Hematopoietic stem cell (HSC)°ú ºÐÈµÈ ¼¼Æ÷·ù (¿¹, primary T cell, macrophage µî) ¿Í °°Àº ´ëºÎºÐÀÇ hematopoietic stem cellÀº virus¸¦ ÀÌ¿ëÇÑ À¯ÀüÀÚ Àü´ÞÀÌ ¾î·Æ±â ¶§¹®¿¡ ´Ù¾çÇÑ Ç÷¾×°ü·Ã Áúº´ÀÇ
À¯ÀüÀÚ Ä¡·á¸¦ À§ÇÑ ¿¬±¸¿¡ °É¸²µ¹ÀÌ µÉ ¼ö ÀÖ´Ù.
Lentivirus, retrovirus¸¦ ÀÌ¿ëÇÑ hematopoietic stem cellÀÇ À¯ÀüÀÚ µµÀÔÈ¿À²Àº Ÿ°Ù ¼¼Æ÷ÀÇ Áֱ⠻óÅÂ, ¼¼Æ÷Ç¥¸éÀÇ receptor ¹ßÇö Á¤µµ µî ´Ù¾çÇÑ ¿äÀÎ
Enhanced virus-cell colocalization
RetroNectin
¢çÀº human fibronectin protein¿¡¼ À¯·¡ÇÑ 3°¡Áö µµ¸ÞÀÎÀ¸·Î ±¸¼ºµÇ¾î ÀÖ´Â polypeptideÀÌ´Ù. Virus ÀÔÀÚ°¡ RetroNectin
¢çÀÇ heparin binding domain°ú °áÇÕÇÔÀ¸·Î½á virus ÀÔÀÚ¿Í
Á¶Ç÷¸ð¼¼Æ÷°£ÀÇ À§Ä¡¸¦ ¹°¸®ÀûÀ¸·Î °¡±õ°Ô ÇÏ¿© transduction È¿À²À» ³ôÀÌ´Â ¿ø¸®·Î, Á¶Ç÷¸ð¼¼Æ÷´Â ÁÖ·Î ¼¼Æ÷ Ç¥¸éÀÇ integrin receptorÀÎ VLA-4¿Í RetroNectin
¢çÀÇ CS-1 »çÀÌÆ®°¡ °áÇÕÇÑ´Ù.
±×¸² 1. Hypothesized mechanism of RetroNectin¢ç-mediated enhancement of transduction
High-efficiency transduction of hematopoietic cells
RetroNectin
¢çÀº ¹°¸®ÀûÀ¸·Î ¼¼Æ÷¿Í ¹ÙÀÌ·¯½º ÀÔÀÚ¸¦ °¡±õ°Ô ÇÏ¿© ¼¼Æ÷µ¶¼ºÀº ÃÖ¼ÒÈÇϸç À¯ÀüÀÚÀÇ µµÀÔÈ¿À²À» ³ôÀ̹ǷÎ, ´Ù¾çÇÑ Á¶Ç÷¸ð¼¼Æ÷¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Ù.
Gene transfer efficiency for hematopoietic cells1 |
Cell type |
Efficiency (%) |
Human CD34+ CD38- BMC2 |
95.5 |
Human PBMC3 |
91.2 |
TF-1 |
97.9 |
SupT1 |
97.3 |
Jurkat |
80.1 |
K-562 |
90.4 |
HL-60 |
86.1 |
Monkey CD34+ BMC |
72.0 |
Monkey CD4+ T-cell |
85.0 |
1 Transduction˼ RetroNectin
¢ç Á¦Ç°¸Å´º¾ó¿¡ ±âÀçµÈ RetroNectin
¢ç-Bound Virus (RBV) MethodÀ» »ç¿ëÇÔ.
ÀÚ¼¼ÇÑ ³»¿ëÀº Á¦Ç°¸Å´º¾ó (¸µÅ©) ÂüÁ¶.
2 Bone marrow cells.
3 Peripheral blood mononuclear cells.
Improved transduction compared with conventional methods
RetroNectin
¢çÀ» ÀÌ¿ëÇÏ¸é ±âÁ¸ÀÇ PolybreneÀ» »ç¿ëÇÑ transduction ¹æ¹ý¿¡ ºñÇØ È¿À²ÀÌ ³·Àº ¼¼Æ÷ ŸÀÔ¿¡µµ À¯ÀüÀÚ µµÀÔÈ¿À²À» ³ôÀÏ ¼ö ÀÖ´Ù.
Human CD34
+¼¼Æ÷¿¡¼ RetroNectin
¢çÀº transduction È¿À²À» 50~70% ³ôÀÏ ¼ö ÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó (±×¸² 1), ¼¼Æ÷µ¶¼ºµµ °ÅÀÇ ¾ø´Â °ÍÀ» È®ÀÎÇÏ¿´´Ù.
±×¸² 2. RetroNectin¢ç°ú PolybreneÀÇ retrovirus transduction È¿À² ºñ±³
Retrovirus µµÀÔÈ¿À²À» ºñ±³Çϱâ À§ÇØ, Polybrene°ú RetroNectin
¢çÀÇ °¢ ¸Å´º¾ó¿¡ µû¶ó Retrovirus transductionÀ» ÁøÇàÇÏ¿© human CD34
+ HSC¿¡ GFP À¯ÀüÀÚ¸¦ ¹ßÇö½ÃÅ°°í, flow cytometry·Î Çü±¤À» ÃøÁ¤ÇÏ¿´´Ù.
GFP ¹ßÇö·®À» ±×·¡ÇÁÀÇ XÃà¿¡, GFP¸¦ ¹ßÇöÇÏ´Â CD34
+ ¼¼Æ÷ÀÇ ºñÀ²À» %·Î Ç¥±âÇÏ¿´´Ù.
±× °á°ú, RetroNectin
¢çÀ» ÀÌ¿ëÇÑ °æ¿ì Polybrene º¸´Ù transduction È¿À²ÀÌ 86¹è ³ôÀº °ÍÀ» È®ÀÎÇÏ¿´´Ù (°¢°¢ 25.8%, 0.3%).
ÀÌ¿Í °°ÀÌ ±âÁ¸¿¡ À¯ÀüÀÚ µµÀÔÈ¿À²ÀÌ ³·¾Æ ¾î·Á¿òÀ» °Þ¾ú´ø Á¶Ç÷¸ð ¼¼Æ÷ ¿¬±¸¿¡ RetroNectin
¢çÀ» ÀÌ¿ëÇÔÀ¸·Î½á Á¶Ç÷¸ð¼¼Æ÷¿Í lentivirus ¶Ç´Â retrovirusÀÇ co-localizationÀ» ÃËÁøÇÏ¿© ¼¼Æ÷ µ¶¼º ¾øÀÌ ³ôÀº È¿À²·Î
À¯ÀüÀÚ¸¦ µµÀÔÇÒ ¼ö ÀÖ´Ù.
Comparison of retroviral transduction of a K562 cell line using transduction enhancers from different companies
±×¸² 3. ´Ù¾çÇÑ transduction enhancerÀÇ retroviral transduction È¿À² ºñ±³
´Ù¾çÇÑ Transduction enhancer¸¦ ÀÌ¿ëÇÏ¿© K562 cell lineÀÇ retrovirus µµÀÔÈ¿À²À» ºñ±³ÇÑ °á°ú,
RetroNectin
¢çÀ» ÀÌ¿ëÇÏ¿´À» ¶§, Polybrene, protamine ¹× Ÿ»ç human fibronectinº¸´Ù ¿ùµîÈ÷ ³ôÀº È¿À²·Î transduction µÇ´Â °ÍÀ» È®ÀÎÇÏ¿´´Ù.