Exact Contract Story

NEW ANIMAL MODEL FOR LATE-ONSET FUCHS ENDOTHELIAL CORNEAL DYSTROPHY - ABSTRACT FUCHS’ ENDOTHELIAL CORNEAL DYSTROPHY (FECD) IS AN INHERITED CORNEAL DISEASE LEADING TO BLINDNESS. LOSS OF CORNEAL ENDOTHELIAL CELLS AND GUTTAE (BUMPS IN DESCEMET’S MEMBRANE) ARE PATHOLOGICAL FECD FEATURES. A DEFICIENCY OF CORNEAL ENDOTHELIAL CELLS (CEC) CAUSES CORNEAL OPACITY DUE TO LOSS OF PUMPING FUNCTION. IN DEVELOPED COUNTRIES, INCLUDING THE U.S., FECD PREVALENCE IS ESTIMATED TO BE 4%. CORNEAL TRANSPLANTATION IS THE ONLY TREATMENT FOR ADVANCED FECD PATIENTS. ALTHOUGH CORNEAL TRANSPLANTATION IS HIGHLY SUCCESSFUL, THE SHORTAGE OF DONOR CORNEAS MAKES PHARMACOLOGIC THERAPIES FOR FECD AN UNMET MEDICAL NEED, YET THE LACK OF ANIMAL MODELS FOR LATE-ONSET FECD (WHICH ACCOUNTS FOR A MAJORITY OF CASES) HAS HAMPERED THE DEVELOPMENT OF NEW THERAPIES. IN THIS PROJECT, WE WILL DEVELOP NEW ANIMAL MODELS FOR LATE-ONSET FECD. RECENT GENOMIC ANALYSES OF PATIENTS’ DNA HAVE DEMONSTRATED EXPANSION OF (CTG)N TRINUCLEOTIDE REPEATS IN THE INTRON OF THE TCF4 GENE. IN CAUCASIAN POPULATIONS, NEARLY 80% OF LATE-ONSET FECD PATIENTS HAVE LONG (CTG)N (N>30-40) REPEATS, AND THE LENGTH OF (CTG)N IS THE MOST PREDICTABLE GENETIC MUTATION FOR LATE-ONSET FECD. NEVERTHELESS, THE MECHANISM OF HOW THE LONG (CTG)N REPEATS LEAD TO FECD IS UNKNOWN. ONE HYPOTHESIS IS THAT LONG (CUG)N RNA SEQUESTERS RNA SPLICING FACTORS THAT LEAD TO ALTERATION OF RNA SPLICING AND MALFUNCTION OF CECS. THE OTHER HYPOTHESIS IS THAT THE LONG (CTG)N REPEAT INDUCES ALTERATION OF TCF4 GENE EXPRESSION THAT LEADS TO THE MALFUNCTION OF CECS. FROM VARIOUS REPORTS AND EVIDENCE, WE HYPOTHESIZE THAT LONG (CUG)N REPEAT RNA INDUCES THE FECD PHENOTYPE INDEPENDENT OF THE TCF4 GENE. TO PROVE THIS, WE WILL PRODUCE TWO DIFFERENT FECD MOUSE MODELS. IN AIM 1, WE WILL OVEREXPRESS LONG (CUG)N RNA IN THE CORNEAL ENDOTHELIUM USING (CTG)960 MICE. THE (CTG)960 MICE WERE INITIALLY DEVELOPED AND USED AS A MODEL FOR MYOTONIC DYSTROPHY 1 AND EXPRESS LONG (CUG)960 TRINUCLEOTIDE RNA UNDER A TETO (TETRACYCLINE OPERATOR) PROMOTER USING THREE DIFFERENT STRATEGIES. IN AIM 2, WE WILL OVEREXPRESS LONG (CUG)N RNA IN CORNEAL ENDOTHELIUM EXOGENOUSLY BY ADENOVIRUS, A MODEL THAT COULD BE APPLICABLE TO LARGER ANIMALS RELEVANT TO HUMANS. WE WILL MONITOR IN VIVO CEC DENSITY USING A ROSTOCK MICROSCOPE AND IN VIVO CORNEAL THICKNESS BY CORNEAL OPTICAL COHERENCE TOMOGRAPHY FOR UP TO 10 MONTHS. ALSO, EX VIVO CORNEAS WILL BE USED TO EVALUATE (CUG)N RNA ACCUMULATION BY IN SITU HYBRIDIZATION, MEASUREMENT OF CEC DENSITY BY ALIZARIN RED STAINING, AND OBSERVATION OF CEC ULTRASTRUCTURE BY PLASMA-FOCUSED ION BEAM SCANNING ELECTRON MICROSCOPE. WHEN ESTABLISHED, OUR ANIMAL MODELS WILL BE USEFUL NOT ONLY FOR THERAPY DEVELOPMENT BUT ALSO FOR UNDERSTANDING EARLY EVENTS IN THE DEVELOPMENT OF LATE-ONSET FECD, SUCH AS GUTTAE FORMATION AND ALTERATION OF MRNA EXPRESSION. THESE STUDIES WILL DETERMINE THE RELATIONSHIP OF TCF4 TO LATE-ONSET FECD, ELUCIDATE THE PATHOGENIC MECHANISMS, AND CREATE ANIMAL MODELS BENEFICIAL FOR THERAPEUTIC DEVELOPMENT.

NEW ANIMAL MODEL FOR LATE-ONSET FUCHS ENDOTHELIAL CORNEAL DYSTROPHY - ABSTRACT FUCHS’ ENDOTHELIAL CORNEAL DYSTROPHY (FECD) IS AN INHERITED CORNEAL DISEASE LEADING TO BLINDNESS. LOSS OF CORNEAL ENDOTHELIAL CELLS AND GUTTAE (BUMPS IN DESCEMET’S MEMBRANE) ARE PATHOLOGICAL FECD FEATURES. A DEFICIENCY OF CORNEAL ENDOTHELIAL CELLS (CEC) CAUSES CORNEAL OPACITY DUE TO LOSS OF PUMPING FUNCTION. IN DEVELOPED COUNTRIES, INCLUDING THE U.S., FECD PREVALENCE IS ESTIMATED TO BE 4%. CORNEAL TRANSPLANTATION IS THE ONLY TREATMENT FOR ADVANCED FECD PATIENTS. ALTHOUGH CORNEAL TRANSPLANTATION IS HIGHLY SUCCESSFUL, THE SHORTAGE OF DONOR CORNEAS MAKES PHARMACOLOGIC THERAPIES FOR FECD AN UNMET MEDICAL NEED, YET THE LACK OF ANIMAL MODELS FOR LATE-ONSET FECD (WHICH ACCOUNTS FOR A MAJORITY OF CASES) HAS HAMPERED THE DEVELOPMENT OF NEW THERAPIES. IN THIS PROJECT, WE WILL DEVELOP NEW ANIMAL MODELS FOR LATE-ONSET FECD. RECENT GENOMIC ANALYSES OF PATIENTS’ DNA HAVE DEMONSTRATED EXPANSION OF (CTG)N TRINUCLEOTIDE REPEATS IN THE INTRON OF THE TCF4 GENE. IN CAUCASIAN POPULATIONS, NEARLY 80% OF LATE-ONSET FECD PATIENTS HAVE LONG (CTG)N (N>30-40) REPEATS, AND THE LENGTH OF (CTG)N IS THE MOST PREDICTABLE GENETIC MUTATION FOR LATE-ONSET FECD. NEVERTHELESS, THE MECHANISM OF HOW THE LONG (CTG)N REPEATS LEAD TO FECD IS UNKNOWN. ONE HYPOTHESIS IS THAT LONG (CUG)N RNA SEQUESTERS RNA SPLICING FACTORS THAT LEAD TO ALTERATION OF RNA SPLICING AND MALFUNCTION OF CECS. THE OTHER HYPOTHESIS IS THAT THE LONG (CTG)N REPEAT INDUCES ALTERATION OF TCF4 GENE EXPRESSION THAT LEADS TO THE MALFUNCTION OF CECS. FROM VARIOUS REPORTS AND EVIDENCE, WE HYPOTHESIZE THAT LONG (CUG)N REPEAT RNA INDUCES THE FECD PHENOTYPE INDEPENDENT OF THE TCF4 GENE. TO PROVE THIS, WE WILL PRODUCE TWO DIFFERENT FECD MOUSE MODELS. IN AIM 1, WE WILL OVEREXPRESS LONG (CUG)N RNA IN THE CORNEAL ENDOTHELIUM USING (CTG)960 MICE. THE (CTG)960 MICE WERE INITIALLY DEVELOPED AND USED AS A MODEL FOR MYOTONIC DYSTROPHY 1 AND EXPRESS LONG (CUG)960 TRINUCLEOTIDE RNA UNDER A TETO (TETRACYCLINE OPERATOR) PROMOTER USING THREE DIFFERENT STRATEGIES. IN AIM 2, WE WILL OVEREXPRESS LONG (CUG)N RNA IN CORNEAL ENDOTHELIUM EXOGENOUSLY BY ADENOVIRUS, A MODEL THAT COULD BE APPLICABLE TO LARGER ANIMALS RELEVANT TO HUMANS. WE WILL MONITOR IN VIVO CEC DENSITY USING A ROSTOCK MICROSCOPE AND IN VIVO CORNEAL THICKNESS BY CORNEAL OPTICAL COHERENCE TOMOGRAPHY FOR UP TO 10 MONTHS. ALSO, EX VIVO CORNEAS WILL BE USED TO EVALUATE (CUG)N RNA ACCUMULATION BY IN SITU HYBRIDIZATION, MEASUREMENT OF CEC DENSITY BY ALIZARIN RED STAINING, AND OBSERVATION OF CEC ULTRASTRUCTURE BY PLASMA-FOCUSED ION BEAM SCANNING ELECTRON MICROSCOPE. WHEN ESTABLISHED, OUR ANIMAL MODELS WILL BE USEFUL NOT ONLY FOR THERAPY DEVELOPMENT BUT ALSO FOR UNDERSTANDING EARLY EVENTS IN THE DEVELOPMENT OF LATE-ONSET FECD, SUCH AS GUTTAE FORMATION AND ALTERATION OF MRNA EXPRESSION. THESE STUDIES WILL DETERMINE THE RELATIONSHIP OF TCF4 TO LATE-ONSET FECD, ELUCIDATE THE PATHOGENIC MECHANISMS, AND CREATE ANIMAL MODELS BENEFICIAL FOR THERAPEUTIC DEVELOPMENT.