dube syndrome is a novel ciliopathy.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

UNLABELLED: Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose-dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas. CONCLUSION: This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals.

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Libechov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

NLGN4X TCR transgenic T cells to treat gliomas.

BACKGROUND: Neuroligin 4 X-linked (NLGN4X) harbors a human leukocyte antigen (HLA)-A*02-restricted tumor-associated antigen, overexpressed in human gliomas, that was found to induce specific cytotoxic T cell responses following multi-peptide vaccination in patients with newly diagnosed glioblastoma. METHODS: T cell receptor (TCR) discovery was performed using droplet-based single-cell TCR sequencing of NLGN4X-tetramer-sorted T cells postvaccination. The identified TCR was delivered to Jurkat T cells and primary human T cells (NLGN4X-TCR-T). Functional profiling of NLGN4X-TCR-T was performed by flow cytometry and cytotoxicity assays. Therapeutic efficacy of intracerebroventricular NLGN4X-TCR-T was assessed in NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice bearing NLGN4X-expressing experimental gliomas. RESULTS: An HLA-A*02-restricted vaccine-induced T cell receptor specifically binding NLGN4X131-139 was applied for preclinical therapeutic use. Reactivity, cytotoxicity, and polyfunctionality of this NLGN4X-specific TCR are demonstrated in various cellular models. Intracerebroventricular administration of NLGN4X-TCR-T prolongs survival and leads to an objective response rate of 44.4% in experimental glioma-bearing NSG MHC I/II KO mice compared to 0.0% in control groups. CONCLUSION: NLGN4X-TCR-T demonstrate efficacy in a preclinical glioblastoma model. On a global scale, we provide the first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.Keywords cell therapy | glioblastoma | T cell receptor | tumor antigen.

pH-triggered nanoparticle mediated delivery of siRNA to liver cells in vitro and in vivo.

Recently, we reported for the first time the development of pH-triggered nanoparticles for the functional delivery of small interfering RNA (siRNA) to liver for treatment of hepatitis B virus infections in vivo. Here, we report on systematic formulation and biophysical studies of three different pH-triggered nanoparticle formulations looking for ways to improve on the capabilities of our previous nanoparticle system. We demonstrate how pH-triggered, PEGylated siRNA nanoparticles stable with respect to aggregation in 80% serum can still release siRNA payload at pH 5.5 within 30 min. This capability allows functional delivery to cultured murine hepatocyte cells in vitro, despite a high degree of PEGylation (5 mol %). We also demonstrate that pH-triggered, PEGylated siRNA nanoparticles typically enter cells by clathrin-coated pit endocytosis, but functional delivery requires membrane fusion events (fusogenicity). Biodistribution studies indicate that >70% of our administered nanoparticles are found in liver hepatocytes, post intravenous administration. Pharmacodynamic experiments show siRNA delivery to murine liver effecting maximum knockdown 48 h post administration from a single dose, while control (nontriggered) nanoparticles require 96 h and two doses to demonstrate the same effect. We also describe an anti-hepatitis C virus (HCV) proof-of-concept experiment indicating the possibility of RNAi therapy for HCV infections using pH-triggered, PEGylated siRNA nanoparticles.

Development of an Orthotopic HPV16-Dependent Base of Tongue Tumor Model in MHC-Humanized Mice.

Head and neck squamous cell carcinomas (HNSCC) caused by infections with high-risk human papillomaviruses (HPV) are responsible for an increasing number of head and neck cancers, particularly in the oropharynx. Despite the significant biological differences between HPV-driven and HPV-negative HNSCC, treatment strategies are similar and not HPV targeted. HPV-driven HNSCC are known to be more sensitive to treatment, particularly to radiotherapy, which is at least partially due to HPV-induced immunogenicity. The development of novel therapeutic strategies that are specific for HPV-driven cancers requires tumor models that reflect as closely as possible the characteristics and complexity of human tumors and their response to treatment. Current HPV-positive cancer models lack one or more hallmarks of their human counterpart. This study presents the development of a new HPV16 oncoprotein-dependent tumor model in MHC-humanized mice, modeling the major biologic features of HPV-driven tumors and presenting HLA-A2-restricted HPV16 epitopes. Furthermore, this model was developed to be orthotopic (base of tongue). Thus, it also reflects the correct tumor microenvironment of HPV-driven HNSCC. The cancer cells are implanted in a manner that allows the exact control of the anatomical location of the developing tumor, thereby homogenizing tumor growth. In conclusion, the new model is suited to study HPV16-specific therapeutic vaccinations and other immunotherapies, as well as tumor-targeted interventions, such as surgery or radiotherapy, or a combination of all these modalities.

Corneal gene delivery: chitosan oligomer as a carrier of CpG rich, CpG free or S/MAR plasmid DNA.

BACKGROUND: Corneal gene therapy can potentially treat acquired and inherited corneal disorders that otherwise lead to blindness. In a previous study on the development of effective vectors for corneal gene delivery, we showed that a particular formulation of chitosan-DNA nanoparticles, based on ultrapure chitosan oligomers injected into rat corneas, led to transgene expression that was 5.4-fold higher than that obtained using polyethylenimine-DNA nanoparticles. METHODS: In the present study, we investigate the same formulation of chitosan-DNA nanoparticles as carriers of six different plasmids for corneal gene delivery. Size, zeta potential, the ability to condense plasmid DNA, and transfection efficiency in cell cultures and in rat corneas, were all investigated. RESULTS: Size, zeta potential, the ability to condense plasmid DNA, and transfection efficiency in cell cultures did not substantially vary for nanoparticles based on different plasmids. One day post-injection of nanoparticles into rat corneas, we found that a CpG-free plasmid DNA, pCpG-Luc, which has an EF1α promoter, led to transgene expression that was 7.1-fold higher than that for gWiz-Luc, a commercially available plasmid DNA with a cytomegalovirus (CMV) promoter used in our previous study; 116.8-fold higher than that for pEPI-CMV, a commercially available plasmid that has a scaffold/matrix attachment region (S/MAR) sequence and a CMV promoter; and 76.8-fold higher than that for pEPI-UbC, an experimental plasmid that has an S/MAR sequence and a ubiquitin C promoter. CONCLUSIONS: The present study reveals the potential of comparing various plasmids as an approach for enhancing transgene expression. The delivery system designed in the present study represents the next step in the development of effective vectors for corneal gene therapy.

Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs.

Disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk invoked at perivascular sites is still rudimentary. Here, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in the lung. We show that specific secreted factors, induced in metastasis-associated endothelial cells (ECs), promote metastasis in mice by enhancing stem cell properties and the viability of cancer cells. Perivascular macrophages, activated via tenascin C (TNC) stimulation of Toll-like receptor 4 (TLR4), were shown to be crucial in niche activation by secreting nitric oxide (NO) and tumor necrosis factor (TNF) to induce EC-mediated production of niche components. Notably, this mechanism was independent of vascular endothelial growth factor (VEGF), a key regulator of EC behavior and angiogenesis. However, targeting both macrophage-mediated vascular niche activation and VEGF-regulated angiogenesis resulted in added potency to curb lung metastasis in mice. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.

Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection.

Hepatic gene therapy by delivering non-integrating therapeutic vectors in newborns remains challenging due to the risk of dilution and loss of efficacy in the growing liver. Previously we reported on hepatocyte transfection in piglets by intraportal injection of naked DNA vectors. Here, we established delivery of naked DNA vectors to target periportal hepatocytes in weaned pigs by hydrodynamic retrograde intrabiliary injection (HRII). The surgical procedure involved laparotomy and transient isolation of the liver. For vector delivery, a catheter was placed within the common bile duct by enterotomy. Under optimal conditions, no histological abnormalities were observed in liver tissue upon pressurized injections. The transfection of hepatocytes in all tested liver samples was observed with vectors expressing luciferase from a liver-specific promoter. However, vector copy number and luciferase expression were low compared to hydrodynamic intraportal injection. A 10-fold higher number of vector genomes and luciferase expression was observed in pigs using a non-integrating naked DNA vector with the potential for replication. In summary, the HRII application was less efficient (i.e., lower luciferase activity and vector copy numbers) than the intraportal delivery method but was significantly less distressful for the piglets and has the potential for injection (or re-injection) of vector DNA by endoscopic retrograde cholangiopancreatography.

An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny.

The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.

Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors.

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells.

The compelling need to provide adoptive cell therapy (ACT) to an increasing number of oncology patients within a meaningful therapeutic window makes the development of an efficient, fast, versatile, and safe genetic tool for creating recombinant T cells indispensable. In this study, we used nonintegrating minimally sized DNA vectors with an enhanced capability of generating genetically modified cells, and we demonstrate that they can be efficiently used to engineer human T lymphocytes. This vector platform contains no viral components and is capable of replicating extrachromosomally in the nucleus of dividing cells, providing persistent transgene expression in human T cells without affecting their behavior and molecular integrity. We use this technology to provide a manufacturing protocol to quickly generate chimeric antigen receptor (CAR)-T cells at clinical scale in a closed system and demonstrate their enhanced anti-tumor activity in vitro and in vivo in comparison to previously described integrating vectors.

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

BACKGROUND: The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP) and directed into a 2000 bp long matrix attachment region sequence (MARS) derived from the human beta-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. RESULTS: 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 in vitro and in vivo. 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 in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element that is known to be less affected by epigenetic silencing events. CONCLUSIONS: The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae.

Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.

Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models.

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

The expression of genes contributing to pancreatic adenocarcinoma progression is influenced by the respective environment.

Pancreatic adenocarcinoma is a highly aggressive malignancy with dismal prognosis and limited curative options. We investigated the influence of organ environments on gene expression in RNU rats by orthotopic and intraportal infusion of Suit2-007luc cells into the pancreas, liver and lung respectively. Tumor tissues from these sites were analyzed by chip array and histopathology. Generated data was analyzed by Chipster and Ingenuity Pathway Analysis (±1.5 expression fold change and p<0.05). Further analysis of functional annotations derived from IPA, was based on selected genes with significant modulation of expression. Comparison of groups was performed by creating ratios from the mean expression values derived from pancreas and respective in vitro values, whereas those from liver and lung were related to pancreas, respectively. Genes of interest from three functional annotations for respective organs were identified by exclusion-overlap analyses. From the resulting six genes, transglutaminase2 (TGM2) was further investigated by various assays. Its knockdown with siRNA induced dose dependent inhibitory and stimulatory effects on cell proliferation and cell migration, respectively. DNA fragmentation indicated apoptotic cell death in response to TGM2 knockdown. Cell cycle analysis by FACS showed that TGM2 knockdown induced G1/S blockade. Therefore, TGM2 and its associated genes may be promising therapeutic targets.