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.

The present invention provides a kit of vectors comprising: (i) a first vector comprising a nucleic acid sequence encoding a first marker component; and (ii) a second vector comprising a nucleic acid sequence encoding a second marker component, wherein, when a cell is transduced with both the first and second vectors, the first and second marker components are expressed by the cell and associate forming a hetero-multimeric marker which is recognised by a cell sorting reagent whereas, when a cell is transduced with either the first or second vector alone, expression of the first or second marker component alone is not recognised by the cell sorting reagent.

Aspects of the disclosure relate to methods for co-administration of recombinant AAVs (rAAVs). In some embodiments, the methods comprise administering one or more rAAVs engineered to express a transgene encoding β-galactosidase (GLB1), Cathepsin A (CTSA), and/or β-galactosidase (GLB1) and Cathepsin A (CTSA). In some embodiments, the disclosure provides methods for treating lysosomal storage disorders, such as GM-1 gangliosidosis, using compositions described by the disclosure.

A triple-plasmid system for producing recombinant adeno-associated viruses is disclosed.

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.

Disclosed herein is a composition including a recombinant nucleic acid sequence that encodes an antibody to an HIV antigen. Also disclosed herein is a method of generating a synthetic antibody in a subject by administering the composition to the subject. The disclosure also provides a method of preventing and/or treating an HIV infection in a subject using said composition and method of generation.

Disclosed herein, in part, are methods for the expression and production of a trivalent, bispecific antibody using a recombinant nucleic acid comprising multiple, different expression cassettes in a specific and defined sequence, which are stably integrated into the genome of a mammalian cell.by targeted integration.

The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

The present invention discloses a SmartBac baculovirus expression system and application thereof. The system can comprise a acceptor plasmid (containing fragment A or fragments B and C) and a donor plasmid (containing fragment D); the fragment A contains a promoter, a sequence encoding a protease, a protease cleavage site, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment B contains a promoter, a sequence encoding a protease and a termination sequence; the fragment C contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment D contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence. The present invention also provides three cloning strategies to achieve the expression of protein complexes with molecular weights of less than 600 kDa and the expression of protein complexes with molecular weights of no less than 600 kDa and efficient screening of a subunit most suitable for adding a purification tag. The present invention is of great significance for recombinantly expressing protein complexes with complex components and large molecular weights in insect cells.