Abstract: The present disclosure relates generally to genetic engineering of cells to perform specific and complex functions. In particular, the present disclosure relates to engineered mammalian cells and methods of engineering mammalian cells, as well as novel multi-functional proteins integrating both transcriptional and post-translational control effectively linking genetic circuits with sensors for multi-input evaluations.

Abstract: Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes (i) an affinity agent, such as a single-chain variable fragment of an antibody (scFv), which is expressed on the surface of the extracellular vesicles and (ii) a transmembrane domain, and may include additional domains. Exemplary extracellular vesicles may include but are not limited to exosomes or microvesicles.

Abstract: Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein.

Abstract: Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes a ligand, an engineered glycosylation site, and an exosome-targeting domain. Exemplary extracellular vesicles may include but are not limited to exosomes.

Abstract: Disclosed are exosomes that include a packaging protein and a cargo RNA in which the packaging protein binds specifically to the cargo RNA. The packaging protein is a fusion protein that includes an RNA-binding domain and an exosome-targeting domain. The cargo RNA includes an RNA-motif that the RNA-binding domain of the fusion protein binds specifically such that the cargo RNA is packaged in the lumen of the exosomes.

Abstract: Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein.

Abstract: Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes a ligand, an engineered glycosylation site, and an exosome-targeting domain. Exemplary extracellular vesicles may include but are not limited to exosomes.

Abstract: Disclosed are systems, components, and methods for sensing a ligand in a cell or a reaction mixture. The disclosed systems, components, and methods may include and/or utilize a fusion protein comprising a ligand-binding protein and a DNA-binding protein. The fusion protein binds the ligand of the ligand-binding protein and modulates expression of a reporter gene operably linked to a promoter that is engineered to include specific binding sites for the DNA-binding protein. The difference in expression of the reporter gene in the presence of the ligand versus expression of the reporter gene in the absence of the ligand can be correlated to the concentration of the ligand in a reaction mixture.

Abstract: Disclosed are systems and methods for detecting extracellular ligands and/or inducing expression of an exogenous or endogenous gene. The disclosed systems and methods typically include and/or utilize (i) first and second exogenous extracellular sensors, and third and fourth exogenous extracellular sensors; and (ii) an expression vector comprising a target gene operably linked to a hybrid promoter sequence. The hybrid promoter sequence of the expression vector includes a minimal promoter for inducing transcription and the hybrid promoter sequence further includes interspaced transcription regulator binding sites upstream of the minimal promoter that bind two or more transcription regulators of the extracellular sensors that are released from the extracellular sensors when the extracellular sensor bind an extracellular ligand.

Abstract: Disclosed are systems and methods that include or utilize composable mammalian elements of transcription (COMET) including engineered recombinant proteins that regulate transcription and engineered DNA promoter sequences that are regulated by the engineered recombinant proteins. The elements may be composed to form logic gates, gene expression cascades and programs, and cell-based biosensors.

Abstract: Disclosed are exosomes that include a packaging protein and a cargo RNA in which the packaging protein binds specifically to the cargo RNA. The packaging protein is a fusion protein that includes an RNA-binding domain and an exosome-targeting domain. The cargo RNA includes an RNA-motif that the RNA-binding domain of the fusion protein binds specifically such that the cargo RNA is packaged in the lumen of the exosomes.

Abstract: Disclosed are systems, components, and methods for sensing a ligand in a cell or a reaction mixture. The disclosed systems, components, and methods may include and/or utilize a fusion protein comprising a ligand-binding protein and a DNA-binding protein. The fusion protein binds the ligand of the ligand-binding protein and modulates expression of a reporter gene operably linked to a promoter that is engineered to include specific binding sites for the DNA-binding protein. The difference in expression of the reporter gene in the presence of the ligand versus expression of the reporter gene in the absence of the ligand can be correlated to the concentration of the ligand in a reaction mixture.

Abstract: Disclosed are systems, components of systems, and methods for sensing extracellular ligands and/or modulating expression of an endogenous or exogenous gene in a cell. In some embodiments, the disclosed systems and methods comprise or utilize first and second exogenous extracellular sensors and/or nucleic acid sequences encoding the first and second exogenous extracellular sensors, wherein the first exogenous extracellular sensor comprises: a) a ligand binding domain; b) a transmembrane domain; c) a protease cleavage site; and d) a functional domain comprising an RNA-binding subdomain fused to a transcription regulatory subdomain; and the second exogenous extracellular sensor comprises: e) a ligand binding domain, f) a transmembrane domain, and g) a protease domain that cleaves the protease cleavage site of the first exogenous extracellular receptor.

Abstract: Disclosed are extracellular vesicles (EVs), such as exosomes, comprising a heterologous cell membrane transporter protein, such as the sodium iodide symporter (NIS). Also disclosed are methods of using the disclosed EVs for delivering agents to recipient cells and methods for measuring efficacy of delivery by the EVs to the recipient cells. Also disclosed are method of making the disclosed EVs and cell lines for producing the EVs.

Abstract: Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein.

Abstract: The present invention provides modular extracellular sensors, nucleic acids encoding such sensors, and cells expressing such sensors, and methods of employing such sensors and cells for detecting extracellular ligands. In certain embodiments, the extracellular sensors comprise a ligand binding domain, a transmembrane domain, a protease domain, a protease cleavage site, and a transcription factor. In other embodiments, a pair of extracellular receptors is provided where both receptors contain a ligand binding domain and transmembrane domain, and one receptor contains a protease cleavage site and a transcription factor and the other receptor contains a protease domain.

Abstract: Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes a ligand, an engineered glycosylation site, and an exosome-targeting domain. Exemplary extracellular vesicles may include but are not limited to exosomes.

Abstract: Disclosed are exosomes that include a packaging protein and a cargo RNA in which the packaging protein binds specifically to the cargo RNA. The packaging protein is a fusion protein that includes an RNA-binding domain and an exosome-targeting domain. The cargo RNA includes an RNA-motif that the RNA-binding domain of the fusion protein binds specifically such that the cargo RNA is packaged in the lumen of the exosomes.

Abstract: The present invention provides modular extracellular sensors, nucleic acids encoding such sensors, and cells expressing such sensors, and methods of employing such sensors and cells for detecting extracellular ligands. In certain embodiments, the extracellular sensors comprise a ligand binding domain, a transmembrane domain, a protease domain, a protease cleavage site, and a transcription factor. In other embodiments, a pair of extracellular receptors is provided where both receptors contain a ligand binding domain and transmembrane domain, and one receptor contains a protease cleavage site and a transcription factor and the other receptor contains a protease domain.

Abstract: The present invention provides methods of purifying encapsidated virus, e.g., viral particles comprising viral nucleic acid, from compositions comprising encapsidated viral nucleic acid and viral particles that lack viral nucleic acid; methods for reducing the particle:genome ratio in a preparation of encapsidated viral nucleic acid; and methods for selectively inactivating viral particles that lack viral nucleic acid in a liquid composition comprising encapsidated viral nucleic acid and the viral particles that lack viral nucleic acid. The methods generally involve subjecting the composition to hydrostatic pressure such that the viral particles lacking viral nucleic acid are selectively inactivated.