Ttsa Phase I Awards

Cutaneous wounds of a chronic nature affect ~6.5 million patients in the US, and the clinical treatment of these wounds imposes a >$25 billion annual burden on the healthcare system [1]. Patients with diabetes are at increased risk for developing non-healing wounds. A variety of factors likely contribute to the predisposition of diabetic patients to develop non-healing wounds including neuropathy, vasculopathy, as well as the underlying endocrine dysfunction that results in elevated glucose levels. Both published and preliminary data from the Rubenstein lab suggests that increased intracellular O-glycosylation of proteins catalyzed by the nucleocytoplasmic enzyme O-GlcNAc transferase (OGT) contributes to delayed wound healing in diabetic skin wounds and that OGT knockdown accelerates wound healing. The Bear lab has developed a model of cutaneous wound healing that can be monitored in detail at the cellular and subcellular level using multiphoton microscopy to perform intravital serial imaging of wounded skin in animal models. We propose to combine forces to bring a new technical approach (intravital imaging) to an important and understudied clinical problem and test the hypothesis that inhibiting OGT is an effective means to accelerate cutaneous wound healing in diabetic animal models. Translational Impact: This is a high risk, high impact study that has the potential to lead directly to translational studies in patients with diabetic skin wounds. We will test the hypothesis that down-regulating OGT activity by topical application of OGT antisense oligonucleotides (OGT antisense ODNs) to skin wound sites will accelerate healing in normal and diabetic wounds. If this treatment does indeed accelerate cutaneous wound healing, it could positively impact millions of patients and reduce an enormous drain on the healthcare system. miR-29 as a Novel Biomarker and Regulator of Aging Mohanish Deshmukh, PhD (Cell Biology and Physiology), Charles Cairns, MD (Emergency Medicine), Praveen Sethupathy, PhD (Genetics), Ned Sharpless, MD (Medicine and Genetics) Abstract: Aging is a complex process that invariably leads to dysregulation and loss of function of various tissues and eventual death. Despite its clinical significance, Aging is a complex process that invariably leads to dysregulation and loss of function of various tissues and eventual death. Despite its clinical significance, however, only a relatively small number of genes that play a direct role in aging have been identified. The focus of our proposal is to investigate microRNA-29 (miR-29) both as a biomarker of aging and as a key regulator of aging. miR-29 is one of very few genes that is upregulated during normal and accelerated aging. miR-29 levels are also elevated during cellular senescence, a cellular marker of aging, and miR-29 inhibition reduces senescence in cell culture studies. Our hypothesis is that miR-29 could be developed not only as a valuable biomarker of aging, but its inhibition could also delay aging in patients with progeria (accelerated aging). However, pursuing these goals requires a team that brings interdisciplinary expertise. Our lab (Deshmukh) has teamed with the Sharpless, Cairns and Sethupathy labs and we are collectively committed to achieving the following in Phase I of this TTSA proposal: i) Evaluating miR-29 as a biomarker of aging in mice and humans; and ii) Determining whether miR-29 knockdown delays aging in models of progeria. Translational Impact: Aging syndromes cause considerable morbidity and mortality with no known cures. Our focus is to investigate the specific importance of a microRNA, miR29, in aging. Our study has significant translational impact for both normal and premature aging because it addresses two important questions: Is miR-29 a reliable and robust biomarker for aging? Can miR-29 inhibition delay pathological aging? Discovering the Mechanisms of Targeted Enhancement of Brain Activity and Cognition by BrainStimulation using Intracranial Electrophysiology in Epilepsy Patients Flavio Frohlich PhD (Psychiatry) and Haewon Shin MD (Neurology) Abstract: Brain stimulation to modulate brain network dynamics represents a promising Brain stimulation to modulate brain network dynamics represents a promising alternative to pharmacological approaches for the treatment of disorders of the central nervous system. In this novel approach, electric current is used to directly interact and shape the activity of large networks of neurons that are engaged in aberrant dynamics mediating neurological and psychiatric symptoms. Today’s best brain stimulation paradigms are static and mostly determined by clinical intuition instead of being based on a mechanistic understanding of how the applied stimulation interacts with brain activity. With this proposal, we will open the door to a new area of clinical brain stimulation that is based on rational design through mechanistic understanding of the interaction between applied stimulation, brain activity, and cognition. To this end, we propose a novel collaboration between a basic scientist and a clinical scientist to leverage invasive intracranial monitoring of brain activity in patients with intractable epilepsy as a unique window into (1) normal and pathological brain function and (2) the response to brain stimulation and its effect on cognition. Our long-term goal is to understand the oscillation structure of human cortical network activity during cognition and to use rational design to develop brain stimulation paradigms that modulate neuronal network oscillations for the treatment of cognitive impairment. Translational Impact: The goal of the proposed project is to understand the mechanisms by which brain stimulation alters cortical network activity and how such perturbations modulate cognition. Such mechanistic understanding of the interaction between brain stimulation, network dynamics, and cognition will enable the rational design of (non-invasive) brain stimulation paradigms that direct target network-level pathologies that play a causal role in a large number of neurological and psychiatric disorders such as schizophrenia, autism, and epilepsy. Stationary Chest Tomosynthesis for Imaging Young Cystic Fibrosis Patients Yueh Z. Lee, MD, PhD (Radiology), Otto Zhou, PhD (Physics & Astronomy), Jianping Lu, PhD (Physics & Astronomy), Marianne Muhlebach, MD (Pediatric Pulmonology), Scott Donaldson, MD (Pulmonary Medicine), Jennifer Goralski, MD (Pulmonary Medicine), Richard Boucher, MD (Pulmonary Medicine), Scott Randell, PhD (Cell Biology and Physiology) Abstract: Although cystic fibrosis disease presents at an early age, a very limited tool set is available to assess young patients. Some have used or proposed the use of computed tomography (CT) in intervention trials, however, the significant radiation dose limits generalizability to other patients and the potential for longitudinal studies. Digital chest tomosynthesis (DCT) is an imaging technique that improves the visibility of the anatomical structures such as the airways and lung parenchyma. Multiple projections of the patient are acquired at different angles and reconstructed into a pseudo-3D volume, which has better resolution than CT in the slice plane, but at a significantly lower radiation dose. Commercially available DCT systems require the translation of a bulky x-ray tube across the length of the patient’s torso, resulting in motion blur and requiring significant imaging time. Our team has invented a multi-beam x-ray source that enables the rapid firing of multiple, individually controllable x-ray beams distributed over a long linear array. The carbon nanotube (CNT) based xray beams may also be readily synchronized to any external signal, such as a respiration trace. Thus the CNT x-ray source offers significant potential advantages for chest tomosynthesis, especially in a technically challenging population such as infants, at a potentially significantly lower radiation dose and lower cost as compared to conventional CT. The goal of this phase I study is to refine the physiological phantom models and evaluate the existing CNT linear source array in these models and ex-vivo human lungs of cystic fibrosis patients. Although cystic fibrosis disease presents at an early age, a very limited tool set is available to assess young patients. Some have used or proposed the use of computed tomography (CT) in intervention trials, however, the significant radiation dose limits generalizability to other patients and the potential for longitudinal studies. Digital chest tomosynthesis (DCT) is an imaging technique that improves the visibility of the anatomical structures such as the airways and lung parenchyma. Multiple projections of the patient are acquired at different angles and reconstructed into a pseudo-3D volume, which has better resolution than CT in the slice plane, but at a significantly lower radiation dose. Commercially available DCT systems require the translation of a bulky x-ray tube across the length of the patient’s torso, resulting in motion blur and requiring significant imaging time. Our team has invented a multi-beam x-ray source that enables the rapid firing of multiple, individually controllable x-ray beams distributed over a long linear array. The carbon nanotube (CNT) based xray beams may also be readily synchronized to any external signal, such as a respiration trace. Thus the CNT x-ray source offers significant potential advantages for chest tomosynthesis, especially in a technically challenging population such as infants, at a potentially significantly lower radiation dose and lower cost as compared to conventional CT. The goal of this phase I study is to refine the physiological phantom models and evaluate the existing CNT linear source array in these models and ex-vivo human lungs of cystic fibrosis patients. Translational Impact: Successful adaptation of the stationary digital chest tomosynthesis imaging system would enable significantly earlier diagnosis of lung pathology in the very young pediatric cystic fibrosis population. Our tomosynthesis system will significantly reduce radiation dose with potentially higher in-plane image quality as compared to conventional CT. This, in turn, could serve as a basis for future clinical trials with focused interventions at a much earlier age. Furthermore, the technology could improve evaluation of disease exacerbations with lower radiation to the patient. Intestinal epithelial cell culture systems to advance personalized research and therapy in Cystic Fibrosis (CF) and other gastrointestinal (GI) diseases. P. Kay Lund, PhD (Cell Biology and Physiology), Ric Boucher, MD (Pulmonary Medicine), Scott Randell, PhD (Cell Biology and Physiology, Evan Dellon, MD, MPH (Gastroenterology), Shengli Ding, PhD (Cell Biology and Physiology), Raluca Dumitru, MD, PhD (Genetics), Martina Gentzsch, PhD (Cell Biology and Physiology) Abstract: Cystic Fibrosis (CF) affects approximately one in 3,500 children born in the USA and leads to lifelong, chronic airway disease and clinical manifestations in other organs including the gastrointestinal (GI) tract. CF results from multiple classes of mutation in CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Particular mutations differentially affect CFTR expression levels, trafficking, function, or turnover. Multiple classes of CFTR modulating drugs that target mutation-specific deficits in CFTR are approved or in clinical trial. However, there Cystic Fibrosis (CF) affects approximately one in 3,500 children born in the USA and leads to lifelong, chronic airway disease and clinical manifestations in other organs including the gastrointestinal (GI) tract. CF results from multiple classes of mutation in CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Particular mutations differentially affect CFTR expression levels, trafficking, function, or turnover. Multiple classes of CFTR modulating drugs that target mutation-specific deficits in CFTR are approved or in clinical trial. However, there is still a critical need for improved, personalized therapies for CF and systems to test them. Intestinal epithelial cells (IEC) derived from rectal biopsies may meet this need. Rectal biopsies are easily obtained by minimally invasive procedures. Compared with airway epithelia, IEC express much higher levels of CFTR offering advantages of greater signal to noise and lower technical variability in assays of CFTR function or drug responses (1, 2). Rectal biopsies are still limited however, by the small amounts of tissue. The ability to culture IEC from individual CF patients would permit previously impossible testing of multiple drugs in the same patient, and molecular or biochemical studies to define mechanisms underlying inter-patient variability in CFTR expression, function or drug response. This proposal will test the hypothesis that rectal biopsy-derived human intestinal epithelial cells (hIEC) provide a powerful new platform to accelerate personalized research and therapeutic approaches in CF. This Phase-1 proposal aims to provide proof of principle that human IEC derived from rectal biopsies using new epithelial culture systems, provide a valid new platform to study CFTR function and drug responses in individual patients. Translational Impact: The CF phenotype is highly variable across individual patients. Variability reflects different classes of mutations in the CFTR gene, inter-patient differences in the impact of the same CFTR mutation and complex effects of significant modifier genes or environmental exposures. Consequently, disease variability and genotype-drug response phenotypes are difficult to predict a priori. Thus, there is a critical need to tailor optimal drug therapy to individual CF patients. Because of difficulties in studying diseased (or healthy) epithelial cells in culture, such an approach has been previously impossible. This study will test new methods to generate cultures of human intestinal epithelial cells (hIEC) from rectal biopsies from individual CF patients, or healthy controls. Molecular and functional studies will provide proof of principle that hIEC permit valid and sensitive testing of responses to approved or experimental CFTR modulating drugs. Success in these studies will provide a new platform to overcome a previously impassable roadblock in our ability to implement and improve individualized approaches to treat CF. If successful in this feasibility study, we will utilize hIEC as a new platform for mechanistic research and testing of novel individualized therapies in larger cohorts of CF patients, and other debilitating gastrointestinal diseases linked to genetic or functional defects in IEC. Identifying Geneticand Microbial-based Molecular Markers of Inflammatory Bowel Disease Shehzad Sheikh, MD (Gastroenterology) and Terry Furey, PhD (Genetics) Abstract: Inflammatory Bowel Diseases (IBD), composed of Crohn’s disease (CD) and ulcerative colitis (UC), result from an inappropriately regulated inflammatory response to the enteric microbiota in a genetically susceptible host. Converging human genetic and functional findings have linked many single nucleotide polymorphisms (SNPs) to Inflammatory Bowel Disease (IBD). However, which of these SNPs is causal and how they influence IBD pathogenesis is unknown. Most IBD-associated SNPs fall into non-coding regions of the genome and are completely unannotated with respect to function. The overall objective of this proposal is to understand how genetic variation affects chromatin structure leading to changes in gene regulatory activity in the presence of particular microbial communities in colon tissue obtained from patients with CD. Using the Inflammatory Bowel Diseases (IBD), composed of Crohn’s disease (CD) and ulcerative colitis (UC), result from an inappropriately regulated inflammatory response to the enteric microbiota in a genetically susceptible host. Converging human genetic and functional findings have linked many single nucleotide polymorphisms (SNPs) to Inflammatory Bowel Disease (IBD). However, which of these SNPs is causal and how they influence IBD pathogenesis is unknown. Most IBD-associated SNPs fall into non-coding regions of the genome and are completely unannotated with respect to function. The overall objective of this proposal is to understand how genetic variation affects chromatin structure leading to changes in gene regulatory activity in the presence of particular microbial communities in colon tissue obtained from patients with CD. Using the FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) chromatin accessibility assay, we will catalog regulatory elements in colon tissue from patients with CD. We will also generate gene expression (RNA-seq) and microbiome data in the same samples and will test all of these for relationships with SNPs to elucidate the functional significance of these regions in maintaining intestinal immune homeostasis. In Aim 1, we will determine chromatin status using FAIRE-seq and gene expression using RNA-seq throughout the genome in colon tissue (N=10) and lamina propria immune cells (CD3+ T cells, CD20+ B cells, CD33+ Monocytes) and EpCAM Epithelial cells harvested from genotyped patients with CD. We will also determine the microbial composition from these biopsy samples using metagenomics studies. In Aim 2, we will develop computational tools to integrate the results from these experiments with clinical patient phenotypes. Our central hypothesis is that profiling open chromatin will identify genetically driven regulatory elements that are active in these tissues and that are relevant to CD pathology. SNPs that occur within these are candidate causal SNPs that are more likely to directly mediate chronic intestinal inflammation. This project is the first step in a continuum of research that is expected to study the influence these nucleotide sequences have on the phenotype of human CD as individual variations in disease extent, severity and response to therapy. Translational Impact: Current therapies for IBD are limited, largely due to our lack of understanding of the genetic and molecular contributions to this disease. Through this proposal, we expect to reveal how the intestinal microenvironment in concert with specific nucleotide sequences (coding and non-‐coding) induces chromatin modification leading to individual variations in IBD phenotype in terms of disease extent, severity and response to therapy. Histone modifications in the clearance of chronic viral infections Maureen Su, MD (Microbiology and Immunology),Mike Cohen, MD (Infectious Disease), George Fedoriw, MD (Pathology), Terry Magnuson, PhD (Genetics), Jason Whitmire, PhD (Genetics) Abstract: Histone modifications play a central role in regulating gene expression; the goal of this project is to understand how histone modifications may alter immune function and chronic viral clearance. To elucidate the role of histone modifications in T cells, our team engineered mice with T cell-specific histone demethylase deficiency and showed that these mice have an inability to clear chronic viral infection. Viral persistence was associated with altered germinal centers and deficiency of serum immunoglobulin necessary for viral clearance. These findings result from a primary defect in T follicular helper (Tfh) cells, a CD4+ T cell subset specialized to provide B cell help. Tfh cells are reduced in number and lack expression of ICOS, SLAMF6, and IL6R, which demonstrates a Histone modifications play a central role in regulating gene expression; the goal of this project is to understand how histone modifications may alter immune function and chronic viral clearance. To elucidate the role of histone modifications in T cells, our team engineered mice with T cell-specific histone demethylase deficiency and showed that these mice have an inability to clear chronic viral infection. Viral persistence was associated with altered germinal centers and deficiency of serum immunoglobulin necessary for viral clearance. These findings result from a primary defect in T follicular helper (Tfh) cells, a CD4+ T cell subset specialized to provide B cell help. Tfh cells are reduced in number and lack expression of ICOS, SLAMF6, and IL6R, which demonstrates a critical role for histone modifications in the control of Tfh signature gene expression. However, the specific mechanism by which histone modfications regulate Tfh gene expression is unclear, and it is unknown whether histone modifications in Tfh cells underlie viral persistence in humans. Based on our preliminary data, we hypothesize that histone demethylases are important in Tfhmediated viral clearance by altering Tfh transcription factor activity in both mice and humans. This hypothesis will be tested in patients with persistent viral infections and in patients with mutations in histone demethylase genes. Translational Impact: Our preliminary studies in mice delineate a novel link between UTX and Tfh function in the clearance of chronic virus infection and have a number of important translational implications. First, chronic viral infections such as HBV, HCV, and HIV, remain a major public health threat and our findings in mouse models point to UTX as a key regulator of immune-mediated viral clearance. Recent analyses show a direct correlation between the frequency of Tfh cells and the presence of broadly neutralizing antibody responses against HIV; conversely, patients with poor immune control of HIV show diminished Tfh formation. The studies proposed here will provide clinical data that UTX may also impact viral clearance in humans. Second, inhibitors of jumonji family members are currently in development for the treatment of autoimmune and inflammatory diseases. Our studies will provide important information regarding how blocking UTX activity impacts viral clearance. Finally, our studies will clarify whether immune dysfunction exists in Kabuki Syndrome and provide a potential molecular mechanism for recurrent ear infections in Kabuki Syndrome. Thus, we believe that this proposal will have direct clinical impact on multiple levels.