Abstract: Compositions and methods are disclosed for producing adeno-associated virus (AAV) in insect cells in vitro. Recombinant baculovirus vectors include an AAV Capsid gene expression cassette (Cap), an AAV Rep gene expression cassette (Rep), and a baculovirus homologous region (hr) located up to about 4 kb from a start codon in an AAV expression cassette. Production levels of baculovirus and AAV in insect cells harboring recombinant baculovirus comprising a Cap, a Rep, and an hr are higher compared to controls comprising a Cap and a Rep but no hr. Furthermore, levels of baculovirus and AAV production in insect cells infected with recombinant baculovirus comprising a Cap, a Rep, and an hr are comparatively stable over serial passages of cells, whereas levels of baculovirus and AAV production decline over serial passages of insect cells comprising recombinant baculovirus comprising a Cap and a Rep, but no hr.

Abstract: A method of purifying a viral vector is provided. The method includes loading a nonionic density gradient into a continuous flow centrifuge rotor; loading a material including the viral vector and a predictable contaminant into the continuous flow centrifuge rotor; rotating the continuous flow centrifuge rotor in a manner sufficient to separate the viral vector from the predictable contaminant and the material in the nonionic density gradient; and unloading the separated viral vector from the continuous flow centrifuge rotor.

Abstract: The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5? to 3? order, a) a first insect cell-operable promoter, b) a 5? portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3? portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.

Abstract: The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5? to 3? order, a) a first insect cell-operable promoter, b) a 5? portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3? portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.

Abstract: A method of purifying a viral vector is provided. The method includes loading a nonionic density gradient into a continuous flow centrifuge rotor; loading a material including the viral vector into the continuous flow centrifuge rotor; rotating the continuous flow centrifuge rotor in a manner sufficient to separate the viral vector and a predictable contaminate from the material in the nonionic density gradient; and unloading the separated viral vector and the predicable contaminant from the continuous flow centrifuge rotor.

Abstract: The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5? to 3? order, a) a first insect cell-operable promoter, b) a 5? portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3? portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.

Abstract: The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5? to 3? order, a) a first insect cell-operable promoter, b) a 5? portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3? portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.