Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors

Peer reviewe

Dual Roles of Interferon Stimulated Gene-15 During Respiratory Virus Infection

ISG15 is a diubiquitin-like posttranslational modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins have been identified as targets of ISG15 conjugation after interferon stimulation, and a number of viral proteins have been shown to be modified by ISG15 during infection. Given the known importance of posttranslational protein modification in the regulation of cellular biology, understanding how ISG15 affects the interferon response and virus replication has been of considerable interest. ISG15 deficient mice have previously been shown to exhibit increased susceptibility to a number of different viruses, including influenza A and influenza B viruses. In these studies we set out to characterize how ISG15 mediates protection against respiratory virus infections. ISG15 conjugation has been shown to inhibit influenza B virus propagation in vivo, however the mechanism by which it reduces viral loads has not been determined. Here we show that ISG15 can inhibit virus replication in a primary trachea epithelial cell culture, suggesting that ISG15 can directly antagonize some step of the virus lifecycle. Furthermore, increasing ISG15 conjugation through inhibition of the ISG15 deconjugating enzyme, UBP43, promotes increased antiviral activity against influenza B virus. Interestingly, increasing the pool of ISG15 conjugates in this manner results in an upregulation of interferon stimulated gene expression, demonstrating a previously undescribed role of ISG15 deconjugation in dampening the interferon response. We found that ISG15 also protects mice from influenza A virus and Sendai virus induced lethality by a conjugation dependent mechanism. However, in these infection models, ISG15 conjugation had little effect on virus burden in vivo or virus replication in tissue culture. We also did not observe any major differences in the acute immune response of ISG15 deficient mice after infection. These results demonstrate a novel non-antiviral role for ISG15 conjugation in promoting survival after viral infection

Nonsegmented negative strand RNA viruses : viral RNA cap methylation and potential applications as an anticancer therapy

The viruses of the order Mononegavirales include important human, animal, and plant pathogens and additionally, have great potential as vaccine, oncolytic and gene therapy vectors. This dissertation focuses on two prototypic Mononegavirales, vesicular stomatitis virus (VSV) and Sendai virus (SeV), their virus-encoded cap methylation function, and potential applications as an anticancer therapy. The L protein of Mononegavirales has six conserved domains postulated to constitute the specific enzymatic activities of this multifunctional protein. We conducted a comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four infectious L mutants. Our analysis identified several residues required for successful cap methylation and virus replication. This study confirms structural and functional similarity of this domain across different families of the order Mononegavirales. Additionally, the oncolytic potential of VSV was analyzed for the first time in a panel of human pancreatic ductal adenocarcinoma (PDA) cell lines and compared to other oncolytic viruses. VSV showed superior oncolytic abilities; however, cells were heterogeneous in their susceptibility to virus-induced oncolysis and several cell lines were resistant to all tested viruses. Four cell lines that varied in their permissiveness to VSV were tested in mice, and in vivo results closely mimicked those in vitro. While our results demonstrate VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance to oncolytic virotherapy

Improving our understanding of autosomal dominant Retinitis Pigmentosa using PRPF31 patient-specific induced pluripotent stem cells (iPSCs)

Ph. D. Thesis.Retinitis pigmentosa (RP) is a genetic condition in which degeneration of photoreceptors, especially rods, gradually leads to visual loss. Patients with RP present symptoms such as dark adaptation or "night blindness," followed by “tunnel vision” and loss of central vision later on in the disease. PRPF31, a widely expressed splicing factor gene causative of RP, encodes a component of the U4/U6.U5 small trinuclear ribonucleic protein (tri-snRNP) complex, which is a constituent of the premRNA processing spliceosome. Mechanisms correlating mutations in splicing factors and retina-specific cell death are still poorly understood. To address this, induced pluripotent stem cells (iPSCs), which can differentiate into any cell types of the three germ layers and are capable of self-renewal, were used to generate patient-specific retinal pigment epithelium (RPE) models to investigate the pathogenesis of the PRPF31 form of RP. PRPF31-RPE patient-derived cells presented defects that impaired normal RPE structure and function, including disrupted apical-basal polarity, reduced transepithelial resistance and phagocytic capacity. Transcriptome profiles from PRPF31- RPE and other cell types revealed that disrupted alternative splicing was more pronounced in the RPE splicing programme. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific RPE and retinal cells. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with decreased cilia length and incidence, which in turn may have contributed with the severe RPE defects described above. In situ gene correction of a PRPF31 mutation rescued protein expression and key cellular phenotypes in RPE, providing proof-of-concept for future therapeutic strategies. In summary, the results generated by this study highlighted the applicability and importance of patient-specific iPSCs in disease modeling, unraveling more of the underlying molecular and cellular mechanisms responsible for causing RP

Abstracts from the 25th Fungal Genetics Conference

Abstracts from the 25th Fungal Genetics Conferenc

Estudio comparativo del splicing alternativo del gen NCR3 en diferentes especies de mamíferos y sus posibles implicaciones funcionales

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-12-2013Alternative splicing (AS) is a major source of transcriptome and proteome diversity in higher eukaryotes allowing the generation of several structurally and functionally distinct mRNAs and protein isoforms from a single gene. Differential AS variants expression has been implicated in tissue and cellular differentiation, which in an evolutionary context could account for the phenotypic divergence of mammals that share a common repertoire of genes. On the other hand, the miss-regulation of AS is one of the mayor sources of human disease. However, there are not many detailed comparative analyses showing the alternative splice forms generated from particular genes among different organisms. The work presented here is a deep study of the splice variants expressed by the “Natural Cytotoxicity Receptor 3” (NCR3) gene and its comparative analysis among 13 mammals. NCR3 is a member of the NCR family, which represents the major NK cells triggering receptors. It has been involved in the recognition and killing of tumoral and infected cells, and in the maturation of dendritic cells. By using a combination of nested RT-PCR and RNA-seq analysis, it was possible to detect the expression of several NCR3 transcripts in all the analysed mammalian species, except in Mus musculus, where NCR3 is a pseudogen. It was observed an increase in the number of coding variants in primates, mainly by the internal splicing of exon 2 and the presence of exons 4II and 4III, which are only expressed in higher primates (Hominoidea). In contrast, the diversity of non-coding variants is similar among all analysed species, although there are some conserved ones, which could have a potential regulatory role. The nine human splice variants, six coding and three non-coding, were only detected at high expression levels in immune-related tissues, being the coding A, B y C the most abundant ones. The predicted human protein isoforms are potential transmembrane type I receptors, which extracellular domains are predicted to be Immunoglobulin type V (IgV) or type C (IgC). The interaction of these potential NCR3 extracellular domains with the ligand B7-H6 was analysed using flow citometry assays. Firstly, they were over-expressed in insect cells finding that defective glycosylation avoids binding. In a second approach, it was used a mammalian system detecting only specific binding of IgV domain to B7H6, indicating that B7H6 is not a ligand for IgC containing isoforms. In conclusion, all the presented data suggest a potential role of AS in the NCR3 functions and immune system regulation and evolution

Développement et caractérisation de la gésifection comme méthode de livraison d'acides nucléiques dans les cellules de mammifères

La livraison d’acides nucléiques a révolutionné la recherche biomédicale depuis des décennies. Il existe de nombreuses manières de livrer des acides nucléiques dans des cellules de mammifères. Chaque méthode possède ses avantages et ses inconvénients. Parmi elles, celles dites hybrides, représentent une alternative très intéressante puisqu’elles résultent de la combinaison de méthodes de livraison traditionnelles pour en obtenir une nouvelle aux propriétés améliorées. Cette thèse se consacre à l’étude d’une de ces méthodes de livraison hybride appelée gésifection. Cette dernière consiste à combiner des nanovésicules biologiques appelées « gésicules » avec un agent chimique nommé bromure d’hexadiméthrine pour livrer des acides nucléiques dans des cellules de mammifères. Bien que cette méthode ait fait l’objet de peu de recherches, les résultats publiés jusqu’à présent apparaissent suffisamment intéressants pour poursuivre son développement et sa caractérisation. Cette thèse est divisée en quatre chapitres. Le chapitre 1, est dédié à la revue de la littérature. Il présente un tour d’horizon des méthodes de livraison d’acides nucléiques, puis une présentation des mécanismes impliqués dans la pénétration cellulaire, et enfin, un état des lieux concernant les travaux sur les gésicules. Les chapitres 2, 3 et 4, présentent les travaux expérimentaux réalisés dans le cadre de cette thèse. Le chapitre 2 est consacré au développement d’une méthode optimisée pour produire et utiliser les gésicules pour la livraison d’acides nucléiques. Les gésicules se sont révélées être des particules très résistantes puisque leur capacité de livraison a été maintenue après plusieurs semaines de conservation à différentes températures, ainsi qu’après plusieurs cycles de congélation décongélation. Par ailleurs, les expériences de gésifection ont démontré la capacité de cette méthode à livrer de l’ADN dans des cellules primaires (connues pour être réfractaires) mais également des acides ribonucléiques (ARN) et des plasmides de grandes tailles. Le chapitre 3 est consacré à des expériences de caractérisation des gésicules au niveau morphologique et de leur contenu protéique. Les gésicules ont été visualisées par microscopie électronique avec la confirmation de la présence de la protéine VSV-G à leur membrane. Enfin, une analyse par spectrométrie de masse a permis de fournir la première description du contenu protéique des gésicules. Cela a permis de faire apparaître toute la complexité de ces nanovésicules. Le dernier chapitre de cette thèse (chapitre 4) est dédié à l’identification des mécanismes de pénétration et de transport cellulaire impliqués dans la gésifection. Tout d’abord, une série d’expériences préliminaires a permis d’établir des conditions expérimentales garantissant la fiabilité des résultats (efficacité des lavages, efficacité et innocuité des inhibiteurs métaboliques). Les résultats ont démontré que les complexes de gésifection pénètrent dans les cellules HeLa par macropinocytose et endocytose dépendante des clathrines. Leur parcours intracellulaire jusqu’au noyau de la cellule implique quant à lui, les dynéines et les microtubules. En conclusion, cette thèse contribue significativement au développement de la méthode de gésifection en décrivant un procédé de production et d’utilisation efficaces, et en apportant une meilleure compréhension de la composition et du fonctionnement des gésicules pour livrer des acides nucléiques.The delivery of nucleic acids has revolutionized the biomedical field for decades, for research as well as for therapeutic applications. There are many nucleic acids delivery methods for mammalian cells. Each has its advantages and disadvantages. Hybrid methods represent a very interesting alternative for the delivery of nucleic acids since it consists in combining elements of existing vectors to develop more efficient new ones. This thesis is devoted to the study of one of these hybrid delivery methods called gesifection. Gesifection involves virus-like particles, pseudotyped with the VSV glycoprotein, able to deliver nucleic acids when combined with a chemical agent named polybrene. Even though the gesifection’s efficiency has been stated in only a few publications, the results were interesting enough for us to investigate the depth of its potential. This thesis is divided into 4 main parts. The chapter 1 is dedicated to a review of the literature and presents the main methods of nucleic acid delivery, followed by a presentation of the mechanisms involved in cell penetration and finally, an inventory of the gesicles and their uses. The chapter 2 of this thesis presents the work that allowed us to optimize the production of gesicles, to determine the best way to use them, to demonstrate their great robustness as well as their capacity to transfect primary cells, large plasmids and also interfering RNAs. The chapter 3 is dedicated to the characterization of gesicles. We have highlighted the formation of gesifection complexes, successfully carried out the purification of gesicles efficient for nucleic acid delivery, visualized the gesicles with their membrane composed of VSV-G proteins and finally, provides the first description of their protein content, showing all the complexity of these particles. Finally, the chapter 4 studies the mechanisms of penetration and cell transport involved during the gesifection. Our results revealed for the first time, that the gesifection complexes enter in HeLa cells by macropinocytosis and clathrin-dependent endocytosis. Furthermore, their intracellular course involves dyneins and microtubules. All of the results presented in this thesis provide a better understanding of gesifection for future developments

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%), p\u3c0.01. Among 218 non-smokers, 130 (59%) were male, 142 (65%) were Caucasian; 60 (28%) reported SHS exposure compared to 158 (72%) with no exposure. Non-smoker adolescents with SHS exposure were compared to those without SHS exposure. There was no racial, age, or gender differences between both groups. Baseline creatinine, diastolic hypertension, C reactive protein, lipid profile, GFR and hemoglobin were not statistically different. Significantly higher protein to creatinine ratio (0.90 vs. 0.53, p\u3c0.01) was observed in those exposed to SHS compared to those not exposed. Exposed adolescents were heavier than non-exposed adolescents (85th percentile vs. 55th percentile for BMI, p\u3c 0.01). Uncontrolled casual systolic hypertension was twice as prevalent among those exposed to SHS (16%) compared to those not exposed to SHS (7%), though the difference was not statistically significant (p= 0.07). Adjusted multivariate regression analysis [OR (95% CI)] showed that increased protein to creatinine ratio [1.34 (1.03, 1.75)] and higher BMI [1.14 (1.02, 1.29)] were independently associated with exposure to SHS among non-smoker adolescents. These results reveal that among adolescents with CKD, cigarette use is low and SHS is highly prevalent. The association of smoking with hypertension and SHS with increased proteinuria suggests a possible role of these factors in CKD progression and cardiovascular outcomes