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Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines)

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National Institutes of Health (NIH), PHS, DHHS.


Notice of final actions under the NIH Guidelines and notice of an addition to Appendix D of the NIH Guidelines.


A proposal to certify Kluyveromyces lactis as a host-vector 1 system has been reviewed by the NIH Start Printed Page 27654Recombinant DNA Advisory Committee (RAC) and approved by the NIH Director. This decision is based upon the determination that the K. lactis host-vector 1 system affords a moderate degree of biological containment equal to other certified host-vector 1 systems presently listed in the NIH Guidelines.

Moreover, it has been determined that certain research with this host-vector system does not present a significant risk to health and the environment and therefore will be exempt from the NIH Guidelines (See Section III-F-6 and Appendix C). Appendix C has been modified to indicate the nature of the research that is exempt when performed in a K. lactis certified host-vector 1 system.

In addition, the Office of Biotechnology Activities is updating Appendix D of the NIH Guidelines to include additional lines of experimentation approved by the NIH Director; in this case an experiment involving the introduction of tetracycline resistance into Chlamydia trachomatis that falls under Section III-A-1-a of the NIH Guidelines.


The final action regarding certification of a new host-vector 1 system is effective April 14, 2011.

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Background documentation and additional information can be obtained from the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, Maryland 20892; e-mail at, or telephone at 301-496-9838. The NIH OBA Web site is located at:​oba/​.

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Under the NIH Guidelines, certification of a host-vector 1 system is based on assessment of the biological containment provided by the recombinant DNA vector (plasmid or virus) and the host (bacterial or lower eukaryote) in which the vector is propagated in the laboratory. Per the NIH Guidelines, a combination of vector and host can be certified based on the ability of this system to provide biological containment so that “the following types of `escape' are minimized: (i) Survival of the vector in its host outside of the laboratory and (ii) transmission of the vector from the propagation host to other non-laboratory hosts” (see Appendix I-I). Host-vector 1 systems provide a moderate level of containment (Appendix I). Most low volume (< 10 liters) research with host-vector 1 systems is exempt from the NIH Guidelines and therefore does not require registration with the IBC. High volume (> than 10 liters) research with a certified host-vector 1 system requires IBC review; however, the required containment practices are not as comprehensive as those required for similar research not involving a host-vector 1 system. For example, because of the biological containment provided by the host-vector 1 system, large volume research in a host-vector 1 system does not require all recombinant material to be handled in a closed system.

In making a determination regarding whether a host-vector combination provides moderate biological containment and therefore can appropriately be certified as a host-vector 1 system, the following information is considered: the host's natural habitat and growth requirements; its physiologic properties, particularly reproduction, survival, and mechanisms for exchange of genetic information; and the history of the particular strains and vectors to be used, including mutations that render this organism less able to survive or transmit genetic information.

The Office of Biotechnology Activities received a request from New England BioLabs (NEB) to certify K. lactis as a host-vector 1 system under the NIH Guidelines and to exempt certain research with this host-vector system. Specifically, NEB requested certification of two parental, laboratory-adapted lineages of K. lactis. One is a strain selected by the K. lactis research community for genome sequencing and the second is a K. lactis strain originally isolated in the 1980s from a dairy process. This latter strain has been used as a host strain in the food industry for heterologous protein expression. In addition, NEB has created host strains that are genetically modified through deletion of one or more genes, including genes involved in the synthesis of cellular components (e.g., those involved in biosynthetic activities and modification of host proteins: proteases and glycosylases). Most of the vectors proposed by NEB are plasmids derived from pGBN1, a K. lactis-Escherichia coli shuttle plasmid containing selectable markers for growth in both organisms. The pGBN1-derived plasmids stably integrate into the LAC4 region of the K. lactis chromosome. Other available plasmids for use in K. lactis are derived from the Saccharomyces cerevisiae 2-micron plasmid and remain stable in an episomal state with a copy number of about 50.

NEB noted that K. lactis has a 20+ year history of safe use as an expression system in regulated food industry processes. NEB further stated that they had found K. lactis to provide an excellent system for heterologous protein expression. With regards to its appropriateness for certification as a host-vector 1 system, K. lactis is closely related to S. cerevisiae, which is certified as a host-vector 1 system. K. lactis, however, is unable to exchange genetic material with this closely related yeast. K. lactis can exchange genetic material only with itself and in rare cases with other Kluyveromyces species, thus affording an additional degree of biologic containment.

On November 15, 2010, background information on these actions and instructions for submitting public comment were published in the Federal Register (75 FR 69687). No public comments were received regarding the proposal to certify K. lactis as a host-vector 1 system. On December 7, 2010, the RAC discussed whether there are sufficient data as outlined in Appendix I-II-B-1 of the NIH Guidelines to certify K. lactis and its associated plasmids as a host-vector 1 system.

In its assessment of the request, the RAC considered a number of factors, including that: (1) K. lactis is a natural and indispensable component of cultured dairy processes (including yogurt, cheese and buttermilk) and has been used widely in the food industry to express heterologous proteins (e.g. lactase which has been used to treat lactose intolerance); (2) its optimum growth is at 30 °C, thereby limiting its survival within humans and most other warm-blooded animals; (3) genetic exchange is limited to a few Kluyveromyces species, which rarely cause any disease in humans (with the exception of rare reports of superficial skin disease with K. marxianus, a closely related yeast), and (4) there are no documented cases of disease or toxicity attributed to K. lactis. With regard to the plasmids proposed for certification, the Committee noted that plasmids that can replicate in K. lactis exist in low copy number, will not be efficiently transferred between K. lactis strains, and are not transferrable to other yeast via mating (K. lactis is unable to mate with other yeast). In sum, it was determined that K. lactis meets the requirements of a host-vector 1 system.

The RAC recommended that laboratory-adapted strains of K. lactis should be certified as a host-vector 1 system, as it was the Committee's assessment that laboratory adapted strains grown under optimized laboratory conditions will be at a selective disadvantage, and thus less competitive compared to strains isolated from the wild. Of note, even wild-type K. lactis has a limited natural habitat and growth requirement, mainly Start Printed Page 27655restricted to lactose-rich environments. Plasmids that can be used in K. lactis are not capable of replicating to high copy numbers. Due to the additional containment and corresponding vastly reduced risk to either human health or the environment afforded by host-vector 1 systems, research with this system will be exempt from the NIH Guidelines unless the system is: (i) Capable of producing a molecule that is toxic to vertebrates; (ii) contains DNA from a Risk Group 3 or 4 organism (see Appendix B of the NIH Guidelines); or (iii) will be employed for large scale (> 10 liters) experimentation.

The following new appendix (C-IV) will be added to Appendix C of the NIH Guidelines. The current Appendices C-IV through C-VIII (and sub-appendices) will be renumbered to Appendices C-V through C-IX, respectively.

Appendix C-IV Kluyveromyces Host-Vector Systems

Experiments involving Kluyveromyces lactis host-vector systems, with the exception of experiments listed in Appendix C-IV-A, are exempt from the NIH Guidelines provided laboratory-adapted strains are used (i.e. strains that have been adapted to growth under optimal or defined laboratory conditions). For these exempt experiments, BL1 physical containment is recommended. For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the host organism unmodified by recombinant DNA techniques; the Institutional Biosafety Committee may specify higher containment if deemed necessary.

Appendix C-IV-A Exceptions

The following categories are not exempt from the NIH Guidelines: (i) Experiments described in Section III-B, which require NIH/OBA and Institutional Biosafety Committee approval before initiation; (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval; (iii) large-scale experiments (e.g., more than 10 liters of culture), and (v) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

Additions to Appendix D of the NIH Guidelines

In accordance with Section III-A of the NIH Guidelines, Appendix D of the NIH Guidelines will be modified as follows to reflect a recent approval for the transfer of a drug resistance trait to a microorganism.

Appendix D-118. Dr. Harlan Caldwell at the Rocky Mountain Laboratories may conduct experiments to deliberately introduce a gene encoding tetracycline resistance into Chlamydia trachomatis serovar L2. This approval is specific to Dr. Caldwell and research with this resistant organism may only occur under the conditions as specified by the NIH Director. This approval was effective as of April 26, 2010.

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Dated: May 6, 2011.

Jacqueline Corrigan-Curay,

Acting Director, Office of Biotechnology Activities, National Institutes of Health.

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[FR Doc. 2011-11668 Filed 5-11-11; 8:45 am]