gU)4MARY OF THE PROPOSED GUIDELINES FOR RRSEARGH ON RECOHJUNANT DNA MOLECULES . f. INTRODUGTION pp/-3 1. Robert Sinsheimer: In the advent of a new biological research, the research process is changing from one of analysis to one of synthesis. The complications are great. The process is an irreversible one. Vectors will escape and there will be no control. We will not be able to. stop production as we could with DDT. 2, Daniel Gallahan: The underlying question is how to establish a proper policy bias. There is no moral obligation to do this research although it certainly is commendable but there is a moral obligation to do no harm; and given the uncertainty of the hazards, one should incline to caution. Does the burden of proof lie with the scientist ko'show no harm or with the public to show that there is harm? The benefit of the doubt should be to the "worriers" and not the researchers. This should be the policy bias, and the guidelines should be reviewed accordingly. With this in mind, the guidelines seem to be reasonable and prudent in the conduct of this research. A. Definition of Experiments Included Within the Guidelines The proposed guidelines concern experiments in which different segments of DNA chains are joined by biochemical techniques and the resulting recombined DNA molecules are then inserted into living cells in which the molecules can be reproduced. "hosts." The recipient cells are called the In general, one segment of the recombined DNA molecule will be derived from an extra-chromosomal genetic element common to the "host" species. That segment of DNA is referred to as the "vector." The other segment of the recombined DNA is referred to as "foreign" DNA, since it is not normally chemically linked to the vector. "Host" cells will usually be either single-celled microorganisms, such as bacteria, or single cells of higher organisms (plants and animals) grown under special laboratory conditions. In nature, exchange and recombination of genetic information (that is, DNA segments)w a consequence of the sexual mode of reproduction and generally occurs only between individiial organisms of the same species. The unique feature of recombinant DNA'experiments is that they provide a means for combining genes from diverse species. This unique feature promises revolutionary potential both for the investigation of basic biological processes and for approaches to important practical problems in medicine and agriculture. The same unique feature is the basis for concern over the potential biological hazards that might result from such novel genetic.recombinants.- B. Principles Upon Which the Guidelines Are Based The guidelines are consistent with conclusions formulated at the International Conference on Recombinant DNA Molecules held at Asilomar Conference Center, Pacific Grove, California, in February 1975. 1. Certain experiments are judged to present such serious potential hazard that they should not be attempted at this time. ,% k&- 2. Other experiments can be undertaken provided that thevlafford new knmr benefits not readily obtained by konventional methodology and provided that appropriate safeguards are incor- porated into the experimental design. Such safeguards should protect laboratory workers, the community, and the environment from possible infection with potentially hazardous agents. The proposed barriers to dissemination of the agents are twofold: '(1) physical containment of organisms containing recombinant DNA; and (2) biological containment, that is the use of "hosts" and "vectors" with limited ability to survive in natural environments. The two types of barriers are complementary and are to be used in combination. ,lHrv & e@ 3. Recognizing that present relevant knowledge is limited,/,the various types of experiments m with regard to expected degree of potential hazard. The level of containment achieved by combined physical and biological barriers should be chosen to match the estimated potential hazard. Containment should be high at the start and should be modified subsequently only if there is a substantiated change in the assessed risks. 4. The guidelines should be reviewed at least annually and modified to reflect new knowledge. II. METHODS OF CONTAINMENT- 1. David Baltimore: He asks whether the proposed guidelines are an appropriate response to the hazard posed by recombinant DNA molecules and whether the process that led to the formulation of guidelines was a responsible one. He notes that recombination of DNA molecules has gone on for aeons; he urges the focus must be on those DNAs that are a potential hazard, not focus on the mere joining of DNA molecules. The hazard is defined as the potentiality of artificially joined DNA molecules escaping from the laboratory and disseminating in the general population. He believes the guidelines are appropriate to meet that hazard. He urges that the guidelines be promulgated as quickly as possible. He cites the scientific interest and the medical justification for these experiments to go on. 2. Robert Sinsheimer:. Too little attention has been given to hazards that might result from new combinations of genes that shall be created in this research. Research Increases the opportunities for potentially dangerous gene combinations by many, many orders of magnitude that might occur sporadically in nature. In this regard, biological containment may not be adequate. The key element of concern is the irreversibility of the process that will become magnified as more investigators in laboratories pursue research in this area. Rather than going the global approach, the guidelines might be better served by approaching each desired objective in as specific and safe a manner as possible. A. and B. Physical Containment 1. Peter Barton Hutt: The concept of physical containment is both too imprecise and'too subject to the vagaries of human fallibilities. Thus physical containment can only be applied when risk is non-existent. When there is any potential risk, protection must rest with biological containment. Consideration of the Pl through P3 levels should be undertaken. Perhaps Pl and P2 should be combined into a single level of physical containment. Another possibility is to upgrade some of the experiments to a P3 or P4 level that are currently under Pl and P2 levels. The intended requirements for Pl through P3 levels should be spelled out. Perhaps Dr. Barkley who raised several points in his presentation could review this section for additional comments. Description of acceptable practices should be as clear as possible and might well include stated minimum levels of scientific training and experience necessary to conduct the experiments. 1.- Philip Handler: What does physical containment mean? Are Pl and P2 levels really physical containment? Should we not consider P3 as the minimum level incorporating Pl and P2? This research must go forward but the pace must be set by Dr. Fredrickson. Four levels are defined and designated Pl, P2, P3, and P4 in order of increasing containment. Each higher level assumes the practices of the lower ones. The specifications reflect existing technology as developed for work with known pathogenic organisms. It is anticipated that technical developments will lead to novel alternate procedures for achieving physical containment. Pl (minimal): Strict adherence to the standard microbiological practices widely used in-research and clinical laboratories for work with moderately pathogenic organisms. Appropriate training of all personnel is required. P2 (low): Access to the laboratory is limited to authorized and informed personnel when potentially hazardous organisms ark being used and.until after appropriate decontamination. Pipetting by mouth is prohibited, and specific precautions are required for procedures expected to release aerosols containing potentially hazardous material. o P3 (moderate): Laboratories.are separated from areas used for less hazardous experiments and access is limited to those who work therein. Laboratories should have air pressure lower than that of the surrounding areas to limit the flow of airborne organisms into surrounding areas. Exhaust air from laboratories should be appropriately. discharged or decontaminated prior to recirculation. Biological safety cabinets,shouid be used for all transfer operations and for all procedures likely to produce aerosols. Gloves are to be worn and all vacuum lines protected by filters. Alternate procedures, affording at least equivalent physical containnwnt, are suggested for situations in which laboratory air conditions cannot be controlled in the specified manner. > o P4 (high): Special facilities designed to contain highly infectious and hazardous microorganisms are required. Such facilities involve isolation by means of airlocks, negative pressure environments, clothing changes and showers by personnel, biological safety cabinets, and decontamination of all air as well as all liquid and solid wastes. Only a limited number of such facilities exist in the United States. c. Biological Containment The nature of these barriers will depend on the particular "host" and "vector" used in each experiment since the barrier is defined by the limited ability of the "host" and its resident recombined DNA to survive in natural environments. In general, the choice of "host" and "vector" will be determined both by the nature of the experiment and by the required containment. Therefore, experiments are grouped below according to possible "host-vector" systems. D. Publication The committee strongly recommends-that all publications dealing with recombinant DNA work include a description of the physical and biological containment procedures used. III. EXPERIMENTAL GUIDEIINES pp )3-./2, A. Experiments That Should Not Be Performed at This Time 1. Any experiments in which a portion of the DNA to be joined is derived from highly pathogenic organisms (classes 3, 4, and 5 of the "Classification of Etiologic Agents on the Basis of Hazard," Center for Disease Control, USPHS, Atlanta, Georgia). 2. Experiments in which a portion of the DNA to be joined contains genes for production of highly toxic agents. 3. Experiments in which a portion of the DNA to be joined is derived from a plant pathogen if the resulting host may acquire increased virulence or the ability to infect previously unsusceptible species. 4. Widespread or uncontrolled release into the environment of any organism containing a recombinant DNA molecule unless a series of controlled tests leave no reasonable doubt of safety. 1. LeRoy Walters: The guidelines are least clear in -defining prohibition on experiments where antibiotic resistance may occur, Further clarification is necessary. 5. Transfer of genes conferring drug resistance to micro- organisms not known to acquire such resistance naturally when such resistance may compromise clinical use of the drug in medicine or agriculture. 6. Large-scale experiments with recombinant DNAs known to result in the formation of harmful products. Exceptions are permissible for specific experiments of direct societal benefit, provided that they are expressly approved by the NIH Recombinant DNA Molecule Program Advisory Committee. B. Guidelines for Permissible.Experiments 5. 6. 1. Wallace Rowe: The risks are real. Tests are necessary to determine the risks including a disturbance in the E. coli, the introduc- tion of DNA from E. cells and the dispersion of.toxic products from the E. ~011. Roy Curtiss: He outlines possible tests for the risks as the probability of escape, the probability of survival in the outside environment; and the probability of the manifestation of adverse consequences. In this regard we are not only talking about the protection of man but the protection of the entire biosphere. Philip Handler: The NIH should undertake to develop a program of study of the most important hazardous experiments This should be done at P4 facilities at the NIH or Fort Detrick. p' These experiments would provide scientific evidence to defuse the debate on the potential hazards. Guidelines can be modified accordingly based on that evidence. 1. Criteria for use of Escher&& coi!i, strain K-12 as "host" 2. Robert Sinsheimer: Experiments should be done with micro- organisms that have no association with man or other animals. These organisms should have a very restricted range of genetic exchange,at a very limited biological niche.of viability. 1. Paul Berg: Because we are-so familiar with the metabolism and genetic systems of E. coli, it provides the best opportunity to achieve the benefits in this research. Further, it's the most likely candidate for providing the safetest host and vector systems. Developing an entirely new host-vector system will not answer questions of risk. The ominous and catastrophic scenarios raised concerning E. coli could equally be raised with any known organism. ii .i 3. Philip Leder: He has constructed an EK2 host-vector system and it has been recently announced in the nucleic acid recombinant scientific memorana. Testing of this system is currently underway. He expects to submit it shortly to the Advisory Committee for certification. Peter Barton Hutt: What would be the cost in not using E. coli? Can incentives be created to foster the development of IX2 and EK3 systems. Should there be a 2-year limit on EKl to force development of EK2 and EK3? If EKl is to be used, there must be as detailed an explanation as possible of that system and its use. The cornnon bacterium Escherichia boi!i, strain K-12, is the "host" of choice at the-present time: Well-studied extrachromosomal DNA molecules known to reside and reproduce in this "host" are avail- able for use as Vectors." These include plasmids and bacterio- phages. Extensive knowledge of Escerichia coli and its plasmids and bacteriophages currently affords the most fruitful approach to the design of containable "host-vector" systems. . Three containment classes for 6. coli strain K-12 "host-vector" systems are defined. The classes are named EK (for E. cozi K-12) 1, 2, and 3 in order of decreasing ability to survive in natural environments (that is, increasing containment). EKl: This class includes most of the currently known and available E. coti K-12 "host-vector? systems. Present knowledge suggests a low probability for dissemination of plasmid or bacteriophage "vectors" carrying recombined "foreign" DNA. This conclusion is based in part on the following information: (1) E. coZi K-12 is not known to colonize the normal bowel; (2) Certain plakmids known to be useful as "vectors" have only limited ability to be transferred to other E. coZi strains commonly found in animal (including human) bowels and sewerage; (3) Bacteriophages which specifically infect E. cozi, and vhich lend themselves well to use in these experiments, are available. Some of these bacteriophages have limited ability either to escape the "host" cell or to reinfect common (not K-12) E. coZi strains. 1. Biological containment criteria using E. coli K-12 host-vectors EKl -host vectors - These are host-vector systems that can be esti- mated to already provide a moderate level of containment, and include most of the presently available systems. The host is always k. coli K-12, and the vectors include nonconjugative plasmids [e.g., pSC101, ColEl or derivatives thereof (17-24)] and variants of bacteriophage x (25-27). The g. coZi K-12 nonconiugative p Zasmid sys tern is taken as an example to iZZust&te the approtimate level of contain- ment referred to here. The avaiZabZe data from experiments involving the feeding of bacteria to humans and calves (28-301 indicate that g. coZi K-12 did not usuaZZy colonize the norma bowel, and exhibited little, if any, multiplication while passing through the alimentary trait even after feeding high -c&es (i.e., 10' to lOlo bacteria per hwnan or calf). However, generaZ extrapolation of these results may not be warranted SP because the imphzntation of bacteria into the intestinaz tract P- _-,.. _a. -. 4 ~~`++--~2epends on a nwZ&-o~, such as the nature of the in- test&a2 fzora present in a given invidivuat and the physiologica state of the inoculia. ti Moreover, since viable E. coZi K-12 can be found in the feces after humans are fed lo7 bacteria in broth (28) gr 3 x lo4 bacteria protected by suspension in miZk _ _.__._" ._I--- \ (2s)~~-trasductiona:~~ and conjugational! transfer of the plasmid vectors f?om E. coZi K-12 to resident bacteria in the fecal -- w matter before and after excretion must aZso be conside'red. . - -- Since most conjugative ptasmids in nature are repressed for expression of donor fertility, the frequency at which noncorj'ugative ptasmids are mobiZized and transferred by this sequence of events in vivo -- is difficuZt to estimate. However, in'caZves fed on an w antibiotic-suppzemented diet, it has been estimated that such triparenta2 nonconjugative R pZasmid transfer occurs * at frequencies of no more than 10m10 to lo-l2 per 24 hours per caZf (30). c In terms of considering other means for pZasmid transmission in nature, it shouZd be noted that transduction does operate in vivo for StaphyZococcus -- aureus (32) and probabZy for E. coZi as weZZ. -- However, no data are avaiZabZe to indicate the frequencies of pkzsmid transfer in vivo by either transduction or transformation. -- These ob.servations indicate the low probabilities for possible dissemination of such.pZasmid vectors by accidenta ingestion, which wouZd probably involve only a feu hundred or thousand bacteria provided that at least the standard practices (Section II-A above) are followed, particularly . the avoidance of mouth pipetting. The possibility of coloni- w -TJa \ - zation and hence of transfer are increased, however, if the normal flora in the bowel is disrupted by, for example, anti- biotic therapy (33). For this reason, persons receiving such therapy should not work with DNA recombinants formed with any g. coli K-12 host-vector system during the therapy period and for seven days thereafter; simitarly, persons who have achlorhydria or who have had surgical removal of part of the stomach or bowel should avoid such work, as should those who require large doses of antacids. / - The observations on the fate of g. coli K-12 in the hwnon alimentary tract are also relevant to the containment of recombinant DNA formed with bacteriophage h variants. Bacteriophage can escape from the laboratory either as mature infectious phage particZes or in bacterial host cells in which the phage genome is carried as a plasmid or prophage. The fate of g. coli K-12. host cells carrying the phage genome as a plasmid or prophage is similar to that for plasmid- containing host cells as discussed above. The~survival of the h phage genome when reZeased as infectiousparticles depends on their stability in nature, their infectivity and on the probability of subsequent encounters with naturally occurring X-sensiti.ve E. coli strains. -- AZthough the pro- babiZity of survival of X and its infection of resident intestinal * g. coZi in animals and humans has not been measured, it is estimated to be small given the high sensitivity of X to the Zow pH of the stomach, the insusceptibility to X infection of smooth g. coli cells (the type that normally resides in the gut),.the infrequency of naturally occurring A-sensitive g. coli (34) and the failure to detect infective A partkles in humax feces after ingestion of up to 1011 X particles (35). Moreover, X part&zles are very sensitive to dessication. Establishment of A as a stable lysogen is a frequent 3 event (loo to 10B1) for the att+ in-t+ cp phage so that this --- mode of escape would be the preponderant laboratory hazard; While not exact, the estimates for containment afforded by using these host-vectors are at least as accurate as those for physical containment, and are \ sufficient to indicate that currently employed plasmid and x vector systems provide a moderate level of bio- 2ogicaZ containment. Other nonconjugative pZasmids and bacteriophages that, in association with E. coli -- K-12 can be estimated to provide the same approtimate level of moderate containment are included in the EKI class, KK2: This class consists of combinations of modified E. coli K-12 "hosts" with modified plasmid or bacteriophage "vectors." The modifications will be achieved by classical genetic manipulation and must be such that the survival rate of the recombined "foreign" DNA fragment, in natural environments, is less than one in 108. Testing procedures for verification of the proper- ties of EK2 systems are described. (EK2 systems are being developed and tested and it is anticipated that they will be available shortly. Responsibility for certification of putative EK2 systems presently lies with the NIH Recombinant DNA Molecule Program Advisory Committee.) EK2 host-vectors - These are host-vector systems that have been genetically constructed and shown to provide a high level of biological containment as demonstrated by data from suitable tests performed in . the laboratory, The genetic modifications of the r. coli K-12 host and/or the plasmid or phage vector should not permit survival of the cloned DNA fragment in other than specially designed and carefully . regulated laboratory environments at a frequency qreater than 10e8& This absolute measure of biological containment has been selected because it is a realistic measurable entity. Indeed, by testing L the contributions of preexisting and newly introduced genetic pro- perties of vectors and hosts, individually or in various combinations, if not prove, that the specially designed host-vector system can provide a margin of biological containment in excess of that required. For EK2 plasmid vectors, no more than one in lo8 host cells con- *taining a chimeric plasmid should be able to perpetuate the cloned DNA fragment under nonpermissive conditions designed to represent the natural environment either by survival of the original host or as a consequence of transmission of the cloned DfiA by transformation, transduction or conjugation to a host with properties common to those in the natural environment. In the construction of EK2 plam`id-host systems it is important to use the most stabZe mutations avaitable, preferab2; deletions. Obviously, the presence of a21 mutations contributing to higher degrees of bio ZogicaZ containment must be verified periodicalZy by appropriate tests. In testing the level of biologica containment afforded by a proposed EK2 plasmid-host system, it is important to design relevant tests to evakate the survival of the cloned DNA under conditions that are possible in nature and that are aZso most advantageous for its perpetuation. For exaqle, one might conduct a t&parental mating +th a primary. donor possessing a derepressed F-type or I-type conjugative plasmid, the safer host with Abiofl-asd, dapD8, A&-chlr, AthyA, deoc, &and hsdS mutations and a plasmid vector carrying an easily detectable inserted gene such as for ampicillin resistance or &+, and a Secondary recipient that is Su+ h&S trp (i.e., permissive for the recombinant plasmid). Such matings wouzd be conducted in a mediwn lacking diaminopimelic acid and thymine and survival of the Apr Or z+ marker in any of the three strains followed as a function of time. Survival of the cloned marker by transduction could also be evaluated by introdL*ing a knam generalized transducing phage into the system. similar experiments should also be oTone using a secondary recipient that is restrictive for the recombinant plasmid as well as with primary dcnors possess- ing repressed conjugative plasm$ds vi th incompatibility group properties like those commonly found in enteric microorganisms. Since a common route of escape of plasmid- host systems in the laboratory-might be by accidental ingestion, it is suggested that the same types of experi- ments be conducted in suitable animal-model systems. In addition to these tests on survival of the cloned DNA, it would be useful to determine the survival of the host strain under nonorowth conditions such as in water and as a function of drying time after a culture has been spilled on a lab bench. For EK2 phage vectors, no more than one in lo8 recombinant phage particles should be able to perpetuate the cloned DNA fragment under non-permissive conditions designed to represent the natural environ- ment either (a) as a prophage or pl,asmid in the laboratory host used for phage propagation or.(b) by surviving in natural environments and transferring the cloned DNA to a host (or its resident lambdoid prophage) with properties common to those in the natural environment. !l'he phenotypes and genetic stabilities of the mutations and chromosome azterations included in these X-host systems indicate that containment we21 in excess of the required 1O-8 or lower survival frequency for the cloned DNA fragment should be attained. ObviousZy the presence of all mutations contributing to this high degree of biological containment must be verified periodicaZly by appropriate tests. w Laboratory tests should be performed with the bactetial host to measure all possible routes of escape of cZoned DNA such as the . L frequency of lysogen formation, the frequency of plasm-id a formation and the survival'of the Zysogen or carrier bacterim. SimilarZy, the potential for perpetuation of the cloned DNA fragment carried by infectious phage particles could be tested by challenging typical wild-type g. coli strains or a A-sensitive nonpermi&ive laboratory K-12 strain, especially one lysogenic . for a lmbdoid phage.. -: These are EK2 systems for which the increased containment independently confirmed by appropriate tests in EK3 systems are not presently avaihbk. 2. Classification of experiments using the E. coli K-12 containment systems In the following classification of containment criteria for different kinds of recombinant DNAs, the stated levels of physical and biological . containment are minimums. It is recommended that higher levels of : biological containment (EK3 > EK2 7 and are equally appropriate for the + Shotgun Experiments EKl) be used if they are available purposes of the experiment. These experiments involve the production of recombinant DNAs between the vector and the total DNA or (preferably) any partially purified fraction thereof from the specified cellular source. 2. 1. Donald Brown: The guidelines are too strict and inflexible. Requiring EK2 containment for plant, vertebrate and viral -DNAs places in effect an indefinite moratorium on research. Basis for this is that there are at present no EK2 vectors tems will be ce eriments in its P4 facilities. Robert Sinsheimer: Shotgun experiments with eukaryotic DNA incorporated into E. coli are especially troublesome. d safer and potentially reversible method is to incorporate such DNA into animal viruses where there is already in place a viable defense. One such example that comes to mind is cowpox. Here vaccination is possible and if the hazard were perceived, all infected animals and tissue culture cells could be destroyed. 4. Allen Silverstone: He recommends banning of all shotgun experiments with mammals and birds. For all of the eukaryotes P4 containment is recommended. He believes the possibility of cancer-causing DNA in lower organisms is a potential hazard which requires the highest physical containment. In the case of mammals he also recommends a ban on the use of chemically purified DNA. (5) Eukaryotic DNA recombinants: Primates - P3 physical containment + an EK3 host-vector; or P4 physical containment + an EK2 host-vector, except for DNA from embryonic tissue or primary tissue cultures therefrom, and germ- line cells for which P3 physical containment + an EK2 host-vector *can be used. The baSis for the lower estimated hazard in the case . of DNA from the latter tissues is their relative freedom from horizontally acquired adventitious viruses. Other mammals - P3 physical containment + an EK2 host-vector. 8irds - P3 physical containment + an EK2 host-vector. Cold-blooded vertebrates - P2 physical containment + an EK2 except for embryonic or germ-line DNA which resuire P2 - containment + an EKl host-vector. Other cold-blooded animals and lower eukaryotes - P2 physical containment + an EKJ host-vector. If the eukaryote in this class is a known pathogen (i.e., an agent listed in Class 2 of ref. 5 or a plant pathogen) or carries such an agent, to P3 + EKZ. Higher plants - P2 physical the containment should be increased containment + an EK2 host-vector. ". If the plant carries a known pathogenic agent or makes a product known to be dangerous to any species, c the containment should be raised to P3 physical containment + an EK2 host-vector. *, Prokaryotic DNA recombinants ~====.-----"..~ ,. _r _ Prokaryotes that exchange genetic information with E. coli&- The level of physical containment is directly determined by the rule of the most dangerous component (see introduction to Section III). Thus Pl conditions can be used for DNAs from those bacteria in Class 1 of ref. 5 ("Agents of no or minimal hazard....") which naturally ex- change genes with L. coli; and P2 conditions should be used for such bacteria if they fall in Class 2 of ref. 5 ("Agents of ordinary potential hazard...."), or are plant pathogens. EKl host-vectors can be used for ^ . B efined as observable under optimal laboratory conditions by transformation, transduction , phage infection and/or conjugation with transfer of phage, plasmid and/or chromosomal genetic information. all experiments requiring only Pl physical containment; in fact, ex- periments in this category can be performed with 5. coli K-12 vectors exhibiting a lesser containment (e.g., conjugative plasmids) than EKl vectors. Experiments with DNA from species requiring P2 physical con- tainment which are of low pathogenicity (for example, enteropathogenic Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae) . o can use EKl host-vectors, but those of moderate pathogenicity (for example, Salmonella typhi, Shigella dysenteriae type I, and Vibrio cholerae) should use EK2 host-vectors.3 3 The bacteria which constitute Class 2 of ref. 5 ("Agents of ordinary potential hazard...." ) represent a broad spectrum of etiologic agents which possess dif- ferent levels of virulence and degrees of communicability. We think it appro- priate for our specific purpose to further subdivide the agents of Class 2 into those which we believe to be of relatively low pathogenicity and those which are moderately pathogenic. The several specific examples given may suffice to illustrate the principle. The Committee has asked the Center for Disease Control to help prepare a complete list of Class 2 agents subdivided into low and moderate pathogenicity that could be distributed to interested parties. Prokaryotes' that do not exchange genetic information with E. coli - The minimum containment conditions for this class consist of PZ physical containment + an EKl host-vector, and apply when the risk that the recombinant DNAs will increase the patho- genicity or ecological potential of the host is judged to be minimal. Experiments with DNAs from pathogenic species (Class 2 ref. 5 plus . *plant pathogens) should use P3 + EK2. Experiments extending the range of resistance to therapeutically useful drugs and disinfectants should use P2 + EK2 containment or higher depending on the virulence of the donor.. . Gv- (iii) Characterized clones of DNA recombinants derived from shotgun experiments 4. The Environmental Defense Fund: They too urge'that E. coli not be used. They also VLb -c~-wLz ,--zrned about use of the terms, "?;armful" and "harmless," in describing genes and their products. They point out that accidents will occur and would like as broad a definition for "harmful" ,s possible to minimize possible accidents. They recc-&..- that EKl strains never be used except,in circumstances where there is no possible risk to humans. When a cloned DNA recombinant has been rigorously characterized4 and there is sufficient evidence that it is free of harmful genes,4 then experiments involving this recombinant DNA can be carried out under Pl + EKl conditions if the inserted DNA is from a species that exchanges genes with r. coli, and under P2 + EKl conditions if not. 4 The terms "characterized" and "free of harmful genes" are unavoidably . vague. But, in this instance, before containment conditions lower than fhees used to clone the DNA can be adopted, the investigator must obtain approval from the granting agency. Such approval would be contingent upon data concerning: (a) th e absence of potentially harmful genes (e.g., sequences contained in indigenous tumor viruses or which code for toxic substances), (b) the relation between the recovered and desired segment (e.g., hybridization and restriction endonuclease fragmentation analysis where applicable), and (c) maintenance of the biological properties of the vector. Purified.cellular DNAs other 'than plasmids, bacteriophages, - and other viruses 1. Donald Brown : The guidelines for ver)tebrate "shotgun" experiments are too stringent and rigid, They establish a 'moratorium on gene isolation from all vertebrates. He urged the use of hybridization methods at reduced levels of physical and biological cant ainment. Further, L the requirements for chemically purified DNA are not achievable. There can be no guarantees for 99% purity. The Committee did not kmsider how purified DNA components differ from bxilk DNA. m The formation of DNA recombinants from cellular DNAs that have been enriched' by physical and chemical techniques (i.e., not by cloning) and which are free of harmful genes can be carried out under ?ower contain- ment conditions than used for the corresponding shotgun experiment. In general, the containment can be decreased one step in physical containment (P4 -+ P3 + P2 + Pl) while maintaining the biological containment specified for the shotgun experiment, or one step in biological containment (EK3 + EK2 + EKl) while maintaining the specified physical contain- ment--provided that the new condition is not less than that specified above for characteriied clones from shotgun experiments (Section -- - -..- iii). A DNA preparation is defined as enriched if the desired DNA represents at least 99% (w/w) of the total DNA in the preparation. The reason for lowering the con- tainment level when this degree of enrichment has been obtained is based on the fact that the total number of clones that must be examined to obtain the desired clone is markedly reduced. Thus, could, for example, the probability of cloning a harmful gene be reduced by more that lo5 -fold when a nonrepetitive gene from mammals was being sought. Furthermore, the level of purity specified here makes it easier to establish that the desired DNA does not contain harmful genes. The following classifications refer to cases where the 3-y "foreign' DNA is itself derived from plasmids, bacterio- 6 phages, or viruses. w "Foreign" DNA from viruses that infect animals. P4 and EK2, or P3 and EK3 are to be used. P3 and EK2 may be used after purification by cloning and demon- stration that only harmless viral genes are present. (if.) "Foreign" DNA from viruses that infect plants. "Foreign" DNA from purified organelle DNAs. From primates: P3 and EKl or P2 and EK2. Other: P2 and EKl.-. (iv) "Foreign" DNA from prokaryotic plasmids or bacteriophages. . If the "j foreign" DNA is from a species that exchanges required. Experiments with prokaqot-c "host-vector" systems other than E. coZi 1. Richard Goldstein : As previously noted he recomends E* C"li be banned as a host-vector with* the next two years. He urges that the development of alternative prokaryote system be developed and suggests one possibility is B. subtilisa Other prokaryotic host-vector systems are at the speculative, planning, or developmental stage, and consequently do not warrant detailed treatment here at this time. However, the containment criteria for different types of DNA recombinants formed with g. coli K-12 host-vectors can, with the aid of some general principles . Qven here, serve as a guide for containment conditions with other host-vectors when appropriate adjustment is made for their different habitats and characteristics. In general, the strain 'of any prokaryotic species used as the host should conform,to the definition of Class 1 etiologic agents given in ref. 5 (i.e., "Agents of no or minima? hazard...."), and the plasmid or phage vector should not make the host more hazardous. In addition, it is recommended that the newly developed host-vector systems offer some distinct advantage over the g. coli K-12 host- vectors--for instance, thermophilic organisms or other host-vectors whose major habitats do not include humans and/or economically im- portant animals and plants. Appendix A gives a detailed discussion of the B. subtilis system, - the most promising alternative to date. At the initial stage, the host-vector should exhibit at least a moderate level of biological containment comparable to EKl systems, and be capable of modification to obtain high levels of containment comparable to EK2 and EK3. The type of confirmation test(s) required to move a host-vector from an EK2-type classification to an EK3-type will clearly depend upon the preponderant habitat of the host-vector. : For example, if the unmodified host-vector propagates mostly in, on, or around higher plants, but not appreciably in warm-blooded animals, modification should be designed to reduce the probability that the host-vector can escape to and propagate in, on, or around such plants, or transmit recombinant DNA to other bacterial hosts that are able to occupy these ecological niches , and it is these lower probabilities which should be confirmed. The following principles should be.followed in using the containment criteria given for experiments with E. coli K-12 host-vectors as a guide for other prokaryotic systems. Experi- ments with DNA from prokaryotes (and their plasmids or viruses) should be classified according to whether the prokaryote in question exchanges genetic information with the host-vector or not, and the con- tainment conditions given for these two classes with r. coli K-12 host-vectors applied. Transfer of retombihant DNA to plant pathogens can be made safer by using nonreverting, doubly auxotrophic , non- pathogenic variants. Experiments using a plant pathogen that affects elements of a local flora will require more stringent containment than if carried out in areas where they are not common. Experiments with DNAs from eukaryotes (an 4 their plasmids or viruses) can also follow the criteria for the corresponding experi- ments with E. coli K-12 vectors if the major habitats of the qiven - host-vector overlap those of g. eoli. 'If the host-vector has a major habitat that does not overlap those of k. ~01.5 (e.g., root nodules in plants), then the containment conditions for some eukaryotic recombinant DNAs should be increased (for instance, higher plants and their viruses in the preceding example), while others may be reduced. 4. Experiments with eukaryotic "host-vector" Systems 1. Maxine Singer: The use of SV40 virus is troublesome. If it is not to be banned altogether, it should probably be used only at P4 levels. 2. Peter Barton Hutt: In the case ofSV40,if it is to be used.at all, it should be done only at the P4 level. Because this virus is known to cause cancer in animals, the burden is on the scientist to show there is no possibility whatever of harm to man when it is used in these experiments. If it is to be used, its use should be fully defended in the preamble in the guidelines. 1 `3Fz / Animal host-vector systems - Because host cell lines generally have little if any capacity for propagation outside the laboratory, the primary focus for containment is the vector, although cells should also be derived from cultures expected to be'of minimal hazard. Given good micro- biological practices, the most likely mode of escape of recombinant DNAs from a physically contained%boratory is carriage by humans; thus vectors should be chosen that have little or no ability to replicate in human cells. To .be used as a vector in a eukaryotic host, a DNA W-/` molecule should display all of the following properties: (1) It should not consist of the whole qenome of any agent that is infectious for humans or that replicates to a significant extent in human cells in tissue culture. (2) Its'functional anatomy should be known--that is, there should be a clear idea of the1location within the molecule of: a) the sites at which DNA synthesis originates and terminates, b) the sites that are cleaved by restriction endonucleases, c) the template regions for the major gene products. (3) It should be well studied genetically. It is desirable 4 that mutants be available in adequate number and variety, and that quantitative studies of recombination have been performed. . (4) The recombinant should be defective, that is, its pro- pagation as a virus is dependent upon the presence of a comple- menting helper genome. This helper should either (a) be integrated into the genome of a stable line of host cells (a situation that would effectively limit the growth of the vector to that particular cell line) or (b) consist of a defective genome or an appropriate conditional lethal mutant virus (in which case the experiments would be done under non-permissive conditions), making vector and helper dependent upon each other for propagation. However, if none of these is available, the use of a non-defective genome as helper would be acceptable. Currently only two viral DNAs can be considered as meeting these requirements: these are the genomes of polyoma virus and sv40. Of these, polyoma virus is highly to be preferred. SV40 is known to propagate in human cells, both in vivo and in vitro, -- and to infect laboratory personnel, as evidenced by the frequency of their conversion to producing SV40 antibodies. Also, SV40 and related viruses have been found in association with certain human neurological and malignant diseases. SV40 shares many properties, and gives complementation, with the common human papova viruses. By contrast, there is no evidence that polyoma infects humans, nor does it replicate to any significant extent in human cells in vitro. -- However, this system still needs to be studied more extensively. Appendix B gives further details and documentation. Taking account of all these factors, it is proposed that: 1. Polyoma Virus 5 Recombinant DNA molecules consisting of defective polyoma virus genomes plus DNA sequences of any non- pathogenic organism , including Class 1 viruses (5), can be propagated in or used to transform cultured cells in P3 conditions; appropriate helper virus can be used if needed. Whenever there is a choice, it is urged that mouse cells, derived preferably from embryos, be used as the source of eukaryotic DNA. Polyoma virus is a mouse virus and recombinant DNA molecules contain- \ ing both viral and cellular sequences are already known to be present in virus stocks grown at a high multiplic- ity. Thus, recombinants formed in vitro between polyoma -- virus DNA and mouse DNA are presumably not novel from an evolutionary point of view. b. Such experiments can be done, under P4 conditions, if the * recombinant.DNA contains segments of the genomes of Class 2 animal viruses (5). Once it has been shown by suit- . * able biochemical and biological tests that the cloned recombinant contains only harmless regions of the viral genome (see Section IIIB-2-c-i) and that the host rang . of the polyoma virus vector has not been altered, periments can be continued under P3 conditions. 2. SV40 Virus a. / ( .,- Defective SV40 genomes, with appropriate helper, can be used in P4 conditions as a vector for recombinant DNA molecules containing sequences of any non-pathogenic organism or Class I virus (5), (i.e., a shotgun type experiment); established lines of cultured cells should be used. Such experiments can be carried out in P3 conditions if the non-SV40 DNA segment is (a) a purified9 segment of prokaryotic DNA lacking toxigenic genes, or (b) a segment 'The DNA preparation is defined as purified if the desired DNA represents at least 99% (w/w> of the total DNA in the preparation, provided that it was achieved or verified by more than one procedure. of eukaryotic DNA whose function has been established, Which does not code for a toxic product, and which has been previously cloned in a prokaryotic host-vector system. c. A recombinant DNA molecule consisting of defective SV40 . DNA lacking substantiai segments of the late region, plus DNA from non-pathogenic organisms or Class I viruses (5), can be propagated as an autonomous cellular element in established lines of cells under P3 conditions provided that there is no exogenous or endogenous helper, and that it is demonstrated that no infectious virus - particles are being produced. Until this has been / 3 i demonstrated, the appropriate containment conditions specified in 2. 5. and 2. b. shall be used. Recombinant DNA molecules consisting of defective SV40 DRA and sequences from non-pathogenic prokaryotic or eukaryotic organisms or Class I viruses (5) can be used to transform established lines of non-permissive cells under P3 conditions. ft must be demonstrated that fro infectious virus particles are being produced; rescue of SV40 from such transformed cells by co-cultivation or trahsfection techniques must be carried out in P4 conditions. gi Efforts should be made to ensure that all celi lines are free of virus particles and mycoplasma. Since SV46 and polyoma are limited in their scope to act as vectors, chiefly because the amount of foreign DNA that the normal virions can carry probably cannot exceed 2 x lo6 daltons, we urge,that consideration be given to the development of systems in which recombinants can be cloned and propagated purely in the form of DNA, rather than in t)e coats of infectious agents. Plasmid forms of viral genomes or organelle DNA should be explored as possible cloning vehicles in eukaryotic cells. Plant host-vector systems 1. r dk Milton Zaitlin: In the guidelines plants have only a peripheral role; however, they could become extraordinarily important in recombinant DNA studies. Cloning of higher plant DNA and incorporating this DNA into bacteria is well covered by the guidelines. However, the guidelines are silent on the incorporation of foreign DNA into the genone of higher plants. There are five DNA plant viruses that could play a significant role here. These viruses are transmitted by aphids and are a problem with regard to physical containment. Physical containment in the guidelines is designed to prevent bacteria from getting out of the facility and to to this, negative air pressures are specified. But positive air pressures are required to avoid drawing insects into the facility. Guidelines might be modified to specify positive air pressures under these circumstances. The guide- lines also are silent on how to deal with a new plant once it is produced. Procedures need to be outlined for testing new plants for any undesirable characteristics they might have. The guidelines, therefore, should include restrictions f __ on the release of plants from containment facilities until they can be tested through several generations. Plants should be tested to insure they are not toxic to animals which might be likely to eat them. In plants where the capacity for nitrogen fixation is introduced, testing must be done to insure that these plants don't have a competitive advantage overcther - plants and become pests in themselves. Cells in tissue cultures, seedlings, or plant parts (e.g., tubers, stems, fruits, and detached leaves) or whole mature plants of small species (e.g., Arabidopsis) can be handled under the Pl-P4 containment conditions that we have specified previously. However, work in most plants poses additional problems. P2 physical containment conditions Zcan be provided by: (i) the best insect-proof greenhouses, (ii) appro- priate disinfection of contaminated plants, pots, soil, and runoff water, and (iii) adoption of the other standard practices for microbio- logical work. P3 physical containment can be sufficiently approximated by confining the operations with whole plants to growth chambers like those used for ,work with radioactive . topes, provided that (i)'such chambers 7% 0 are modified to produce a negativ pressure environment with the exhaust air appropriately filtered, and (ii) that other operations with infectious materials are carried out under the specified P3 conditions. The P2 and P3 conditions specified earlier are therefore extended to include these cases for work on higher plants. The host cells for experiments on recombinant DNAs may be cells in culture, in seedling or plant parts, or in whole plants. Cells in .whole plants that cannot be adequately contained should not be used as hosts for shotgun experiments at this time, and attempts to infect whole plants with DNA recombinants cloned elsewhere should not be initiated until their effects on host cells in culture, seedlings, or plant parts have been studied. Organelle or plasmid DNAs or DNAs-of viruses of low pathogenicity to plants may be used as vectors. In general, the same preference criteria for selecting host-vectors given in the preceding section on animal systems apply to plant systems, where organelle and plasmid DNAs can be grouped together as offering the potential of highly contained vectors that should be investigated. Experiments on recombinant DNAs formed between the initial moderately contained vectors and DNA from cells of species in which the vector DNA can replicate, either autonomously or as an integrated segment of the cell's genome, should use P2 physical containment--provided that the source of the DNA is itself not pathogenic or known to carry pathogenic agents, or to produce products dangerous to plants. In the latter cases, of if the vector is periments should be and has Experiments on DNAs from other been purified" 10 The DNA preparation is an unmodified virus of low pathogenicity, the ex- carried out under P3 conditions. recombinant DNAs formed between the above vectors species can also be carried out under P2 if that DNA - -- and determined not to contain harmful genes. Other- defined as purified if the desired DNA represents at least 99% (w/w> of the total DNA in the preparation, provided that it was achieved or verified by more than one procedure. wise, the experiments should be carried out under P3 conditions if the source of the inserted DNA is not itself a pathogen, or known to carry such pathogenic agents, or to produce harmful products--and under P4 conditions if these conditions are not met. The development and use of host-vector systems that exhibit a high level of biological containment permit a decrease of one step in the physical containment specified above (P4 -f P3 + P2 -+ Pl). KC> Fungal or similar lower eukaryotic host-vector systems The containment criteria for experiments on recombinant DNAs using these host-vectors most closely resemble those for prokaryotes, rather . than those for the preceding eukaryotes, in that the host cells usually exhibit a capacity for dissemination outside the laboratory that is similar to that for bacteria. We therefore consider that the contain- ment guidelines given for experiments with r. coli K-12 and other pro- karyotic host-vectors (Sections IIIB-1 and -2, respectively) provide adequate direction for experiments with these lower eukaryotic host- vectors. This is particularly true at this time since the,development of these host-vectors is presently in the speculative stage. IV. IMPLEMENTATION