Steadman Philippon Research Institute

THE USE OF SENOLYTIC AND ANTI-FIBROTIC AGENTS TO IMPROVE THE BENEFICIAL EFFECT OF BONE MARROW STEM CELLS FOR OSTEOARTHRITIS - ABSTRACT: OSTEOARTHRITIS (OA) IS A PROGRESSIVE JOINT DISEASE LEADING TO CARTILAGE DAMAGE, PAIN AND LOSS OF FUNCTION. WHILE MANY STEM CELL THERAPIES FOR OA ARE UNDER INVESTIGATION, NONE ARE CURRENTLY FDA-APPROVED FOR MODIFYING THE COURSE OF THE DISEASE. OF THE MANY ADULT STEM CELL TYPES POTENTIALLY APPLICABLE TO OA, BONE MARROW STEM CELLS (BMSCS) FROM BONE MARROW ASPIRATE CONCENTRATE (BMC) ARE THE MOST CLINICALLY TRANSLATABLE (AND ARE ALREADY IN CLINICAL USE) SINCE THEY CAN BE HARVESTED USING MINIMALLY INVASIVE TECHNOLOGY AND DO NOT REQUIRE IN VITRO EXPANSION. THERE IS, HOWEVER, SIGNIFICANT POTENTIAL FOR IMPROVING EFFICACY OF BMSC TREATMENT FOR OA. THE NUMBER OF SENESCENT CELLS IN BMC INCREASES WITH AGE AND OA, AND THESE CELLS RELEASE PRO-INFLAMMATORY CYTOKINES/CHEMOKINES, PROTEASES, AND OTHER SENESCENCE-ASSOCIATED SECRETORY PHENOTYPES (SASP) THAT CAN IMPAIR STEM CELL FUNCTION AND LIKELY CONTRIBUTE TO OA DEVELOPMENT/PROGRESSION. DR. KIRKLAND’S LABORATORY (CO- INVESTIGATOR) HAS IDENTIFIED COMPOUNDS THAT SPECIFICALLY KILL SENESCENT CELLS, ABROGATING SYSTEMIC SASP FACTORS. WE HAVE SHOWN THAT THESE SENOLYTIC AGENTS CAN DELAY OA IN A PRECLINICAL MODEL. WE HAVE ALSO SHOWN THAT BLOCKING FIBROSIS WITH LOSARTAN (A TGF-SS1BLOCKER) CAN IMPROVE CARTILAGE REPAIR BY PROMOTING REGENERATION OF HYALINE CARTILAGE WHILE REDUCING THE AMOUNT OF FIBROCARTILAGE. THUS, WE HYPOTHESIZE THAT ADMINISTRATION OF SENOLYTIC AND/OR ANTI-FIBROTIC AGENTS WILL ENHANCE THE BENEFICIAL EFFECT OF BMSCS FOR TREATING OA. WE PROPOSE TO PERFORM, A RANDOMIZED CLINICAL TRIAL AT THE STEADMAN CLINIC (TSC) AND STEADMAN PHILIPPON RESEARCH INSTITUTE (SPRI). THIS PHASE I/II TRIAL WILL EVALUATE THE SAFETY AND EFFICACY OF FISETIN (A SENOLYTIC DIETARY SUPPLEMENT) AND LOSARTAN (AN ANTI-FIBROTIC DRUG), USED EITHER INDIVIDUALLY OR IN COMBINATION, FOR IMPROVING THE CLINICAL EFFICACY OF BMSCS IN THE TREATMENT OF KNEE OSTEOARTHRITIS. THE SENOLYTIC (FISETIN 1000MG/DAY, PREVIOUSLY FDA IND APPROVED) REGIMEN WILL BE CYCLES OF 2 DAYS ON/28 DAYS OFF, ADMINISTERED BEFORE AND 3 MONTHS AFTER BMSC TREATMENT. THE ANTI-FIBROTIC (LOSARTAN, 25MG/DAY, PREVIOUSLY IND-EXEMPTED) WILL BE ADMINISTERED FOR 30 DAYS STARTING IMMEDIATELY AFTER BMSC TREATMENT. OA KNEE JOINTS WILL UNDERGO MRI AT BASELINE AND 18 MONTHS POST- TREATMENT TO ASSESS CHANGES IN CARTILAGE MORPHOLOGY AND STRUCTURE OVER TIME. PATIENT-REPORTED OUTCOMES FOR PAIN AND FUNCTION WILL BE COLLECTED AT BASELINE AND 3, 6, 12 & 18 MONTHS. JOINT AND CARTILAGE FUNCTION WILL BE ASSESSED USING VIDEO-MOTION ANALYSIS AT BASELINE AND 18 MONTHS. OA BIOMARKERS RELATED TO CARTILAGE DEGENERATION, INFLAMMATION AND PAIN WILL BE ASSESSED AT BASELINE AND 18 MONTHS. BLOOD AND SYNOVIAL FLUID WILL BE EVALUATED AT BASELINE, 4 DAYS AND 18 MONTHS AFTER TREATMENT TO ASSESS CHANGES IN CELLULAR SENESCENCE AND OA BIOMARKERS. THIS TRIAL WILL BUILD UPON A CURRENTLY ACTIVE CLINICAL TRIAL ON ORTHOBIOLOGICS FOR OA TREATMENT AT SPRI (UTILIZING THE SAME PATIENT POPULATION AND OUTCOMES ASSESSMENTS), DEMONSTRATING THE ABILITY OF OUR TEAMS TO PERFORM THE PROPOSED STUDY AND ALSO LEVERAGING THE COMBINED TRIALS TO EFFECTIVELY PROVIDE A 6-ARM, COMPREHENSIVE ASSESSMENT OF BIOLOGICAL THERAPIES FOR IMPROVING TREATMENT OF OA.

THE USE OF SENOLYTIC AGENT TO IMPROVE THE BENEFIT OF PLATELET-RICH PLASMA AND LOSARTAN FOR TREATMENT OF FEMOROACETABULAR IMPINGEMENT AND LABRAL TEAR: A PILOT STUDY

BONE ABNORMALITIES & HEALING DEFECT IN MUSCULAR DYSTROPHY

ARTICULAR CARTILAGE TISSUE ENGINEERING WITH HUMAN PLURIPOTENT STEM CELLS - ABSTRACT IMPAIRMENT OF ARTICULAR CARTILAGE FUNCTION AFTER INJURY AND DISEASE LIKE OSTEOARTHRITIS (OA), REMAINS A MAJOR HEALTH PROBLEM. ONE OF THE MAJOR DRAWBACKS OF TISSUE ENGINEERING-BASED THERAPIES FOR DAMAGED JOINT ARTICULAR CARTILAGE IS THAT THE CARTILAGINOUS REPAIR TISSUE FORMED BY IMPLANTED MESENCHYMAL STROMAL CELLS OR ENDOGENOUS PROGENITORS DOES NOT RESEMBLE ARTICULAR CARTILAGE, LIKELY DUE TO FIBROCHONDROGENESIS AND THE ENDOCHONDRAL OSSIFICATION PROCESS. JOINT CARTILAGE IS GENERATED DURING EMBRYOGENESIS BY SPECIALIZED GDF5+ CELLS CALLED ‘INTERZONE’ CELLS OR ‘JOINT PROGENITORS’. THEY ARE DISTINCT FROM PROGENITORS THAT GIVE RISE TO GROWTH PLATE CHONDROCYTES. IN THIS PROPOSAL, WE AIM TO DEFINE THE MOLECULAR TARGETS THAT CONTROL ARTICULAR-LIKE PERMANENT CHONDROCYTE FORMATION VERSUS GROWTH PLATE-LIKE TRANSIENT CHONDROCYTE FORMATION, BY USING NOVEL GDF5+ MESENCHYMAL CELLS DEVELOPED FROM HUMAN PLURIPOTENT STEM CELLS (HPSCS). DURING IN VITRO CHONDROGENESIS, SUCH CELLS EXPRESS SIGNS OF PRIMITIVE (OR EMBRYONIC) ARTICULAR CHONDROCYTES BUT NOT OF CHONDROCYTE HYPERTROPHY. SIGNIFICANTLY, AFTER TRANSPLANTATION OF THE CARTILAGE THEY DEVELOP, NO MINERALIZATION WAS OBSERVED FOR 8 WEEKS (I.E., PERMANENT CARTILAGE). THEREFORE, THE HPSC-DERIVED GDF5+ CELLS MAY SHARE THE ACTIVITY OF JOINT PROGENITORS, ALTHOUGH GENOME-WIDE RNA-SEQUENCING (SEQ) ANALYSES SUGGESTED ASSOCIATION WITH DEVELOPING TENOCYTES OR LIGAMENTOCYTES. IN CONTRAST, ALTERNATIVE HPSC-DERIVED CHONDROPROGENITORS, SOX9+ CELLS, GENERATED CARTILAGE THAT READILY UNDERWENT COMPLETE MINERALIZATION, MIMICKING GROWTH-PLATE CHONDROPROGENITORS. INTERESTINGLY, WHEN MIXED WITH GDF5+ CELLS, THE SOX9+ CELL-DERIVED CARTILAGE BEHAVED AS PERMANENT CARTILAGE IN A GDF5+ CELL-DOSE-DEPENDENT MANNER, SUGGESTING THE INVOLVEMENT OF A NON-CELL AUTONOMOUS MECHANISM. THEREFORE, WE FIRST PROPOSE TO TEST IF THE GDF5+ CELLS HAVE A JOINT PROGENITOR-LIKE ACTIVITY, CHARACTERIZE THE (PERMANENT) CARTILAGE DEVELOPED FROM THEM, AND SHED LIGHTS ON HOW THE CELLS GENERATE PERMANENT CARTILAGE (AIM 1). SECOND, WE PLAN TO IDENTIFY GENES POTENTIALLY INVOLVED IN PERMANENT CARTILAGE FORMATION FROM GDF5+ CELLS THROUGH COMPARATIVE RNA-SEQ ANALYSES OF CARTILAGE PELLETS DEVELOPED FROM THE GDF5+ AND SOX9+ CELLS (AIM 2). WE WILL THEN FUNCTIONALLY VALIDATE THE CANDIDATE GENES (AND THEIR ENCODED PROTEINS, INHIBITORS AND ACTIVATORS IF COMMERCIALLY AVAILABLE) FOR THEIR ABILITY TO ENABLE SOX9+ CELLS TO GENERATE ARTICULAR-LIKE PERMANENT CHONDROCYTES USING GENE KNOCKOUT AND OVEREXPRESSION TECHNIQUES (AIM 3). WE WILL THEN EXAMINE WHETHER GDF5+ CELLS AND SUCH GENE-MODIFIED SOX9+ CELLS INDUCE MORE SUSTAINED REPAIR OF DAMAGED JOINT CARTILAGE THAN SOX9+ CELLS (AIM 4). LASTLY, ANY OF THE GENES DEFINED IN THESE STUDIES WILL BE MANIPULATED SIMILARLY IN THERAPEUTICALLY RELEVANT ADULT MESENCHYMAL STROMAL CELLS TO CONFIRM THAT TARGETING THE SAME MECHANISMS WILL CONVERT THE ADULT STEM CELLS TO ARTICULAR CARTILAGE-FORMING CELLS (AIM 4). THUS, SUCCESS OF THE PROPOSED RESEARCH WILL PROVIDE MECHANISTIC INSIGHTS INTO HOW ARTICULAR-LIKE PERMANENT CARTILAGE CAN BE SELECTIVELY FORMED FROM VARIOUS CHONDROGENIC CELLS, POTENTIALLY LEADING TO NOVEL THERAPEUTIC STRATEGIES FOR EFFECTIVE, SUSTAINED REPAIR OF DAMAGED CARTILAGE.

STRATEGIES FOR IMPROVING THE QUALITY AND EFFICACY OF AUTOLOGOUS EXTRACELLULAR VESICLES FOR TREATING MUSCULOSKELETAL INJURIES AND DISEASE

THE USE OF SENOLYTIC AND ANTI-FIBROTIC AGENTS TO IMPROVE THE BENEFICIAL EFFECT OF BONE MARROW STEM CELLS FOR OSTEOARTHRITIS

MUSCLE STEM CELLS REPROGRAMMED THROUGH GENOME ENGINEERING FOR AUTONOMOUSLY REGULATED ANTI-FIBROTIC THERAPY

SMART STEM CELLS THAT AUTONOMOUSLY DOWN-MODULATE TFG-? SIGNALING FOR ARTICULAR CARTILAGE REPAIR - ABSTRACT ARTICULAR CARTILAGE IS AN IMPORTANT HYPOVASCULAR TISSUE STRUCTURE THAT, ONCE DAMAGED, DOES NOT SPONTANEOUSLY REGENERATE AND OFTEN LEADS TO OSTEOARTHRITIS. CONSIDERABLE EFFORTS HAVE BEEN MADE TO ESTABLISH THERAPIES THAT BIOLOGICALLY REPAIR DAMAGED ARTICULAR CARTILAGE, WHICH RELY HEAVILY ON ENDOGENOUS OR EXOGENOUS CHONDROGENIC STEM/PROGENITOR CELLS (CSPCS). ONE MAJOR DRAWBACK OF CURRENT BIOLOGICAL THERAPIES IS THAT FIBROCARTILAGE TENDS TO BE REGENERATED, WHICH SHOWS INFERIOR BIOMECHANICAL PROPERTIES COMPARED WITH THE HEALTHY HYALINE ARTICULAR CARTILAGE. ALTHOUGH A NUMBER OF THERAPIES HAVE BEEN DEVELOPED TO IMPROVE THE SITUATION, A REPRODUCIBLE METHOD TO REGENERATES HYALINE CARTILAGE THAT RESISTS ENDOCHONDRAL OSSIFICATION IS YET TO BE DEVELOPED. RECENTLY, WE HAVE DEMONSTRATED THAT ORAL ADMINISTRATION OF TYPE 1 ANGIOTENSIN II RECEPTOR ANTAGONIST, LOSARTAN, REGENERATES MOSTLY HYALINE CARTILAGE AFTER MICROFRACTURE IN RABBITS, AND CONCOMITANTLY REDUCES TRANSFORMING GROWTH FACTOR-BETA 1 (TGF-B1) EXPRESSION. THESE RESULTS SUGGEST THAT A PROPER SPATIOTEMPORAL SUPPRESSION OF TGF-B1 MAY BE CRITICAL TO PREVENT FIBROCARTILAGE FORMATION AND ALLOW HYALINE CARTILAGE REGENERATION. HOWEVER, TGF-B IS A CHONDROGENIC FACTOR FOR CSPCS, AND INVOLVED IN THE MAINTENANCE OF ARTICULAR CARTILAGE. FURTHERMORE, PHARMACOLOGICAL ANTI-TGF- B THERAPIES CAN CAUSE SIGNIFICANT UNWANTED SIDE EFFECTS. THEREFORE, WE HYPOTHESIZE THAT EFFECTIVE HYALINE CARTILAGE REGENERATION WITHOUT OVERT SIDE EFFECTS MAY BE ACHIEVED BY A CELL THERAPY THAT ALSO INHIBITS TGF-B1 SIGNALING LOCALLY AS NEEDED. USING THE CRISPR/CAS9 TECHNOLOGY, DR. FARSHID GUILAK (MPI) HAVE REPORTED A NOVEL APPROACH THAT REPROGRAMS STEM CELLS (CALLED STEM CELLS MODIFIED FOR AUTONOMOUS REGENERATIVE THERAPY OR SMART) TO MAKE IT POSSIBLE TO DELIVER ANTI-INFLAMMATORY FACTOR IN AN AUTO-REGULATED, FEEDBACK-CONTROLLED MANNER, AND DEMONSTRATED ITS UTILITY FOR MUSCULOSKELETAL REGENERATIVE MEDICINE. IN THIS PROPOSAL, WE AIM TO REPROGRAM THERAPEUTIC CELLS TO BE ABLE TO SUPPRESS TGF-B1 ACTION LOCALLY AROUND THE CELLS BY INDUCING TGF-B INHIBITOR FROM THEM WHENEVER TGF-B1 IS PRESENT IN THE ENVIRONMENT (I.E., AUTONOMOUS SUPPRESSION OF FIBROTIC ENVIRONMENT). WE CONSEQUENTLY PROPOSE TO TEST WHETHER SUCH SMART CELLS MAY IMPROVE CARTILAGE REPAIR WHEN COMPARED TO CONVENTIONAL CELLS. FOR THIS PURPOSE, WE WILL USE MUSCLE-DERIVED STEM CELLS (MDSCS) AND MESENCHYMAL STROMAL CELLS (MSCS) TO REPROGRAM DECORIN (DCN) AS THE TGF-B1 INHIBITOR, AND THE TGF-B-INDUCIBLE SMAD7 GENE AS THE SITE TO KNOCK-IN DCN (DCN-KI), USING THE CRISPR/CAS9 TECHNOLOGY. WE HAVE ALREADY REPROGRAMMED MDSCS, AND OUR PRELIMINARY IN VITRO RESULTS INDICATE THAT DECORIN IS INDUCED IN A TIME & DOSE DEPENDENT MANNER AFTER TGF-B1 EXPOSURE, AND CAN SUPPRESS THE FIBROTIC CASCADE. WE PROPOSE TO REPROGRAM MSCS USING A SIMILAR TACTIC, AND TEST WHETHER THESE SMART CELLS (DCN-KI MDSCS, AIM1; DCN-KI MSCS AIM 2) MITIGATE THE EFFECTS OF TGF- B1 AUTONOMOUSLY AND INDUCE LONG-TERM REPAIR OF HYALINE ARTICULAR CARTILAGE, WHEN COMPARED WITH CONTROL UNMODIFIED CELLS. THUS, RESULTS OF THIS STUDY WILL PROVIDE A PROOF-OF-CONCEPT ON THE UTILITY OF THE INNOVATIVE AUTOREGULATORY GENE CIRCUIT SYSTEM FOR DEVELOPMENT OF EFFECTIVE & SAFE CELLULAR TOOLS FOR ARTICULAR CARTILAGE REPAIR.

EFFECTS OF CIRCULATING FACTORS AND PROGENITORS ON WOUND HEALING DURING PREGNANCY