Technical Specification:A Biosynthetic Approach to

Replacing Scarred post-Infarct Tissue With Healthy Cardiac Tissue

20.020

April 9, 2008

Anna ShcherbinaDerek Ju

Prarthna DesaiAmber Lin

Aditya KohliRobbie Barbero

Michael Oh

We'd like to thank Professor Gredzinsky for his valuable advice

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)

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

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.

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.

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

Device Diagram

Scar DigestionDevice

Scar binding device

Trigger

RegeneratorDevice

Scar tissue moleculeScar digesting molecule

Heart regeneration molecule

Bacterial Cell

A

B

C

D

E

F

Regulatory Device

G

H

Timing Diagram

A

B

C

D

E

F

G

H

E. coli binds to scar

Trigger mechanism is fully activated

Scar has been fully digested

Bacteria cell

Scar binding device

Trigger

Scar digesting Device

Regenerator Device

Scar Tissue Molecule

Scar Digesting Molecule

Regenrating Molecule

regulatory device

A

B

C

D

E

F

G

H

Scar digestingdevice concentration builds

System Parts

List Of Parts

ScarTissue

Monoclonalantibody SMAD 3 DAXX HIPK2 ASK-1 MAP2k3

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 Binding Device

Parts Diagram (Left 1/3 of cell)

RBS

B0034

RBS

B0034

TT

B0015

Asp

Scar Digesting Sensor Device

Tar/envz

C0082

Tar/envz

C0082

E. coli binds to one end of monoclonal antibody

OmpRR0082

RBS

B0034

RBS

B0034

hk022CI

C0050

hk022CI

C0050

TT

B0015 pRR0050

HKCI

RBS

B0034

RBS

B0034

λ CI

C00051

λ CI

C00051

λCl

pBadI13453

RBS

B0034

RBS

B0034

hk022CI

C0051

hk022CI

C0051

TT

B0015

cI pRR0051

RBS

B0034

RBS

B0034

hk022CI

C0050

hk022CI

C0050

HKCIλCl

Trigger

MAP2k3

Parts Diagram (Middle 1/3 of cell)

Asp

To periostin secreting device

λ CI

C00051

λ CI

C00051

λCl

hk022CI

C0050

hk022CI

C0050

HKCI

TRIGGER

mOrange

E0030

mOrange

E0030

RBS

B0034

RBS

B0034

Checkpoint 2

Glow orange

collagenasecollagenaseRBS

B0034

RBS

B0034

Scar digesting device

Collagenase

AspA

C0083

AspA

C0083

RBS

B0034

RBS

B0034

Asp producer

AspA

Asp

ECFP

E0022

ECFP

E0022

RBS

B0034

RBS

B0034

Reporter 1

TT

B0015

Glow cyan

TT

B0015

periostinperiostinRBS

B0034

RBS

B0034

Heart regeneration device

periostin

GFP

J52028

GFP

J52028

RBS

B0034

RBS

B0034

Reporter 2

TT

B0015

Glow green

Parts Diagram (Right 1/3 of cell)

From scar binding device

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

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?

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

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

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)

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)

In Vivo Debugging: Fluorescent Markers

Glow orange Trigger “Off”

Glow cyan Scar Digesting Device “On”

Glow green

Heart Regeneration Devices “On”

Reporter Debugging Function

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.

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

Thank You.

Are there any questions?

Referenceshttp://en.wikipedia.org/wiki/Monoclonal_antibodies

Information about monoclonal antibodies

http://www.ncbi.nlm.nih.gov/pubmed/16883602scar and healthy tissue binding

http://herkules.oulu.fi/isbn9514267214/html/x1329.htmlcardiac extracellular matrix

http://www.ncbi.nlm.nih.gov/pubmed/9521338 expression of cardiac proteins

http://www.ncbi.nlm.nih.gov/pubmed/10198196SMAD 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=8monoclonal antibodies

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=b622660c2a4b7ab53b1f77

0f463c8979myocardial infarction description

http://www.medicalnewstoday.com/articles/92139.phpFibroblast/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). 5223407http://www.freepatentsonline.com/5223407.html

References (cont.)

http://www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET=F&LANGUAGE=ENG&KEY=05%2F019471&IA=05%2F019

471&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