Reprogramming Cellular Behavior with RNA Controllers Responsive to

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 of transcript to make mature m. RNA 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 = MS 2 • 3 and 10 increase expression • 6 decreases expression • Flow cytometry (top right) • q. RT-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) • p 65 -3 increased gene expression (exon exclusion) • P 50 -3 decreased gene expression (exon inclusion)

Modularity: Sensor integrated into β-catenin pathway responds to LTD 4 stimulation • β-catenin: part of Wnt signalling pathway • Stimulated with leukotriene D 4 (LTD 4) • Position 3 did not respond to stimulation • Position 6 increased expression

Multiple Inputs • • MS 2 -Ds. Red 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 MS 2 -Ds. Red and endogenous NF-κB p 50 • • • TNF-α stimulation led to a decrease in gene expression and exon exclusion, whereas expression of MS 2 -Ds. Red 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 (p 65 -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? P 50 -3 decreased GOI expression while p 65 -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 SMN 1 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)

q. RT-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. ”