Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

R1745 farshid guilak

56 views

Published on

Basic Award

Published in: Health & Medicine
  • Be the first to comment

  • Be the first to like this

R1745 farshid guilak

  1. 1. Engineering New Biologic Therapies For Osteoarthritis Farshid Guilak, Ph.D. Departments of Orthopaedic Surgery, Developmental Biology, and Biomedical Engineering Center of Regenerative Medicine and Shriners Hospitals for Children – St. Louis Washington University St. Louis MO Disclosures: Cytex Therapeutics Inc., Active Implants Corp.
  2. 2. Soft tissue Injury and repair Obesity and OA Tissue Engineering and Stem Cells Mechanobiology Synthetic Biology Post-traumatic Arthritis
  3. 3. • Small molecule compounds that block or simulate mechanical loading • Diet, drug, and gene therapies for obesity-induced OA • Total biological resurfacing of the joint • Autoregulated designer stem cells for arthritis therapy Mechanobiology Tissue Engineering and Stem Cells Synthetic Biology Obesity and OA
  4. 4. Abnormal Stress Normal Biology Normal Stress Abnormal Biology Inflammation Aging Sepsis Genetic mutations Immune responses Altered joint loading Malalignment Trauma Obesity Joint shape Aberrant repair response Increased catabolism Matrix damage Mechanical failure Joint Destruction Pain Disability Osteoarthritis: A Family of Diseases Mechanically and biologically mediated
  5. 5. Abnormal Stress Normal Biology Normal Stress Abnormal Biology Inflammation Aging Sepsis Genetic mutations Immune responses Altered joint loading Malalignment Trauma Obesity Joint shape Aberrant repair response Increased catabolism Matrix damage Mechanical failure Joint Destruction Pain Disability Osteoarthritis: A Family of Diseases Mechanically and biologically mediated
  6. 6. Body (100 m) Understanding Mechanotransduction: From Whole Body to Cell Membrane Organ (10-2 m) Tissue (10-3 m) Cell (10-5 m) Nucleus (10-6 m) Membrane (10-8 m)
  7. 7. Physiologic Magnitudes of Cartilage Strain Torzilli et al., 1999; Eckstein et al., 2001; D’Lima et al., 2001; Lee et al., 2005; Bischof et al., 2010; Widmyer et al., 2013; Stolberg et al., 2013; Rolauffs et al., 2013 Reviewed in Sanchez-Adams et al., Curr Rheum Rep, 2014
  8. 8. Free-swelling Collagen Fiber PGFixed Charges Mobile Ions oo + + + ++ + + + + + + + - -- - + + + + + - - - - - - - - - - - - + oo + + + + - -- - + + + ++ + + + + + + + + - - - - - - - - - - - - Mechanical Stress Biophysical Signals at the Cellular Level: How do Cells Sense Mechanical Loading? Mechanically-induced Ca++ Signaling Uncompressed Compressed (15%) Guilak, et al., 1995; Roberts et al., 2001; Pingguan-Murphy et al., 2006; Wann et al., 2012; Han et al. 2012;
  9. 9. The ECM and PCM in Mechanotransduction Type VI Collagen Normal Cartilage PCM OA Cartilage PCM Mechanical Properties of the PCM
  10. 10. Chondrocytes are Highly Sensitive to Osmolarity but Not Stretch 0 25 50 75 100 Hypo-osmotic Stress * Mean ± s.e.prop. N=30-40 per group PercentResponding Hyper-osmotic Stress Iso-osmotic Stretch CC pC CP = 2c + cF pP s P p* C* = 2c* pressure Vc VCh -VC PCM Cell potentials NaCl solution mixture stress s* sC
  11. 11. Transient Receptor Potential (TRP) Channels: The Cellular Sensors of the Body Nilius (2003), Strotmann (2000) Melastatin Vanilloid Canonical MucolipinPolycystin with Sanjay Kumar and Bart Votta, Articular Cartilage
  12. 12. 40x Osmotically-induced Calcium Signaling in TRPV4 WT and Knockout Mice Clark et al., A&R, 2010
  13. 13. Loss of Mechanotransduction via TRPV4 leads to Osteoarthritis Control (+/+) 6 months Knockout (-/-) 12 months 12 months 6 months 12 months
  14. 14. Inhibiting TRPV4 blocks the biosynthetic effect of mechanical loading * GSK205 GSK205 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Control GSK205 Loading Loading+GSK205 Collagencontent(mg/disk) Day 28 Total Collagen O’Conor et al., PNAS, 2014
  15. 15. Inhibiting TRPV4 blocks the biosynthetic effect of mechanical loading * GSK205 GSK205 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Control GSK205 Loading Loading+GSK205 Collagencontent(mg/disk) Day 28 Total Collagen O’Conor et al., PNAS, 2014
  16. 16. “Artificial” Loading with TRPV4 Agonist Enhances Matrix Accumulation * GSK205 GSK205 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Control GSK205 Loading Loading+GSK205 Collagencontent(mg/disk) Day 28 Total Collagen O’Conor et al., PNAS, 2014 Control GSK101 Safranin-O (s-GAG)CollagentypeII Control GSK101
  17. 17. GSK101 (TRPV4 Activator) Loading Gene Array Shows Highly Similar “Mechanome” in Response to Loading or Chemical TRPV4 Activation Upregulated Downregulated Amy McNulty and Chris O’Conor TGF-b family BMP inhibitors
  18. 18. Kanju et al., in review Novel Compounds Activate or Block TRPV4: Mechanotransduction-based DMOADs? Phan et al., A&R, 2009 Fluorescence Imaging Plate Reader (FLIPR) Antagonist Dual Inhibitor of TRPA1 and TRPV4
  19. 19. Artificial Mechanotransduction: Can a Drug Simulate or Block Mechanical Loading? Role of Mechanotransduction • Disuse • Altered joint loading and PTOA • Overload • Channelopathies (mutations) • Tissue engineering • Obesity
  20. 20. Obesity and OA: Leptin, Loading, or Lard? High-fat diet Osteoarthritis Altered Joint Loading Pain Sensing & Coping Behavior Adiposity & Systemic Inflammation Strength & Motor Performance Anxiety & Hyperalgesia Saturated fat content Altered mechanical signaling Adiposity & Leptin Macrophage Infiltration Joint Injury & inflammation
  21. 21. Diet-induced Obesity Results in a Dysfunctional Stem Cell Population Wu et al., IJO, 2014 • ASCs or Bone marrow MSCS • CD45- TER119- PDGFRα+ Sca-1+ A B C D Lean Obese Adipogenesis Osteogenesis E F Lean Obese A B C D A B C D E Sca-1 CD45/TER119 MSCs 1% 5%
  22. 22. 2 4 6 8 10 12 14 16 18 -log10(pvalue) Intergenic 2 4 6 8 10 12 14 16 18 -log10(pvalue) Differentially Methylated CpG Sites in Obese Stem Cells 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 1819 X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 X Gene Regions (<5000bp to TSS) Chromosome Number
  23. 23. Stem Cells and Tissue Engineering: Can We Treat OA?
  24. 24. Moutos et al, Nature Materials, 2007 3D Woven Composite Scaffolds
  25. 25. Anatomically Shaped Scaffolds for Total Joint Resurfacing CAD Model MRI Tissue Engineered Construct for Whole Joint Resurfacing Molded Scaffold
  26. 26. Liposuction Digest and separate fat cells from stem cells Cartilage and Bone FormationImplantation Clinical Paradigm for Treating OA Custom fit scaffold Osteoarthritis
  27. 27. Inflammatory Processes in OA Poole, Guilak, and Abramson, 2007
  28. 28. Interleukin 1 Prevents MSC Chondrogenesis Buhrmann et al., 2010; Felka et al., 2009; Wehling et al., 2009; Ousema et al., 2012; Ferreira et al., 2013 100 pg/ml IL-1 Control Type II Collagen Chondroitin-4-Sulfate
  29. 29. Site-Specific Protein or Virus Delivery for Cell-Instructive Scaffolds Lentivirus Poly-L-lysine Brunger et al, PNAS, 2014
  30. 30. Anti-Cytokine Tissue Engineered Cartilage: Dox-Inducible IL-1Ra Expression via Lentivirus 5’ LTR CMVie cPPT/cTS IRES Puro 3’ LTR psi WPRE IL-1Ra rtTA2S-M2hPGKTRE Glass et al., Biomaterials, 2014 ~1 µg/ml IL-1Ra
  31. 31. Can We Program Intelligence into Cells to Autoregulate Gene Expression? Gene Therapy: Constitutive promoter Dox-Inducible Promoter Smart cell? Protein delivery
  32. 32. Can We Custom-Design a “Smart” Stem Cell to Express Transgenes only when Needed? Cytokine stimulus Inflammatory response Cytokine Inhibitors
  33. 33. CCL2 Garcia-Bonilla, 2014 Autoimmune Diseases and TNF-a • Episodic disease flares • Anti-TNF therapy successful • Given continuously at high dose • Potential side effects
  34. 34. 5’ UTREndogenous Ccl2 Promoter 5’ UTR sTNFRI Exon 1 CDS Exon 1 CDS Intron Homologous Donor + Targeted GOI TNFa TNFa TNFa Brunger et al., ORS, 2016
  35. 35. TNF-a Induces sTNFR1 Production and Shuts off NF-kB Added TNF
  36. 36. TNF-a Induces sTNFR1 Production and Shuts off NF-kB
  37. 37. TNF-a Induces sTNFR1 Production and Shuts off NF-kB
  38. 38. Autoregulated sTNFR1 Production Prevents TNF-a Effects 0 ng/ml TNF-a 20 ng/ml TNF-a ControlsTNFR1
  39. 39. Custom-Designed Cells for Cell, Gene, or Tissue Engineering Therapies Stimulus Functional properties Programmed cell response
  40. 40. Mechanobiology Tissue Engineering and Stem Cells Synthetic Biology Obesity and OA • Control of mechanotransduction may provide a novel target for treating musculoskeletal diseases • The role of obesity in OA is really complicated: • Complex scaffolds can provide mechanical function as well as cell-instructive signals • Tools of synthetic biology and genome editing can be used to create “smart” cells that can provide autoregulated drug delivery for OA
  41. 41. Faculty Lou Defrate, Ph.D. Amy McNulty, Ph.D. Dianne Little, BVSc, Ph.D. Post-docs and Staff Brian Diekman, Ph.D. Brad Estes, Ph.D. Frank Moutos, Ph.D. Johannah Sanchez, Ph.D. Vincent Willard, Ph.D. Chris Gilchrist, Ph.D. Holly Leddy, Ph.D. Oswaldo Lozoya, Ph.D. Natasha Case, Ph.D. Ruhang Tang, Ph.D. Orthopaedic Bioengineering Lab Staff Elaine Campbell, M.S. Bridgette Furman, B.S. Steve Johnson, C.V.T. Dawn Chasse, B.S. Undergraduate Students Ananya Zutshi Dutch Waanders Laurel Kaye Dan Sykora Dharshan Sivaraj Jacob Ruprecht Adarsh Ettyreddy Graduate Students Jonathan Brunger, M.S. Shaunak Adkar, B.S. Nguyen Hyunh, B.A. Katie Glass, B.S. Shannon O’ Connor, B.S. Kelly Kimmerling, M.S. Chris O’Conor, B.S. Chris Rowland, B.S. Chia-Lung Wu, M.S. Alison Ross, B.S.
  42. 42. Acknowledgments Lori Setton, Ph.D. Dept. of Biomed. Eng. Virginia Kraus, M.D., Ph.D. Dept. of Medicine Janet Huebner, M.S. Dept. of Medicine David Pisetsky, M.D., Ph.D. Dept. of Medicine Brice Weinberg, M.D. Dept. of Medicine Charlie Gersbach, Ph.D. Dept. of Biomed. Eng. Wolfgang Liedtke, M.D., Ph.D. Dept. of Medicine Stefan Zauscher, Ph.D. Dept. of Biomed. Eng. Steve Olson, Ph.D. Dept. of Orthop. Surg. Kam Leong, Ph.D. Columbia Univ. Lisa Freed, M.D., Ph.D. MIT NIH R42 AR66439 Arthritis Foundation NIH R42 AR55042 AO Foundation NIH R01 AG46927 DOD W81XWH NIH R01 AG15768 NSF 1445792 NIH R01 AR48182 Nancy Taylor Foundation NIH R01 AR48852 NIH R21 AR65956

×