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AR123924A1 - Células nk con receptores de antígeno quimérico (car) y usos de estas - Google Patents

Células nk con receptores de antígeno quimérico (car) y usos de estas

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Publication number
AR123924A1
AR123924A1 ARP210102971A ARP210102971A AR123924A1 AR 123924 A1 AR123924 A1 AR 123924A1 AR P210102971 A ARP210102971 A AR P210102971A AR P210102971 A ARP210102971 A AR P210102971A AR 123924 A1 AR123924 A1 AR 123924A1
Authority
AR
Argentina
Prior art keywords
cell
car
polynucleotide sequence
crispr
plasmid
Prior art date
Application number
ARP210102971A
Other languages
English (en)
Inventor
Meisam Naeimi Kararoudi
Dean Anthony Lee
Original Assignee
Res Institute At Nationwide Children’S Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Res Institute At Nationwide Children’S Hospital filed Critical Res Institute At Nationwide Children’S Hospital
Publication of AR123924A1 publication Critical patent/AR123924A1/es

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    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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Abstract

Reivindicación 1: Un plásmido para usar con sistemas asociados a repeticiones palindrómicas cortas agrupadas y espaciadas de forma regular (CRISPR) / proteína 9 asociada a CRISPR (Cas9) caracterizado porque el plásmido comprende en orden un brazo de homología izquierdo, una secuencia polinucleotídica que codifica un polipéptido del receptor de antígeno quimérico (CAR) y un brazo de homología derecho; donde los brazos de homología izquierdo y derecho tienen cada uno 1000 bp de longitud o menos. Reivindicación 16: Un vector viral adenoasociado (AAV) caracterizado porque comprende el plásmido de una cualquiera de las reivindicaciones 1 a 15. Reivindicación 22: Una célula modificada que comprende el plásmido de una cualquiera de las reivindicaciones 1 a 15 o el vector AAV de una cualquiera de las reivindicaciones 16 a 21. Reivindicación 27: Un método para modificar genéticamente una célula caracterizado porque comprende a) obtener un complejo de ribonucleoproteína (RNP) que comprende una endonucleasa CRISPR / Cas de clase 2 (Cas9) complejada con un RNA guía CRISPR / Cas correspondiente y un vector AAV que comprende un plásmido que comprende una secuencia polinucleotídica que codifica un polipéptido del receptor de antígeno quimérico (CAR); donde la secuencia de polinucleótidos está flanqueada por brazos de homología; y donde los brazos de homología tienen una longitud de 800 bp o menos; y b) introducir la secuencia de polinucleótidos que codifica el polipéptido CAR y el complejo RNP en la célula; donde la secuencia de polinucleótidos que codifica el polipéptido CAR se introduce en la célula a través de la infección con el AAV en la célula; donde el complejo RNP se hibrida con una secuencia dirigida dentro del DNA genómico de la célula y las enzimas de reparación del DNA de la célula insertan la secuencia polinucleotídica que codifica el polipéptido CAR en el genoma del hospedador en la secuencia dirigida dentro del DNA genómico de la célula creando así una célula modificada. Reivindicación 51: Un método para crear una célula citolítica natural (NK) del receptor de antígeno quimérico (CAR) o una célula T CAR NK caracterizado porque comprende a) obtener un complejo de ribonucleoproteína (RNP) que comprende una endonucleasa CRISPR / Cas de clase 2 (Cas9) complejada con un RNA guía CRISPR / Cas correspondiente y un vector AAV que comprende un plásmido que comprende una secuencia polinucleotídica que codifica un polipéptido del receptor de antígeno quimérico (CAR); donde la secuencia de polinucleótidos está flanqueada por brazos de homología; y donde los brazos de homología tienen una longitud de 1000 bp o menos; e b) introducir la secuencia de polinucleótidos que codifica el polipéptido CAR y el complejo RNP en una célula NK o una célula T NK; donde la secuencia de polinucleótidos que codifica el polipéptido CAR se introduce en la célula NK o célula T NK a través de la infección con el AAV en la célula NK o célula T NK; donde el complejo RNP se hibrida con una secuencia dirigida dentro del DNA genómico de la célula NK o célula T NK y las enzimas reparadoras del DNA de la célula NK o célula T NK insertan la secuencia de polinucleótidos que codifica el polipéptido CAR en el genoma del hospedador en la secuencia dirigida dentro del DNA genómico de la célula creando así una célula CAR NK o una célula T CAR NK. Reivindicación 97: Un plásmido para usar con sistemas asociados a repeticiones palindrómicas cortas agrupadas y espaciadas de forma regular (CRISPR) / proteína 9 asociada a CRISPR (Cas9) caracterizado porque el plásmido comprende una secuencia de polinucleótidos que codifica un polipéptido del receptor de antígeno quimérico (CAR); donde la secuencia de polinucleótidos es adyacente a un motivo adyacente protoespaciador (PAM) y una secuencia de polinucleótidos que codifica RNA crispr (crRNA) o flanqueada por dos PAM y dos secuencias de polinucleótidos que codifican crRNA. Reivindicación 105: Un vector viral adenoasociado (AAV) caracterizado porque comprende el plásmido de una cualquiera de las reivindicaciones 97 a 104. Reivindicación 109: Una célula modificada que comprende el plásmido de una cualquiera de las reivindicaciones 97 a 104 o el vector AAV de una cualquiera de las reivindicaciones 105 a 108. Reivindicación 114: Un método para crear una célula asesina natural (NK) o célula T NK del receptor de antígeno quimérico (CAR) caracterizado porque comprende a) obtener un complejo de ribonucleoproteína (RNP) que comprende una endonucleasa CRISPR / Cas de clase 2 (Cas9) complejada con un RNA guía CRISPR / Cas correspondiente y un vector AAV que comprende un plásmido que comprende una secuencia de polinucleótidos que codifica un receptor de antígeno quimérico (CAR); donde la secuencia de polinucleótidos es adyacente a un motivo adyacente protoespaciador (PAM) y una secuencia de polinucleótidos que codifica RNA crispr (crRNA) o flanqueada por dos PAM y dos secuencias de polinucleótidos que codifican crRNA; y b) introducir la secuencia de polinucleótidos que codifica el polipéptido CAR y el complejo RNP en la célula NK o célula T NK; donde el plásmido se introduce en la célula a través de la infección con el virus adenoasociado (AAV) en una célula efectora; donde el complejo de ribonucleoproteína (RNP) se hibrida con una secuencia dirigida dentro del DNA genómico de la célula, y las enzimas de reparación del DNA de la célula insertan el polinucleótido que codifica el CAR en el genoma del hospedador en la secuencia dirigida, creando así una célula CAR NK o célula T CAR NK. Reivindicación 116: Un método para modificar genéticamente una célula asesina natural (NK) o una célula T NK caracterizado porque comprende a) obtener un complejo de ribonucleoproteína (RNP) que comprende una endonucleasa CRISPR / Cas de clase 2 (Cas9) complejada con un RNA guía CRISPR / Cas correspondiente y un vector AAV que comprende un plásmido que comprende una secuencia de polinucleótidos que codifica un receptor de antígeno quimérico (CAR); donde la secuencia de polinucleótidos es adyacente a un PAM y una secuencia de polinucleótidos que codifica crRNA o flanqueada por dos PAM y dos secuencias de polinucleótidos que codifican crRNA; y b) introducir la secuencia de polinucleótidos que codifica el polipéptido CAR y el complejo RNP en la célula NK o célula T NK; donde la secuencia de polinucleótidos que codifica el polipéptido CAR se introduce en la célula a través de la infección con el virus adenoasociado (AAV) en una célula efectora; donde el complejo de ribonucleoproteína (RNP) se hibrida con una secuencia dirigida dentro del DNA genómico de la célula, y las enzimas de reparación del DNA de la célula insertan la secuencia polinucleotídica que codifica el receptor de antígeno quimérico (CAR) en el genoma del hospedador en la secuencia dirigida, creando así una célula modificada.
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