Geraerts Martine, Michiels Martine, Baekelandt Veerle, Debyser Zeger, Gijsbers Rik
Laboratory for Molecular Virology and Gene Therapy, K.U. Leuven, Belgium.
J Gene Med. 2005 Oct;7(10):1299-310. doi: 10.1002/jgm.778.
HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods.
In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation.
Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively.
With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.
源自HIV-1的载体是将基因导入大脑的有前景的工具。将这些载体应用于基因治疗或用于创建神经退行性疾病的动物模型需要对慢病毒载体生产方法进行标准化和扩大规模。
在本研究中，通过使用细胞工厂并在离心前引入切向流过滤（TFF），有效地扩大了无血清HIV-1载体的生产规模。
在细胞工厂中生产并在离心前使用TFF的无血清HIV-1载体的载体滴度（TU/ml）和p24值（pg p24/ml）与小规模生产的相当。TFF可使载体浓缩66倍且载体完全回收。通过低速离心或超速离心可进一步浓缩载体（30倍）。TFF与超速离心相结合导致载体回收率为90 - 100%，转导单位和p24浓度的滴度分别增加1800倍和900倍。
采用这种新的慢病毒载体生产和浓缩的标准化方法，从2升细胞培养基开始，常规生产出1毫升浓缩载体，滴度为10(9)-10(10) TU/ml。此外，将这种载体立体定向注射到小鼠纹状体中，在没有任何免疫反应的情况下导致了大量的大脑转导体积。
