Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.
Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.
Transfiguracion
Julia
J
Biotechnology Research Institute/National Research Council of Canada, BioProcess Sector, Department of Animal Cell Technology, Montreal, Province of Quebec, H4P 2R2 Canada.
Jaalouk
Diana E
DE
Ghani
Karim
K
Galipeau
Jacques
J
Kamen
Amine
A
D002478:Cells, Cultured; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000302:isolation & purification; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000302:isolation & purification*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D017154:Stromal Cells / Q000378:metabolism; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation; D014462:Ultrafiltration; D014759:Viral Envelope Proteins / Q000235:genetics*; D014771:Virion / Q000302:isolation & purification
D004279:DNA, Viral; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D003851:Deoxyribonucleases