A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector
A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector
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DOI：
10.1053/jhep.2003.50265
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发表时间：
2003-07-01
期刊：
影响因子：
13.5
通讯作者：
Charneau, P
中科院分区：
文献类型：
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作者：
Giannini, C;Morosan, S;Charneau, P
同种异体肝细胞移植或转基因肝细胞的自体移植已成功用于纠正先天性或获得性肝病，并可被视为原位肝移植的替代方案。然而，肝细胞既不容易在培养物中维持，也不能有效地进行基因修饰，并且在重新植入受体之前对解离非常敏感。这些困难极大地限制了离体方法在临床试验中的使用。在本研究中，我们证明了 FLAP 慢病毒载体可以有效转导原代人和大鼠肝细胞，而无需铺板和培养。通过与载体颗粒的短时间接触，实现了非贴壁原代肝细胞的有效转导，不改变肝细胞活力，并且使用减少量的载体。我们还表明，载体构建体中 DNA FLAP 的存在对于达到高水平的转导至关重要。此外，移植到uPA/SCID小鼠肝脏中后，慢病毒转导的原代人肝细胞在其肝脏中广泛繁殖，并通过在动物血清中稳定检测人白蛋白和抗胰蛋白酶数月来评估其维持分化和功能表型。总之，使用FLAP慢病毒载体可以在短时间内实现人类功能性和可再移植肝细胞的高转导效率。因此，这项工作为基于肝脏定向离体基因治疗的人体临床试验的发展开辟了新的视角。
Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.