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

Sensing and Actuation: 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 - Actuation - RNA splicing = introns cut out of transcript to make mature mRNA Input: Endogenous Protein 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) Why is there p50(1) and p50(2)?

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 Mutatns +/- input as normaization S J Culler et al. Science 2010;330:1251-1255

Experiment: Detect heterologous MS2-DsRed and endogenous NF-κB p50 Fig. 3 RNA devices implement combinatorial control schemes through multi-input processing. Fig. 3 RNA devices implement combinatorial control schemes through multi-input processing. 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. Leukotriene D4 at position 3, b catenin at location 6. increases ganciclovir (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 cell-survival percentages for the β-catenin– and NF-κB–responsive devices fused to HSV-TK indicate a decrease in cell survival RNA devices detect endogenous markers of disease and trigger targeted cell death. (A) Functional representation of a targeted therapeutic device that integrates across two therapeutic inputs—disease biomarker and an exogenously applied, inactive pro-drug—to 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. (D) Dose-response curves of cell-survival percentages for the β-catenin– and NF-κB–responsive devices fused to HSV-TK indicate a decrease in cell survival as a result of increased signaling through the targeted pathway and the presence of GCV. For all reported data, the mean cell survival levels from two independent experiments are shown. Error bars indicate ±SD from mean values. **P < 0.01. S J Culler et al. Science 2010;330:1251-1255

Critiques It’s “modular”, but… But is it really modular? 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 First study of intron locations: 3, 10 up Gcv concentrations Many proteins don’t bind rna!!! Can’t pick up every intracellular signal

Relevance to 20.385 Beyond engineering at the transcriptional level Scalable Beyond engineering at the transcriptional level Growing body of synthetic RNA molecules Offers additional biological control and functionality

Significance Applications: Gene and cellular therapy Diagnostics “a new synthetic device based on RNA can respond sensitively to alterations in the levels of endogenous signaling molecules, and so can potentially link disease-associated 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.”