Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into the human PPP1R12C locus, for example, for expression of a polypeptide of interest.

An expression system for expressing a herpesvirus glycoprotein complex including a vector inserted with two or more nucleic acid sequences that encode two or more subunits of a herpesvirus glycoprotein complex linked by one or more linking sequences such that the subunits are co-expressed simultaneously and self-processed to assemble into a glycoprotein complex. The expression system or the vector can be included in a vaccine composition. The vaccine composition can be used for preventing or treating herpesvirus infections.

This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

Methods and compositions for rapid development of reporter lines utilizing safe harbor sites in iPSCS, as well as other progenitor cells, pluripotent and multipotent stem cells and differentiated cells, and multiple Lox sites are provided.

The disclosure provides methods and compositions for the integration (insertion) of a donor DNA molecule into a target DNA molecule. In general, the methods include contacting a target DNA molecule with a linear donor DNA molecule and a Cas 1 protein, where the target DNA molecule includes an AT-rich region (e.g., in some cases positioned 5 and within 50 nucleotides of a region that forms a DNA cruciform structure), where the contacting is not in a bacterial or archaeal cell (e.g., the contacting is in vitro outside of a cell, inside of a eukaryotic cell, etc.), and provides for integration of the donor DNA molecule into the target DNA molecule.

The present invention provides a method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, as well as a kit for use in said method. Also provided is a method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3.

Synthetic regulation of gene expression is provided. In some embodiments, synthetic regulatory constructs are provided. In some embodiments, a synthetic regulatory construct expresses a heterologous gene in a selected cell type. In some embodiments, methods of expressing a heterologous gene in a selected cell type are provided.

Compositions containing MiniVectors and gene therapy uses, including long term repeated gene therapy uses, to treat liver fibrosis or liver cancer.

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.

The present disclosure provides recombinant adeno-associated virus virions with variant capsid protein, where the recombinant AAV (rAAV) virions exhibit one or more of increased ability to cross neuronal cell barriers, increased infectivity of a neural stem cell, increased infectivity of a neuronal cell, and reduced susceptibility to antibody neutralization, compared to a control AAV, and where the rAAV virions comprise a heterologous nucleic acid. The present disclosure provides methods of delivering a gene product to a neural stem cell or a neuronal cell in an individual. The present disclosure also provides methods of modifying a target nucleic acid present in a neural stem cell or neuronal cell.