RNA-based Logic Circuits with RNA Binding Proteins, Aptamers and Small Molecules

Technology #16631

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RNA-only multi-input microRNA sensor is able to differentiate between Hela, HEK 293 and MCF7 cell lines as demonstrated with transient DNA transfection. (A) Implementation of the L7Ae-based miRNA sensor that specifically recognizes Hela cells based on specific miRNA profile (highly expressed miR21 and low levels of 141, 142(3p) and 146a). (B) Expression scheme of the sensor inputs, operator and output in Hela cells. Operation of the circuit results in high expression of the output only in Hela cells, but not other cell types. (C) Differential expression of the output protein, EGFP, in HEK, MCF7 and Hela cells. When output is not regulated by endogenous microRNA the EGFP fluorescence is high in all three cell types (set to 1, not shown). Control of the EGFP expression by the sensor circuit results in over 9-fold higher output in Hela cells with respect to HEK cells and almost 11-fold higher output as compared with MCF7 cells. (0) Specific induction of apoptosis in Hela cells by expression of circuit-controlled hBax protein as determined with Annexin V staining and pDNA transfection.
Categories
Inventors
Professor Darrell Irvine
Department of Biological Engineering, MIT
External Link (irvine-lab.mit.edu)
Professor Ron Weiss
Department of Biological Engineering, MIT
External Link (groups.csail.mit.edu)
Liliana Wroblewska
Department of Biological Engineering, MIT
Maria Foley
Department of Biological Engineering, MIT
Velia Siciliano
Department of Biological Engineering, MIT
Tasuku Kitada
Department of Biological Engineering, MIT
Katie Bodner
Department of Biological Engineering, MIT
Jacob Becraft
Department of Biological Engineering, MIT
Tyler Wagner
Boston University
Hirohide Saito
Kyoto University
Kei Endo
Kyoto University
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Rna-based logic circuits with rna binding proteins, aptamers and small molecules

PCT Patent Application WO 2016-040395

Applications

This invention is an RNA-based genetic circuit which can produce antigens or numerous proteins including: therapeutic, cell death, fluorescent, and selection proteins. This technology may be used for a number of applications including, but not limited to, selective stem cell reprogramming or vaccination.

Problem Addressed

Synthetic biology, which has the potential to provide genetic circuits with greatly improved output control over traditional pharmaceuticals, has remained DNA-centered, and genetic circuit design always relies exclusively or partially on transcriptional regulation. However, messenger RNA (mRNA), as a platform for gene transfer, has numerous advantages over plasmid DNA, including the lack of requirement for crossing the nuclear envelope and has negligible risk of genomic integration, making it a significantly safer alternative. However, no control mechanisms have been developed to regulate replicon-based expression. Efforts to engineer post-transcriptional devices based on microRNA, aptamers, or aptazymes have been characterized to have a very low dynamic range and have resulted in devices not suitable for construction of scalable circuits. This technology is the successful construction of synthetic circuits using RNA and RNA binding proteins (RBPs).

Technology

Devices based on RBPs can be easily wired together to create synthetic circuits of various complexities or to interconnect cellular and synthetic signaling pathways. The design to control protein expression includes two translational repressors, L7Ae, which blocks ribosome scanning if placed in the 3' ultrasound region (3'UTR), and a fusion protein MS2-CNOT7. MS2 is another RNA binding coat protein from bacteriophage, MS2, and CNOT7 is a human deadenylase that can efficiently repress translation of mRNA. This platform provides a plug-and play post-transcriptional regulation framework through an engineered set of diverse regulatory circuits including a multi-input cell type classifier, a cascade, and a two state switch. For instance, the Inventors have designed a circuit that recognizes a microRNA profile that is specific for Hela cells and it only triggers a response if the profile is matched, which if the response includes a pro-apoptotic gene, can selectively induce apoptosis in Hela cells. Tunable expression of RNA agents can also be achieved using aptamers or a cascade and a switch between two different therapeutic agents.

Advantages

  • Minimal risk of harmful genomic integration
  • Tunable/controllable circuit behavior