Abstract: Provided herein are feedback controller circuits and cell state classifiers for tunable phosphorylation-based transcriptional regulation of gene expression in cells based on intracellular miRNA profiles and degradation by small molecules. Also provided are methods of using feedback controller circuits and cell state classifiers for determining the cell state, and methods of treating cells and subjects using feedback controller circuits and cell state classifiers encoding therapeutic output molecules.

Abstract: Provided herein are genetic circuits and cell state classifiers for detecting the microRNA profile of a cell. The cell state classifiers of the present disclosure utilize phosphorylation state of a transcription factor to control classifier output. Kinases and phosphatase pairs that function in phosphorylating or dephosphorylating the transcription factor are integrated into the circuit, their expression tuned by the presence of microRNAs of interest (e.g., in a cell). The genetic circuits and cell state classifiers may be used in various applications (e.g., therapeutic or diagnostic applications).

Abstract: The present disclosure, at least in part, relates to an engineered incoherent feed forward loop (iFFL) comprising a first transcription unit encoding an endoribonuclease and a second transcription unit encoding an output molecule and an endoribonuclease target site located in the 5? UTR of the output molecule coding sequence. The engineered iFFL, at least in part, can be used for sustained expression of an output molecule despite of transcription resource disturbance in a given environment.

Abstract: The present disclosure, at least in part, relates to an engineered incoherent feed forward loop (iFFL) comprising a first transcription unit encoding an endoribonuclease and a second transcription unit encoding an output molecule and an endoribonuclease target site located in the 5? UTR of the output molecule coding sequence. The engineered iFFL, at least in part, can be used for sustained expression of an output molecule despite of transcription resource disturbance in a given environment.

Abstract: Provided herein are engineered HSV-1 vectors comprising a modified HSV-1 genome. The engineered HSV-1 vectors can be used to deliver genetic circuits (e.g., up to 100 kb) to cells in vitro or in vivo. Methods of treating or diagnosing a disease (e.g., cancer) using the engineered HSV-1 vectors described herein are also provided.

Abstract: Provided herein are genetic circuits and cell state classifiers for detecting the microRNA profile of a cell. The cell state classifiers of the present disclosure utilize phosphorylation state of a transcription factor to control classifier output. Kinases and phosphatase pairs that function in phosphorylating or dephosphorylating the transcription factor are integrated into the circuit, their expression tuned by the presence of microRNAs of interest (e.g., in a cell). The genetic circuits and cell state classifiers may be used in various applications (e.g., therapeutic or diagnostic applications).