Safety

General Lab Safety

Before we started our project, all of the team members had received lab safety training, including waste disposal, information on hazardous chemicals, proper use of biosafety cabinets. Team members working in specific labs went through orientations with mentors or managers detailing proper equipment usage and safety before gaining access to that lab. The risks to team members were minimized by wearing appropriate PPEs in the lab all the time and following lab safety regulations of Tsinghua University. Molecular cloning was carried out in BL1 lab, while experiments related to AAV vectors, production of adeno-associated virus and cell transfection and transduction were conducted in BL2 lab. The E. Coli we used to amplify vectors was lab safe strain DH5α. Hazardous chemicals such as ethidium bromide were carefully used according to protocols.

Risk assessment of Adeno-associated virus for medical use

Adeno-associated virus (AAV) vectors have been extensively studied for gene therapy. They are favorably chosen by researchers not only because their ability to infect both dividing and non dividing cells with persistent expression, but also the safety concerns. These vectors can insert DNA fragment at a specific site on chromosome 19 with nearly 100% certainty. A study published by Nowrouzi et al. measuring how often insertion to other chromosomes occurs showed it was a rare event with 1 in 10,000 cells. For patients who are injected with gene-therapy viruses, the chance of fail to locate insertion to chromosome 19 and interruption of other gene’s expression would be extremely low.

The serotype AAV2 in our study was isolated from humans and lacks apparent pathogenicity. In fact, as most people carry this virus, it’s safer that other viral delivery systems such as retrovirus, and normally would not cause disease. However, concerning the potential risks raised from the nature of gene therapy, medical use of this virus with modified vector should be restricted and closely regulated.

Risk assessment of Adeno-associated virus on researchers’ safety

We used Stratagene’s AAV Helper-Free System, in which genes required for the production of infective AAV particles, including the AAV-2 ITR sequences and rep/cap genes, are supplied on the plasmid pHelper, thus the vectors along are noninfectious and have minimal risks to researchers. The helper plasmids lack homology, and prevent production of recombinant wild type virus. However, considering the risk of infection by virus particles and introducing extra insulin gene into researchers’ body, all the experiments involving virus were carried out with precautions and following strict regulations. Disposable pipets or pipettes with filter tips were used to prevent the transfer of contaminated aerosols. Cell transfection and transduction were carried out in a laminar flow hood designated for use with virus in BL2 lab. All items that may be contaminated by virus were disposed in separate waste bag, immediately sealed, and autoclaved. Cells used in virus related experiments were cultured in separate incubators. In addition, insulin gene was replaced by fluorescence proteins when testing vectors in mammalian cells to minimize the danger to researchers.

Risk assessment of Adeno-associated virus on environment

Potential danger to environment is minimal, as this virus has no viability outside living organisms and has been deprived of its ability to reproduce.