These condensed guidelines are provided to assist researchers in determining whether or not their research requires Institutional Biosafety Committee (IBC) approval. If so, the researcher should submit a registration form to the IBC. This abstract is not all inclusive and if the researcher is not sure if his/her program needs review, he/she should contact the IBC. Also, see the complete NIH Guidelines for Research Involving Recombinant DNA Molecules (http://oba.od.nih.gov/rdna/nih_guidelines_oba.html) for further details.
PLEASE NOTE: Those researchers proposing human gene therapy protocols must submit additional information. Specific points to consider can be found on a separate form available through Research Administration.
Recombinant DNA is defined as: (1) Molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell. (2) DNA molecules that result from the replication of these molecules.
NIH has implemented three major levels of review: (1) Institutional Biosafety Committee (IBC), the local recombinant DNA review board of the university; (2) Office of Recombinant DNA Activities (ORDA), an office within NIH; and (3) Recombinant DNA Advisory Committee (RAC), a national committee that advises the Secretary, Assistant Secretary for Health, and the Director of the NIH on recombinant DNA research.
NIH has defined six classifications for various types of experiments. These classifications define the level of review required and are as follows: (III-A) Experiments that require specific RAC review and NIH/ORDA and IBC approval before initiation of the experiment; (III-B) Experiments that require IBC and NIH/ORDA approval before initiation of the experiment; (III-C) Experiments that require IBC approval and NIH/ORDA registration before initiation; (III-D) Experiments that require IBC approval before initiation; (III-E) Experiments that require IBC notification at the time of initiation of the experiment; (III-F) Experiments that are exempt from the NIH Guidelines, but that still require submission to the IBC. Note that it is UTHSC policy that the IBC must make the determination as to whether the experiments can be classified as exempt under the NIH Guidelines.
Note: If an experiment falls into sections III-A, III-B, or III-C and one of the other sections, the rules pertaining to sections III-A, -B, or -C will be followed. If an experiment falls into section III-F and into either section III-D or III-E as well, the experiment is considered exempt from the NIH Guidelines.
III-A. Experiments that require IBC approval, RAC review, and NIH director approval before initiation.
1. Deliberate release into the environment of any organisms containing recombinant DNA, except certain plants.
2. Deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire it naturally.
3. Deliberate transfer of certain recombinant DNA molecules into a human subject that are deemed Major Actions by the NIH (see Appendix D of the NIH Guidelines for examples).
III-B. Experiments that require both NIH/ORDA and IBC approval before initiation.
1. Experiments involving the cloning of toxin molecules with LD50 of less than 100 nanograms per kilogram body weight. Examples: botulinum, tetanus, and diphtheria toxins; S. dysenteriae neurotoxin.
III-C. Experiments that require IBC approval and NIH/ORDA registration before initiation.
1. Experiments involving the deliberate transfer of recombinant DNA or DNA or RNA derived from recombinant DNA into a human subject.
Note: IBC approval must be obtained from each institution at which recombinant DNA material will be administered to human subjects (as opposed to each institution involved in the production of vectors for human application and each institution at which there is ex vivo transduction of recombinant DNA material into target cells for human application).
III-D. Experiments that require IBC approval before initiation of the experiment (generally BL2 or higher containment required).
1. Work using human or animal pathogens (risk groups 2, 3, 4 or restricted agents) as host-vector systems.
a. introduction of recombinant DNA into risk group 2 agents can be carried out at BL2.
b. introduction of recombinant DNA into risk group 3 agents can be carried out at BL3.
c. introduction of recombinant DNA into risk group 4 agents can be carried out at BL4.
d. introduction of recombinant DNA into restricted agents is a case-by-case situation to be decided after NIH/ORDA review.
e. in all cases, whole animal experiments will require containment levels equivalent to the risk group.
2. Work in which DNA from risk group 2, 3, 4, or restricted human or animal pathogens is cloned in nonpathogenic prokaryotic or lower eukaryotic host-vector systems.
a. cloning of DNA from risk group 2 or 3 agents can be carried out at BL2.
b. cloning of DNA from risk group 4 agents can be carried out at BL4 unless a totally and irreversibly defective fraction of the genome was cloned (BL2).
c. cloning of DNA from restricted agents is a case-by-case situation.
d. specific lowering of containment to BL1 for particular experiments can be approved by IBC.
e. many of these experiments may be deemed exempt by the IBC.
3. Work involving the use of infectious viruses or defective viruses in the presence of helper virus in tissue culture systems.
a. risk group 2 agent work can be carried out at BL2.
b. risk group 3 agent work can be carried out at BL3.
c. risk group 4 agent work can be carried out at BL4.
d. restricted agent work is a case-by-case situation.
Note: Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.
4. Experiments involving the generation of transgenic animals and experiments involving viable recombinant DNA-modified microorganisms tested on whole animals (not lower than BL2 containment).
5. Introduction of recombinant DNA (ie, naked DNA injections) into a non-human vertebrate or invertebrate organism (BL1), unless the DNA represents greater than two-thirds of a eukaryotic viral genome.
6. Work involving more than 10 liters of culture (IBC will determine containment level).
III-E. Experiments that require IBC notification at the time of initiation. (BL1 containment required).
1. Work involving no more than two-thirds of any eukaryotic viral genome (except risk group 3, 4, or restricted agents; see III-D) when performed in tissue culture in the absence of helper virus.
2. Experiments involving the generation of transgenic rodents judged to require only BL1 containment.
III-F. Experiments that are exempt (BL1 containment suggested). It is UTHSC policy that all research utilizing recombinant DNA must be registered, even if it meets the exempt criteria. The UTHSC IBC will make the determination as to whether the research will be classified as exempt.
1. Those experiments involving recombinant DNA molecules that:
a. are not in organisms or viruses.
b. consist entirely of DNA segments from a single nonchromosomal or viral DNA source, though one or more of the segments may be a synthetic equivalent.
c. 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), or when transferred to another host by well established physiological means.
d. consist entirely of DNA from a eukaryotic host including its mitochondria or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain).
e. consist entirely of DNA segments from different species that exchange DNA by known physiological processes.
f. contain less than one-half of any eukaryotic viral genome from risk groups 1 or 2, and are propagated and maintained in cells in tissue culture. However, experiments that involve the deliberate introduction of genes coding for the biosynthesis of molecules toxic to vertebrates or whose other aspects warrant a section.
2. Experiments which use E. coli K-12 host-vector systems provided that the E. coli host contains no conjugation-proficient plasmids or generalized transducing phages, and that lambdoid or Ff phages or non-conjugative plasmids are used as vectors.
3. Experiments involving S. cerevisiae or S. uvarum host-vector systems.
4. Experiments involving any asporogenic B. subtilis or asporogenic B. licheniformis host-vector system.
5. Experiments involving recombinant DNA molecules derived entirely from extrachromosomal elements and maintained in the natural host from a number of Bacillus, Listeria, Pediococcus, Staphylococcus, and Streptococcus species (see NIH Guidelines for a specific listing).
6. The purchase or transfer of transgenic rodents for experiments that require BL1 containment.
In addition to submitting a registration form to the IBC, those researchers proposing human gene therapy protocols must submit information addressing each of the following points. The submission may be in narrative form, but must address each point in the following order:
1. What is the structure of the cloned DNA that will be used?
a. Describe the gene (genomic or cDNA), the bacterial plasmid or phage vector, and the delivery vector (if any). Provide complete nucleotide sequence analysis or a detailed restriction enzyme map of the total construct.
b. Describe the regulatory elements the construct contains (e.g., promoters, enhancers, polyadenylation sites, replication origins, etc.). Name the source from which these elements are derived. Summarize what is currently known about the regulatory character of each element.
c. Describe the steps used to derive the DNA construct.
2. What is the structure of the material that will be administered to the patient?
a. Describe the preparation, structure, and composition of the materials that will be given to the patient or used to treat the patient's cells.
i. If DNA, describe the purity (both in terms of being a single DNA species and in terms of other contaminants) and the sensitivity of the assays that will be used to determine this.
ii. If a virus, describe any special features of the cell lines, media, or sera used to propagate it. Describe the purification methods and assays with their sensitivity that will be used to detect and eliminate any contaminating materials (including helper virus or other organisms) that may have biological effects.
iii. If co-cultivation is employed, describe the cells used for co- cultivation. Describe the purification methods and assays with their sensitivities that will be used to detect and eliminate any contaminating materials. Specifically, describe the tests used to assess the material to be returned to the patient for the presence of live or killed donor cells or other non-vector materials originating from those cells.
iv. If other methods are to be used, describe the purification methods and assays with their sensitivities that will be used to detect and eliminate any contaminating materials. Name the possible sources of contamination.
b. Describe any other material to be used in the preparation of the material to be administered to the patient.
i. If a viral vector is proposed, describe the nature of the helper virus or cell line.
ii. Describe the nature of any carrier particles that are to be used.
3. What cells are the intended targets of the recombinant DNA?
a. Describe how ex vivo targeted cells will be characterized before and after treatment.
b. Describe the theoretical and practical basis for assuming that only the target cells will incorporate the DNA.
c. Provide the percentage of target cells that contain the added DNA.
d. Describe whether the added DNA is extrachromosomal or integrated and whether it is unrearranged.
e. Describe the assays with their sensitivities to monitor this.
f. Provide the number of copies of added DNA present per cell and describe the stability of the added DNA both in terms of its continued presence and its structural stability.
4. How efficient and specific is gene transfer and expression?
a. Describe the animal and cultured cell models used to assess the in vivo and in vitro efficacy of the gene transfer system, comparing and contrasting these to the proposed human treatment.
b. Provide the minimal level of gene transfer and/or expression that is estimated to be necessary for the gene transfer protocol to be successful in humans. How was this determined?
c. Explain in detail all results from animal and cultured cell model experiments which assess the effectivenessof the delivery system in achieving the minimally required level of gene transfer and expression.
d. Describe to what extent expression is only from the desired gene (and not from the surrounding DNA).
e. Describe to what extent the insertion modifies the expression of other genes.
f. Provide the percentage of cells that express the desired gene, whether the product is biologically active, and if so, the percentage of normal activity that results from the inserted gene.
g. Describe the extent to which the gene is expressed in cells other than the target cells.
5. Is a retrovirus delivery system being used?
a. Describe the cell types that have been infected with the retroviral vector preparation and describe which cells, if any, produce infectious particles.
b. Describe the stability of the retroviral vector and resulting provirus in terms of loss, rearrangement, recombination, or mutation. Describe steps taken in designing the vector to minimize instability or variation and any assays, with their sensitivities, used to measure stability. Provide information on how much rearrangement or recombination with endogenous or other viral sequences is likely to occur in the patient's cells.
c. Describe laboratory evidence that is available concerning potential harmful effects of the transfer (eg, development of neoplasia, harmful mutations, regeneration of infectious particles, or immune responses).
d. Describe steps taken in the design of the vector to minimize its pathogenicity and describe assays with their sensitivities to determine this.
e. Provide any evidence from animal studies that vector DNA has entered untreated cells, particularly germ-line cells.
f. Provide whether a similar protocol has been conducted in non-human primates and/or other animals. If so, describe the results. Specifically, provide any evidence that the retroviral vector recombined with any endogenous or other viral sequences in the animals.
6. Is a non-retrovirus delivery/expression system being used?
a. Describe animal studies that have been conducted to determine if there are pathological or other undesirable consequences of the protocol (including insertion of DNA into cells other than those treated, particularly germ-line cells).
b. Provide how long the animals have been studied after treatment.
c. Describe any safety studies that have been conducted, including data about the level of sensitivity of such assays.
Randall J. Nelson, Ph.D.
Professor and Associate Vice Chancellor for Research
Memphis, TN 38163
Phone: (901) 448-3533
Fax: (901) 448-5222