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.

Method of generating a barcoded library, comprising delivering a polynucleotide into a cell, each polynucleotide comprising: (i) a sequence encoding a barcoding construct operably linked to a first promoter that is an antisense promoter, wherein the barcoding construct comprises a trans-splicing element and a barcode sequence; and a sequence encoding a perturbation element operably linked to a second promoter; generating RNA transcripts of the polynucleotide delivered into the cell, wherein the RNA transcripts comprise the barcoding construct and the perturbation element; and splicing the barcoding sequence onto endogenous RNA molecules in the cell, thereby generating a barcoded library, each member of the barcoded library comprising the barcode sequence and the endogenous RNA molecule attached with the barcode sequence.

Described herein are methods for treating a subject having or at risk of developing a neurocognitive disorder, such as frontotemporal lobar degeneration or neuronal ceroid lipofuscinosis, by administering cells that contain a transgene encoding a progranulin (PGRN) or a granulin (GRN) or cells that express the PGRN or the GRN to the subject. Also disclosed are compositions comprising cells containing the transgene encoding the PGRN or the GRN.

The 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 by using an improved hybrid dual recombinant AAV vector system.

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 present disclosure provides an adeno-associated viral (AAV) vector system for expressing a human ABCA4 protein in a target cell, the AAV vector system comprising a first AAV vector comprising a first nucleic acid sequence and a second AAV vector comprising a second nucleic acid sequence; wherein the first nucleic acid sequence comprises a 5′ end portion of an ABCA4 coding sequence (CDS) and the second nucleic acid sequence comprises a 3′ end portion of an ABCA4 CDS, and the 5′ end portion and the 3′ end portion together encompass the entire ABCA4 CDS; wherein the first nucleic acid sequence comprises a sequence of contiguous nucleotides corresponding to nucleotides 105 to 3597 of SEQ ID NO: 1; wherein the second nucleic acid sequence comprises a sequence of contiguous nucleotides corresponding to nucleotides 3806 to 6926 of SEQ ID NO: 1; wherein the first nucleic acid sequence and the second nucleic acid sequence each comprise a region of sequence overlap with the other; and wherein the region of sequence overlap comprises at least about 20 contiguous nucleotides of a nucleic acid sequence corresponding to nucleotides 3598 to 3805 of SEQ ID NO: 1. Also provided are uses of AAV vector systems in the prevention or treatment of disease.

The invention relates to an expression construct for the expression of polypeptides in host cells using alternative splicing. The expression construct can be used for the expression of polypeptides such as antibodies, antibody fragments and bispecific antibodies by expressing the gene products required for protein expression at the ratio leading to the highest titres or the best product quality profile.

Provided herein are methods and compositions for expressing Otoferlin, e.g., utilizing adeno-associated viral (AAV) particles. Such methods and compositions may be useful for treatment of diseases such as Deafness, Autosomal Recessive 9 (DFNB9).

Disclosed herein are compositions and methods that can be used to express a nucleotide sequence in a specific cell type. The compositions can comprise nucleic acid constructs comprising a start codon; and an intron cassette. The intron cassette can comprise a cell specific exon sequence, a splice donor site, a branch site, and an acceptor site. The cell specific exon sequence is out of frame with the start codon and comprises one or more frameshift mutations. The compositions can be used to treat human diseases.

Disclosed herein are systems, compositions and methods for using a split dCas protein system to modify the epigenetic profile of a gene of interest. The systems, compositions, and methods are useful for modifying the epigenetic profile of a particular gene within a cell, based on the discovery that effective expression of a larger-sized recombinant protein can be successfully achieved using two separate expression cassettes each encoding a half of the protein fused with a half of an intein, utilizing the unique feature of an intein system to ultimately rejoin the two halves to form one larger fusion protein with the intein spliced out.