gU)4MARY OF THE PROPOSED GUIDELINES FOR RRSEARGH
     ON RECOHJUNANT DNA MOLECULES
         .

f. INTRODUGTION pp/-3

1.  Robert Sinsheimer:  In the advent of a new biological
  research, the research process is changing from one
  of analysis to one of synthesis.  The complications are
  great.  The process is an irreversible one. Vectors will
  escape and there will be no control.  We will not be able
  to. stop production as we could with DDT.

2,  Daniel Gallahan:  The underlying question is how to
  establish a proper policy bias.  There is no moral
  obligation to do this research although it certainly is
  commendable but there is a moral obligation to do no harm;
  and given the uncertainty of the hazards, one should incline
   to caution.  Does the burden of proof lie with the scientist
  ko'show no harm or with the public to show that there is
  harm? The benefit of the doubt should be to the "worriers"

and not the researchers.  This should be the policy bias,
and the guidelines should be reviewed accordingly. With
this in mind, the guidelines seem to be reasonable and
prudent in the conduct of this research.

A. Definition of Experiments Included Within the Guidelines

The proposed guidelines concern experiments in which different segments
of DNA chains are joined by biochemical techniques and the resulting
recombined DNA molecules are then inserted into living cells in which
the molecules can be reproduced.
"hosts."                The recipient cells are called the
      In general, one segment of the recombined DNA molecule will
be derived from an extra-chromosomal genetic element common to the
"host" species.  That segment of DNA is referred to as the "vector."
The other segment of the recombined DNA is referred to as "foreign"
DNA, since it is not normally chemically linked to the vector.  "Host"
cells will usually be either single-celled microorganisms, such as
bacteria, or single cells of higher organisms (plants and animals)
grown under special laboratory conditions.


In nature, exchange and recombination of genetic information (that is,
DNA segments)w a consequence of the sexual mode of reproduction and
generally occurs only between individiial organisms of the same species.
The unique feature of recombinant DNA'experiments is that they provide
a means for combining genes from diverse species.  This unique feature
promises revolutionary potential both for the investigation of basic
biological processes and for approaches to important practical problems
in medicine and agriculture.  The same unique feature is the basis for
concern over the potential biological hazards that might result from
such novel genetic.recombinants.-

B.  Principles Upon Which the Guidelines Are Based

The guidelines are consistent with conclusions formulated at the
International Conference on Recombinant DNA Molecules held at
Asilomar Conference Center, Pacific Grove, California, in February
1975.

1.  Certain experiments are judged to present such serious potential
hazard that they should not be attempted at this time.           ,%
                                                                      
                   k&-
2.  Other experiments can be undertaken provided that thevlafford
new knmr benefits not readily obtained by konventional
methodology and provided that appropriate safeguards are incor-
porated into the experimental design.  Such safeguards should
protect laboratory workers, the community, and the environment
from possible infection with potentially hazardous agents. The
proposed barriers to dissemination of the agents are twofold:
'(1) physical containment of organisms containing recombinant
DNA; and (2) biological containment, that is the use of "hosts"
and "vectors" with limited ability to survive in natural
environments.  The two types of barriers are complementary
and are to be used in combination.                   ,lHrv
                                       & e@
3. Recognizing that present relevant knowledge is limited,/,the
various types of experiments m with regard to
expected degree of potential hazard. The level of containment
achieved by combined physical and biological barriers should
be chosen to match the estimated potential hazard. Containment
should be high at the start and should be modified subsequently
only if there is a substantiated change in the assessed risks.

4.  The guidelines should be reviewed at least annually and
modified to reflect new knowledge.


II.  METHODS OF CONTAINMENT-

1.

David Baltimore:  He asks whether the proposed guidelines
are an appropriate response to the hazard posed by
recombinant DNA molecules and whether the process  that
led to the formulation of guidelines was a responsible
one.  He notes that recombination of DNA molecules has gone
on for aeons; he urges the focus must be on those DNAs that
are a potential hazard, not focus on the mere joining of
DNA molecules.  The hazard is defined as the potentiality
of artificially joined DNA molecules escaping from the
laboratory and disseminating in the general population.
He believes the guidelines are appropriate to meet that
hazard.  He urges that the guidelines be promulgated as
quickly as possible.  He cites the scientific interest
and the medical justification for these experiments to
go on.

2.  Robert Sinsheimer:. Too little attention has been given
  to hazards that might result from new combinations of
  genes that shall be created in this research. Research
Increases the opportunities for potentially dangerous
  gene combinations by many, many orders of magnitude that
  might occur sporadically in nature.  In this regard,
biological containment may not be adequate. The key element
of concern is the irreversibility of the process that will
become magnified as more investigators in laboratories
pursue research in this area. Rather than going the global
approach, the guidelines might be better served by approaching
each desired objective in as specific and safe a manner as
  possible.


A. and B.  Physical Containment

1.  Peter Barton Hutt:  The concept of physical containment
  is both too imprecise and'too subject to the vagaries
  of human fallibilities.  Thus physical containment can
  only be applied when risk is non-existent.  When there
  is any potential risk, protection must rest with biological
   containment.  Consideration of the Pl through P3 levels
  should be undertaken.  Perhaps Pl and P2 should be combined
  into a single level of physical containment. Another
possibility is to upgrade some of the experiments to a
  P3 or P4 level that are currently under Pl and P2 levels.
  The intended requirements for Pl through P3 levels should
  be spelled out.  Perhaps Dr. Barkley who raised several
  points in his presentation could review this section for
  additional comments.  Description of acceptable practices
  should be as clear as possible and might well include
  stated minimum levels of scientific training and experience
  necessary to conduct the experiments.

1.-  Philip Handler:  What does physical containment mean?
  Are Pl and P2 levels really physical containment? Should
  we not consider P3 as the minimum level incorporating
  Pl and P2? This research must go forward but the pace
  must be set by Dr. Fredrickson.

Four levels are defined and designated Pl, P2, P3, and P4 in order of
increasing containment.  Each higher level assumes the practices of
the lower ones.  The specifications reflect existing technology as
developed for work with known pathogenic organisms. It is anticipated
that technical developments will lead to novel alternate procedures
for achieving physical containment.

Pl (minimal):  Strict adherence to the standard microbiological
practices widely used in-research and clinical laboratories
for work with moderately pathogenic organisms. Appropriate
training of all personnel is required.

P2 (low):  Access to the laboratory is limited to authorized
and informed personnel when potentially hazardous organisms
ark being used and.until after appropriate decontamination.
Pipetting by mouth is prohibited, and specific precautions
are required for procedures expected to release aerosols
containing potentially hazardous material.


o P3 (moderate):  Laboratories.are separated from areas used
for less hazardous experiments and access is limited to those
who work therein.  Laboratories should have air pressure
lower than that of the surrounding areas to limit the flow
of airborne organisms into surrounding areas.  Exhaust air
from laboratories should be appropriately. discharged or
decontaminated prior to recirculation. Biological safety
cabinets,shouid be used for all transfer operations and
for all procedures likely to produce aerosols. Gloves are
to be worn and all vacuum lines protected by filters.

Alternate procedures, affording at least equivalent physical
  containnwnt, are suggested for situations in which laboratory
air conditions cannot be controlled in the specified manner.
                                                                     >

o P4 (high):  Special facilities designed to contain highly
infectious and hazardous microorganisms are required. Such
facilities involve isolation by means of airlocks, negative
pressure environments, clothing changes and showers by
personnel, biological safety cabinets, and decontamination
of all air as well as all liquid and solid wastes.  Only a
limited number of such facilities exist in the United States.

c.  Biological Containment

The nature of these barriers will depend on the particular "host" and
"vector" used in each experiment since the barrier is defined by the
limited ability of the "host" and its resident recombined DNA to survive
in natural environments. In general, the choice of "host" and "vector"
will be determined both by the nature of the experiment and by the
required containment.  Therefore, experiments are grouped below
according to possible "host-vector" systems.

D.  Publication

The committee strongly recommends-that all publications dealing with
recombinant DNA work include a description of the physical and
biological containment procedures used.


III.  EXPERIMENTAL GUIDEIINES pp )3-./2,

A.  Experiments That Should Not Be Performed at This Time

1.  Any experiments in which a portion of the DNA to be joined is
derived from highly pathogenic organisms (classes 3, 4, and 5 of
the "Classification of Etiologic Agents on the Basis of Hazard,"
Center for Disease Control, USPHS, Atlanta, Georgia).

2.  Experiments in which a portion of the DNA to be joined
contains genes for production of highly toxic agents.

3.  Experiments in which a portion of the DNA to be joined is
derived from a plant pathogen if the resulting host may acquire
increased virulence or the ability to infect previously
unsusceptible species.

4.  Widespread or uncontrolled release into the environment of
any organism containing a recombinant DNA molecule unless a
series of controlled tests leave no reasonable doubt of safety.

1.  LeRoy Walters:  The guidelines are least clear in -defining
  prohibition on experiments where antibiotic resistance
  may occur,  Further clarification is necessary.

5.  Transfer of genes conferring drug resistance to micro-
organisms not known to acquire such resistance naturally when
such resistance may compromise clinical use of the drug in
medicine or agriculture.

6.  Large-scale experiments with recombinant DNAs known to
result in the formation of harmful products.  Exceptions
are permissible for specific experiments of direct societal
benefit, provided that they are expressly approved by the NIH
Recombinant DNA Molecule Program Advisory Committee.


B.  Guidelines for Permissible.Experiments

5.

6.

1.

Wallace Rowe:  The risks are real.  Tests are necessary
to determine the           risks including a
disturbance in            the E. coli, the introduc-
tion of DNA from E.           cells and the dispersion
of.toxic products from the E. ~011.

Roy Curtiss: He outlines possible tests for the risks as
the probability of escape, the probability of survival
in the outside environment; and the probability of the
manifestation of adverse consequences.  In this regard
we are not only talking about the protection of man but
the protection of the entire biosphere.

Philip Handler:  The NIH should undertake to develop a
program of study of the most important hazardous experiments
This should be done at P4 facilities at the NIH or
Fort Detrick.             p'
        These experiments would provide scientific
evidence to defuse the debate on the potential hazards.
Guidelines can be modified accordingly based on that
evidence.


1. Criteria for use of Escher&& coi!i, strain K-12 as "host"

2.  Robert Sinsheimer:  Experiments should be done with micro-
  organisms that have no association with man or other
   animals.  These organisms should have a very restricted
range of genetic exchange,at a very limited biological
  niche.of viability.

1.  Paul Berg:  Because we are-so familiar with the metabolism
and genetic systems of E. coli, it provides the best
  opportunity to achieve the benefits in this research.
Further, it's the most likely candidate for providing
the safetest host and vector systems.  Developing an
entirely new host-vector system will not answer questions
  of risk.  The ominous and catastrophic scenarios raised
  concerning E. coli could equally be raised with any
  known organism.

  ii  .i
3. Philip Leder:  He has constructed an EK2 host-vector
  system and it has been recently announced in the nucleic
  acid recombinant scientific memorana.  Testing of this
  system is currently underway.  He expects to submit it
  shortly to the Advisory Committee for certification.

Peter Barton Hutt:  What would be the cost in not using
E. coli?  Can incentives be created to foster the
development of IX2 and EK3 systems. Should there be a
2-year limit on EKl to force development of EK2 and EK3?
If EKl is to be used, there must be as detailed an
explanation as possible of that system and its use.


The cornnon bacterium Escherichia boi!i, strain K-12, is the "host"
of choice at the-present time: Well-studied extrachromosomal DNA
molecules known to reside and reproduce in this "host" are avail-
able for use as Vectors."  These include plasmids and bacterio-
phages.  Extensive knowledge of Escerichia coli and its plasmids
and bacteriophages currently affords the most fruitful approach
to the design of containable "host-vector" systems.

.

Three containment classes for 6. coli strain K-12 "host-vector"
systems are defined.  The classes are named EK (for E. cozi K-12)
1, 2, and 3 in order of decreasing ability to survive in natural
environments (that is, increasing containment).

EKl:  This class includes most of the currently known and available
E. coti K-12 "host-vector? systems.  Present knowledge suggests
a low probability for dissemination of plasmid or bacteriophage
  "vectors" carrying recombined "foreign" DNA.  This conclusion
is based in part on the following information:  (1) E. coZi
K-12 is not known to colonize the normal bowel; (2) Certain
plakmids known to be useful as "vectors" have only limited
ability to be transferred to other E. coZi strains commonly
found in animal (including human) bowels and sewerage;
(3) Bacteriophages which specifically infect E. cozi, and
vhich lend themselves well to use in these experiments, are
  available.  Some of these bacteriophages have limited ability
either to escape the "host" cell or to reinfect common (not K-12)
  E. coZi strains.

1.

Biological containment criteria using E. coli K-12 host-vectors

EKl

-host vectors - These are host-vector systems that can be esti-

mated to already provide a moderate level of containment, and include
most of the presently available systems.  The host is always k. coli K-12,
and the vectors include nonconjugative plasmids [e.g., pSC101, ColEl or
derivatives thereof (17-24)] and variants of bacteriophage x (25-27).
    The g. coZi K-12 nonconiugative p Zasmid sys tern is taken
  as an example to iZZust&te the approtimate level of contain-

  ment referred to here.  The avaiZabZe data from experiments
  involving the feeding of bacteria to humans and calves (28-301
  indicate that g. coZi K-12 did not usuaZZy colonize the norma
  bowel, and exhibited little, if any, multiplication while


passing through the alimentary trait even after feeding high
-c&es (i.e., 10' to lOlo bacteria per hwnan or calf).  However,

generaZ extrapolation of these results may not be warranted

SP  because the imphzntation of bacteria into the intestinaz tract
P-                  _-,.. _a. -.
4 ~~`++--~2epends on a nwZ&-o~, such as the nature of the in-

test&a2 fzora present in a given invidivuat and the physiologica

state of the inoculia.
ti            Moreover, since viable E. coZi K-12 can
be found in the feces after humans are fed lo7 bacteria in

broth (28) gr 3 x lo4 bacteria protected by suspension in miZk
             _ _.__._"

._I--- \

(2s)~~-trasductiona:~~ and conjugational! transfer of the plasmid

vectors f?om E. coZi K-12 to resident bacteria in the fecal
              --
w

matter before and after excretion must aZso be conside'red.

            .
- --  Since most conjugative

ptasmids in nature are repressed for expression of donor

fertility, the frequency at which noncorj'ugative ptasmids are

mobiZized and transferred by this sequence of events in vivo
                                                             --

is difficuZt to estimate.  However, in'caZves fed on an
                              w
antibiotic-suppzemented diet, it has been estimated that

such triparenta2 nonconjugative R pZasmid transfer occurs
*
at frequencies of no more than 10m10 to lo-l2 per 24 hours

per caZf (30).
c        In terms of considering other means for
pZasmid transmission in nature, it shouZd be noted that

transduction does operate in vivo for StaphyZococcus
                             --
aureus (32) and probabZy for E. coZi as weZZ.
                                --       However,
no data are avaiZabZe to indicate the frequencies of pkzsmid

transfer in vivo by either transduction or transformation.
         --


  These ob.servations indicate the low probabilities for

possible dissemination of such.pZasmid vectors by accidenta

ingestion, which wouZd probably involve only a feu hundred

or thousand bacteria provided that at least the standard

practices (Section II-A above) are followed, particularly
.
the avoidance of mouth pipetting.  The possibility of coloni-
                                       w

-TJa
\ -

zation and hence of transfer are increased, however, if the

normal flora in the bowel is disrupted by, for example, anti-

biotic therapy (33).  For this reason, persons receiving such

therapy should not work with DNA recombinants formed with any

g. coli K-12 host-vector system during the therapy period and

for seven days thereafter; simitarly, persons who have achlorhydria

or who have had surgical removal of part of the stomach or bowel

should avoid such work, as should those who require large doses

of antacids.

/ -

  The observations on the fate of g. coli K-12 in the

hwnon alimentary tract are also relevant to the containment

of recombinant DNA formed with bacteriophage h variants.

Bacteriophage can escape from the laboratory either as

mature infectious phage particZes or in bacterial host cells

in which the phage genome is carried as a plasmid or prophage.

The fate of g. coli K-12. host cells carrying the phage genome

as a plasmid or prophage is similar to that for plasmid-


containing host cells as discussed above.  The~survival of

the h phage genome when reZeased as infectiousparticles

depends on their stability in nature, their infectivity and

on the probability of subsequent encounters with naturally

occurring X-sensiti.ve E. coli strains.
                        --       AZthough the pro-

babiZity of survival of X and its infection of resident intestinal
*

g. coZi in animals and humans has not been measured, it is

estimated to be small given the high sensitivity of X to the

Zow pH of the stomach, the insusceptibility to X infection of

smooth g. coli cells (the type that normally resides in the

gut),.the infrequency of naturally occurring A-sensitive
g. coli (34) and the failure to detect infective A partkles
in humax feces after ingestion of up to 1011 X particles (35).

Moreover, X part&zles are very sensitive to dessication.

  Establishment of A as a stable lysogen is a frequent
     3

event (loo to 10B1) for the att+ in-t+ cp phage so that this
                               ---

mode of escape would be the preponderant laboratory hazard;

  While not exact, the estimates for containment

afforded by using these host-vectors are at least as

accurate as those for physical containment, and are \

sufficient to indicate that currently employed plasmid

and x vector systems provide a moderate level of bio-

2ogicaZ containment.  Other nonconjugative pZasmids

and bacteriophages that, in association with E. coli
                                                  --

K-12 can be estimated to provide the same approtimate

level of moderate containment are included in the

EKI class,


KK2:  This class consists of combinations of modified E. coli K-12
"hosts" with modified plasmid or bacteriophage "vectors." The
modifications will be achieved by classical genetic manipulation
and must be such that the survival rate of the recombined
"foreign" DNA fragment, in natural environments, is less than
one in 108.  Testing procedures for verification of the proper-
  ties of EK2 systems are described.  (EK2 systems are being
developed and tested and it is anticipated that they will
be available shortly.  Responsibility for certification
of putative EK2 systems presently lies with the NIH
Recombinant DNA Molecule Program Advisory Committee.)

   EK2 host-vectors - These are host-vector systems that have been

genetically constructed and shown to provide a high level of biological
containment as demonstrated by data from suitable tests performed in
                                                                 .
the laboratory,  The genetic modifications of the r. coli K-12 host
and/or the plasmid or phage vector should not permit survival of the

cloned DNA fragment in other than specially designed and carefully
.

regulated laboratory environments at a frequency qreater than 10e8&

This absolute measure of biological containment has been selected

because it is a realistic measurable entity. Indeed, by testing
L

the contributions of preexisting and newly introduced genetic pro-

perties of vectors and hosts, individually or in various combinations,

                            if not prove, that

the specially designed host-vector system can provide a margin of
biological containment in excess of that required.

  For EK2 plasmid vectors, no more than one in lo8 host cells con-

*taining a chimeric plasmid should be able to perpetuate the cloned DNA
fragment under nonpermissive conditions designed to represent the natural

environment either by survival of the original host or as a consequence
of transmission of the cloned DfiA by transformation, transduction or

conjugation to a host with properties common to those in the natural

environment.


  In the construction of EK2 plam`id-host systems it is
important to use the most stabZe mutations avaitable, preferab2;

deletions.  Obviously, the presence of a21 mutations contributing
to higher degrees of bio ZogicaZ containment must be verified
periodicalZy by appropriate tests. In testing the level of

biologica containment afforded by a proposed EK2 plasmid-host

system, it is important to design relevant tests to evakate
the survival of the cloned DNA under conditions that are possible
in nature and that are aZso most advantageous for its perpetuation.
For exaqle, one might conduct a t&parental mating +th a primary.
donor possessing a derepressed F-type or I-type conjugative plasmid,
the safer host with Abiofl-asd, dapD8, A&-chlr, AthyA, deoc, &and
hsdS mutations and a plasmid vector carrying an easily detectable

inserted gene such as for ampicillin resistance or &+, and a
Secondary recipient that is Su+ h&S trp (i.e., permissive for the
recombinant plasmid).  Such matings wouzd be conducted in a mediwn
lacking diaminopimelic acid and thymine and survival of the Apr
Or z+ marker in any of the three strains followed as a function

of time.  Survival of the cloned marker by transduction could also
be evaluated by introdL*ing a knam generalized transducing phage

into the system.  similar experiments should also be oTone
using a secondary recipient that is restrictive for the
recombinant plasmid as well as with primary dcnors possess-
ing repressed conjugative plasm$ds vi th incompatibility
group properties like those commonly found in enteric


microorganisms.  Since a common route of escape of plasmid-

host systems in the laboratory-might be by accidental

ingestion, it is suggested that the same types of experi-

ments be conducted in suitable animal-model systems. In

addition to these tests on survival of the cloned DNA, it

would be useful to determine the survival of the host strain

under nonorowth conditions such as in water and as a function

of drying time after a culture has been spilled on a lab bench.

For EK2 phage vectors, no more than one in lo8 recombinant phage
particles should be able to perpetuate the cloned DNA fragment under
non-permissive conditions designed to represent the natural environ-

ment either (a) as a prophage or pl,asmid in the laboratory host used
for phage propagation or.(b) by surviving in natural environments
and transferring the cloned DNA to a host (or its resident lambdoid
prophage) with properties common to those in the natural environment.

  !l'he phenotypes and genetic stabilities of the mutations
and chromosome azterations included in these X-host systems
indicate that containment we21 in excess of the required
1O-8 or lower survival frequency for the cloned DNA fragment
should be attained.  ObviousZy the presence of all mutations

contributing to this high degree of biological containment
must be verified periodicaZly by appropriate tests.
w                            Laboratory
tests should be performed with the bactetial host to measure
all possible routes of escape of cZoned DNA such as the      .
L
frequency of lysogen formation, the frequency of plasm-id
a


formation and the survival'of the Zysogen or carrier bacterim.

SimilarZy, the potential for perpetuation of the cloned DNA

fragment carried by infectious phage particles could be tested

by challenging typical wild-type g. coli strains or a A-sensitive

nonpermi&ive laboratory K-12 strain, especially one lysogenic
                 .

for a lmbdoid phage..

-: These are EK2 systems for which the increased containment
      independently confirmed by appropriate tests in
       EK3 systems are not presently avaihbk.


2. Classification of experiments using the E. coli K-12

  containment systems

  In the following classification of containment criteria for different
kinds of recombinant DNAs, the stated levels of physical and biological
                       .
containment are minimums.  It is recommended that higher levels of

: biological containment (EK3 > EK2 7
and are equally appropriate for the

   + Shotgun Experiments

EKl) be used if they are available
purposes of the experiment.

  These experiments involve the production of recombinant DNAs
between the vector and the total DNA or (preferably) any partially

purified fraction thereof from the specified cellular source.

2.









1.

Donald Brown:  The guidelines are too strict and inflexible.
Requiring EK2 containment for plant, vertebrate and viral
-DNAs places in effect an indefinite moratorium on research.
Basis for this is that there are at present no EK2 vectors
                             tems will be ce
                           eriments in its P4
facilities.

Robert Sinsheimer:  Shotgun experiments with eukaryotic
DNA incorporated into E. coli are especially troublesome.
d safer and potentially reversible method is to incorporate
such DNA into animal viruses where there is already in
place a viable defense.  One such example that comes to
mind is cowpox.  Here vaccination is possible and if the
hazard were perceived, all infected animals and tissue
culture cells could be destroyed.

4. Allen Silverstone:  He recommends banning of all shotgun
  experiments with mammals and birds.  For all of the
  eukaryotes P4 containment is recommended.  He believes
  the possibility of cancer-causing DNA in lower organisms
  is a potential hazard which requires the highest physical
   containment.  In the case of mammals he also recommends
  a ban on the use of chemically purified DNA.


  (5) Eukaryotic DNA recombinants:
    Primates - P3 physical containment + an EK3 host-vector; or
P4 physical containment + an EK2 host-vector, except for DNA from

embryonic tissue or primary tissue cultures therefrom, and germ-
line cells for which P3 physical containment + an EK2 host-vector

*can be used. The baSis for the lower estimated hazard in the case
.
of DNA from the latter tissues is their relative freedom from

horizontally acquired adventitious viruses.

Other mammals - P3 physical containment + an EK2 host-vector.

8irds - P3 physical containment + an EK2 host-vector.

Cold-blooded vertebrates - P2 physical containment + an EK2
except for embryonic or germ-line DNA which resuire P2
     -
containment + an EKl host-vector.

  Other cold-blooded animals and lower eukaryotes - P2 physical
containment + an EKJ host-vector.  If the eukaryote in this class is a
known pathogen (i.e., an agent listed in Class 2 of ref. 5 or a plant

pathogen) or carries such an agent,

to P3 + EKZ.

Higher plants - P2 physical

the containment should be increased

containment + an EK2 host-vector.

". If the plant carries a known pathogenic agent or makes a product known

to be dangerous to any species,
c             the containment should be raised to P3
physical containment + an EK2 host-vector.
*,


          Prokaryotic DNA recombinants
~====.-----"..~ ,.    _r _
     Prokaryotes that exchange genetic information with E. coli&-

The level of physical containment is directly determined by the rule
of the most dangerous component (see introduction to Section III).

Thus Pl conditions can be used for DNAs from those bacteria in Class 1

of ref. 5 ("Agents of no or minimal hazard....") which naturally ex-

change genes with L. coli; and P2 conditions should be used for such

bacteria if they fall in Class 2 of ref. 5 ("Agents of ordinary potential

hazard...."), or are plant pathogens.  EKl host-vectors can be used for

^ .

B efined as observable under optimal laboratory conditions by transformation,
transduction , phage infection and/or conjugation with transfer of phage, plasmid
and/or chromosomal genetic information.


all experiments requiring only Pl physical containment; in fact, ex-

periments in this category can be performed with 5. coli K-12 vectors

exhibiting a lesser containment (e.g., conjugative plasmids) than EKl

vectors.  Experiments with DNA from species requiring P2 physical con-

tainment which are of low pathogenicity (for example, enteropathogenic

Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae)
.
o can use EKl host-vectors, but those of moderate pathogenicity (for

example, Salmonella typhi, Shigella dysenteriae type I, and Vibrio
cholerae) should use EK2 host-vectors.3

3

The bacteria which constitute Class 2 of ref. 5 ("Agents of ordinary potential
hazard...." ) represent a broad spectrum of etiologic agents which possess dif-
ferent levels of virulence and degrees of communicability. We think it appro-
priate for our specific purpose to further subdivide the agents of Class 2 into
those which we believe to be of relatively low pathogenicity and those which are
moderately pathogenic.  The several specific examples given may suffice to
illustrate the principle.  The Committee has asked the Center for Disease Control
to help prepare a complete list of Class 2 agents subdivided into low and moderate
pathogenicity that could be distributed to interested parties.


Prokaryotes' that do not exchange genetic information with

     E. coli - The minimum containment conditions for this class

consist of PZ physical containment + an EKl host-vector, and apply

when the risk that the recombinant DNAs will increase the patho-

genicity or ecological potential of the host is judged to be minimal.

Experiments with DNAs from pathogenic species (Class 2 ref. 5 plus
.

*plant pathogens) should use P3 + EK2.

Experiments extending the range of resistance to therapeutically

useful drugs and disinfectants should use P2 + EK2 containment or higher

depending on the virulence of the donor.. .


Gv-  (iii) Characterized clones of DNA recombinants derived from

        shotgun experiments

4.

The Environmental Defense Fund: They too urge'that E. coli

not be used.  They also VLb -c~-wLz ,--zrned about use
of the terms, "?;armful" and "harmless," in describing genes
and their products.  They point out that accidents will
occur and would like as broad a definition for "harmful"

,s possible to minimize possible accidents.  They recc-&..-
that EKl strains never be used except,in circumstances
where there is no possible risk to humans.

When a cloned DNA recombinant has been rigorously characterized4

and there is sufficient evidence that it is free of harmful genes,4
then experiments involving this recombinant DNA can be carried out
under Pl + EKl conditions if the inserted DNA is from a species that

exchanges genes with r. coli, and under P2 + EKl conditions if not.

4 The terms "characterized" and "free of harmful genes" are unavoidably
.
vague.  But, in this instance, before containment conditions lower than
fhees used to clone the DNA can be adopted, the investigator must obtain
approval from the granting agency.  Such approval would be contingent upon
data concerning: (a)  th e absence of potentially harmful genes (e.g.,
sequences contained in indigenous tumor viruses or which code for toxic
substances), (b) the relation between the recovered and desired segment
(e.g., hybridization and restriction endonuclease fragmentation analysis
where applicable), and (c) maintenance of the biological properties of
the vector.


<b> Purified.cellular DNAs other 'than plasmids, bacteriophages,
-
  and other viruses

1.  Donald Brown :  The guidelines for ver)tebrate "shotgun"
  experiments are too stringent and rigid,  They
  establish a 'moratorium on gene isolation from all
   vertebrates.  He urged the use of hybridization
  methods at reduced levels of physical and biological
   cant ainment.  Further,
                     L     the requirements for chemically
  purified DNA are not achievable. There can be no
  guarantees for 99% purity. The Committee did not
  kmsider how purified DNA components differ from
  bxilk DNA.
    m

  The formation of DNA recombinants from cellular DNAs that have been
enriched' by physical and chemical techniques (i.e., not by cloning) and

which are free of harmful genes can be carried out under ?ower contain-

ment conditions than used for the corresponding shotgun experiment. In

general, the containment can be decreased one step in physical containment
(P4 -+ P3 + P2 + Pl) while maintaining the biological containment specified

for the shotgun experiment, or one step in biological containment
(EK3 + EK2 + EKl) while maintaining the specified physical contain-

ment--provided that the new condition is not less than that specified

above for characteriied clones from shotgun experiments (Section <a>--
                                                                          -

-..-  iii).

A DNA preparation is defined as enriched if the desired DNA represents at least
99% (w/w) of the total DNA in the preparation.  The reason for lowering the con-
tainment level when this degree of enrichment has been obtained is based on the
fact that the total number of clones that must be examined to obtain the desired
clone is markedly reduced.  Thus,
could, for example,         the probability of cloning a harmful gene
           be reduced by more that lo5 -fold when a nonrepetitive gene
from mammals was being sought. Furthermore, the level of purity specified here
makes it easier to establish that the desired DNA does not contain harmful genes.


   
 The following classifications refer to cases where the
3-y    "foreign' DNA is itself derived from plasmids, bacterio-

6        phages, or viruses.

        w  "Foreign" DNA from viruses that infect animals.

P4 and EK2, or P3 and EK3 are to be used. P3 and EK2
may be used after purification by cloning and demon-
stration that only harmless viral genes are present.

(if.) "Foreign" DNA from viruses that infect plants.

"Foreign" DNA from purified
organelle DNAs.

From primates:  P3 and EKl or P2 and EK2.
Other:  P2 and EKl.-.

(iv)  "Foreign" DNA from prokaryotic plasmids or
   bacteriophages.                           .

If the "j

foreign" DNA is from a species that exchanges

required.


Experiments with prokaqot-c "host-vector" systems other than
E. coZi

1.  Richard Goldstein : As previously noted he recomends E* C"li
be banned as a host-vector with* the next two years. He
urges that the development of alternative prokaryote system
be developed and suggests one possibility is B. subtilisa

  Other prokaryotic host-vector systems are at the speculative,
planning, or developmental stage, and consequently do not warrant
detailed treatment here at this time. However, the containment
criteria for different types of DNA recombinants formed with g.
coli K-12 host-vectors can, with the aid of some general principles
.
Qven here, serve as a guide for containment conditions with other
host-vectors when appropriate adjustment is made for their different

habitats and characteristics.

  In general, the strain 'of any prokaryotic species used as the
host should conform,to the definition of Class 1 etiologic agents
given in ref. 5 (i.e., "Agents of no or minima? hazard...."), and
the plasmid or phage vector should not make the host more hazardous.
In addition, it is recommended that the newly developed host-vector
systems offer some distinct advantage over the g. coli K-12 host-
vectors--for instance, thermophilic organisms or other host-vectors
whose major habitats do not include humans and/or economically im-
portant animals and plants.  Appendix A gives a detailed discussion
of the B. subtilis system,
       -          the most promising alternative to date.


At the initial stage, the host-vector should exhibit at least

a moderate level of biological containment comparable to EKl systems,
and be capable of modification to obtain high levels of containment

comparable to EK2 and EK3.  The type of confirmation test(s) required

to move a host-vector from an EK2-type classification to an EK3-type
will clearly depend upon the preponderant habitat of the host-vector.

: For example, if the unmodified host-vector propagates mostly in, on,

or around higher plants, but not appreciably in warm-blooded animals,
modification should be designed to reduce the probability that the
host-vector can escape to and propagate in, on, or around such plants,
or transmit recombinant DNA to other bacterial hosts that are able to
occupy these ecological niches , and it is these lower probabilities

which should be confirmed.  The following principles should be.followed
in using the containment criteria given for experiments with E. coli
K-12 host-vectors as a guide for other prokaryotic systems. Experi-
ments with DNA from prokaryotes (and their plasmids or viruses)

should be classified according to whether the prokaryote in question
exchanges genetic information with the host-vector or not, and the con-
tainment conditions given for these two classes with r. coli K-12
host-vectors applied.  Transfer of retombihant DNA to plant pathogens
can be made safer by using nonreverting, doubly auxotrophic , non-

pathogenic variants.  Experiments using a plant pathogen that affects
elements of a local flora will require more stringent containment
than if carried out in areas where they are not common.
  Experiments with DNAs from eukaryotes (an 4 their plasmids or
viruses) can also follow the criteria for the corresponding experi-
ments with E. coli K-12 vectors if the major habitats of the qiven
            -


host-vector overlap those of g. eoli. 'If the host-vector has a major

habitat that does not overlap those of k. ~01.5 (e.g., root nodules
in plants), then the containment conditions for some eukaryotic

recombinant DNAs should be increased (for instance, higher plants

and their viruses in the preceding example), while others may be

reduced.


4. Experiments with eukaryotic "host-vector" Systems

1. Maxine Singer:  The use of SV40 virus is troublesome.
  If it is not to be banned altogether, it should probably
  be used only at P4 levels.

2.  Peter Barton Hutt:  In the case ofSV40,if it is to
be used.at all, it should be done only at the P4 level.
Because this virus is known to cause cancer in animals,
  the burden is on the scientist to show there is no
  possibility whatever of harm to man when it is used in
  these experiments.  If it is to be used, its use should
be fully defended in the preamble in the guidelines.

                                                                   1
`3Fz
/    <a> Animal host-vector systems - Because host cell lines generally
have little if any capacity for propagation outside the laboratory, the
primary focus for containment is the vector, although cells should also be
derived from cultures expected to be'of minimal hazard. Given good micro-

biological practices, the most likely mode of escape of recombinant DNAs
from a physically contained%boratory is carriage by humans; thus vectors

should be chosen that have little or no ability to replicate in

human cells.  To .be used as a vector in a eukaryotic host, a DNA
             W-/`

molecule should display all of the following properties:

(1) It should not consist of the whole qenome of

any agent that is infectious for humans or that replicates

to a significant extent in human cells in tissue culture.


(2) Its'functional anatomy should be known--that is,

there should be a clear idea of the1location within the

molecule of:

a) the sites at which DNA synthesis originates

  and terminates,
b) the sites that are cleaved by restriction

  endonucleases,

c) the template regions for the major gene products.

(3) It should be well studied genetically. It is desirable
    4

that mutants be available in adequate number and variety, and

that quantitative studies of recombination have been performed.
                                                           .
  (4) The recombinant should be defective, that is, its pro-

pagation as a virus is dependent upon the presence of a comple-

menting helper genome.  This helper should either (a) be integrated
into the genome of a stable line of host cells (a situation that
would effectively limit the growth of the vector to that particular
cell line) or (b) consist of a defective genome or an appropriate

conditional lethal mutant virus (in which case the experiments
would be done under non-permissive conditions), making vector and
helper dependent upon each other for propagation. However, if
none of these is available, the use of a non-defective genome

as helper would be acceptable.

  Currently only two viral DNAs can be considered as meeting

these requirements: these are the genomes of polyoma virus and

sv40.


  Of these, polyoma virus is highly to be preferred. SV40
is known to propagate in human cells, both in vivo and in vitro,
                                                --

and to infect laboratory personnel, as evidenced by the frequency

of their conversion to producing SV40 antibodies. Also, SV40

and related viruses have been found in association with certain

human neurological and malignant diseases. SV40 shares many

properties, and gives complementation, with the common human

papova viruses.  By contrast, there is no evidence that polyoma

infects humans, nor does it replicate to any significant extent

in human cells in vitro.
                --  However, this system still needs to

be studied more extensively.  Appendix B gives further details

and documentation.

Taking account of all these factors, it is proposed that:

1. Polyoma Virus

5 Recombinant DNA molecules consisting of defective
polyoma virus genomes plus DNA sequences of any non-

pathogenic organism , including Class 1 viruses (5), can

be propagated in or used to transform cultured cells

in P3 conditions; appropriate helper virus can be used

if needed.  Whenever there is a choice, it is urged
that mouse cells, derived preferably from embryos, be
used as the source of eukaryotic DNA. Polyoma virus
is a mouse virus and recombinant DNA molecules contain-
               \

ing both viral and cellular sequences are already known
to be present in virus stocks grown at a high multiplic-
ity.  Thus, recombinants formed in vitro between polyoma
                                    --
virus DNA and mouse DNA are presumably not novel from

an evolutionary point of view.


b. Such experiments can be done, under P4 conditions, if the
    *
recombinant.DNA contains segments of the genomes of Class
2 animal viruses (5).  Once it has been shown by suit-
  .

* able biochemical and biological tests that the cloned
recombinant contains only harmless regions of the viral
genome (see Section IIIB-2-c-i) and that the host rang
.

of the polyoma virus vector has not been altered,

periments can be continued under P3 conditions.

2. SV40 Virus

    a.



/
( .,-

Defective SV40 genomes, with appropriate helper, can

be used in P4 conditions as a vector for recombinant DNA

molecules containing sequences of any non-pathogenic
organism or Class I virus (5), (i.e., a shotgun type
experiment); established lines of cultured cells should

be used.

Such experiments can be carried out in P3 conditions if
the non-SV40 DNA segment is (a) a purified9 segment of
prokaryotic DNA lacking toxigenic genes, or (b) a segment

'The DNA preparation is defined as purified if the desired DNA represents
at least 99% (w/w> of the total DNA in the preparation, provided that it
was achieved or verified by more than one procedure.


of eukaryotic DNA whose function has been established,
Which does not code for a toxic product, and which has
been previously cloned in a prokaryotic host-vector

system.

c. A recombinant DNA molecule consisting of defective SV40
                  .

DNA lacking substantiai segments of the late region,

plus DNA from non-pathogenic organisms or Class I
viruses (5), can be propagated as an autonomous cellular

element in established lines of cells under P3 conditions

provided that there is no exogenous or endogenous helper,

and that it is demonstrated that no infectious virus
                                    -

particles are being produced. Until this has been /     3
                                                                      i

demonstrated, the appropriate containment conditions

specified in 2. 5. and 2. b. shall be used.

Recombinant DNA molecules consisting of defective SV40

DRA and sequences from non-pathogenic prokaryotic or

eukaryotic organisms or Class I viruses (5) can be
used to transform established lines of non-permissive
cells under P3 conditions. ft must be demonstrated that

fro infectious virus particles are being produced; rescue
of SV40 from such transformed cells by co-cultivation

or trahsfection techniques must be carried out in P4

conditions.

gi Efforts should be made to ensure that all celi lines are

free of virus particles and mycoplasma.
   Since SV46 and polyoma are limited in their scope to

act as vectors, chiefly because the amount of foreign DNA


that the normal virions can carry probably cannot exceed
2 x lo6 daltons, we urge,that consideration be given

to the development of systems in which recombinants can
be cloned and propagated purely in the form of DNA, rather

than in t)e coats of infectious agents. Plasmid forms
of viral genomes or organelle DNA should be explored as

possible cloning vehicles in eukaryotic cells.

Plant host-vector systems

1.

r
dk

Milton Zaitlin: In the

guidelines plants have only a

peripheral role; however, they could become extraordinarily
important in recombinant DNA studies. Cloning of higher
plant DNA and incorporating this DNA into bacteria is well
covered by the guidelines.  However, the guidelines are
silent on the incorporation of foreign DNA into the genone
of higher plants.  There are five DNA plant viruses that
could play a significant role here.  These viruses are
transmitted by aphids and are a problem with regard to physical
containment.  Physical containment in the guidelines is
designed to prevent bacteria from getting out of the facility
and to to this, negative air pressures are specified.  But
positive air pressures are required to avoid drawing insects
into the facility.  Guidelines might be modified to specify
positive air pressures under these circumstances. The guide-
lines also are silent on how to deal with a new plant once
it is produced.  Procedures need to be outlined for testing
new plants for any undesirable characteristics they might
have.  The guidelines, therefore,  should include restrictions

f
__  on the release of plants from containment facilities until they

can be tested through several generations.  Plants should be
tested to insure they are not  toxic to animals which might be
likely to eat them.  In plants where the capacity for nitrogen
fixation is introduced, testing must be done to insure that
these plants don't have a competitive advantage overcther -
plants and become pests in themselves.


  Cells in tissue cultures, seedlings, or plant parts (e.g., tubers,
stems, fruits, and detached leaves) or whole mature plants of small

   species (e.g., Arabidopsis) can be handled under the Pl-P4 containment
   conditions that we have specified previously. However, work in most

   plants poses additional problems.  P2 physical containment conditions
  Zcan be provided by: (i) the best insect-proof greenhouses, (ii) appro-
   priate disinfection of contaminated plants, pots, soil, and runoff
   water, and (iii) adoption of the other standard practices for microbio-

   logical work.  P3 physical containment can be sufficiently approximated

   by confining the operations with whole plants to growth chambers like those

    used for ,work with radioactive .  topes, provided that (i)'such chambers
7%     0
   are modified to produce a negativ  pressure environment with the exhaust
   air appropriately filtered, and (ii) that other operations with infectious

   materials are carried out under the specified P3 conditions.  The P2

   and P3 conditions specified earlier are therefore extended to include

   these cases for work on higher plants.

  The host cells for experiments on recombinant DNAs may be cells
in culture, in seedling or plant parts, or in whole plants.  Cells in

.whole plants that cannot be adequately contained should not be used
as hosts for shotgun experiments at this time, and attempts to infect
whole plants with DNA recombinants cloned elsewhere should not be
initiated until their effects on host cells in culture, seedlings,
or plant parts have been studied.


Organelle or plasmid DNAs or DNAs-of viruses of low pathogenicity

to plants may be used as vectors.  In general, the same preference
criteria for selecting host-vectors given in the preceding section on

animal systems apply to plant systems, where organelle and plasmid DNAs

can be grouped together as offering the potential of highly contained

vectors that should be investigated.

  Experiments on recombinant DNAs formed between the initial moderately
contained vectors and DNA from cells of species in which the vector DNA

can replicate, either autonomously or as an integrated segment of the

cell's genome, should use P2 physical containment--provided that the

source of the DNA is itself not pathogenic or known to carry pathogenic

agents, or to produce products dangerous to plants.  In the latter cases,

of if the vector is

periments should be

and

has

Experiments on

DNAs from other
been purified"

10 The DNA preparation is

an unmodified virus of low pathogenicity, the ex-

carried out under P3 conditions.

recombinant DNAs formed between the above vectors

species can also be carried out under P2 if that DNA
         - --
and determined not to contain harmful genes.  Other-


defined as purified if the desired DNA represents

at least 99% (w/w> of the total DNA in the preparation, provided that it
was achieved or verified by more than one procedure.


wise, the experiments should be carried out under P3 conditions if the

source of the inserted DNA is not itself a pathogen, or known to carry

such pathogenic agents, or to produce harmful products--and under P4
conditions if these conditions are not met.

  The development and use of host-vector systems that exhibit a

high level of biological containment permit a decrease of one step in
the physical containment specified above (P4 -f P3 + P2 -+ Pl).

KC> Fungal or similar lower eukaryotic host-vector systems

  The containment criteria for experiments on recombinant DNAs using

these host-vectors most closely resemble those for prokaryotes, rather

                                                               .
than those for the preceding eukaryotes, in that the host cells usually

exhibit a capacity for dissemination outside the laboratory that is

similar to that for bacteria.  We therefore consider that the contain-

ment guidelines given for experiments with r. coli K-12 and other pro-
karyotic host-vectors (Sections IIIB-1 and -2, respectively) provide

adequate direction for experiments with these lower eukaryotic host-

vectors.  This is particularly true at this time since the,development

of these host-vectors is presently in the speculative stage.


IV.  IMPLEMENTATION