Virovek, Inc.

Virovek Incorporation

Genovo, Inc.

Alfa Wassermann, Inc.

Haifeng Chen

Gary J Kurtzman

Sandra Patricia Meriño

Matthew A Gonda

Siyamak Rasty

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.

AAV production in insect cells, methods and compositions therefor

Methods of purifying a virus from a virus-infected cell lysate using three-phase partitioning (TPP) are disclosed. The methods comprise a first round of TPP, including mixing a cell lysate comprising a virus with ammonium sulfate and t-butanol, and separating the mixture, thereby forming a first aqueous phase, a first organic phase, and a first interphase. The first aqueous phase can comprise the virus, which can be subjected to a second round of TPP, resulting in a second aqueous phase, a second organic phase, and a second interphase. The second interphase can comprise highly purified virus. The methods can comprise subjecting a first aqueous phase to further purification by column chromatography or density gradient centrifugation. Purification of AAV, including AAV2, AAV5 and AAV6, from lysates of infected insect cell cultures is demonstrated. TPP-purified AAV particles infect at least as well as those prepared by standard methods.

Three phase partitioning (TPP) method for virus purification

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&#x2032; order, a) a first insect cell-operable promoter, b) a 5&#x2032; 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&#x2032; 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.

Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor

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.

Methods of purifying adeno-associated virus (AAV) and/or recombinant adeno-associated virus (rAAV) and gradients and flow-through buffers therefore

The present disclosure describes nucleic acids, and viruses comprising such nucleic acids, for growing a toxic gene in an insect cell. These nucleic acids comprise a sequence encoding a toxic polypeptide, and an intron that interrupts the sequence, whereby the intron is spliced in mammalian cells but not in insect cells. Infection of mammalian cells but not insect cells with the nucleic acids or viruses can lead to expression of toxic levels of the toxic polypeptide in mammalian cells but not in insect cells. Viruses, such as an AAV or a baculovirus comprising a nucleic acid can be grown in insect cell lines in vitro and can be administered to a subject in need of therapy, such as a subject in need of cancer therapy.

Vectors harboring toxic genes, methods and uses therefor

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA insert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogenous preparation and composition of rAAV.

Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

This invention relates to novel nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector (&ldquo;carrier vector&rdquo;) is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous preparation, compositions to produce the recombinant virus, and novel recombinant viruses.

Growing community of inventors

Piedmont, CA, United States of America

Haifeng Chen