Adeno-associated Virus Biology And Utilization For Gene Transfer

National Institute Of Dental & Craniofacial Research

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Abstract

Application of these vectors for diseases with unmet clinical need are ongoing. By utilizing phylogenetic analysis we have begun to identify AAVs with distinct tropisms and transduction activities. One recent isolate, 44.9, has shown low levels of neutralizing antibodies in the general population as well as transduction of salivary gland acinar cells of humans and mice (DiPasquale 2020). In collaboration with Drs. Sanford and Shannon Boye we have reported that follow subretinal injections we can use this vector for effective gene delivery to treat mouse models of LCA1 deficiency (Boye et al 2020). Many viruses are able to infect salivary glands, be shed into saliva, and induce pathology. The cellular factors that drive the infection are poorly defined. Given the global impact of the pandemic we worked to develop single-cell RNA sequencing data-sets from human minor salivary glands and gingiva to identify cell clusters that might aid in understanding transmission of SARS-CoV-2. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae and infection could be confirmed in COVID-19 autopsy tissues and virus could be detected in saliva. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission. The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. In collaboration with researchers at the FDA we worked to use Crispr-Cas9 technology to remove the T-antigen from popular producer cells and tests their utility in AAV vector production (Bae 2020). Despite the absence of the SV40 T antigen, we found that the knockout clones retained the high-titer phenotype of HEK293T cells for lentiviral vector production. When producing adeno-associated virus (AAV)2 vectors, the HEK293T knockout clones performed similarly to their parental cell clones. A limiting factor in the treatment of prostate specific membrane antigen (PSMA) positive prostate cancer is the secondary toxicity associated with PSMA expression on other tissues such as the salivary gland. In order to address this limitation, we have worked to study animal models of salivary gland PSMA expression with the goal of using them for preclinical studies aimed at limiting this toxicity (Roy et al 2021). Sjogrens syndrome (SS) 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. Cell death is often found associated with autoimmune disease development but our understanding of the mechanism associated with this aspect of disease is poorly understood. Furthermore, the balance between cell death and survival is a critical parameter in the regulation of cells and the maintenance of homeostasis in vivo. Autophagy is emerging as a regulator of this balance and whether autophagy functions primarily in cell survival or cell death is a critical question yet to be answered. We have begun to investigate the role of autophagy and potential regulators of this process first in literature review (Noguchi et al 2020) and moving experimentally with the development of RNA aptamers that regulate the interaction of phosphoinositides that are critical for the initiation of autophagy (Donia et al 2019). In an ongoing project we have used this aptamer as a consensus sequence for identifying cellular long noncoding RNA molecules that could regulate this process and affect the development of autoimmune diseases. Aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjogrens syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjogrens syndrome cases suggesting a role for the lysosome in Sjogrens syndrome. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and -fodrin protein, common autoantigens in Sjogrens syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease. Furthermore, additional work in mice using AAV vectors that encode LAMP3 introduced into the salivary gland showed that LAMP3 expression could initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS (Nakamura 2021). This model is currently being used to test novel therapies for Sjogrens syndrome as well as investigate the potential for better targeting of current therapies to specific patient sub-groups. Although many genome-wide association studies have revealed disease-associated risk alleles, the functions of the majority of these alleles are unclear. In collaboration, we have investigated the role of one risk allele GTF2I (Shimoyama et al 2021). We found that the risk alleles of GTF2I SNPs increased GTF2I expression and enhanced nuclear factor-kappa B (NF-B) activation in human salivary gland cells via the NF-B p65 subunit. Indeed, the knockdown of GTF2I suppressed inflammatory responses in mouse endothelial cells and in vivo. Conversely, the over-expression of GTF2I enhanced NF-B reporter activity depending on its p65-binding N-terminal leucine zipper domain. GTF2I is highly expressed in the human salivary gland cells of SS patients expressing the risk alleles. Consistently, the risk alleles of GTF2I SNPs were strongly associated with activation of the IL-6 amplifier, which is hyperactivation machinery of the NF-B pathway, and lymphoid infiltration in the salivary glands of SS patients. These results demonstrated that GTF2I expression in salivary glands is increased in the presence of the risk alleles of GTF2I SNPs, resulting in activation of the NF-B pathway in salivary gland cells.