Large-Scale Production Means for the Manufacturing of Lentiviral Vectors

Abstract

Lentiviral vectors become more and more famous for the use as gene vector for gene therapy purposes for the treatment of acquired or inherited diseases. In this review, the present state of the art of the production of lentiviral vectors is presented with particular emphasis on the large scale production of these vectors for preclinical and clinical purposes. In contrast to oncoretroviral vectors which are produced using stable producer cell lines, clinical grade lentiviral vectors are essentially produced by transient transfection of 293 or 293T cells grown in Cell Factories. The main reason is that these production processes have been developed when good and safe LV producer cell lines were not available. With respect to the purification of lentiviral and in agreement with actual developments in the biotech industry, rather sophisticated downstream processing protocols have been established in order to remove any potentially dangerous process derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review presents large scale production means for LV vectors, the different downstream processing steps as used for the purification of LV vectors as well as LV specific safety issues. Published large scale production and purification processes of lentiviral vectors and their process performances are compared.

A Simple and Effective Method to Generate Lentiviral Vectors forEx VivoGene Delivery to Mature Human Peripheral Blood Lymphocytes

“…However, this method is extremely sensitive to variations in pH, as well as Ca 2 + and phosphate concentrations, which can affect titer and reproducibility between production runs. Transfection reagents such as polyethylenimine (PEI) are being developed for large-scale, serum-free transfection, which should help overcome the challenges associated with transfection using Ca-Phos (Ansorge et al, 2009;Schweizer and Merten, 2010;Segura et al, 2010). An additional challenge hindering the widespread use of LVV is the significant amount of downstream processing required for concentration and purification of the LVV in order to remove contaminating DNA and other impurities.…”

“…4A). Column chromatography is also routinely being used for vector purification as it allows for clean separation of LVV from other impurities (Schweizer and Merten, 2010;Segura et al, 2010). To simplify the manufacture of LVV for ex vivo ACT protocols, we performed large-scale transient transfection of 293T cells followed by Benzonase treatment (50 U/ml) and clarification by modified step filtration (Fig.…”

Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus

“…Most of non-HIV derived lentiviral vectors have been reported to be tat and sometimes rev independent, thus falling in the 3 rd or 4 th generation of packaging systems. For clinical trials purposes, both second and third generation lentiviral vector systems were reported although only HIV-1 and EAIV derived vectors have been used (Schweizer and Merten 2010). Contrarily to simple retroviral vectors, the cytotoxicity of some of the lentiviral proteins has hampered the establishment of stable cell lines constitutively expressing vector components.…”

“…Except for the systems reported by and Ni et al (2005), all the packaging cell lines for lentiviral vector production have been based on human 293 cells transformed with oncogenes such as the SV40 (simian vacuolating virus 40) large T antigen -293T -or the Nuclear Antigen of Epstein-Barr Virus -293EBNA. For clinical application human 293 and 293T cells have been the exclusive cell substrates (Schweizer and Merten 2010). However, safety concerns arise from the fact that 90% of noncoding mobile sequences of the human genome are endogenous retrovirus and although most of them are defective, because of mutations accumulation, some are still active (Zwolinska 2006).…”

“…Transient production, makes use of transfection methods to introduce the viral constructions, commonly cationic agents that complex with the negatively charged DNA, thus allowing it to be up-taken by the cell via endocytosis (Al-Dosari and Gao 2009). From those, polyethylenimine (PEI) (Boussif et al 1995) is probably the less expensive, one of the most efficient and the most widely used in the current protocols (Schweizer and Merten 2010;Segura et al 2010;Toledo et al 2009). Others methods such as calcium phosphate precipitation (Jordan and Wurm 2004;Mitta et al 2005) and cationic lipids complexation including LipofectAMINE® and FuGENE®, have also been used, although at small-scale production or for research purposes only since, these are either difficult to scale-up or very expensive.…”