The present disclosure provides a novel genetically modified high productivity VVK-V432C insect cell line derived from the ATCC CRL-1711 cell line. Methods for obtaining this high productivity cell line are also provided.
The present disclosure relates to a heterologous recombinant baculovirus (rBV) expression system for the production of foreign heterologous proteins in insect cells. This system comprises a recombinant baculovirus backbone within a genome with a deletion in the cathepsin gene into which foreign gene cassettes can be integrated, and an insect cell that can be infected by the Δv-cath-rBV, and in which the foreign proteins and/or viral vectors or particles are expressed.
The present disclosure relates to a heterologous recombinant baculovirus (rBV) expression system for the production of foreign heterologous proteins in insect cells. This system comprises a recombinant baculovirus backbone within a genome with a deletion in the cathepsin gene into which foreign gene cassettes can be integrated, and an insect cell that can be infected by the ?v-cath-rBV, and in which the foreign proteins and/or viral vectors or particles are expressed.
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.
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.
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.
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.
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.
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.
C12N 15/00 - Techniques de mutation ou génie génétiqueADN ou ARN concernant le génie génétique, vecteurs, p. ex. plasmides, ou leur isolement, leur préparation ou leur purificationUtilisation d'hôtes pour ceux-ci
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.
G01N 33/53 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
12.
Methods of purifying adeno-associated virus (AAV) and/or recombinant adeno-associated virus (rAAV) and gradients and flow-through buffers therefore
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.
B01D 21/26 - Séparation du sédiment avec emploi de la force centrifuge
H01B 1/06 - Conducteurs ou corps conducteurs caractérisés par les matériaux conducteurs utilisésEmploi de matériaux spécifiés comme conducteurs composés principalement d'autres substances non métalliques
C12N 7/00 - Virus, p. ex. bactériophagesCompositions les contenantLeur préparation ou purification
13.
Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor
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.
C12N 15/00 - Techniques de mutation ou génie génétiqueADN ou ARN concernant le génie génétique, vecteurs, p. ex. plasmides, ou leur isolement, leur préparation ou leur purificationUtilisation d'hôtes pour ceux-ci
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.
C07H 21/00 - Composés contenant au moins deux unités mononucléotide comportant chacune des groupes phosphate ou polyphosphate distincts liés aux radicaux saccharide des groupes nucléoside, p. ex. acides nucléiques
A61K 31/7088 - Composés ayant au moins trois nucléosides ou nucléotides
C07K 14/34 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Corynebacterium (G)
C07K 14/21 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Pseudomonadaceae (F)
C12N 9/24 - Hydrolases (3.) agissant sur les composés glycosyliques (3.2)
A61K 48/00 - Préparations médicinales contenant du matériel génétique qui est introduit dans des cellules du corps vivant pour traiter des maladies génétiquesThérapie génique
15.
VECTORS HARBORING TOXIC GENES, METHODS AND USES THEREFOR
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.
C12N 15/31 - Gènes codant pour des protéines microbiennes, p. ex. entérotoxines
C12N 15/113 - Acides nucléiques non codants modulant l'expression des gènes, p. ex. oligonucléotides anti-sens
C12N 15/79 - Vecteurs ou systèmes d'expression spécialement adaptés aux hôtes eucaryotes
C12N 15/64 - Méthodes générales pour la préparation du vecteur, pour son introduction dans la cellule ou pour la sélection de l'hôte contenant le vecteur
C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
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.
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.
C12N 15/00 - Techniques de mutation ou génie génétiqueADN ou ARN concernant le génie génétique, vecteurs, p. ex. plasmides, ou leur isolement, leur préparation ou leur purificationUtilisation d'hôtes pour ceux-ci
C12N 15/85 - Vecteurs ou systèmes d'expression spécialement adaptés aux hôtes eucaryotes pour cellules animales
C12N 7/00 - Virus, p. ex. bactériophagesCompositions les contenantLeur préparation ou purification
C07K 14/005 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de virus
18.
EXPRESSION IN INSECT CELLS OF GENES WITH OVERLAPPING OPEN READING FRAMES, METHODS AND COMPOSITIONS THEREFOR
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-operabie 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.
C12N 5/00 - Cellules non différenciées humaines, animales ou végétales, p. ex. lignées cellulairesTissusLeur culture ou conservationMilieux de culture à cet effet
C12N 15/00 - Techniques de mutation ou génie génétiqueADN ou ARN concernant le génie génétique, vecteurs, p. ex. plasmides, ou leur isolement, leur préparation ou leur purificationUtilisation d'hôtes pour ceux-ci
brevets
