Daily Rules, Proposed Rules, and Notices of the Federal Government
These changes also clarify the criteria for determining whether an experiment to introduce drug resistance into a microorganism raises sufficient public health issues to warrant the experiment being reviewed by the RAC and approved by the NIH Director under Section III-A-1-a of the
In March 2009, NIH/OBA also proposed changes to Section III-E-1 of the
As discussed in more detail in the March 2009
The impetus for these changes to the
In that report, the NSABB noted that practitioners of synthetic genomics or researchers using synthetic nucleic acids in the emerging field of synthetic biology are not necessarily biologists and, therefore, may not have been trained in biosafety. These researchers may be uncertain about how to conduct a risk assessment, as required for research currently subject to the
The recommendation on the need for examination of existing biosafety guidance was accepted by the U.S. Government with the understanding that implementation would be through examination and modification of the
As stated in the March 2009
All of the comments submitted in response to the
Most of the comments regarding synthetic nucleic acids and the
Many of the respondents to this question were involved in developing such products to be used as therapeutics or represent companies and investigators involved in such research. As discussed in more detail herein, the respondents argued that small non-replicating synthetic nucleic acids used as therapeutics are more akin to small molecule drugs than traditional gene transfer agents. A session at the June 23, 2009, public consultation focused on whether certain non-replicating synthetic nucleic acids used in human clinical trials should be exempted from the
The second set of comments focused on the proposed changes to Section III-A-1-a, which addresses certain experiments that involve the introduction of drug resistance into microorganisms. The comments uniformly disagreed with the proposed changes stating that the new proposed criteria were too broad and would lead to federal review of experiments that did not raise public health issues warranting heightened scrutiny. Moreover, they stated that there is no evidence that the current language had failed to serve the public health and therefore the changes were not warranted given the potential problems raised by expanding such review. As discussed herein, the III-A-1-a language in the current
The following paragraphs review (1) The specific comments received on each section of the
In order to ensure that biosafety considerations of synthetic biology research are addressed appropriately, changes are being made to the following sections of the
As discussed herein, the
In addition to the changes being made specifically to address research with synthetic nucleic acids, the following sections are also being revised:
The title of the document will be changed from the
To clarify the applicability of the
The purpose of the
Section I-A was proposed to be changed to:
The purpose of the
NIH/OBA received one comment regarding the use of the term “constructing” in reference to synthetic nucleic acids. The concern was that the
NIH/OBA also received comments requesting a definition of the term “functional equivalents of nucleotides.” This term was intended to capture synthetic nucleic acids that contain nucleotides that have been chemically modified and do not have the same chemical structure as the nucleotides in naturally occurring nucleic acids (see, for example, S. Benner, Redesigning Genetics.
Thus, the amended Section 1-A Purpose will state:
Section 1-A. Purpose
The purpose of the
As a result of these modifications, the
The current definition of a recombinant DNA molecule in the
Section I-B also contains a paragraph that states:
Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (
A second paragraph in the definition states:
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
The final changes eliminate the first paragraph above, referring to synthetic DNA segments, because the
With respect to the definition of recombinant and synthetic nucleic acids, NIH/OBA received several comments with suggestions to use a single definition for recombinant and synthetic nucleic acids. NIH/OBA considered these proposals carefully but decided instead to largely retain the original definition of recombinant DNA, with clarification that it applies to both DNA and RNA and to add a new definition of synthetic nucleic acids. This was done because the definition of recombinant DNA will not change with this revision to the
Section I-B is changed as follows:
Section I-B. Definition of Recombinant and Synthetic Nucleic Acid Molecules:
In the context of the
(i) molecules that a) are constructed by joining nucleic acid molecules and b) can replicate in a living cell,
(ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules,
(iii) molecules that result from the replication of those described in (i) or (ii) above.
In the March 2009
Section I-C-1. The
Section I-C-1-a. All recombinant DNA research within the United States (U.S.) or its territories that is within the category of research described in either Section I-C-1-a-(1) or Section I-C-1-a-(2).
Section I-C-1-a-(1). Research that is conducted at or sponsored by an institution that receives any support for recombinant DNA research from NIH, including research performed directly by NIH. An individual who receives support for research involving recombinant DNA must be associated with or sponsored by an institution that assumes the responsibilities assigned in the
Section I-C-1-a-(2). Research that involves testing in humans of materials containing recombinant DNA developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects. Participation includes research collaboration or contractual agreements, not mere provision of research materials.
Section I-C-1-b. All recombinant DNA research performed abroad that is within the category of research described in either Section I-C-1-b-(1) or Section I-C-1-b-(2).
Section I-C-1-b-(1). Research supported by NIH funds.
Section I-C-1-b-(2). Research that involves testing in humans of materials containing recombinant DNA developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects. Participation includes research collaboration or contractual agreements, not mere provision of research materials.
Section I-C will now read:
Section I-C-1. The
Section I-C-1-a. All recombinant or synthetic nucleic acid research within the United States (U.S.) or its territories that is within the category of research described in either Section I-C-1-a-(1) or Section I-C-1-a-(2).
Section I-C-1-a-(1). Research that is conducted at or sponsored by an institution that receives any support for recombinant or synthetic nucleic acid research from NIH, including research performed directly by NIH. An individual who receives support for research involving recombinant or synthetic nucleic acids must be associated with or sponsored by an institution that assumes the responsibilities assigned in the
Section I-C-1-a-(2). Research that involves testing in humans of materials containing recombinant or synthetic nucleic acids developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects. Participation includes research collaboration or contractual agreements, not mere provision of research materials.
Section I-C-1-b. All recombinant or synthetic nucleic acid research performed abroad that is within the category of research described in either Section I-C-1-b-(1) or Section I-C-1-b-(2).
Section I-C-1-b-(1). Research supported by NIH funds.
Section I-C-1-b-(2). Research that involves testing in humans of materials containing recombinant or synthetic nucleic acids developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects. Participation includes research collaboration or contractual agreements, not mere provision of research materials.
In March 2009, NIH/OBA proposed the following change to the definition of human gene transfer:
For an experiment involving the deliberate transfer of recombinant and/or synthetic nucleic acids into one or more 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).
NIH/OBA had proposed exempting from the
The proposal to exempt basic research with non-replicating synthetic nucleic acids but not to extend that exemption to human gene transfer research was based on the differences in the potential health risk from inadvertent exposure during basic or preclinical work versus intentional exposure in a clinical setting. The doses and routes of administration used in human gene transfer generally increase the safety risks as compared to exposures that may occur in a basic research setting. Moreover, the clinical safety risks to be considered for human gene transfer are not limited to the replicative nature of the vector but include transgene effects, risks of insertional mutagenesis, immunological responses, and potential epigenetic changes. Human gene transfer also raises scientific, medical, social, and ethical considerations that warrant special attention and public discussion.
NIH/OBA received a number of comments from industry, including several comments from the Oligonucleotide Safety Working Group (OSWG), which represents 70 pharmaceutical and regulatory professionals involved in the clinical development of oligonucleotide-based therapies. The OSWG stated that synthetic nucleic acid oligonucleotides that are less than 100 nucleotides and are not delivered in a bacterial or viral vector are more analogous to small molecule drugs than to the agents currently used in human gene transfer. They noted that these constructs can be distinguished from the recombinant agents currently used in human gene transfer by their inability to integrate into the genome or replicate in cells, their lack of a transgene that can be transcribed into RNA or translated into a protein, and their transient nature, i.e., they are degraded within days. They recognized that the review of gene transfer protocols by the RAC is useful to address such risks in gene transfer, but they did not believe that review should be extended to these constructs merely because they are synthetic nucleic acids. They noted that no significant safety issues have arisen in the ongoing Phase I and Phase II clinical trials using short-interfering RNA oligonucleotides (siRNAs). In addition to these trials, there is significant interest in developing clinical applications directed at microRNAs (miRNAs). For recent reviews of the field see K. Tiemann, J. Rossi, RNAi-based therapeutics-current status, challenges and prospects.
While this clinical data is reassuring, several preclinical investigations raised important questions regarding the current understanding about the mechanisms underlying the clinical action of these constructs. For example, clinical trials using a siRNA against vascular endothelial growth factor-A (VEGFA) or its receptor (VEGFR1) in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration have demonstrated promising results. The hypothesis is that the siRNAs that are specific for VEGFA or its receptor are responsible for the clinical responses seen. In 2008, M.E. Kleinman,
In addition to questions regarding the mechanisms of action and potential off target effects raised by these publications, the RAC discussed whether administration of these synthetic RNAs could potentially lead to long-term gene silencing and phenotypic changes. As stated by the OSWG in their comments, one of the reasons for the RAC oversight of recombinant research is to assess the potential for alteration of a research participant's DNA, which could have unknown and unintended consequences. Recent research indicates that siRNA and miRNAs may be involved in long-term gene silencing (A. Verdel,
After considering the comments by the OSWG and other interested stakeholders, as well as the available literature, the RAC initially recommended that NIH/OBA consider an exemption for certain well characterized synthetic oligonucleotides, such as synthetic DNA
The RAC, however, continued to reflect upon the data and considered additional stakeholder input. Further discussions were held with leading experts on RNAi, including Noble Prize laureates Dr. Phillip Sharp and Dr. Craig Mello. The RAC carefully considered the differences between synthetic nucleic acids that are not delivered in vectors and those delivered in bacterial or viral vectors, taking into account their inability to replicate, integrate, or be transcribed or translated. Finally, given the uncertain significance of preclinical data in the absence of adverse effects in the ongoing clinical trials, the RAC concluded that oversight is not warranted at this time. NIH/OBA concurs with this assessment, and the
Therefore, Section III-C-1 will be revised as follows:
Section III-C-1. Experiments Involving the Deliberate Transfer of Recombinant or Synthetic Nucleic Acid Molecules, or DNA or RNA Derived from Recombinant or Synthetic Nucleic Acid Molecules, into One or More Human Research Participants
Human gene transfer is the deliberate transfer into human research participants of either:
1. Recombinant nucleic acid molecules, or DNA or RNA derived from recombinant nucleic acid molecules, or
2. Synthetic nucleic acid molecules, or DNA or RNA derived from synthetic nucleic acid molecules, that meet any one of the following criteria:
a. Contain more than 100 nucleotides; or
b. Possess biological properties that enable integration into the genome (e.g.,
c. Have the potential to replicate in a cell; or
d. Can be translated or transcribed.
No research participant shall be enrolled (see definition of enrollment in Section 1-E-7) until the RAC review process has been completed (see Appendix M-1-B,
Modifications were proposed to augment or clarify experiments that are exempt from the
As stated in the March 2009
To emphasize that research exempt from the
The following recombinant DNA molecules are exempt from the
This portion is amended to read:
The following recombinant or synthetic nucleic acid molecules are exempt from the
A new entry under Section III-F was proposed to exempt from the
NIH/OBA received a number of comments on this proposed exemption. Most of the comments questioned whether this exemption should be extended to certain non-replicating nucleic acids used in human gene transfer because such constructs are likely to pose quantitatively different risks than vector-based gene transfer. The response to these comments is articulated in the prior section of this notice that focuses on Section III-C-1.
With respect to basic research, NIH/OBA received comments questioning whether all non-replicating synthetic nucleic acids used in basic research pose sufficiently low biosafety risks to be exempt from the
NIH/OBA carefully considered all of these comments. With respect to making this exemption apply generally to all nucleic acid constructs, recombinant and synthetic, NIH/OBA notes that the definition of recombinant DNA molecules, which remains unchanged, only includes molecules that can replicate in a living cell or molecules that result from the replication of those described above. Therefore, to include them in the exemption under III-F-1 would be redundant, as this exemption only applies to nucleic acids that cannot replicate and are not derived from those that can replicate. NIH/OBA acknowledges that research with an integrating vector could raise biosafety considerations even if the vector does not replicate. With respect to toxins, a non-replicating expression cassette can only express the toxin in a single cell and the toxin cannot spread from cell to cell, thereby limiting its toxic effect. Nonetheless, NIH/OBA agrees that constructs expressing toxins that are currently reviewed under Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less Than 100 Nanograms per Kilogram Body Weight, should remain subject to the NIH Guidelines. Indeed, under the current NIH Guidelines, even if an experiment falls under a Section III-F exemption, it may still be subject to review under Section III-B-1. For clarity, NIH/OBA therefore decided to specify that toxin-producing expression cassettes that would fall under Section III-B-1 will not be exempt under III-F.
Synthetic constructs that have the potential to integrate will not likewise be exempted because they could inadvertently activate an oncogene, or an integrating sequence containing an oncogene could inadvertently be integrated into a cell and persist and transform that cell and its progeny.
In the March 2009
Section III-F-1 is changed to exempt the following experiments:
Section III-F-1. Those synthetic nucleic acids that: (1) Can neither replicate nor generate nucleic acids that can replicate in any living cell (
Section III-F-1 will now be renumbered to III-F-2 and is amended to clarify that replicating nucleic acids that are not in cells, organisms, or viruses are exempt. The current NIH Guidelines only mentions organisms and viruses, and for clarity the term “cells” has been added. In addition, if a molecule is modified to facilitate entry into a cell, this will also not be exempt. Nucleic acids that are not in a biological system that will permit replication and that have not been modified to enable improved penetration of cell membranes are unlikely to have associated biosafety risks. NIH/OBA received no comments on this change.
The current Section III-F-1 states: “Those that are not in organisms or viruses.”
Section III-F-1 is re-numbered to III-F-2 and will exempt the following experiments:
Section III-F-2. Those that are not in organisms, cells, or viruses and that have not been modified or manipulated (
Revised Sections III-F-3 through III-F-7 retain exemptions that were in the current version of NIH Guidelines (October 2011) with minor revisions. There were no comments to the minor changes made in Sections III-F-3 through III-F-7. The following changes will be made for these Section III-F exemptions.
Section III-F-2 exempts nucleic acid sequences that are essentially copies of those found in nature. The language has been modified as discussed in the March 2009
Section III-F-2 will be re-numbered to III-F-3 and will exempt the following experiments:
Section III-F-3. Those that consist solely of the exact recombinant or synthetic nucleic acid sequence from a single source that exists contemporaneously in nature.
The current Section III-F-3 exempts nucleic acids that are being propagated only in a prokaryotic host that is either the natural host or a closely related strain of the natural host. Again such constructs may already exist outside of a laboratory. It is renumbered to Section III-F-4 and no amendment to the language is made. It exempts the following experiments:
Section III-F-4. Those that consist entirely of nucleic acids 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.
The current Section III-F-4 exempts nucleic acids that are being propagated
Section III-F-5. Those that consist entirely of nucleic acids 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).
Research that falls under Section III-F-6 (formerly Section III-F-5) is exempt because the manipulation of these nucleic acids in a laboratory setting would be equivalent to processes that occur in nature when certain organisms exchange genetic material via physiological processes (
Section III-F-6. 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-6—Sublists of Natural Exchangers, for a list of natural exchangers that are exempt from the
Additionally, Appendix A will be amended to reference Section III-F-6 rather than III-F-5.
Research that falls under the proposed Section III-F-7 exemption also involves a natural physiological process,
Section III-F-7. Those genomic DNA molecules that have acquired a transposable element, provided the transposable element does not contain any recombinant and/or synthetic DNA.
The current Section III-F-6 provides a mechanism by which other experiments that do not raise significant biosafety risks can be exempted from the NIH Guidelines after review by the RAC and approval by the NIH Director. The language has not been amended but, due to the insertion of two additional exemptions, it is being renumbered to Section III-F-8 and will exempt the following experiments:
Section III-F-8. 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-8 for other classes of experiments which are exempt from the
Additionally, Appendix C will be amended to reference Section III-F-8 rather than III-F-6.
Section IV-A addresses the roles and responsibilities of local institutions and investigators in implementing the
The safe conduct of experiments involving recombinant DNA depends on the individual conducting such activities. The
Section IV-A is amended to read:
The safe conduct of experiments involving recombinant or synthetic nucleic acid molecules depends on the individual conducting such activities. The
It is the responsibility of the institution and those associated with it to adhere to the intent of the
Currently, the risk assessment framework of the NIH Guidelines uses the Risk Group (RG) of the parent organism as a starting point for determining the necessary containment level. For example, genetic modifications of a Risk Group 3 organism (defined as agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available) would generally be carried out at Biosafety Level 3 (BL3) containment, but the containment level might be raised or lowered depending on the specific construct and the experimental manipulations. The RAC concluded that the current risk assessment framework under the
NIH/OBA received one comment on its proposed revisions to Section II-A-3. The comment asked for clarification of the meaning of the term “chimera” because it is not currently used in the
Section II-A-3 Comprehensive Risk Assessment currently states:
In deciding on the appropriate containment for an experiment, the initial risk assessment from Appendix B,
Careful consideration should be given to the types of manipulation planned for some higher Risk Group agents. For example, the RG2 dengue viruses may be cultured under the Biosafety Level 2 (BL2) containment (see Section II-B); however, when such agents are used for animal inoculation or transmission studies, a higher containment level is recommended. Similarly, RG3 agents such as Venezuelan equine encephalomyelitis and yellow fever viruses should be handled at a higher containment level for animal inoculation and transmission experiments.
Individuals working with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or other bloodborne pathogens should consult the applicable Occupational Safety and Health Administration (OSHA) regulation, 29 CFR 1910.1030, and OSHA publications, e.g., OSHA 3186-06R (2003 revised). 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 BL2 facility using the additional practices and containment equipment recommended for BL3. Activities involving industrial scale volumes or preparations of concentrated HIV are conducted in a BL3 facility, or BL3 Large Scale if appropriate, using BL3 practices and containment equipment.
Exotic plant pathogens and animal pathogens of domestic livestock and poultry are restricted and may require special laboratory design, operation and containment features not addressed in
The first paragraph is being revised to clarify that the assignment of an organism to a Risk Group in Appendix B,
In deciding on the appropriate containment for an experiment, the first step is to assess the risk of the agent itself. Appendix B,