A recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses two functional antibody constructs in a cell is described. Also described are antibodies comprising a heavy chain and a light chain from a heterologous antibody. In one embodiment, the antibodies are co-expressed from a vector containing: a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof; and a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF for a second and third immunoglobulin construct. The vector co-expressing these two antibody constructs is in one embodiment an AAV, in which the 5 and 3 ITRs flank the expression cassettes and regulatory sequences.

Methods and compositions for modifying the expression of endogenous genes or modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases.

Provided are a protein complex in which estrogen receptor 2 (ERT2) is fused to CRISPR associated protein 9 (Cas9), and a recombinant vector carrying a gene coding the protein complex, wherein ERT2 is bonded to the N-terminus and C-terminus of nuclear localization sequence (NLS)-removed Cas9 and the complex has the advantage of translocating from the cytosol into the nucleus at a certain time point upon treatment with tamoxifen and modifying a specific DNA with the aid of guide RNA (gRNA), ultimately enabling a more elaborate DNA modification operation in a desired part at a desired time point.

Described herein are methods for treating a retinal degeneration in a subject, such as Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma. Also provided herein are methods of altering expression of one or more gene products in a cell, such as a retinal ganglion cell. Such methods may comprise utilizing a modified nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising a bidirectional HI promoter and gRNAs directed to retinal degeneration related genes, packaged in a single, compact adeno-associated virus (AAV) particle.

The invention provides recombinant adenovirus (rAd) and rAd vectors comprising a bidirectional mouse CMV (mCMV) promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction. The invention also provides methods of making and using such rAd and rAd vectors.

The presently disclosed subject matter provides compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter. In particular, compositions and methods are provided for the use of the H1 promoter to express CRISPR guide RNA (gRNA) with altered specificity of the 5 nucleotide, as well as use of the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease and the gRNA simultaneously. Compositions and methods are also provided for the expression and regulation of gRNA expression in vivo through the use of RNA ribozymes and regulatable aptazymes.

The presently disclosed subject matter provides compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter. In particular, compositions and methods are provided for the use of the H1 promoter to express CRISPR guide RNA (gRNA) with altered specificity of the 5 nucleotide, as well as use of the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease and the gRNA simultaneously. Compositions and methods are also provided for the expression and regulation of gRNA expression in vivo through the use of RNA ribozymes and regulatable aptazymes.

A recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses two functional antibody constructs in a cell is described. Also described are antibodies comprising a heavy chain and a light chain from a heterologous antibody. In one embodiment, the antibodies are co-expressed from a vector containing: a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof; and a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF for a second and third immunoglobulin construct. The vector co-expressing these two antibody constructs is in one embodiment an AAV, in which the 5 and 3 ITRs flank the expression cassettes and regulatory sequences.

The presently disclosed subject matter provides compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter. In particular, compositions and methods are provided for the use of the H1 promoter to express CRISPR guide RNA (gRNA) with altered specificity of the 5 nucleotide, as well as use of the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease and the gRNA simultaneously. Compositions and methods are also provided for the expression and regulation of gRNA expression in vivo through the use of RNA ribozymes and regulatable aptazymes.

The invention provides a bidirectional hCMV-CAG4 promoter and recombinant vectors and recombinant virus comprising the bidirectional hCMV-CAG4 promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction. The invention also provides methods of making and using such recombinant vectors and recombinant virus.