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Biosynthetic Approach for Replacing Scarred Heart Tissue

This project aims to develop a new system for replacing scarred cardiac tissue caused by heart attacks, using a biosynthetic approach. Monoclonal antibodies, engineered bacteria, and growth factors will be used to digest scar tissue and stimulate the formation of healthy cardiac tissue.

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Biosynthetic Approach for Replacing Scarred Heart Tissue

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  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

  2. Impact of the Project • Almost 100 percent of living organisms (humans included) have scars of some sort (topical, internal, etc.) • Topical scars are a commonplace imperfection with a variety of already-existing treatment options • Of all scars, scarring in the heart is one of the most perilous • Heart scar tissue forms after heart attacks • The formation of heart scars after heart attacks results in inefficiency of blood pumping and higher occurrences of arrhythmias

  3. Impact (continued)‏ • What if we could find a way to actually eliminate the precarious tissue build-up that forms after heart attacks and re-form healthy tissue? • Our project would propose a new system of dealing with formed scars • Being able to give back someone his heart tissue could, both literally and figuratively, give back his life

  4. The General Idea: • Monoclonal antibodies are injected into the bloodstream. • One end binds to cardiac scar tissue. • The other end binds to a SMAD receptor on the engineered bacteria. • Engineered bacteria are injected into the bloodstream and bind to the monoclonal antibody on the cardiac tissue. • The bacteria secrete collagenase to digest scar tissue.

  5. The General Idea (ctd.): • At the same time, they secrete periostin, a growth factor that stimulates the formation of healthy cardiac tissue. • When the scar tissue has been digested, the bacteria bind to healthy tissue and continue secreting periostin. • The bacteria begin to secrete Verapamil, a synthetic drug that prevents the reformation of scar tissue. • Periodic injections of bacteria are given to the patient until scar tissue has been fully replaced by healthy tissue.

  6. Device Diagram Scar digesting molecule B Bacterial Cell Scar digesting molecule Scar tissue molecule D Scar Digesting Molecule Sensor A Scar binding device M Trigger 1 K Scar Digestion Device N C L Scar Repressor Device J G Trigger 2 H Healthy Cell Binding Device Regenerator Device F Scar Repressor molecule I E Heart regeneration device

  7. Timing Diagram

  8. System Parts

  9. OR GATE Part Name Gene Name/Code (If Applicable)‏ Promoter I13453 HEALTHY TISSUE BINDING DEVICE I14032 RBS B0034 Heterotrimeric G protein LacI C0012 Alpha-syntropin TT B0015 Gs-alpha Gb-y tetR C0040 pBAD promoter SCAR DIGESTING DEVICE SCAR BINDING DEVICE RBS B0034 SMAD 3 receptor protein DAXX protein Collagenase HIPk2 protein Aspa C0083 ASK-1 Map2k3 SCAR PREVENTION DEVICE pBAD SCAR DIGESTING SENSOR DEVICE RBS B0034 Verapamil ASP RBS B0034 tar/env C0082 TISSUE REGENERATION DEVICE TT B0015 RBS B0034 NAND GATE periostin RBS B0034 nk022CI C0050 TT B0015 CHECKPOINT Promoter R0082 EYEP E0030 Promoter R0030 morange E2050 Promoter I13453 Promoter R0031 mcherry E2060 HK022CI C0050 RFP E1010 Lambda CI C0051 ECFP E0022 GFP I52028

  10. 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 TGF- Scar Binding Device beta- 1 SMAD 3 DAXX HIPK2 ASK-1 MAP2k3 Monoclonal antibody P Phosphorylation Healthy Tissue Binding Device Healthy Heart Tissue Allosteric inhibitor laminin Gs-alpha Ca 2+ Alpha-syntropin Stimulates release of Ca 2+ ions Heterotrimeric G-protein G-Beta-y Parts Diagram (Left 1/3 of cell)‏

  11. OmpRR0082 pR R0050 pBad I13453 cI pR R0051 I pBad 13453 pTet R0040 pBad I13453 pLac I14032 hk022CI C0050 TT B0015 λ CI C00051 RBS B0034 RBS B0034 Asp λCl HKCI TRIGGER 1 λCl HKCI hk022CI C0051 TT B0015 hk022CI C0050 RBS B0034 RBS B0034 MAP2k3 lacI C0012 TT B0015 RBS B0034 lacI C0012 RBS B0034 LacI LacI Ca 2+ Ca 2+ TRIGGER 2 Ca 2+ lacI tetR lacI C0012 TT B0015 RBS B0034 tetR C040 RBS B0034 Parts Diagram (Middle 1/3 of cell)‏

  12. 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 NAND Gate Collagenase Asp HKCI Checkpoint 2 hk022CI C0050 mOrange E0030 TT B0015 RBS B0034 Parts Diagram (Right 1/3 of cell)‏ Glow orange Glow green Scar prevention device Heart regeneration device Reporter 2 lacI C0012 Verapamil synthesizer periostin GFP J52028 TT B0015 RBS B0034 RBS B0034 RBS B0034 LacI periostin OR Gate Verapamil Glow red tetR Checkpoint 4 tetR C040 RFP E1010 TT B0015 RBS B0034

  13. Unknowns • How will the MAP kinase bind to pBAD? • Is TGF-Beta specific enough of an antigen to make sure that the monoclonal antibody binds only to scar tissue and not to healthy tissue as well? • SMAD Protein presents a similar problem. Is it specific enough? • The protein kinases look good on paper; but will they function as predicted in vivo? • How will we synthesize Verapamil, a synthethic molecule? • Is there a way to incorporate a specific sequence so that the bacterium • synthesizes Verapamil, or will this prove to be inefficient?

  14. In Vivo Debugging: Fluorescent Markers Reporter Debugging Function Glow orange NAND Gate “Off” Glow cyan Scar Digesting Device “On” Glow red OR Gate “Off” Glow green Scar Prevention AND Heart Regeneration Devices “On”

  15. 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.

  16. Part Type Cost Amount Needed Total Cost Antibody $37 /mg 1 mg $37 DNA $.80 /bp 13 parts, 1000 bp/part $10,400 E. Coli Promoters Free 6 types $0 COSTS:

  17. Total Estimated Cost: • Approximately $11,000 • May be lower if we are able to attain DNA for the reduced iGEM price • Overall, the project appears cost efficient.

  18. FINAL VERDICT: IT’S A GO!

  19. References for Targeting and Binding 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

  20. References for Targeting and Binding 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

  21. References for Targeting and Binding 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

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