ES2632497T3 - Mutantes de la polimerasa de HBV - Google Patents

Mutantes de la polimerasa de HBV Download PDF

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ES2632497T3
ES2632497T3 ES12733748.3T ES12733748T ES2632497T3 ES 2632497 T3 ES2632497 T3 ES 2632497T3 ES 12733748 T ES12733748 T ES 12733748T ES 2632497 T3 ES2632497 T3 ES 2632497T3
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mutated
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hbv polymerase
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Perrine Martin
Nathalie Silvestre
Jean-Baptiste Marchand
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Transgene SA
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    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12Y301/26—Endoribonucleases producing 5'-phosphomonoesters (3.1.26)
    • C12Y301/26004—Ribonuclease H (3.1.26.4)
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus

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Abstract

Polipéptido mutante que comprende un dominio de polimerasa de HBV mutado con una eliminación interior que interrumpe funcionalmente la actividad de la polimerasa, en el que dicha eliminación interior es de a lo sumo 30 residuos de aminoácidos, comprendiendo dicho dominio de polimerasa mutado la secuencia de aminoácidos representada en SEC ID nº: 1, pero que carece de por lo menos el residuo Tyr en la posición 203, el residuo Met en la posición 204, el residuo Asp en la posición 205, el residuo Asp en la posición 206, el residuo Val en la posición 207, el residuo Val en la posición 208 y el residuo Leu en la posición 209, en el que dicho polipéptido mutante comprende además un dominio de RNasaH mutado que comprende una eliminación de por lo menos 8 aminoácidos y de a lo sumo 60 aminoácidos que incluye por lo menos la parte de la SEC ID nº: 3 que se extiende desde el residuo Glu (E) en la posición 39 al residuo Ala (A) en la posición 46.

Description

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manera no limitativa la cepa de Copenhague (Goebel et al., 1990, Virol. 179: 247; Johnson et al., 1993, Virol.
196: 381), la cepa de Wyeth y particularmente la cepa modificada de Ankara (MVA) (Antoine et al., 1998, Virol.
244: 365). Las condiciones generales para construir los poxvirus recombinantes son bien conocidas en la técnica. La molécula de ácido nucleico de la presente invención se inserta preferentemente en el genoma poxvírico en un lugar no esencial. El gen de la timidina cinasa es particularmente apropiado para la inserción en los vectores vaccinia de Copenhague y la eliminación II o III para la inserción en el vector MVA. Preferentemente, la molécula de ácido nucleico de la invención se inserta en la eliminación III del vector MVA y se coloca bajo el control de vaccinia 7.5K o promotor pH5R.
Otros vectores víricos en el contexto de la invención son morbillivirus que se pueden obtener a partir de una familia de paramyxoviridae, con preferencia específica para el virus del sarampión. Diversas cepas atenuadas están disponibles en la técnica (Brandler et al, 2008, CIMID, 31 : 271; Singh et al, 1999, J. virol. 73(6): 4823), tal como y sin limitación, las cepas Edmonston A y B (Griffin et al., 2001, Field's in Virology, 1401-1441), la cepa de Schwartz (Schwarz A, 1962, Am J Dis Child, 103: 216), la cepa S-191 o C-47 (Zhang et al, 2009, J Med Virol. 81 (8): 1477). La inserción entre los genes P y M es particularmente apropiada.
De acuerdo con la presente invención, la molécula(s) de ácido nucleico comprendida en el vector de la invención está en una forma adecuada para la expresión en una célula u organismo hospedador, lo que significa que la molécula de ácido nucleico es colocada bajo el control de las secuencias reguladoras apropiadas. Como se usa en la presente memoria, el término "elementos reguladores" se refiere a cualquier elemento que permite, contribuye o modula la expresión de una molécula de ácido nucleico en una célula o organismo hospedador determinado, incluyendo la replicación, duplicación, transcripción, empalme, traducción, estabilidad y/o transporte del ácido nucleico o su derivado (es decir, ARNm).
Es apreciado por los expertos en la materia que la elección de las secuencias reguladoras puede depender de tales factores como el propio vector, la célula hospedadora, el nivel de expresión deseado, etc. El promotor es de especial importancia. En el contexto de la invención, puede ser constitutivo que dirige la expresión de la molécula de ácido nucleico en muchos tipos de células hospedadoras o específica para ciertas células hospedadoras (por ejemplo, secuencias reguladoras específicas del hígado) o reguladas en respuesta a eventos específicos o factores exógenos (por ejemplo, por la temperatura, el aditivo nutriente, la hormona, etc.) o de acuerdo con la fase de un ciclo vírico (por ejemplo, temprano o tardío). También se pueden usar promotores que son reprimidos durante la etapa de producción en respuesta a eventos específicos o factores exógenos, con el fin de optimizar la producción del vector y eludir la toxicidad potencial del polipéptido(s) expresado.
Los promotores adecuados para la expresión constitutiva en células de mamíferos incluyen, de manera no limitativa citomegalovirus (CMV), el promotor temprano inmediato (Boshart et al, 1985, Cell 41: 521), el promotor RSV, el promotor tardío principal de adenovirus, el promotor de la fosfoglicerato cinasa (PGK) (Adra et al., 1987, Gene 60:65), el promotor de la timidina cinasa (TK) del virus del herpes simple (HSV)-1 y el promotor de la polimerasa de T7. Los promotores de virus vaccinia son particularmente adaptados para la expresión en los vectores poxvirus. El ejemplo representativo incluye de manera no limitativa, vaccinia 7.5 K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15:18), TK, p28, p11 y el promotor K1L, así como promotores sintéticos, tales como los descritos en Chakrabarti et al. (1997, Biotechniques 23: 1094), Hammond et al. (1997, J. Virological Methods 66: 135) y Kumar y Boyle (1990, Virology 179: 151), así como promotores quiméricos tempranos/tardíos. Los promotores adecuados para la expresión sarampión mediado incluyen de manera no limitativa cualquier promotor que dirige la expresión de las unidades de transcripción del sarampión (Brandler y Tangy, 2008, CIMID 31: 271). Los promotores específicos del hígado incluyen de manera no limitativa los de la HMG-CoA reductasa (Luskey, 1987, Mol Cell Biol. 7: 1881); el elemento regulador de esterol 1 (SRE-1; Smith et al, 1990, J. Biol. Chem. 265: 2306); albúmina (Pinkert et al, 1987, Genes Dev. 1: 268); fosfoenolpiruvato carboxiquinasa (PEPCK) (Eisenberger y otros, 1992, Mol. Cell Biol. 12: 1396); alfa-1 antitripsina (Ciliberto et al., 1985, Cell 41: 531); transferrina humana (Mendelzon et al, 1990, Nucleic Acids Res. 18: 5717); y genes FIX (patente US nº 5.814.716).
Los expertos en la materia apreciarán que los elementos reguladores que controlan la expresión de la molécula de ácido nucleico de la invención pueden comprender además elementos adicionales para la iniciación, la regulación y/o terminación de la transcripción apropiada (por ejemplo, las secuencias de terminación de la transcripción polyA), el transporte de ARNm (por ejemplo, las secuencias de señal de localización nuclear), el procesamiento (por ejemplo, las señales de empalme), y la estabilidad (por ejemplo, los intrones y lass secuencias 5' y 3' no codificantes), la traducción (por ejemplo, un iniciador Met, secuencias líder tripartitas, sitios de unión de ribosoma IRES, secuencias de Shine-Dalgarno, etc) en la célula u organismo hospedador y etapas de purificación (por ejemplo, una etiqueta).
Las formas de realización particularmente preferidas de la invención están dirigidas a vectores (o partículas víricas) seleccionadas de entre el grupo que consiste en:
-Un vector de Ad defectuoso que comprende insertado en lugar de la región E1 una molécula de ácido nucleico colocada bajo el control de un promotor tal como el promotor CMV, y que codifica un polipéptido
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Claims (1)

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ES12733748.3T 2011-07-12 2012-07-12 Mutantes de la polimerasa de HBV Active ES2632497T3 (es)

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EP11305909 2011-07-12
EP11305909 2011-07-12
EP12305450 2012-04-18
EP12305450 2012-04-18
PCT/EP2012/063640 WO2013007772A1 (en) 2011-07-12 2012-07-12 Hbv polymerase mutants

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