MANF Produced by MRL Mouse-Derived Mesenchymal Stem Cells Is Pro-regenerative and Protects From Osteoarthritis

The super healer Murphy Roths Large (MRL) mouse represents the “holy grail” of mammalian regenerative model to decipher the key mechanisms that underlies regeneration in mammals. At a time when mesenchymal stem cell (MSC)-based therapy represents the most promising approach to treat degenerative diseases such as osteoarthritis (OA), identification of key factors responsible for the regenerative potential of MSC derived from MRL mouse would be a major step forward for regenerative medicine. In the present study, we assessed and compared MSC derived from MRL (MRL MSC) and C57BL/6 (BL6 MSC) mice. First, we compare the phenotype and the differentiation potential of MRL and BL6 MSC and did not observe any difference. Then, we evaluated the proliferation and migration potential of the cells and found that while MRL MSC proliferate at a slower rate than BL6 MSC, they migrate at a significantly higher rate. This higher migration potential is mediated, in part, by MRL MSC-secreted products since MRL MSC conditioned medium that contains a complex of released factors significantly increased the migration potential of BL6 MSC. A comparative analysis of the secretome by quantitative shotgun proteomics and Western blotting revealed that MRL MSC produce and release higher levels of mesencephalic astrocyte-derived neurotrophic factor (MANF) as compared to MSC derived from BL6, BALB/c, and DBA1 mice. MANF knockdown in MRL MSC using a specific small interfering RNA (siRNA) reduced both MRL MSC migration potential in scratch wound assay and their regenerative potential in the ear punch model in BL6 mice. Finally, injection of MRL MSC silenced for MANF did not protect mice from OA development. In conclusion, our results evidence that the enhanced regenerative potential and protection from OA of MRL mice might be, in part, attributed to their MSC, an effective reservoir of MANF.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth’s Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. However, only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins (SASP) in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

AB0377 COMPUTATIONAL DISCOVERY AND PRECLINICAL VALIDATION OF THERAPEUTIC LEADS WITH NOVEL MOAS FOR SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

Background:Lupus is a heterogeneous, systemic disease that affects millions of patients globally with a high unmet medical need. We present results from our powerful and efficient computational drug discovery platform that identifies hits with first-in-class mechanisms of action that can advance rapidly and successfully through preclinical validation studies. The twoXAR discovery platform uses an artificial-intelligence framework to integrate diverse patient-derived biomedical data sets to build holistic and unbiased models of human disease biology. The utilization of diverse, proprietary algorithms and deep learning principles provides a highly sensitive platform to elucidate complex disease-specific associations between biology and biomedical data that are integrated with a library of existing drug molecules. This enables the identification of novel, high-value drug discovery hits with known pharmacological properties. The twoXAR platform also preserves interpretable data-driven links to disease biology to facilitate efficient validation and optimization studies.Objectives:Apply twoXAR’s computational drug discovery platform for the discovery of first-in-class lupus therapy hits and perform preclinical characterization of selected hits to identify drug discovery lead molecules.Methods:Using clinical SLE patient data, we employed the twoXAR platform to build anin-silicoSLE disease model. Nine molecules with novel mechanisms of action (not previously tested as candidate clinical therapies for lupus) were identified as drug discovery hits and then characterized in preclinical efficacy studies using the MRL mouse model of lupus.Results:In preclinical validation studies with the MRL mouse model, 2 compounds were differentiated by significant efficacy and excellent tolerability. TXR-711 and TXR-712 increased renal function, decreased renal inflammation and decreased inflammation compared to vehicle-treated control mice. In particular, TXR-711 and TXR-712 significantly decreased serum blood urea nitrogen (BUN) levels, decreased proteinuria levels, and significantly improved kidney histology readouts such as glomerulonephritis and tubule basophilia. Additionally, TXR-711 and TXR-712 treatment resulted in significantly decreased inguinal lymph node weight.Conclusion:TXR-711 and TXR-712 were identified as SLE drug discovery leads with novel MOAs for further preclinical development. Ongoing studies with TXR-711 and TXR-712 includes pharmacokinetic, pharmacodynamic, and additional MRL mouse efficacy characterization.Disclosure of Interests:Isaac Hakim Employee of: twoXAR, Inc, Sana Mujahid Employee of: twoXAR, Inc., Aaron C. Daugherty Employee of: twoXAR, Inc., Timothy S. Heuer Employee of: twoXAR, Inc