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Principal Investigator: Jay Chiorini
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2020
Award: $2,447,071
Funding agency: National Institute of Dental and Craniofacial Research
Application of these vectors for disease with unmet clinical need are ongoing. We have previously shown that in vitro transduction with a novel AAV referred to as 44.9 has unique cell tropism and can transduce acinar cells in the parotid and sublingual glands (DiPasquale manuscript under revision). 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). Interesting this vector shows enhanced distribution in nonhuman primates and is being evaluated for rapid advancement into clinical trials. Ongoing projects are looking at liver gene transfer as well as the glycan binding requirements for transduction.
As the field of gene therapy advances into new disease models depending on the transgene product, this could induce proapoptotic, cytostatic, or other unknown effects that interfere with producer cell function and, therefore, reduce viral vector yield. This can be a major limitation when trying to characterize poorly described genes. We have developed a novel vector packaging approach that uses shRNA encoding plasmids cotransfected during packaging to limit the expression of the cytotoxic transgene product (Guimaro et al 2020). The approach is simple, versatile, does not require modification of the vector plasmid, and should be easily adaptable to almost any transgene with minimal cost. We are currently applying this technology to produce AAV vectors that encoded proteins associated with the induction of cell death in Sjgrens patients so we can better study there function and develop therapeutic interventions.
Our clinical trial to use localized gene therapy for radiation induced xerostomia is progressing and the technology that is being developed is having a clinical impact on the treatment of other diseases. A limiting factor in the treatment of 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 2020). Future interventional studies will be led by Blake Warner in his new position as an ACI and will focus on using retroductal cannulation of the salivary glands.
Sjgrens 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. 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 (Vivio et al 2019). Although the underlying trigger(s) of SS remain unknown, genetic susceptibility when combined with environmental exposures (e.g. bacterial or viral infections) likely contributes to disease initiation. In 2017 we were awarded a DDIR innovation award which was renewed in 2019 to investigate the role of non-coding DNA in the regulation of gene expression on a genome level scale. This was a collaborative effort with both NEI and NHGRI. As part of this project we utilized the NIH Biowulf supercomputing cluster to assess the impact of parameter selection on biological reproducibility and ChIP-seq recovery by analyzing 4560 pipeline configurations. We have worked to develop an optimized pipeline for analysis and made this available for other researchers (Pranzatelli et al 2018). In 2020 we have begun to apply this pipeline to well defined genetic disorders with the goal of moving to more complex diseases like SS. Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction (Schnappauf et al. 2020). However, in a case study of a 14 yr old patient seen by colleagues at NHGRI, exome sequencing identified heterozygosity for the known pathogenic variant but no second pathogenic variant could be identified. Subsequent genome analysis with our current pipeline identified a canonical splice site variant within the 5UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. Although genomic sequencing is a powerful tool for investigating genetic disease, tools for identifying pathogenic variants are still needed and may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods, such as our pipeline, to make a definitive molecular diagnosis. We are currently investigating unexplained cases with a similar rare genetic disease, cystinosis, a recessive disease that results in an inability to transport cystine.
In order to understand the environment of the salivary gland that might contribute to SS we have developed the first epigenetic road maps of the salivary gland (Michael et al 2019) and have recently competed RNAseq data as well as ATAC sequencing on minor salivary glands from SS and HV samples and will work to integrate the data to look for potential regulatory variants and genes predictive of specific clinical phenotypes.
Our previous work on differentially expressed genes associated with SS identified that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction (Yin et al 2013). In our recent work we examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested obtained from the SICCA organization and the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models (Yin et al 2020). Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.
Cell death is an 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 to 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).
This project involves research on novel coronavirus, COVID-19, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.
Terms: <2019 novel coronavirus><2019-nCoV><5 year old><5 years of age><AAV vector><ATAC><Acinar Cell><Aciner Cells><Address><Adeno-Associated Viruses><Affect><Angiitis><Animal Model><Animal Models and Related Studies><Apoplexy><Asialia><Autoimmune Diseases><Autophagocytosis><Autoregulation><Award><BMP-6><Bacterial Infections><Binding><Biological><Biology><Body Tissues><Brain Vascular Accident><COVID-19><COVID19><Cannulations><Case Study><Cell Body><Cell Communication and Signaling><Cell Death><Cell Death Induction><Cell Function><Cell Growth and Maintenance><Cell Maintenance><Cell Process><Cell Signaling><Cell Survival><Cell Viability><Cell physiology><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cerebral Stroke><Cerebrovascular Apoplexy><Cerebrovascular Stroke><ChIP Sequencing><ChIP-seq><Chromosome Mapping><Chronic><Clinical><Clinical Trials><CoV emergence><Collaborations><Complex><Cystine><Cystinosis><Cytostatic Agents><Cytostatic Drugs><Cytostatics><DNA><DNA Therapy><Data><Deoxyribonucleic Acid><Dependoparvovirus><Dependovirus><Development><Diagnostic Method><Diagnostic Procedure><Diagnostic Technique><Diamond-Blackfan anemia><Dideoxy Chain Termination DNA Sequencing><Disease><Disease Vectors><Disease model><Disorder><Dysfunction><Environment><Environmental Exposure><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Equilibrium><Exocrine Glands><Exons><FOLH><FOLH1><FOLH1 gene><Fathers><Female><Fever><Folate Hydrolase 1><Functional RNA><Functional disorder><Future><GCP2><Gene Action Regulation><Gene Delivery><Gene Expression><Gene Expression Regulation><Gene Localization><Gene Mapping><Gene Mapping Genetics><Gene Regulation><Gene Regulation Process><Gene Transfer><Gene Transfer Clinical><Genes><Genetic Diseases><Genetic Intervention><Genetic Predisposition><Genetic Predisposition to Disease><Genetic Susceptibility><Genome><Genomics><Glutamate Carboxypeptidase II><Glycans><Goals><Hematology><Heterozygote><Homeostasis><Hyposalivation><Immune Cell Activation><In Vitro><Infiltration><Inherited Predisposition><Inherited Susceptibility><Inositide Phospholipids><Inositol Phosphoglycerides><Inositol Phospholipids><Intervention Studies><Intracellular Communication and Signaling><Investigators><L-Cystine><LPTN><Lacrimal Glands><Lacrimal gland structure><Linkage Mapping><Liver><Lymphoid Cell><Malignant Tumor of the Prostate><Malignant neoplasm of prostate><Malignant prostatic tumor><Manuscripts><Maps><Mice><Mice Mammals><Minor Salivary Glands><Minor salivary gland structure><Modification><Molecular Diagnosis><Molecular Interaction><Mouth Dryness><Murine><Mus><N-Acetylated Alpha-Linked Acidic Dipeptidase 1><NAALAD1><NAALADase I><NHGRI><NIH><National Center for Human Genome Research><National Human Genome Research Institute><National Institutes of Health><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><PSM><PSMA><Parotid><Parotid Gland><Pathogenicity><Pathway interactions><Patients><Phosphatidyl Inositol><Phosphatidylinositols><Phosphoinositides><Phosphorylation><Physiological Homeostasis><Physiopathology><Plasmid Cloning Vector><Plasmid Vector><Plasmids><Polysaccharides><Population><Position><Positioning Attribute><Prevalence><Process><Prostate CA><Prostate Cancer><Prostate-Specific Membrane Antigen><Prostatic Cancer><Protein Phosphorylation><Proteins><PtdIns><Pyrexia><RNA><RNA Gene Products><RNA Seq><RNA Splicing><RNA sequencing><RNAseq><Recovery><Regulation><Reporting><Reproducibility><Research><Research Personnel><Researchers><Review Literature><Ribonucleic Acid><Role><SARS Virus><SARS corona virus><SARS coronavirus><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><SCM-1><SCM-1a><SCM1><SCYC1><Saliva><Salivary><Salivary Glands><Salivary Glands Head and Neck><Sampling><Sanger Sequencing><Serotyping><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe acute respiratory syndrome coronavirus 2><Siblings><Signal Transduction><Signal Transduction Systems><Signaling><Sister><Site><Splicing><Stroke><Subcellular Process><Sublingual Gland><Sublingual Salivary Gland><Submandibular gland><Submaxillary Gland><Supercomputing><Symptoms><Syndrome><Technology><Testing><Therapeutic Intervention><Tissues><Total Human and Non-Human Gene Mapping><Toxic effect><Toxicities><Transgenes><Treatment Efficacy><Tropism><United States><United States National Institutes of Health><Untranslated RNA><Variant><Variation><Vasculitis><Viral Diseases><Viral Vector><Virus><Virus Diseases><Woman><Work><Wuhan coronavirus><XCL1><XCL1 gene><Xerostomia><Yin><adeno associated virus group><adeno-associated viral vector><adeno-associated virus vector><adenosine deaminase deficiency><age 5 years><analysis pipeline><aptamer><aptyalism><autoimmune disorder><autophagy><bacteria infection><bacterial disease><balance><balance function><biological signal transduction><bone morphogenetic protein 6><brain attack><case report><cerebral vascular accident><cerebrovascular accident><chromatin immunoprecipitation-sequencing><clinical phenotype><cohort><computational tools><computerized tools><corona virus disease 2019><coronavirus disease 2019><coronavirus emergence><cost><cystine storage disease><cytotoxic><developmental><differential expression><differentially expressed><disorder model><dry eye><dry mouth><early onset><emergent CoV><emergent coronavirus><emerging CoV><emerging coronavirus><exome sequencing><exome-seq><eye dryness><febrile><febris><five year old><five years of age><functional genomics><gene testing><gene therapy><gene-based testing><gene-based therapy><gene-radiation therapy><genetic condition><genetic disorder><genetic etiology><genetic mapping><genetic mechanism of disease><genetic testing><genetic therapy><genetic vulnerability><genetically predisposed><genome analysis><genomic therapy><hepatic body system><hepatic organ system><heterozygosity><immune activation><in vivo><inflammation marker><inflammatory marker><inhibitor><inhibitor/antagonist><innovate><innovation><innovative><intervention efficacy><intervention research><intervention therapy><interventional research><interventional study><interventions research><loss of function><model of animal><model organism><mouse model><murine model><nCoV><necrocytosis><new CoV><new coronavirus><non-human primate><noncoding><nonhuman primate><novel><novel CoV><novel coronavirus><pathophysiology><pathway><pre-clinical study><preclinical study><rare genetic disease><rare genetic disorder><severe acute respiratory syndrome-CoV><shRNA><short hairpin RNA><small hairpin RNA><social role><subretinal injection><super computing><therapeutic efficacy><therapeutically effective><therapy efficacy><tool><transcriptional differences><transcriptome sequencing><vasculitides><vector><vgr-1 protein><viral infection><virus infection><virus-induced disease>