Reliable RT-qPCR-based titration of retroviral and lentiviral vectors via quantification of residual vector plasmid DNA in samples.

ABSTRACT

OBJECTIVES: To develop a method for reliable quantification of viral vectors, which is necessary for determining the optimal dose of vector particles in clinical trials to obtain the desired effects without severe unwanted immune responses. RESULTS: A significant level of vector plasmid remained in retroviral and lentiviral vector samples, which led to overestimation of viral titers when using the conventional RT-qPCR-based genomic titration method. To address this problem, we developed a new method in which the residual plasmid was quantified by an additional RT-qPCR step, and standard molecules and primer sets were optimized. The obtained counts were then used to correct the conventionally measured genomic titers of viral samples. While the conventional method produced significantly higher genomic titers for mutant retroviral vectors than for wild-type vectors, our method produced slightly higher or equivalent titers, corresponding with the general idea that mutation of viral components mostly results in reduced or, at best, retained titers. CONCLUSION: Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.