Characterization of complete particles (VSV-G/SIN-GFP) and empty particles (VSV-G/EMPTY) in human immunodeficiency virus type 1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety.
Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.
Zhao
Yuan
Y
Biotherapeutics Group, National Institute for Biological Standards and Control, Potters Bar EN6 3QG, United Kingdom. yzhao@nibsc.ac.uk
Keating
Kenneth
K
Dolman
Carl
C
Thorpe
Robin
R
D002460:Cell Line; D002850:Chromatography, Gel; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D046529:Microscopy, Electron, Transmission; D010316:Particle Size; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D014759:Viral Envelope Proteins / Q000032:analysis / Q000235:genetics; D014771:Virion / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins