Infectious Titration

Although recombinant adeno-associated virus (rAAV) vectors are currently gaining popularity in clinical trials related to gene therapy, there is still a need to develop appropriate quality control methods for accurate vector characterization. At Creative Biolabs's vector service platform, titration of infectious particles is critical to determine the delivery efficacy. We have developed different methods to measure rAAV infectivity in vitro based on the detection of vector genome replication in adenovirus-infected trans-complementing cells and detection of transgene expression in cells.

Introduction of rAAV

AAVs were first discovered when observing adenoviruses by electron microscopy and were considered as contaminants in adenovirus preparations. It quickly attracted the attention of researchers after being discovered and was used as a vector in various gene therapy experiments. AAV belongs to the parvovirus family, specifically the dependoparvovirus subfamily. The members of this subfamily require a helper virus, such as the adenovirus (Ad) or herpes simplex virus (HSV), to facilitate productive infection and replication. Although an estimated 90% of the population is AAV-positive, these viruses do not cause known human diseases, which is an important safety standard for their use in gene therapy.

Clinical trials using rAAV vectors have shown impressive results for Leber congenital amaurosis, hemophilia B, spinal muscular atrophy, and other diseases. Commercial products based on rAAV were first approved by European medical institutions for the treatment of lipoprotein lipase deficiency in 2012. Subsequently, other drugs have been also performed well in clinical trials and are expected to be listed. Nonetheless, one of the barriers to the commercialization of these drugs is how to produce rAAV on a large scale according to production practices (cGMPs), and the production of rAAV in human cells (HEK293) by transient transfection with plasmids is probably the most common method. Given that quality attributes of rAAV stocks could be different depending on the manufacturing platform, it is important, as FDA emphasizes, to accurately analyze the characterization. Among the quality attributes, infection titer is crucial to ensure the efficacy of the product.

Figure 1. Recombinant adeno-associated virus.

rAAV Infectious Titration

AAV infection does not cause cytopathic effects, therefore, plaque detection cannot be used to determine infectious titers; however, in the presence of helper virus, replication of the AAV genome can be induced, and its infection efficiency can be tested. Infectious titers are typically quantified by cell transduction assays. Wild-type AAV2 has been reported to have a near-perfect physical-to-infectious particle ratio of 1:1. However, for recombinant AAV2, the same study reported a physical-to-infectious particle ratio of 50:1. The specific infectivity of viral preparations is defined by the ratio of physical viral particles to infectious viral particles. Creative Biolabs has developed some solutions for titrating infective AAV vector particles for our customers through long-term research accumulation, including but not limited to:

• Infectious AAV Particle Titration by the TCID50 assay. TCID50 is the tissue culture infectious dose, defined as the virus dilution required to infect 50% of the cell monolayer. There are several statistical methods that can be used to analyze generated data (such as Probit formula or Spearman-Karber analysis). TCID50 is of greatest value when experimental material is lacking or the cost of analysis excludes extensive repeated titer determination, but a good estimate of titer and treatment difference is necessary.
• Infectious AAV Particle Titration by the ICA. Creative Biolabs uses the Center of Infection (ICA) as a complement and alternative to the standard AAV quantification method with higher sensitivity, reproducibility and shorter cycle times. The AAV vector ICA assay based on serotype discrimination can assess the relative potency of a particular formulation, but does not predict in vivo performance, especially when comparing different AAV serotypes.

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.