E1A of human adenovirus is the first gene product expressed during viral infection and serves as a preliminary step for efficient viral replication. Other early gene products include early proteins designated as E2, E3, and E4 are also made, which together with E1A prepare the infected cell for replication of the viral genome. Various studies have elucidated that E1A functions largely as a transcriptional regulator and can interact with a host of cellular modulators to enhance replication of the virus. RuvBL1, a chromatin remodeling protein involved in a host of cellular functions such as transcriptional regulation and host cell immune response has been shown to bind to E1A. My results identify RuvBL1 as an E1A binding protein and show that E1A is a direct binding partner of RuvBL1. I demonstrate that RuvBL1 plays a role in the growth of adenovirus, as knockdown of RuvBL1 negatively affects the growth of the virus and reveals that RuvBL1 functions as a viral growth enhancer. Finally, I identify a possible role of E1A’s inhibition of interferon response in a RuvBL1 dependent manner. / February 2017

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron protein (SMN), leading to progressive deterioration of α-motor neurons, onset of muscle atrophy and, in severe disease, death. We investigated whether reducing the size of Adenovirus (Ad) vectors, through use of a short fibre protein, could enhance delivery of a transgene to muscle and motor neurons after systemic delivery in vivo. Unfortunately, the biodistribution of the smaller Ad vector was unaltered compared to wildtype Ad, with most of the virus localizing to the liver. However, we determined Ad-derived SMN was efficiently packaged into cellular exosomes, suggesting a novel approach to protein delivery. We showed that exosomes naturally contain SMN both in vitro and in vivo and that exosomes can be used to deliver SMN to recipient cells. Further testing is required to establish if SMN-containing exosomes can function as an SMA therapeutic.

No description available.

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Thesis / Master of Science (MS)

No description available.

No description available.

Human adenoviruses (Ads) as vectors have been studied for cancer gene therapy for several decades due to their ability to shut down host cell replication and lyse tumour cells. Ad5 of species C is commonly used as a replication-defective or a replication-competent vector. However, many tumour cells are relatively refractory to infection by Ad5 since the cells lack the viral receptor CAR. Thus, species B Ads are becoming more important as alternative vectors since they use CD46 as primary receptor, the expression of which is up-regulated on many tumour cell surfaces, and they also have low seroprevalence in humans. Although Ad3, Ad7, Ad11 and Ad35 have been altered to become replication-defective vectors, investigations based on replicating adenovirus vectors are still warranted.   The major aim of this thesis has been to characterize the transduction efficacy and oncolytic effect of the replication-competent adenovirus 11pGFP vector (RCAd11pGFP) in human solid tumour cell lines. Evaluation of the vector would ultimately help us to understand whether the tumour cells affect virus replication and whether the vector replicates differently in tumour cells and in untransformed diploid cells, and would eventually lead to development of more potent oncolytic adenoviruses for treatment of human cancers. The Ad11-based vector RCAd11pGFP consists of the entire Ad11p genome with a green fluorescence protein (GFP) expression cassette inserted. RCAd11pGFP shows all the characteristics of the wild-type virus and expresses GFP in cells four hours p.i. Antisera raised against Ad11p virions and hexons were able to neutralize RCAd11pGFP infection but antiserum raised against the Ad11p fibre knob could not. The infection is reduced by 90% but the fibre knob antiserum cannot completely block virus infection. Initial screening of the infection capacity of five wild-type adenoviruses in four colon cancer cell lines revealed that Ad11p, Ad11a and Ad35 of species B, showed similar replication kinetics but Ad5 showed delayed onset of virus replication in comparison to species B Ads. These data support the use of Ad11p as an alternative vector for treatment of colon cancer. The transduction efficiency of RCAd11pGFP in colon cancer and prostate cancer cell lines was studied using flow cytometry assay (FACS), and this showed that the cytolytic effect was not always in accordance with GFP expression. Toxicity assay and virus one-step replication assay showed that RCAd11pGFP replicates in highly tumorigenic cell lines (HT29, T84 and PC-3) to a greater extent than less tumorigenic cell lines (LS174T, HCT-8, DU145 and LNCaP cells), even though the latter showed relatively high GFP expression. This initial finding led to the subsequent discovery of CEACAM-family molecules, which were highly expressed in HT29 and T84 cells. Interestingly, the Ad5 wild-type virus did not manifest the same tumour-specific replication that RCAd11pGFP did in the cell lines studied. Furthermore, we investigated the influence of tumour markers for RCAd11pGFP replication in colon cancer cells. A double-staining FACS assay for detecting members of CEACAM-family molecules was established and we found that the levels of CEACAM6 were up-regulated in the cells infected by RCAd11pGFP or Ad11pwt relative to uninfected cells. However, this virus replication could not be suppressed by CEACEA6 siRNA. Our results indicate that several tumour markers or factors might be involved in promoting propagation of the virus. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumours in xenograft mice treated with either RCAd11pGFP or Ad11pwt, compared to untreated controls. Furthermore, the role of the anti-tumour effect of RCAd11pGFP was also confirmed in PC3 prostate tumours in BALB/c mice. In conclusion, the novel RCAd11pGFP vector was shown to have an anti-tumour effect in vitro and in vivo. This tumour-killing effect could be enhanced in highly tumorogenic cells through virus replication. Consequently, RCAd11p may lead to development of a more potent and useful vector for human cancer therapy.