The present invention relates to CRISPR-mediated means and methods, e.g., for adjustably induction of multiple gene expression and subsequent cell reprogramming. Particularly, the CRISPR-mediated means and methods of the present invention relate to conversion of endogenous glial cells into GABAergic neurons representing an effective method for cell reprogramming.

The present disclosure provides constructs comprising a coding sequence operably linked to a promoter, wherein the coding sequence encodes a clarin 1 protein. Exemplary constructs include AAV constructs. Also provided are methods of using disclosed constructs for the treatment of hearing loss and/or deafness. Also provided are methods of using disclosed constructs for the treatment of vision loss.

This invention relates to an RNA trans-splicing molecule (RTM) that targets human endogenous retrovirus (HERV) pre-mRNA. The RTM comprises (i) a binding region specific for a HERV pre-mRNA, (ii) a trans-splicing splice domain and (ill) a coding sequence for a suicide protein. The binding region of the RTM binds to HERV pre-mRNA in a cell, such that the coding sequence is trans-spliced through the trans- splicing domain with the HERV pre-mRNA, resulting in a chimeric mRNA causing the suicide protein to be expressed in the cell. RTMs of the invention may be useful in selectively killing cells that express HERV genes, for example cancer cells. RTMs, encoding nucleic acids, methods of treatment and associated methods and uses are provided.

We disclose dumbbell-shaped vectors adapted for efficient expression in mammalian cells. We also disclose a novel method allowing the efficient synthesis of dumbbell-shaped vectors at low cost for delivery of recombinant DNA and RNA into host cells; and the use of dumbbell-shaped vectors for transient expression in, for example, primary human cells.

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

The invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5Kb by using an improved hybrid dual recombinant AAV vector system.

The purpose of the present invention is to provide a method for producing a very stable, cyclized mutant protein such that high cyclization efficiency is achieved while the number of amino acids added is minimal and the biological properties of an original protein are maintained. In view of conformational information about the original protein, secondary structure-free regions at N/C terminal portions are deleted. Then, a protein database is screened for proteins with secondary structures similar to those of N/C terminal residues of a secondary structure-forming portion after the deletion. The screening results are used to determine the amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected. A cyclized mutant protein is finally produced having a loop structure with the determined amino acid length.

The present invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5 Kb.

The present invention relates to a recombinant adenovirus with increased in-vivo safety, tissue specificity, and anticancer activities, and a use thereof. Specifically, the recombinant adenovirus comprising: a promoter of the liver tissue-specific phosphoenolpyruvate carboxykinase (PEPCK) gene; a trans-splicing ribozyme which is operably linked to the promoter and acts on a cancer-specific gene; a therapeutic gene or a reporter gene which is linked to the 3 exon of the ribozyme; and a serotype 35 fiber knob and a serotype 5 shaft, in which the orf4 gene is deleted from adenovirus E1, E3 and E4 orf1, shows remarkable in-vivo safety, high specificity for a target tissue, and remarkable anticancer effects, and thus can be useful for an anticancer drug or a cancer diagnostic agent as a gene delivery vector.