Viral shedding assays presents unique challenges. The analytes released are typically found in very low quantities, and the matrices involved in shedding assays often contain significant levels of PCR inhibitory agents and endogenous DNA.
The FDA requires a shedding assay to assess the risk of transmission to individuals in close contact with subjects receiving gene therapy treatments. This assay monitors whether products are shed and, if so, whether they are capable of causing infection.
Viruses such as AAV and Ad are not replication-competent; therefore, shedding is expected to be extremely low. This necessitates a PCR assay capable of quantifying very low copy numbers of the test item.
Highly sensitive assays
At TATAA Biocenter, we develop, optimize, qualify, validate, and run assays tailored explicitly for each viral shedding study, including studies on oncolytic viruses. With over 20 years of experience in extraction and validation, we always include spikes to determine recovery and efficiency. Our validations adhere to COU or regulatory requirements, including GLP and GCLP.
At TATAA Biocenter, we primarily use digital PCR for shedding studies because of its increased sensitivity, ability for absolute quantification, and reduced susceptibility to PCR inhibition. PCR is the recommended method for detecting shed products in gene therapy assessments. PCR identifies fragments of the viral genome; however, this detection does not confirm the presence of the entire genome or determine its infectious potential. Thus, while PCR provides a necessary initial screening for genetic material from shed products and further evaluates the infectious potency, particularly of replication-competent products, a cell-based assay is employed.
Despite AAV viruses having their replication-responsible rep and cap genes depleted, the shedding of viral particles still poses an infection risk to individuals in close contact with the treated patient. These genes are supplied separately on helper vectors during AAV production, since the vector itself cannot replicate independently. Regulatory agencies mandate shedding studies because of the potential scenario where a patient sheds a virus that has regained the ability to infect. The risk associated with shedding is significantly higher with replication-competent viruses than with replication-incompetent ones.
Shedding is most likely to occur shortly after product administration. Replication-competent products may show a second peak of shedding days or weeks after administration due to the product’s multiplication within the body.
To monitor the immune system’s effect on the drug before and after dosing, we employ Olink’s Target 48 Cytokine panel. This panel offers absolute quantification of key cytokines at the protein level using only microliters of plasma or blood from either mice or humans, helping to understand how the immune system has been primed by initial and subsequent doses.
Assay design
Our primers and probes are designed to quantify the test item without detecting background DNA. Whenever possible, at least one of the primers targets non-natural sequences, such as vector backbones, non-natural exon junctions, promoter regions, endogenous genes, or codon-optimized sequences.
To monitor the recovery of the target, we spike the sample before extraction, and to monitor the interference in the PCR, we spike the PCR reaction. This is to ensure the highest standards of accuracy.
Assay validation
Each assay is validated according to regulatory requirements, which include a comprehensive description of performance parameters such as sensitivity and reproducibility.
Whether GLP/GCLP is required or not, as a GLP-accredited laboratory, we conduct assays in a GLP-like manner. This means we handle samples using the same processes, control the work environment according to GLP standards (including instrument service programs, temperature control, staff training, IT security, and more), and ensure that sample handling is time-stamped to maintain the integrity of both the samples and the data.
