Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The current standard-of-care treatment including surgery, radiation and temozolomide (TMZ) chemotherapy does not prolong the survival satisfactorily. Here we have tested the feasibility, efficacy and safety of a potential gene therapy approach using AAV as gene delivery vehicle for treatment of GBM. Interferon-beta (IFNβ) is a cytokine molecule also having pleiotropic anticancerous properties. Previously it has been shown by our group that AAV mediated local (intracranial) gene delivery of human IFNβ (hIFNβ) could be an effective treatment for non-invasive human glioblastoma (U87) in orthotopic xenograft mouse model.But as one of the major challenges to treat GBM effectively in clinics is its highly invasive property, in the current study we first sought to test the efficacy of our therapeutic model in a highly invasive human GBM (GBM8) xenograft mouse model. One major limitation of using the xenograft mouse model is that these mice are immune-compromised. Moreover, as IFNβ does not interact with cross-species receptors, the influence of immune systems on GBM remains largely untested. Therefore to test the therapeutic approach in an immune-competent mouse model, we next treated a syngeneic mouse GBM model (GL261) in an immune-competent mouse (C57B6) with the gene encoding the species-matched IFNβ (mIFNβ). We also tested if combination of this IFNβ gene therapy with the current standard chemotherapeutic drug (TMZ) is more effective than any one of the therapeutic modes alone. Finally, we tested the long term safety of the AAV-mIFNβ local gene therapy in healthy C57B6 mice. Next, we hypothesized that global genetic engineering of brain cells expressing secretory therapeutic protein like hIFNβ could be more beneficial for treatment of invasive, migratory and distal multifocal GBM. We tested this hypothesis using systemic delivery of AAV9 vectors encoding hIFNβ gene for treatment of GBM8 tumor in nude mice. Using in vivo bioluminescence imaging of tumor associated firefly luciferase activity, long term survival assay and histological analysis of the brains we have shown that local treatment of AAV-hIFNβ for highly invasive human GBM8 is therapeutically beneficial at an early growth phase of tumor. However, systemic delivery route treatment is far superior for treating multifocal distal GBM8 tumors. Nonetheless, for both delivery routes, treatment efficacy is significantly reduced when treated at a later growth phase of the tumor. In syngeneic GL261 tumor model study, we show that local AAV-mIFNβ gene therapy alone or in combination with TMZ treatment can provide significant survival benefit over control or only TMZ treatment, respectively. However, the animals eventually succumb to the tumor. Safety study in the healthy animals shows significant body weight loss in some treatment groups, whereas one group shows long term survival without any weight loss or any noticeable changes in the external appearances. However, histological analysis indicates marked demyelinating neurotoxic effects upon long term exposures to mIFNβ over-expressions in brain. Overall, we conclude from this study that AAV-IFNβ gene therapy has great therapeutic potential for GBM treatment in future, but the therapeutic window is small and long term continuous expression could have severe deleterious effects on health.

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Bakgrund - Hemofili A och B är X-kromosombundna blödarsjukdomar, som beror på genetiska avvikelser i de gener som kodar för koagulationsfaktor VIII respektive IX. I dagsläget förlitar sig hemofilipatienter på kontinuerliga intravenösa injektioner med faktorkoncentrat, för att förhindra att potentiellt livshotande blödningar uppstår. Genterapi med rekombinanta adeno-associerade virus (AAV) skulle kunna erbjuda ett kurativt behandlingsalternativ, genom införandet av friska arvsanlag i hepatocyter. Syfte - Syftet med den här litteraturstudien var att undersöka huruvida genterapi medierad av AAV-vektorer är en effektiv och säker behandling mot hemofili A och B ur ett långsiktigt perspektiv. Metod - Studien är genomförd som en systematisk litteraturstudie och är baserad på sex originalartiklar framsökta via databasen PubMed, med sökorden "hemophilia AND gene therapy". Specificerade sökkriterier användes för att underlätta relevansbedömning och valet av artiklar. Resultat - En ökad endogen koagulationsfaktorproduktion kunde påvisas hos majoriteten av studiedeltagarna efter genterapibehandlingarna. Sammantaget observerades också en väsentlig blödningsreducering och en minskad faktorkoncentratanvändning. Störst förbättring noterades i de kohorter som erhållit högre genterapidoser eller den muterade faktor IX Padua-genen. Ingen immunrespons mot transgenprodukten detekterades i någon studie. Däremot sågs ett humoralt immunsvar mot AAV-kapsiden hos samtliga studiedeltagare. En mycket stor variation i T-cellssvar mot AAV-kapsiden kunde noteras. Förhöjda nivåer av alaninaminotransferas (ALAT) var den vanligast förekommande incidenten, men samtliga fall kunde framgångsrikt behandlas med glukokortikoidpreparat. Slutsats - Genterapibehandling med rekombinanta AAV-vektorer mot hemofili A och B förefaller effektiv och säker. Förhöjda ALAT-nivåer återstår dock som en behandlingsproblematik. Längre uppföljningar av fler genterapibehandlade hemofilipatienter krävs, för att kunna dra några definitiva slutsatser, väga risker mot nytta, samt optimera och individanpassa doser. / Background - Hemophilia A and B are X-linked bleeding disorders, resulting from defects in the genes encoding coagulation factors VIII and IX respectively. The current treatment for hemophiliacs entails frequent intravenous injections of coagulation factor concentrates, to prevent potentially life-threatening hemorrhages. Gene therapy utilizing recombinant adeno-associated viruses (AAV) could offer a potentially curative treatment option through the introduction of healthy genes into hepatocytes. Aim - The aim of this literature study was to investigate the long-term efficacy and safety of AAV vector-mediated gene therapy for the treatment of hemophilia A and B. Methods - The study is conducted as a systematic literature study and is based on six original articles retrieved from the search engine PubMed, using the key words "hemophilia AND gene therapy". Specific search criteria were used to facilitate the relevance assessment and selection of articles. Results - An increased endogenous coagulation factor synthesis was noted in the majority of the study participants after the gene therapy. Overall, a significant reduction in bleeding episodes and the use of factor concentrates were observed. The greatest improvements were noted in the cohorts that received the higher gene therapy doses or the mutated factor IX Padua gene. None of the study participants had an immunologic response to the transgene product. A humoral immune response against the AAV capsid was seen in all participants though. Large differences in AAV capsid-specific T-cell activation were observed. The most common adverse event was an elevation in the alanine aminotransferase (ALT) level. However, these events could be controlled with glucocorticoids. Conclusions - AAV vector-mediated gene therapy for the treatment of hemophilia A and B had a positive efficacy and safety profile. Although increased ALT levels remain a concern. Monitoring of larger numbers of study participants for longer follow-up periods is necessary for any definite conclusions to be drawn, to weigh risks against benefits and to optimize individual dosing.