Browsing by Subject "onkolyyttinen adenovirus"

Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.

Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.