Viral Gene Therapy

The development of technologies that allow targeting of specific cells has progressed substantially in recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. This book is designed to present the most recent advances in viral gene therap

Regulation of Phenotypic Expressions in Lung Cancer Models In Vitro and In Vivo

The objectives were to investigate the relationship between lung cancers and to determine the effects of micro-environmental and genetic manipulation on regulation of phenotypic expression. A small cell lung carcinoma (SCLC) cell line, NCI-H69 was selected as a model for the study of heterogeneity. Two adherent sublines (H69V and H69VZ) have been isolated, and characterized, for species and lineage confirmation, and for expression of neuroendocrine markers. They have also been characterized for their growth, morphological properties, and invasiveness in vitro, radiation- and chemo-sensitivity. The cell lines have also been investigated for tumorigenicity, invasion and metastases formation in vivo. The cell lines were also investigated for c-myc expression by immunostaining. Significant morphological differences have been found in culture, between the parental and the derivative lines. While the parental line grew as densely packed free floating cellular aggregates, the sublines grew as a monolayer of epithelioid cells. Both the derivative lines either expressed very low levels or did not express neuroendocrine cell markers including L-dopa-decarboxylase, creatine kinase-BB isoenzyme, bombesin-like immunore activity, neuron specific enolase, and neurosecretory type granules, compared to the parental line. All the lines stained positively for epithelial markers. Karyotypic analysis of the derivative lines showed features of SCLC. Differences have also been observed in their growth characteristics, both in vitro and in vivo. The growth rates of both the derivative lines are faster than the parental line, with doubling times closer to non-small cell lung carcinoma (NSCLC) cell lines. In vitro invasion studies of the cell lines showed early, single cell invasion in the parental cells, while solitary invasion was absent in both the derivative lines. Both the parental and the derivative lines were tumorigenic in nude mice. The parental line was both invasive locally, and formed multiple metastatic lesions in visceral organs, while local invasion was not found in either of the derivative lines, and they were non-metastatic. Xenograft histology of all the lines revealed an anaplastic, pleomorphic and rather large cell type tumours. However, the derivative lines showed focal spindle cell morphology, and spaces filled with an amorphous material, which was alcianophilic, and some of the cells contained glycogen. These features were not found in the parental line. Therefore, it seems likely that H69 cell line contains cells of both small and non-small cell like phenotypes, suggesting a common origin for these two cell types. Further studies showed that H69 clones were able to give rise to a heterogeneous cell population similar to the parental line. These data suggest a common origin for both derivative and parental lines. Having established that phenotypic changes can occur spontaneously in H69 cells, they were also investigated further to establish whether similar phenotypic changes could be induced in these cells by micro-environmental manipulation. The cells were exposed to different chemical inducers, and investigated for various parameters as above. The specific aim was to see whether malignant cells still respond to changes in microenvironment, and if they do, whether it was possible to alter the malignant behaviour of cells by micro-environmental manipulation. Cells treated with hexamethylene bisacitamide (HMBA), dibutyryl cyclic adenosine monophosphate (dbcAMP), and sodium butyrate (Na-But) showed cytostatic effects on cell growth in culture, changes in culture morphology, inhibition of invasion in vitro, decrease in final tumour volume in vivo, changes in tumour histology, and reduction in expression of some neuroendocrine markers e.g. L-dopa decarboxylase, suggesting that the H69 cell phenotype can be regulated be microenvironmental alterations. A NSCLC cell line (WIL) derived from an adenocarcinoma of lung was selected for the study of phenotypic expression in this group of lung cancer, caused by varying the micro-environmental influences. Growth of cells in conventional monolayer, with and without stromal interactions, was compared with the growth in a histotypic tissue culture model and xenografts in vivo. The malignant cells grown in optimum conditions (histotypic culture) also were exposed to various chemical agents, and effects on morphology both at light and electronmicroscopic level, and on the production of mucin-like glycoproteins, and immunohistochemical markers were analyzed. Cells grown on collagen in histotypic culture showed increased production of mucin-like glycoproteins and formation of surface microvilli, and intracellular mucin-like inclusions

Sam68, Stress Granules, and translational control of HIV-1 nef mRNA

Indiana University-Purdue University Indianapolis (IUPUI)More than 20 million people have died of AIDS since the early eighties, while nearly 34 millions are currently infected with the HIV. Anti-retroviral therapy (ART) directed at key viral enzymes has changed AIDS from uniformly fatal to a manageable chronic disease. However, ART-associated drug resistance and toxicity have posed a great challenge for long-term management of the disease and have called for development of new therapeutics. In this study, we focused on the viral factor Nef and the host factor Sam68. Nef is a major pathogenic viral determinant for HIV-1, and no therapeutics have been targeted to this factor. Sam68 is indispensible for HIV-1 propagation. We revealed that Sam68 variants were very potent in preventing Nef expression. We found that these effects were associated with their ability to form a macromolecular structure called stress granules (SG). In addition, we demonstrated that these variants bound to nef mRNA in a sequence-specific manner. Furthermore, we showed that these variants co-localized with nef mRNA in SG. Importantly, we validated these findings in the context of HIV-1 infection of its natural target cells and found significant loss of Nef function in these cells. Taken together, these results demonstrate that SG induction and nef mRNA sequestration account for translational suppression of Nef expression and offer a new strategy for development of anti-HIV therapeutics. Sam68 is implicated in a variety of other important cellular processes. Our findings that Sam68 variants were able to induce SG formation prompted us to investigate whether wild-type Sam68 was also recruited to SG. We found that Sam68 was increasingly recruited into SG under oxidative stress, and that its specific domains were involved. However, Sam68 knockdown had no effects on SG assembly, suggesting that Sam68 is not a constitutive component of SG assembly. Lastly, we demonstrated that Sam68 complexed with TIA-1, an essential SG component. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1, and suggest that SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions

Gene therapy for retinal degeneration due to a defect in the retinal pigment epithelium.

The Royal College of Surgeons (RCS) rat is a well characterised model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding Mertk, a receptor tyrosine kinase found in RPE cells, which is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. Recently, MERTK has been established as a human retinal dystrophy gene. Retinal dystrophies are the most common cause of visual impairment in the Western World, for which no effective treatment exists. In order to evaluate the efficacy of virus mediated gene replacement therapy in the RCS rat, we produced recombinant adeno-associated viruses (AAV) and lentiviruses containing murine Mertk cDNA. Vectors were subretinally injected into the right eye of 10 day old RCS rats the left eye was left untreated as an internal control. Animals were examined at various time points by light and electron microscopy, electroretinography, and ophthalmoscopy. A detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits is presented in this thesis. AAV-2-mediated gene therapy resulted in preservation of retinal function for more than 9 weeks, when there is no activity in untreated eyes. Photoreceptors were still present at this time point and debris layer thickness was reduced. After subretinal delivery of human immunodeficiency virus type 1 (HIV-1) based lentiviral vectors carrying a functional copy of Mertk to the RCS rat eye, correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function was observed for up to 7 months, the latest time point evaluated. Whilst this was an improvement of the rescue compared to that achieved with AAV-2, lentiviral vectors raise more safety concerns regarding clinical application. Due to these biosafety issues, gene therapy vectors based on non-human lentiviruses and integration-deficient vectors have been developed. As part of this project, the potential of equine infectious anemia virus (EIAV) and non-integrating HIV-1-based vectors for the management of retinal degenerative disorders has been evaluated. The results presented in this thesis support the use of viral vectors for the treatment of retinal dystrophies. However, the development of an efficient therapy depends on the identification of patients and characterisation of pathological changes. Therefore, a panel of DNA samples from patients with autosomal recessive and sporadic forms of RP was screened for mutations in the MERTK gene. A new homozygous frame- shifting deletion was identified in four affected members of a family with RP. Clinical examination of these patients showed distinctive clinical signs that may improve the chances of identifying further patients and families in the future