Among its various objectives, the National Institute of Standards and Technology (NIST) Genome Editing Consortium aims to evaluate and qualify robust analytical methods to assess genome editing outcomes. As a member of this global cooperative, we recently completed a lentiviral integration project, during which our proprietary TLA-based solutions were applied for the complete genetic characterization and QC of 5 transgenic human cell lines. Furthermore, lentiviral vector copy number (VCN) was determined by coupling TLA-based data with ddPCR results.

Our Scientist Assay Developer (Gabrielle Dijksteel, PhD) reveals “we were excited to partake in this recent NIST study! This project confirms that ddPCR serves as a reliable orthogonal method to validate vector copy number. This is particularly relevant in the context of gene-modified cellular products, where thorough characterization is recommended by the FDA to safeguard the safety, efficacy, and consistency of manufactured ATMPs. Therefore, we now offer our customers ddPCR in conjunction with our TLA-based services in order to accurately quantify copy number and thus, further validate our robust TLA-based data for the genetic characterization and quality control of ATMPs.”

The outcome of variant detection and quantitation from this first NIST Genome Editing Consortium Interlab Study was presented by Samantha Maragh (Leader, NIST Genome Editing Program) at the 2023 AGBT General Meeting, which was held in Florida. In case you missed, but wish to learn more about the details of this important work make sure to watch our latest webinar entitled: 

Accurate VCN determination in gene-modified cellular products
via ddPCR results and TLA-based data

Lentiviral GFP-transduced human Jurkat cell lines

With the growing popularity and use of lentiviral vectors (LV) as vehicles for therapeutic gene delivery, product characterization has become increasingly important. Here, the study sought to evaluate the copy number of vector specific RRE element and of the breakpoint sequence(s) between vector and genome.

Although Paugh, et al. previously reported integration sites in the analyzed clones, one appears to have been missed. In fact, rather than the previously observed integration on chromosome 4 in VCN4, our in-house experts detected integration on chromosome 16 (results are summarized in Table 1B), thanks to the broad coverage generated across the integration locus via our NGS-based approach (see Figure 1A).

The identified vector-genome breakpoint sequences were then independently validated by ddPCR. Ultimately, ddPCR results were found to be concordant with our TLA-based data. Indeed, at each integration site of the LV 1 copy per cell was found, which corresponds to 1 to 4 copies per cell for VCN1-4.

Furthermore, for cell line VCN1, -2, and -4 a single small sequence variant was identified in the integrated vector sequence. Based on the mutation frequency, it was concluded that these variants are only present in one of the integrated vector copies.

Interestingly, a 648-kb genomic deletion near the integration site (specified by the green arrow below) on chromosome 14 in cell line VCN3 was detected (as shown below). No genomic rearrangements were observed at other integration sites.

TetO-PiggyBac transfected near-haploid cell line

Generating lentiviral vector producer cell lines via sequential stable transfection or transduction of DNA encoding each vector component at separate genomic loci often leads to lengthy cell line development campaigns. Therefore, some manufacturers favor instead a strategy in which all vector components are expressed from a single large DNA construct, e.g. bacterial artificial chromosome (BAC), which is then introduced into host cells in a single stable transfection manner. In a separate project, our experts characterized the genetic outcome of TetO-PiggyBac transfected near-haploid cell line.

Our whole genome coverage plot revealed many integration sites of PiggyBac transfected cell line (see Figure 2A). In fact, vector-genome breakpoint analysis disclosed 85 integration sites, of which 49 were found in intragenic regions. The NCG7.0-network of cancer genes indicated that 17 of the integration sites are within an intronic region of a cancer-related gene (see Figure 2B). The integration sites were independently confirmed by capture NGS.

Finally, we demonstrated that our TLA-based solutions could genetically QC transposons integrations with a 1/1000 sensitivity.

Complete genomic characterization and QC of modified cells

In conclusion, TLA-based solutions enabled complete genomic characterization of 2 popular vector integration systems by shedding light into (1) sequence and structural variants in integrated vector sequences and integration sites as well as (2) insertional mutagenesis. Secondly, our genomic-identity assays revealed a QC sensitivity of 1/1000 for the detection of transposon integrations. Finally, (3) the use of ddPCR proved to be an elegant quantification method to validate our TLA-based findings on VCN in gene-modified cellular products.

To learn more about this new service, please reach out to us at sales@cergentis.com or watch our latest expert-led webinar below!