An inducible expression cassette for controlled expression of multiple genes includes the DNA sequences of a bidirectional promoter inserted between two DNA sequences. Transfection into a host cell and the two DNA sequences encode genes of interest, provides one DNA sequence expressed constitutively and manipulation of the expression of the other DNA sequence. A regulatory DNA cassette containing another bidirectional promoter and two DNA sequences that encode a marker and a regulatory expression product is also disclosed. Both cassettes can be incorporated in a non-viral vector, like the Sleeping Beauty transposon, or a viral vector to induce controlled expression of multiple genes into host cells. A kit containing a package of each above vector type is also disclosed, as is a method of transforming a host cell.

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 present disclosure describes a next-generation antisense transfer vector along with methods for a high titer lentivirus production, allowing efficient transduction of T cells with a constitutively expressed tumor-targeting receptor along with the activation-induced expression of various gene cargos. The disclosed antisense transfer vector and the methods can reduce virus production costs as well as enhance the efficacy and safety of next-generation CAR- or TCR-T cells reaching the clinic.

Reagents and methods are provided that allow for an improved expression of a recombinant protein in an insect, More specifically, the introduction of recombinant DNA elements into an insect larva allows for the increased expression of a recombinant protein, an improvement of the correct folding of said protein and an increase in the survival rate after infection of the insect These recombinant DNA elements can be introduced, for example, into insect larvae via a recombinant baculovirus, which has incorporated said elements. The recombinant DNA elements include nucleic acids encoding transcriptional regulators, such as IE-0 and IE-1, transcriptional, enhancer elements, such as the homologous region (hr) and promoters.

The present invention relates to a viral vector for silencing a gene specifically in astrocytes comprising: an astrocyte-specific viral envelope protein, a first nucleic acid sequence encoding a transcription activator and at least one target sequence of a neuron-specific miR under the control of an astrocyte-specific promoter, anda second nucleic acid sequence encoding a RNA for silencing the gene under the control of a promoter inducible by the transcription activator.

Some aspects of this invention provide nucleic acid constructs for transgene expression. Some aspects of this invention provide multicistronic nucleic acid constructs, for example, comprising an expression cassette encoding a hairpin RNA and a reporter expression cassette. Some aspects of this invention provide nucleic acid constructs comprising two or more self-complementary nucleic acid sequences, for example, hairpin RNA encoding nucleic acid sequences and AAV inverse terminal repeats. Methods for the use of the constructs in therapy and research are also provided.

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 alight 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 encode 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.

Delivery of glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is currently being tested in a Phase 1/2a clinical trial for ALS patients. However, chronic trophic factor delivery prohibits dose adjustment or shut off in the event of side effects. To address this, the Inventors engineered a stably integrating, third-generation doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule Human iPSC-derived neural progenitors were stably transfected with the vector, expanded and transplanted into the adult mouse brain. The Inventors observed that the addition and withdrawal of doxycycline led to GDNF expression that could be induced and reversed multiple times, demonstrating that doxycycline can penetrate the graft and regulate transgene expression in vivo. The Inventors' findings provide a proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.

Provided herein, inter alia, are compositions including viruses and nucleic acids having promoters capable of expressing multiple heterologous nucleic acids. The compositions are particularly useful for delivering and expressing heterologous nucleic acids in neurons. With respect to the nervous system, neural tissue, and neurons, the compositions listed herein are contemplated to be effective for treatment of retinal neurodegenerative diseases.

Some aspects of this invention provide nucleic acid constructs for transgene expression. Some aspects of this invention provide multicistronic nucleic acid constructs, for example, comprising an expression cassette encoding a hairpin RNA and a reporter expression cassette. Some aspects of this invention provide nucleic acid constructs comprising two or more self-complementary nucleic acid sequences, for example, hairpin RNA encoding nucleic acid sequences and AAV inverse terminal repeats. Methods for the use of the constructs in therapy and research are also provided.