The invention relates to a viral expression construct and related viral vector and composition and to their use wherein said construct and vector comprise elements a) and b): a) a nucleotide sequence encoding an insulin operably linked to a first promoter, b) a nucleotide sequence encoding a glucokinase operably linked to a second promoter and said viral expression construct and related viral vector comprise at least one of elements c), d) and e): c) the first and the second promoters are positioned in reverse orientation within the expression construct, d) the first and the second promoters are positioned in reverse orientation within the expression construct and are located adjacent to each other and e) the first promoter is a CMV promoter, preferably a mini CMV promoter.

The present disclosure provides methods for treating or preventing Huntington's disease (HD) in a subject in need thereof, comprising administering a therapeutic agent that activates mTORC 1 function and/or increases Ras Homolog Enriched in Striatum (Rhes) level in the subject's brain as compared to the function or level in the subject prior to treatment, and methods for modulating mHTT-associated metabolic phenotypes and/or reversal of striatal atrophy by administering a therapeutic agent that activates mTORC1 function and/or increases Ras Homolog Enriched in Striatum (Rhes) level in the subject's brain as compared to the function or level in the subject prior to treatment.

An optimized coding sequence of human blood clotting factor eight (VIII) and a promoter may be used in vectors, such as rAAV, for introduction of factor VIII, and/or other blood clotting factors and transgenes. Exemplary of these factors and transgenes arc alpha-1-antitrypsin, as well as those involved in the coagulation cascade, hepatocye biology, lysosomal storage, urea cycle disorders, and lipid storage diseases. Cells, vectors, proteins, and glycoproteins produced by cells transformed by the vectors and sequence, may be used in treatment.

In alternative embodiments, the invention provides methods for treating, ameliorating or protecting (preventing) an individual or a patient against a disease, an infection or a condition responsive to an increased paracrine polypeptide level in vivo comprising: providing a paracrine polypeptide-encoding nucleic acid or gene operatively linked to a transcriptional regulatory sequence; or an expression vehicle, a vector, a recombinant virus, or equivalent, having contained therein a paracrine-encoding nucleic acid or gene, and the expression vehicle, vector, recombinant virus, or equivalent can express the paracrine-encoding nucleic acid or gene in a cell or in vivo; and administering or delivering the paracrine polypeptide-encoding nucleic acid or gene operatively linked to a transcriptional regulatory sequence, or the expression vehicle, vector, recombinant virus, or equivalent, to an individual or a patient in need thereof, thereby treating, ameliorating or protecting (preventing) the individual or patient against the disease, infection or condition responsive to an increased paracrine polypeptide level.

The invention involves inducing 3-50 or more mutations (e.g., any whole number between 3 and 50 of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs, e.g., 32 or even 48 different guides delivered by one vector) in transgenic Cas9 eukaryotes to model genetic disease, e.g. cancer. The invention comprehends testing putative treatments with such models, e.g., testing putative chemical compounds that may be pharmaceutically relevant for treatment or gene therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic diseases and putative treatments to better understand and alleviate a genetic disease or a condition, e.g., cancer.

Delivery devices, systems, and methods related thereto may be used in humans for spinal delivery of cells, drugs or vectors. Thus, the system enables subpial delivery, which leads to a near complete spinal parenchymal AAV9-mediated gene expression or ASO distribution in both white and grey matter.

The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.

A method of preparing a recombinant adeno-associated virus (rAAV) including: (1) preparing a shuttle plasmid and a corresponding recombinant bacmid including a baculovirus genome, where the shuttle plasmid includes at least an rAAV gene of interest flanked by inverted terminal repeats (ITR-GOI) integrated with a heterologous functional gene fragment, and the recombinant bacmid includes an expression cassette of functional protein components necessary for assembly of the rAAV; (2) integrating the rAAV ITR-GOI and the expression cassette of functional protein components by using the shuttle plasmid and the recombinant bacmid, to yield a recombinant bacmid including a recombinant baculovirus genome; and (3) transfecting, with the recombinant bacmid, a host cell line.

The present invention provides nucleic acid constructs, expression vectors, transgenic cell and methods of making and using the same, wherein the nucleic acid construct includes a synthetic promoter designed from the endogenous promoter of BIRC5 and LAMC2. In illustrative working embodiments of the invention, an exogenous nucleic acid fragment encoding thymidine kinase is operably linked to the synthetic promoter which is then shown to regulate the expression of this polypeptide.

Disclosed herein are methods and compositions for inactivating mutant genes associated with LCA, using engineered nucleases comprising a DNA binding domain and a cleavage domain or cleavage half-domain in conditions promoting the cleavage of the mutant genes. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases, and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided.