Controlled inactivation of recombinant viruses with vitamin B2
Introduction
Recombinant adenoviruses are one of the most widely used vectors for gene transfer in the clinic (No Authors Cited, 2007). Their use, however, is limited by strong innate and adaptive immune responses that occur soon after administration of a single dose by the systemic route (Sakurai et al., 2007). A single dose of virus also significantly alters the expression and function of several key cytochrome P450 (CYP) enzymes in the liver and the kidney (Callahan et al., 2006, Le et al., 2006). It is not clear if this effect is related to the innate immune response against the virus or if certain components of the viral genome are responsible for this effect. In order to investigate this further, a preparation of inactive, yet intact, virus capsids is needed. Exposure to 8-methoxy posoralen (8-MOP) and ultraviolet light is one of the most commonly used methods for inactivation of pathogens in blood products (Lin et al., 2005, Seghatchian, 2006, Wu and Snyder, 2003) and has often been used to inactivate adenovirus to create biologically inert, immunologically active controls in pre-clinical gene transfer and vaccination protocols (Borgland et al., 2000, Cotten et al., 1994, Cotten et al., 1992, Kafri et al., 1998, Schnell et al., 2001, Thomas et al., 2000).
Psoralens are photochemically active tricyclic compounds initially used in ancient Egypt and India to treat depigemented lesions (Pathak and Fitzpatrick, 1992). They continue to be used for this indication today and are an important tool in nucleic acid research. Their chemical structure allows them to easily fit between adjacent thymine base pairs so that, when exposed to UV radiation, they irreversibly link two DNA strands. In an initial pilot study, the combined treatment of 8-MOP and UV light effectively and efficiently inactivated the virus, however, administration of 8-MOP alone significantly altered the expression and activity of hepatic CYP3A2 as early as 6Ā h after administration in the male Sprague Dawley rat (Fig. 1). The human correlate of this isoform, CYP3A4, responsible for metabolizing approximately 40% of all currently marked medications (Plant, 2006), can convert 8-MOP to metabolites that irreversibly bind to and alter the expression and function of many CYP isoforms (Fouin-Fortunet et al., 1986, Mays et al., 1989, Tinel et al., 1987, Yang and Yan, 2007). This effect would skew results obtained from animals treated with virus inactivated in this manner unless excess 8-MOP was removed from the preparation by size-exclusion chromatography or dialysis. Although each of these methods would most likely remove the compound from the preparation, it is difficult to confirm since 8-MOP absorbs light within the same range as the virus itself.
Riboflavin, vitamin B2, is a molecule capable of intercalation and oxidation of DNA and RNA in the presence of UV light (Kumar et al., 2004). Unlike psoralen, riboflavin is an elemental dietary component with a low toxicity profile (Corbin, 2002, Unna and Greslin, 1942). To date, changes in the expression and function of CYP in the presence of riboflavin have not been described. Use of riboflavin in combination with UV light has effectively inactivated a wide array of pathogens such as extra- and intracellular human immunodeficiency virus (HIV), pseudorabies virus, West Nile virus, parvovirus, Gram positive bacteria (Straphylococcus epidermidis), Gram negative bacteria (Escherichia coli) and Leishmania protozoa without compromising blood components, producing toxic byproducts or inducing adverse side effects (Cardo et al., 2006, Corbin, 2002, Pelletier et al., 2006, Ruane et al., 2004). In this report, the effect of riboflavin and UV light on the infectivity of a first generation recombinant adenovirus, a non-enveloped virus with a double stranded DNA genome was assessed. Aggregation of virus particles was monitored throughout the inactivation process by field emission scanning electron microscopy (FESEM) and dynamic light scattering. Capsid integrity was confirmed by transmission electron microscopy (TEM). The in vivo performance of the inactive virus was compared to the same dose of active virus by histochemical staining for the beta-galactosidase transgene in liver sections obtained from treated animals 6Ā h after administration. Serum cytokines (IL-6, IL-12 and IFN-Ī³) and CYP activity were also assessed. The utility of this combination for inactivation of adeno-associated virus, a non-enveloped virus with a single-stranded DNA genome and lentivirus, an enveloped virus with a RNA genome is also described.
Section snippets
Materials
All chemicals were purchased in the highest purity available from EMD Chemicals (Gibbstown, NJ), unless specified otherwise. Phosphate-buffered saline (PBS), riboflavin, 8-methoxypsoralen (8-MOP), and dimethylsulfoxide (DMSO) were purchased from SigmaāAldrich (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM) was purchased from Mediatech (Herndon, VA). 5-Bromo-4-chloro-3-indolyl-Ī²-d-galactoside (X-gal) was purchased from Gold Biotechnology (St. Louis, MO). Tissue-tekĀ® optimal cutting
Effect of riboflavin on the expression and function of hepatic CYP3A2 and 2C11 in the male Sprague Dawley rat
Five hundred microliters of either riboflavin (50Ā Ī¼M, effective dose 36.2Ā Ī¼g/kg) and 8-MOP (psoralen, 1.5Ā mM, effective dose 625Ā Ī¼g/kg) were administered intravenously to male Sprague Dawley rats at concentrations documented to significantly inactivate several different pathogens (Cotten et al., 1994, Cotten et al., 1992, Ruane et al., 2004). The activity of CYP3A2 and 2C11 was assessed by separation and quantification of isoform-specific testosterone metabolites, 6Ī²-hydroxytestosterone and
Discussion
Initial observations that a single dose of recombinant adenovirus expressing the beta-galactosidase transgene significantly compromised hepatic cytochrome P450 (CYP) enzymes 3A2 and 2C11 and renal 2E1 for a period of 14 days led to additional studies to identify components of a recombinant virus that might be responsible for these effects (Callahan et al., 2006, Callahan et al., 2005, Le et al., 2006). In order to determine how the immune response against virus proteins in vivo affects the
Acknowledgements
The authors would like to thank Dr. Gary Kobinger of the National Microbiology Laboratory, Public Health Agency of Canada, in Winnipeg, Manitoba for kindly providing the plasmids necessary for producing the recombinant lentivirus used in these studies. We also thank Ms. Hong Le of The University of Texas at Austin College of Pharmacy for assistance with production of recombinant adeno-associated virus and Dr. Gary Vellekamp of SPRI for assistance in the compilation of this manuscript. This work
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Current address: Bristol-Myers Squibb Bioanalytical Sciences, Pennington, NJ, USA.