published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids. We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI). DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells. Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).

Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system.

Clonally derived liver tumours and an ovarian cyst developed in mice following EIAV and FIV delivery in utero. LAM PCR and 454 sequencing was used to retrieve proviral insertion sites. TaqMan analysis revealed gene expression changes in lentiviral infected tumours. STRING and IPA networks identified links between genes flanking the lentivirus provirus and oncogenic pathways supporting the role of insertional mutagenesis in Hepatocellular Carcinoma (HCC). Global methylation analysis demonstrated increased relative methylation levels in lentivirus (EIAV, FIV, and HIV) infected normal and tumour samples. This provided strong evidence for host defence against lentivirus infection by epigenetic means. Microarray data showed altered expression of Dnmt1 and Dnmt3b and TaqMan analysis revealed specific changes in Dnmts levels when compared to uninfected liver. The evidence found for involvement of DNA methylation associated with lentivirus infection and possibly tumour development required that this study be repeated in vitro. DNA methylation was investigated at early time points after lentivirus and retrovirus infection in HepG2 cells. Results revealed sharp increases in global methylation and Dnmt levels at 24 and 30hrs post infection. E2F targets play a key role in the regulation of gene expression and aberrations result in the development of cancer. Of the 94 E2F target genes analysed 77.7% were involved in DNA damage and repair mechanisms, 21.3% were known oncogenes or shown to exert oncogenic activity and 80.9% were categorised as HCC target genes. The fact that all lentiviral/retroviral vectors used in this study were found to cause changes in methylation and gene expression in vivo and in vitro suggests that these vectors, at least in the mouse, are genotoxic. Findings here support the use of the fetal animal model to identify vector genotoxicity and the mechanisms of lentiviral vector-induced tumorigenesis. This model may be a valuable tool to evaluate the safety of lentiviruses for gene therapy.

<div>Utilization of nucleic acids to manipulate genetic information within a cell is known as gene therapy. It has provided researchers with unprecedented opportunities in treatment and mitigation of several life-threatening diseases. Gene therapy is an attractive alternative to conventional chemotherapy or radiation therapy due to its high efficiency, minimal side effects, and potential to evade drug resistance. The versatility of gene therapy makes it useful for the treatment of diseases dangerous disease like cancer. However, delivery of nucleic acid payloads to the intended target has been the bottleneck in clinical translation of such therapies. Here, we have developed and evaluated three different delivery systems (lipid based, polymer based and lipid-polymer hybrid) which can complex nucleic acid payloads, able to target specific cell types and get dissembled on cellular internalization to release the therapeutic payload. Our lipid and lipid-polymer hybrid delivery systems utilize a novel bacterial peptide sequence which enables these vectors to “stick” to fibronectin present in tumor extracellular matrix making them attractive for intravesical administration in bladder cancer management. Additionally, these systems have pH responsive modalities which aids in vector dissemble under acidic endosomal pH conditions for efficient release of therapeutic cargos after internalization into target cells.</div><div><br></div><div>In our efforts to develop an ideal delivery system with a tunable the assembly/disassembly properties, we synthesized a library of pendent polymer with biodegradable polycarbonate backbone. The ability of the pendent groups to form host-gest interaction with hydrophobic core of cationic cyclodextrins determined the stability of the delivery system. We demonstrate the capability of such polymer systems to form nano-dimensinal complexes with nucleic acid and transfect cancer cells. The above-mentioned property of cyclodextrins to form host-guest interaction with hydrophobic molecules also forms the basis of its utilization in the treatment of a rare metabolic disorder called Niemann Pick type C disease where there the cells loses the ability to remove stored cholesterol from endo-lysosomal compartments. Cyclodextrin forms host-gest complexes with aberrantly stored cholesterol and helps normalization of cellular cholesterol level. However, the soluble nature and small size of the cyclodextrin causes very rapid clearance for the body necessitating dosage levels as high as 9000mg/kg for therapeutic benefit. We have also developed a library of polyrotaxanes, where multiple cyclodextrins are threaded onto linear polymer chains and end-capped with bulky groups to prevent slippage. This kind to assembly drastically increases the systemic circulation time by preventing rapid renal clearance. We evaluated the ability of these constructs to serve as a long circulation delivery system for delivery of β-cyclodextrins for potential treatment of Niemann Pick type C disease. Finally, we have studied the structure-function relationships of these supramolecular assemblies to aid in rational design of therapeutic polyrotaxanes.</div>

The Gangliosidoses, to include GM1 gangliosidosis and Sandhoff disease are a class of incurable lysosomal storage disorders characterized by an abnormal accumulation of gangliosides leading to progressive neurodegeneration and eventually death. GM1 gangliosidosis is caused by a genetic defect in the lysosomal-specific acid β-galactosidase, which results in the massive accumulation of ganglioside GM1 primarily in the central nervous system (CNS). Sandhoff disease (SD) results from a defect in the β-subunit of β- Hexosaminidase A and leads to the accumulation of ganglioside GM2 and its asialo derivative (GA2). As there are no effective therapies for these glycosphingolipid (GSL) storage disorders, I studied substrate reduction therapy (SRT), stem cell therapy, and adeno-associated viral (AAV) gene therapy in neonatal mice as early intervention therapies and were effective in reducing CNS GSL storage. In addition, AAV gene therapy was also evaluated in the adult GM1 gangliosidosis mice. Furthermore, analysis of the brain lipids in mice, cats, and humans with Sandhoff disease revealed that the SD cat model is intermediate between the SD mouse and the SD patient with respect to GM2 and GA2 accumulation. These findings are the first to compare the different therapies and provide valuable information for the translation of mouse studies to clinical trials in patients. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Eve Spangler / This study seeks to better understand the "public face" of human gene therapy through an examination of coverage of the technology in mainstream U.S. newspapers, news magazines, and online news sites from 1989 to 2011. By conducting a qualitative content analysis that employs a constant comparative method and uses the computer-assisted qualitative data analysis software HyperRESEARCH, prevailing images and metaphors about human gene therapy are identified. These images and metaphors are analyzed through the lens of the sociology of technology, with particular attention given to technological determinism, geneticization, and the sociology of expectations. Further, their connection to issues of self and identity, embodiment, and illness meanings is explored. Four main types of images and metaphors emerge from this analysis: essentialist, fatalistic, expectant, and conflictive. While these types present an array of diverse (and sometimes conflicting) characterizations of human gene therapy, they all contribute to a positive, hopeful public face of the technology, despite its limited successes and sometimes tragic failures over the past three decades. The study considers the broader implications of these findings and addresses the role sociologists could play in helping the public to navigate the media discourse surrounding human gene therapy and other emerging medical technologies. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Sociology.

L’administration intraveineuse d’ADN thérapeutique rencontre de nombreux obstacles liés à sa dégradabilité, ainsi qu’à sa difficulté à pénétrer les cellules en raison de sa taille importante et de son hydrophilie. Des lipoplexes conjugués à de l’acide hyaluronique (HA) de haut poids moléculaire ont été développés afin de délivrer de l’ADN plasmidique à l’intérieur de cellules cancéreuses exprimant le récepteur membranaire CD44, récepteur clé du développement tumoral. L’emploi d’HA conjugué au phospholipide DOPE (HA-DOPE) et d’un plasmide modèle GFP a permis d’obtenir des lipoplexes d’environ 250 nm, chargés négativement, protégeant efficacement l’ADN contre les nucléases et activant peu la fraction C3 du système du complément. Dans un modèle cellulaire exprimant CD44, la transfection optimale a été obtenue par l’utilisation de lipides avec 10% d’HA-DOPE complexés à de l’ADN selon un rapport 2:1. Ces lipoplexes sont internalisés par la voie des cavéoles et de façon dépendante du récepteur CD44. Cette formulation a été appliquée à la vectorisation d’un gène thérapeutique, codant pour le récepteur des estrogènes β (ERβ), qui est un potentiel suppresseur de tumeur. Sur un modèle in vivo de xénogreffes de cellules humaines de cancer du sein estrogéno-dépendant et exprimant CD44, la diminution du volume tumoral, ainsi que de l’indice de prolifération Ki67 ont permis de montrer l’effet anticancéreux par voie intraveineuse des lipoplexes conjugués à l’HA. / Intravenous administration of therapeutic DNA faces many obstacles related to its degradability and its difficulty to penetrate into the cells due to its large size and its hydrophilicity. Lipoplexes conjugated with high molecular weight hyaluronic acid (HA) have been designed in order to deliver plasmid DNA inside cancer cells expressing the membrane receptor CD44, a key receptor in the development of tumors. The use of HA conjugated to the phospholipid DOPE (HA-DOPE) and of the GFP model plasmid lead to obtain lipoplexes around 250 nm, negatively charged, which efficiently protect the DNA against nucleases and slightly stimulate the C3 fraction of the complement system. In a cellular model expressing CD44, the optimal transfection was obtained by using lipids containing 10% of HA-DOPE complexed to DNA at a 2:1 ratio. Internalization of these lipoplexes is mediated by the caveolae pathway and involves the CD44 receptor. This formulation was applied to the delivery of a therapeutic gene encoding the estrogen receptor β (ERβ), which is a potential tumor suppressor. On an in vivo xenograft model of estrogen-dependent breast cancer cells expressing CD44, decrease of the tumor volume, as well as decrease of the Ki67 proliferation index, have shown the anticancer activity of the lipoplexes conjugated to HA following intravenous administration.