175. Titering Gene Delivery Vectors by QPCR: Application to rSV40-Derived Vectors

Abstract

Several methods have been used for quantification of SV40 gene transfer vectors (rSV40). Each technique measures a different parameter of recombinant virus structure or function, and each has its own advantages and disadvantages. Titering by A260/A280 ratios, as is commonly used for Adenovirus, is unreliable because full-length rSV40 genome is indistinguishable from rSV40 fragments and non-viral DNA. Quantifying non-replicating viruses like rSV40 by plaque assay is difficult and not reproducible. In situ PCR has been used to determine rSV40 titers, but this is very time consuming and is subject to interpretative errors. Consequently, a quantitative real-time PCR (QPCR) assay was developed to titer rSV40 preparations. This method was fast, simple and generated highly reproducible virus titers.Primer sets specific for the rSV40 vectors were coupled with SYBR green detection of PCR products. Virus titers were determined by comparison to a standard curve of purified rSV40 plasmid DNA. Several commercially available SYBR green PCR mixes and a lab-made formulation were tested to determine which mix performed best.Prior to performing the QPCR, rSV40 samples were dialyzed to remove detergents (deoxycholate, Triton X-100) that were used during virus purification, and digested with DNase and RNase to eliminate any non-encapsidated nucleic acids. Experiments that compared DNase-digested and undigested aliquots from the same virus preparations demonstrated that 50%|[ndash]|100% of the viral DNA was encapsidated (depending on the preparation). Treatment with proteinase K to remove the virus capsid before QPCR was determined to be unnecessary: the hot start step required to activate the Taq polymerase was sufficient to break the virus capsids and to release the viral DNA for amplification. Primer sets that amplify different regions of the rSV40 genome were tested. All the primer sets gave comparable amplification profiles and generated similar titers, indicating that the virus genomes were largely complete|[ndash]|without systematic large deletions or rearrangements. We have employed QPCR to titer rSV40 preparations for use in gene delivery experiments, and to monitor virus replication in SV40 packaging cell lines to determine when the maximal progeny virions are produced. With the design of appropriate primers, this QPCR assay is readily adaptable for quantifying other DNA viruses. We are developing a QRT-PCR protocol that will be useful for titering RNA viruses.