Technical Specification:

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 IdeaBinding

• 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.

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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

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Device Diagram
Bacterial Cell
G
Scar digesting molecule
Scar tissue molecule
Regulatory Device
A
E
Scar Digestion Device
C
H
Trigger
B
Scar binding device
Regenerator Device
D
F
Heart regeneration molecule
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Timing Diagram
A B C D E F G H
Bacteria cell
Scar Digesting Molecule
Scar Tissue Molecule
G
E
regulatory device
C
H
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
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System Parts
10
List Of Parts
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Asp
Scar Digesting Sensor Device
TT B0015
Tar/envz C0082
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
Trigger
Asp
TT B0015
hk022CI C0050
RBS B0034
? CI C00051
RBS B0034
MAP2k3
HKCI
?Cl
HKCI
?Cl
TT B0015
hk022CI C0050
hk022CI C0051
RBS B0034
RBS B0034
To periostin secreting device
Parts Diagram (Middle 1/3 of cell)?
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Scar digesting device
Asp producer
Reporter 1
? CI C00051
collagenase
AspA C0083
ECFP E0022
TT B0015
RBS B0034
RBS B0034
RBS B0034
?Cl
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
RBS B0034
RBS B0034
From scar binding device
periostin
Parts Diagram (Right 1/3 of cell)?
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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
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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?

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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
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• 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.

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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

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Thank You. Are there any questions?
25
References
http//en.wikipedia.org/wiki/Monoclonal_antibodies
Information about monoclonal antibodies http//w
ww.ncbi.nlm.nih.gov/pubmed/16883602 scar and
healthy tissue binding http//herkules.oulu.fi/is
bn9514267214/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/10198
196 SMAD protein research http//www.ingentaconn
ect.com/content/ap/mc/1999/00000031/00000003/art00
902jsessionidcw3x1rheoz22.alice?formatprint SMA
D and TGF -beta protein research
http//books.nap.edu/openbook.php?record_id9450
page8 monoclonal antibodies
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References (cont.)
http//content.nejm.org/cgi/content/full/344/23/17
85engineered cardiomycotes, myocardial
infarction http//www.sciencedirect.com/science?_
obArticleURL_udiB6T13-4CNCNXW-5_user501045_r
doc1_fmt_origsearch_sortdviewc_acctC000
022659_version1_urlVersion0_userid501045md5
b622660c2a4b7ab53b1f770f463c8979myocardial
infarction description http//www.medicalnewstoda
y.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/52
23407.html
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References (cont.)
http//www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET
FLANGUAGEENGKEY052F019471IA052F019471DISP
LAYDESC 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.childrenshospit
al.org/newsroom/Site1339/mainpageS1339P1sublevel31
9.html article on how periostin was used
to induce mature heart cells to grow