We have continued to develop AAV4 and AAV5 as vectors for gene transfer and they are both actively being evaluated 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 beta glucaronidase for the correction of learning defects in MPS VII mice (Liu et al. 2007), transfer TGFβto protect transplanted islets in diabetes (Craig et al 2009), transduce primary salivary epithelial cells to repair damage to the gland or produce therapeutic proteins (Tran et al. 2007, Voutetakis et al. 2007, Hai B et al. 2009), and deliver a truncated CFTR for primary airway epithila from cystic fibrosis patients and correct the underlying defect in chloride transport associated with this disease (Ostegaard et al 2005). Vectors based on BAAV, which we have previously cloned and characterized, also demonstrating utility in defining the underlying biology associated with signal transduction and channel formation in the inner ear (Ortolano et al. 2008, Shibata et al 2009) and may also be useful in treating hereditary deafness and balance disorders. We also are working with both intramural and extramural researches to test the effect of local expression of immunomodulatory proteins on animal models of Sjogrens Syndrome to evaluate them as potential therapies for this disease. In addition through funding received from the office of the scientific director as part of the salivary gland biology initiative, we have begun to collaborate with researchers in the Laboratory of Cell and Developmental Biology, NIDCR at identifying AAV vectors for gene transfer to the developing salivary gland. We believe that just as our characterizations of AAV4, AAV5, and BAAV have advanced the field of gene therapy, the development of new vectors also will have an impact on other gene therapy applications, as well as our understanding of parvovirus biology. The first isolate we have extensively characterized with tropism for secretory epithelia is an isolate found as a contaminant in a Simian Adenovirus 17 (SV17) sample obtained from vervet monkeys. We have termed this isolate AAV12. In contrast to all other reported AAVs, AAV12 cell attachment and transduction does not require cell surface sialic acids or heparan sulfate proteoglycans (HSPG). Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. Feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications (Schmidt et al. 2008). In addition to providing new vectors for gene transfer, study of these new isolates has provided important information regarding structural domains on the surface of the virus While natural isolates have served as a rich source of vectors for gene transfer, an alternative approach would be to develop vectors with defined tropism. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. Previously we have reported the structure of the AAV5 capsid and more recently we have solved the structure for one of the most antigenically distinct serotypes, AAV4, to 3- resolution. Structural comparison of AAV4 to AAV2 shows conservation of core beta strands and helical secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations, some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes. To date over 100 AAV capsid sequences have been cloned from a variety of sources but we have very little understanding of their host cell requirements for entry and even less of an understanding of which regions on the surface of the particles are involved in entry. We recently reported the identification of a new heparin dependent transduction domain on the surface of AAV(VR-942) that is distinct from the heparin binding site in AAV2 (Schmidt 2008). This is only the second domain on the surface of any AAV particle for which a receptor function has been mapped. Mapping the heparin binding domain on AAV2 has allowed for this region to be deleted and the tropism of these particles to be retargeted. This finding has had a major impact on the field and now knowledge routinely used by several labs. Further analysis of the AAV(VR-942) heparin transduction region is likely to also lead to improvements in gene transfer vectors. 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. We have demonstrated that delivery of immunomodulatory proteins to the salivary glands of a mouse model of Sjogrens Syndrome can amelorate the gland dysfunction in these animals. In the past we have used female non-obese diabetic (NOD) mouse to model the human disease. While these mice develop exocrine gland infiltrates and, as in SS, a loss of salivary flow that is age and gender dependent, they also develop insulin dependent type 1 diabetes which complicates the study of Sjogrens Syndrome. Furthermore, maintenance of longitudinal stability with respect to onset of the Sjogrens Syndrome phenotype has been reported as problematic. Currently other 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 developed and study the salivary gland activity, histopathology, and autoantibody levels in a mouse that is transgenic for expression of Il12 in the thyroid (Vosters et al 2009). 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. 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. We will continue to identify the domains critical for these interaction on the virus surface, as well as to identify new AAV isolates that maybe useful for gene therapy applications.

Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2009
Total Cost
$1,907,272
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
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Corden, A; Handelman, B; Yin, H et al. (2017) Neutralizing antibodies against adeno-associated viruses in Sjögren's patients: implications for gene therapy. Gene Ther 24:241-244
Alevizos, I; Zheng, C; Cotrim, A P et al. (2017) Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction. Gene Ther 24:176-186
Zeng, Mei; Szymczak, Mitchell; Ahuja, Malini et al. (2017) Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice. Gastroenterology 153:1148-1159
Alevizos, I; Zheng, C; Cotrim, A P et al. (2017) Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands. Oral Dis 23:337-346
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Fong, Shelley; Chiorini, John A; Sneyd, James et al. (2017) Computational modeling of epithelial fluid and ion transport in the parotid duct after transfection of human aquaporin-1. Am J Physiol Gastrointest Liver Physiol 312:G153-G163
Chiorini, John A (2016) And one to bind them all. Oral Dis :
Lai, Zhennan; Yin, Hongen; Cabrera-Pérez, Javier et al. (2016) Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice. Proc Natl Acad Sci U S A 113:5694-9

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