Lentiviral standards to determine the sensitivity of assays that quantify lentiviral vector copy numbers and genomic insertion sites in cells

With an increasing number of gene therapy clinical trials and drugs reaching the market, it becomes important to standardize the methods that evaluate the efficacy and safety of gene therapy. We herein report the generation of lentiviral standards which are stable, cloned human cells prepared from the diploid HCT116 cell line and which carry a known number of lentiviral vector copies in their genome. These clones can be used as reference cellular materials for the calibration or qualification of analytical methods that quantify vector copy numbers in cells (VCN) or lentiviral vector genomic integration sites (IS). Cellular standards were used to show the superior precision of digital droplet PCR (ddPCR) over quantitative PCR (qPCR) for VCN determination. This enabled us to develop a new sensitive and specific VCN ddPCR method specific for the integrated provirus and not recognizing the transfer plasmid. The cellular standards, were also useful to assess the sensitivity and limits of a ligation-mediated PCR (LM-PCR) method to measure IS showing that at least 1% abundance of a single IS can be detected in a polyclonal population but that not all IS can be amplified with similar efficiency. Thus, lentiviral standards should be systematically used in all assays that assess lentiviral gene therapy efficacy and safety.

Automated summary

With the increasing number of gene therapy clinical trials and drugs, it is important to standardize the methods used to evaluate the safety and effectiveness of gene therapy. This study reports the creation of stable lentiviral standards, which are cloned human cells with a known number of lentiviral vector copies in their genome. These standards can be used as reference materials for calibrating and qualifying analytical methods used to measure vector copy numbers and lentiviral vector integration sites. The study shows that digital droplet PCR (ddPCR) is more precise than quantitative PCR (qPCR) for determining vector copy numbers, and a new sensitive and specific ddPCR method was developed. Lentiviral standards should be used in all assays that assess the efficacy and safety of lentiviral gene therapy.