ES2623780T3 - Sistemas de expresión de baculovirus mejorados - Google Patents
Sistemas de expresión de baculovirus mejorados Download PDFInfo
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- ES2623780T3 ES2623780T3 ES12745446.0T ES12745446T ES2623780T3 ES 2623780 T3 ES2623780 T3 ES 2623780T3 ES 12745446 T ES12745446 T ES 12745446T ES 2623780 T3 ES2623780 T3 ES 2623780T3
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- baculovirus
- raav
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- 241000701447 unidentified baculovirus Species 0.000 title abstract description 18
- 101710181863 Structural DNA-binding protein p10 Proteins 0.000 abstract description 13
- 101710086987 X protein Proteins 0.000 abstract description 13
- 102000005600 Cathepsins Human genes 0.000 abstract description 6
- 108010084457 Cathepsins Proteins 0.000 abstract description 6
- 108010022172 Chitinases Proteins 0.000 abstract description 5
- 102000012286 Chitinases Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 102100038982 Exosome complex component RRP40 Human genes 0.000 description 12
- 230000000694 effects Effects 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 239000013608 rAAV vector Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000013607 AAV vector Substances 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 102100032106 Transmembrane emp24 domain-containing protein 3 Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14051—Methods of production or purification of viral material
- C12N2710/14052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14144—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Un genoma baculovírico recombinante que comprende los genes baculovíricos p26 y p74, en el que se interrumpen los genes baculovíricos catepsina, quitinasa y p10.
Description
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La ausencia de quitinasa y catepsina, en combinación con una deleción del ORF de p10 o una deleción de los genes p26, p10 y p74 tenía un efecto beneficioso sobre la integridad del vector de AAV. Lo más probablemente, la ausencia de actividad proteasa (catepsina) derivada del baculovirus conducía a una degradación de partícula vectorial reducida como se muestra por PAGE-SDS y análisis de transferencia Western (WB) (Figura 3). Estos métodos analíticos indicaban claramente la desaparición de al menos tres “bandas de degradación contaminantes” específicas de VP. La banda de degradación contaminante principal de las tres bandas se localiza cerca de VP3 (Figura 3). El uso de los baculovirus ∆CC∆p10 o ∆CC∆p26p10p74 en lugar de baculovirus wt conduce por tanto a una degradación del vector de rAAV reducida y a la desaparición de varios productos de degradación de VP.
Las partículas de rAAV producidas usando un baculovirus con deleción de los genes quitinasa, catepsina yp10 o p26, p10, p74 exhibe mayor infectividad in vivo.
La desaparición de ciertas bandas de proteína de tamaño menor en WB cuando se usan los baculovirus ∆CC∆p10 o ∆CC∆p26p10p74 significa que la partícula del vector de rAAV se degrada menos y puede tener una mejor integridad, sugiriendo que puede mejorarse la infectividad/potencia in vivo. De hecho, cuando se inyectan partículas de rAAVmSeAP purificadas producidas con los tres esqueletos baculovíricos diferentes (wt, ∆CC∆p10 y ∆CC∆p26p10p74) por vía intramuscular en ratones (C57Black6), se observó actividad de mSeAP en el suero aproximadamente 1 semana después de la inyección. La actividad aumentaba hasta niveles de meseta de aproximadamente 5,8 ng/ml, 23,2 ng/ml y 9,3 ng/ml cuando se usaba el esqueleto de wt y los esqueletos de ∆CC∆p10 y ∆CC∆p26p10p74 para la producción de AAV, respectivamente, a las 3 semanas después de la inyección (Figura 4A). La diferencia es del orden de un factor de 4 cuando se usa el esqueleto baculovírico de ∆CC∆p10 para la producción de rAAV en comparación con el esqueleto baculovírico de wt (p= 0,01). La diferencia es del orden de un factor de 2 cuando se usa el esqueleto de ∆CC∆p26p10p74 en lugar del esqueleto de wt (p= 0,05).f
Treinta y cinco días después de la inyección, se sacrificaron los ratones y se analizaron histológicamente los músculos inyectados. En cuanto a los niveles séricos aumentados de actividad de mSeAP, los ratones inyectados con rAAV producido con el baculovirus ∆CC∆p10 mostraron una actividad de mSeAP considerablemente aumentada en el tejido muscular transducido en comparación con aquellos ratones inyectados con rAAV producido con el sistema baculovírico wt. La actividad de mSeAP en el tejido muscular transducido con rAAV producido con ∆CC∆p26p10p74 se encontraba en un intervalo medio en comparación con el rAAV producido con los otros dos esqueletos baculovíricos (Figure 4B). La actividad de mSeAP aumentada observada con rAAV producido con el baculovirus ∆CC∆p10 se correlaciona con un aumento del número de copias del genoma de rAAV suministradas a las células de músculo TA como se muestra por PCR cuantitativa (Figura 4C), ilustrando que se suministraban 3,25 veces más copias en comparación con el sistema de producción wt. De manera similar, la evaluación del número de copias del genoma de rAAV suministrado a las células de músculo TA después de la producción con baculovirus ∆CC∆p26p10p74 conducía a un aumento de 2 veces en comparación con el uso de rAAV producido con el esqueleto baculovírico de wt (Figura 4C). Estos valores están bien de acuerdo con los niveles de actividad de mSeAP obtenidos con los diversos vectores baculovíricos.
Referencias
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Carbonell LF, Hodge MR, Tomalski MD, Miller LK (1988). “Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors”. Gene 73, 409-18.
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9
LISTADO DE SECUENCIAS
Tabla 1: Secuencias cebadoras usadas en este estudio
- Cebador
- Secuencia 5’ a 3’ Fin*
- CC-KO-F
- Inactivación génica de quitinasa/catepsina nt 105771-107700
- CC-KO-R
-
imagen7
- Quitinasa105625F
- Verificación
- Catepsina107849R
- Verificación
- p10-KO-F
- Inactivación de la secuencia de codificación de p10 (codón de inicio a codón de terminación) nt 118839-119121
- P10-KO-R
- p10118725-F
-
imagen8 Verificación
- p10119259-R
- Verificación
- M13 PUC F
- Verificación de bácmidos transpuestos
- M13 PUC R
- Verificación de bácmidos transpuestos
- Genta
-
imagen9 Verificación de bácmidos transpuestos
* La numeración de baculovirus es según Ayres et al. 1994
10
Claims (1)
-
imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161512142P | 2011-07-27 | 2011-07-27 | |
| US201161512142P | 2011-07-27 | ||
| PCT/EP2012/064843 WO2013014294A2 (en) | 2011-07-27 | 2012-07-27 | Improved baculovirus expression systems |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2623780T3 true ES2623780T3 (es) | 2017-07-12 |
Family
ID=46640007
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES12745446.0T Active ES2623780T3 (es) | 2011-07-27 | 2012-07-27 | Sistemas de expresión de baculovirus mejorados |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US10480010B2 (es) |
| EP (1) | EP2737071B1 (es) |
| JP (1) | JP6122430B2 (es) |
| CN (1) | CN103748229A (es) |
| BR (1) | BR112014001863A2 (es) |
| CA (1) | CA2842392C (es) |
| DK (1) | DK2737071T3 (es) |
| ES (1) | ES2623780T3 (es) |
| IN (1) | IN2014CN00688A (es) |
| WO (1) | WO2013014294A2 (es) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015191508A1 (en) | 2014-06-09 | 2015-12-17 | Voyager Therapeutics, Inc. | Chimeric capsids |
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| RU2020108189A (ru) | 2014-11-14 | 2020-03-11 | Вояджер Терапьютикс, Инк. | Композиции и способы лечения бокового амиотрофического склероза (als) |
| MX2017006217A (es) | 2014-11-14 | 2018-05-02 | Voyager Therapeutics Inc | Polinucleotidos moduladores. |
| EP3230441A4 (en) | 2014-12-12 | 2018-10-03 | Voyager Therapeutics, Inc. | Compositions and methods for the production of scaav |
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| AU2017267665C1 (en) | 2016-05-18 | 2023-10-05 | Voyager Therapeutics, Inc. | Modulatory polynucleotides |
| BR112018073472A2 (pt) | 2016-05-18 | 2019-08-27 | Voyager Therapeutics, Inc. | composições e métodos de tratamento da doença de huntington |
| WO2018044933A1 (en) | 2016-08-30 | 2018-03-08 | The Regents Of The University Of California | Methods for biomedical targeting and delivery and devices and systems for practicing the same |
| CA3061652A1 (en) | 2017-05-05 | 2018-11-08 | Voyager Therapeutics, Inc. | Compositions and methods of treating amyotrophic lateral sclerosis (als) |
| CN111108198A (zh) | 2017-05-05 | 2020-05-05 | 沃雅戈治疗公司 | 治疗亨廷顿病的组合物和方法 |
| JOP20190269A1 (ar) | 2017-06-15 | 2019-11-20 | Voyager Therapeutics Inc | بولي نوكليوتيدات aadc لعلاج مرض باركنسون |
| CN111132626B (zh) | 2017-07-17 | 2024-01-30 | 沃雅戈治疗公司 | 轨迹阵列引导系统 |
| WO2019028306A2 (en) | 2017-08-03 | 2019-02-07 | Voyager Therapeutics, Inc. | COMPOSITIONS AND METHODS FOR ADMINISTRATION OF ADENO-ASSOCIATED VIRUSES |
| AU2018338728B2 (en) | 2017-09-29 | 2025-01-02 | Centre National De La Recherche Scientifique (Cnrs) | Rescue of central and peripheral neurological phenotype of Friedreich's Ataxia by intravenous delivery |
| US20200237799A1 (en) | 2017-10-16 | 2020-07-30 | Voyager Therapeutics, Inc. | Treatment of amyotrophic lateral sclerosis (als) |
| CN111479924B (zh) | 2017-10-16 | 2024-06-14 | 沃雅戈治疗公司 | 肌萎缩性侧索硬化症(als)的治疗 |
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| EP3856762A1 (en) | 2018-09-28 | 2021-08-04 | Voyager Therapeutics, Inc. | Frataxin expression constructs having engineered promoters and methods of use thereof |
| WO2020072844A1 (en) * | 2018-10-05 | 2020-04-09 | Voyager Therapeutics, Inc. | Engineered nucleic acid constructs encoding aav production proteins |
| CN113166781A (zh) * | 2018-10-15 | 2021-07-23 | 沃雅戈治疗公司 | 在杆状病毒/Sf9系统中大规模生产rAAV的表达载体 |
| CN109609552B (zh) * | 2018-12-28 | 2020-04-14 | 中国科学院武汉物理与数学研究所 | 重组腺相关病毒的制备方法、系统及重组杆粒 |
| CN113728108A (zh) | 2019-02-15 | 2021-11-30 | 桑格摩生物治疗股份有限公司 | 用于生产重组aav的组合物和方法 |
| KR102272651B1 (ko) * | 2019-03-28 | 2021-07-02 | 충북대학교 산학협력단 | 외래단백질의 발현량과 발현시간이 증대된 배큘로바이러스 |
| AU2020302861A1 (en) * | 2019-06-26 | 2022-02-24 | Virovek, Inc. | Baculovirus expression system |
| WO2022079082A1 (en) | 2020-10-15 | 2022-04-21 | F. Hoffmann-La Roche Ag | Nucleic acid constructs for simultaneous gene activation |
| CN114317608B (zh) * | 2020-12-28 | 2023-08-22 | 陕西杆粒生物科技有限公司 | 一种基因敲除型杆状病毒表达载体 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4745051A (en) | 1983-05-27 | 1988-05-17 | The Texas A&M University System | Method for producing a recombinant baculovirus expression vector |
| EP0127839B1 (en) | 1983-05-27 | 1992-07-15 | THE TEXAS A&M UNIVERSITY SYSTEM | Method for producing a recombinant baculovirus expression vector |
| ZA848495B (en) | 1984-01-31 | 1985-09-25 | Idaho Res Found | Production of polypeptides in insect cells |
| FR2664905B1 (fr) * | 1990-07-18 | 1994-08-12 | Agronomique Inst Nat Rech | Baculovirus modifie, son procede d'obtention, et vecteurs d'expression obtenus a partir dudit baculovirus. |
| CN1205741A (zh) * | 1995-12-22 | 1999-01-20 | 纳幕尔杜邦公司 | 重组杆状病毒的生产 |
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| US20140356904A1 (en) | 2014-12-04 |
| EP2737071A2 (en) | 2014-06-04 |
| IN2014CN00688A (es) | 2015-04-03 |
| WO2013014294A2 (en) | 2013-01-31 |
| JP2014522658A (ja) | 2014-09-08 |
| BR112014001863A2 (pt) | 2017-02-21 |
| CA2842392C (en) | 2020-09-15 |
| CA2842392A1 (en) | 2013-01-31 |
| US10480010B2 (en) | 2019-11-19 |
| CN103748229A (zh) | 2014-04-23 |
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