NIH Recombinant DNA Guidelines

Excerpted from http://www4.od.nih.gov/oba/rac/guidelines/guidejan01.htm

SECTION III.  EXPERIMENTS COVERED BY THE NIH GUIDELINES

This section describes six categories of experiments involving recombinant DNA:  

Note: experiments involving transgenic animals are in Section III-D-3-4.

Section III-A.  Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation (See Section IV-C-1-b-(1), Major Actions).

Section III-A-1.  Major Actions under the NIH Guidelines

Experiments considered as Major Actions under the NIH Guidelines cannot be initiated without:

The containment conditions or stipulation requirements for such experiments will be recommended by RAC and set by NIH at the time of approval.  Such experiments require Institutional Biosafety Committee approval before initiation.  Specific experiments already approved are included in Appendix D, Major Actions Taken under the NIH Guidelines, which may be obtained from the Office of Biotechnology Activities.

Section III-A-1-a.  The deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally (see Section V-B, Footnotes and References of Sections I-IV), if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture, will be reviewed by RAC.  

Back to Section III

Section III-B.  Experiments That Require NIH/OBA and Institutional Biosafety Committee Approval Before Initiation

Experiments in this category cannot be initiated without submission of relevant information on the proposed experiment to NIH/OBA.  The containment conditions for such experiments will be determined by NIH/OBA in consultation with ad hoc experts.  Such experiments require Institutional Biosafety Committee approval before initiation (see Section IV-B-2-b-(1), Institutional Biosafety Committee).

Section III-B-1.  Experiments Involving the Cloning of Toxin Molecules with LD₅₀ of Less than 100 Nanograms per Kilogram Body Weight

Deliberate formation of recombinant DNA containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD₅₀ of less than 100 nanograms per kilogram body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin).  Specific approval has been given for the cloning in Escherichia coli K-12 of DNA containing genes coding for the biosynthesis of toxic molecules which are lethal to vertebrates at 100 nanograms to 100 micrograms per kilogram body weight.  Specific experiments already approved under this section may be obtained from the Office of Biotechnology Activities.  

Back to Section III

Section III-C.  Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approvals and RAC Review Before Research Participant Enrollment

Section III-C-1.  Experiments Involving the Deliberate Transfer of Recombinant DNA, or DNA or RNA Derived from Recombinant DNA, into One or More Human Research Participants

For an experiment involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into human research participants (human gene transfer), no research participant shall be enrolled (see definition of enrollment in Section I-E-7) until the RAC review process has been completed (see Appendix M-I-B, RAC Review Requirements).

In its evaluation of human gene transfer proposals, the RAC will consider whether a proposed human gene transfer experiment presents characteristics that warrant public RAC review and discussion (See Appendix M-I-B-2).  The process of public RAC review and discussion is intended to foster the safe and ethical conduct of human gene transfer experiments.  Public review and discussion of a human gene transfer experiment (and access to relevant information) also serves to inform the public about the technical aspects of the proposal, meaning and significance of the research, and any significant safety, social, and ethical implications of the research.

Public RAC review and discussion of a human gene transfer experiment may be: (1) initiated by the NIH Director; or (2) initiated by the NIH OBA Director following a recommendation to NIH OBA by:  (a) three or more RAC members; or (b) a Federal agency other than NIH.  After a human gene transfer experiment is reviewed by the RAC at a regularly scheduled meeting, NIH OBA will send a letter within 10 working days to the NIH Director, the Principal Investigator, the sponsoring institution, and other DHHS components, as appropriate, summarizing the RAC recommendations.

For a clinical trial site that is added after the RAC review process, no research participant shall be enrolled (see definition of enrollment in Section I-E-7) at the clinical trial site until the following documentation has been submitted to NIH OBA:  (1) Institutional Biosafety Committee approval (from the clinical trial site); (2) Institutional Review Board approval; (3) Institutional Review Board-approved informed consent document; (4) curriculum vitae of the principal investigator(s) (no more than two pages in biographical sketch format); and (5) NIH grant number(s) if applicable.

In order to maintain public access to information regarding human gene transfer protocols (including protocols that are not publicly reviewed by the RAC), NIH OBA will maintain the documentation described in Appendices M-I through M-V.  The information provided in response to Appendix M should not contain any confidential commercial information or trade secrets, enabling all aspects of RAC review to be open to the public.

Note:  For specific directives concerning the use of retroviral vectors for gene delivery, consult Appendix B-V-1, Murine Retroviral Vectors.

Back to Section III

Section III-D.  Experiments that Require Institutional Biosafety Committee Approval Before Initiation

Prior to the initiation of an experiment that falls into this category, the Principal Investigator must submit a registration document to the Institutional Biosafety Committee which contains the following information:  (i) the source(s) of DNA; (ii) the nature of the inserted DNA sequences; (iii) the host(s) and vector(s) to be used; (iv) if an attempt will be made to obtain expression of a foreign gene, and if so, indicate the protein that will be produced; and (v) the containment conditions that will be implemented as specified in the NIH Guidelines.  For experiments in this category, the registration document shall be dated, signed by the Principal Investigator, and filed with the Institutional Biosafety Committee.  The Institutional Biosafety Committee shall review and approve all experiments in this category prior to their initiation.   Requests to decrease the level of containment specified for experiments in this category will be considered by NIH (see Section IV-C-1-b-(2)-(c), Minor Actions).

Section III-D-1.  Experiments 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-1-a.  Experiments involving the introduction of recombinant DNA into Risk Group 2 agents will usually be conducted at Biosafety Level (BL) 2 containment.  Experiments with such agents will usually be conducted with whole animals at BL2 or BL2-N (Animals) containment.

Section III-D-1-b.  Experiments involving the introduction of recombinant DNA into Risk Group 3 agents will usually be conducted at BL3 containment.  Experiments with such agents will usually be conducted with whole animals at BL3 or BL3-N containment.

Section III-D-1-c.  Experiments involving the introduction of recombinant DNA into Risk Group 4 agents shall be conducted at BL4 containment.  Experiments with such agents shall be conducted with whole animals at BL4 or BL4-N containment.

Section III-D-1-d.  Containment conditions for experiments involving the introduction of recombinant DNA into restricted agents shall be set on a case-by-case basis following NIH/OBA review.  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G and V-L, Footnotes and References of Sections I-IV).  Experiments with such agents shall be conducted with whole animals at BL4 or BL4-N containment.

Section III-D-2.  Experiments 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

Section III-D-2-a.  Experiments in which DNA from Risk Group 2 or Risk Group 3 agents (see Section II-A, Risk Assessment) is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BL2 containment.  Experiments in which DNA from Risk Group 4 agents is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BL2 containment after demonstration that only a totally and irreversibly defective fraction of the agent's genome is present in a given recombinant.  In the absence of such a demonstration, BL4 containment shall be used.  The Institutional Biosafety Committee may approve the specific lowering of containment for particular experiments to BL1.  Many experiments in this category are exempt from the NIH Guidelines (see Section III-F, Exempt Experiments).  Experiments involving the formation of recombinant DNA for certain genes coding for molecules toxic for vertebrates require NIH/OBA approval (see Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD₅₀ of Less than 100 Nanograms Per Kilogram Body Weight) or shall be conducted under NIH specified conditions as described in Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates.

Section III-D-2-b.  Containment conditions for experiments in which DNA from restricted agents is transferred into nonpathogenic prokaryotes or lower eukaryotes shall be determined by NIH/OBA following a case-by-case review (see Section V-L, Footnotes and References of Sections I-IV).  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

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

Caution:  Special care should be used in the evaluation of containment levels for experiments which are likely to either enhance the pathogenicity (e.g., insertion of a host oncogene) or to extend the host range (e.g., introduction of novel control elements) of viral vectors under conditions that permit a productive infection.  In such cases, serious consideration should be given to increasing physical containment by at least one level.

Note:  Recombinant DNA or RNA molecules derived therefrom, which contain less than two-thirds of the genome of any eukaryotic virus (all viruses from a single Family (see Section V-J, Footnotes and References of Sections I-IV) being considered identical (see Section V-K, Footnotes and References of Sections I-IV), are considered defective and may be used in the absence of helper under the conditions specified in Section III-E-1, Experiments Involving the Formation of Recombinant DNA Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic Virus.

Section III-D-3-a.  Experiments involving the use of infectious or defective Risk Group 2 viruses (see Appendix B-II, Risk Group 2 Agents) in the presence of helper virus may be conducted at BL2.

Section III-D-3-b.  Experiments involving the use of infectious or defective Risk Group 3 viruses (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) in the presence of helper virus may be conducted at BL3.

Section III-D-3-c.  Experiments involving the use of infectious or defective Risk Group 4 viruses (see Appendix B-IV-D, Risk Group 4 (RG4) - Viral Agents) in the presence of helper virus may be conducted at BL4.

Section III-D-3-d.  Experiments involving the use of infectious or defective restricted poxviruses (see Sections V-A and V-L, Footnotes and References of Sections I-IV) in the presence of helper virus shall be determined on a case-by-case basis following NIH/OBA review.  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

Section III-D-3-e.  Experiments involving the use of infectious or defective viruses in the presence of helper virus which are not covered in Sections III-D-3-a through III-D-3-d may be conducted at BL1.  

Back to Section III

Section III-D-4.  Experiments Involving Whole Animals

This section covers experiments involving whole animals in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic animals) and experiments involving viable recombinant DNA-modified microorganisms tested on whole animals.  For the latter, other than viruses which are only vertically transmitted, the experiments may not be conducted at BL1-N containment.  A minimum containment of BL2 or BL2-N is required.

Caution - Special care should be used in the evaluation of containment conditions for some experiments with transgenic animals.  For example, such experiments might lead to the creation of novel mechanisms or increased transmission of a recombinant pathogen or production of undesirable traits in the host animal.  In such cases, serious consideration should be given to increasing the containment conditions.

Section III-D-4-a.  Recombinant DNA, or DNA or RNA molecules derived therefrom, from any source except for greater than two-thirds of eukaryotic viral genome may be transferred to any non-human vertebrate or any invertebrate organism and propagated under conditions of physical containment comparable to BL1 or BL1-N and appropriate to the organism under study (see Section V-B, Footnotes and References of Sections I-IV).  Animals that contain sequences from viral vectors, which do not lead to transmissible infection either directly or indirectly as a result of complementation or recombination in animals, may be propagated under conditions of physical containment comparable to BL1 or BL1-N and appropriate to the organism under study.  Experiments involving the introduction of other sequences from eukaryotic viral genomes into animals are covered under Section III-D-4-b, Experiments Involving Whole Animals.  For experiments involving recombinant DNA-modified Risk Groups 2, 3, 4, or restricted organisms, see Sections V-A, V-G, and V-L, Footnotes and References of Sections I-IV.  It is important that the investigator demonstrate that the fraction of the viral genome being utilized does not lead to productive infection.  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

Section III-D-4-b.  For experiments involving recombinant DNA, or DNA or RNA derived therefrom, involving whole animals, including transgenic animals, and not covered by Sections III-D-1, Experiments Using Human or Animal Pathogens (Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems, or III-D-4-a, Experiments Involving Whole Animals, the appropriate containment shall be determined by the Institutional Biosafety Committee.

Section III-D-4-c.  Exceptions under Section III-D-4, Experiments Involving Whole Animals

Section III-D-4-c-(1).  Experiments involving the generation of transgenic rodents that require BL1 containment are described under Section III-E-3, Experiments Involving Transgenic Rodents.

Section III-D-4-c-(2).  The purchase or transfer of transgenic rodents is exempt from the NIH Guidelines under Section IIIF, Exempt Experiments (see Appendix C-VI, The Purchase or Transfer of Transgenic Rodents).  

Back to Section III

=========================

Section III-E-3.  Experiments Involving Transgenic Rodents

This section covers experiments involving the generation of rodents in which the animal's genome has been altered by stable introduction of recombinant DNA, 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.

============================

Section III-F.  Exempt Experiments

The following recombinant DNA molecules are exempt from the NIH Guidelines and registration with the Institutional Biosafety Committee is not required:

Section III-F-1.  Those that are not in organisms or viruses.

Section III-F-2.  Those that consist entirely of DNA segments from a single non-chromosomal or viral DNA source, though one or more of the segments may be a synthetic equivalent.

Section III-F-3.  Those that consist entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well established physiological means.

Section III-F-4.  Those that consist entirely of DNA from a eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species).

Section III-F-5.  Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent.  A list of such exchangers will be prepared and periodically revised by the NIH Director with advice of the RAC after appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), Major Actions).  See Appendices A-I through A-VI, Exemptions Under Section III-F-5--Sublists of Natural Exchangers, for a list of natural exchangers that are exempt from the NIH Guidelines. 

Section III-F-6.  Those that do not present a significant risk to health or the environment (see Section IV-C-1-b-(1)-(c), Major Actions), as determined by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public comment.  See Appendix C, Exemptions under Section III-F-6 for other classes of experiments which are exempt from the NIH Guidelines. 

Back to Section III

©1999, Janet Becker Rodgers, DVM, MS, DipACLAM, MRCVS

All rights reserved.

Comments? Send an email to janet.rodgers@vet.ox.ac.uk