Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.

During the past 12 years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.

[1]  R. Levy,et al.  Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts. , 2001, International journal of pharmaceutics.

[2]  J. Mallet,et al.  Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[3]  Mark A. Kay,et al.  Progress and problems with the use of viral vectors for gene therapy , 2003, Nature Reviews Genetics.

[4]  D. Trono,et al.  Production and titration of lentiviral vectors. , 2006, Current protocols in neuroscience.

[5]  Hideki Mochizuki,et al.  High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells , 1998, Journal of Virology.

[6]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  L. Naldini,et al.  Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy , 2000, The journal of gene medicine.

[8]  Y. Durocher,et al.  High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. , 2002, Nucleic acids research.

[9]  Jianzhu Chen,et al.  Cross-linked Small Polyethylenimines: While Still Nontoxic, Deliver DNA Efficiently to Mammalian Cells in Vitro and in Vivo , 2005, Pharmaceutical Research.

[10]  Qing Ge,et al.  Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Karlsson,et al.  Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[12]  H. Luthman,et al.  High efficiency polyoma DNA transfection of chloroquine treated cells. , 1983, Nucleic acids research.

[13]  B. Nuttin,et al.  Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain , 2003, Gene Therapy.

[14]  S. Reed,et al.  Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. , 2006, Journal of virological methods.

[15]  R. Kutner,et al.  A comparative analysis of constitutive and cell‐specific promoters in the adult mouse hippocampus using lentivirus vector‐mediated gene transfer , 2008, The journal of gene medicine.

[16]  K. Cornetta,et al.  Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods , 2002, Gene Therapy.

[17]  J. Reiser.  Production and concentration of pseudotyped HIV-1-based gene transfer vectors , 2000, Gene Therapy.

[18]  Y. Durocher,et al.  Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification , 2007, Biotechnology and bioengineering.

[19]  Xian-Yang Zhang,et al.  Transduction of Bone-Marrow-Derived Mesenchymal Stem Cells by Using Lentivirus Vectors Pseudotyped with Modified RD114 Envelope Glycoproteins , 2004, Journal of Virology.

[20]  V. Baekelandt,et al.  Upscaling of lentiviral vector production by tangential flow filtration , 2005, The journal of gene medicine.

[21]  B. Karolewski,et al.  Comparison of transfection conditions for a lentivirus vector produced in large volumes. , 2003, Human gene therapy.

[22]  J. Nalbantoglu,et al.  Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells. , 2007, Journal of virological methods.

[23]  D. Anson,et al.  An improved β-galactosidase reporter gene , 2004 .

[24]  Amine Kamen,et al.  Large-Scale transfection of mammalian cells for the fast production of recombinant protein , 2006, Molecular biotechnology.

[25]  R. Corriveau,et al.  Gap junctions are required for NMDA receptor dependent cell death in developing neurons. , 2007, Journal of neurophysiology.

[26]  Y. Durocher,et al.  Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. , 2005, Biotechnology and bioengineering.

[27]  J. Reiser,et al.  Genetically selected stem cells from human adipose tissue express cardiac markers. , 2007, Biochemical and biophysical research communications.

[28]  M. Federico.  Lentivirus Gene Engineering Protocols , 2010, Methods in Molecular Biology.

[29]  R. Kutner,et al.  Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells. , 2008, Stem cells and development.

[30]  M. Welsh,et al.  Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates , 1999, Gene Therapy.

[31]  M. DeCoster,et al.  Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining. , 2004, Brain research. Brain research protocols.

[32]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.

[33]  K. Cornetta,et al.  Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy. , 1989, Journal of virological methods.