Genome Titration
Recombinant adeno-associated virus has been widely applied to the field of gene therapy as one of the most promising gene vectors. The clinical starting dose and dose escalation studies of AAV vectors are the top priority for the commercialization of related products. Creative Biolabs has the world's leading vector technology service platform to provide customers with accurate AAV carrier quantitative services.
Introduction to rAAV
Recombinant AVV is a complex biological preparation developed from non-pathogenic virus in adenovirus preparation. It is composed of protein-like virus capsid and single-stranded DNA (ssDNA) vector genome, which produces replication defects by eliminating open reading frame. AAV is generally composed of a viral capsid protein and a ssDNA, and a specific gene can be introduced into a target cell. Its genetic structure is very flexible, and most of the genetic components can be adjusted according to customer needs. These AAV2-based ITR genomes can be packaged into excess natural or engineered AAV capsids, thereby establishing methods for producing high purity rAAV vector particles. The initially produced rAAV vector particles must undergo various quantification and quality assessments in order to continue to provide reliable data in subsequent experiments. Virus titration is one of the more commonly used evaluation methods and is designed to quantify the number of vector particles in a given volume.
Figure 1. Production of rAAV. (Zhang, 2016)
rAAV Genome Titration
For most experiments, the vector genome in rAAV preparation is the main active part. Therefore, most of the dose criteria for clinical use of AAV are determined by titration to detect the number of viral genomes (vg) or genomic particles (GC) in a given volume. Genomic titration is primarily determined by quantitative PCR (qPCR) or droplet digital PCR (ddPCR). Our technical service process is rough as follows:
- qPCR: We performed titration of the rAAV vector using validated qPCR to determine its infectious potency. Viral titers were quantified by comparison to a standard curve of known concentrations of plasmid samples. For AAV and adenovirus, physical titers were measured as genomic copy (GC) and viral particles (VP), respectively.
- ddPCR: ddPCR offers significant advantages over other qPCR methods, allowing absolute, but not relative, quantification of target DNA molecules present in the sample, which is critical for a range of analytical assays necessary for viral vector production and characterization. Creative Biolabs uses this technology to obtain more accurate analysis results for a variety of viral vectors.
If you have any questions about our vector titration service, you can contact us by email or send us an inquiry to find a complete solution.
Reference
- Zhang, C.; et al. (2016). Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery. Biomaterials. 80: 134-145.
