Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells

Abstract

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

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

“…Our laboratory has previously cloned HT1080 cells transduced with a green fluorescent protein (GFP)-expressing lentiviral vector and containing 1, 3, or 8 copies of integrated vector [ 9 ]. Such clones demonstrated a high correlation between VCN and transgene expression measured by GFP mean fluorescence intensity in flow cytometry, as reported by others [ 10 ]. Such clones served in preclinical studies to assess the sensitivity of several DNA extraction and PCR methods to detect VCN in individual CFU-C and to validate VCN results in clinical gene therapy trials [ 4 , 6 , 13 ].…”

“…Already, ddPCR has been applied to lentiviral vector titration showing a broad dynamic range for the quantification between 30 to 7700 copies of HIV sequence per reaction as measured on a plasmid [ 11 ]. The ddPCR technique was also used by others for VCN measures and reportedly provides similar results as qPCR [ 10 ]. In our case, we show that ddPCR with PSI-ALB sequences enables a VCN measure on human cell gDNA using 20–40 ng per reaction with a LOQ at 0.01 VCN and LOD at 0.005 VCN.…”

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products

“…Droplet digital PCR (ddPCR) was performed using the Bio-Rad QX200 system according to the manufacturer’s protocols (Bio-Rad). The ddPCR reaction mixture consisted of 10 μl of 2 × ddPCR master mix (Bio-Rad), 60 ng genomic DNA, and 20× primers/probes mix targeting RRE (FAM), a Rev response element sequence located upstream of the MSCV promoter in the integrated lentiviral genome ( 26 ). Primers and probes targeting the reference gene RPP30 (HEX) were added in a final volume of 21 μl in all reactions.…”

Clinical grade lentiviral vector purification and quality control requirements

“…Meanwhile, defined copy numbers of a reference LV integrated cell line have been published for accurate validation and optimization of assays that can determine integration of the LV copy number in transduction. [71].…”