Under physiological conditions, cells communicate with one another by activating receptors on the cell surface, which convey the signals enrolling proteins located in the cytoplasm and subsequently, activating transcription factors in the nucleus. The stimulation of this thick network of signaling is responsible for the control of expression of genes that mediate the cellular response. The aberrant production of growth factors and the broken communication cell to cell triggers an uncontrolled cellular transformation interfering with the normal functions of tissues. The concept of gene delivery is based on the introduction of genetic material into the cells to compensate the abnormal genes and restore the function of misfolded proteins. The intracellular delivery needs genetically engineered carriers, commonly called vectors, which facilitate gene transfer to targeted cells without degradation of the delivered gene. To date, it is possible to refer to two principals families of vectors, viral and non-viral, used in clinical and research studies, responsible for the success of gene therapy in the treatment and prevention of several diseases. In the following chapters, viral and non-viral vectors will be described highlighting the advantages and disadvantages correlated to the use of the ones or the others one for the treatment of cancer diseases. In addition, an overview of the mechanisms and of the improvements of the vectors in several years will be reported, emphasizing that the acquired scientific knowledge on the use of several types of viral and non-viral vectors was helpful to improve target efficiency, cellular bioavailability and to limit the cytotoxicity effect preferentially to tumor environment. Therefore, the aim of this work was to analyze several cellular pathways, which are influenced, manipulated and controlled by a potential engineered HSV-viral and non-viral vectors. The success of gene therapy realized with viral and non-viral vectors, depends on a variety of factors including the interactions between host and vector. The viral model used in this thesis is Herpes Simplex Virus type 1 (HSV-1), whereas, the non-viral vector is represented by graphene-based platforms. The HSV-1 genome is around 150 kilobases (kbp) in length and encodes for about 84 genes. Many genes are not essential for its productive growth and for this reason is possible to manipulate the HSV-1 genome without critically damaging the ability of the virus to replicate. The efficient viral replication depends on delicate balance between host immune surveillance and production of viral proteins, which occurs with an accurate program of gene transcription. In this regard, and in order to create oncolytic HSVs (oHSVs), it is important to analyze cellular signals, including innate immune response, activated by HSV-1 and by involved viral proteins. The fundamental mission of all viruses is to replicate and spread to persist in the host environment. In order to perform these functions, is essential to confiscate the protein synthetic machinery and reduce the host immune response. An important mechanism, that regulate translation initiation, involves phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF2α) mediated by protein kinase R (PKR). The modulation of PKR was analyzed after infection with HSV-1 and mutant HSV-1-viruses, deleted in tegument proteins closely connected with immunological escape. The model “host shut off” adopted by VHS protein of HSV-1, represents an immune evasion mechanism, which affects the activation of PKR. However, to date, the real mechanism used by the viral protein VHS to block PKR is not perfectly clear. Several years ago, it was proposed a new mechanism based on potential engagement of the mitogen-activated extracellular signal-regulated kinase (MEK), normally involved in the cell survival, in the PKR control. Indeed, Sciortino M.T. and collegues 2013 have demonstrated that, in HSV-1 infected cells, MEK protein, a well-characterized kinase in the cancer pathway, blocks phospho-PKR accumulation. Otherwise, in VHS, mutant virus (ΔVHS) infected cells, MEK protein is not able to control phospho-PKR accumulation. This finding highlights the important involvement of VHS tegument protein of HSV-1 in the regulation of PKR phosphorylation and led to investigate the role of additional teguments proteins Ser/Thr kinases US3 and UL13 on VHS/PKR control. The study of viral proteins and their contribution in PKR regulation could contribute to develop new HSV-1-based vector for cancer therapy. Based on this, infection and transfection experiments were performed on several tumor cell lines to evaluate the expression of PKR active form and the transcriptional levels of PKR after HSV-1 (F) wild-type virus, R2621 (∆VHS), R7356 (∆UL13) or R7041 (∆US3) mutant viruses infection and after pVHS, pUS3 and pUL13 plasmids transfection. In addition, PKR-/- cell lines have been used to analyze the role of PKR in the HSV-1 replication, in the spread cell to cell, in the regulation of transcriptional and translational levels of the viral proteins. Finally, preliminary results have been obtained by comparing the quantitative expression of p53 in HEp-2 and PKR-/- cells after infection with HSV-1 and all mutant virus considered. The tumor suppressor p53 protein supports a transcriptional response involved in cell cycle arrest or apoptosis and its gene mutation is most frequent in human cancer diseases. Further studies are necessary to analyze the signaling network linking HSV-1 to p53 and PKR and the associated molecular mechanisms. In the second part of this thesis work, two potential non-viral vectors have been used as a platform to delivery anticancer and antiviral drugs, plasmid DNA and miRNAs. Carbon nanotubes (CNTs) and graphene are two representative carbon-based nanomaterials used as a platform in this work. The first used carbon-based nanovector, nanotube namely MWCNT, was modiﬁed with branched polyethylenimine to form a MWCNTCD-PEI platform consequently conjugated with Rhodamine (MWCNT-CD-PEI-Rhod) to track in vitro the nanocarrier and to elucidate the mechanisms of cellular uptake. In order to explore the potentiality of the nanoplatform as drug delivery system, the cytotoxicity of the platform was evaluated at different concentrations on Vero cell line (kidney epithelial cells extracted from an African green monkey). To investigate the mechanisms involved in the cellular internalization, clathrin-dependent and caveole-dependent endocytosis inhibitors have been used. In addition, the endocytic compartmentation of MWCNT-CD-PEI-Rhod was studied by Cell Light lysosomes-GFP. Finally, the antiviral drug, cidofovir, was assembled with MWCNTCD-PEI-Rhod platform, with or without pCMS-EGFP to investigate the drug and gene delivery by inhibition of plaque formation assay and expression of the green ﬂuorescent protein, respectively. The obtained results, published by Mazzaglia A. and collaborators 2018 2 , have shown the high drug delivery efficiency mediated by nanotubes but inefﬁcient delivery of nucleic acids. For this reason, further studies were performed by using another type of graphene-based platform and all experiments were carried out by comparing HEp-2 tumor cell lines with Vero non-tumor cell lines. The Graphene-based vector was modified with cationic cyclodextrins producing a G-CD platform which, in combination with a fluorescent probe, such as rhodamine, (G-CD@ADA-Rhod) was used for cellular internalization study. In a first phase, the biological evaluation has been carried out on G-CD platform assembled with the anticancer drug, doxorubicin, G-CD@Doxo, in order to evaluate the regulation of proteins, which, by acting as transcriptional factors, are involved in the control of cell cycle and tumor suppression. The communication cell to cell is guaranteed by molecular signals involved in the formation of signalosome complex. Signalosome consists in a set of several proteins able to respond to extracellular signals activating an intracellular signalling network in order to trigger an appropriate response. Therefore, the protein expression levels of p53 and its downstream and upstream regulators, transcription factor E2F and nuclear kinase Wee-1 have been investigated by western blot analysis. In the second phases, the regulation of autophagy pathway was explored as a response to the activation of intracellular signals driven by G-CD@Doxo. To monitor autophagy flux, transfection experiments were performed by using the tandem monomeric RFP-GFP-tagged LC3. The GFP signal is sensitive to the acidic and/or proteolytic conditions of the lysosome lumen, whereas mRFP is more stable; therefore, colocalization of both GFP and RFP fluorescence indicates a compartment that has not fused with a lysosome, such as the phagophore or an autophagosome; in contrast, an mRFP signal without GFP, corresponds to autolysosome. In addition, the regulation of autophagy was evaluated by western blot analysis of expression of LC3 and p62, key proteins of autophagy process. Finally, G-CD platform was assembled with miRNA-15a, involved in tumorigenesis, and its delivery was analyzed by studing the expression levels of BCL-2 target. This thesis work, underlines the importance of molecular research in the design of biological vectors. The study of HSV-1 viral proteins and of the interplay between viral teguments proteins and host immune response could be useful to define new retargeting approaches in the HSV-1 oncolytic virus.
|Titolo:||Study of intracellular signaling network triggered by HSV- 1 and graphene based nanomaterials: their use as potential tools in gene therapy|
|Data di pubblicazione:||15-nov-2018|
|Appare nelle tipologie:||Tesi di dottorato|