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

Abstract

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.

Evaluation of diversity indices to estimate clonal dominance in gene therapy studies

“…In order to evaluate the behavior of the Simpson and Pielou indices in experimental conditions, we used a dataset of ISs with different amounts of clonal dominance from a previously reported study. 27 To artificially increase clonal dominance, a DNA from a polyclonal population of LV-transduced cells was spiked with known and increasing quantities of DNA extracted from stable cell clones containing either one, two, or three copies of LV, generating increasing amounts of multiple detectable ISs. Under these conditions, an average of 900 ISs (683–920) for 2,500 estimated cells per condition (2,388–3,502) were analyzed.…”

“…Figure S4 confirmed that the Simpson index is less sensitive to the increase in dominance than Pielou, especially when multiple ISs contribute to the change in evenness.

Figure 4 Comparison of Pielou and Simpson indices in polyclonal populations with increasing contribution of a subset of insertion sites Vector insertion site (IS) clonal contributions is analyzed from a published dataset 27 in which DNA from a polyclonal population of cells transduced with a lentiviral vector (1.5 average vector copy number) is spiked with increasing amounts of DNA from lentiviral standard clones. ISs were obtained by Illumina sequencing following DNA sonication, LM-PCR, and analyzed by the “soniclength” R package (see Corre et al.

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“…ISs were obtained by Illumina sequencing following DNA sonication, LM-PCR, and analyzed by the “soniclength” R package (see Corre et al. 27 for details). Under each condition represented in the bar graph, “Spiked #” represents the number of unique ISs added in the population, and “Expected %” represents their cumulated expected abundance.…”

“… 17 , 18 , 19 , 20 , 22 , 23 , 26 We modeled the performance of these indices upon the occurrence of clonal dominance and found differential impacts of sample size on the results obtained. Using an already published experimental IS dataset with spiked clones, 27 we show that Pielou’s index provides a sensitive measure of clonal dominance. Using published gene therapy studies, we also show that the Pielou index enables inter-study comparisons and can be used to define a safety threshold in trials where leukemias occurred.…”

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

“…DNA was digested using DraI restriction enzyme (NEB) at 37°C for 30 min and then mixed with the ddPCR reaction mix composed of 2X ddPCR SuperMix for probes (no dUTP) (Bio-Rad), forward (for) and reverse (rev) primers (at a final concentration of 900 nM) and probes (at a final concentration of 250 nM). We used probes and primers specific for: (i) albumin (VIC-labeled ALB probe with a QSY quencher, 5'-CCTGTCATGCCCACACAAATCTCTCC-3'; FOR ALB primer, 5'-GCTGTCATCTCTTGTGGGCTGT-3'; REV ALB primer, 5'-ACTCATGGGAGCTGCTGGTTC-3'), and for (ii) the LV (FAM-labeled LV probe with an MGB quencher, 5'-CGCACGGCAAGAGGCGAGG-3'; FOR LV primer 5'-TCCCCCGCTTAATACTGACG-3'; REV LV primer 5'-CAGGACTCGGCTTGCTGAAG-3' or FAMlabeled PRO-LV probe with an MGB quencher 5'-TCTCTAGCAGTGGCGCCCGAACAGG-3' ; FOR PRO-LV primer: 5'-CACTCCCAACGAAGACAAGA-3'; REV PRO-LV primer: 5'-TCTGGTTTCCCTTTCGCTTT-3' to measure VCN in BM cells (Corre et al, 2022). The albumin gene was chosen as reference locus to calculate the VCN per genome.…”