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 PDF

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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
delivery system
drug delivery
system based
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Victoria Demina
Heiko Manninga
Armin Götzke
Alexander Glassmann
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Life Science Inkubator Betriebs GmbH and Co KG
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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)

  1. imagen1
ES13714195.8T 2012-03-06 2013-03-06 Un sistema novedoso de suministro de fármacos basado en JCV-VLP Active ES2658941T3 (es)

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

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ES2658941T3 true ES2658941T3 (es) 2018-03-13

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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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EP3031821A1 (en) * 2014-12-08 2016-06-15 Life Science Inkubator polyomavirus VLPs with a fusion protein
EP3031820A1 (en) 2014-12-08 2016-06-15 Life Science Inkubator JC Polyomavirus VLP (virus-like particle) with a targeting peptide
WO2017072303A1 (en) * 2015-10-28 2017-05-04 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
BR112019014209A2 (pt) * 2018-12-18 2021-09-28 Neuway Pharma Gmbh Sistema de liberação de fármaco, método para provimento do mesmo, partículas semelhantes a vírus (vlp), composição e uso do sistema de liberação de fármaco
EP3670652A1 (en) * 2018-12-18 2020-06-24 NEUWAY Pharma GmbH Vlp for the treatment of a lysosomal storage disease

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