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March 2013 October 2011 NIH rDNA Guidelines (and numerous rDNA and IBC support documents) ◦ http://www4.od.nih.gov/oba/rdna Changes effective in March 2013 ◦ Scope expanded to include synthetic nucleic acids ◦ Title of Guidelines will change (…recombinant or synthetic nucleic acid molecules…) National Science Advisory Board for Biosecurity ◦ Dual use, synthetic biology, personnel reliability, biosecurity of select agents Scientifically responsive document PI is responsible for full compliance with the NIH Guidelines in the conduct of rDNA research Continue to evolve (current edition October 2011, next update March 2013) Guidelines = term/condition of funding from NIH! ◦ Consequences of non-compliance can be significant May not cover every experiment – call OBA for Guidance Scope and Applicability ◦ Practices for constructing and handling Recombinant and Synthetic Nucleic Acid Molecules Organisms and viruses containing recombinant and synthetic nucleic acid molecules ◦ Definition Constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell Molecules resulting from the replication of those described above Section I-C-1-a-(1). Research that is conducted at or sponsored by an institution that receives any support for research involving recombinant and synthetic nucleic acid molecules from NIH, including research performed directly by NIH. An individual who receives support for research involving recombinant DNA must be associated with or sponsored by an institution that assumes the responsibilities assigned in the NIH Guidelines. 5 Section I-D-1. All NIH-funded projects involving recombinant DNA techniques must comply with the NIH Guidelines. Noncompliance may result in: (i) suspension, limitation, or termination of financial assistance for the noncompliant NIH-funded research project and of NIH funds for other recombinant DNA research at the institution, or (ii) a requirement for prior NIH approval of any or all recombinant DNA projects at the institution. 6 Identify if Research is subject to the Guidelines and Identify the Guideline Section Propose an appropriate Containment Level (BSL) Obtain Yale Biological Safety Committee Approval before starting work subject to the NIH Guidelines Train lab staff in safe practices and emergency response procedures Alert staff of any special medical surveillance restrictions or immunizations Supervise and monitor lab staff for adherence to safety protocols Request authorization for significant updates of your rDNA experiments from the Yale Biological Safety Committee Report any significant problems, such as violations of the NIH Guidelines or any significant research related accidents, exposures and illnesses to: ◦ Yale Biological Safety Committee (203-7853550) ◦ Yale EHS Biosafety Office (203-785-3550) ◦ NIH Office of Biotechnology Activities (301496-9838 Risk Group 1 (RG1) Risk Group 2 (RG2) Agents that are not associated with disease in healthy adult humans Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available Risk Group 3 (RG3) Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk) Risk Group 4 (RG4) Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk) Classification of parent agent Toxins Antibiotic resistance genes Altered host range or tropism Replication competency Integration into host genome Interference with cell cycle Toxicity, allergenicity, other Level of Review Example of rDNA Experiment NIH Guideline Section IBC, RAC review and NIH Director Approval Experiments that compromise the control of disease agents in medicine through deliberate transfer or a drug resistance trait III-A IBC Approval and NIH review Deliberate formation of rDNA containing genes of a toxin with an LD50 < 100 ng/kg III-B IBC and IRB Approval and NIH review Introduction of rDNA into human subjects (Human gene transfer) III-C IBC approval before initiation Wide range (rDNA exp’s involving pathogens, defective vectors, animals, plants, large scale III-D IBC Notice at initiation Creating transgenic rodents, low risk rDNA Plant experiments III-E Exempt – registration not required Those do not represent a significant risk to health or the environment III-F 12 III-A-1: Deliberate transfer of a drug resistance trait that could compromise disease control III-B-1: Cloning of toxins with low LD50 (<100 ng/kg) rDNA experiments involving restricted agents ◦ DNA from restricted agents into host cells ◦ Experiments introducing DNA into restricted agents Experiments involving restricted poxviruses 13 III-C-1: Human gene transfer experiments Experiments not explicitly covered by the NIH rDNA Guidelines (NIH OBA establishes containment) Changes in containment level that varies from what has been specified in the NIH rDNA Guidelines Protocols that have been approved at another institution as a Major Action by the NIH ◦ i.e. Location B cannot approve the same work that has been approved at Location A by the NIH (w/out NIH permission) (will change March 2013) 14 Section III-D ◦ Use of Risk Group 2 or higher pathogens as host or vector ◦ Cloning DNA or RNA from Risk Group 2 pathogens ◦ Use of defective pathogen vectors with helper virus or packaging cells ◦ rDNA experiments involving whole animals (arthropods) ◦ rDNA experiments involving whole plants ◦ Large Scale (> 10 Liters) rDNA experiments ◦ Experiments involving high risk Influenza viruses Section III-E (Experiments that can be initiated at the time of registration) ◦ rDNA Experiments that involve the formation of rDNA molecules that contain less than 2/3’s of any Eukaryotic Virus ◦ Experiments involving whole plants (not covered under III-D) ◦ Creation of Transgenic animals Section III-F (Exempt rDNA Experiments) ◦ Appendix C Vector ◦ Risk Group of parent agent ◦ Defective/Replication incompetent Chance to recover missing portions Test for replication competency prior to use ◦ Percentage of the genome utilized ◦ Shedding of the vector in animal experiments ◦ Risks associated with exposure to vector components ◦ Integration into host cells or transient infection Inserted DNA ◦ Structural genes ◦ Cell cycle/regulatory genes ◦ Oncogenes Human, animal ◦ Toxins ◦ Genomes from other pathogens Chapter 28 Cell Lines: Biosafety and Viral Gene Transfer Vectors (Tom Kost, Patrick Condreay and Claudia Mickelson) ASM Biosafety: Principles and Practices, 4th Edition Fleming, D.F. and Hunt, D. Editors Risk of host cell line starting point Consider insert? ◦ ◦ ◦ ◦ ◦ Viral sequences Pathogenic function Virulence factors Transactivation of endogenous viruses Production of highly reactive bioactive molecules Growth factors Growth factor receptors Recombinant gene products Replication defective vectors ◦ Competent virus may arise in cultures – must monitor Host Cells ◦ Human or Non-human primate Primary or continuous Transformed Shedding human pathogens Oncogenic ◦ Other mammalian cells (rodent, dog, pig, etc.) ◦ Non mammalian cells (plant, insect, etc. Chapter 11 Cell Lines: Applications and Bioafety (Otto Doblhoff-Dier and Gwyn Stacey) ASM Biosafety: Principles and Practices, 4th Edition Fleming, D.F. and Hunt, D. Editors Yale EHS Office ◦ ◦ ◦ ◦ Biosafety Officers The Safety Advisor Assigned to your lab Back-up Safety Advisor(s) 203-785-3550 Yale EHS website ◦ www.yale.edu/ehs NIH Office of Biotechnology Activities ◦ Webpage http://oba.od.nih.gov/oba/index.html