1. UNIVERSITY OF COLORADO COLORADO SPRINGS Recombinant and Synthetic Nucleic Acid Molecules (rsNA) Training University of Colorado Colorado Springs Environmental Health and Safety www.uccs.edu/~pusafety/environmental/index.shtml

2. Introduction Recombinant and Synthetic Nucleic Acid Molecule Training This training is designed to familiarize the Principal Investigator (PI) and lab personnel with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). NIH Guidelines are the standards for containment and safe research practices involving recombinant or synthetic nucleic acid molecule (rsNA) activities. When finished, please complete the rsNA Quiz and e-mail it to cnorton@uccs.edu. cnorton@uccs.edu

3. Introduction Links have been provided to direct you to specific Sections of these guidelines or to reference materials that can provide a more detailed description or explanation of the topic discussed. Principal Investigators are responsible for knowing the NIH Guidelines and training lab personnel as it pertains to your research. Recombinant and Synthetic Nucleic Acid Molecule Training

4. Purpose of NIH Guidelines The purpose of the NIH Guidelines is to specify practices for constructing and handling: i.recombinant nucleic acid molecules ii.Synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules iii.cells, organisms and viruses containing such molecules. Divided into 5 sections (I-V) and fifteen Appendices (A-Q) Recombinant and Synthetic Nucleic Acid Molecule Training

5. Definition of Recombinant and Synthetic Nucleic Acid Molecules In the context of the NIH Guidelines, recombinant and synthetic nucleic acids (rsNA) molecules are defined as: (i) molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a living cell, i.e. recombinant nucleic acids; (ii) Nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids, or (iii) Molecules that result from the replication of those described in (i) or (ii) above. Recombinant and Synthetic Nucleic Acid Molecule Training

6. What are the NIH Guidelines ? Created in 1976 Outlines principles for the safe conduct of research employing rsNA Details practices and procedures for containment of various forms of rsNA research safe conduct of research involving genetically modified plants and animals, and safe conduct of human gene transfer research. Periodically revised and may be found at http://oba.od.nih.gov/rsNA/nih_guidelines_oba.html From NIH Office of Biotechnology Activities (NIH-OBA ) Recombinant and Synthetic Nucleic Acid Molecule Training

7. What is the NIH Office of Biotechnology Activities (NIH-OBA)? The NIH Office of Biotechnology Activities (NIH-OBA) promotes science, safety, and ethics in biotechnology through the advancement of knowledge, enhancement of public understanding, and development of sound public policies. A core responsibility of NIH-OBA is to foster awareness of, and adherence to, the standards and practices set forth in the NIH Guidelines. Recombinant and Synthetic Nucleic Acid Molecule Training What is the NIH Office of Biotechnology Activities (NIH-OBA)?

8. NIH Recombinant DNA Advisory Committee (RAC) Federal advisory committee providing advice and recommendations to the NIH Director regarding rsNA research Unique public forum for the discussion of science, safety, and ethics of rsNA research Reviews and analyzes unique clinical gene transfer protocols and safety information Observations and findings of general importance to the field Not an approved process Recombinant and Synthetic Nucleic Acid Molecule Training

9. What is an Institutional Biosafety Committee (IBC)? Provide local review and oversight of nearly all forms of research utilizing rsNA Reviews and approves all research subject to the NIH Guidelines. Approved applications are valid for 3 years. Ensures that rsNA research conducted at or sponsored by the institution is in compliance with the NIH Guidelines. Recombinant and Synthetic Nucleic Acid Molecule Training

10. University of Colorado Colorado Springs IBC Reviews all UCCS activities involving biological agents or materials including educational and research laboratories, and field research Reviews and approves research that is conducted by UCCS faculty and students at other institutions and non-CU entities. Ensures that employees, students, the public and the environment are protected from biohazards associated with UCCS operations. Meets at least annually, more frequently as needed Recombinant and Synthetic Nucleic Acid Molecule Training

11. Who Must Comply with the NIH Guidelines All institutions that receive NIH funding for rsNA research must comply with the NIH Guidelines. Researchers at institutions that are subject to the NIH Guidelines must comply with the requirements even if their individual projects are not funded by NIH. Recombinant and Synthetic Nucleic Acid Molecule Training

12. What are Potential Consequences of Noncompliance with the NIH Guidelines for NIH and Non-NIH Funded Projects? For NIH and Non-NIH funded projects involving rsNA techniques: suspension, limitation, or termination of NIH funds for the noncompliant NIH-funded research project and of NIH funds for other rsNA research at the institution, or that of the Principal Investigator, or a requirement for prior NIH approval of any or all rsNA projects at the institution.

13. Recombinant and Synthetic Nucleic Acid Molecule Training What are Potential Consequences of Noncompliance with the NIH Guidelines for Non-NIH Funded Projects? For Non-NIH funded projects involving rsNA techniques conducted at or sponsored by an institution that receives NIH funds for projects involving such techniques: suspension, limitation, or termination of NIH funds for rsNA research at the institution, or that of the Principal Investigator, or a requirement for prior NIH approval of any or all rsNA projects at the institution.

14. Risk Assessment and Risk Groups (RG) NIH Guidelines require that the PI make an initial assessment of risk based on the Risk Group (RG) of an agent (see Appendix B of the Guidelines, Classification of Human Etiologic Agents on the Basis of Hazard). Appendix B is based on the World Health Organization (WHO) Risk Classification. Recombinant and Synthetic Nucleic Acid Molecule Training

15. Basis for the Classification of Biohazardous Agents by Risk Group (RG) Risk Group 1 (RG1) Agents that are not associated with disease in healthy adult humans Risk Group 2 (RG2) Agents that are associated with human disease which is rarely serious and for which preventative 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) Recombinant and Synthetic Nucleic Acid Molecule Training

16. Containment Requirements The Biosafety Officer (BSO) and the IBC will review the agent itself and the proposed experiment procedures to determine the appropriate level of containment. The levels of containment or Biosafety Levels are described in Appendix G of the Guidelines http://oba.od.nih.gov/oba/rac/guidelines_02/APPENDIX_G.htm and are also found in the Centers for Disease Control and Prevention (CDC) guideline, Biosafety in Microbiological and Biomedical Laboratories, 5 th ed http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm Recombinant and Synthetic Nucleic Acid Molecule Training

17. Section III-A Require IBC Approval, RAC Review, and NIH Director Approval Before Initiation Section III-B Require NIH/OBA and IBC Approval Before Initiation Section III-C Require IBC and Institutional Review Board Approvals and RAC Review Before Research Participant Enrollment Section III-D Require IBC Approval Before Initiation Section III-E Require IBC Approval Simultaneous with Initiation Section III-F Exempt Experiments Recombinant and Synthetic Nucleic Acid Molecule Training

18. Section III-A The deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the ability to control disease in humans, veterinary medicine or agriculture, will be reviewed by the RAC. Consideration should be given as to whether the drug resistance trait to be used in the experiment would render that microorganism resistant to the primary drug available to and/or indicatd for certain populations, for example children or pregnant women. These experiments require IBC approval, RAC Review, and NIH Director approval before initiation and is considered a Major Action. Recombinant and Synthetic Nucleic Acid Molecule Training

19. Section III-B Experiments Involving the Cloning of Toxin Molecules with LD 50 of Less than 100 Nanograms per Kilogram Body Weight Require NIH-OBA and IBC approval before initiation (may require CDC Select Agent registration if exempt quantities are not used). Recombinant and Synthetic Nucleic Acid Molecule Training Section III-C Experiments Involving the Deliberate Transfer of rsNA, or DNA or RNA Derived from rsNA, into One or More Human Research Participants UCCS is not conducting in-vivo human gene therapy.

20. Section III–D There are 7 subcategories for Section III-D: Section III-D-1Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems (see Section II-A, Risk Assessment) Section III-D-2Experiments in which DNA from Risk Group 2, Risk Group 3, Risk Group 4 or restricted agents is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems Recombinant and Synthetic Nucleic Acid Molecule Training Experiments in this category require IBC approval before initiation

21. Section III–D Recombinant and Synthetic Nucleic Acid Molecule Training Section III-D-3 Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems Section III-D-4 Experiments involving whole animals in which the animal’s genome has been altered by the stable introduction of rsNA, or nucleic derived therefrom, into the germ line (transgenic animals), and experiments involving viable rsNA-modified microorganisms tested on whole animals

22. There are 7 subcategories for Section III-D, continued: Recombinant and Synthetic Nucleic Acid Molecule Training Section III-D-5 Experiments to genetically engineer plants by rsNA methods, to use such plants for other experimental purposes (e.g., response to stress), to propagate such plants, or to use plants together with microorganisms or insects containing rsNA Section III-D-6 Experiments involving more than 10 liters of culture. The appropriate containment will be determined by the IBC Section III-D-7 Experiments involving influenza viruses.

23. Section III–E Recombinant and Synthetic Nucleic Acid Molecule Training This category includes experiments that are not included in Sections III-A through III-D, III-F and their subcategories are considered in Section III-E. These experiments may be conducted at BL-1 containment. These experiments require IBC approval simultaneous with initiation.

24. There are 3 subcategories for Section III–E Recombinant and Synthetic Nucleic Acid Molecule Training Section III-E-1 Experiments involving the formation of rsNA molecules containing no more than two-thirds of the genome of any eukaryotic virus. Section III-E-2 This section covers experiments involving nucleic acid molecule-modified whole plants, and/or experiments involving rsNA-modified organisms associated with whole plants, except those experiments that fall under Section III-A, III-B, III-D or III-F.

25. There are 3 subcategories for Section III-E, Recombinant and Synthetic Nucleic Acid Molecule Training Section III-E-3 Experiments involving the generation of rodents in which the animal's genome has been altered by stable introduction of rsNA, or DNA derived therefrom, into the germ-line (transgenic rodents). Only experiments that require BL1 containment are covered under this section; experiments that require BL2, BL3, or BL4 containment are covered under Section III-D-4, Experiments Involving Whole Animals.

26. Section III–F Recombinant and Synthetic Nucleic Acid Molecule Training Experiments involving rsNA molecules included in this category are exempt from these Guidelines. The University of Colorado requires that an IBC application be submitted for review and approval. There are 6 subcategories for Section III-F. These experiments require IBC approval simultaneous with initiation.

27. Other Institutional Committees and the IBC Remember that if you submit a protocol to the IACUC or IRB and it involves rsNA techniques or other biological materials, you must also submit an IBC application for review and approval of this work. Recombinant and Synthetic Nucleic Acid Molecule Training IRB IBC IACUC

28. IBC Responsibilities Requirement Reference Adequate IBC Training Section IV-B-1-h Annual Report To NIH Section IV-B-2-a-(3) No Conflict of Interest Section IV-B-2-a-(4) Write Meeting Minutes Section IV-B-2-a-(7) Recombinant and Synthetic Nucleic Acid Molecule Training

29. IBC Responsibilities Assignment Reference Assess Lab Facilities Section IV-B-2-b-(1) Notify PI of Approval Section IV-B-2-b-(2) Set Containment Level Section IV-B-2-b-(3-4) Monitoring ProjectsSection IV-B-2-b-(5) Establish Emergency Plans Section IV-B-2-b-(6) Notify NIH-OBA & UCB of Section IV-B-2-b-(7) Accidents, Illnesses, & NIH Violations within 30 days Ensure NIH reviewSection IV-B-2-b-(8)

30. Biosafety Officer (BSO) Responsibilities Assignment Reference Conduct Inspections Section IV-B-3-c-(1) Notify IBC & UCCS of Section IV-B-3-c-(2) Accidents, Illnesses, & NIH Violations Investigate Accidents Section IV-B-3-c-(3) Establish Emergency Plans Section IV-B-3-c-(3) Advise on Lab Security Section IV-B-3-c-(4) Provide Technical Advice Section IV-B-3-c-(5) Recombinant and Synthetic Nucleic Acid Molecule Training

31. Principal Investigator Responsibilities Responsible for full compliance with the NIH Guidelines during the conduct of rsNA research. The PI should: Be adequately trained in good microbiological techniques. Provide laboratory research staff with protocols describing potential biohazards and necessary precautions. Instruct, train and supervise laboratory staff in: (i) the practices and techniques required to ensure safety, and (ii) the procedures for dealing with accidents. Inform the laboratory staff of the reasons and provisions for any occupational health/medical surveillance (e.g., vaccinations or restricted work duties). Recombinant and Synthetic Nucleic Acid Molecule Training

32. Principal Investigator Responsibilities Correct work errors and conditions that may result in the release of recombinant or synthetic nucleic acid molecule materials. Ensure the integrity of physical containment (e.g., biological safety cabinets) and biological containment (e.g., purity and genotypic and phenotypic characteristics). Comply with permit and shipping requirements for recombinant or synthetic nucleic acid molecule materials. Adhere to IBC-approved emergency plans for handling accidental spills and personnel contamination. Recombinant and Synthetic Nucleic Acid Molecule Training

33. Principal Investigator Responsibilities Before initiating research subject to the NIH Guidelines, the PI must: Determine whether the research is subject to Section III-A, III- B, III-C, III-D, III-E, or III-F of the NIH Guidelines. Propose physical and biological containment levels in accordance with the NIH Guidelines when registering research with the IBC. Propose appropriate microbiological practices and laboratory techniques to be used for the research. Submit an IBC application to the IBC for review and approval. Recombinant and Synthetic Nucleic Acid Molecule Training

34. Principal Investigator Responsibilities While conducting research subject to the NIH Guidelines, the PI must: Determine the need for IBC review before modifying rsNA research already approved by the IBC. Submit any subsequent changes (e.g., changes in the source of DNA or host-vector system) to the IBC for review and approval or disapproval. Remain in communication with the IBC throughout the duration of the project. Report any significant problems pertaining to the operation and implementation of containment practices and procedures, violations of the NIH Guidelines, or any significant research- related accidents (e.g. needlestick) and illnesses to the Biological Safety Officer, the IBC, NIH-OBA, Animal Facility Director (where applicable), and other appropriate authorities within 30 days. Recombinant and Synthetic Nucleic Acid Molecule Training

35. Appendix B CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD Includes those biological agents known to infect humans and selected animal agents that may pose theoretical risks if inoculated into humans. Includes lists of representative genera and species known to be pathogenic Mutated, recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are excluded. Recombinant and Synthetic Nucleic Acid Molecule Training

36. Appendix G PHYSICAL CONTAINMENT Specifies physical containment for standard laboratory experiments and defines Biosafety Levels, consistent with the CDC Guideline, Biosafety in Microbiological and Biomedical Laboratories. Use the following appendices to determine containment: For large-scale (over 10 liters) research or production, Appendix K shall supersede Appendix G. For certain work with plants, Appendix P shall supersede Appendix G. For certain work with animals, Appendix Q supersedes Appendix G. Recombinant and Synthetic Nucleic Acid Molecule Training

37. Appendix K LARGE SCALE CONSIDERATIONS Specifies physical containment guidelines for large-scale (greater than 10 liters of culture produced at one time) research or production involving viable organisms containing rsNA as specified in Section III-D-6, Experiments Involving More than 10 Liters of Culture. Four biosafety levels of containment are –BL1-Large Scale (LS) –BL2-LS –BL3-LS –Good Large Scale Practice (GLSP)

38. Recombinant and Synthetic Nucleic Acid Molecule Training Appendix Q PHYSICAL AND BIOLOGICAL CONTAINMENT FOR rsNA RESEARCH INVOLVING ANIMALS Specifies containment and confinement practices for research involving whole animals Four levels of containment are established: –BL1-Animals (N) –BL2-N –BL3-N –BL4-N.

39. WHAT DOES ALL OF THIS MEAN TO ME? Apply To The IBC Recombinant and Synthetic Nucleic Acid Molecule Training If you are conducting any work with rsNA molecules, then you must complete an IBC application, regardless of your source of funding and even if it meets the “Exempt” classification of the Guidelines. The University reviews alI lab, field, and class activities involving biological agents and materials. Currently, UCCS does not allow possession or use of Risk Group 3 or 4 agents Currently, UCCS does not allow possession or use of Select Agents or Toxins that would require registration with the CDC or USDA.

40. IBC Application Research Description The “Research Description” is one of the most important parts of the IBC Application because it helps the IBC assess the appropriate level of containment to safely work with the agents and materials listed. It’s important that you include details about how each biological agent or material will be used, e.g. experiment procedure(s), agent strain(s), etc. Recombinant and Synthetic Nucleic Acid Molecule Training

41. IBC Application Which Section(s) of the Guidelines does my research fall under? Based on the agent used, e.g. adenovirus 5, determine what risk group it is by referring to Appendix B of the Guidelines. In our example of adenovirus, adenovirus is a Risk Group 2 organism. Once the Risk Group of the agent is known, determine the type of experiment described in Section III of the NIH Guidelines (e.g. Section III-D-1, the use of Risk Group 2 agents as part of a host vector system). Recombinant and Synthetic Nucleic Acid Molecule Training

42. IBC Application Which level of containment is required? Based on the agent used, e.g. adenovirus 5, and the experiment type (in our example, Section III-D-1), determine the level of containment or biosafety level you propose for this experiment by reviewing Appendix G. Usually a RG2 agent will be used at Biosafety Level 2 but depending on the procedures, the biosafety level may be lower or higher. The IBC will review the Research Description narrative to review the procedures performed and make the final determination of the Biosafety Level. Recombinant and Synthetic Nucleic Acid Molecule Training

43. Importance of IBC and BSO to Investigators The IBC and Biosafety Officer can help you to: –Ensure that you are working with rsNA safely –Meet all compliance requirements associated with NIH funding for research involving recombinant or synthetic nucleic acid moleculres –Avoid preventable accidents and incidents that might cause harm or undermine public confidence in your research activities –Obtain biosafety advice on an ongoing basis Recombinant and Synthetic Nucleic Acid Molecule Training

44. QUESTIONS? Contact Information Cynthia Norton Institutional Biosafety Officer Department of Environmental Health and Safety cnorton@uccs.edu (719)255-3212 Recombinant and Synthetic Nucleic Acid Molecule Training

45. THANK YOU You are now ready to take the required rsNA Quiz