AAV8 Shedding Assay to Support Gene Therapy Clinical Trials

Gene therapy has already demonstrated its potential of bringing life-changing therapies to patients with devastating diseases and a growing number of clinical trials have been approved in recent years. One of the requirements in clinical development is to monitor vector shedding to control the potential environmental risk associated with the therapy. The assay read-out is qualitative, providing the sponsor and investigator with the relevant information about vector-shedding clearance after therapy. A sensitive and reliable method is needed in several different matrices, typically including blood, plasma, faeces, semen, urine and saliva. Viral shedding assays involve the extraction of viral DNA from patient secretions and excreta, the presence of which is then measured by selective and sensitive molecular methods (qPCR or ddPCR).

Here we present the development of a shedding method for the detection of a AAV8 vector in several matrices, including whole blood, plasma, faeces, semen, urine and saliva. The method development is split into 3 distinct phases: primer/probe design, PCR optimization and extraction. In first part of method development, we designed 5 sets or primers/probes. In the second phase we optimised the best qPCR conditions by comparing the five primers/probe sets and four master mixes to identify the combination, showing the best amplification efficiency and linearity and the lowest limit of quantification (LLOQ), limit of detection (LOD), and background noise. In the third phase, we compared seven commercially available DNA extraction kits and evaluated them based on the viral DNA recovery efficiency and, most importantly, the sensitivity, measured in genome copies per µl (or mg) of starting material.

While we chose a single combination of primer/probe set and master mix, we observed that different DNA extraction kits have to be used depending on the matrix of interest in order to achieve the best sensitivity which ranged from 0.6 to 7.4 GC/µl (mg) obtained from 50-200 µl (mg) of starting material.

In this presentation we share the unique requirements and challenges in developing vector shedding assays, based on the results of a streamlined AAV8 shedding assay method development. We are sharing an extensive comparison of most commercially available extraction kits. We conclude with our recommendation for validation parameters, acceptance criteria and results reporting to support clinical and preclinical gene therapy studies.