L’adénovirus possède plusieurs caractéristiques faisant de ce virus un candidat de choix pour la construction de vecteurs utiles dans les études de génomique fonctionnelle. Dans la majorité de ces applications, on a recours à un vecteur adénoviral de première génération délété de sa région E1. L’utilisation de vecteurs adénoviraux comprend deux maillons faibles : la construction du vecteur et la production subséquente de ce dernier. Le développement de méthodes alternatives est donc nécessaire pour renforcer ces deux maillons, permettant ainsi une utilisation étendue de ces vecteurs. Ce développement va s’articuler sur deux axes : l’ingénierie du vecteur de transfert pour la construction de l’adénovirus recombinant et l’ingénierie d’une lignée cellulaire pour la production du vecteur. En utilisant un vecteur de transfert adénoviral co-exprimant, à partir d’un promoteur régulable à la tétracycline, la protéase de l’adénovirus et une protéine de fluorescence verte (GFP) par l’intermédiaire d’un site d’entrée ribosomal interne (IRES), notre groupe a établi que la sélection positive, via l’expression ectopique de la protéase, est un processus efficace pour la création de librairie d’adénovirus recombinants. Par contre, la diversité atteinte dans ce premier système est relativement faible, environ 1 adénovirus recombinant par 1 000 cellules. Le travail effectué dans le cadre de cette thèse vise à construire un nouveau transfert de vecteur dans lequel l’expression de la protéase sera indépendante de celle du transgène permettant ainsi d’optimiser l’expression de la protéase. Ce travail d’optimisation a permis de réduire le phénomène de transcomplémentation du virus parental ce qui a fait grimper la diversité à 1 virus recombinant par 75 cellules. Ce système a été mis à l’épreuve en générerant une librairie adénovirale antisens dirigée contre la GFP. La diversité de cette librairie a été suffisante pour sélectionner un antisens réduisant de 75% l’expression de la GFP. L’amplification de ce vecteur adénoviral de première génération doit se faire dans une lignée cellulaire exprimant la région E1 telle que les cellules 293. Par contre, un adénovirus de première génération se répliquant dans les cellules 293 peut échanger, par recombinaison homologue, son transgène avec la région E1 de la cellule créant ainsi un adénovirus recombinant réplicatif (RCA), compromettant ainsi la pureté des stocks. Notre groupe a déjà breveté une lignée cellulaire A549 (BMAdE1) exprimant la région E1, mais qui ne peut pas recombiner avec le transgène du virus. Par contre, le niveau de réplication de l’adénovirus dans les BMAdE1 est sous-optimal, à peine 15-30% du niveau obtenu dans les cellules 293. Le travail fait dans le cadre de cette thèse a permis de mettre en évidence qu’une expression insuffisante d’E1B-55K était responsable de la mauvaise réplication du virus dans les BMAdE1. Nous avons produit de nouveaux clones à partir de la lignée parentale via une transduction avec un vecteur lentiviral exprimant E1B-55K. Nous avons confirmé que certains clones exprimaient une plus grande quantité d’E1B-55K et que ces clones amplifiaient de manière plus efficace un vecteur adénoviral de première génération. Ce clone a par la suite été adapté à la culture en suspension sans sérum. / The adenovirus has numerous interesting characteristics making this particular virus an ideal candidate for the construction of vector for conducting studies in functional genomics. The vast majority of those applications rely on a so-called “first-generation vector” in which the E1 region is replaced by a transgene. Despite all their advantages, there are 2 weak links associated with first-generation vector: the efficient construction of the actual vector and its production. Therefore, the development of alternative methods for construction and production is necessary to ensure their usefulness. The development will involve 2 axes: the reengineering of the transfer vector for the construction of recombinant adenovirus and the reengineering of the cell line capable of producing the vector. Using a transfer vector co-expressing the adenoviral protease (PS) gene and GFP by using an IRES under the control of a tetracycline-regulated promoter, our laboratory previously established the proof of concept that positive selection of recombinant adenovirus through ectopic expression of the PS gene was an efficient approach to generate adenoviral libraries. However, the diversity achieved was quite low, around 1 recombinant adenovirus per 1,000 cells. The goal of this thesis was to design a new transfer vector in which the PS expression was independent from the expression of the transgene in order to be able to optimize its expression independently. We also improved library diversity by lowering the amount of PS in order to reduce the the trans-complementation from the transfer vector. Using this method, at least 1 recombinant adenovirus per 75 cells was generated with 100% of the plaques being recombinant. This system was successfully used to generate an antisense library targeting GFP. The diversity of the library was high enough to allow the selection of an antisense that inhibited 75% of GFP expression. Amplification of those first-generation recombinant adenoviruses must take place in an E1-expressing cell such as 293 cells. However, when replicating in 293 cells, the recombinant adenovirus can exchange their transgene with the E1 region of the cell by homologous recombination, which results in the generation of a fully replicative adenovirus (RCA), a situation that compromises the purity of the viral preparation. Our laboratory has previously patented an A549 cell line expressing the E1 region and producing RCA-free recombinant adenovirus (BMAdE1). However, the replication of E1-deleted adenovirus in BMAdE1 cells was sub-optimal, in the range of 15-30% the level obtained in 293 cells. The work done in this thesis establishes that the low level of E1B-55K could be responsible for the lower productivity of BMAdE1 cells. Thus, we have derived new clones following lentiviral transduction in order to increase E1B-55K expression. Western blot confirmed that some clones expressed more E1B-55K than BMAdE1, and this correlated with a more robust replication of a recombinant adenovirus in those clones. This newly optimized BMAdE1 cell line was adapted to serum-free suspension culture.

Els tractaments actuals pel càncer de pàncreas presenten un eficàcia limitada de manera que es necessari el desenvolupament de noves teràpies antitumorals. La teràpia gènica pel càncer de pàncreas basada en l’ús d’adenovirus es troba limitada per la baixa capacitat dels virus d’arribar a les masses tumoral, de distribuir-se pel tumor i d’infectar les cèl·lules tumorals. Nosaltres hem observat que l’administració intraductal d’adenovirus al ducte biliar de ratolins Ela-myc permet arribar als tumors pancreàtics de manera més eficient que per la via sistèmica. A més a més permet transduir la majoria de la massa tumoral restringint l’expressió adenoviral al teixit pancreàtic. D’altre banda, l’administració intraductal del tractament AduPARTat8TK/GCV retarda significativament el creixement tumoral i disminueix la toxicitat associada al tumor. El nou adenovirus AdTATMMP és activat per les MMP2/9 restaurant la capacitat de transducció de l’AdYTGRE in vitro, i incrementant 7,3 vegades la infecció del tumor pancreàtic. El tractament combinat de l’AduPARTat8TK/GCV amb gemcitabina presenta un efecte sinèrgic in vitro, però no millora la eficàcia antitumoral de les teràpies simples. D’altre banda el tractament de l’electroporació irreversible presenta efectes antitumorals significatius en tumors ortotòpics de la línia cel·lular BxPC-3-Luc i allarga la supervivència dels ratolins provocant una toxicitat mínima. / Novel therapies are needed to overcome the limited efficacy of current treatments in pancreatic cancer. Adenoviral gene therapy against pancreatic tumors is challenged by the limitation of viruses to reach the tumor mass, poorly distribute within the tumor and inefficiently transduce tumor cells. We show that intraductal administration of adenoviruses into the common bile duct of Ela-myc mice targets pancreatic tumors more efficiently than systemic delivery with relevant transduction of the bulk of the tumor and restricts expression to pancreatic tissue. Moreover, intraductal administration of AduPARTat8TK/GCV treatment significantly delayed tumor growth ameliorating tumor-associated toxicity. Noticeable the new generated MMP-activatable adenovirus AdTATMMP was susceptible to MMP2/9 activation, restored the transduction capacity of AdYTRGE in vitro, and increased 7.3 times tumor pancreas transduction. The multimodal treatment AduPARTat8TK/GCV and gemcitabine showed synergistic effects in vitro; however, did not enhance the antitumoral efficacy of single therapies. Interestingly, IRE treatment exhibited significant antitumor effects in BxPC-3-Luc orthotopic tumors and prolonged mice survival with minimal toxicity.

RNA splicing is a process where introns are removed and exons are joined together. Human adenovirus type 2 pre-mRNAs undergoes intensive alternative splicing and produce more than 40 differently spliced mRNAs.  This thesis work is focused on the identification of new splice sites in adenovirus. By virtue of Illumina mRNA sequencing technology we have identified 255 splice sites. Splice site analysis of the introns revealed the presence of three types of splice sites GT-AG (61.2%), GC-AG (25.9%) and AT-AC (12.9%). Among 255 splice sites, 224 were new. Significantly, more than 50% of the new splice sites were located in the major late transcription unit on the positive strand of adenovirus DNA. Three new splice sites; 17452-29489 (GC-AG) located on the negative strand of adenovirus DNA in the E2 region, 9668-20346 (AT-AC) and 9699-30505 (GC-AG) on the positive strand of adenovirus DNA in the major late transcription unit were further confirmed by PCR analysis. / Adenovirus replication and transcriptome

A presença de Ad com competência para replicar (RCA, replication-competent adenovirus) nas preparações é um dos maiores problemas para a produção de Ad em larga escala. RCAs são gerados pela recombinação entre seqüência do vetor e seqüência homóloga do gene E1 presente nas células helper. Objetivo: desenvolver uma nova linhagem auxiliar para produção de Ad livre de RCA - LEY79 - derivada da linhagem de retinoblastoma humano Y79, tratando-se da primeira linhagem empacotadora de adenovírus com inativação mutacional da proteína supressora de tumor pRb, que crescem em suspensão. Células Y79 foram infectadas com o retrovírus pCLDE1A/E1BSN, selecionadas com G418. A eficiência de produção de AdeGFP na linhagem LEY79 foi testada e comparada com a HEK293A. Células Y79 foram adaptadas em meio livre de soro. Esperamos com a linhagem LEY79SF inovar no campo de processos para a produção de Ad recombinante. / The presence of Ad with the ability to replicate (RCA, replication-competent adenovirus) in preparations is a major problem in the large-scale production of Ad. RCAs are generated by recombination between the vector sequence and sequence of the homologous gene in E1 helper cells. Objective: To develop a new helper cell line for the production of RCA-free Ad., called LEY79, derived from the Y79 of human retinoblastoma line, the first line Packer adenovirus with mutational inactivation of the tumor suppressor protein pRb, which are adapted to grow in suspension. Y79 cells were infected with the retrovirus pCLDE1A/E1BSN, selected with G418. The efficiency of production of AdeGFP in the LEY79 was tested and compared with the HEK293A. Y79 cells were adapted to grow in serum-free medium. We hope that use of the the LEY79SF cell line will promote innovation in the processing and production of recombinant Ad.

No description available.

Since their discovery in 1953, adenoviruses have significantly contributed to the understanding of virus-host cell interactions, including mechanistic details of cellular processes such as cell cycle control and alternative RNA splicing. Among the first characterized adenoviral genes were the virus-associated RNAs (VA RNAI/II), which are produced in massive amount during a lytic infection. The VA RNAs perform multiple functions and are required for a successful adenovirus life cycle. More recently, it was shown that the VA RNAs are processed into small viral miRNAs, so-called mivaRNAs, which interfere with the function of the cellular RNAi/miRNA machinery. In papers I and II, we focused on a structural and functional characterization of the mivaRNAs using two approaches. Firstly, we created a model system where the predicted miRNA-like function of mivaRNAI could be investigated, without interfering with other VA RNA functions. This was accomplished by construction of recombinant adenoviruses, in which the seed sequence of mivaRNAI was altered. The results showed that in cell culture experiments the mivaRNAI seed sequence mutants grew as the wild type virus, suggesting that the mivaRNAs are not required during the lytic phase of an adenovirus infection. Secondly, we showed that the VA RNAs from different human adenoviruses (Ad4, Ad5, Ad11 and Ad37) undergo the same type of Dicer-dependent processing into mivaRNAs, which subsequently are loaded onto the RNA induced silencing complex (RISC), albeit with different efficiencies. In paper III, we demonstrated that the promoter proximal region of the adenovirus major late promoter (MLP) produces a novel non-canonical class of small RNAs, which we termed the MLP-TSS-sRNAs. Surprisingly the MLP-TSS-sRNA maintains the m7G-cap structure while bound to Ago2 containing RISC. These complexes are functional suppressing expression of target mRNAs with complementary binding site. Most importantly, the MLP-TSS-sRNA limits the efficiency of viral DNA replication probably through a targeting of the E2B mRNAs, which are transcribed in the antisense orientation. In conclusion, the MLP-TSS-sRNA represents the first viral small RNA, which has been shown to have a function as a regulator of an adenovirus infection.

Η επιπεφυκίτιδα (φλεγμονή του επιπεφυκότα) είναι η πιο συχνή ασθένεια των οφθαλμών, η οποία εκδηλώνεται σε παγκόσμια κλίμακα με τη μορφή σποραδικών κρουσμάτων ή επιδημίας. Μπορεί να είναι λοιμώδους (βακτήρια, ιοί, παράσιτα) ή μη λοιμώδους αιτιολογίας. Η κύρια αιτία της οξείας ιογενούς αιτιολογίας επιπεφυκίτιδας είναι οι αδενοϊοί. Περίπου το 15- 70% του συνόλου των κρουσμάτων της επιπεφυκίτιδας οφείλονται στους αδενοϊούς. Σκοπός της μελέτης είναι η χρήση επιδημιολογικών δεδομένων προκειμένου να πραγματοποιηθεί επιδημιολογική παρακολούθηση των κρουσμάτων επιπεφυκίτιδας στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών (ΠΓΝΠ) από ασθενείς οι οποίοι επισκέφθηκαν την οφθαλμιατρική κλινική και τα εξωτερικά ιατρεία του νοσοκομείου τη χρονική περίοδο 2 Ιανουαρίου – 29 Ιουλίου 2012 (εβδομάδες 1- 30), ο καθορισμός της συχνότητας της ιογενούς αιτιολογίας επιπεφυκίτιδας και ο εντοπισμός πιθανής ύπαρξης επιδημίας. Ταυτόχρονα, πραγματοποιήθηκε μοριακή ανίχνευση και τυποποίηση αδενοϊών από ασθενείς με κλινική εικόνα ιογενούς επιπεφυκίτιδας το χρονικό διάστημα μεταξύ 27 Φεβρουαρίου και 17 Ιουνίου. Όλα τα κρούσματα καταγράφηκαν από τα ιατρικά αρχεία του ΠΓΝΠ για το χρονικό διάστημα Ιανουαρίου- Ιουλίου του 2012 και για το ίδιο χρονικό διάστημα το προηγούμενο έτος (2011). Καταγράφηκαν 231 κρούσματα επιπεφυκίτιδας (47,1% άνδρες και 52,8% γυναίκες), από τα οποία τα 205 ήταν ιογενούς αιτιολογίας, τα 4 βακτηριογενούς αιτιολογίας και 22 ήταν απροσδιόριστης αιτιολογίας από τους ιατρούς. Για την ίδια χρονική περίοδο το προηγούμενο έτος (2011), σύμφωνα με τα αρχεία του ΠΓΝΠ καταγράφηκε ένα σύνολο από 156 κρούσματα επιπεφυκίδας (38,5% άνδρες και 61,5% γυναίκες), από τα οποία τα 135 ήταν ιογενούς αιτιολογίας, τα 3 βακτηριογενούς αιτιολογίας και 18 ήταν απροσδιόριστης αιτιολογίας. Ο αριθμός κρουσμάτων επιπεφυκίτιδας τους δυο πρώτους μήνες καθώς και τον Ιούλιο του 2012 ήταν στα ίδια επίπεδα με τους αντίστοιχους μήνες το 2011 και παρατηρείται επιδημία που πραγματοποιήθηκε μεταξύ Μαρτίου- Ιουνίου 2012. Οι ασθενείς κατανέμονταν σε όλες τις ηλικίες και στα δυο φύλα. Το χρονικό διάστημα μεταξύ 27 Φλεβάρη- 17 Ιουνίου του 2012 (εβδομάδες 9- 24), 48 επιχρίσματα επιπεφυκότα ασθενών με κλινική εικόνα αδενικής επιπεφυκίτιδας συλλέχθηκαν από τους ιατρούς του ΠΓΠΝ και μεταφέρθηκαν υπό κατάλληλες συνθήκες στο εργαστήριο Υγιεινής της Ιατρικής Σχολής του Πανεπιστημίου Πατρών. Ταυτόχρονα συμπληρώθηκε από τους ιδίους ερωτηματολόγιο με δημογραφικά και κλινικά στοιχεία. Το DNA του ιού απομονώθηκε με Qiagen και ενισχύθηκε με nested PCR. Τα θετικά αποτελέσματα επιβεβαιώθηκαν με αλληλούχιση του PCR προϊόντος. Για τον προσδιορισμό της συγγένειας μεταξύ των διαφόρων απομονωμένων αλληλουχιών του DNA, φυλογενετική ανάλυση πραγματοποιήθηκε. Από το σύνολο των δειγμάτων που αναλύθηκαν με μοριακές τεχνικές, DNA αδενοϊού ανιχνεύθηκε σε 40 δείγματα (83%). Στα σαράντα θετικά δείγματα καθορίστηκε η αλληλουχία του DNA τους, από τα οποία τα 29 (72,5%) προσδιορίστηκαν ως τύπος HAdV17 και τα 5 (12,5%) ως τύπος HAdV-54. Σε 6 θετικά δείγματα (15%) ο τύπος του ιού δεν προσδιορίστηκε. Τέλος, με τη βοήθεια των μοριακών τεχνικών προκύπτει το συμπέρασμα ότι το στέλεχος αδενοϊού 17 (Αd 17) είναι η αιτία της εμφάνισης επιδημίας μεταξύ Μαρτίου- Ιουνίου 2012. Η έρευνα αυτή είναι από τις λίγες πιυ έχουν πραγματοποιηθεί στον Ελλαδικό χώρο σε κρούσματα επιπεφυκίτιδας με αποτέλεσμα να εμπλουτίζει τα φτωχά επιδημιολογικά και μοριακά δεδομένα για την συγκεκριμένη ασθένεια και το συγκεκριμένο τύπο ιών. Παράλληλα μέσω της έρευνας υπογραμμίζεται η ανάγκη για εθνικό σύστημα επιτήρησης της επιπεφυκίτιδας. / Conjunctivitis (inflammation of the conjunctiva) is the most common eye disease that occurs worldwide in both sporadic and epidemic form. There are infectious conjunctivitis, which is caused by a variety of microorganisms (such as bacteria, viruses and parasites) and noninfectious conjunctivitis, which is caused by an allergic reaction. The leading cause of acute viral conjunctivitis in clinical practice includes human adenoviruses (HAdVs). About 15- 70% of all conjunctivitis cases worldwide are associated with AdVs. The aim of the study is the performance of epidemiological surveillance of cases of conjunctivitis using epidemiological data from patients who visited the ophthalmic clinic and the outpatient ophthalmic department of the University General Hospital of Patras (UGHP) in the period from January 2nd to July 29th in 2011 and 2012 (weeks 1st-30th), in order to determine the frequency of viral conjunctivitis and to determine a potential epidemic. An additional task of the study is the molecular detection and typing of adenoviruses for cases of patients with clinical viral conjunctivitis in the period from February 27th to June 17th 2012. All conjunctivitis cases referred to UGHP in the period between January and July 2012 as well as between January and July 2011 were ascertained using medical records. 231 conjunctivitis cases were reported (47.1% male and 52.8% female), in which 205 were virological conjunctivitis, 4 bacteriological conjunctivitis and 22 were undefined conjunctivitis. For the same period the previous year (2011), according to the records of UGHP recorded a total of 156 conjunctivitis cases (38.5% male and 61.5% female), of which 135 were of viral origin, 3 bacteriogenic orogin and 18 were undetermined etiology. The number of conjunctivitis cases in the first two months and in July 2012 was at the same level as the corresponding period in 2011 and there is an epidemic that took place between March and June 2012. Patients were allocated to all age groups and both sexes. In the period from February 27th ,2012 to June 17th , 2012 (weeks 9th – 24th), 48 conjunctival swabs were collected from cases which were clinically suspected of having adenoviral conjunctivitis and transported under appropriate conditions to the laboratory of Hygiene, Medical School, University of Patras. At the same time, the patients were asked to answer a structured questionnaire with demographic and clinical data. The viral DNA was isolated with Qiagen and amplified by nested PCR. The positive results were confirmed by sequencing the PCR product. To determine the relatedness between the different isolated sequences, a phylogenetic analysis was constructed. Of the total samples, which were analyzed with molecular techniques, adenovirus DNA was detected in 40 samples (83%). Of the positive samples which were confirmed by sequencing, 29 samples (72.5%) were typed as AdV17 and 5 samples (12.5%) as AdV54. For 6 positive samples (15%) the serotype was not determined. Finally, it was concluded that the strain Adenovirus 17 (Ad 17) was the cause of the epidemic between March and June 2012. There are poor epidemiological and molecular data for this particular disease in Greece. This study is one of the very few on conjunctivitis determination in Greece. This research underscores the need for a national surveillance system for conjunctivis outbreaks.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

The impact of Ad.mda-7 on the survival of renal cell carcinoma lines (RCC), primary renal epithelial cells, glioblastoma multiforme lines (GBM), and primary rodent astrocytes is unknown. The present studies examine whether the GST fusion protein, GST-MDA-7, and the adenovirus, Ad.mda-7, altered the growth and survival of the A498 and UOK121N RCC lines or radiosensitized GBM, respectively. Due to previous findings that the RCC lines, but not primary renal epithelial cells, were resistant to type 5 adenoviral infection, we used purified GST-MDA-7 protein to show that GST-MDA-7, but not GST, caused a dose-dependent reduction in A498 and UOK121N proliferation but not that of primary renal epithelial cells. Free radical species, generated by clinically relevant concentrations of arsenic trioxide, synergized with subnanomolar concentrations of GST-MDA-7 to inhibit the proliferation, viability, and long-term survival of RCC. We also found that MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, exerted anti-proliferative effects on GBM cells, an effect found to be enhanced in a greater than additive fashion when combined with ionizing radiation. These findings argue that MDA-7, in combination with agents that generate free radicals, such as arsenic trioxide and ionizing radiation, may have potential in the treatment of RCC and GBM. Geldanamycins are currently being used in a number of clinical trials in different tumor cell types, such as hepatocellular carcinoma (HCC), targeting the inhibition of the heat shock protein and molecular chaperone Hsp90. Previous studies have demonstrated that geldanamycins have dose limiting toxicity in vivo due to their actions in promoting normal liver dysfunction. These studies show that the geldanamycin derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG), interacts with the secondary bile acid, deoxycholic acid (DCA), to kill primary rat hepatocytes and HuH7 human hepatoma cells. An effect abolished by the addition of the ROS-quenchers, NAC and Trolox. Collectively, these findings argue that geldanamycins may not be a viable therapy for HCC treatment and that 17-AAG toxicity in primary hepatocytes may be, at least upon initial drug exposure, due to ROS generation and mechanisms independent of Hsp9O inhibition and the down-regulation of "classical" Hsp90 client proteins.