Salivary Glands and Viral Pathogenesis.

The oral cavity is an epidemiologically relevant route of viral transmission due to the shedding of viruses in saliva. With advancements in salivary diagnostics, an increasing number of viruses have been detected. However, the anatomic source of virus in saliva is still largely unknown. Some viruses have a well-established tropism for the salivary glands (SGs), and recent studies have emphasized the importance of the glands as potential reservoirs for infectious viruses. Viral infections of the SGs have been linked to acute and chronic SG pathology and may be associated with SG dysfunction, with phenotypes similar to those seen in SjÖgren's disease (SjD), an autoimmune condition that affects the salivary and lacrimal glands. Understanding the breadth of viruses that infect the SG and the conserved or distinct host responses to these infections may provide insights into the pathogenesis of virus-mediated SG diseases. There is a need for further research to fully understand the molecular mechanisms by which viruses enter and replicate in the glands, their physiologic impact on SG function, and whether the SGs can serve as a long-term reservoir for infectious viral particles. The purpose of this review is to highlight a group of viruses that infect the salivary gland: hepatitis C virus, hepatitis D virus, severe acute respiratory syndrome coronavirus 2, enteric viruses, human T-cell leukemia virus type I, human immunodeficiency virus, human cytomegalovirus, and BK polyomavirus. We focus on the effects of viral infection on salivary gland (SG) inflammation, function, and its association with SjD.

Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab.

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

Profiling Autoantibodies against Salivary Proteins in Sicca Conditions.

Salivary gland dysfunction occurs in several autoimmune and immune-related conditions, including Sjögren syndrome (SS); immune checkpoint inhibitor-induced sicca (ICIS) that develops in some cancer patients and is characterized by severe, sudden-onset dry mouth; and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Although subjects with these conditions present with oral dryness and often exhibit inflammatory infiltration of the salivary gland, little is known about the B-cell humoral responses directed against salivary gland protein targets. In this study, autoantibodies were evaluated against Ro52, Ro60, and La, as well as against a panel of 22 proteins derived from the salivary proteome. The tested cohort included healthy volunteers and subjects with SS, ICIS, and APECED without and with sicca. As expected, a high percentage of autoantibody seropositivity was detected against Ro52, Ro60, and La in SS, but only a few ICIS patients were seropositive for these autoantigens. A few APECED subjects also harbored autoantibodies to Ro52 and La, but only Ro60 autoantibodies were weakly associated with a small subset of APECED patients with sicca. Additional testing of the salivary panel failed to detect seropositive autoantibodies against any of the salivary-enriched proteins in the SS and ICIS subjects. However, APECED subjects selectively demonstrated seropositivity against BPI fold containing family A member 1 (BPIFA1), BPI fold containing family A member 2 (BPIFA2)/parotid salivary protein (PSP), and lactoperoxidase, 3 salivary-enriched proteins. Moreover, high levels of serum autoantibodies against BPIFA1 and BPIFA2/PSP occurred in 30% and 67% of the APECED patients with sicca symptoms, respectively, and were associated with an earlier age onset of oral dryness (P = 0.001). These findings highlight the complexity of humoral responses in different sicca diseases and provide new insights and biomarkers for APECED-associated sicca (ClinicalTrials.gov: NCT00001196; NCT00001390; NCT01425892; NCT01386437).

Integrated Epigenetic Mapping of Human and Mouse Salivary Gene Regulation.

Significant effort has been applied to identify the genome-wide gene expression profiles associated with salivary gland development and pathophysiology. However, relatively little is known about the regulators that control salivary gland gene expression. We integrated data from DNase1 digital genomic footprinting, RNA-seq, and gene expression microarrays to comprehensively characterize the cis- and trans-regulatory components controlling gene expression of the healthy submandibular salivary gland. Analysis of 32 human tissues and 87 mouse tissues was performed to identify the highly expressed and tissue-enriched transcription factors driving salivary gland gene expression. Following RNA analysis, protein expression levels and subcellular localization of 39 salivary transcription factors were confirmed by immunohistochemistry. These expression analyses revealed that the salivary gland highly expresses transcription factors associated with endoplasmic reticulum stress, human T-cell lymphotrophic virus 1 expression, and Epstein-Barr virus reactivation. DNase1 digital genomic footprinting to a depth of 333,426,353 reads was performed and utilized to generate a salivary gland gene regulatory network describing the genome-wide chromatin accessibility and transcription factor binding of the salivary gland at a single-nucleotide resolution. Analysis of the DNase1 gene regulatory network identified dense interconnectivity among PLAG1, MYB, and 13 other transcription factors associated with balanced chromosomal translocations and salivary gland tumors. Collectively, these analyses provide a comprehensive atlas of the cis- and trans-regulators of the salivary gland and highlight known aberrantly regulated pathways of diseases affecting the salivary glands.

Neutralizing antibodies against adeno-associated viruses in Sjögren's patients: implications for gene therapy.

One potential setback to the use of gene therapy for the treatment of Sjögren's syndrome is the presence of neutralizing antibodies (nAb) against adeno-associated virus (AAV) serotypes. In order to evaluate the efficacy of this treatment option, nAb titers were measured in both healthy individuals and Sjögren's patients. Several serotypes with known transduction activity in mouse salivary glands were tested and only AAV5 showed a statistically significant change in the prevalence of nAbs between Sjögren's and healthy participants. Both groups showed a higher rate of nAbs for AAV2 compared with most of the other serotypes tested, except for bovine AAV (BAAV). Although a similar rate of seropositivity was seen against BAAV and AAV2, the percentage of samples with high titer was significantly lower with BAAV. Furthermore, the majority of positive samples exhibited low nAb titers in the primary Sjögren's syndrome (pSS) group for all serotypes except for AAV2. AAV5 was the only serotype that showed a statistically significant shift in the percentage of medium or high neutralizing titer. Based on these results, many serotypes are viable vectors in a gene therapy approach and pSS patients do not have a statistically significant higher rate of seropositivity or titer compared with healthy donors.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

Targeting the splicing of mRNA in autoimmune diseases: BAFF inhibition in Sjögren's syndrome as a proof of concept.

BAFF (B-cell-activating factor of the tumor necrosis factor family), a pivotal cytokine for B-cell activation, is overexpressed by salivary gland (SG) epithelial cells in primary Sjogren's syndrome (pSS). ΔBAFF, a physiological inhibitor of BAFF, is a minor alternative splice variant of BAFF. A U7 RNA was reengineered to deliver antisense sequences targeting BAFF splice regions. A major decrease of BAFF messenger RNA (mRNA) and protein secretion, concomitantly with the increase of ΔBAFF mRNA, was observed in vitro. In vivo, SG retrograd instillation of nonobese diabetic mice by the modified U7 cloned into an adeno-associated virus vector significantly decreased BAFF protein expression and lymphocytic infiltrates and improved salivary flow. This study offers a rationale for localized therapeutic BAFF inhibition in pSS and represents a proof of concept of the interest of exon skipping in autoimmune diseases.

TACI-Fc gene therapy improves autoimmune sialadenitis but not salivary gland function in non-obese diabetic mice.

OBJECTIVE: Patients with Sjögren's syndrome (SS) 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Bovine AAV transcytosis inhibition by tannic acid results in functional expression of CFTR in vitro and altered biodistribution in vivo.

Bovine adeno-associated virus (BAAV) can enter a cell either through a transcytosis or transduction pathway. We previously demonstrated that particles entering via the transcytosis pathway can be redirected to transduce the cell by blocking particle exocytosis with tannic acid (TA). To investigate whether this approach is useful in lung gene therapy applications, we tested the effect of TA on BAAV transduction in cystic fibrosis airway epithelia in vitro, and in mouse lung in vivo. Our findings suggest that BAAV transcytosis can occur in vivo and that treatment with TA reduces transcytosis and increases lung transduction. TA treatment did not impair the sorting and the activity of the BAAV expressed cystic fibrosis transmembrane regulator membrane protein.

Temporal changes in salivary glands of non-obese diabetic mice as a model for Sjögren's syndrome.

OBJECTIVE: Non-obese diabetic (NOD) mice develop an autoimmune exocrinopathy that shows similarities with Sjögren's syndrome. They provide an experimental model to study the pathoetiogenesis of this disease. MATERIALS AND METHODS: Salivary gland (SG) function and salivary sodium content were measured in 8-, 12-, 16- and 20-week-old NOD and age-matched CB6 mice. In NOD mice, SG expression of phenotypic cell markers, B cell-stimulating and costimulatory molecules were evaluated. Cytokine levels were measured in serum and SG homogenates. RESULTS: Microscopically evident SG inflammation in NOD mice was preceded by expression of intercellular adhesion molecule 1 on epithelial cells in the presence of macrophages and relatively high levels of cytokines. Next, an influx consisting of mainly T, B, natural killer, plasma and dendritic cells was seen. Most cytokines, except for interleukin (IL)12/IL23p40 and B cell-activating factor, decreased or remained stable over time, while glandular function deteriorated from 16 weeks of age onward compared with CB6 mice. CONCLUSION: Sjögren's syndrome-like disease in NOD mice occurs in multiple stages; immunological and physiological abnormalities can be detected before focal inflammation appears and salivary output declines. Extrapolating this knowledge to human subjects could help in understanding the pathogenesis and aid the identification of potential therapeutic targets.

Viral gene transfer to developing mouse salivary glands.

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.

AAV2-mediated transfer of the human aquaporin-1 cDNA restores fluid secretion from irradiated miniature pig parotid glands.

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands.

OBJECTIVES: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval. RESULTS: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Sjögren syndrome: advances in the pathogenesis from animal models.

Sjögren syndrome is an autoimmune disease characterized by hyposecretion of the lacrimal and salivary glands, resulting in dryness of the eyes and mouth. Individuals may experience primary Sjögren syndrome or a secondary form accompanying another rheumatic autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus. The pathogenic mechanisms of Sjögren syndrome remain largely unknown, in part a consequence of the heterogeneity of the disease. Animal models have shed light on the connections between specific pathways and symptoms, but an ideal system is wanting. Improved disease models will enable a better understanding of Sjögren syndrome, including how immune tolerance is lost and potential therapeutic interventions. Most importantly, an optimal model will enable detection of disease biomarkers, since injury to the salivary glands may precede lymphocytic infiltration. This review aims to characterize available mice models of Sjögren syndrome, including advantages and disadvantages, from the researcher's perspective.

Gene transfer using bovine adeno-associated virus in the guinea pig cochlea.

Gene transfer into the cells of the cochlea is useful for both research and therapy. Bovine adeno-associated virus (BAAV) is a new viral vector with potential for long-term gene expression with little or no side effects. In this study, we assessed transgene expression using BAAV with beta-actin-GFP as a reporter gene, in the cochleae of normal and deafened guinea pigs. We used two different routes to inoculate the cochlea: scala media (SM) or scala tympani (ST). Auditory brainstem response assessments were carried out before inoculation, 7 days after inoculation and immediately before killing, to assess the functional consequences of the treatment. We observed threshold shifts because of the surgical invasion, but no apparent pathology associated with the virus. Fourteen days after the injection, animals were killed and cochleae assessed histologically. Epi-fluorescence showed that BAAV transduced the supporting cells of both normal and deafened animals through SM and ST inoculations. Transgene expression in cells of the membranous labyrinth after ST inoculation is an important outcome because of the greater feasibility of this route for future clinical application. BAAV facilitates efficient transduction of the membranous labyrinth epithelium with minimum pathogenicity and may become clinically applicable for inner ear gene therapy.

Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Enhanced transduction of mouse salivary glands with AAV5-based vectors.

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.

Adeno-associated virus type 4 (AAV4) targets ependyma and astrocytes in the subventricular zone and RMS.

The subventricular zone (SVZ) is one of the neurogenic niches in the adult mammalian brain. The SVZ is of interest for studies on neurogenesis and stem cell therapy. Here, we report specific transduction of ependyma and/or astrocytes by recombinant adeno-associated virus type 4 (AAV4) viral vectors. AAV4 vectors encoding beta-galactosidase or eGFP were injected into the lateral ventricles of neonatal and adult C57BL/6 mouse brains. In addition, SVZ injections were conducted on adult mice. AAV4 vectors show a characteristic transduction of the ependyma independent of delivery route. However, AAV4 virus injected into the SVZ targeted GFAP positive astrocytes forming the glial tube in the SVZ and rostral migratory stream (RMS). Our results introduce AAV4 as a new tool by which to manipulate glial cells in the RMS.