Liquid-Phase Capture Plasmid Integration Analysis

Liquid-Phase Capture Plasmid Integration Analysis

1. Background

Cell therapy, as an integral component of modern precision medicine, has achieved breakthrough progress in recent years. As of the first quarter of 2025, more than ten CAR-T cell therapy products have received marketing approval worldwide, with over 800 related clinical trials underway, signaling that cell therapy has entered an accelerated phase of clinical application. Plasmid vectors, owing to their ease of operation, large cargo capacity, and low cost, have become the primary tools for introducing CAR sequences and gene-editing elements into target cells. Typically, plasmid vectors mediate targeted integration of the functional sequences they carry (such as CAR constructs) into specific genomic loci via homology-directed repair (HDR) or transposase systems (such as Sleeping Beauty or PiggyBac).

However, the safety profile of plasmid integration warrants careful attention. Research has demonstrated that, in addition to the expected targeted integration, plasmid vectors can undergo random integration into the host genome through non-specific pathways. These unintended integration events may trigger insertional mutagenesis, dysregulation of gene expression, or chromosomal structural abnormalities, potentially leading to clonal expansion or malignant transformation—representing a key safety challenge in the evaluation of cell therapy products. The regulatory framework continues to tighten in response to this concern. In August 2023, the NMPA Technical Guidelines for Non-Clinical Research of Cell Therapy Products explicitly required "integration site analysis and insertional mutagenesis risk assessment" to be conducted; in March 2024, the NMPA-CDE Good Manufacturing Practice for Cell and Gene Therapy Products formally listed "integration site analysis and genomic integrity assessment" as key quality indicators, requiring the establishment of comprehensive detection methods.

Based on the liquid-phase hybrid capture principle, ZhuHai GeneRulor has developed a high-precision plasmid integration site analysis platform, with detection strategies specifically optimized for homology-directed CAR sequence integration, providing a reliable solution for the integration safety evaluation of cell therapy products. Compared to conventional methods, this technology enables comprehensive detection of the chromosomal coordinates and functional features of plasmid integration sites, along with oncogenic risk assessment, effectively meeting increasingly stringent regulatory requirements and product safety evaluation needs.

2. Principle of Liquid-Phase Hybrid Capture

The fundamental principle of liquid-phase hybrid capture detection technology is based on nucleic acid base complementary pairing. A panel of biotinylated capture probes designed to cover the full length of the exogenous plasmid sequence is used to enrich and detect exogenous plasmid integration events. When the exogenous plasmid integrates into the host genome, it forms a chimeric DNA molecule containing both the exogenous plasmid sequence and the host genomic sequence at the integration site. After shearing the extracted genomic DNA into fragments, hybridization with the probes followed by streptavidin–magnetic bead capture enables specific enrichment of DNA fragments containing plasmid sequences.

The detection workflow is as follows: the DNA sample to be analyzed is first subjected to library construction including end repair, dA-tailing, and adapter ligation; full-length strand-specific probes are then custom-synthesized based on the full-length exogenous plasmid sequence provided by the client, and the library is hybridized with the probes followed by streptavidin–magnetic bead capture to enrich integration-positive fragments containing both plasmid and host genomic sequences; finally, integration site signals are amplified by PCR and a sequencing library is constructed. Compared to other conventional methods that rely on sequence-specific primers, the liquid-phase hybrid capture approach enables more comprehensive capture of exogenous plasmid integration events, significantly improving the accuracy, comprehensiveness, and sensitivity of plasmid vector integration site detection.

Figure 1. Schematic Diagram of the Liquid-Phase Hybrid Capture Workflow for Plasmid Vector Integration Detection

3. Technical Innovations and Advantages of Liquid-Phase Hybrid Capture for Plasmid Vector Integration Site Detection

3.1 Core Technical Innovations

3.1.1 Full-Length Probe Design Strategy

A probe design strategy optimized for the specific characteristics of plasmid vectors:

Coverage of the full-length plasmid sequence, including the backbone region, regulatory region, and functional region;

Particularly enhanced coverage of regions prone to integration hotspots (such as replication origins and promoter regions);

Tiling probe design is employed to ensure accurate capture of integration breakpoints.

3.1.2 High-Efficiency Targeted Enrichment Technology

Optimized hybridization conditions and magnetic bead purification workflows are employed to build a highly efficient enrichment system:

Long-duration hybridization in a liquid-phase environment substantially improves probe sequence capture efficiency;

Stringent washing steps effectively remove non-specific binding, reducing background noise;

Supports parallel processing of multiple samples, improving throughput and experimental efficiency.

3.1.3 Multi-Dimensional Bioinformatic Analysis

A comprehensive professional bioinformatic analysis pipeline is integrated to achieve:

Precise characterization of the chromosomal coordinates and functional features of integration sites;

Genomic functional element annotation, oncogenic risk assessment, and diversity analysis;

Multi-dimensional visualization of integration events, supporting in-depth interpretation.

3.2 Method Validation and Performance Metrics

ZhuHai GeneRulor has conducted a comprehensive systematic validation. Comparative validation results across WGS, nrLAM-PCR, LM-PCR, and liquid-phase hybrid capture demonstrate that liquid-phase hybrid capture performs optimally across all technical metrics:

4. Application Scenarios and Service Advantages

4.1 Application Scenarios

Liquid-phase hybrid capture plasmid integration site detection technology has broad applications in gene therapy and cell therapy product development:

Cell therapy product safety assessment: evaluation of the plasmid integration risk in CAR-T, TCR-T, and other engineered cell therapy products;

Gene-editing efficiency and specificity assessment: detection of unintended integration of CRISPR/Cas9 and other gene-editing system plasmid vectors;

Stable cell line construction: assessment of integration sites and copy numbers during stable transfection processes;

Pre-clinical safety research: provision of genomic integration safety data required for IND submissions;

Plasmid vector development: optimization of plasmid design to reduce integration risk.

4.2 Service Advantages

Technical leadership: ZhuHai GeneRulor's proprietary liquid-phase hybrid capture technology overcomes the limitations of conventional methods, providing more accurate and comprehensive integration site analysis;

Certified quality management: the laboratory operates concurrently under an ISO 9001 quality management system and meets ISO/IEC 17025 testing and calibration laboratory competency accreditation standards, ensuring data reliability and complete traceability;

Comprehensive method validation: full method validation encompassing sensitivity, specificity, accuracy, and precision has been completed; validation reports can directly support IND submissions;

Standardized reporting system: integration site analysis reports conforming to the latest CDE and FDA guidance requirements, fully supporting drug review and regulatory inspection;

Extensive track record: ZhuHai GeneRulor has assisted numerous pioneering companies in completing AAV product integration safety assessments, accumulating rich project experience.

5. Liquid-Phase Hybrid Capture Plasmid Integration Site Analysis — Sample Report

ZhuHai GeneRulor provides comprehensive plasmid vector integration site analysis reports that meet regulatory requirements, including detailed sequencing data quality assessment, per-sample sequencing depth statistics, reference genome alignment analysis, vector sequence variant analysis, and integration site count statistics as foundational information. In addition, the report includes the following core content:

(1) Integration Site Detection Results and Genomic Distribution Analysis: Precise chromosomal coordinates, gene annotations, reads support counts, and other detailed information are provided for each integration site. The system displays the chromosomal location of each detected integration site, the associated gene names, genomic functional regions (such as UTR5, intergenic, intronic, etc.), as well as the positional information of the exogenous sequence within the vector. Circos circular plots are simultaneously employed to intuitively visualize the distribution pattern of integration sites across chromosomes, enabling visualization of the association between the vector and host genome.

Figure 2. Plasmid Vector Integration Site Detection Results by Liquid-Phase Hybrid Capture

(2) Plasmid–Host Junction Sequence Validation Analysis: The precise junction information between viral sequences and host genomic sequences at each integration site is displayed in detail. The exogenous sequence fragment and corresponding host sequence fragment at each integration site are provided, enabling authenticity and accuracy verification of integration events through sequence alignment, furnishing critical sequence-level evidence for molecular mechanism analysis and safety assessment of integration sites.

Figure 3. Plasmid–Host Junction Sequence Validation Results for Plasmid Integration Sites Detected by Liquid-Phase Hybrid Capture

(3) Oncogene Annotation: Integration events associated with tumor-related genes are screened based on ONCOGENE and TSG databases, with integration sites related to proto-oncogenes and tumor suppressor genes specifically highlighted and labeled, and safety risk assessment provided.

Figure 4. Oncogene Risk Assessment Annotation Table for Plasmid Integration Sites Detected by Liquid-Phase Hybrid Capture

(4) Integration Site Diversity Analysis: Statistical indices including Chao Index, Shannon Index, and Pielou's Index are employed to quantitatively assess integration site distribution characteristics, identify integration hotspots and clonal dominance phenomena, and evaluate the randomness of exogenous plasmid vector integration.

Figure 5. Integration Site Diversity Statistical Analysis Table for Plasmid Integration Sites Detected by Liquid-Phase Hybrid Capture

(5) Gene Functional Analysis: GO functional enrichment and KEGG pathway analysis are performed on genes harboring integration events, evaluating the potential impact of integration events on cellular biological processes and signaling pathways, and providing scientific evidence for risk control strategies.

Figure 6. GO and KEGG Pathway Enrichment Analysis (Example)

6. Liquid-Phase Hybrid Capture Plasmid Vector Integration Site Detection Service Contents

*Standard service turnaround: 20–30 business days

7. Sample Requirements

*Notes: ① All samples must comply with the above standards to ensure the accuracy and reliability of detection results; ② Clients may also submit tissue or cell samples for DNA extraction, with a tissue requirement of > 50 mg and a cell requirement of > 2×10⁷ cells per site; ③ For special sample types, please contact ZhuHai GeneRulor's technical team in advance (Tel: 400-6309596; Product Orders/Technical Support: service@generulor.com).

8. References

[1] Center for Drug Evaluation, NMPA. (2023). Technical Guidelines for Non-Clinical Research of Cell Therapy Products. Center for Drug Evaluation, NMPA.

[2] Kebriaei, P., et al. (2023). CAR T cells: New approaches to improve safety, efficacy, and persistence. Blood Reviews, 57, 101023.

[3] Fraietta, J.A., et al. (2022). Integration site analysis in CAR T cell therapy: Current methods, applications and limitations. Nature Reviews Clinical Oncology, 19(6), 392–408.

[4] Center for Drug Evaluation, NMPA. (2024). Good Manufacturing Practice for Cell and Gene Therapy Products. Center for Drug Evaluation, NMPA.

[5] Nobles, C.L., et al. (2022). High-throughput analyses of retroviral integration sites in clinical samples: From raw sequence files to genotoxicity risk assessment. Current Protocols, 2(4), e427.