We have continued to develop AAV vectors for gene transfer and we are actively engaged in evaluating 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 lacrimal gland gene transfer (Rocha et al. 2010), produce therapeutic proteins from the salivary gland for the treatment of diabetes (Rowzee et al 2011), transduce cells in the inner ear of guinea pigs for the treatment of hereditary deafness and balance disorders (Crispino et al. 2011), gene transfer to the nasal epithelia for use as a vaccine (Quinn et al. 2011), and the mapping of neutralizing antibodies to the surface of AAV particles (Harbison et al. 2012). 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 (Zheng et al 2011). In addition through funding received from the office of the scientific director as part of the salivary gland biology initiative, we are collaborating with researchers in the Laboratory of Cell and Developmental Biology, NIDCR at identifying AAV and adenovirus vectors for gene transfer to the developing salivary gland (Hsu et al. 2011). 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 (Di Pasquale et al. 2003). 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 and Axl, a largely unstudied regulator of this pathway (Quinn et al. 2009, Brindley et al. 2011). We have continued this project and have recently reported the identification of a new class of receptor molecules for Ebola (TIM1) (Kondratowicz 2011). TIM-1 is also recognized at the receptor for hepatitis A virus and its expression was shown to be critical for Ebola virus entry. TIM-1 was not expressed in all permissive cell lines, suggesting that additional, currently unidentified cellular proteins may also enhance filovirus entry. Although highly effective for understanding the complex interaction necessary for vector transduction, CGA has limitation and therefore we have developed an alternative genetics based screen for genes associated with virus transduction (Pilz 2012). By combining the gene transfer activity of AAV vectors and toxin selection we have developed a powerful complementary forward genetics approach and validated the system with an AAV5 serotype, which confirmed the importance of PDGFRαin the transduction of AAV5. Sjogrens syndrome is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes. In order to understand the environment of the salivary gland that might contribute to the gender bias associated with primary Sjgrens syndrome (pSS) we compared the transcriptome of male and female salivary glands from healthy individuals (Michael et al. 2012). Comparison of the transcriptome of minor salivary glands from normal male and female volunteers with that of salivary glands and other secretory epithelia identified a number of gender, species, and tissue-specific gene expression patterns. These differences include, but are not limited to, a diverse set of genes involved in immune modulation, chemotactic control, inhibition of complement, metabolism, and neurogenesis. Analysis of these changes provides insight into the protective and predisposing molecular factors that may be involved in the development of Sjgren's syndrome. Some of the gene changes observed in this study correlate with previously observed sexual dimorphisms in salivary gland function and also illustrate several new targets for further investigation. Patients with pSS show aberrant expression of the B cell-related mediators, B cell-activating factor (BAFF), and a proliferation-inducing ligand (APRIL) in serum and salivary glands (SGs). We studied the biological effect of neutralizing these cytokines by local gene transfer of the common receptor transmembrane activator and CAML interactor (TACI) in an animal model of SS (Vosters et al. 2012). A recombinant serotype 2 adeno-associated virus (rAAV2) encoding TACI-Fc was constructed, and its efficacy was tested in the SGs of non-obese diabetic mice. Ten weeks later, SG inflammation was evaluated and serum and SG tissue were analyzed for inflammatory markers including immunoglobulins (Ig) and cytokines. AAV2-TACI-Fc gene therapy significantly reduced the number of inflammatory foci in the SG, owing to a decrease in IgD+ cells and CD138+ cells. Moreover, IgG and IgM levels, but not IgA levels, were reduced in the SG. Overall expression of mainly proinflammatory cytokines tended to be lower in AAV2-TACI-Fc-treated mice. Salivary flow was unaffected. Although local expression of soluble TACI-Fc reduced inflammation and immunoglobulin levels in the SG, further research will have to prove whether dual blockade of APRIL and BAFF by TACI-Fc can provide a satisfying treatment for the clinical symptoms of patients. Results with an alternative co-stimulatory molecule are more promising (Yin et al 2012). CTLA4 is expressed on the surface of Helper T cells and can transmit an inhibitory signal by interacting with B7 receptor. In contrast, interaction of B7 with CD28 transmits a stimulatory signal. Recently it is noted that epithelial cells in the minor SGs of pSS patients express costimulatory molecules B7.1 (CD80) and B7.2 (CD86). Correspondingly, different haplotypes of CTLA-4 were found to be associated with increased susceptibility to pSS. Expression of soluble CTLA4IgG blocked B7 expression on macrophages in vitro. In vivo, localized expression of CTLA4IgG in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice inhibited the loss of salivary gland activity and decreased T and B cell infiltration as well as dendritic cells and macrophages in the glands compared with control mice. In addition a decrease in several proinflammatory cytokines and an increase in transforming growth factor beta-1 (TGF-β1) expression were also observed. Our findings of both functional and immunological improvement in the mice warrant further investigation of CTLA4 mediated immunomodulation as a therapeutic pathway for treatment of pSS patients. 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.
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