ES2582091T3 - Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas - Google Patents
Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas Download PDFInfo
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- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
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- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
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- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
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Abstract
Una meganucleasa recombinante que comprende: un polipéptido que tiene una similitud de al menos el 85 % con los restos 2-153 de la meganucleasa I-CreI de SEQ ID NO: 1; en la que la afinidad de unión a ADN se ha aumentado mediante al menos una modificación de un resto que es parte de la superficie de contacto de la estructura de la meganucleasa I-CreI de SEQ ID NO: 1, correspondiendo dicha modificación a una sustitución seleccionada entre el grupo que consiste en: sustitución de E80 por H, N, Q, S, T, K o R.
Description
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reconocen específicamente pero que constituyen el sitio de escisión real (que tiene un saliente de 4 pares de bases). Por lo tanto, las secuencias de reconocimiento combinadas de los dímeros de meganucleasa I-CreI, I-MsoI e I-CeuI normalmente abarcan 22 pares de bases, incluyendo dos semi-sitios de 9 pares de bases que flanquean un sitio de escisión de 4 pares de bases. Los pares de bases de cada semi-sitio se designan de -9 a -1, siendo la posición -9 la más distal del sitio de escisión y siendo la posición -1 adyacente a los 4 pares de bases centrales, que se designan N1-N4. La hebra de cada semi-sitio que se orienta de 5' a 3' en la dirección de -9 a -1 (es decir, hacia el sitio de escisión), se designa la hebra “sentido” y la hebra opuesta se designa la “hebra antisentido”, aunque ninguna de las hebras puede codificar proteína. Por lo tanto, la hebra “sentido” de un semi-sitio es la hebra antisentido del otro semi-sitio. Véase, por ejemplo, la Figura 1 (A). En el caso de la meganucleasa I-SceI, que es un monómero de meganucleasa di-LAGLIDADG, la secuencia de reconocimiento es una secuencia no palindrómica de aproximadamente 18 pb y no hay pares de bases centrales que no se reconozcan específicamente. Por convención, una de las dos hebras se denomina la hebra “sentido” y la otra la hebra “antisentido”, aunque ninguna de las hebras puede codificar proteína.
Como se usa en el presente documento, el término “especificidad” significa la capacidad de una meganucleasa para reconocer y escindir moléculas de ADN bicatenarias solamente en una secuencia particular de pares de bases denominada secuencia de reconocimiento, o solamente en un conjunto particular de secuencias de reconocimiento. El conjunto de secuencias de reconocimiento compartirá ciertas posiciones conservadas o motivos de secuencia, pero pueden degenerarse en una o más posiciones. Una meganucleasa altamente específica es capaz de escindir solamente una o muy pocas secuencias de reconocimiento. La especificidad puede determinarse en un ensayo de escisión como se describe en el Ejemplo 1. Como se usa en el presente documento, una meganucleasa tiene especificidad “alterada” si se une a y escinde una secuencia de reconocimiento que no está unida a y escindida por una meganucleasa de referencia (por ejemplo, de tipo silvestre) o si la tasa de escisión de una secuencia de reconocimiento aumenta o se reduce por una cantidad estadísticamente significativa (p < 0,05) en relación con una meganucleasa de referencia.
Como se usa en el presente documento, el término “degeneración” significa lo opuesto de “especificidad”. Una meganucleasa altamente degenerada es capaz de escindir un gran número de secuencias de reconocimiento divergentes. Una meganucleasa puede tener degeneración de secuencia en una posición sencilla dentro de un semisitio o en múltiples, incluso todas las, posiciones dentro de un semi-sitio. Dicha degeneración de secuencia puede resultar de (i) la incapacidad de cualquier aminoácido en el dominio de unión a ADN de una meganucleasa para realizar un contacto específico con cualquier base en una o más posiciones en la secuencia de reconocimiento, (ii) la capacidad de uno o más aminoácidos en el dominio de unión a ADN de una meganucleasa para realizar un contacto específico con más de una base en una o más posiciones en la secuencia de reconocimiento y/o (iii) suficiente afinidad de unión a ADN no específico para actividad. Una posición degenerada “completamente” puede ocuparse por cualquiera de las cuatro bases y puede designarse con una “N” en un semi-sitio. Una posición degenerada “parcialmente” puede ocuparse por dos o tres de las cuatro bases (por ejemplo, purina (Pu), pirimidina (Py) o no G).
Como se usa en el presente documento con respecto a meganucleasas, la expresión “afinidad de unión a ADN” o “afinidad de unión” significa la tendencia de una meganucleasa a asociarse de forma no covalente con una molécula de ADN de referencia (por ejemplo, una secuencia de reconocimiento o una secuencia arbitraria). La afinidad de unión se mide por una constante de disociación, KD (por ejemplo, la KD de I-CreI para la secuencia de reconocimiento WT es de aproximadamente 0,1 nM). Como se usa en el presente documento, una meganucleasa tiene afinidad de unión “alterada” si la KD de la meganucleasa recombinante para una secuencia de reconocimiento de referencia aumenta o se reduce en una cantidad estadísticamente significativa (p < 0,05) en relación con una meganucleasa de referencia.
Como se usa en el presente documento con respecto a monómeros de meganucleasa, la expresión “afinidad para formación de dímeros” significa la tendencia de un monómero de meganucleasa para asociarse de forma no covalente con un monómero de meganucleasa de referencia. La afinidad para formación de dímeros puede medirse con el mismo monómero (es decir, formación de homodímeros) o con un monómero diferente (es decir, formación de heterodímeros) tal como una meganucleasa de tipo silvestre de referencia. La afinidad de unión se mide por una constante de disociación, KD. Como se usa en el presente documento, una meganucleasa tiene afinidad “alterada” para formación de dímeros si la KD del monómero de meganucleasa recombinante para un monómero de meganucleasa de referencia aumenta o se reduce en una cantidad estadísticamente significativa (p < 0,05) en relación con un monómero de meganucleasa de referencia.
Como se usa en el presente documento, el término “palindrómico” se refiere a una secuencia de reconocimiento que consiste en repeticiones invertidas de semi-sitios idénticos. En este caso, sin embargo, la secuencia palindrómica no necesita ser palindrómica con respecto a los cuatro pares de bases centrales, que no entran en contacto con la enzima. En el caso de meganucleasas diméricas, las secuencias de ADN palindrómicas se reconocen por homodímeros en los que los dos monómeros realizan contactos con semi-sitios idénticos.
Como se usa en el presente documento, la expresión “pseudo-palindrómico” se refiere a una secuencia de reconocimiento que consiste en repeticiones invertidas de semi-sitios palindrómicos no idénticos o imperfectos. En este caso, la secuencia pseudo-palindrómica no solamente no necesita ser palindrómica con respecto a los cuatro pares de base centrales, sino que también puede desviarse de una secuencia palindrómica entre los dos semi-sitios.
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con un ácido glutámico en la posición 26 (véase Figura 1(D)); una A en la hebra sentido podría realizar un enlace de hidrógeno con una glutamina en la posición 77 y una T en la hebra antisentido podría formar contactos hidrófobos con una alanina en la posición 26 (véase Figura 1 (E)). Si el contacto específico de base se proporciona por la posición 77, entonces el contacto de tipo silvestre, Q26, puede sustituirse (por ejemplo, con un resto de serina) para reducir o retirar
5 su influencia en la especificidad. Como alternativa, pueden combinarse mutaciones complementarias en las posiciones 26 y 77 para especificar un par de bases particular (por ejemplo, A26 especifica una T en la hebra antisentido y Q77 especifica una A en la hebra sentido (Figura 1 (E)). Todas estas sustituciones de restos predichas se han validado experimentalmente.
10 Por lo tanto, de acuerdo con la invención, se ha identificado una variedad sustancial de modificaciones de aminoácidos para el dominio de reconocimiento de ADN de la meganucleasa I-CreI que, individualmente o en combinación, dan como resultado meganucleasas recombinantes con especificidades alteradas en bases individuales dentro del semi-sitio de secuencia de reconocimiento de ADN, de modo que estas meganucleasas diseñadas racionalmente tienen semi-sitios diferentes de la enzima de tipo silvestre. Las modificaciones de aminoácidos de I-CreI y el cambio
15 resultante en la especificidad del semi-sitio de secuencia de reconocimiento se muestran en la Tabla 1:
TABLA 1
- Base de hebra sentido favorecida
- Posn.
- A C G T A/T A/C A/G C/T G/T A/G/T A/C/G/T
- -1
- Y75 L75* C75* Y139* C46* A46* R70* H75* R75* H46* K46* R46* K70 E70* E75* E46* D46* Q70* C70 L70 Y75* Q75* H75* H139 Q46* H46* T46* G70 A70 S70 G46*
- -2
- Q70 T44* A44* V44* I44* L44* N44* E70 D70 K44* R44* H70 D44* E44* Q44* C44*
- -3
- Q68 C24* I24* E68 F68 K24* R24* R68 M68 C68 L68 F68 H68 Y68 K68
- -4
- A26* Q77 E77 K26* R77 E26* S77 Q26* S26*
- -5
- E42 R42 K28* C28* Q42 M66 K66
- -6
- Q40 C28* E40 R28* R40 C40 I40 V40 C79 I79 V79 Q28* A40 A79 A28* H28* S40 S28*
- -7
- N30* Q38 E38 K30* R30* K38 R38 E30* I38 L38 C38 H38 N38 Q30*
- -8
- F33 Y33 E33 D33 F33 H33 L33 V33 I33 F33 C33 R32* R33
16
Claims (1)
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imagen1 imagen2
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72751205P | 2005-10-18 | 2005-10-18 | |
US727512P | 2005-10-18 |
Publications (1)
Publication Number | Publication Date |
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ES2582091T3 true ES2582091T3 (es) | 2016-09-09 |
Family
ID=37963286
Family Applications (9)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ES13164628.3T Active ES2582091T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES06826293T Active ES2425022T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES17161899T Active ES2829549T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164623.4T Active ES2539616T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasa diseñada racionalmente con afinidad de formación de dímeros alterada |
ES12162075.1T Active ES2440801T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas racionalmente diseñadas con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES10191888T Active ES2384440T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164585.5T Active ES2626025T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES10191904.1T Active ES2533370T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164564.0T Active ES2602184T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
Family Applications After (8)
Application Number | Title | Priority Date | Filing Date |
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ES06826293T Active ES2425022T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES17161899T Active ES2829549T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164623.4T Active ES2539616T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasa diseñada racionalmente con afinidad de formación de dímeros alterada |
ES12162075.1T Active ES2440801T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas racionalmente diseñadas con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES10191888T Active ES2384440T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164585.5T Active ES2626025T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES10191904.1T Active ES2533370T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
ES13164564.0T Active ES2602184T3 (es) | 2005-10-18 | 2006-10-18 | Meganucleasas diseñadas racionalmente con especificidad de secuencia y afinidad de unión a ADN alteradas |
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Country | Link |
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US (19) | US8021867B2 (es) |
EP (13) | EP2341135A3 (es) |
JP (9) | JP5937292B2 (es) |
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