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As cell and gene therapy (CGT) transitions from laboratory research to clinical and commercial applications, the integration behavior of viral vectors (such as lentiviral, retroviral, and AAV vectors) in cellular genomes has become a "double-edged sword" for therapeutic efficacy and safety:
Positive aspects
Integration enables persistent expression and long-term therapeutic efficacy.
Integration near oncogenes or critical regulatory regions may induce aberrant expression or clonal proliferation, potentially leading to serious consequences such as secondary malignancies.
Risk factors
Therefore, Integration Site Analysis (ISA) has become one of the mandatory safety assessment components required by global regulatory agencies (FDA, EMA, CDE), particularly applicable to therapeutic models with long-term persistence such as CAR-T, iPSC, and stem cell therapies.
Integration site analysis has advanced from early methods like Southern blotting and inverse PCR to high-throughput approaches such as NGS and CRISPR/Cas9-based techniques. LM-PCR significantly improved site recovery efficiency and, when combined with NGS, enabled large-scale, high-precision mapping. CRISPR/Cas9 further enhanced accuracy by reducing PCR bias. Among these, LAM-PCR remains a widely used and essential method in current ISA workflows.
Creative Biogene, with over a decade of experience in viral vectors and genomic analysis, provides three technical approaches, comprehensive sample type support, high-resolution sequencing, and in-depth bioinformatics annotation to establish a complete integration risk assessment framework, helping you advance product development and clinical applications earlier, more stably, and more safely.
A sensitive method relies on the use of restriction enzymes, introducing a bias into the capacity of IS, and only semi-quantitative measurement of individual integration sites/clonal contributions.
EPTS LM PCR
comparable sensitivity to LAM PCR, but with less bias and much more accurate quantitative measurement of each clonal contribution.
HES
Lower sensitivity than the above two technologies, but more accurate for quantitative analysis. Using this method, both integration sites and vector copy number could be studied.
Supported Sample Types
PBMC, whole blood, bone marrow, tumor tissues, peripheral T/NK cells, cell lines (such as CHO, HEK293)
Innovative Extensions
For detection of structural rearrangements, fusion genes, and novel transcripts, customized introduction of Cas9 cleavage and Nanopore long-read platforms are available.
Our analysis goes beyond identifying integration loci—we provide comprehensive insights into the clinical significance of each event, including assessments of insertional safety, clonal dominance, and vector structural integrity.
Standard Analysis Modules
Optional Extension Modules
Human pluripotent stem cell-derived skeletal muscle progenitor cells (hPSC-SMPCs) hold promise for regenerative therapies by forming functional human–mouse chimeric myofibres. However, their inefficient colonization of native satellite cell (SC) niches has posed a major barrier to long-term engraftment. Researchers developed a satellite cell ablation (SC-ablated) mouse model to overcome this competition. Integration site analysis using LAM-PCR performed by Creative Biogene was applied to confirm safe and stable lentiviral transduction of hPSC clones expressing luciferase or PAX7-GFP reporters before in vivo engraftment.
Figure 1. PAX7⁺ hPSC-derived SMPCs showed limited occupancy of chimeric satellite cell niches compared to adult satellite cells, indicating reduced niche integration efficiency. (Hicks MR, et al., 2023)
Creative Biogene is your trusted partner for regulatory-compliant, high-quality integration site analysis services. Our experienced team of scientists and bioinformaticians is committed to supporting your research and clinical development needs with cutting-edge technologies and personalized solutions.
Q1: Which analysis technology should I choose? Can Creative Biogene help me decide?
A1: Absolutely. You only need to provide the following basic information:
Our technical experts will recommend the most suitable detection approach based on the above information, and multiple approaches can be combined for cross-validation as needed.
Table 1. Comparison of Integration Site Analysis Technologies
| Analysis Method | Basic Principle | Main Advantages | Limitations | Application Scenarios |
| Southern Blotting | DNA digestion with restriction endonucleases, hybridization with viral probes | Direct integration verification, standardized operation | Low sensitivity, time-consuming, and imprecise localization | Preliminary integration verification, basic research |
| Inverse PCR | Amplification of self-ligated fragments using outward primers | Simple operation, unknown sequence detection, low cost | Strong PCR bias, high false positive rate, limited detection depth | Known sequence detection, rapid screening |
| HES | gDNA fragmentation followed by library construction, probe hybridization for target sequence enrichment | High quantitative accuracy, simultaneous copy number analysis, and good specificity | Relatively low sensitivity, high probe design requirements | Vector copy number analysis, integration site quantification |
| LM-PCR/LAM-PCR | Adapter ligation and specific primer amplification | High-throughput, high-sensitivity, NGS compatible | Slightly complex operation | Clinical sample analysis, safety assessment |
| nrLAM-PCR | Ultrasonic fragmentation replacing enzymatic digestion | No enzymatic digestion bias, high coverage, simple operation | Heterogeneous fragments, complex analysis | Genome-wide scanning, clonal analysis |
| AFIS-Seq | Cas9-mediated long fragment enrichment | No PCR bias, long reads, lower cost | Requires sgRNA design, limited efficiency, and throughput | Preclinical assessment, safety studies |
| CReVIS-seq | CRISPR-directed cleavage combined with sequencing | Multi-site detection, high specificity, broad applicability | Complex operation, high analytical requirements, high cost | Multi-target analysis, complex sample detection |
Q2: How much sample is needed for integration site analysis? What are the sample requirements?
A2: We generally recommend ≥5 μg of high-quality gDNA per sample, with OD260/280 ratios between 1.8-2.0. If sample sources are limited, we can accept as little as 2 μg using low-input protocols. We support various sample types, including frozen PBMC, cell pellets, tissue blocks (fresh or FFPE), cell banks, or CHO suspension cultures. We provide sample shipping recommendations and preprocessing protocols to ensure smooth testing procedures.
Q3: How is clonal expansion or abnormal integration determined? Will this be reflected in the report?
A3: Yes, it will be reflected. We not only annotate the chromosomal position of each integration site but also calculate relative clonal frequencies based on sequencing depth and read distribution, with time-point comparisons (if provided). We also specifically annotate whether integration occurs near known oncogenes and provide risk scoring and annotation for potential risks. If abnormal clonal enrichment is detected, we provide recommendations for subsequent dynamic tracking.
Q4: Is ISA service data confidential? How is client data protected?
A4: Creative Biogene strictly enforces Non-Disclosure Agreements (NDAs) for all client projects.
If needed, we can provide GxP-compliant data access, audit, and compliance procedures to support client compliance inspections.
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