Kinetic analyses of stability of simple and complex retroviral vectors.
Simple and complex retroviral vectors derived from Moloney murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), respectively, are useful tools for gene transfer studies. However, factors affecting the stability of these vectors have not been carefully investigated. Here we studied the stability factors on vesicular stomatitis viral envelope glycoprotein (VSV-G)-pseudotyped MLV- and HIV-1-derived vectors. Analysis of the ratio of defective particles versus infectious units using electron microscopy and a functional transduction assay revealed that both vectors consisted of high numbers of defective particles ( approximately 100-350:1), which could be reduced ( approximately 10-20:1) by centrifugation. Frequent freeze-and-thaw rapidly decreased vector titer in the first three to five cycles and stabilized thereafter. Both viral vectors were sensitive to temperatures above 37 degrees C but more stable at temperatures below 37 degrees C, exhibiting a two-phase inactivation kinetic starting with a steep inactivation phase, followed by a more leveled phase. Interestingly, HIV-1-derived vectors were significantly more stable than MLV-derived vectors at higher temperatures (>37 degrees C). Both vectors were rapidly destabilized at pH either below or above 7.0. Incubation with human or mouse serum significantly inhibited VSV-G-pseudotyped vector activities. Preheated human serum still reduced vector half-lives to approximately 50% (150 min), suggesting that certain inactivation factors are not heat-labile. Analyses of these stability factors may improve future production and applications of retroviral and lentiviral vectors.
Higashikawa
F
F
Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Gainesville, Florida 32610-0266, USA.
Chang
L
L
D000818:Animals; D003673:Defective Viruses; D005615:Freezing; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000502:physiology*; D008562:Membrane Glycoproteins; D051379:Mice; D013696:Temperature; D014407:Tumor Cells, Cultured; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins