RNA-Based Regulatory Technologies for Improved miRNA Sensors

Technology

The first technology is a system in which a DNA launched self-replicating RNA replicon (DREP) is used to increase the signal robustness of a miRNA circuit. In this circuit design, a transcription factor drives expression of a DREP replicon which contains the output. Additionally, the DREP replicon can be negatively regulated by expression of the nuclease Csy4. In order for the output to be expressed the miRNA which targets the transcription factor and output sequences must not be expressed. Additionally, a second miRNA must be expressed at high levels to repress the negative regulator Csy4. This is an improvement over traditional miRNA circuits because in traditional expression systems circuit function is directly related to how much DNA payload a given cell received. However, by using a self-replicating output the robustness of the system is greatly increased in cells that received even low levels of the output replicon. Additionally, including the negative regulator Csy4 ensures very low levels of background activation by destroying the output DREP replicon.

The second technology is an expression system that serves as an all-in-one miRNA circuit that senses both lowly and highly expressed miRNAs. This system uses lowly expressed miRNA target sequences on output expression, and highly expressed miRNA target sequences on output repressors, all expressed on the same piece of DNA. This results in output expression only in the presence of the particular high-miRNA and absence of the low-miRNA. The all-in-one vector system is made possible by inserting splitter sequences that sever, protect, and provide internal ribosome entry sites in the mRNA transcript. Current circuit technologies require that many different DNA vectors be transferred into the same cell to function correctly, and often most of the cells only receive a subset of the circuit components. By combining all of the components into a single DNA vector this technology greatly increases the efficiency of successfully introducing the circuit into cells.

Problem Addressed

Sensing cellular biomarkers to drive a desired output allows cells to be used as miniature computational circuits. These cellular circuits can be used to generate outputs such as identifying the cell of interest with a marker, producing a desired secreted protein, or having a therapeutic effect only in cells that meet all of the circuit requirements. For example, sensing cell-type can allow for targeted killing of cancer cells that express particular markers. MicroRNA (miRNA) expression profiles differ widely across cell types, including cancer, and can therefore be used as cell-specific identifiers. Additionally, new multiplexing techniques in which one miRNA needs to be lowly expressed and another highly expressed conveys added specificity to these miRNA circuits. These inventors describe two technologies that improve upon current miRNA circuit designs by increasing system robustness using a self-replicating RNA replicon and designing an all-in-one vector for circuit control.

Advantages

• Increased circuit robustness through use of self-replicating output
• All-in-one vector increases efficiency of introducing circuit into cells