Gene Therapy Using rAAV Vector Against Hematological Disorders : From Basic Science To Clinical Application.

使用 rAAV 载体对抗血液疾病的基因治疗：从基础科学到临床应用。

• 批准号: 09671105
• 负责人: TANIMOTO Mitsune
• 金额: $ 2.05万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671105/
• 关键词: gene therapy; adeno associate virus; gene transduction; hematopoetic cell; cell therapy; cell culture; GVL effect; hematopoietic neoplasms; 造血幹細胞移植; GLV効果; ドナーリンパ球輸注; リコンビナント; AAV; gene therapy; adeno associate virus; gene transduction; hematopoetic cell; cell therapy; cell culture; GVL effect; hematopoietic neoplasms; 造血幹細胞移植; GLV効果; ドナーリンパ球輸注; リコンビナント; AAV

Adeno associated virus (AAV) has been thought to be the promising vector for gene therapy because of its wide range of human tissues and low toxicity. We have recently initiated the study of rAAV for hematopoietic cells. Preliminary results revealed that the transduction efficiencies assessed by colony formation assay employing NeoR gene into various cell lines, were CML->AML->ALL-derived cell lines by log differences. Present study was performed using peripheral T cell as a target tissue. After purification and concentration of rAAV, 27% efficiency was observed in CML line at an MOI of 7 and a linear relationship between MOI and efficiency was confirmed, suggesting that this syatem may be useful for gene transduction into leukemia cells. Integration of the NeoR gene into the host genome was assessed by Southern blotting, that revealed various sizes of DNA fragments. A fluorescent in situ hybridization (FISH) analysis was perfomed on 11 clones, in all of which the NeoR gene was integrated out of chromosome 19. In five clones, chromosome painting probes showed that the integration sites were chromosome 1q, 2q, 2q, 11p, 12p and l3q.

由于其广泛的人体组织和低毒性，因此人们认为Adeno相关病毒（AAV）被认为是基因治疗的有前途的载体。我们最近启动了造血细胞的RAAV研究。初步结果表明，通过对数差异，通过cml--> aml->全源性细胞系评估，通过菌落形成分析评估的转导效率。目前的研究使用外周T细胞作为目标组织进行。在RAAV的纯化和浓度之后，在CML线的MOI处观察到27％的效率，MOI为7，MOI与效率之间的线性关系得到了证实，这表明该SYATEM对于基因转导向白血病细胞可能很有用。通过Southern印迹评估了Neor基因在宿主基因组中的整合，该印迹揭示了各种大小的DNA片段。将荧光原位杂交（FISH）分析在11个克隆上呈现，其中所有神经基因均从19号染色体中整合。在5个克隆中，染色体绘画探针表明，整合位点是1q，2q，2q，2q，11p，12p，12p和L3Q。