Titration of rAAV Genome Copy Number

Creative Biolabs's gene therapy platform provides a range of viral vectors services including the detection of recombinant adeno-associated virus (rAAV) titration. Our scientific researchers have rich experience and advanced technical equipment in the detection of virus concentration and titer, which enables us to accurately judge the ability of virus vectors to infect cells. So, titration services of AAV are of great significance for the reliability of subsequent experiments and the efficiency of product development.

Introduction of rAAV

rAAV is one of the most promising gene delivery vectors in the development of gene therapy. Its safety, site-specificity and wide range of infections are considerable advantages. To date, rAAV vectors have been evaluated in clinical studies such as Parkinson's disease, cystic fibrosis and hemophilia B. The treatment of lipoprotein lipase deficiency by liposome tiparvovec (AAV1-LPLS447X) is currently the only commercially available rAAV-based commercial gene therapy drug in humans. The basic premise for planning and applying rAAV vectors in clinical studies is the quantification of recombinant viral particles. The best titration analysis must be fast, reliable and easy to operate.

Currently, most assays are based on the ability of viral transduction to culture cells. After transduction, intracellular replication of the recombinant viral genome or expression of an easily detectable transgene is measured. Other assays are independent of the infectivity of the virus. They rely on the cleavage of core units and the release of single-stranded DNA. Serial dilutions of dot blot DNA are compared to plasmid probes and then the number of particles is calculated. The assays can be influenced by various parameters and are time-consuming procedures. Creative Biolabs quickly and reliably quantifies rAAV particles by real-time PCR, which is a powerful tool for customers to standardize rAAV formulations in clinical studies.

Recombinant adeno-associated virus. Figure 1. Recombinant adeno-associated virus.

Titration of rAAV Genome Copy Number

  • Production and purification of rAAV vector. We can provide customers with expanded production of specific rAAV products, or design your vectors from scratch. Our molecular tool library has a large selection of promoters and labels that can be used to customize your viral vectors in AAV clones.
  • Determination of infectious virus particles by fluorescence activated cell sorting (FACS). We use FACS to distinguish infectious particles from purified AAV preparations from inactivated viruses with shorter detection times and higher separation efficiency.
  • CMV promoter-specific quantitative real-time PCR (qPCR). We have developed qPCR to determine the potency of genomic rAAV using the Light-Cycler technique. Since the CMV promoter is the most commonly used promoter in gene therapy methods, primers that hybridize to the human CMV promoter sequence are designed. The PCR product is detected by adding SYBR green.

In addition to simplifying titration and standardization of rAAV formulations for clinical large-scale use, Creative Biolabs can also quantitatively monitor the rAAV genome present in human tissues treated with rAAV 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.

For research use only. Not intended for any clinical use.