Provided are methods for screening a desired variant of a target gene in a eukaryotic system. Compositions for screening a desired variant of a target gene are also provided.

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AA V) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.

Provided are a preparation method and system for a recombinant adeno-associated virus (rAAV) and a recombinant bacmid. The method comprises: first reconstructing a recombinant bacmid containing a recombinant baculovirus genome that produces essential functional elements for an rAAV, at least one of the essential functional elements being inserted into the N-terminal or C-terminal of a locus of an essential gene of the recombinant baculovirus genome; and then transfecting the obtained recombinant bacmid containing the recombinant baculovirus genome that produces the rAAV into a host cell line for culturing to prepare an rAAV. Compared with recombinant baculoviruses obtained by conventional Tn7 recombinant preparations of recombinant bacmid, the recombinant baculovirus obtained by inserting a core element containing Cap, Rep and ITR into two sides of a baculovirus essential gene has a more stable rAAV serial passage production level in a cell and has a higher rAAV yield.

The present disclosure relates to nucleic acid sequences encoding components of adeno-associated virus (AAV), such as those that have been codon optimized for expression in plants, and the proteins that are expressed from these nucleic acid sequence. Also disclosed are methods of producing functional AAV particles using these nucleic acid sequences in plants. Production of AAV in plants as disclosed herein offer many advantages over conventional processes, such as efficiency, cost, yield, scalability, and safety.

The instant technology relates to a production system to produce AAV vectors in a serum free suspension platform and at high titers. This technology uses reagents comprising media, cells, transfection reagent, AAV enhancer, and a lysis buffer, each of which is designed to provide maximal AAV production from suspension culture of mammalian cells, e.g. HEK293 cells. With this new system we are able to deliver up to about 2×10.sup.11 viral genomes per milliliter (vg/mL) of unconcentrated AAV vectors.

The invention provides hybrid and recombinant phagemid vectors for expressing a transgene in a target cell transduced with the vector. A recombinant phagemid particle comprises at least one transgene expression cassette which encodes an agent which exerts a biological effect on the target cell, characterised in that the phagemid particle comprises a genome which lacks at least 50% of its bacteriophage genome. The invention extends to the use of such phagemid expression systems as a research tool, and for the delivery of transgenes in a variety of gene therapy applications, DNA and/or peptide vaccine delivery and imaging techniques. The invention extends to in vitro, in vivo or in situ methods for producing viral vectors, such as recombinant adeno-associated viruses (rAAV) or lentivirus vectors (rLV), and to genetic constructs used in such methods.

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus (AAV) particle comprising a viral gene and a capsid, wherein the viral genome comprises at least one Vectorized Editing of Nucleic acids to correct Overt Mutations (VENOM) element.

The invention provides methods for the production of recombinant adeno-associated virus vectors (rAAV), comprising contacting a host cell with a solution comprising at least one compound of formula (I), (I-A), (I-B), (II), (III), or (IV), or a salt thereof, or a vitamin B, or any combination(s) thereof. Also provided are methods for increasing the production of rAAV by a host cell, comprising contacting a host cell with a solution comprising at least one compound of formula (I), (I-A), (I-B), (II), (III), or (IV), or a salt thereof, or a vitamin B, or any combination(s) thereof.

The present disclosure provides engineered E. coli host cells that combine a knockout of SbcC, SbcD, or both without certain other mutations that can be used to propogate vectors. Methods of improved vector production using such engineered E. coli host cells are also provided.

Closed-ended, linear, duplex (CELiD) DNA molecules, recombinant AAV (rAAV), particles comprising CELiD DNA, methods of making such molecules and particles, and therapeutic applications of such particles.