Real-time quantitative PCR for the design of lentiviral vector analytical assays

Abstract

From the recent and emerging concerns for approving lentiviral vector-mediated gene transfer in human clinical applications, several analytical methods have been applied in preclinical models to address the lentiviral vector load in batches, cells or tissues. This review points out the oldest generation methods (blots, RT activity, standard PCR) as well as a full description of the newest real-time quantitative PCR (qPCR) applications. Combinations of primer and probe sequences, which have worked in the lentiviral amplification context, have been included in the effort to dress an exhaustive list. Also, great variations have been observed from interlaboratory results, we have tempted to compare between them the different analytical methods that have been used to consider (i) the titration of lentiviral vector batches, (ii) the absence of the susceptible emerging replicative lentiviruses or (iii) the lentiviral vector biodistribution in the organism.

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

“…The observed titers in transducing units/ml (TU/ml) were roughly 1 log lower compared with the ones obtained as genomic RNA (RNAs/ml). This finding, being in agreement with earlier observations reported in the literature, [37][38][39] validates our real-time PCR method. The higher RNA titers are most likely due to the presence of defective interfering particles and do not appear to be due to plasmid DNA, as we conducted the PCR analysis of supernatants in the presence and absence of reverse transcription to exclude DNA background.…”

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy

“…Although the RTqPCR titration has been shown to overestimate the number of infectious particles (for review see Delenda and Gaillard, 2005) and does not allow an absolute quantification of the viral titer, it provides a more accurate relative assessment of infectivity than the p24 titer. We have observed that viral supernatants can vary substantially in the ratio of p24 to genome content, and successfully used this titering technique as a surrogate for infectivity with a number of different lentiviral constructs in addition to the three shown in the present study (data not shown).…”

Gene delivery by lentivirus vectors

“…Direct comparison of integrated vector DNA, vector particle RNA, and marker expression assays demonstrated that quantitative PCR to measure integrated vector DNA in transduced cells is the most reliable method [139]. Various researchers have employed a number of primers, probes, and standards to measure integrated vector DNA, as well as vector episomal forms, many of which are described in Delenda et al and references therein [138]. Moreover, quantitative RT-PCR techniques were recently employed to measure transgene mRNA expression from an integrated lentiviral vector [140,141].…”

“…Within the last 6 years the use of real time PCR-based methods to determine vector titers has become commonplace, reviewed in Delenda and Gaillard [138]. Direct comparison of integrated vector DNA, vector particle RNA, and marker expression assays demonstrated that quantitative PCR to measure integrated vector DNA in transduced cells is the most reliable method [139].…”