Viral vector integration site analysis for cell and gene therapy applications

As cell and gene therapies (CGTs) expand to meet new challenges in human disease, risk management through monitoring of vector-integration events will be increasingly important. Current approaches for monitoring vector integration lack applicability across therapeutics, require extended laboratory time, or carry extensive computational needs. To address the future need of CGT trials, new assays are required, offering improved sensitivity, a priori detection of integration events, and applicability or adaptability to diverse vectors and therapeutic sequences. Additionally, these integration site analysis (ISA) assays will need to enable detection of clonal expansion or reduction of cells containing integration events in order to monitor integration that results in either excessive cell proliferation or loss of cell viability, respectively.

Development of ISA assays for the most frequently used viral vector—lentivectors (LVs)—presents several challenges. First, LV platforms vary, with LVs split into generations (first, second and third) utilizing different components for creation of the viral vector.³ Third-generation LVs offer an improved safety profile compared to earlier generations and are widely used in CGTs. A typical example is the use of LVs to deliver chimeric antigen receptor "CAR" genes to T-cells to generate CAR T-cells for oncology and other indications. However, an ideal ISA would not only allow detection of third-generation vectors but would be compatible with all LVs, including potential future designs, to support both preclinical and clinical therapeutic development stages. Second, LVs can be either integration-competent or integration-deficient. Additionally, in both cases, it’s possible for LVs to form non-integrated stable DNA structures called episomes^4–6. As such, "ISA" assays must differentiate between viral sequences present in an episome and those present via integration in the patient genome. Third, the ISA assay must permit for differentiation between the therapeutic cargo of the LV and the site of integration. 

At Labcorp, we developed a novel assay to address these challenges. The LV ISA assay meets the unique requirements of therapeutics that are delivered with LVs, including: 

• Reduced assay run time
• Detection of all three LV generations
• Differentiation between episomal LVs and integrated LVs
• Characterization against an international standard reagent and additional custom reagents
• Monitoring of clonal expansion, including relative quantification of different integration events.

We characterized the assay against custom samples and an integration standard available commercially through the National Institute for Biological Standards and Control (WHO 1st Reference Reagent for Lentiviral Vector Integration Site Analysis, code 18/144).⁷

Results

LV ISA performance: Assay accuracy

To determine the assay’s accuracy in detecting integration events, we tested the LV ISA assay on the WHO 1st Reference Reagent for Lentiviral Vector Integration Site Analysis (code 18/144).^7,8 This standard contains 10 consensus-confirmed integration sites, where the sites were detected via sequencing-based methods by four separate labs. Additional sites were detected by a subset of the confirming labs. The LV ISA was able to detect the 10 consensus-confirmed sites and an 11th site detected by 3 of 4 of the confirmatory labs (Figure 1). Importantly, the LV ISA assay provides quantitation of each integration site detected for longitudinal or replicate comparison. 

Figure 1. LV ISA assay successfully detects 11 integration sites in the WHO 1st Reference Reagent for Lentiviral Vector Integration. *Chr9p11.2 was detected by a subset of the confirming labs.

LV ISA Performance: Assay sensitivity

To characterize sensitivity of the assay, we generated admixtures of integration-negative human gDNA with engineered plasmids containing mock integration sites at varying percentages. These plasmids encode a sequence consisting of an LV vector sequence bracketed by fragments of a human gene and allows us to determine sensitivity against a known percentage of genome copies containing integration. The LV ISA assay demonstrates sensitivity to 0.1% of genome copies containing integration events (Figure 2), corresponding to integration detection in 1 out of every 1,000 cells.

Figure 2. LV ISA detects vector integration to 0.1% of genomic material present in a sample. This corresponds to detection of an integration event in 1 out of every 1,000 cells. Samples were run in duplicate; error bars represent SD.

Comparison to alternative approaches

To establish accuracy and sensitivity against alternative assays, we compared LV ISA to whole genome sequencing. Whole genome sequencing is typically considered too expensive for frequent monitoring and can exhibit gaps in detection in certain genomic regions. Both assays were performed on the WHO 1^st Reference Reagent for Lentiviral Vector Integration Site Analysis.^7,8 Where WGS detected 10 of 11 sites, despite achieving 113x average sequencing depth across the genome, LV ISA successfully detected 11 sites (Figure 3 a,b). The number of reads obtained at each site for WGS versus LV ISA is shown in Table 1. 

Table 1. Comparison of read depth over integration sites between WGS on 100% WHO #18/144 standard and LV ISA with 25% of input WHO #18/144 standard. 250 ng of WHO #18/144 standard was prepared using a Watchmaker WGS library preparation kit, using 4 PCR cycles to amplify libraries. Final libraries were sequenced on a NovaSeq 6000. 125 ng of WHO #18/144 was mixed with 375 ng of integration-negative gDNA and the resulting admixture was prepared using Labcorp’s LV ISA protocol and sequenced on an Illumina MiSeq. For both library preparation approaches, 100% integration-negative gDNA samples were included as negative controls. No integration sites were detected in the negative controls (data not shown).

Figure 3. Comparison of LV ISA to WGS. (a) High-coverage WGS was performed on a 100% sample of the WHO standard (250 ng input). (b-d) LV ISA assay was performed on varying admixtures of integration-negative gDNA with the WHO #18/144 standard. Detection of 11 sites was maintained to 25% of input material consisting of the WHO standard, with all 10 consensus sites detected at 10% of input material consisting of the WHO standard. Integration sites detected at lower levels are lost to detection first, with sites showing greater abundance detected with as little as 0.1% of input material consisting of the WHO standard.

 LV ISA: Modeling real-world applications

To meet the need for monitoring of clonal expansion, the ISA assay provides relative quantification of integration sites by tracking unique initial molecules detected in a sample at each integration site. To demonstrate this capability, we created a hypothetical expansion of an integration event, with one integration site expanding while a second is observed at a constant level (Figure 4). The LV ISA demonstrates detection of the expansion of a given integration site from 0.1% of genome copies to 2% of genome copies, without impacting detection of a second non-expanding integration event.

Figure 4. LV ISA provides relative quantification of integration events to support detection of clonal expansion. Admixtures of two modeled integration events in an integration-negative background were created. T1 = 0.1% Chr 7, 0.1% Chr 12; T2 = 0.1% Chr 7, 0.5% Chr 12; T3 = 0.1% Chr 7, 1% Chr 12; T4 = 0.1% Chr 7, 2% Chr 12. Hypothetical time points are shown on the x-axis. Samples were run in duplicates. Comparison between integration sites and across time points informs determination of clonal expansion and additional monitoring or intervention steps for clinical trial participants.

Conclusions

Viral vectors offer many advantages for delivery of CGT, but they require monitoring for both safety and efficacy. Here, we present a vector integration detection assay designed to detect, monitor and characterize lentivector integration events that may occur with LV-delivered CGTs. The reduced assay run time, ability to monitor expansion over time, and sensitive detection of integration events make this assay a powerful tool in guiding development and clinical trial testing of LV-based CGTs.