Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable proteins, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and, in certain instances, cleavage activity of RNA-programmable proteins (e.g., RNA-programmable endonucleases). By incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, a set of aptazyme-embedded guide RNAs was developed that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

The invention provides polynucleotide constructs for the regulation of gene expression by aptamer-based modulation of self-cleaving ribozymes and methods of using the constructs to regulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The invention further provides methods for making and using riboswitches that decrease target gene expression in response to an aptamer ligand as well as riboswitches that increase target gene expression in response to an aptamer ligand.

The invention provides polynucleotide constructs for the regulation of gene expression by aptamer-based modulation of self-cleaving ribozymes and methods of using the constructs to regulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The invention further provides methods for making and using riboswitches that decrease target gene expression in response to an aptamer ligand as well as riboswitches that increase target gene expression in response to an aptamer ligand.

The present invention provides compositions, systems and methods for using ribozyme-mediated cis-cleavage and trans-splicing of RNA molecules to express proteins or fusion proteins of interest.

Aspects of the application relate to methods and compositions for delivering therapeutic nucleic acids to neural cells or tissue in a subject. Additional aspects of the application relate to therapeutic nucleic acids, for example therapeutic ribozymes, that are useful for inhibiting viral reactivation in a subject.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.

Novel processes and compositions are described which use viral capsid proteins resistant to hydrolases to prepare virus-like particles to enclose and subsequently isolate and purify target cargo molecules of interest including nucleic acids such as siRNAs and shRNAs, miRNAs, messenger RNAs, small peptides and bioactive molecules.

Molecular target for healing or treating wounds and, in particular chronic, human wounds, are described. The molecular target is PTPRK, or a protein 50% homologous therewith, and which retains the same activity as PTPRK protein. Further, methods and novel therapeutics are described for treating said wounds.

Non-conventional yeasts are disclosed herein comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5′-cap. This uncapped RNA component comprises a sequence complementary to a target site sequence in a chromosome or episome in the yeast. The RGEN can bind to, and optionally cleave, one or both DNA strands at the target site sequence. An example of an RGEN herein is a complex of a Cas9 protein with a guide RNA. A ribozyme is used in certain embodiments to provide an RNA component lacking a 5′-cap. Further disclosed are methods of genetic targeting in non-conventional yeast.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and/or NK cells. The methods can include inhibitory RNA molecule(s) and/or engineered signaling polypeptides that can include a lymphoproliferative element, and/or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and/or genetically modified T cells and/or NK cells are provided, such as, for example, those including riboswitches, MRB-CARs, recognition domains, and/or pH-modulating agents.