Ri Bio

RSK3 ANCHORING DISRUPTOR THERAPY FOR HEART FAILURE - NOVEL THERAPEUTIC APPROACHES ARE DESPERATELY NEEDED IN AN EXPANDING PATIENT POPULATION TO IMPROVE BOTH THE SURVIVAL AND QUALITY OF LIFE FOR PATIENTS WITH OR SUSCEPTIBLE TO HEART FAILURE. RESEARCH OVER THE LAST TWO DECADES HAS ESTABLISHED P90 RIBOSOMAL S6 KINASE TYPE 3 (RSK3) AS A PIVOTAL REGULATOR OF PATHOLOGICAL CARDIAC REMODELING, INCLUDING IN MOUSE MODELS OF PRESSURE OVERLOAD AND GENETIC CARDIOMYOPATHY. RSK3 IS REQUIRED FOR PATHOLOGICAL REMODELING EVEN THOUGH RSK3 IS LESS ABUNDANT IN THE CARDIAC MYOCYTE THAN OTHER MEMBERS OF THE RSK PROTEIN KINASE FAMILY. THE EFFICIENCY AND SPECIFICITY OF INTRACELLULAR SIGNAL TRANSDUCTION CAN BE ENHANCED BY THE ANCHORING AND CO-LOCALIZATION OF KEY ENZYMES AND THEIR UPSTREAM ACTIVATORS AND SUBSTRATE EFFECTORS BY SCAFFOLD PROTEINS. RSK3’S UNIQUE N-TERMINAL DOMAIN CONFERS HIGH AFFINITY BINDING TO THE SCAFFOLD PROTEIN MUSCLE A-KINASE ANCHORING PROTEIN SS (MAKAPSS) IN THE CARDIAC MYOCYTE. RECENTLY PUBLISHED DATA SHOW THAT BLOCKADE OF RSK3 ASSOCIATION WITH MAKAPSS BY ADENO-ASSOCIATED VIRUS (AAV) MEDIATED EXPRESSION OF THE MAKAPSS-DERIVED RSK3 BINDING DOMAIN (RBD) ANCHORING DISRUPTOR POLYPEPTIDE WILL INHIBIT THE DEVELOPMENT OF PATHOLOGICAL REMODELING AND HEART FAILURE DURING CHRONIC PRESSURE OVERLOAD IN THE MOUSE. NEWER DATA DEMONSTRATE THAT RBD EXPRESSION WILL ALSO PREVENT REMODELING FOLLOWING MYOCARDIAL INFARCTION (MI) IN THE MOUSE. CARDIAC RSK3 INHIBITORS, LLC (CRI BIOTECH) IS A COMPANY DEVELOPING PATENT-PROTECTED THERAPEUTICS TARGETING CARDIAC MYOCYTE MAKAPSS SIGNALOSOMES FOR THE PREVENTION AND/OR TREATMENT OF HEART FAILURE. TO TARGET MAKAPSS-RSK3-DEPENDENT SIGNALING VIA BLOCKADE OF THE MAKAPSS-RSK3 PROTEIN-PROTEIN INTERACTION, CRI BIOTECH PROPOSES TO TREAT PATIENTS AT RISK FOR HEART FAILURE WITH THE NEW SELF-COMPLEMENTARY, SEROTYPE 9 AAV GENE THERAPY VECTOR AAV9SC.RBD. AS A DOSE SUFFICIENT TO DELIVER AAV9SC.RBD WITH HIGH PENETRANCE TO THE SWINE MYOCARDIUM HAS BEEN DETERMINED, CRI BIOTECH WILL NOW TEST IN THIS DIRECT TO PHASE II SBIR PROJECT THE EFFICACY OF AAV9SC.RBD IN A CLINICALLY RELEVANT SWINE MODEL OF ISCHEMIC CARDIOMYOPATHY. SPECIFIC AIM 1: EFFICACY OF RSK3 ANCHORING DISRUPTOR PEPTIDE FOR POST-MYOCARDIAL INFARCTION HEART FAILURE IN A LARGE ANIMAL MODEL. THE CORE OF THIS PROJECT IS TO TEST WHETHER RBD PEPTIDE EXPRESSION WILL REDUCE PATHOLOGICAL REMODELING INDUCED BY MI IN SWINE, PREVENTING HEART FAILURE. SWINE WILL BE SUBJECTED TO ISCHEMIA-REPERFUSION INJURY TO INDUCE MI AND THEN TREATED IMMEDIATELY AFTERWARDS WITH AAV9SC.RBD BY ANTEGRADE INTRACORONARY INFUSION. CARDIAC FUNCTION WILL BE ASSESSED SERIALLY FOR 12 WEEKS POST- MI. SPECIFIC AIM 2: AAV9SC.RBD-MEDIATED INHIBITION OF THE MOLECULAR AND CELLULAR PATHOLOGY ASSOCIATED WITH HEART FAILURE. TAKING ADVANTAGE OF TISSUE COLLECTED FROM THE SAME ANIMALS USED IN SPECIFIC AIM 1, THE BENEFITS OF THE RBD ANCHORING DISRUPTOR BIOLOGIC IN SWINE WILL BE FURTHER DEMONSTRATED BY GRAVIMETRIC, HISTOLOGICAL, AND MOLECULAR ANALYSES FOR FIBROSIS AND OTHER MARKERS OF CARDIAC REMODELING AND HEART FAILURE. IN ADDITION, INITIAL TOXICOLOGY SCREENS WILL BE DONE TO TEST AAV9SC.RBD SAFETY.

PP2A ANCHORING DISRUPTOR THERAPY IN HEART FAILURE - PATHOLOGICAL CARDIAC REMODELING CONSTITUTES A COMMON PATHWAY TO HEART FAILURE IN DISEASE. DESPITE CURRENT PHARMACOLOGIC THERAPY AND OTHER ADVANCES THAT ATTENUATE REMODELING, MORBIDITY AND MORTALITY DUE TO HEART FAILURE REMAIN HIGH. NOVEL THERAPEUTIC APPROACHES ARE DESPERATELY NEEDED IN AN EXPANDING PATIENT POPULATION TO IMPROVE BOTH THE SURVIVAL AND QUALITY OF LIFE FOR PATIENTS WITH OR SUSCEPTIBLE TO HEART FAILURE. RESEARCH OVER THE LAST TWO DECADES, MAINLY IN THE ACADEMIC LABORATORY OF DR. MICHAEL S. KAPILOFF, HAS ESTABLISHED THE 230 KDA SCAFFOLD PROTEIN MUSCLE A-KINASE ANCHORING PROTEIN SS (MAKAPSS) AS THE ORGANIZER OF MULTIMOLECULAR SIGNALING COMPLEXES CRITICAL IN THE CARDIAC MYOCYTE FOR THE INDUCTION AND PROGRESSION OF PATHOLOGICAL CARDIAC REMODELING. ONE CONSTITUENT OF MAKAPSS “SIGNALOSOMES” IS PROTEIN PHOSPHATASE 2A (PP2A) THAT CONTAINS THE B56D (PPP2R5D) REGULATORY SUBUNIT. NEW PRELIMINARY DATA SHOW THAT MAKAPSS-BOUND PP2A REGULATES MYOCYTE ELONGATION AND VENTRICULAR DILATION IN DISEASE. OBSERVATIONS THAT B56D EXPRESSION IS ELEVATED IN HUMAN AND CANINE HEART FAILURE SUPPORT THE CANDIDACY OF MAKAPSS-BOUND PP2A AS A TARGET FOR INTERVENTION IN THE TREATMENT OF NON-ISCHEMIC AND ISCHEMIC DILATED CARDIOMYOPATHIES. CARDIAC RSK3 INHIBITORS, LLC (CRI BIOTECH) IS A COMPANY FOUNDED BY DR. KAPILOFF THAT IS DEVELOPING PATENT-PROTECTED THERAPEUTICS TARGETING CARDIAC MYOCYTE MAKAPSS SIGNALOSOMES FOR THE PREVENTION AND/OR TREATMENT OF HEART FAILURE. TO TARGET MAKAPSS-PP2A COMPLEXES VIA BLOCKADE OF PP2A-MAKAPSS PROTEIN-PROTEIN INTERACTION, CRI BIOTECH IS DEVELOPING A NEW SELF-COMPLEMENTARY, ADENO-ASSOCIATED VIRUS SEROTYPE 9 (AAV9SC) GENE THERAPY VECTOR. PRELIMINARY DATA IN MICE SHOWS THAT TREATMENT WITH THIS VECTOR RESULTS IN RESTORATION AND LONG TERM PRESERVATION OF CARDIAC STRUCTURE AND FUNCTION FOLLOWING MYOCARDIAL INFARCTION. CRI BIOTECH PROPOSES THAT THIS GENE THERAPY CONSTITUTES A NOVEL APPROACH FOR THE PREVENTION AND/OR TREATMENT OF PATHOLOGICAL VENTRICULAR DILATION AND ECCENTRIC HYPERTROPHY, WITH POTENTIALLY BROAD EFFICACY ACROSS DIVERSE CARDIOVASCULAR DISEASES. IN THIS FAST-TRACK SBIR APPLICATION, CRI BIOTECH WILL TEST THE EFFICACY OF THE NEW BIOLOGIC IN A CLINICALLY RELEVANT SWINE MODEL OF ISCHEMIC CARDIOMYOPATHY, AS A PIVOTAL EFFICACY STUDY AND KEY STEP ON THE PATH TO FIRST-IN-HUMAN CLINICAL TRIALS. PHASE I - THE MILESTONE FOR THIS AIM IS THE DETERMINATION OF THE MINIMUM AAV9SC DOSE REQUIRED FOR CONSISTENT INHIBITION OF PP2A ANCHORING TO MAKAPSS IN THE HEART, THEREBY DEFINING THE APPROPRIATE BIOLOGIC DOSE TO BE USED FOR EFFICACY TESTING IN PHASE II. PHASE II - SPECIFIC AIM 1: EFFICACY OF THE NEW GENE THERAPY FOR POST-MYOCARDIAL INFARCTION HEART FAILURE IN A LARGE ANIMAL MODEL. THE CORE OF THIS PROJECT IS TO TEST WHETHER THE NEW AAV9SC-BASED GENE THERAPY WILL REDUCE PATHOLOGICAL REMODELING INDUCED BY MI IN SWINE, PREVENTING HEART FAILURE. SPECIFIC AIM 2: TAKING ADVANTAGE OF TISSUE COLLECTED FROM THE SAME ANIMALS USED IN AIM 1, THE BENEFITS OF THE NEW GENE THERAPY WILL BE FURTHER DEMONSTRATED BY GRAVIMETRIC, HISTOLOGICAL, AND MOLECULAR ANALYSES.

SHRNA-BASED TARGETING OF MAKAP?? IN HEART FAILURE

A CROSS-SPECIES PRECLINICAL PLATFORM TO ENHANCE THE TRANSLATION OF NEW MEDICINES - PROJECT DESCRIPTION DUCHENNE MUSCULAR DYSTROPHY (DMD) IS A LETHAL, X-LINKED RECESSIVE DISORDER WITH NO KNOWN CURE THAT AFFLICTS 1 IN 5,000 NEWBORN MALES. PATIENTS CARRY A MUTATION IN THE DYSTROPHIN (DMD) GENE, RESULTING IN ABERRANT OR ABSENT EXPRESSION OF THE DYSTROPHIN PROTEIN. AFFECTED INDIVIDUALS EXPERIENCE PROGRESSIVE WASTING OF SKELETAL MUSCLES AND CARDIAC DYSFUNCTION LEADING TO LOSS OF AMBULATION AND PREMATURE DEATH, PRIMARILY DUE TO CARDIAC OR RESPIRATORY FAILURE. ONLY PALLIATIVE TREATMENTS ARE AVAILABLE, ALTHOUGH GENE THERAPY APPROACHES FOR DMD HAVE BEEN EFFECTIVELY APPLIED IN DYSTROPHIC ANIMAL MODELS BY EITHER DIRECTLY TARGETING A CLASS OF MUTATIONS (AS WITH EXON SKIPPING OR GENE EDITING) OR BY DELIVERING A SYNTHETIC VERSION OF THE DYSTROPHIN GENE. THE LACK OF SCALABLE, HUMAN- BASED PRE-CLINICAL SCREENING MODELS IS A SIGNIFICANT ROADBLOCK TO DEVELOPING NEW THERAPIES FOR THESE PATIENTS. SIMPLE SINGLE MOLECULE ASSAYS CAN BE READILY SCALED, BUT LACK THE COMPLEXITY NEEDED TO MODEL MUSCLE CONTRACTION WHICH RELIES ON A SYMPHONY OF MANY BIOLOGICAL SYSTEMS WORKING IN CONCERT TO PRODUCE MOVEMENT. AT THE OTHER END OF THE SPECTRUM, ANIMAL (PARTICULARLY MOUSE) MODELS HAVE GENERATED MUCH THAT IS KNOWN ABOUT THE DISEASE, BUT THEY ARE COSTLY, SLOW, AND IN NOTABLE INSTANCES HAVE GIVEN FALSE POSITIVES FOR NEW TREATMENTS THAT FAILED TO TRANSLATE TO HUMANS. IN THIS FASTTRACK SBIR PROPOSAL, CURI BIO AND ITS PARTNERS WILL DEVELOP A NOVEL PRECLINICAL SCREENING PLATFORM FOR DMD THAT DIRECTLY MEASURES THE CONTRACTION OF STEM CELL-DERIVED MUSCLE TISSUE CONSTRUCTS FROM BOTH HUMAN AND MURINE CELLS. BECAUSE THE CONTRACTILITY OF TISSUE CONSTRUCTS IS DIRECTLY MEASURED, THERAPIES CAN BE TESTED IN THE COMPLEX CONTEXT OF THE MOSAIC OF PHENOTYPES THAT CONSTITUTE MUSCLE CONTRACTION. FURTHER, THE USE OF BOTH MURINE AND HUMAN CELLS WILL ALLOW DIRECT COMPARISON OF RESULTS TO UNDERSTAND SPECIES- SPECIFIC BIOLOGY AS WELL AS TO TRANSLATE PAST RESULTS FROM ANIMAL MODELS. SUCCESSFUL COMPLETION OF THIS PROPOSAL WILL VALIDATE THE PLATFORM’S ABILITY TO MODEL CONTRACTILE DYSFUNCTION IN THE DISH, AND TO MEASURE THE DEGREE OF RECOVERY AFTER APPLICATION OF A NOVEL THERAPEUTIC STRATEGY THAT HAS BEEN SHOWN TO RESTORE THE EXPRESSION OF HEALTHY DYSTROPHIN IN PATIENT CELLS. THE COMPANY WILL ALSO LEVERAGE THIS PLATFORM BY MEASURING THE THERAPEUTIC EFFECT OF A REVOLUTIONARY ADENO-ASSOCIATED VIRUS-BASED GENE THERAPY THAT WILL BE SUPERIOR IN BOTH SAFETY AND EFFICACY COMPARED TO CURRENT APPROACHES. THE DELIVERABLES OF THIS PROJECT WILL GREATLY IMPROVE THE FIELD’S ABILITY TO PRECLINICALLY TEST NOVEL THERAPEUTICS AND WILL SPEED TO MARKET NEW LIFESAVING DRUGS FOR DEVASTATING DISEASES.

PREDICTIVE ASSESSMENT OF ACUTE AND CHRONIC CARDIOTOXICITY USING COMBINATORIALLY MATURED HPSC-CMS

LIVE MICROBIAL THERAPEUTICS: AN ENHANCED TREATMENT PARADIGM FOR CLASSICAL HOMOCYSTINURIA - PROJECT SUMMARY THE GOAL OF THIS PROPOSAL IS TO CONTINUE THE DEVELOPMENT OF A SYNTHETIC LIVE BACTERIAL THERAPEUTIC FOR HOMOCYSTINURIA (HCU), AN INBORN METABOLIC DISORDER LEADING TO ACCUMULATION OF HOMOCYSTEINE (HCY), AN INTERMEDIARY OF THE AMINO ACID METHIONINE. THIS CONDITION IS ESTIMATED TO OCCUR AT AN ESTIMATED PREVALENCE OF 1 IN 100,000 TO 200,000. THE CONDITION MAY EVADE CLINICAL DETECTION UNTIL SPECIFIC HALLMARKS MANIFEST, INCLUDING LENS DETACHMENT FROM THE CENTER OF THE EYE OR INCREASED INCIDENCE OF STROKE AND OTHER THROMBOTIC CONDITIONS. PETRI BIO’S APPROACH IS INTENDED TO BREAK DOWN METHIONINE IN THE GUT TO SUBSEQUENTLY REDUCE SYSTEMIC LEVELS OF HCY. CURRENT TREATMENT STRATEGIES FOR PYRIDOXINE NON-RESPONSIVE HCU TYPICALLY ATTEMPT TO LOWER PLASMA AND TISSUE LEVELS OF HCY BY A COMBINATION OF RESTRICTING DIETARY INTAKE OF THE HCY PRECURSOR METHIONINE AND DIETARY SUPPLEMENTATION WITH TRIMETHYLGLYCINE, MORE COMMONLY REFERRED TO AS BETAINE. BOTH STRATEGIES ARE OF LIMITED EFFICACY DUE TO A LACK OF ADHERENCE TO THE DIET, AND UNPLEASANT SIDE EFFECTS FROM TAKING BETAINE (E.G., DIARRHEA, NAUSEA, ODOR). PETRI BIO, INC. EMPLOYS PROKARYOTIC STRAINS COMPATIBLE WITH THE HUMAN GUT MICROBIOME TO SERVE AS EXPRESSION VECTORS FOR THERAPEUTIC PROTEINS CAPABLE OF TARGETED MODULATION OF METABOLIC PATHWAYS, SUCH AS THE METHIONINE CYCLE. IN A SEARCH FOR ENZYMES CAPABLE OF IMPACTING METHIONINE LEVELS, WE CARRIED OUT AN IN SILICO SCREEN FOR POTENTIAL METHIONASE ENZYMES IN MICROBIAL GENOMIC DATASETS. DURING THE PHASE I PROGRAM, LEADING CANDIDATE ENZYMES WERE SUBSEQUENTLY CLONED, EXPRESSED, AND TESTED IN VITRO FOR METHIONINE CATALYSIS CAPABILITIES. THE BEST PERFORMING ENZYMES WERE ENGINEERED FOR EXPRESSION IN A BACTERIAL STRAIN WHICH IS KNOWN TO READILY ENGRAFT INTO THE HUMAN GUT MICROBIOME. THESE STRAINS WERE TESTED IN A GENETIC MODEL SYSTEM OF HCU AND MITIGATED SYSTEMIC LEVELS OF HCY AS INTENDED. DURING PHASE II, STRAINS WILL BE ADDITIONALLY OPTIMIZED AND SCALED UP TO DEVELOP A COMMERCIAL-SCALE FERMENTATION METHOD. SAFETY, TOXICOLOGY, AND MAXIMUM TOLERABLE DOSE TESTING WILL BE UNDERTAKEN IN A RANGE OF STANDARDIZED TESTS (BOTH IN VITRO AND IN VIVO). THE LEAD STRAIN WILL THEN BE TESTED FOR EFFICACY IN THE HCU MODEL EMPLOYED IN PHASE I AS WELL AS IN A MURINE MODEL OF HCU TO MEASURE METABOLIC EFFECTIVENESS AS WELL AS MITIGATION OF COGNITIVE DEFICITS DUE TO HCU. PENDING SUCCESSFUL COMPLETION OF PHASE II, THE DATA WILL BE SUBMITTED TO THE FDA IN A PRE-IND MEETING, THE NEXT STEP TOWARDS EVENTUAL CLINICAL EVALUATION AND AVAILABILITY TO PATIENTS.

HIGH-THROUGHPUT NMJ ASSAY FOR BOTOX POTENCY SCREENING - PROJECT SUMMARY THE BOTULINUM TOXIN POTENCY ASSAY USING TISSUE CHIPS PROGRAM FROM FDA AND NCATS HIGHLIGHTS THE NEED FOR NOVEL ENGINEERED SYSTEMS CAPABLE OF RELIABLY EVALUATING BOTULINUM TOXIN (BOT) POTENCY. HUMAN INDUCED PLURIPOTENT STEM CELL (IPSC)-DERIVED MOTOR NEURONS AND MUSCLE CELLS HAVE BEEN PREVIOUSLY MAINTAINED IN CO- CULTURE AND SHOWN TO FORM FUNCTIONAL SYNAPTIC CONTACTS REPRESENTING IN VIVO NEUROMUSCULAR JUNCTIONS (NMJS). HOWEVER, THE ABILITY TO EFFECTIVELY MODEL NMJ FUNCTIONAL RESPONSES TO BOT USING IN VITRO PLATFORMS AMENABLE TO HIGH-THROUGHPUT SCREENING HAS YET TO BE ACHIEVED DUE TO THE COMPLEXITY OF GENERATING MATURE AND FUNCTIONALLY COMPETENT NMJS IN CULTURE. THE DEVELOPMENT OF A HIGH-THROUGHPUT PLATFORM CAPABLE OF PROMOTING NMJ DEVELOPMENT ACROSS A MULTIPLEXED ASSAY WILL HAVE A SUBSTANTIAL POSITIVE IMPACT ON BOT PRODUCTION, AS WELL AS ADVANCED THERAPY DEVELOPMENT, DRUG EFFICACY/TOXICITY SCREENING, AND MECHANISTIC STUDIES OF NEURONAL AND NMJ PATHOPHYSIOLOGY IN NEURODEGENERATIVE DISEASES. BASED ON PRELIMINARY WORK AND PUBLISHED DATA, WE POSIT THAT A CULTURE PLATFORM INTEGRATING ELECTRODE-BASED STIMULATION OF NEURONAL FIRING AND MAGNET-BASED MEASUREMENT OF ENGINEERED MUSCLE CONTRACTION WILL ENABLE REAL-TIME ANALYSIS OF NMJ DEVELOPMENT AND FUNCTION AT BASELINE AND IN RESPONSE TO BOT EXPOSURE. USING IPSC-DERIVED MOTOR NEURONS AND MUSCLE CELLS, WE WILL ESTABLISH ORGANIZED CO-CULTURES WITHIN A CULTURE PLATE THAT IS COMPATIBLE WITH OUR COMPANY’S EXISTING MUSCLE CONTRACTILITY ASSAY, MANTARRAYTM. MICROCHANNELS IN THE WALLS SEPARATING THE CELLS WILL ALLOW NEURONAL PROCESSES TO GROW INTO THE MUSCLE COMPARTMENT, FACILITATING SYNAPTIC CONTACT. TESTS WITH REFERENCE BATCHES OF BOT, IN TERMS OF THEIR ABILITY TO ALTER SYNAPTIC COMMUNICATION BETWEEN OUR CELL POPULATIONS, WILL THEN BE USED TO DEMONSTRATE THE SUITABILITY OF THIS MODEL FOR ASSAYING NMJ FUNCTION AND BOT POTENCY IN VITRO (PHASE 1). ONCE VALIDATED, OUR NMJ ASSAY WILL BE FURTHER EVALUATED TO DETERMINE ITS ACCURACY, PRECISION, SPECIFICITY, AND REPRODUCIBILITY IN MODELING BOT RESPONSES IN HUMAN TISSUES (PHASE 2). REFERENCE BATCHES OF BOT WILL BE TESTED ACROSS A WIDE RANGE OF DONOR CELL SOURCES AND IN COMPARISON TO AN ARRAY OF REFERENCE COMPOUNDS WITH KNOWN AND PREDICTABLE EFFECTS ON NMJ FUNCTION. THE CENTRAL HYPOTHESIS OF THIS WORK IS THAT DIFFERENCES IN NMJ FUNCTION BETWEEN ENGINEERED SKELETAL MUSCLE AND MOTOR NEURON CO-CULTURES TREATED WITH DIFFERENT DOSES OF BOT WILL ENABLE STRATIFICATION OF PHENOTYPES THAT CAN BE SUCCESSFULLY USED TO PLOT DOSE RESPONSE CURVES COMPARABLE TO OUTPUT DATA FROM MOUSE LETHALITY BIOASSAYS (THE CURRENT GOLD STANDARD FOR BOT POTENCY SCREENING). SUCCESSFUL COMPLETION OF THIS STUDY WILL PROVIDE A NEW PROTOTYPE HUMAN-BASED PLATFORM FOR MODELING HUMAN PERIPHERAL NEUROPATHIES AS WELL AS A VALUABLE PRECLINICAL SCREENING TOOL FOR ASSESSING NOVEL THERAPEUTICS. THE CONSUMABLE PLATE WILL BE INTEGRATED WITH CURI’S EXISTING HARDWARE/SOFTWARE PACKAGES AND SO CAN BE QUICKLY DISSEMINATED TO CUSTOMERS UPON VALIDATION.

BIOENGINEERING MATURE HUMAN PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES FOR PREDICTIVE DRUG-INDUCED TOXICITY TESTING

HIGH-THROUGHPUT NANOMEA-BASED PROARRHYTHMIA ASSAY

SBIR PHASE II: VASOREACTIVE PERFUSED IN VITRO VASCULAR NETWORK

USING ACT01 - DHA COMBINATION TO IMPROVE CONDITIONS OF FXS

LIVE MICROBIAL THERAPEUTICS FOR ENZYME REPLACEMENT THERAPY AGAINST HOMOCYSTINURIA - PROJECT SUMMARY THE GOAL OF THIS PROPOSAL IS TO CONTINUE THE DEVELOPMENT OF A SYNTHETIC LIVE BACTERIAL THERAPEUTIC FOR HOMOCYSTINURIA, AN INBORN METABOLIC DISORDER LEADING TO ACCUMULATION OF THE AMINO ACID METHIONINE, AND RESULTS IN DRAMATICALLY INCREASED RISK OF STROKE AND OTHER THROMBOTIC CONDITIONS. PETRI BIO’S APPROACH WILL BE CAPABLE OF BREAKING DOWN METHIONINE IN THE GUT TO REDUCE OR ELIMINATE DEPENDENCE ON A METHIONINE RESTRICTED DIET AND RESULT IN DECREASED PLASMA AND TISSUE HOMOCYSTEINE RESULTING IN SUPERIOR CLINICAL OUTCOMES. THE CONDITION IS ESTIMATED TO OCCUR AT AN INCIDENCE OF 1 IN 250,000, HOWEVER SOME REPORTS INDICATE A POTENTIAL INCIDENCE OF 1 IN 65,000 WHEN ACCOUNTING FOR THE CURRENT IMPRECISE DIAGNOSTIC ASSAYS AND OFTEN SUBTLE SYMPTOMS WHICH MAY EVADE CLINICAL DETECTION UNTIL THEY BECOME SEVERE OR OBVIOUS, SUCH AS LENS DETACHMENT FROM THE CENTER OF THE EYE. CYSTATHIONINE BETA-SYNTHASE (CBS), THE GENE MUTATED IN CLASSICAL HOMOCYSTINURIA, IS LOCALIZED AT A KEY REGULATORY BRANCH POINT IN THE EUKARYOTIC METHIONINE CYCLE. CBS CATALYZES A PYRIDOXAL 5'-PHOSPHATE DEPENDENT BETA-REPLACEMENT REACTION CONDENSING SERINE AND HOMOCYSTEINE (HCY) INTO CYSTATHIONINE THAT IS SUBSEQUENTLY CONVERTED TO CYSTEINE IN A REACTION CATALYZED BY CYSTATHIONINE--LYASE (CGL, EC 4.4.1.1). INACTIVATION OF CBS BY MUTATION RESULTS IN CLASSICAL HOMOCYSTINURIA (HCU) WHICH IN HUMAN SUBJECTS, IS CHARACTERIZED BY A RANGE OF CONNECTIVE TISSUE DISTURBANCES INCLUDING MARFANOID HABITUS AND LENS DISLOCATION, INTELLECTUAL IMPAIRMENT, AND A DRAMATICALLY INCREASED INCIDENCE OF VASCULAR DISORDERS PARTICULARLY THROMBOEMBOLIC COMPLICATIONS SUCH AS STROKE. TREATMENT STRATEGIES FOR PYRIDOXINE NON-RESPONSIVE HCU TYPICALLY ATTEMPT TO LOWER PLASMA AND TISSUE LEVELS OF HCY BY A COMBINATION OF RESTRICTING DIETARY INTAKE OF THE HCY PRECURSOR METHIONINE AND DIETARY SUPPLEMENTATION WITH TRIMETHYLGLYCINE, MORE COMMONLY REFERRED TO AS BETAINE. PETRI BIO, INC. HAS DEVELOPED A NOVEL STRATEGY FOR ENZYME THERAPY, EMPLOYING PROKARYOTIC STRAINS COMPATIBLE WITH THE HUMAN GUT MICROBIOME TO SERVE AS EXPRESSION VECTORS FOR THERAPEUTIC PROTEIN ADMINISTRATION. AFTER IN SILICO SCREENING OF BACTERIALLY-DERIVED METHIONASES FOR A NUMBER OF DESIRABLE CHARACTERISTICS OF THERAPEUTIC ENZYMES, TEN HAVE BEEN CLONED, EXPRESSED, AND SHOWN TO REDUCE METHIONINE CONCENTRATIONS IN VITRO. DURING THIS PHASE I PROGRAM, WE WILL EXTEND THESE STUDIES BY SCREENING HUNDREDS OF BACTERIALLY-DERIVED METHIONASES IN SILICO AND SUBSEQUENTLY CLONING, EXPRESSING, AND TESTING IN VITRO METHIONINE CATALYSIS CAPABILITIES. IN VITRO TESTS WILL BE UNDERTAKEN TO MEASURE METHIONASE ACTIVITY OF THESE BACTERIAL STRAINS. AFTER OPTIMIZATION OF STRAINS WITH HIGH METHIONASE ACTIVITY, WE WILL EVALUATE THEIR ABILITY TO REDUCE METHIONINE CONCENTRATIONS IN VIVO, AS WELL AS AMELIORATE THE EFFECTS OF METHIONINE ACCUMULATION IN A MURINE MODEL OF HCU. FUTURE STUDIES WILL OPTIMIZE THE BACTERIAL METHIONASE TRANSGENES TO ENSURE MAXIMUM ACTIVITY AND BIOCOMPATIBILITY AS WELL AS SELECT A LEAD CANDIDATE BACTERIAL STRAIN FOR PRECLINICAL DRUG DEVELOPMENT.

A VASCULARIZED BONE MARROW MODEL FOR IN VITRO TESTING OF DRUG TOXICITY - PROJECT SUMMARY THE PRIMARY FUNCTION OF THE BONE MARROW IS HEMATOPOIESIS -- THE PRODUCTION OF LEUKOCYTES (WHITE BLOOD CELLS), ERYTHROCYTES (RED BLOOD CELLS), AND PLATELETS TO PERFORM THE ESSENTIAL FUNCTIONS OF BLOOD. BLOOD CELLS HAVE A LIMITED LIFESPAN RANGING FROM DAYS (LEUKOCYTES) TO MONTHS (ERYTHROCYTES), AND THUS HAVE TO BE REGULARLY REPLACED. THIS FUNCTION IS ACCOMPLISHED BY HEMATOPOIETIC STEM/PROGENITOR CELLS (HSPCS) WHICH RAPIDLY AND REGULARLY DIFFERENTIATE TO PRODUCE NEARLY 200 BILLION CELLS EACH DAY, BUT ALSO EXISTS IN SPECIALIZED NICHES WITHIN THE BONE MARROW TO MAINTAIN THEIR STEM CELL STATE. BECAUSE OF THEIR REGULAR AND RAPID DIFFERENTIATION, HSPCS ARE SUSCEPTIBLE TO DRUGS WHICH TARGET RAPIDLY DIVIDING CELLS, SUCH AS CHEMOTHERAPEUTICS FOR CANCER, AS WELL AS A HOST OF OTHER NON-CANCER DRUGS. AS SUCH, ASSESSING POTENTIAL BONE MARROW TOXICITY IS CRITICAL IN THE DRUG DEVELOPMENT PIPELINE. TO ASSESS TOXICITY (AND EFFICACY), THE EMERGING FIELD OF MICROPHYSIOLOGICAL SYSTEMS (MPS) OR “ORGAN-ON-A-CHIP” PROVIDES HUMAN CELL/TISSUE MODELS TO ENHANCE THE EFFICIENCY OF PRE-CLINICAL TESTING DURING DRUG DEVELOPMENT. THE PRIMARY GOAL OF THIS PHASE I SBIR APPLICATION IS TO DEMONSTRATE THE FEASIBILITY OF A PROPRIETARY TECHNOLOGY OF ARACARI, AN EARLY STAGE MEDICAL DEVICE COMPANY, TO MODEL APPROPRIATE DRUG RESPONSES OF HUMAN BONE MARROW IN VITRO. THE PROJECT LEVERAGES ARACARI’S PATENTED CORE TECHNOLOGY – 3D PERFUSABLE LIVING HUMAN VASCULAR NETWORKS – TO CREATE A VASCULARIZED MICRO-BONE MARROW, OR VMBMTM. THE 3D VASCULAR NETWORK IN THE VMBM RECREATES THE PERIVASCULAR NICHE OF THE BONE MARROW, THOUGHT TO BE CRITICAL TO MAINTAIN A SMALL POPULATION OF HSPCS AS WELL AS GUIDE DIFFERENTIATION INTO MYELOID AND ERYTHROID LINEAGES. THE SPECIFIC AIMS ARE TO: 1) TEST THE ABILITY OF THE VMBM TO CAPTURE BONE MARROW TOXICITY USING A SMALL PANEL OF DRUGS (TENIPOSIDE, ALEMTUZUMAB, AND PACLITAXEL) THAT DEMONSTRATE WELL-DOCUMENTED, AS WELL AS UNEXPECTED AT THE TIME OF HUMAN TRIALS, TOXICITY TO BONE MARROW MANIFESTED AS NEUTROPENIA, BUT ACT THROUGH DIFFERENT MECHANISMS; AND, 2) TEST THE ABILITY OF THE VMBM TO MODEL THE EFFECT OF BONE MARROW PROTECTIVE DRUGS USING A SMALL PANEL OF COMMERCIALLY AVAILABLE DRUGS THAT ARE KNOWN TO STIMULATE PRODUCTION OF NEUTROPHILS (NEUPOGENÒ (FILGRASTIM)), AND RED BLOOD CELLS (ERYTHROPOIETIN) BY THE BONE MARROW. THE DEVELOPMENT OF ARACARI’S VMBM REPRESENTS A SIGNIFICANT ADVANCE IN TECHNOLOGY TO ASSESS BONE MARROW TOXICITY TO NOT ONLY CHEMOTHERAPY DRUGS FOR CANCER, BUT A MUCH WIDER RANGE OF DRUGS THAT POTENTIALLY IMPACT BONE MARROW FUNCTION. UPON COMPLETION OF THE PROPOSED PHASE I FEASIBILITY STUDY, THE PLANS FOR A PHASE II APPLICATION WOULD FOCUS ON A BROADER RANGE OF VARIABLES THAT COULD IMPACT DRUG RESPONSE (E.G., SEX, AGE, RACE) AS WELL AS THE CRITICAL NEXT STEP IN THE COMMERCIALIZATION PROCESS – PRODUCTION AND SCALE-UP.

A PROGRAM TO COMMERCIALIZE AND EXPAND LIFE SCIENCE START-UP ACTIVITY IN RHODE ISLAND'S ENTREPRENEURIAL ECOSYSTEM THROUGH THE RI BIO NETWORK

A VASCULARIZED BLOOD-BRAIN BARRIER MODEL FOR IN VITRO TESTING OF DRUG AND IMMUNOTHERAPY DELIVERY - PROJECT SUMMARY THE BRAIN IS A HIGHLY-SPECIALIZED, FINELY-TUNED ORGAN THAT DEPENDS FOR ITS FUNCTION ON MAINTAINING HOMEOSTASIS WITHIN NARROWLY-DEFINED LIMITS. THIS IS ACHIEVED IN LARGE PART BY THE BLOOD-BRAIN BARRIER (BBB), WHICH TIGHTLY REGULATES WHAT CAN AND CAN’T GET INTO THE BRAIN. FUNCTIONALLY, THE BBB IS THE COMBINED WORK OF ENDOTHELIAL CELLS (EC), PERICYTES AND ASTROCYTES, THE LATTER TWO OF WHICH ACT ON THE EC TO MAINTAIN AN EXTENSIVE NETWORK OF TIGHT JUNCTIONS, PROMOTE THE EXPRESSION OF SPECIALIZED TRANSPORTERS, AND LIMIT THE RATE OF TRANSCELLULAR PINOCYTOSIS. THIS LACK OF FREE TRANSFER BETWEEN BLOOD AND BRAIN FOR NON-LIPOPHILIC SPECIES SEVERELY LIMITS THE ENTRY OF MANY, OTHERWISE USEFUL, SMALL MOLECULE DRUGS. FOR EXAMPLE, CISPLATIN (<3% BRAIN/PLASMA RATIO), IS EFFECTIVE IN NON-BRAIN TUMORS, BUT HAS POOR PENETRANCE AND LOW EFFICACY AGAINST BRAIN TUMORS SUCH AS GLIOMA. SIMILARLY, DELIVERY OF IMMUNOTHERAPEUTICS SUCH AS BEVACIZUMAB (0.2%) AND BI-SPECIFIC ANTIBODIES IS ALSO LIMITED, AND MUCH IS STILL TO BE UNDERSTOOD REGARDING THE DELIVERY OF IMMUNE CELLS TO THE BRAIN, INCLUDING CAR-TS, NK CELLS, D T CELLS AND THE PATIENT’S OWN MONOCYTES AND LYMPHOCYTES. FINALLY, THERE ARE SIGNIFICANT DIFFERENCES BETWEEN MOUSE AND HUMAN PHYSIOLOGY INCLUDING IN THE IMMUNE SYSTEM. MANY DRUG DEVELOPERS ARE THEREFORE TURNING TO HUMAN CELL- BASED SYSTEMS RATHER THAN MOUSE MODELS. THE PRIMARY GOAL OF THIS PHASE I SBIR APPLICATION IS TO DEMONSTRATE THE FEASIBILITY OF ARACARI BIOSCIENCES’ PROPRIETARY TECHNOLOGY TO MODEL APPROPRIATE DRUG AND IMMUNOTHERAPEUTIC DELIVERY ACROSS THE HUMAN BBB. ARACARI’S PATENTED CORE TECHNOLOGY IS A PERFUSABLE HUMAN VASCULAR NETWORK WITHIN A MICROFLUIDIC DEVICE THAT FITS IN A CONVENIENT 96-WELL PLATE FORMAT. THIS TECHNOLOGY HAS BEEN SUCCESSFULLY DEVELOPED INTO COMMERCIALIZED PRODUCTS INCLUDING THE VASCULARIZED MICRO-ORGAN (VMOTM) AND THE VASCULARIZED MICRO-TUMOR (VMTTM). IMPORTANTLY, LEUKOCYTES CAN BE PERFUSED THROUGH THE VESSELS AND ON STIMULATION THEY WILL ADHERE AND EXTRAVASATE AS THEY DO IN VIVO. WE HAVE ALSO DEVELOPED A VASCULARIZED MICRO-BRAIN (VMB), WHICH IS THE FOCUS OF THIS APPLICATION. THIS INCORPORATES A PERFUSED NEUROVASCULAR UNIT (NVU) COMPRISED OF HUMAN BBB EC, PERICYTES AND ASTROCYTES, ALL EMBEDDED IN A BRAIN-MIMICKING EXTRACELLULAR MATRIX. THE VESSELS SHOW UPREGULATION OF BBB TRANSPORTERS AND JUNCTIONAL PROTEINS, AND GREATLY-REDUCED PERMEABILITY COMPARED TO NON- NVU VESSELS. TUMOR CELLS (GLIOMA) CAN BE ADDED TO MODEL BRAIN TUMORS (VMB-T) AND THE POTENTIAL COMPROMISE OF THE BBB. THIS MODEL WILL PROVIDE UNPRECEDENTED OPPORTUNITIES TO STUDY DRUG RESPONSES OF GLIOMA AND OTHER BRAIN TUMORS IN A MORE NATURAL ENVIRONMENT. OUR PRELIMINARY DATA SHOW THE FEASIBILITY OF OUR GOALS WHICH ARE: SPECIFIC AIM 1: CHARACTERIZE PERMEABILITY OF A SMALL PANEL OF BRAIN-TARGETED DRUGS IN THE VMB AND VMB-T SPECIFIC AIM 2: CHARACTERIZE DELIVERY OF A SMALL PANEL OF IMMUNOTHERAPEUTIC DRUGS IN THE VMB AND VMB-T SPECIFIC AIM 3: CHARACTERIZE DELIVERY OF IMMUNE CELLS IN THE VMB AND VMB-T COMPLETION OF THESE AIMS WILL PROVIDE US WITH A UNIQUE TOOL WE CAN OFFER TO PHARMACEUTICAL COMPANIES FOR STUDIES ON BBB PROPERTIES, AND DELIVERY OF NEUROACTIVE DRUGS AND IMMUNO-ONCOLOGY DRUGS AND CELLS.

CRYOPRESERVING STEM CELL-DERIVED CARDIOMYOCYTES IN READY-TO-USE ASSAY PLATES FOR IMPROVED REPRODUCIBILITY OF DRUG TOXICITY TESTING

MATURATION OF STEM CELL-DERIVED CARDIOMYOCYTES USING A SIMULATED MICROGRAVITY BIOREACTOR AND COMBINATIONS OF EXTRACELLULAR DEVELOPMENTAL CUES

A COMBINATORIAL BIOREACTOR FOR ELECTROMECHANICAL CONDITIONING OF DIFFERENTIATED STEM CELLS

MULTIPLEXED QUANTITATIVE MEASUREMENTS OF FIELD POTENTIAL AND CONTRACTILITY ON BIOMIMETICALLY-MATURED HPSC-CMS

A NANOPATTERNED INTERDIGITATED ELECTRODE ARRAY DEVICE FOR ANTI-MIGRATORY CANCER DRUG SCREENING

SBIR PHASE I: VASOREACTIVE PERFUSED IN VITRO VASCULAR NETWORK

LOW-COST, ROLL-TO-ROLL MANUFACTURING OF NANOSCALE BIOMIMETIC SCAFFOLDS FOR ENHANCED BIOLOGICAL CELL CULTURE

HIGH-THROUGHPUT NANOMEA-BASED PROARRHYTHMIA ASSAY

ASSESSMENT OF DOPAMINERGIC IMPACT AND DEVELOPMENT OF CLINICAL BIOMARKER FOR FRAGILE X SYNDROME

MOLECULARLY DEFINED MODELS FOR HYPOPHARYNGEAL CANCER PATIENT SUBSETS