Lentiviral Integration Insertion Site Standards Expansion

Lentiviral vectors (LVs) are modified from the human immunodeficiency virus (HIV-1). They can deliver large amounts of genetic information into host cells and integrate into the cell genome, enabling stable expression for generations. Furthermore, because their virulence genes have been removed, they can be used as pseudotyped viruses in the laboratory with relative safety. Lentiviral vectors can transfect a variety of cell types, including difficult-to-transfect cells such as primary cells.

Application of Lentivirus in CAR-T Cell Therapy

Lentiviral virus is currently the mainstream transduction method for CAR-T (approximately 90% of FDA-approved CAR-T products use LV). Lentiviral integration in cells is semi-random (preferring transcriptionally active regions).

1.Lentiviruses recognize host surface receptors and transport RNA and proteins into cells;

2.Viral RNA is reverse transcribed to form linear double-stranded cDNA (containing LTR sequences at both ends)

3.Integrase recognizes LTR, and the cDNA is integrated into the host genome;

4.The exogenous gene continues to be expressed as the host cell divides.

Integration site analysis (ISA) is a critical step in the clinical development and post-marketing monitoring of gene therapy products (such as CAR-T). Regulatory requirements are established by national regulatory agencies (e.g., the FDA and EMA) and aim to assess the potential risks of genomic integration (e.g., insertional mutagenesis and clonal expansion).

Current ISA detection methods include LM-PCR, NGS, and single-cell sequencing. To ensure accuracy, CobiBio has developed a series of lentiviral integration site standards for use by testing institutions, serving as a quality benchmark for assessing detection capabilities. In the previous article, "Lentiviral Integration Site Standards Are Coming," we provided product descriptions and data presentation for monoclonal multiple insertion sites, monoclonal single insertion sites, and sex chromosome insertion sites. CB-Gene recently launched a lentiviral polyclonal multi-integration site reference standard with an integration frequency of 5% to 2%. This reference standard, derived from a mixture of multiple monoclonal integration site products, covers 20 insertion sites from 20 genes on 11 different chromosomes, as shown in the table below.

Figure 1. Gene Distribution

Figure 2. Chromosome Distribution