[Federal Register Volume 76, Number 92 (Thursday, May 12, 2011)]
[Pages 27653-27655]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 2011-11668]



National Institutes of Health

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

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

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


SUMMARY: A proposal to certify Kluyveromyces lactis as a host-vector 1 
system has been reviewed by the NIH

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Recombinant 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 

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

FOR FURTHER INFORMATION CONTACT: 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 [email protected], or 
telephone at 301-496-9838. The NIH OBA Web site is located at: http://oba.od.nih.gov/oba/.

SUPPLEMENTARY INORMATION: 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 [deg]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

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restricted 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, 

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 
    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.

    Dated: May 6, 2011.
Jacqueline Corrigan-Curay,
Acting Director, Office of Biotechnology Activities, National 
Institutes of Health.
[FR Doc. 2011-11668 Filed 5-11-11; 8:45 am]