IVD Technical Analysis｜LAM-PCR

LAM-PCR is a technique used to detect and sequence the adjacent sequences of unknown DNA and known DNA, and is mainly used to analyze the integration sites of retroviral vectors in the host genome in gene therapy. In a clinical trial for the treatment of X-SCID, the researchers used LAM-PCR technology to conduct a comprehensive and precise analysis of the vector integration sites in patients, and found the distribution, association and influence of the vector integration sites in the genome, And the prediction of the safety and efficacy of gene therapy.

LAM-PCR technology provides an effective method for the monitoring and evaluation of vector integration in gene therapy.

Technology Introduction

The principle of LAM-PCR is to use linear amplification and magnetic bead separation to enrich the vector-genome junction, followed by double-strand synthesis, restriction enzyme digestion, linker ligation and exponential PCR on a semi-solid streptavidin phase and other steps, and finally identify the unknown genome sequence by electrophoresis or sequencing. LAM-PCR can select appropriate primers and restriction enzymes according to different vectors and genomes to improve specificity and sensitivity. LAM-PCR can also be combined with next-generation sequencing (NGS) technology for high-throughput and high-resolution analysis of a large number of ISs. LAM-PCR is a flexible, reliable, and effective method for detecting vector integration sites.

LAM-PCR has many application scenarios, mainly for studying vector integration and clonal composition in gene therapy. For example, LAM-PCR can be used to examine the cloning kinetics and integration-related side effects of genetically modified blood cells in patients, or to assess the integration behavior and biosafety of novel vector systems. In addition, LAM-PCR can also be used to analyze the integration patterns of other types of integrating vectors (such as transposons, lentiviral vectors) or passive integrating vectors (such as adeno-associated virus or lentiviral vectors lacking integrase) in the host genome . LAM-PCR is a technique widely used in the fields of gene therapy and genomics.

An example of the use of LAM-PCR technology in gene therapy is: In a clinical trial for the treatment of immunodeficiency, researchers used LAM-PCR to analyze the integration sites of blood cells transduced with retroviral vectors in patients to identify Assess the safety and efficacy of the vector. They found that the distribution of vector integration sites in the genome is non-random and has a certain preference. They also found that vector integration sites are associated with patient blood reconstitution and cloning dynamics, and may lead to the activation of leukemia-associated genes. Through LAM-PCR technology, they can conduct a comprehensive and accurate analysis of the vector integration in patients, as well as predict the long-term effects and risks of gene therapy.

Pollution Problem

The contamination problem in LAM-PCR experiments means that non-target DNA sequences may be detected when analyzing integration sites, resulting in biased and inaccurate results. There are several ways to solve the pollution problem:

1.Choose appropriate restriction enzymes and primers to improve the specificity and sensitivity of LAM-PCR.

2.Use a dual barcoding strategy to reduce collisions and confusion between integration site sequences.

3.Use non-restriction LAM-PCR (nrLAM-PCR) method to avoid inhomogeneity and loss of DNA fragments caused by restriction enzyme digestion.

4.Before the PCR reaction, use enzyme treatment to remove possible microbial DNA contamination.

How to solve the pollution problem in LAM-PCR experiment?

An example of the use of other technologies in gene therapy: In a clinical trial to treat cancer, researchers used oligonucleotide-based therapy to block the survival process of cancer cells. They used a special oligonucleotide called AS1411 to recognize and bind to the nuclear protein A (Nucleolin) on the surface of cancer cells, thereby preventing the proliferation and angiogenesis of cancer cells. They found that AS1411 was able to effectively inhibit several types of cancer cells, including kidney, breast and lung cancers. Using oligonucleotide technology, they were able to specifically and selectively target cancer cells.

Evaluation and effect of genetic safety

There are several ways to assess the safety and effectiveness of gene therapy:

Through three stages of clinical trials, the safety, effectiveness and advantages of gene therapy are tested respectively. Clinical trials need to follow strict ethical and legal norms to protect the rights and health of participants.

Predict the effects and risks of gene therapy in humans through animal models and in vitro experiments. Animal models and in vitro experiments can provide information on the mechanism and pharmacokinetics of gene therapy to guide the design and execution of clinical trials.

The delayed effects and adverse reactions of gene therapy were monitored through long-term follow-up. Gene therapy may lead to potential consequences such as genome instability, immune response or carcinogenesis, so long-term observation and evaluation of the treated patients are required.

The advantages of gene therapy are as follows:

1.Gene therapy can treat or prevent some inherited or acquired diseases that are difficult to treat with traditional medicines or surgery, such as cystic fibrosis, hemophilia, cancer and AIDS.

2.Gene therapy can directly target the root cause of a disease, the abnormal or missing gene, to repair or replace damaged cellular function.

3.Gene therapy can improve the quality of life and survival of patients, and may even achieve a cure or immunity.

4.Gene therapy can reduce dependence on other drugs or treatments, thereby reducing side effects and costs.

5.Gene therapy can use the body's own cells and molecules, thereby improving specificity and selectivity, and reducing immune rejection or allergic reactions.

Product and Service

1.Creative Biogene provides customized LAM-PCR services for analyzing the integration sites of retroviral vectors in the host genome, including linear PCR, magnetic bead capture, double-stranded DNA synthesis, restriction enzyme digestion, linker ligation, embedding A set of steps such as PCR, PCR product purification and sequencing.

2.Bio-Rad provides a PCR instrument for LAM-PCR analysis, called CFX96 Touch Real-Time PCR Detection System, which can be used to detect and quantify PCR products quickly, accurately and sensitively, with high throughput and high resolution rate features.

3.Thermo Fisher Scientific provides a sequencing instrument for LAM-PCR analysis, called the Ion Torrent S5 System, which can be used for high-throughput and high-quality sequencing of the integration site sequence amplified by LAM-PCR, with high speed and Low cost feature.

4.Elchrom Scientific provides a precast gel for LAM-PCR analysis, called Spreadex EL1200, which can be used to separate and detect DNA fragments of different lengths, with high resolution and high sensitivity.

Analysis process case

ISA analysis of a small number of sites based on the LAM-PCR method:

Blood or bone marrow samples containing transduced cells are collected from treated patients or animals, and total DNA is extracted using a DNA extraction kit.

Linear PCR with primers specific for the retroviral vector amplifies the DNA fragment at the vector-genome junction.

Use magnetic beads and streptavidin to capture biotin-labeled PCR products and remove non-target DNA.

Double-stranded DNA synthesis was performed on magnetic beads, and complementary strands were randomly synthesized using Klenow enzyme and hexanucleotide mixture.

Digest the double-stranded DNA with a restriction enzyme, choosing a restriction enzyme that is different from the vector sequence to avoid cutting the vector sequence.

T4 DNA ligase is used to ligate linkers, which contain a biotin tag and a specific primer-binding site, to the cleaved genome ends.

Exponential PCR is performed on magnetic beads, amplified with vector-specific primers and linker-specific primers to generate double biotin-labeled DNA fragments.

The PCR products are separated and detected by gel electrophoresis, and the appropriate length range is selected for cutting and recovery.

Purify the recovered DNA fragments with a PCR purification kit, use sequencing primers for sequencing reactions, and use NGS technology for high-throughput sequencing.

The sequencing data was analyzed with bioinformatics software, compared with the genome database, and the precise position of the integration site and surrounding genes were determined.

ISA analysis of a large number of loci based on the LAM-PCR method:

LAM-PCR technique was used to amplify the integration site of the vector to obtain the amplified product containing ITR and flanking DNA sequence.

The amplified products were sequenced using next-generation sequencing technology to obtain sequencing data of the ITR and flanking DNA sequences.

Use an algorithm to analyze the sequencing data, and decompose the data into mutually independent subspaces, each subspace corresponds to a feature vector of a carrier integration site.

The subspaces were further analyzed using clustering or classification methods, and the vector integration sites were classified into different categories or groups according to the degree of similarity or difference between the subspaces.

Use visualization methods to display the results of ISA analysis, such as using scatter diagrams, heat maps, tree diagrams, etc., to display information such as the distribution, association, and diversity of vector integration sites.