Aspects of the disclosure relate to bicistronic AAV nucleic acid constructs comprising a transgene encoding hexosaminidase A (HEXA) and hexosaminidase (HERB) proteins. In some embodiments, the disclosure provides methods for treating or preventing lysosomal storage disorders, such as Tay-Sachs disease and Sandhoff disease, using bicistronic nucleic acid constructs described by the disclosure.

The invention provides bidirectional expression vectors comprising Chinese hamster ovary elongation factor 1-a (CHEF1) transcriptional regulatory DNA elements, a gene of interest (GO I), a minimal cytomegalovirus (minCMV) and a selectable marker (SM) and/or a human adenovirus tripartite leader (AdTPL) sequence. The invention also provides method for increasing heterologous protein expression in a host cell comprising culturing the host cell the bidirectional expression vector(s).

The present invention relates to compositions and methods comprising a single viral vector comprising both a first polynucleotide comprising a constitutive promoter operably linked to a nucleic acid encoding at least one transgene, wherein one of the at least one transgenes encodes a receptor or receptor subunit, a receptor fusion protein or a fluorescent marker; and a second polynucleotide comprising an inducible promoter operably linked to a nucleic acid encoding an effector. Also provided are engineered cells comprising the viral vector and methods for generating the engineered cells comprising the viral vector. Also provided is site-specific integration of the genetic element into the a gene locus by means of a CRISPR-related system. Further provided are methods for treating a patient having a disease, a disorder or condition associated with expression of an antigen, the method comprising administering to the patient an effective amount of a composition comprising the engineered cell.

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.

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.

The present invention relates to a nucleic acid expression cassette, in particular for the expression of a human liver-specific and/or liver-expressed protein and/or preferably physiologically active domains and/or fragments thereof in a patient suffering from a monogenetic disorder caused by a mutation in the gene coding for the liver-specific and/or liver-expressed protein.

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 present invention is related to a dual promoter lentiviral vector and methods of use for the treatment of diseases and disorders, specifically lysosomal storage disorders.

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.