“viral smart bombs”

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“Viral Smart Bombs” Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm

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“Viral Smart Bombs”. Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm. Outline. Why viruses for iGEM? Project outline: targeting viruses to specific cells. Technical point: An in vivo aptamer selection scheme to increase specificity. Viruses?. Pro: - PowerPoint PPT Presentation

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Page 1: “Viral Smart Bombs”

“Viral Smart Bombs”

Joe Levine

April 27, 2007

Caltech iGEM Team Brainstorm

Page 2: “Viral Smart Bombs”

Outline

• Why viruses for iGEM?

• Project outline: targeting viruses to specific cells.

• Technical point: An in vivo aptamer selection scheme to increase specificity.

Page 3: “Viral Smart Bombs”

Viruses?

• Pro:– Certain systems (, M13, T7) are well understood.– Both molecular biology and biochemistry well characterized.– Wide variety of useful mutants available.– Short life cycle, short iteration times?– Even used for undergraduate lab classes (e.g. MIT 20.109,

http://openwetware.org/wiki/20.109)

• Con:– Hard to visualize– Usually assayed through interaction with other organisms– Is there an interested resident faculty virus expert?

• Opinion: The relative ease and speed of viral manipulations may fit the limitations of iGEM, but lack of local expertise is a real worry.

Page 4: “Viral Smart Bombs”

Virus Life Cycle

Page 5: “Viral Smart Bombs”

Outline

• Why viruses for iGEM?

• Project outline: targeting viruses to specific cells.

• Technical point: A completely in vivo aptamer selection scheme to increase specificity.

Page 6: “Viral Smart Bombs”

Project Overview: Targeting Viruses Specifically & Synthetically

• Can we engineer viruses to target cells expressing specific proteins or mRNA’s?– mRNA’s: easier?

– Proteins: foreign proteins, or post translationally modified (phosphorylated, prions, etc)?

• Virus infects and lyses cells containing target molecule.

• A construct in the virus prevents it from killing other cells.

Page 7: “Viral Smart Bombs”

Nucleic Acid Based Sensors?

• Detecting mRNA’s:– Stem loop invasion– Tuneable specificity – Designable1?

• Detecting other molecules:– Aptamers2,3,etc

– Protein conformers, foreign molecules, etc.

– In vivo functioning in the presence of confounding molecules may require in vivo selection

Covalent modification Prions

A A* (RBS)

B

B*/2A*

Viral mRNA

B

B*/2

A

A*

A* (RBS)Target mRNA

1. Isaacs et al. 2004.

2. Ellington and Szostak, 1990.

3. Bayer & Smolke, 2005.

Page 8: “Viral Smart Bombs”

The plan and system

• E. coli makes a sensible initial host cell– Well studied phages: , T7, M13 naturally target E. Coli– Easy to induce varying expression of heterologous

target.

• Picking targets of increasing difficulties:– 1st stage: A well defined mRNA (GN?)

– 2nd stage: A heterologous mRNA– 3rd stage: An easy heterologous protein (lysozyme?)– 4th stage: A difficult protein (phosphorylation state of two

component response regulator?)

• Hopefully goals #1, and maybe #2, achievable. #3 would be outstanding. I would be absolutely shocked to see #4 over the summer.

Page 9: “Viral Smart Bombs”

What specific genes to block?

• Early viral life cycle genes– The antiterminator ‘N’ in – virus strains do not infect1

– Other strains (M13, T7) might have similar candidates• These should prevent the virus from beginning its developmental life

cycle• Maybe target aptamers to later stage genes, to allow aptamers time

to mature

Page 10: “Viral Smart Bombs”

Possible problems

• Proteins may be a challenging substrate for aptamers to recognize.

• Specificity (false positives) will be a major issue.

• How will the devices work with the virus?

• Can we address these problems with in-vivo selection schemes?

Page 11: “Viral Smart Bombs”

Selecting aptamers in vivo

• Aptamers are typically selected using in vitro assays.

• These selection methods do not mimic in vivo constraints:– Lots of background molecules

– Unsure if random proteins will trigger aptamers.

• A selection scheme that mimics cellular environment might help.

Page 12: “Viral Smart Bombs”

“In Vivo SELEX” – Positive Selection

88

8

Infect

Lysis by successful infectors

8 8

8

8

8

8

8

Purify phage, repeat if desired.

• Important caveat: Nvirus << Ncell, else cells will get infected by multiple

viruses and unsuccessful aptamers will piggyback on successful

ones.

Page 13: “Viral Smart Bombs”

“In Vivo SELEX” – Negative Selection• Want to select for viruses that do not infect cells. This is hard.• Penalize aptamers opening in the absence of ligand.

88

8

E. Coli counterselection: sacB (sucrose sensitivity), rpsL (streptomycin sensitivity)?

Counterselection N

Promoter

Repressingstructure

N

Gene remains repressed

N

Repression non-specifically relieved

8

8

8

8

N

Lysis

Counterselection kills cells

Page 14: “Viral Smart Bombs”

% selectsim.m%% selection dynamics.% Nij is a vector of the number of viruses that are of type ij,% with:% i: 0/1 if aptamers don't/do open in the present of ligand% j: 0/1 if aptamer don't/do open in the absence of ligand%% Mijk is a number describing the multiplier that virus type ij goes% through on round k. ij are the same indices as above, k is 0 for negative% selection and 1 for positive selection. Nrounds = 100;Ratio = 5; M000 = Ratio; % aptamers not opening in either case multiply during negative selection.M001 = 1; % aptamers not opening in either case do not multiply during positive selection.M010 = 1; % aptamers opening only in the absence of ligand do not multiply during negative selection.M011 = 1; % aptamers opening only in the absence of ligand do not multiply during positive selection.M100 = Ratio; % aptamers opening only in the presence of ligand multiply during negative selection.M101 = Ratio; % aptamers opening only in the presence of ligand multiply during positve selection.M110 = 1; % aptamers opening all the time do not multiply during negative selection.M111 = Ratio; % aptamers opening all the time do multiply during positive selection. N00 = zeros(1,Nrounds); N00(1) = 0.33;N01 = zeros(1,Nrounds); N01(1) = 0.33;N10 = zeros(1,Nrounds); N10(1) = 0.01;N11 = zeros(1,Nrounds); N11(1) = 0.33; for j = 2 : Nrounds if mod(j,2) % even round = negative selection N00_new = M000*N00(j-1); N01_new = M010*N01(j-1); N10_new = M100*N10(j-1); N11_new = M110*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; else N00_new = M001*N00(j-1); N01_new = M011*N01(j-1); N10_new = M101*N10(j-1); N11_new = M111*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; endendplot(1:Nrounds,N00,1:Nrounds,N01,1:Nrounds,N10,1:Nrounds,N11)legend('N_{00}','N_{01}','N_{10}','N_{11}')

Page 15: “Viral Smart Bombs”

Sketch of selection dynamics?

Populations oscillate on alternate rounds due to alternating selection types.

Page 16: “Viral Smart Bombs”

Lots of Details to Work Out!!!

• Which virus to use

• Culture conditions

• How exactly to clone into them.

• Etc…?

• Thanks for your attention!