shRNA

Biosafety

 
MISSION shRNA

How serious are lentiviral biosafety concerns? Is there a realistic risk of HIV infection?
Viral vector systems have been developed with enhanced safety features. It is recommended to use a third generation lentiviral vector system. It is a three plasmid system consisting of:
  1. The packaging vector, which contains the minimal set of lentiviral genes required to generate the virion structural proteins and packaging functions.
  2. The vesicular stomatitis virus G-protein (pCMV-VSV-G) envelope vector, which provides the heterologous envelope for pseudotyping, and
  3. The shRNA transfer vector, which contains the sequence of interest as well as the cis acting sequences necessary for RNA production and packaging. The multi-plasmid approach results in no single plasmid containing all the genes necessary to produce packaged lentivirus.

 

Resulting particles are replication-incompetent and deletion in the U3 portion of the 3’ LTR eliminates the promoter-enhancer region, further negating the possibility of viral replication. The system has also removed virulence genes which are not necessary for shRNA packaging. These features combined have improved biosafety and handling. It is recommended to use a third generation lentiviral system for its enhanced biosafety features. Though there are no known incidents of third generation systems producing replication competent virus, it is important to monitor for replication competency when performing routine lentiviral packaging. NIH guidelines recommend replication-incompetent lentiviral particles be handled as Risk Group-Level 2 (RGL2). Additional precautions may be required based upon local, state, or country regulations.


What are some successful approaches designed to minimize issues related to large scale manufacturability of viral vectors and its impact on environmental risk and regulatory risk?
One safety question regarding the large-scale manufacturability of viral vectors is the generation of replication-competent helper viruses during the packaging of the vector, which can occur by homologous recombination. Good laboratory practices are necessary to prevent any forms of contamination that may result in an opportunity for a replication-competent virus to form. Replication-competent virus testing is necessary. Because viral structural genes have been placed on different genetic units, multiple recombination events must occur before a replication competent helper virus is generated. Furthermore, areas of homology among the units expressing the helper virus proteins have been minimized. Also, heterologous promoters for the helper virus proteins are used. CDC-directed guidelines for BSL-2 + practices should dictate the handling of large amounts of virus and of concentrated virus.


Can the MISSION lentiviral particles be further propagated in the lab?
No, the viral particles cannot be propagated for biosafety reasons. The MISSION TRC lentiviral particles are replication incompetent. The particles are made using features of 2nd and 3rd generation lentiviral packaging systems. Genes for replication and structural proteins are absent in the packaged viral genome since these genes are supplied by other plasmids in the packaging cells. The viral genome contains only the region between the 5’ and 3’ LTRs of pLKO.1. In addition, the lentiviral vector contains a self-inactivating 3’ LTR that renders it unable to produce infectious virus once it integrates into the host chromosome.


Is pLKO.1 vector a HIV-based vector and if so are there any bio-safety issues?
The pLKO.1 vector is a lentiviral (HIV)-based plasmid. The vector is regarded as a bio-safety level 2 material and safe to use due to its modified features (deletion of a number of accessory genes implicated in the virulence of HIV, minimal genome of the viral particles, non-replicating and self-inactivation features), making it incapable of producing virus once infected into the host cell. Please consult with your institution’s biosafety officer on specific requirements.


Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.

MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.

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