ES2658941T3 - Un sistema novedoso de suministro de fármacos basado en JCV-VLP - Google Patents
Un sistema novedoso de suministro de fármacos basado en JCV-VLP Download PDFInfo
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- ES2658941T3 ES2658941T3 ES13714195.8T ES13714195T ES2658941T3 ES 2658941 T3 ES2658941 T3 ES 2658941T3 ES 13714195 T ES13714195 T ES 13714195T ES 2658941 T3 ES2658941 T3 ES 2658941T3
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- vlp
- jcv
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- drug delivery
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- 238000012377 drug delivery Methods 0.000 title 1
- 241001505332 Polyomavirus sp. Species 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 238000000034 method Methods 0.000 abstract 2
- 208000015114 central nervous system disease Diseases 0.000 abstract 1
- 238000003745 diagnosis Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 238000002560 therapeutic procedure Methods 0.000 abstract 1
- 239000013612 plasmid Substances 0.000 description 12
- 108060001084 Luciferase Proteins 0.000 description 11
- 239000005089 Luciferase Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- 238000010361 transduction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 2
- 101710197658 Capsid protein VP1 Proteins 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 2
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710108545 Viral protein 1 Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000013115 immunohistochemical detection Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 241000867607 Chlorocebus sabaeus Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101710169675 Major capsid protein VP1 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
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- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010149 post-hoc-test Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 210000001177 vas deferen Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/01—DNA viruses
- C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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Abstract
Las VLP derivadas de un virus del polioma humano que comprende un fármaco para usar en un método de tratamiento para terapia o un método de diagnóstico in vivo de una enfermedad del CNS.
Description
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Materiales
Se evaluaron sondas deferentes de VP1-VLP sobre la capacidad de empaquetar y suministrar el plásmido reportero en células Cos7 (células de riñón de mono verde) in vitro, ya que se conoce que esta línea celular es transducible por JCV VLP. Había 9 sondas de VP1-VLP precipitadas con sales y 15 sondas de VP1-VLP purificadas por cromatografía. Los experimentos de transducción in vitro se repitieron 5 veces. En estos experimentos se evaluaron la capacidad para trasmitir lamáxima señal deluminiscencia con los sustratos de luciferina durante los experimentos in vivo.
Las VP1-VLP precipitadas con sales se precipitaron toda la noche y se dializaron 24 horas contra el tampón estándar (NaCl 150mM, Tris-HCI pH7.5 10mM). El empaquetamiento del plásmido del gen reportero se logró por disociación química y reasociación como se describió en Goldmann y otros, 1999, Journal of Virology: Molecular cloning and expression of major structural protein VP1 of the human polyoma virus: formation of virus like particles useful for immunological andtherapeutic studies.
El diseño experimental
La inyección intravenosa de las VLP en ratones BALB/c inmunocompetentes se realizó en la vena de la cola bajo anestesia con isofluorano.
Los animales se agruparon dela siguientemanera:
-5 µg de VLP precipitadas con sales (4 animales)
-50 µg de VLP precipitadas con sales (4 animales)
-5 µg de VLP purificadas por cromatografía (4 animales)
-Control de ADN (solo el plásmido del gen reportero) (3 animales)
-Control de VLP (solo cápsides de VP1-VLP) (3 animales)
La bioluminiscencia semidió los días 2, 4, 7, 14 y 22, 12 minutos después de la inyección intraperitoneal del sustrato de Luciferina. Los resultados se agruparon en cada grupo y se calcularon el promedio y la desviación estándar. Los promedios se analizaron con ANOVA de dos vías, con Holm-Sidak como prueba post hoc.
Los resultados semuestran en las Figuras 3 y 4.
3. Eficacia de transducción de células Cos7 (células de riñón de mono verde africano) con ayuda de JC VP1-VLP cargadas con los plásmidos de Luciferasa y JC VP1-VLP mezcladas con plásmidos de Luciferasa
- 1.
- 18 horas antes de la transducción, las células Cos7 se pasaron a la placa de 24 pocillos.
- 2.
- Las VP1-VLP se disociaron con DTT y EGTA, se mezclaron con el plásmido de Luciferasa y se dializaron frente al tampón de reasociación durante toda la noche a +4°C.
- 3.
- Al día siguiente, las VP1-VLP empaquetadas se sacaron de la diálisis.
- 4.
- Las VP1-VLP mezcladas con el plásmido de Luciferasa se prepararon de acuerdo con Krauzewicz (Gene Therapy (2000) 7, 1094-1102):
- a.
- La relación de lamezcla de VLP con respecto al plásmidode Luciferasa fue de 30:1 (w/w)
- b.
- Estamezcla se incubó 15minutos a TA
- c.
- Las mezclas se diluyeron con medio celular DMEM y se pipetearon sobre las células Cos7 en una placa de 24 pocillos.
- 5.
- Las VP1-VLP empaquetadas con el plásmido de Luciferasa se pipetearon sobre las células Cos7; lo mismo se realizó con las VP1-VLP mezcladas con plásmido de Luciferasa.
- 6.
- Después de 72 horas, las células se lisaron y la actividad de la luciferasa se midió por triplicado con la ayuda del ensayo de luciferasa de Promega.
Los resultados de los experimentos de transducción independientes semuestran en la Figura 5
4. Detección inmunohistoquímica de luciferasa y proteína VP1 en el cerebro de ratón después de la aplicación intravenosa de las VLP empaquetadas con un plásmido deluciferasa
Como se mostró anteriormente, las JC VP1-VLP son capaces de suministrar sustancias en las células y órganos en el organismo vivo como se demostró por la detección del ADN del plásmido (por qPCR) o la actividad de luciferasa. Estos resultados experimentales se soportan adicionalmente por la detección inmunohistoquímica de la proteína Luciferasa y la proteína VP1 en el cerebro.
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Claims (1)
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imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP12001507 | 2012-03-06 | ||
| EP12001507.8A EP2636746A1 (en) | 2012-03-06 | 2012-03-06 | A novel drug delivery system based on JCV-VLP |
| PCT/EP2013/000656 WO2013131644A1 (en) | 2012-03-06 | 2013-03-06 | A novel drug delivery system based on jcv-vlp |
Publications (1)
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| ES13001491.3T Active ES2661389T3 (es) | 2012-03-06 | 2013-03-22 | Sistema de administración de un fármaco para uso en el tratamiento o diagnóstico de trastornos neurológicos |
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| ES13001491.3T Active ES2661389T3 (es) | 2012-03-06 | 2013-03-22 | Sistema de administración de un fármaco para uso en el tratamiento o diagnóstico de trastornos neurológicos |
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| EP (3) | EP2636746A1 (es) |
| JP (1) | JP6175080B2 (es) |
| KR (1) | KR102058488B1 (es) |
| CN (1) | CN104395475B (es) |
| AU (1) | AU2013230232B2 (es) |
| BR (1) | BR112014021497A2 (es) |
| CA (1) | CA2865564C (es) |
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| DK (2) | DK2823048T3 (es) |
| EA (1) | EA035308B1 (es) |
| ES (2) | ES2658941T3 (es) |
| HR (1) | HRP20180221T1 (es) |
| HU (2) | HUE038284T2 (es) |
| LT (1) | LT2823048T (es) |
| MX (1) | MX354004B (es) |
| NO (1) | NO2823048T3 (es) |
| NZ (1) | NZ629255A (es) |
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| PT (2) | PT2823048T (es) |
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| SI (1) | SI2823048T1 (es) |
| TR (1) | TR201802849T4 (es) |
| WO (1) | WO2013131644A1 (es) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3031820A1 (en) * | 2014-12-08 | 2016-06-15 | Life Science Inkubator | JC Polyomavirus VLP (virus-like particle) with a targeting peptide |
| EP3031821A1 (en) * | 2014-12-08 | 2016-06-15 | Life Science Inkubator | polyomavirus VLPs with a fusion protein |
| US20180320199A1 (en) * | 2015-10-28 | 2018-11-08 | Life Science Inkubator Gmbh | Use of vlp for the detection of nucleic acids |
| CN106048093A (zh) * | 2016-08-02 | 2016-10-26 | 北京思尔成生物技术有限公司 | Jc病毒的检测方法、试剂盒及其应用 |
| EP3670652A1 (en) * | 2018-12-18 | 2020-06-24 | NEUWAY Pharma GmbH | Vlp for the treatment of a lysosomal storage disease |
| DK3688147T3 (da) * | 2018-12-18 | 2022-12-19 | Neuway Pharma Gmbh | Hidtil ukendt lægemiddelindgivelsessystem og fremgangsmåder til tilvejebringelse heraf |
| WO2020252455A1 (en) | 2019-06-13 | 2020-12-17 | The General Hospital Corporation | Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells |
| CA3189601A1 (en) | 2020-07-24 | 2022-01-27 | The General Hospital Corporation | Enhanced virus-like particles and methods of use thereof for delivery to cells |
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| DE19543553B4 (de) * | 1995-11-22 | 2009-04-09 | Deutsches Primatenzentrum Gmbh | VP-Antigene des JC-Virus |
| AU7220198A (en) | 1997-04-26 | 1998-11-24 | Rpms Technology Limited | Use of papovavirus capsid protein for delivery of genetic material |
| DE19916224C1 (de) * | 1999-04-10 | 2000-06-21 | November Ag Molekulare Medizin | Synthetisches biologisch aktives Molekül |
| DE10131145B4 (de) | 2001-06-28 | 2005-07-14 | Innovent E.V. | Zusammensetzung zum zellspezifischen Transfer von Wirkstoffen |
| AU2003238008A1 (en) * | 2002-06-12 | 2003-12-31 | Temple University Of The Commonwealth System Of Higher Education | Method of cell growth inhibition with agnoprotein |
| EP2036980A1 (de) | 2007-09-14 | 2009-03-18 | Gruber, Jens | Herabregulation der Genexpression mittels Nukleinsäure-beladener virusähnlicher Partikel |
| EP2093780B1 (en) | 2008-02-22 | 2014-11-05 | ABB Technology AG | A spring drive unit, an operating device for an electrical switching apparatus, and method for charging and/or discharging a spring in an operating device for an electrical switching apparatus |
| CN102369436B (zh) | 2009-02-05 | 2014-10-22 | 生物基因Idecma公司 | 用于检测jc多瘤病毒的方法 |
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2012
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2013
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- 2013-03-06 HR HRP20180221TT patent/HRP20180221T1/hr unknown
- 2013-03-06 JP JP2014560272A patent/JP6175080B2/ja not_active Expired - Fee Related
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- 2013-03-06 SG SG10202102293QA patent/SG10202102293QA/en unknown
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- 2013-03-06 LT LTEP13714195.8T patent/LT2823048T/lt unknown
- 2013-03-06 SG SG11201405255SA patent/SG11201405255SA/en unknown
- 2013-03-06 EP EP17200325.3A patent/EP3299468A1/en not_active Withdrawn
- 2013-03-06 HU HUE13714195A patent/HUE038284T2/hu unknown
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- 2013-03-06 DK DK13714195.8T patent/DK2823048T3/en active
- 2013-03-06 NO NO13714195A patent/NO2823048T3/no unknown
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- 2013-03-06 PT PT137141958T patent/PT2823048T/pt unknown
- 2013-03-06 US US14/383,267 patent/US20150045417A1/en not_active Abandoned
- 2013-03-06 SI SI201330942T patent/SI2823048T1/en unknown
- 2013-03-06 KR KR1020147028123A patent/KR102058488B1/ko not_active Expired - Fee Related
- 2013-03-06 ES ES13714195.8T patent/ES2658941T3/es active Active
- 2013-03-06 WO PCT/EP2013/000656 patent/WO2013131644A1/en not_active Ceased
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2018
- 2018-02-05 CY CY20181100133T patent/CY1120150T1/el unknown
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2019
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