Development of novel packaging cells for refrovirus vectors based on stromal cells

基于基质细胞的逆转录病毒载体新型包装细胞的开发

• 批准号: 11670993
• 负责人: ITOH Katsuhiko
• 金额: $ 2.05万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670993/
• 关键词: stroma; retrovirus; vector; packaging cell; 遺伝子治療; ストローマ細胞; レトロウイルス・ベクター; 造血幹細胞

A novel murine stromal cell line, HESS-M28, was established, which supports the expansion of human CD34+CD38- cells more than 300- fold in vitro in the presence of human IL- 3 and SCF.Utilizing this cells, attempt was made to evaluate cis- acting elements of retroviral vectors in human primitive hematopoietic cells.Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector producing cells. The FMEV- type vector, SF/Lyt, contained the spleen focus- forming virus U3 and the MESV pimer binding site (PBS), while MO3/Lyt contained the U3 region ard PBS from McMLV.Following transduction by the FMEV- type and the MonLV- based vectors, expression of the marker gene, murine CD8 (mCD8), was examined in CD34-, CD34+ and CD34+CD38- cells. In CD34+ and CD34+CD38- cells, expression of mCD8 was higher with the FMEV- type vector, SF/Lyt, compared to the cells transduced by the MoMLV- based vector, MO3/Lyt, although the expression was comparable in CD34- cells. Expression of marker genes was also confirmed in long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs).

建立了一种新的小鼠基质细胞系HESS-M28，其支持在人IL- 3和SCF存在下体外扩增人CD 34 + CD 38-细胞超过300倍。尝试评估顺式-逆转录病毒载体在人原始造血细胞中的作用元件将脐带血细胞培养在基质细胞系的混合细胞层上，HESS-M28和逆转录病毒载体产生细胞。FMEV型载体SF/Lyt含有脾病灶形成病毒U3和MESV引物结合位点（PBS），而MO 3/Lyt含有来自McMLV的U3区和PBS。经FMEV型和基于MonLV的载体转导后，在CD 34-、CD 34+和CD 34 + CD 38-细胞中检测标记基因鼠CD 8（mCD 8）的表达。在CD 34+和CD 34 + CD 38-细胞中，与基于MoMLV的载体MO 3/Lyt转导的细胞相比，使用FMEV型载体SF/Lyt的mCD 8表达更高，尽管在CD 34-细胞中表达相当。在长期培养起始细胞（LTC-IC）和SCID再增殖细胞（SRC）中也证实了标记基因的表达。

项目成果

Tsuji T,Itoh K et al.: "Retroviral vector mediated gene expression in human CD34+CD38-cells expanded in vitro: cis-elements of FMEV are superior to those of MoMLV"Human Gene Therapy. 11. 271-284 (2000)

Tsuji T、Itoh K 等人：“体外扩增的人 CD34 CD38 细胞中逆转录病毒载体介导的基因表达：FMEV 的顺式元件优于 MoMLV 的顺式元件”人类基因疗法。

Tsuji T, Itoh K, Nishimura- Morita Y, Watanabe Y, Hirano D, Mori KJ and Yatsunami K.: "CD34high+CD38low/- cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation."Leukemia. 13. 1409-1419 (19

Tsuji T、Itoh K、Nishimura-Morita Y、Watanabe Y、Hirano D、Mori KJ 和 Yatsunami K.：“异种共培养系统中产生的 CD34high CD38low/- 细胞能够长期造血和多重分化。”白血病

Danno S,Itoh K et al.: "Efficient gene transfer by hybrid retroviral vectors to murine spermatogenic cells"Human Gene Therapy. 10. 1819-1831 (1999)

Danno S、Itoh K 等人：“通过杂交逆转录病毒载体将基因有效转移至小鼠生精细胞”人类基因疗法。

Tatsumi K.et al.: "Induction of tryptophan 2,3-Dioxygenase in the mouse endomstrium during implantation"Biochem Biophys Res Commun. 274. 166-170 (2000)

Tatsumi K.等人：“植入期间小鼠子宫内膜中色氨酸2,3-双加氧酶的诱导”Biochem Biophys Res Commun。

Itoh K.: "Transduction of human hematopoietic stem cells. (review, in Japanese)"Ketsueki-Syuyouka. 39. 417-426 (1999)

Itoh K.：“人类造血干细胞的转导。（评论，日语）”Ketsueki-Syuyouka。