Lentiviral/Retroviral Integration Sites Analysis

Introduction to Lentiviral/Retroviral Integration Sites Analysis

Lentiviral vectors are widely recognized as the predominant gene delivery systems for achieving stable genetic modification of cells. These vectors integrate into the host genome to facilitate the expression of therapeutic transgenes. Although the integration process of the lentiviral RNA genome—first reverse-transcribed into DNA—is ostensibly random, existing literature indicates the presence of integration hotspots. Given the potential for lentiviral integration to disrupt cellular function, particularly through proximity to oncogenes or critical genes, it is imperative to identify the precise integration sites post-infection. The selection of integration sites significantly influences both transgene expression and host cell phenotype. Consequently, integration site analysis is an essential tool for evaluating the biosafety of gene therapy vectors and monitoring the clonal dynamics of genetically modified cells in vivo.

Our laboratory has developed an advanced suite of methodologies for comprehensive viral integration site analysis. By integrating PCR and sequencing-based techniques with proprietary bioinformatics pipelines, we provide unparalleled precision and sensitivity in identifying integration site positions and quantifying viral vector frequencies. Our end-to-end service encompasses PCR amplification, library construction, paired-end (PE) sequencing, and rigorous bioinformatics analysis. We are committed to delivering the highest quality data and reliable analytical reports, ensuring robust and reproducible results for our clients.

Methods for Integrative Site Analysis

The initial exploration of viral integration sites employed Southern blotting and genomic libraries to characterize integration site features. Over time, polymerase chain reaction (PCR) techniques have supplanted Southern blotting due to their simplicity and accuracy. Depending on the PCR methodology used, viral integration site analysis can be categorized into Reverse PCR, Ligated Mediated PCR (LM-PCR), and Linear Amplification Mediated PCR (LAM-PCR).

• Reverse PCR

Reverse PCR involves the circularization of DNA fragments following enzymatic digestion. Specific primers are then designed to amplify sequences flanking the viral integration site. However, the low efficiency of circularization with ligase limits its widespread application.

• LM-PCR

In LM-PCR, DNA is fragmented enzymatically, and adaptors are ligated to both ends of the fragments. Primers that recognize both viral DNA and the fixed adaptor sequences are utilized to amplify integration sites, thereby unlocking their positional information. LM-PCR stands out for its straightforward principle and ease of operation, retaining its relevance from inception to the present day.

• LAM-PCR

LAM-PCR begins with the linear amplification of DNA, producing single-stranded products. These single-stranded molecules are then subjected to further amplification via LM-PCR. The introduction of linear amplification significantly enhances both the sensitivity and specificity of LAM-PCR, making it superior to Reverse PCR and LM-PCR and leading to its broad adoption.

• Next-Generation Sequencing for Lentiviral Integration

Currently, high-depth sequencing, or next-generation sequencing (NGS), represents the principal platform for detecting lentiviral integration sites. The most refined and widely utilized method for creating enriched libraries for integration site analysis in industrial applications and clinical research involves mechanical fragmentation and adaptor-based amplicon library preparation (such as LM-PCR techniques like INSPIIRED). This methodology has been applied extensively in the safety evaluation of various CAR-T19 clinical projects as well as in retrospective pharmacodynamic analyses related to CAR-T cell proliferation.

Advantages of Our Lentiviral/Retroviral Integration Sites Analysis Service

• Mature integration site analysis techniques.
• Multiplex samples for cost-effective results.
• Effective workflow and fast turnaround time.
• Qualitative and quantitative analysis.
• Comprehensive bioinformatics analysis.
• Multiple approaches to meet different goals.

Applications of Lentiviral/Retroviral Integration Sites Analysis

Gene Therapy Safety Assessment:

• Avoiding Insertional Mutagenesis
• Monitoring Long-Term Effects

Cancer Research:

• Understanding Tumorigenesis
• Developing Cancer Treatments

Virology Research:

• Understanding Viral Life Cycle
• Developing Vaccines and Antiviral Drugs

Optimizing Transgenic Animal Models

• Enhancing Stability and Expression
• Reducing Adverse Effects

Regenerative Medicine and Cell Therapy

• Improving Therapy Efficiency
• Tracking Cell Fate

Basic Biological Research

• Exploring Genome Structure and Function
• Investigating Genomic Rearrangements

Lentiviral/Retroviral Integration Sites Analysis Workflow

Our highly experienced expert team executes quality management following every procedure to ensure comprehensive and accurate results. Our Lentiviral Integration Sites Analysis workflow is outlined below, including library prep, sequencing, and bioinformatics analysis.

Service Specifications

	Sample Requirements gDNA sample≥500ng Note: Sample amounts are listed for reference only. For detailed information, please contact us with your customized requests.
Click	Sequencing Strategy Illumina Hiseq/NovaSeq PE150 Platform For LM-PCR method (suitable for samples with known LTR sequences): construct 400~600 bp insert size library; 1Gb data output For WGS method (only suitable for monoclonal cell line): construct 350bp library, ~50X coverage
	Bioinformatics Analysis We provide multiple customized bioinformatics analyses: Data quality control Alignment to reference genome and insert sequences Integration site detection Integration site annotation Note: Recommended data outputs and analysis contents displayed are for reference only. For detailed information, please contact us with your customized requests.

Analysis Pipeline

Deliverables

• The original sequencing data
• Experimental results
• Data analysis report
• Details in Lentiviral/Retroviral Integration Sites Analysis for your writing (customization)

CD Genomics' lentiviral integration sites analysis service utilizes the power of Illumina Platform for integration sites detection, and has developed a low-cost high-throughput approach by using target enrichment sequencing or LM-PCR method. We are pleased to use our extensive experience and advanced platform to offer the best service and the most qualified products to satisfy each demand from our customers.

Reference

1. Dawes JC, Webster P, Iadarola B, et al. LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites. Mob DNA. 2020; 11:7.

Partial results are shown below:

1. What is Lentiviral/Retroviral Integration Site Analysis?

Lentiviral/Retroviral Integration Site Analysis is a sophisticated technique used to examine how lentivirus and retrovirus genomes integrate into the host cell genome. By identifying and mapping these integration sites, researchers gain crucial insights into the mechanisms of viral integration, assess the safety and efficacy of gene therapy, and monitor the in vivo behavior of transgenic cells.

2. Why is Integration Site Analysis Necessary?

Integration site analysis is vital for evaluating the safety of gene therapies. It ensures that viral vectors do not insert into critical regions of the host genome, which could potentially trigger oncogenesis or other genetic disorders. Additionally, this analysis aids researchers in understanding the preferences and mechanisms of viral integration, thereby providing essential theoretical support for the development of safer and more effective gene therapy strategies.

3. What are the Basic Steps of Integration Site Analysis?

The fundamental procedure for lentiviral/retroviral integration site analysis includes the following steps:

• Infection of Target Cells: Infect the target cells with the viral vector.
• Isolation of Genomic DNA: Extract the genomic DNA from the infected cells.
• Restriction Enzyme Digestion: Digest the isolated genomic DNA using restriction endonucleases.
• Ligation of Adapter Sequences: Ligate adapter sequences to the digested DNA fragments.
• PCR Amplification: Perform PCR amplification to enrich for DNA fragments containing viral integration sites.
• Library Construction: Construct sequencing libraries from the enriched PCR products.
• High-Throughput Sequencing: Conduct high-throughput sequencing, such as Illumina sequencing.
• Data Analysis and Integration Site Identification: Analyze the sequencing data to identify and characterize the viral integration sites.

4. What Tools and Techniques are Required?

Integration site analysis typically involves the following tools and techniques:

• Restriction Endonucleases: Enzymes used to digest genomic DNA at specific sequences.
• PCR Amplification: Techniques for selective amplification of DNA sequences containing viral integration sites.
• Adapter Ligation: The method of attaching adapter sequences to DNA fragments to facilitate sequencing.
• High-Throughput Sequencing: Platforms such as Illumina sequencing for generating extensive sequence data.
• Bioinformatics Analysis Tools: Software and algorithms for sequence alignment (e.g., BLAST, Bowtie) and data analysis (e.g., R, Python) to elucidate integration site locations and their characteristics.

Integrome signatures of lentiviral gene therapy for SCID-X1 patients

Journal: Science Advances

Impact factor: 14.957

Published: 06 October 2023

Background

Lentiviral vectors are used in gene therapies and CAR T-cell treatments, integrating into the host genome. A study on SCID-X1 infants analyzed over 280,000 integration sites from 273 samples, using high-resolution genomic, epigenomic, and 3D genome data. This research is crucial for improving the safety and effectiveness of gene therapies, especially with recent reports of therapy-related complications.

Materials & Methods

Results

In a study on SCID-X1 patients, infants received autologous HSCs transduced with a lentiviral vector encoding the IL2RG gene. VISs were analyzed using qsLAM-PCR across various cell types and time points, revealing consistent integration patterns and clonal diversity correlated with vector copy number.

Lentiviral integration sites (VIS) in SCID-X1 patients show hotspots and recurrent integration genes (RIGs), predominantly in introns and active gene regions. ChIP-seq data reveal VIS enrichment in active histone marks and depletion in repressive marks, with consistent patterns across patients except for one. These findings highlight the importance of genomic and epigenomic contexts in lentiviral integration.

Fig. 1. Genomic signatures of lentiviral VIS.

The lentiviral integrome signature identified in HSCs of SCID-X1 patients is also present in LV-transduced CAR T cells, showing similar hotspots, histone mark preferences, and nuclear compartment enrichment. VIS patterns in CAR T cells correlate with those in SCID-X1 patients, with both shared and unique recurrent integration genes (RIGs) across cell types.

Fig. 2. The HSC lentiviral integrome signatures are present in lentiviral-transduced T cells.

Lentiviral integration sites differ from γ-retroviral (gRV) sites, with LVs preferring active gene bodies and gRVs targeting promoter regions. Clinical issues with gRVs, like T-ALL and MDS, were linked to integrations near specific gene loci, whereas LVs did not show such patterns. Comparative studies revealed LVs and gRVs have distinct integration site preferences, with LVs showing a lower frequency in compartments associated with cancer-related genes and preferring introns and upstream regions, rather than promoters.

Fig. 3. LV integration sites near cancer genes and LV signatures are distinct from the gRV integrome.

Conclusion

This study found that lentiviral integration sites (VISs) in SCID-X1 patients and CAR T cells share similar patterns influenced by chromatin structure. LVs integrate into active chromatin regions, unlike γ-retroviruses (gRVs) which target promoters. The research highlights the importance of VIS patterns for optimizing gene therapy safety and efficacy.

Reference

1. Yan K K, Condori J, Ma Z, et al. Integrome signatures of lentiviral gene therapy for SCID-X1 patients. Science Advances, 2023, 9(40): eadg9959.