We have continued to develop AAV vectors for gene transfer and we are actively engaged in evaluating in their use in several gene therapy applications including gene transfer to the lung, CNS, eye, and salivary gland. In addition to distributing these vectors to labs throughout the world, we have continued to collaborate with a number of researchers in the field and in several publications describe the use of AAV based vectors to express genes in cancer application, the inner ear of guinea pigs for the treatment of hereditary deafness and balance disorders, primary salivary epithelial cells to repair damage to the gland, or produce therapeutic proteins, as well as develop a universal reference standard to normalize assays between labs . We also are working with both intramural and extramural researches to test the effect of local expression AAV based vectors expressing AQP1 for the treatment of radiation induced xerostomia. We believe that just as our characterizations of AAV4, AAV5, and BAAV have advanced the field of gene therapy, the characterization of other vectors will enhance their utility in gene transfer application. In 2006 we reported that AAV vectors are able to pass through barrier epithelia via transcytosis, a transcelluar transport pathway previously not reported to occur with non-enveloped viruses. Since this time transcytosis has been reported with other AAV isolates and is being exploited for gene transfer to the brain. However, little is know about the pathway(s) used by AAV for transcytosis. By combining glycan microarrays and biochemical techniques we were able to identify two critical molecules in the transcytosis pathway of BAAV (chitotriose, and gp96). In ongoing work, we are testing modulators of these molecules to either enhance or inhibit transcytosis and alter the spread of vector for in vivo applications. In 2003 we reported the development of a microarray based high throughput screening technique for identifying gene expression patterns that correlated with a specific phenotype. This approach was termed comparative gene analysis (CGA). Since our initial publication we have continued to refine the bioinformatics aspect of this approach and recently applied it to several diverse systems. Through funding receive as part of an NIAID biodefense grant we have worked with extramural researchers to identify genes that correlate with Ebola virus entry. Unlike AAV, the entry pathway of enveloped viruses is very complex and may take several routes. We have recently reported a component of Ebola entry involves macropinocytosis and specifically the activation of RhoC, a largely unstudied regulator of this pathway. Ongoing studies have identified a cell surface attachment receptors that may be critical for entry into epithelial cells. In collaboration with NCI researchers we applied CGA to identify both genes that correlated with tumor killing as well as those genes that would block this pathway. By identifying these genes we can better classify tumors and aid in the selection of patients who would benefit from in virally-mediated anti tumor therapies. We have also applied CGA to the study of AAV6. Through the use of CGA we have identified EGFR as a key receptor for AAV6 attachment and entry in epithelial cells. Based on this understanding, and in collaboration with NIDCR researchers, we have demonstrated effective killing of aggressive head and neck tumor cells which was previously unknown. Autoantibodies such as anti Ro or La (SSA or SSB respectively) are common markers of disease. However, the role of these antibodies in disease is not clear. The detection of autoantibodies to the muscarinic receptor type 3 (M3R) in the serum of patients with Sjogrens syndrome (SS) by ELISA is controversial. A study was undertaken to test whether modification of M3R peptides could enhance the antigenicity and increase the detection of specific antibodies using an ELISA. Although some differences in detection were noted between peptides, in general significant variability existed in the assay, making it difficult to reliable detect differences in anti-MR3 autoantibodies between patients with primary Sjogrens and healthy volunteers. Currently several mouse models of this disease are available and we are reviewing and evaluating them as an alternative to the NOD mouse. Cytokines have been reported to play a key role in the development of Sjogrens syndrome. In order to better understand the role of IL-12 in animal models of SS, we studied the salivary gland activity, histopathology, and autoantibody levels in a mouse that is transgenic for expression of IL-12 in the thyroid. Pilocarpine-stimulated salivary flow was significantly lower in IL-12 transgenics than wild type controls, independent of sex, and pilocarpine dose. Salivary glands from transgenic mice showed both a greater number and size of lymphocytic foci than those of age-matched controls. Furthermore, their acini were fewer and larger compared with controls. Anti-Ro and La antibodies showed an age-dependent increase in IL-12 transgenic mice, accompanied by a rise in anti-nuclear antibodies. Our findings indicate that SJL IL-12 transgenic mice express a number of conditions associated with SS and may serve as a useful model for research on multiple aspects of the disease. Other cytokines such as TNFa are over expressed in the gland and have been attractive points of therapy for treating Sjogrens syndrome. Although, TNFa inhibitors have demonstrated great utility in treating other autoimmune diseases, such as rheumatoid arthritis (RA), publications from our clinics study suggested no benefit to the patients following systemic delivery of etanercept and no decrease in the immune activation parameters. The reason for these negative results is poorly understood but some results from this study appear to indicate an increase in systemic inflammatory markers. To investigate the possibility of TNF inhibitors potentially increasing salivary gland dysfunction through a similar paradoxical increase in inflammation we locally expressed the TNFa inhibitor TNFR1:Ig (similar to etanercept) in the salivary glands of a sub-strain of NOD mice that do not develop salivary gland dysfunction and assayed for changes in salivary gland function. We observed that local expression of TNFR1:IgG in the salivary gland can lead to a reduction in salivary gland function compared with untreated or control vector treated mice. The reduction in salivary gland activity was not associated with a change in the incidence of diabetes, the number of inflammatory cell infiltrates, or autoantibody levels but was associated with a change in salivary gland cytokine levels (Vosters et al 2010). These findings suggest that expression of TNFa inhibitors in the salivary gland can have a negative effect on salivary gland function and that other cytokines should be explored as targets for therapeutic intervention. Furthermore, the results of this study support a continued close working relationship with the Sjogrens clinic to evaluate potential therapies. In summary, the future directions for the AAV Biology Section will be to continue examination and development of gene transfer vectors for use in treating disease as well as refine our tools for studying interactions necessary for cellular transduction.
Showing the most recent 10 out of 75 publications
