Most of the lentiviral vectors (LVs) used for therapeutic applications belong to a subclass of the Retroviridae family, the genus Lentivirus. They are approximately 80–100 nm in diameter, and their genome consists of two copies of positive-sense single-stranded RNA inside a conical capsid, which is surrounded by a lipid bilayer. As a popular gene delivery tool, LVs are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptors (CAR) in CAR-T cell therapies.

 

Researchers have made improvements to the vectors to improve biosafety and transgene expression,  which have led to their approval for clinical research. A combination of methods is required to analyze the quality of lentiviral vector production, the efficiency of gene transfer, and the extent of therapeutic gene expression. Typically, virus quantification methods aim to determine either the total physical viral particle (VP) titer or the infectious virus particle titer, given in transducing units (TU) per mL. The infectious titer is more meaningful as it measures the number of virus particles that can infect target cells. HEK293T cells are typically used as target cells for LV infectious titer determination.

 

In the following content, we will introduce some commonly used lentivirus titer detection methods to assess the quality of the virus and maintain experimental consistency.

 

qPCR to Measure Lentiviral Titers

The most common assays for the total viral particle (VP) titer are direct viral RNA via qPCR. Direct measurement of lentiviral RNA with qPCR is a relatively quick method, providing results in less than a day, and is usually less expensive than an ELISA kit. Some qPCR kits currently on the market provide a fast and simple method for measuring lentivirus titer. In this approach, viral RNA is first converted to cDNA and then quantified using qPCR primers targeting specific viral components such as LTRs, gag, WPRE, antibiotic resistance-genes, or the transgene itself.

 

Many researchers prefer to design primers that target the common features of viral vector backbones; once validated, these universal primers can be used to titer any lentiviral prep that shares that specific feature. Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used in the transfection; if preparing several different lentiviruses in parallel, this approach should be considered for validation.

 

Case 1. Lentiviral Titering via qPCR

The studies revealed that lentiviral titer can be easily assessed with the use of qPCR based on the SYBR Green-based detection system. Moreover, the method was rapid and reproducible.

 

Figure 1. Quantitative and qualitative assessment of WPRE fragment relative to single copy gene, albumin [1]. A) standard curve for gene copies for albumin (right) and WPRE (left). Crossing point—a cycle in which the fluorescence level rises above the baseline; B) typical quantitative PCR analysis of reference gene (right) and WPRE (left); C) typical qualitative qPCR analysis based on melting temperature of amplicons: WPRE (left) and single copy gene, albumin (right).

Calculation Schema: Lentiviral copy number per cell = (copy number WPRE/copy number Alb) × 2, Titer (TU/ml) = (Primary number of cells count in day 1 × lentiviral copy number per cell of the sample)/volume of used lentivirus (ml), and qPCR titer = 1.8 × 106 ± 2, 1 × 105

 

Fluorescence-Activated Cell Sorting (FACS) to Measure Lentiviral Titers

 

FACS (Fluorescence-Activated Cell Sorting) provides a method for sorting a mixed population of cells into two or more groups, one cell at a time, based on the specific light scattering and fluorescence of each cell. This method provides fast, objective, and quantitative recording of fluorescent signals from individual cells. FACS can determine the functional titer of lentiviral particles.

 

Case 2. Lentiviral Titer via Flow Cytometry - CD19 Antigen

The lentiviral purification solution expressing the CD19 antigen was diluted in four gradients (0.01, 0.1, 1, 10 μL) and added to the same number of 293T cells separately. After 72 hours, the positive rate of 293T cells was detected by flow cytometry. The percentage of virus-infected 293T cells to the total number of cells), and then calculate the transduction titer of the lentivirus according to the following formula:

A: The percentage of positive cells in the 0.1 μl group.   

B: The percentage of positive cells in the 0.01 μl group.   

N: Number of cells when the virus is added   

V: Volume of virus added in group A

 

Table 1. The lentivirus titer measurement results of flow cytometry detection of CD19 antigen (source: Cyagen)

The number of CD19 antigen-positive cells gradually increases with the increase of the transfection virus gradient, indicating that the lentivirus has infectious activity. The calculated virus titer is 1.4×108 TU/mL.

 

Selection of Lentiviral Vector Titration Methods

For lentivirus vectors lacking a fluorescent marker, or for those carrying promoters that may not be functional in 293T cells, titer can be determined either by real-time polymerase chain reaction (rtPCR) quantification of viral genomes in genomic DNA from transduced cells or a p24 ELISA-based assay.

For lentivirus vectors encoding fluorescent proteins under the control of promoters functional in these cells, titration can be performed using the limiting dilution method or a flow cytometry-based method.

 

Why Choose Cyagen

Cyagen can provide lentivirus (LV), adeno-associated virus (AAV), adenovirus (ADV) packaging services. We ensure the reliability of virus packaging quality from the aspects of virus titer, purity and activity, and may provide these alongside the construction of various cell models, in vivo injection of living animals, pharmacodynamic evaluations, and more. Our services have been used by biomedical researchers across many disciplines and cited in top international journals, including Science and Nature. References and reports in the literature help promote the smooth development of the experiment.