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Home > Learning center > RetroNectin > ´Ù¾çÇÑ ½ÇÇè Àû¿ë »ç·Ê > mRNA¸¦ ÀÌ¿ëÇÏ¿© Á¦ÀÛÇÑ CAR-T ¼¼Æ÷ÀÇ ¼¼Æ÷»óÇØÈ°¼º Æò°¡

mRNA¸¦ ÀÌ¿ëÇÏ¿© Á¦ÀÛÇÑ CAR-T ¼¼Æ÷ÀÇ ¼¼Æ÷»óÇØÈ°¼º Æò°¡

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mRNA¸¦ ÀÌ¿ëÇÏ¿© Á¦ÀÛÇÑ CAR-T ¼¼Æ÷ÀÇ ¼¼Æ÷µ¶¼º Æò°¡

CAR-T´Â ¾Ï¼¼Æ÷°¡ ¼¼Æ÷ Ç¥¸é¿¡¼­ ¹ßÇöÇϴ ƯÁ¤ Ç׿øÀ» ÀνÄÇÏ´Â CAR (chimeric antigen receptor) À¯ÀüÀÚ¸¦ ȯÀÚ À¯·¡ÀÇ ¸²ÇÁ±¸ (T-cell)¿¡ µµÀÔÇÏ¿© ´Ù½Ã ü³»¿¡ ÁÖÀÔÇÏ´Â Ä¡·á¹ýÀÌ´Ù. À̶§ ÀϹÝÀûÀ¸·Î lentivirus¿Í °°Àº ¹ÙÀÌ·¯½º º¤ÅÍ°¡ »ç¿ëµÇÁö¸¸ ¿©·¯ È帱ºÀÇ CAR À¯ÀüÀÚ ±â´ÉÀ» Æò°¡ÇÏ´Â °æ¿ì ¹ÙÀÌ·¯½º º¤Å͸¦ ¸Å¹ø ¸¸µå´Â °ÍÀº Ãʱ⠽ÇÇè ´Ü°è¿¡ Àå¾Ö¹°ÀÌ µÉ ¼ö ÀÖ´Ù. µû¶ó¼­ ÀÌ ¿¬±¸¿¡¼­´Â in vitro transcription (IVT*)À¸·Î ÇÕ¼ºÇÑ mRNA¸¦ È°¿ëÇØ CAR À¯ÀüÀÚ¸¦ T-cell¿¡ µµÀÔÇÏ¿© mRNA CAR-T cellÀ» ÀϽÃÀûÀ¸·Î »ý¼ºÇÑ ÈÄ ±× ±â´ÉÀ» Æò°¡ÇÏ¿´´Ù. mRNAÀÇ delivery¿¡´Â ¹Ì±¹ MaxCyte¿¡¼­ °³¹ßÇÑ Àü±âõ°ø¹ý (electroporation) ÀåÄ¡ÀÎ ExPERT SystemÀ» »ç¿ëÇÏ¿´´Ù.


* ´ç»ç¿¡¼­´Â ½ÃÁß¿¡¼­ °¡Àå ³ôÀº mRNA ÇÕ¼º ¼öÀ²À» ÀÚ¶ûÇÏ´Â Takara IVTpro¢â T7 mRNA Synthesis Kit (Code 6144)¿Í mRNA ÇÕ¼º¿¡¼­ »ç¿ë °¡´ÉÇÑ template plasmid¸¦ Á¦ÀÛÇÏ´Â kitÀÎ Cloning Kit for mRNA Template (Code 6143)À» ÆǸÅÇÏ°í ÀÖ½À´Ï´Ù.

¡á Result
1. ½Ã°£´ëº° CAR À¯ÀüÀÚ ¹ßÇöÀ² º¯È­
MaxCyte ATx¸¦ »ç¿ëÇÏ¿© T-cell*1¿¡ anti-CD19 CAR mRNA*2¸¦ µµÀÔ ÈÄ 3½Ã°£¿¡¼­ 96½Ã°£±îÁö ½Ã°£º°·Î CARÀ¯ÀüÀÚ ¹ßÇöÀ² (¿ø¹®, ¾ç¼ºÀ²)À» ÃøÁ¤ÇÏ¿´´Ù. ±× °á°ú, µµÀÔ ÈÄ 3½Ã°£ºÎÅÍ CAR À¯ÀüÀÚÀÇ °í¹ßÇöÀÌ È®ÀεǾú°í, µµÀÔ 24½Ã°£ ÈÄ 90%ÀÇ ¼¼Æ÷¿¡¼­ CAR À¯ÀüÀÚ¸¦ ¹ßÇöÇß´Ù. ±× ÈÄ CAR À¯ÀüÀÚ´Â Á¡Â÷ÀûÀ¸·Î ¹ßÇöÀÌ °¨¼ÒÇÏ¿© 96½Ã°£ µÚ¿¡´Â °ÅÀÇ ¼Ò½ÇµÇ¾ú´Ù (±×¸² 1A). ¶ÇÇÑ Àü±âõ°ø ÈÄ ¼¼Æ÷ Áõ½ÄÀ²À» È®ÀÎÇÑ °á°ú mRNA ºñµµÀÔ±º°ú À¯»çÇÏ¿´´Ù (±×¸² 1B).
*1 RetroNectin¢ç (Code T100A)¹ýÀ¸·Î À¯µµÇÑ ÈÄ 10ÀÏ°£ È®´ë¹è¾çÇÏ¿© »ç¿ëÇÏ¿´À¸¸ç ¼øµµ´Â ¾à 90%¿´À½.
*2 Takara IVTpro¢â mRNA Synthesis System (Code 6141)À¸·Î ÇÕ¼ºÇÏ¿´À¸¸ç, Capping¿¡´Â CleanCap Reagent AG (TriLink»ç) Á¦Ç°À¸·Î capping¿´À½.



2. ¼¼Æ÷µ¶¼º ÃøÁ¤
Anti-CD19 mRNA¸¦ µµÀÔÇÑ ´ÙÀ½ ³¯ mRNA CAR-T ¼¼Æ÷¸¦ CD19 positiveÀÎ NALM-6¼¼Æ÷ (±Þ¼º ¸²ÇÁ¼º ¹éÇ÷º´ À¯·¡)¿Í °øµ¿¹è¾çÇÏ¿© ¼¼Æ÷µ¶¼ºÀ» ÃøÁ¤ÇÏ¿´´Ù (±×¸² 2A). ±× °á°ú, mRNA CAR-T ¼¼Æ÷´Â lentivirus·Î Á¦ÀÛÇÑ CAR-T ¼¼Æ÷*3¿Í À¯»çÇÑ ¼¼Æ÷µ¶¼ºÀ» º¸¿´´Ù (±×¸² 2B).
*3 Lentivirus (LVpro¢â Packaging Mix (Code 6195)·Î Á¦ÀÛ)¸¦ ÀÌ¿ëÇÏ¿© CAR À¯ÀüÀÚ¸¦ µµÀÔÇÏ¿´À¸¸ç CAR À¯ÀüÀÚÀÇ ¹ßÇöÀ²Àº ¾à 90% ¿´À½.



¡á °ü·ÃÁ¦Ç°
- Çѹø¿¡ 200 §¶ mRNA ÇÕ¼º, °í¼öÀ² in vitro transcription: Takara IVTpro¢â T7 mRNA Synthesis Kit (Code 6144)
- IVT¿ë Plasmid templateÀ» °£´ÜÇÏ°Ô Á¦ÀÛÇÏ´Â Kit: Cloning Kit for mRNA Template (Code 6143)
- Transformation½Ã Poly(A)µîÀÇ ¹Ýº¹¼­¿­À» ¾ÈÁ¤ÀûÀ¸·Î À¯Áö: Takara Stable Competent Cells (Code 9132)
- dsRNA È¥ÀÔÀÌ Àû¾î ÀÓ»óºÐ¾ß ¿¬±¸°³¹ß¿¡ ÀûÇÕ: PrimeCap¢â T7 RNA Polymerase (low dsRNA) (Code 2560A)
- Lentivirus transduction enhancer¿Í T-cell È®´ë¹è¾çÀ» µ¿½Ã¿¡: RetroNectin¢ç (Code T100A ¿Ü)
- HIV-1 À¯·¡ ¼­¿­À» ÃÖ¼ÒÈ­ÇÑ 3¼¼´ë Lentivirus ½Ã½ºÅÛ: LVpro¢â Packaging Mix (Code 6195 ¿Ü)
- Transfection ½Ã¾à Àü¹® ºê·£µå MirusÀÇ mRNA Àü¿ë Transfection ½Ã¾à: TransIT¢ç-mRNA Transfection Kit (Code MIR 2250)
- Cloning½Ã CDS¼­¿­À» Á¤È®ÇÏ°Ô PCR: PrimeSTAR¢ç GXL DNA Polymerase (Code R050A)