Lentiviral vectors (LVs) are efficient vehicles for stable gene transfer in both dividing and non-dividing cells, which makes them a powerful tool in molecular research. However, the use of lentiviruses in research and clinical trials requires precise and validated titration methods. Creative Biolabs has developed a variety of lentiviral titer platforms and proprietary technologies. With in-depth expertise in this field, we provide lentiviral vectors titration service with robust methods.
LVs are efficient vehicles for stable gene transfer in both dividing and non-dividing cells, such as neurons or hepatocytes, and allow long-term expression of transgenes and target specific cell types via simple pseudotyping processes. Based on these advantages, LVs have often been used to deliver genes of interest into mammalian cells in molecular research and preclinical/clinical trials. However, in order to apply lentiviral methods in research and clinical studies, it firstly requires an assessment of titers. Viral titer determination is an essential quality assessment for lentivirus-based basic research and clinical application, especially the accuracy of this assessment is of the utmost importance for therapeutic dosing of patients. To date, lentiviral titration can be determined by two general categories: nonfunctional (physical titer) and functional (infectious titer) methods.
Figure 1. Overview of lentiviral vector titration methods. The non-functional titration methods are carried out on concentrated vector after production and the functional titration methods are determined after transduction on cells. (Farhangi, 2019)
Nonfunctional titration methods include detection of expressed virus proteins (such as p24) or their transcripts using enzyme-linked immunosorbent assay (ELISA), assessment of the reverse transcriptase (RT) activity and determination of the genomic RNA concentration in vector preparations by semi-quantitative northern blotting, dot blot analysis or real-time quantitative polymerase chain reaction (RT-qPCR). LVs are traditionally produced by transient transfection of human embryonic kidney (293T) cells with high concentrations of different plasmids. In general, the nonfunctional titration methods are carried out on the concentrated vector after production.
For clinical studies and other gene therapy applications, it is essential to accurately determine the functional titers of LVs. Functional titer, or infectious titer, is defined as the number of viral particles per certain volume required to transduce a single cell. Functional titration methods permit detection of integrated proviral DNA, transgene mRNA or protein expression levels in target cells. Titration methods based on protein expression are typically based on vector-encoded reporter gene expression or staining with an antibody against the transgene. For instance, vectors expressing the fluorescent reporter gene (GFP, luciferase) can be titrated by fluorescence-activated cell sorting (FACS) analysis or immunofluorescence staining with specific antibodies against protein reporters. For vectors that do not contain a reporter gene, the titers can be determined by the integration of proviral or transgene copies into host cell genomic DNA using quantitative PCR (qPCR). To determine the integrated virus for each cell, standard curves must be prepared with DNA materials that contain a known amount of PCR targets. So far, qPCR-based methods have been recognized as the gold standard for lentivirus infection titers.
Creative Biolabs is a leading commercial supplier of viral vector technologies with state-of-the-art technology and versatile platforms for LVs titration. We provide robust and strong titration methods based on your specific application, such as FACS analysis for your clinical studies and other gene therapy applications. Our robust and optimized processes will save you time and reduce variability in your research. In addition to the titer service, many other customizable options are already available at Creative Biolabs and we will continue to consider your requests to bring you more flexibility.
Along with providing high-quality LVs titration service, Creative Biolabs offers its expertise and support to clients in areas such as LVs project management, scientific design, and project follow-up. If you have any question about our lentivirus services or don’t see the service you’re looking for, please feel free to contact us.
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