The Guidelines for Research Involving Recombinant DNA Molecules. Recombinant DNA Molecules

Transcription

1 The Guidelines for Research Involving Recombinant DNA Molecules Speaker : Dr. M-S M S Ho Jui-Yen Lin Reference The NSC Guidelines for Research Involving Recombinant DNA Molecules The NIH Guidelines for Research Involving Recombinant DNA Molecules html Biosafety in Microbiological and Biomedical Laboratories (BMBL) htm If your research using recombinant DNA (rdna) techniques, you should downloading the entire Guidelines for reference, and carefully study the sections applicable to your proposed work

2 Definition : Recombinant DNA Molecules In the context of the NSC and NIH Guidelines, recombinant DNA molecules are defined as either Molecules that are constructed outside living cells by joining natural or synthetic DNA molecules that can replicated in a living cell, or Molecules that result from the replication of those described above. Definition : Recombinant DNA Molecules (cont.) Synthetic DNA segments which are likely to yield a potentially harmful ploynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent in their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NSC Guidelines Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NSC Guidelines unless the transposon itself contains recombinant DNA.

3 Risk Assessment Risk implies the probability that harm, injury, or disease will occur. In the context of the microbiological and biomedical laboratories, the assessment of risk focuses primarily on the prevention of laboratory-associated associated infections. When addressing laboratory activities involving infectious or potentially infectious material, risk assessment is a critical and productive exercise. BioSafety levels: Facilities, equipment, and practices that can reduce the worker s and the environment s risk of exposure to an agent to an absolute minimum. Comprehensive risk assessment establishes a framework for selecting the appropriate BioSafety level. Risk Assessment (continue) The laboratory director or principal investigator is responsible for assessing risks in order to set the BioSafety level for the work. This should be done in close collaboration with the Biosafety Committee (and/or other Biosafety professionals as needed) to ensure compliance with established guidelines and regulations. The challenge of risk assessment lies in these cases where complete information on these factors is unavailable. A conservative approach is generally advisable when insufficient information forces subjective judgment. Universal precautions are always advisable.

4 Risk Assessment (continue) In performing a qualitative risk assessment, all the risk factors are first identified and explored. There may be related information available, such as the NSC recombinant DNA Guidelines, Biosafety in Microbiological and Biomedical Laboratories (BMBL), the NIH Recombinant DNA Guidelines, the Canadian Laboratory Biosafety Guidelines, or the WHO Biosafety Guidelines. In some cases, one must rely on other sources of information such as field data from subject matter experts. This information is interpreted for its tendency to raise or lower the risk of laboratory-acquired acquired infection. Factors to Consider in Risk Assessment Pathogenicity: The more severe the potentially acquired disease, the higher the risk. However, disease severity should be tempered by other factors Pathogenicity Route of Transmission: Agents with aerosol transmission potential have caused the most laboratory infections- The greater the aerosol potential, the greater the risk Agent Stability: The agent The agent s ability to survive over rime in the environment. Factors such as desiccation, exposure to sunlight or ultraviolet light, or exposure to chemical disinfectants must be considered. Infectious Dose: Infectious dose can vary from one to hundreds of thousands of units. The immune status of workers id directly related to susceptibility when working with an infectious agent Concentration: in most instances, the risk factors increase as the working volume of high titered microorganisms increases, since additional handling of the materials is often required. Concentration

5 Factors to Consider in Risk Assessment (cont.) Origin: may refer to geographic location (e.g., domestic or foreign); host (e.g., infected or uninfected human or animal); or nature of source (potential zoonotic or associated with a disease outbreak). This factor can also consider the potential of agents to endanger American livestock and poultry. Origin Treatment Availability: Most common prophylaxis is immunization. The use of immunizations is part of risk management Medical surveillance: part of risk management, and may include serum banking, employee health monitoring, and post exposure management Personnel Experience/Skill: additional education may be needed to ensure safety of persons working at each biosafety level Categories for Risk Assessment Materials containing known infectious agents: the characteristics of most infectious agents have been well characterized. Materials containing unknown infectious agents: often clinical specimens. The challenge is to establish the most appropriate BioSafety level.

6 Animal studies may present many different kinds of physical, environmental, and biological hazards. Specific hazards present in any particular animal facility are unique, varying according to the species involved and the nature of the research activity. The risk assessment for biological hazard should particularly focus on the animal facility s potential for increased exposure. Both to human pathogens and to zoonotic agents. Categories for Risk Assessment (cont.) Materials containing recombinant DNA molecules: includes microbes that have been genetically modified through recombinant DNA technologies. The nature of the genetic modification and the quantity of virus must be carefully considered when selecting the appropriate BioSafety level for work with a recombinant virus.

7 Comprehensive Risk Assessment In choosing appropriate containment for an experiment, the initial risk assessment from Appendix 2 of the NSC Guidelines. Classification of a thorough consideration of the agent itself and how it is to be manipulated. Factors to be considered in determining the level of containment include agent factors such as: virulence, pathogenicity, infectious dose, environmental stability, route of spread, communicability operations, quantity, availability of vaccine or treatment, and gene product effects such as toxicity, physiological activity, and allergenicity. Comprehensive Risk Assessment (cont.) Any strain that is known to be more hazardous than the parent (wild-type) strain should be considered for handling at a higher containment level. Certain attenuated strains or strains that have been demonstrated to have irreversibly lost known virulence factors may qualify for a reduction of the containment level compared to the Risk Group assigned to the parent strain.

8 Comprehensive Risk Assessment (cont.) A final assessment of risk based on these considerations is then used to set the appropriate containment conditions for the experiment (see Chapter 2-3 of the NSC Guidelines, or Section II-B of the NIH Guidelines, Containment). The containment level required may be equivalent to the Risk Group classification of the agent or it may be raised or lowered as a result of the above considerations. Comprehensive Risk Assessment (cont.) The Biosafety Committee must approve the risk assessment and BioSafety containment level for recombinant DNA experiments described in the following chapters of the NSC Guidelines: All Biological related experiments require Biosafety Committee approval before initiation; Chapter 3 Section 8 and 10: Experiments that require Biosafety Committee approve and related Departments of the Executive Yuan approval before initiation.

9 Comprehensive Risk Assessment (cont.) Careful consideration should be given to the types of manipulation planned for some Risk Group (RG) agents. RG2 dengue viruses may be cultured under the Biosafety Level (BL / P) 2 containment, however, when used for animal inoculation or transmission studies, a higher containment level is recommended. RG3 agents such as Mycobacterium tuberculosis and SARS associated coronaviruses should be handled at a higher containment level for animal inoculation and transmission experiments. Comprehensive Risk Assessment (cont.) P2 (BL2) containment is recommended for activities involving all blood-contaminated clinical specimens, body fluids, and tissues from all humans, or from HIV- or HBV- infected or inoculated laboratory animals. Activities such as the production of research- laboratory scale quantities of HIV or other bloodborne pathogens, manipulating concentrated virus preparations, or conducting procedures that may produce droplets or aerosols, are performed in a P2 facility using the additional practices and containment equipment recommended for P3 (BL3). Activities involving industrial scale volumes or preparations of concentrated HIV are conducted in a P3 facility.

10 Comprehensive Risk Assessment (cont.) Exotic plant pathogens and animal pathogens of domestic livestock and poultry are restricited and may require special laboratory design, operation and containment features not addressed in Biosafety in Microbiological and Biomedical Laboratories. ( Laboratory personnels are not to be mixed with those working on human pathogens. Information regarding the importation, possession, or use of these restricted agents see Chapter 5 and 6 of the NSC Guidelines.(or Sections V-G and V-H, footnotes and references of Sections I through IV of the NIH Guidelines. Classification Rick Group (RG) Risk group 1 (RG1) agents are not associated with disease in healthy adult humans. Risk Group 2 (RG2) agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. Risk Group 3 (RG3) agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

11 Criteria for Risk Groups Classification of agents in appendix 2 of the NSC Guidelines, Classification of Human Etiologic Agents on the Basis of Hazard, is based on the potential effect of a biological agent on a healthy human adult and does not account for instances in which an individual may have increased susceptibility to such agents, e.g., preexisting diseases, medications, compromised immunity, pregnancy or breast feeding (which may increase exposure of agents to some agents). Personnel may need periodic medical surveillance to ascertain fitness to perform certain activities; they may also need to be offered prophylactic vaccines and boosters. Biosafety Levels The CDC has developed four BioSafety levels (BL/P 1-4) which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and the laboratory function or activity. The laboratory director is specifically and primarily responsible for assessing the risks and appropriately applying the recommended BioSafety levels.

12 Biosafety level 1 (BL1/ P1) Suitable for working with well-characterized agents not known to be human pathogens, and of minimal potential hazard to lab personnel and the environment. may be conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Personnel are trained in specific procedures conducted in the lab and are supervised by a scientist with general training in microbiology or a related science. Standard Microbiological Practices (BL1/P1) Access limited at discretion of lab director during agent use Wash hands before leaving lab No eating, drinking or smoking, no contact lenses No mouth pipetting Procedures designed to minimize splashes and aerosols Decon work surfaces at least once a day, or more if needed Insect & rodent control program in effect Cultures, stocks, regulated waste decontaminated prior to disposal by an approved method Durable leak-proof containers needed for transfer out of lab Comply with local & government regulations before removal from facility for decontamination Biohazard sign posted at entrance to lab during infectious agent use Agent name Contact information for investigator

13 Laboratory Biosafety Level 1 (BL1/P1) Safety Equipment-Primary Barriers Special containment devices not usually required Lab coats, gowns, uniforms recommended to prevent contamination or soiling of street clothes Gloves recommended if skin is broken or rash exists Protective eyewear should be worn for anticipated splashes of hazardous material to the face Laboratory facilities-secondary Barriers Hand-washing sink in lab Lab easily cleaned- no rugs or carpets Impervious benchtops Sturdy lab furniture, space easily cleaned windows that can open, screens required should have doors for access control Typical P1 Laboratory Door that can be closed to keep visitors out while work with agents in progress. Hazard warning signs may be posted on the door indicating ing hazards that may be present, including radioactive materials, laser lights, high noise emitting equipment, or toxic chemicals. Hand-washing sink available, preferably near the door. Waste materials segregated according to hazard type, with an appropriate chemical decon tray for collecting contaminated implements. Work is done on the open bench and plastic backed absorbent pads can be placed on the work surface to collect splatter or droplets. Bench tops should be sturdy, and impervious to acid. If there are windows in the lab, they should be fitted with screens.

14 Biosafety Level 2 (BL2/P2) Practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate-risk risk agents that are present in the community and associated with human disease of varying severity. Hepatitis B, Salmonella sp., and HIV are representative microbes assigned to the P2 containment level. Laboratory Biosafety Level 2 (BL2/P2) P2 is similar to P1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from P1 in that: Laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists Access limited to the laboratory when work is being conducted Extreme precautions are taken with contaminated sharp items Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment

15 Standard Microbiological Practices (BL2/P2) No eating, drinking, cosmetics application, or contact lenses in work areas Hand-washing prior to leaving lab Mouth pipetting prohibited Sharps policies in place Minimize aerosol and splash generation Decon at end of day, or if splashes/spoillsspoills occur Proper decon of cultures, stocks, regulated wastes by approved methods Insect and rodent control program in effect Special Practices (BL2/P2) Evaluates at risk persons, ie. immuno-compromised persons, etcimmunizations and/or serum banking may be appropriate Personnel receive training on specific hazards, exposure precautions (PI responsibility) Only personnel informed of the hazard are admitted Biohazard sign required during infectious operations Biosafety manual prepared with SOPs and specific proceduressharps precautions instituted Cultures, tissues, specimens, infectious wastes contained in leak proof containers for handling or transport Equipment and work surfaces decontaminated on a routine basis Spills and/or accidents reported to lab director ASAP Animals not involved in work not permitted in lab

16 Laboratory Biosafety Level 2 (BL2/P2) Safety Equipment (Primary Barriers) Properly maintained biological safety cabinets, personal protective equipment, or physical containment devices are used whenever: Potential for infectious aerosols exists High concentrations or large volumes of infectious agent are used Face/eye protection provided if splashes/sprays possible outside BSC Lab coats, gowns, uniforms are worn in laboratory protective clothing must stay in lab and be cleaned/laundered by institution Gloves used when contact with infectious materials possible Gloves replaced when overtly contaminated Disposable gloves not re-used Alternatives to powdered latex gloves available Hands washed after removal of gloves Laboratory Biosafety Level 2 (BL2/P2) Laboratory Facilities (Secondary Barriers) Lockable doors for facilities that house restricted agents Facility contains hand-washing sink (foot, knee, or auto operated sink recommended) Laboratory easily cleaned rugs or carpets inappropriate Bench tops impervious to water and resistant to decontamination chemicals Lab furniture sturdy enough to support anticipated loading and uses Biological safety cabinets (BSC) not installed so room air fluctuations uations affect safe operation Eyewash station readily available Illumination adequate for operations planned Negative, or inward flowing room air recommended, though not required.

17 Typical P2 Laboratory Some work may be done on the open bench by persons wearing appropriate protective clothing or gear. Any work that may produce splatters or aerosols of infectious materials should be done inside a biological safety cabinet (BSC) or other containment device, such as aerosol- containing centrifuge cups. Waste materials need to be segregated into chemical, radioactive, bio-hazardous, or general waste streams. Infectious waste should be decontaminated (by treating with chemical disinfectants or by steam autoclaving). Biosafety Level 3 (BL3/P3) Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route, Laboratory personnel must have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

18 Typical P3 Laboratory Double-door entry (shown here as an ante-room; other configurations are also used). Because the agents manipulated at P3 are transmissible by the aerosol route, particular attention is given to air movement. Air moves from areas of lesser contamination to areas of higher contamination, and is single pass exhaust air not recirculated to other rooms. Exhaust air should have HEPA filtered, and out to less populated area. All work that may create aerosols or splatter is done inside a biological safety cabinet. Wall ceiling and floor penetrations are sealed to keep aerosols and gaseous decontaminants in, Biosafety Level 4 (BL4/P4) Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk if aerosol-transmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.

19 Biosafety Manual Biosafety procedures outlined Special hazards identified, and mitigation strategies outlined Written practices and procedures for the specific operations using etiologic agents outlined SOPs The laboratory director/pi is responsible for ensuring that the biosafety manual is comprehensive Laboratory Biosafety Level Sign Signs like these three are available at the Office of Academic Affairs. (TEL: )

20 Examples of a biohazard sign. Signs like this one are available at the Office of Academic Affairs. (TEL: ) Biohazard Sign Animal Biosafety Levels If experimental animals are used, institutional management must provide facilities, staff, and established practices that reasonably ensure appropriate levels of environmental quality, safety, and care. As a general principle, the BioSafety level (facilities, practices, and operational requirements) recommended for working with infectious agents in vivo and in vitro are comparable.

21 Containment-Laboratory Practices and Techniques Personnel must: Practice strict adherence to standard microbiological procedures (the most important element of containment) Be trained and proficient in practices and techniques for working with such material safely Each lab should develop or adopt a BioSafety or operations manual that: Identifies potential hazards Specifies practices designed to minimize or eliminate hazards A Scientist trained and knowledgeable in appropriate techniques must: Be responsible for the conduct of work with infectious agents or materials Consult with experts with regard to risk assessment Safety Equipment-Primary Barriers Safety wquipment includes biological safety cabinets (BSCs), enclosed containers, and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of anticipated or unanticipated infectious splashes or aerosols generated by many microbiological procedures. Three General types of Biological Safety Cabinets are available Class I, II, and III. P1, 2 and 3 work can be performed in Class I and II cabinets under proper conditions

22 Class I Biological Safety Cabinet The Class I Biological Safety Cabinet is a negative-pressure, ventilated cabinet usually operated with an open front and a minimum face velocity at the air from the cabinet is exhausted through a HEPA filter either into the laboratory or to the outside. It moderate risk agents. HEPA filters High Efficiency particulate Air (HEPA) Removes particulates from the air Removes at least 99.97% of particles at 0.3 microns in diameter at rated air flow capacity (has greater efficiency against larger and smaller particles) Mechanisms of action Inertial impact Interception (electrostatic) Diffusion Tested with DOP (dioctylphthalate) Alternatives to DOP: Kaydol mineral oil, Emery 3004, Ondina EL mineral oil.

23 Class I Biological Safety Cabinet Open front Negative pressure 75 linear feet per minute (lfpm) HEPA filtered exhaust (into room or outside) Not appropriate for materials vulnerable to airborne contamination Limited manufacture (Class II BSCs replacing) Class II Biological Safety Cabinet The Class II Biological Safety Cabinet (BSC) is designed with inward air flow at a velocity to protect personnel ( lfpm). HEPA-filtered downward vertical laminar airflow for product protection, and HEPA-filtered exhaust air for environmental protection. Class II BSCs are classified into two types (A and B) based on construction, airflow velocities and patterns, and exhaust systems. Type A cabinets are suitable for microbiological research in the absence of volatile or toxic chemicals and radionuclides, since air is recirculated within the cabinet. Type A cabinets may be exhausted into the laboratory or to the outdoors via a thimble connection to the building exhaust system. Type B cabinets are further sub-typed into types B1, B2, and B3. Type B cabinets are hard-ducted ducted to the building exhaust system and contain negative pressure plan. Theese features, plus a face velocity of 100 lfpm, allow work to be done with toxic chemicals or radiomuclides.

24 Class II Type A Biological Safety Cabinet Recirculated air Suitable for microbiological research in the absence of volatile or toxic chemicals and radionuclides.

25 Class II Type B Biological Safety Cabinet One pass air allows work to be done with toxic chemicals or radionuclides.

26 Biological Safety Cabinets It is imperative that Class I and II biological safety cabinets be tested and certified in situ at the time of installation within the laboratory, at any time the BSC is moved, and at least annually thereafter. Certification at locations other than the final site may attest to the performance capability of the individual cabinet ot model but does not supersede the critical certification prior to use in the laboratory. Decontamination / Sterilization There are a number of methods for accomplishing decontamination / sterilization of various infectious agents. It is incumbent upon the principal investigator to know which method, or combination of methods is effective against the agent in question. The method (s) selected for a specific agent should be accurately documented in the laboratory biosafety manual. Some widely used decontamination / sterilization strategies for infectious agents are listed below: Steam autoclaving Ethylene Oxide Liquid disinfectants UV radiation Formaldehyde Peracetic acid Hydrogen peroside

27 Recombinant DNA Containment Effective biological safety programs have been operative in a variety of laboratories for many years. Existing programs rely upon mechanisms that can be divided into two categories: A set of standard practices (SOP) that are generally used in microbiological laboratories barriers that are applied in varying degrees according to the estimated biohazard. Hardware design: Special procedures, equipment, and laboratory installations that provide physical barriers that are applied in varying degrees according to the estimated biohazard. Recombinant DNA Containment (continued) Four biosafety levels are described in Chapter 2 of the NSC Guidelines, Physical Containment. These biosafety levels consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed, and are based on the potential hazards imposed by the agents used and for the laboratory function and activity.

28 Recombinant DNA Containment (continued) Experiments involving recombinant DNA lend themselves to a third containment mechanism, namely, the application of highly specific biological barriers. Natural barriers exist that limit either: The infectivity of a vector or vehicle (plasmid or virus) for specific hosts. Or Its dissemination and survival in the environment. Vectors, which provide the means for recombinant DNA and/or host cell replication, can be genetically designed to decrease, by many orders of magnitude, the probability of dissemination of recombinant DNA outside the laboratory. Recombinant DNA Plant Containment For research with plants, the facilities and procedures provide a modified and protected environment for the propagation of plants and microorganisms associated with plants and containment that adequately controls the potential for release of biologically viable plants, plant parts, and microorganisms associated with them.

29 Recombinant DNA Animal Containment For research with animals that have size or growth requirements precluding conventional primary lab animal containment systems, containment is designed to eliminate possibility of sexual transmission of modified genome or transmission of recombinant DNA-derived viruses known to be transmitted from animal parent to offspring only by sexual reproduction. Containment used for transgenic animals associated with recombinant DNA-derived organisms is designed to eliminate the possibility of horizontal transmission of the agent. vertical transmission occurs only under controlled situation.