Transcript Reprogramming Cellular Behavior with RNA Controllers Responsive to Endogenous Proteins Stephanie J. Culler, Kevin G.

Reprogramming Cellular Behavior with RNA
Controllers Responsive to Endogenous
Proteins
Stephanie J. Culler, Kevin G. Hoff, Christina D. Smolke
SCIENCE Nov. 2010
Presented by:
Andrew Yang and Tina Stutzman
3/2/2011
Introduction: RNA control device couples endogenous
protein level with targeted gene expression through
alternative RNA splicing
- Sensing Aptamers = short pieces of
RNA that specifically bind to
targets, ex. proteins
Input:
Endogenous
Protein
- Actuation RNA splicing = introns cut out
of transcript to make mature
mRNA
Sensing and
Actuation:
Specific
Aptamer
Output:
Gene
Expression
Controller Design: Minigene + Aptamer
+ Gene of Interest
• Three-exon, two-inton
minigene
• Middle exon alternatively
spliced
• Aptamer in intron, protein
binds specific aptamer
• Splicing pattern altered
Optimal location of
input module in introns
• Bacteriophage coat
protein = MS2
• 3 and 10 increase
expression
• 6 decreases expression
• Flow cytometry (top right)
• qRT-PCR (bottom right)
Modularity: Sensor integrated into NF-κB pathway
responds to TNF-α stimulation
• NF-κB: immune responses, cell
adhesion, proliferation,
apoptosis
• Stimulated with tumor necrosis
factor-α (TNF-α)
• p65-3 increased gene
expression (exon exclusion)
• P50-3 decreased gene
expression (exon inclusion)
Modularity: Sensor integrated into β-catenin pathway
responds to LTD4 stimulation
• β-catenin: part of
Wnt signalling
pathway
• Stimulated with
leukotriene D4
(LTD4)
• Position 3 did
not respond to
stimulation
• Position 6
increased
expression
Multiple Inputs
•
•
MS2-DsRed Device
Devices containing the WT
aptamer in either position
displayed significant increases in
gene expression
S J Culler et al. Science 2010;330:1251-1255
Multiple Inputs
Experiment: Detect
heterologous MS2-DsRed
and endogenous NF-κB p50
•
•
•
TNF-α stimulation led to a
decrease in gene
expression and exon
exclusion, whereas
expression of MS2-DsRed
led to a significant increase
“…in both positions showed
a ~30 to 45%
increase…synergistic effect
on the output signal
S J Culler et al. Science 2010;330:1251-1255
Modularity of Output: RNA devices detect endogenous
markers of disease and trigger targeted cell death.
(B and C) Mechanisms of the β-catenin– (β-cat-6) (B) and NF-κB–
responsive (p65-3) (C) devices fused to a suicide gene-therapy
output module (HSV-TK), which controls cell survival in response
to detection of disease markers and GCV, a pro-drug trigger.
S J Culler et al. Science 2010;330:1251-1255
Modularity of Output: RNA devices detect endogenous
markers of disease and trigger targeted cell death.
(D) Dose-response
curves of cellsurvival percentages
for the β-catenin–
and NF-κB–
responsive devices
fused to HSV-TK
indicate a decrease
in cell survival
S J Culler et al. Science 2010;330:1251-1255
Critiques
• It’s “modular”, but…

Aptamer specific behavior?

P50-3 decreased GOI expression while p65-3 increased
 “probably through steric hindrance or recruitment of
components involved in spliceosome…”
Aptamer location in intron?



Can’t predict effects of aptamer location
Must determine “optimal input-module location within
intronic sequence space” for each regulatory device
• But is it really modular?

“We selected the SMN1 minigene because key regulatory
sequences are located in its exon regions, such that insertion of
synthetic sequences into intronic regions is not likely to strongly
affect splicing patterns”
• Many proteins don’t bind RNA
Relevance to 20.385
•
•
•
Beyond engineering at the transcriptional level
Growing body of synthetic RNA molecules
Offers additional biological control and functionality
Significance
•
•
•
•
“a new synthetic device based on RNA can respond
sensitively to alterations in the levels of endogenous
signaling molecules, and so can potentially link diseaseassociated pathways to cell fate decisions.”
Applications:
•
Gene and cellular therapy
•
Diagnostics
•
Cancer treatment
•
Drug discovery
RNA is an apt molecule for such devices, given its
versatile sensing functions and the ease with which RNA
molecules can be designed.
Smolke lab: Designing ‘intelligent’ therapeutic molecules
•
“construction of ligand-regulated RNA-based regulators of
gene expression”
Statistical Information – gene expression
device response (+ligand)
device response (-ligand)
= Relative expression (fold)
mutant device response (+ligand)
mutant device response (-ligand)
qRT-PCR Analysis
• “…ratio of the mean expression levels of the exon 7
excluded isoform to the exon 7 included isoform for the
wildtype device relative to the same ratio for the mutant
control device under the indicated ligand condition + the
average error.”