Technical Specification: A Biosynthetic Approach to Replacing Scarred post-Infarct Tissue

1. Technical Specification: A Biosynthetic Approach to Replacing Scarred post-Infarct Tissue With Healthy Cardiac Tissue 20.020 April 9, 2008 Anna Shcherbina Derek Ju Prarthna Desai Amber Lin Aditya Kohli Robbie Barbero Michael Oh We'd like to thank Professor Gredzinsky for his valuable advice

2. Impact of the Project • All living organisms have scars of some sort (topical, internal, etc.) • Topical scars are a commonplace imperfection with a variety of already-existing treatment options • Scarring in the heart is a lot more perilous • The formation of heart scars after heart attacks results in inefficiency of blood pumping and higher occurrences of arrhythmias cardiac scar tissue (google.com)‏

3. Impact (continued)‏ • What if we could eliminate the tissue build-up that forms after heart attacks and re-form healthy tissue? • Our project presents a new system of treating cardiac scars • Giving someone back his heart tissue could, both literally and figuratively, give back his life

4. The General Idea:Binding • Monoclonal antibodies are injected into the bloodstream. • One end binds to non-phosphorylated light myosin chains in the ECM of cardiac scar cells. • The other end binds to the engineered E. coli bacteria. • Engineered bacteria are injected into the bloodstream and bind to the monoclonal antibody on the cardiac tissue.

5. The General Idea: Digesting and Regenerating • After binding, the E. coli secrete collagenase • Collagenase digests collagen, the main component of scar tissue • A protein tether may be necessary for more targeted digestion • At the same time, the E. coli secrete periostin • A growth factor that stimulates the formation of healthy cardiac tissue.

6. General Idea : Termination • Collagenase and periostin function on a similar time scale and can thus be secreted simultaneously • When the scar tissue has been digested, the E. coli no longer has a binding site • The bacterium is unbound from the heart and carried away by the bloodstream • Several injections may be necessary to ensure complete removal of scar tissue

7. Device Diagram Bacterial Cell G Scar digesting molecule Scar tissue molecule Regulatory Device A E C Scar Digestion Device H B Trigger Scar binding device D Regenerator Device F Heart regeneration molecule

8. Timing Diagram B A B C D E F G H Bacteria cell Scar Digesting Molecule Scar Tissue Molecule G E regulatory device H C Scar digesting Device A Scar binding device Trigger Regenerator Device D F Regenrating Molecule Scar digesting device concentration builds E. coli binds to scar Trigger mechanism is fully activated Scar has been fully digested

9. System Parts

10. List Of Parts

11. Asp Scar Digesting Sensor Device Tar/envz C0082 TT B0015 RBS B0034 Checkpoint 1: Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue Checkpoint 2: Ligand Blot Overlay and Immunoprecipitation Assays are Used to monitor the binding of proteins to one another in the scar binding and healthy tissue binding cascades Scar Tissue Scar Binding Device Monoclonal antibody SMAD 3 DAXX HIPK2 ASK-1 MAP2k3 Parts Diagram (Left 1/3 of cell)‏

12. E. coli binds to one end of monoclonal antibody

13. OmpRR0082 pR R0050 cI pR R0051 pBad I13453 Trigger Asp TT B0015 hk022CI C0050 RBS B0034 λ CI C00051 RBS B0034 MAP2k3 HKCI λCl HKCI λCl TT B0015 hk022CI C0050 hk022CI C0051 RBS B0034 RBS B0034 To periostin secreting device Parts Diagram (Middle 1/3 of cell)‏

14. Scar digesting device Asp producer Reporter 1 λ CI C00051 collagenase AspA C0083 ECFP E0022 TT B0015 RBS B0034 RBS B0034 RBS B0034 λCl AspA Glow cyan TRIGGER Collagenase Asp HKCI Checkpoint 2 hk022CI C0050 mOrange E0030 TT B0015 RBS B0034 Glow orange Glow green Heart regeneration device Reporter 2 periostin GFP J52028 TT B0015 From scar binding device RBS B0034 RBS B0034 periostin Parts Diagram (Right 1/3 of cell)‏

15. Our Highlighted Part: Periostin Secreting Device • Original Gene Sequence taken from Human Chromosome #13 • Coordinates: 13q13.3 • Nucletiodes changed: • 1.EcoRI sites: 2148-2150 from GAA to GAG • ‘2. PstI sites: 1305-1307 from CTG to CTA, 1572-1574 from GCT to GCA • 3. SpeI sites: changed 1653-1655 from ACT to ACC • 4. XbaI sites: changed 2496-2498 from TCT to TCC • For Protein secretion outside E.coli cell, attaching DNA construct • Patent # 5223407 • Inventors: Raymond Wong of Mississauga, CA and Margaret Sutherland of Cambridge, GB • Composed of: • OmpA signal peptide • “Control Region”-lac promoter, tac promoter, and 5'AGGAGGAAAAAATT3' ribosome binding site. Google.com

16. Unknowns • What E. coli receptor proteins are best to use for binding to the monoclonal antibody? • The protein kinases look good on paper; but will they function as predicted in vivo? • Are we correct in assuming that we can synchronize the function of collagenase and periostin so that both can be secreted at the same time? • Dosage and frequency of administration: how many injections are necessary, and how often must they be given?

17. Safety and Security Considerations • It is necessary to prevent the immune system from attacking the E. coli in the bloodstream • Bactoblood chassis • Periostin is a growth factor, however similar clinical usage has not demonstrated carcinogenic side effects • The E. coli bacteria will bind to the extracellular matrix of myocardial cells • It may not be safe to block heart cell receptors by binding directly to them google.com

18. Safety and Security Considerations (Continued)‏ • It is necessary to ensure that collagenase only digests collagen in infarcted cells, not healthy cells • If more accurate targeting is needed than our regulation device provides, a protein “tether” can be used • Initial stages of the project can be performed in BL1 labs • BL2 labs are necessary for subsequent development

19. The Competition: Who Else is Doing This? • Our project was inspired by Gelfoam • A periostin-soaked gel applied directly to the heartOur product is less invasive Gelfoam inserted into the heart (google.com)‏

20. Safety and Security Considerations (Continued)‏ • Scar healing silicone sheets • Limited usage: must be applied daily, immediately after the wound has closed, and not longer than 3 months • Low-energy laser irradiation • Cost prohibitive Scar-healing silicone sheet (google.com)‏

21. In Vivo Debugging: Fluorescent Markers Reporter Debugging Function Glow orange Trigger “Off” Glow cyan Scar Digesting Device “On” Glow green Heart Regeneration Devices “On”

22. Testing and Debugging: • Mechanism 1: • Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue • Mechanism 2: • Immunoprecipitation Assays • Method that uses the antigen-antibody reaction principle to identify a protein that reacts specifically with an antibody from mixture of proteins so that its quantity or physical characteristics can be examined.

23. 6 Month Development Plan 2 Teams of 3 People Each • Clone collagenase and express it in E. coli • Synthesize trigger mechanism for collagenase secretion • Clone periostin and express it in E. coli • Synthesize trigger mechanism for periostin secretion • Synthesize monoclonal antibody GOAL: A successfully engineered plasmid ready for insertion into an E. coli bacterium

24. Thank You. Are there any questions?

25. References http://en.wikipedia.org/wiki/Monoclonal_antibodies Information about monoclonal antibodies http://www.ncbi.nlm.nih.gov/pubmed/16883602 scar and healthy tissue binding http://herkules.oulu.fi/isbn9514267214/html/x1329.html cardiac extracellular matrix http://www.ncbi.nlm.nih.gov/pubmed/9521338 expression of cardiac proteins http://www.ncbi.nlm.nih.gov/pubmed/10198196 SMAD protein research http://www.ingentaconnect.com/content/ap/mc/1999/00000031/00000003/art00902;jsessionid=cw3x1rheoz22.alice?format=print SMAD and TGF -beta protein research http://books.nap.edu/openbook.php?record_id=9450&page=8 monoclonal antibodies

26. References (cont.) http://content.nejm.org/cgi/content/full/344/23/1785engineered cardiomycotes, myocardial infarction http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T13-4CNCNXW-5&_user=501045&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=b622660c2a4b7ab53b1f770f463c8979myocardial infarction description http://www.medicalnewstoday.com/articles/92139.php Fibroblast/tissue buildup description Wong, Raymond W. K. (Mississauga, CA), Sutherland, Margaret L. (Cambridge, GB) 1993. “Excretion of heterologous proteins from E. Coli.” United States ALLELIX INC (CA). 5223407 http://www.freepatentsonline.com/5223407.html

27. References (cont.) http://www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET=F&LANGUAGE=ENG&KEY=05%2F019471&IA=05%2F019471&DISPLAY=DESC periostin a natural response in mice to myocardial infarctions http://www.nature.com/nrd/journal/v6/n9/full/nrd2406.html periostin experiment showing it improves heart function after infarctions by regenerating tissue http://www.nature.com/nm/journal/v13/n8/abs/nm1619.html article on prospects of periostin as treatment http://www.childrenshospital.org/newsroom/Site1339/mainpageS1339P1sublevel319.html article on how periostin was used to induce mature heart cells to grow