Assessing the environmental fate of rAAV in activated sludge and water: Implications for environmental risk assessments and GMO regulatory frameworks.

During the past +20 years, recombinant adeno-associated virus (rAAV) vectors have emerged as the primary vehicle of choice for in vivo gene therapy. rAAV vectors are classified as genetically modified organisms (GMOs), therefore specific biosafety laws apply regarding their use. Environmental agencies participating in the review of clinical trial applications involving viral-based gene therapies (eg based on AAV) focus among other phenomena especially on shedding, a mechanism by which rAAV vectors exit a patient's body and enter the natural environment. For example, following patient use, shed viral particles excreted in urine and feces enter the wastewater treatment facility (WWTF) and subsequently may be released into the natural environment through wastewater effluent discharges. Based on basic molecular biology, it is generally assumed by the scientific community that shed rAAV particles will undergo degradation during the wastewater treatment process. However, despite their importance and increase in use during the last few decades, actual data to support our understanding of the environmental fate of shed rAAV vector particles is unavailable. Data to support this assumption would greatly enhance our knowledge and understanding of degradation kinetics associated with rAAV in the environment. Such data would also provide strong scientific support for changes in current legislation regarding the medicinal use of GMOs. Therefore, the goal of this research was to conduct laboratory experiments to assess the actual environmental fate of rAAV virions. In this study the stability of 4 different rAAV vectors (based on wildtype (wt) AAV serotypes 2,3,6,9) was assessed during incubation in activated sludge (containing live microorganisms). This setting corresponds to conditions as encountered in WWTFs, and has been used in order to assess rAAV fate under environmentally relevant conditions, to gain a better understanding of the general environmental risk posed by shed rAAV particles. The amount of detectable virions in the supernatant, as measured by sensitive and specific qPCR, rapidly decreased within hours and continued to decline, reaching the lower limit of quantitation prior to or by study termination on day 7. Furthermore, a half-life of approximately 7 days for rAAV virions was determined under abiotic conditions, during a room temperature incubation experiment of rAAV vectors in water in the absence of any microbiota or sludge. The findings from this study provide the first insight of its kind into the actual environmental fate of shed rAAV particles, and help the community to better understand the potential impact of rAAVs on the environment. It has become evident now that shed particles are not equipped to remain stable and/or soluble once entering a typical WWTF and therefore do not pose a threat to the natural environment. These findings support a data-driven approach towards a simplified, risk-based regulation of medicinal GMOs in the EU and other regions.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.