Lenti-ISA - CB-Gene

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Lenti-ISA Detection Service

Definition

Lentiviral Integration Site Analysis utilizes a series of advanced molecular biology and bioinformatics techniques to sequence and analyze the genomes of cells successfully transduced with a lentiviral vector, thereby precisely identifying the specific location (i.e., the "integration site") where the viral vector DNA has inserted into the host genome.

Working Principle

Currently, the industry's gold standard method relies primarily on the combination of PCR amplification and next-generation sequencing (NGS). Its core principles are as follows:

1. Anchoring the known, exploring the unknown: All designs are based on a fundamental fact: the long terminal repeats (LTRs) at both ends of the viral vector are known, while the host genome sequences flanking them are unknown. Detection technology is like an explorer armed with a map of the viral LTRs, searching for the "unknown continent" (host DNA) adjacent to them.

2. Key Step - Linker-mediated PCR (LM-PCR):

Fragmentation: The intact genomic DNA is randomly fragmented into small fragments suitable for sequencing. Linker ligation: A linker with a known sequence is attached to the ends of all DNA fragments. This linker acts as a universal "handle."

Nested PCR: Two rounds of precise PCR amplification are performed.

*First round PCR: One primer binds to the end of the viral LTR, and another primer binds to the linker. Only fragments with a viral LTR at one end and a linker at the other are exponentially amplified, efficiently enriching for information on thousands of integration sites.

*Second round (nested) PCR: A second amplification is performed within the first PCR product using another pair of primers, further improving specificity and yield and preparing for the introduction of NGS sequencing adapters.

3. High-throughput sequencing and bioinformatics analysis:

PCR products are sequenced using NGS, generating millions of sequence reads. Using specialized bioinformatics pipelines, these reads are aligned to the human reference genome to precisely locate the chromosome, coordinates, and neighboring genes of each integration event. The frequency of each integration site is then calculated to assess clonality.

Standard Workflow

A complete Integration Site Analysis service typically includes the following steps:

Sample Preparation

The client provides a lentiviral-transduced cell sample (typically >1×10^6 cells) or extracted genomic DNA.

Library Construction and Sequencing

The service provider constructs a sequencing library based on LM-PCR and performs sequencing on a high-throughput sequencer.

Data Analysis

This is the core value of the service. The analysis includes:

Integration Site Identification: Lists all detected unique integration sites and their genomic coordinates.

Gene Annotation: Analyzes whether the site is located within a gene (introns/exons), in an intergenic region, near a promoter, or near a cancer-related gene (e.g., an oncogene).

 Clonality Analysis: Counts the number of "reads" corresponding to each integration site to assess the presence and proportion of dominant clones in the cell population.

 Safety Assessment: Based on databases (e.g., UCSC, NCBI Gene), reports whether the integration site is located at a genomic vulnerable site or adjacent to an oncogene, providing potential risk warnings.

Report Delivery

Provide a detailed, clear analysis report in both Chinese and English, including a list of all integration sites, visual diagrams (such as the distribution of integration sites on chromosomes), and professional interpretation of the conclusions.

Application Scenarios

This service is widely used in the following areas:

Safety Assessment of Gene Therapy/Cell Therapy Products

This is a core application. In preclinical studies and IND filings for products such as CAR-T, CAR-NK, and stem cell therapies, regulatory agencies (such as the National Medical Products Administration (NMPA) and the Food and Drug Administration (FDA)) require analytical data on vector integration characteristics to demonstrate that the therapeutic product does not have the potential risk of insertional mutagenesis.

Basic Research

Study the integration preferences of lentiviruses in specific cell types (such as hematopoietic stem cells and T cells); track the clonal origins and evolution of daughter cells during stem cell differentiation, and conduct lineage tracing studies.

Stability and Efficacy Studies

Verify the long-term stable expression of the target gene and analyze whether its expression level is correlated with integration site or copy number.

Clinical Monitoring

Long-term follow-up of patients receiving gene therapy to monitor the dynamic changes of cell clones in vivo, promptly detect abnormal clonal expansion, and ensure long-term patient safety.

Standard Workflow

Choosing professional testing services can bring multiple benefits to researchers:

High Sensitivity and Accuracy

NGS technology can detect integration events at extremely low concentrations (<0.1%) within a cell population, enabling the identification of rare clones.

Comprehensive and Unbiased

The LM-PCR method does not rely on restriction endonucleases, providing a more genome-wide, unbiased integration map, eliminating missed detections.

Time and Cost Savings

Eliminating the need to build complex and time-consuming experiments and bioinformatics analysis platforms in-house, our professional team provides a one-stop solution, accelerating R&D progress.

Regulatory Compliance

Our service process and report format align with regulatory agency technical guidelines, providing critical supporting data for drug applications.

Professional Interpretation

Receive more than just data analysis; receive in-depth interpretation and risk insights from experienced scientists to inform research decisions.

In summary, lentiviral Integration Site Analysis services serve as a critical bridge connecting basic gene therapy R&D with clinical application. Through precise "genomic navigation," it reveals the behavior of lentiviruses within cells, providing solid data support for therapeutic safety and significantly advancing our understanding of viral vector biology and cellular dynamics. With the advent of the era of precision medicine, the importance of this service will become increasingly prominent.

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