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OKIE.

[Ms. of article submitted to PRISM --AMA policy journal]
JUNE 15 1975

DNA Research: Uncertain Peril and Certain Promise

Joshua Lederberg
Stanford University &
Center for Advanced Study in the Behavioral Sciences
Stanford Calif., 94305

Nucleic acids were first isolated from cells and recognized as distinctive
substances over 100 years ago . However, their crucial role in biology was long obscured
for two reasons:

1) a methodological one -- the lack of a direct biological assay for the
specificity of a nucleic acid; and

2) a doctrinal one -- the fallacy of chemical simplicity that characterized the organic
chemists’ model of nucleic acids as homogeneous polymers devoid of informational variety.

As a result, although nucleic acids were known quite early to be systematically
associated with the chromosomes (indeed these are named from the colorability of nucleic
acids with the basic dyes used in cytology) there seemed to be no way to relate them to the
vital functions of the cell, much less to put them to any utilitarian application in
medicine, in agriculture, or in industry.

The scientific revolution of modern biology may be dated to 1944 when O.T.Avery,
Colin MacLeod and Maclyn McCarty working at the Rockefeller Institute announced that
the "transforming agent" of the pneumococcus consisted of DNA. This agent had
already been speculatively compared with genes transferable from one bacterial strain to
another. The announcement quickly provoked a high level of critical discussion, as befit a
claim of such fundamental import. It was crucial to be certain that DNA itself,rather than a
trace contamination with an active protein, was the active agent. Furthermore, too little
was known of bacterial genetics to be sure that an isolated example of the modification of
a bacterial outer coat really did reflect a gene transfer. However, so many new experiments
were prompted by these new claims that, by 1953, the detailed atomic structure of DNA
had been embodied in the now familiar double-helix model of Watson and Crick; and
a whole new discipline of microbial (and viral) genetics had grown up in an area that a few
years before had been widely thought to be wholly disconnected from Mendelian-
Darwinian biology.

The unprecedented acceleration of fundamental chemical biology since 1945 can be
related to the intellectual stimulation of these findings, and the analytical tools that accompany
them, as well as to the policy of continued federal support of scientific research in the
era since World War IT. We may also note that medical microbiology was just then past its
most heroic era, the identification of all of the principle agents of infectious disease,
the very empirical success of which may have tended to hinder closer cooperation with
academic biology.

Although our scientific understanding of the cell has been completely transformed
in the last 30 years, we have yet to see practical applications of molecular biology of
nearly comparable importance. There is very little indeed in the practice of
contemporary medicine (even of clinical genetics) that depends on the knowledge that
ND

DNA has a bihelical structure, which is an appropriate shibboleth for the underlying doctrine
of this new field. This has not eroded our faith that basic approaches to the
understanding of viruses, of the neoplastic and of the aging cell, of the mechanism of the
immune response, or of aberrant chromosomes, will be instrumental to farreaching changes in
medical technology. The human benefit from these will match the theoretical impact that
DNA study has already demonstrated for cell biology.

These expectations for a possibly long-delayed future fruit have been exalted
within the last year or so by some new findings that give us much greater technical capacity
for the laboratory manipulation of microbial DNA. These new methods of "DNA-splicing”
have already opened up many lines of experimental investigation of the structure of
eukaryotic [higher life forms] chromosomes. For it is now possible to fragment an animal
or human cell’s DNA into perhaps a million segments, and transfer a single one to a bacterial
host wherein it can be studied in a microcosm, or whence large quantities of a specific
DNA can be produced for more elaborate analysis than will ever be possible with the
enormously complex original source material.

This technique of gene implantation can also be used to transfer the genetic
information for a given product from one species of cell to another; and this is the direction
that, in my own view, leads to an early chance for

a technology of untold importance for diagnostic and therapeutic medicine: the ready
production of an unlimited variety of human proteins.

Analogous applications may be foreseen in fermentation processes for cheaply
manufacturing essential nutrients, and in the improvement of microbes for the production of
antibiotics and of special industrial chemicals. It has also been foreseen by many workers
in the field that such technical potency may also be associated with public hazards, and
that we then face a Promethean dilemma.

Public policy decision in this field can only lead to social benefit if it is well
informed,equally about the potential risks as well as benefits of further work on DNA-
splicing. Indeed, if any substantial risk can be identified, there is no question of the need
for ethical and operational standards; the only question must be whether their
actual form and implementation result in a true net advantage to the public health and
safety. Too often, the superficially easy way to cope with such a problem is to invoke a
formal regulatory statute, ignoring how well the actual bureaucratic implementation or
policing of the rules meets the intended balance of risks and benefits. Before elaborating
on the policy issues, it may therefore be incumbent to outline some further detail on 1)
the present state of DNA-splicing technology, 2) some promising applications, and 3) the
sources of risk in further work in this field.

1. How we splice DNA in the laboratory.

DNA recombination as the be-all and end-all of sexual reproduction is of course
one of the major happenings of the natural world of life.

Among higher forms, exchange of DNA is almost always limited to members of the same
or closely related species and is generally associated with sexual differentiation, that is
males and females. Bacteria and viruses exhibit some analogous barriers; but also many
exceptions: which may reflect the fragility of the idea of "species" when applied to these
forms. For example, the entire group of “enteric bacteria" including also such forms as
Shigella, Escherichia coli, Proteus, and Serratia can readily be shown to exchange genetic
fragments without special interventions -- we have to assume that this also occurs in nature,
and that it would be difficult to aggravate the risks already latent in that natural occurrence.

An especially interesting and important level of genetic organization in bacteria is
the plasmid: a bit of circular DNA that behaves like an extra small chromosome, and one
that seems to survive in nature by virtue of its easy transmissibility from one bacterial
strain to another. Many different kinds of plasmids are known; medically, the most
prominent today are those which confer transmissible antibiotic resistance on human
pathogens, notably staphylococci and some enteric pathogens like Shigella. These plasmids
are part of the evolutionary history of their host organisms: the spread of antibiotic-
resistance plasmids is the most formidable ecological response that pathogenic
bacteria have yet discovered to our widespread use of antibiotics. Other plasmids are
undoubtedly involved in altering the pathogenicity and _ host-specificity of various
bacteria;therefore in simple self-defense of our species it is urgently important that we learn
what we can about them.

Plasmids have now achieved special prominence also for a technical reason, that
they are especially convenient vehicles for DNA splicing and for the transmission of DNA
segments from one species to another, especially in conjunction with another elegant tool:
the R-[for restriction] enzyme. The R-nucleases are widely distributed among cell types;
they may be an important mechanism by which a cell fends off any "foreign" DNA _ while
protecting its own. For historical reasons, this particular mode of defense has been called
"restriction", and now that we know the responsible enzymes, they have inherited a name
that may be confusing to bystanders. The important attribute of an R- nuclease is that
it recognizes a special sequence of bases in double-stranded DNA, depending on the
particular enzyme, and that it will then cut that DNA leaving a protruded "sticky" end. The
"stickiness" arises from the fact that the same sequence can be recognized on other
protrusions, and that the Watson-Crick rules for uniting separated DNA strands will then
apply.

For example, Dr. Stanley N. Cohen has used an R-enzyme to simplify a naturally
occurring plasmid to the point where it consisted of a small circle of DNA, embracing the
minimum genetic information needed to be able to replicate, plus a single R- enzyme
recognition site. This artificial plasmid, pSC-101, has been an important tool for DNA-
splicing research. When it is exposed to R-enzyme, the circle is cut into a single open length
with sticky ends. Another enzyme, ligase, can be used to rejoin such ends; However, it is
also possible to insert other sticky- ended pieces of DNA from divers sources into the
plasmid, then close it up with ligase. This is then the key to the convenient design and
construction of new DNA molecules which can then be transferred to a bacterial host -- an
experiment which is today’s counterpart of Avery’s report of 30 years ago.

The new DNA does not have to be of the same bacterial species; for example, Dr.
Cohen and his collaborators have already reported on the effective transfer of DNA from
a toad, Xenopus, into E. coli with evidence of the production of toad- like ribosomal
nucleic acids by the modified bacteria.

Besides these plasmids, bacterial viruses are being used in a similar fashion; less
elegantly, segments of DNA from intact bacteria may also be used both for insertions
and as the acceptors. All of these experiments so far depend on the innate (and poorly
understood) ability of bacterial cells to incorporate DNA furnished from without. There have
been many published claims of similar phenomena with plant and animal cell acceptors, but
none to date have the reproducibility and general acceptance as undoubted fact that pertains
to the bacterial work. There is no immediate substance to the idea that these
techniques are applicable to the "genetic engineering of human beings". (In the long run,
the possibility of such technical capabilities cannot be denied in principle, no more than
we can disprove the possibility of a peaceful world, or of a global morale capable of the
wisest disposition of our existing powers for good and evil.)

The special power of these enzymological techniques is that they depend on the
basic chemical structure of DNA (recall the shibboleth!) rather than on the biological
adaptations of particular microbial species. Laboratory manipulations may then synthesize
constructs that occur rarely, if ever, in the natural world. It is difficult, however, to
assess just what can or cannot occur in the entire immense mass of the earth’s biosphere. R-
enzymes,mixed DNA and acceptor bacteria surely occur with some frequency in natural
habitats. The likelihood of a prevalent natural transmission of plasmids among
“unrelated” forms is also enhanced by recent findings of agents with extraordinarily broad
host range. From a practical standpoint, there can be no doubting the power of DNA-splicing
as a means of acquiring specific gene sequences and studying and applying them.
Possible regulatory strictures against promoting the transfer of plasmids among "species
that do not normally do so" may be mooted by the behavior of the newly found agents
-- unless, of course, we are prevented from making just such discoveries by the restriction of
research.

DNA-splicing is however but the most powerful of a range of techniques and
processes whose end-effect is the bringing together of more or less natural assemblages of
DNA information. Indeed, it may prove to be less powerful than older methods -- of sexual
crossing, of transduction with bacteriophage, of DNA transformation -- for special
constructions that involve larger complexes than the segments yielded by the R-enzymes.
These methods in turn are an extrapolation of the artificial breeding of domestic animals
and plants upon which civilization was founded. In any event, the most efficient
application of DNA- splicing requires intimate knowledge of the genetic structure of both
the donor and the acceptor strains, for which breeding methods are important if not
indispensable.

Perhaps the most important single conclusion is that this technology is just in its
infancy, but has already made great leaps; and that it is simple enough that it can be
practised in any laboratory that can handle pure bacterial cultures. Just this simplicity, which
makes for great convenience and rapidity of experimental advance, has been a source
of concern about the proliferation of the methods in the hands of people with less than
mature professional and ethical judgment and with deficiencies in the skills entailed
in containing bacterial cultures in the laboratory.

2. The promise: biosynthetic human proteins

DNA segmentation and splicing is certain to play a vital role in the further
domestication of microbes for such roles as antibiotic development and production of high
quality protein supplements. However, the unique strength of this procedure is to allow the
large scale production of gene products of a less easily domesticated species: the human.
In particular, human proteins already play a substantial role in medicine but one which is
hindered by scarce supplies.

The most attractive options that are visible are the human antibody globulins.
This preference has a theoretical basis. Of all the body proteins, the immune globulins
have evolved to be variable within and between individuals in the fulfillment of a vital
function. Compared to the rare genetic defects in other proteins (like hemophilia), failures
or errors in production of antibody globulin are quite prevalent, and are known to play a
major role in (1) defense against infectious diseases, (2) autoimmune and allergic disease,
and (3) perhaps also in cancer.

The most comprehensive role of biosynthetic proteins would be in passive
immunization against infectious diseases. Animal antisera were once used but had to be
abandoned because of the anti-animal antibody that they provoked in man. Priority targets
for passive globulin therapy are those diseases where either technical or social factors may
lead to gaps in protection by active immunization. They include influenza, hepatitis,
smallpox, encephalitis virus, rubella, herpes, rabies and perhaps also trypanosomes, malaria,
schistosomes, tuberculosis and leprosy and many others.

I believe there is reason for special urgency to develop a backup capability of passive
immunization to prevent a global catastrophe that may result from our becoming too
complacent about active immunzation against diseases like smallpox and polio, and the
technical inadequacy of vaccines like rubella and hepatitis. Our general posture of defense
against viral pandemic is a feeble one. We have no assurance that the next influenza
epidemic will not be slightly more virulent and cost a million lives for lack of a ready
defense.

A broader need applies to polyvalent prophylaxis for infants. The principal
medical argument for breast-feeding is the provision of colostrum and of a continuing supply
of maternal mixed globulins in the milk. There would be a huge and valid market for
polyvalent gamma globulin supplements to infant dietaries both in industrialized and in
poorer countries. An analogous veterinary demand speaks to further efficiency in food
production.

Specific antibodies are, of course, already very widely applicable as diagnostic
reagents of high specificity and selectivity. Blocking antibodies may also be expected to
play auseful role in protecting transplanted tissues and organs from more aggressive
immunological attack by the new host. Conversely, tissue-specific ligating antibodies,
although not necessarily themselves carrying cytotoxic capability, may also be expected to be
useful in enhancing the cell specific toxicity of cancer controlling compounds. Cell
specific reagents will also be invaluable for diagnostic purposes and for the
specific separation of human cell types to be used further for either diagnostic or
therapeutic applications.

Anti-sperm immunity is also being very seriously proposed as an approach to durable
male contraception. I have been quite uneasy about such proposals that involve the
vaccination of men against their own sperm for fear of unwonted side-effects and also
on account of probable difficulties in reversibility. Passive antibody directed against
sperm flagella is demon- strably able to interfere with fertilization simply by the
immobilization of the sperm and should have a minimum of other side-effects. Such
immunizations would be reversible by the spontaneous decay of passive immunity over
periods of from 3 to 6 months. Comparable possibilities exist for the immunization of
women against sperm.

Besides the specific antibody globulins, a number of important but less specific
proteins (complement, properidin) play an important part in defense against
infection. Fibrinolysin (plasmin) and urokinase (plasminogen-activator) represent a group
of enzymes that are experimentaly promising for the control of embolism. Many protein
hormones are equally scarce for clinical trials. The list could be extended
substantially! The most important products are perhaps those that are invisible by present
methodology.

Microbial biosynthesis may well be supplemented by organic synthesis by the
elaboration of proteins in human and hybrid somatic cell culture and by cell free ribosomal
synthesis with contrived m-RNA. Each of these options has its own peculiar difficulties and
hazards and the whole field will be most rapidly advanced by using the best available methods
for a given problem. -------

3. The risks: will dangerous organisms escape?

At the present time, perhaps a half-dozen bacterial species are well enough
understood to be prime vehicles for laboratory study of DNA-splicing. For safety and
convenience, investigators have preferred not to use pathogenic forms wherever feasible.
Significant concern arises from the possibility that the introduction of new genetic
information may (inadvertently) generate a new pathogen for man, or its analogue, a source
of ecological disruption at some other point in the biosphere. The most likely, but not
necessarily the only, source of such genes for pathogenicity are precisely the organisms that
most urgently need further study -- the subtle and insidious killers that are not now
amenable to medical treatment and prevention. These include slow virus infections that
may be involved in a wide range of chronic diseases and cancer, and more familiar
viruses like herpes for which satisfactory vaccines are not now available.

Public discussion of advances in DNA-splicing indeed have been almost totally
focussed on the possible hazards of escape of new forms of microorganisms, rather than on
their utilitarian merits. The most urgent source of concern has been for the prospect of
introducing potential cancer-causing DNA into common bacteria. While it is recognized how
speculative this hazard is, the general territory is so poorly understood that no one can
argue against the need for cautious laboratory procedures. Having gone so far,a number of
workers -- particularly those not previously experienced or trained in medical microbiology
-- have confessed having given almost no thought in the past to problems of microbial
safety; and some of these are now among the most zealous in demanding tight regulation of
further research. As part of this process, there has been a sincere ,almost frantic, effort to
seek out the most remote conceivable hazards.

Viewed as a rather public soul-searching and self- education, these discussions
are invaluable. The main danger is that tentative questions will be incorporated by some
political imperative into iron-clad regulations that will be with us long after anyone has
forgotten why they were instituted. One can after all raise similar questions about the
widest range of human activities: should it be lawful to keep domestic cats now that they
are under suspicion of harboring toxoplasmosis, and possibly leukemia as well? The same
kinds of questions that are asked of microbiology could be lodged against plant
breeding: what positive assurance can there be against the next artificial pollination
producing the weed that will ruin the wheat crop a decade from now? Closer to home,
should we forbid international travel, given the certain knowledge that our
quarantine procedures are quite unable to hinder the importation of exotic diseases?

For each of these cases, and many more, the apparently innocuous doctrine: "As
long as there is any risk, don’t do it!" can only lead to a loss in human welfare. We must
instead make every feasible effort to assess both the risks and the benefits of a given course
of action -- only then are we in a position to weigh the optimal balance. This in no way
may deny the rights of individuals to make voluntary decisions about their exposure to risk,
even if for public benefit. But individuals can hardly make the best policy about their own
future, including their expectations for what medicine will offer for the infirmities of their
own later years, without expert assessment.

These assessments are difficult, problematical, and controversial but a committee
of the National Academy of Sciences has made some headway in trying to classify different
categories of hazard. Where that hazard is reasonably predictable (in the current
atmosphere, now so highly sensitized) laboratory containment precautions akin to
those appropriate for known pathogens have been recommended, and these will doubtless
be comprehendingly complied with. This applies, for example, to experiments involving
the recombination of known tumor virus DNA with bacterial plasmids.

For more conjectural hazards -- like the introduction of antibiotic resistance into
common, non-pathogenic species -- the requirements for high security laboratories may be an
inordinate burden (who, in fact will pay for them?) in relation to the prospective gains.
The best strategy here seems to be the development of safe vectors: plasmids and
bacteria engineered to have little chance of survival outside the laboratory. In fact, in the
long run this is a safer procedure than relying upon uncertain human compliance with fixed
rules and regulations.

Remaining controversies in this field center upon rather complicated analyses of the
remotest kinds of risks. Given some additional time, and a decision to postpone a rigorous
framework of external regulation, most research institutions will be able to work out their
own reasonable plans based on the national guidelines. A premature crystallization of
such regulation will not only frustrate the achievement of pragmatically useful goals,but
will hinder the research needed to make more refined assessments. Those who regard
themselves as guardians of the public safety must count not only the speculative
hazards of these marginal situations, but also the costs to the public health of impeding
their investigation.

This partly voluntaristic approach will not satisfy a demand for absolute assurance
that no foolish experiment is ever attempted. But the history of human institutions should
suffice to show that NO system of sanctions can have such a perfect outcome. The
human species is constantly and inevitably attended by contaminating and parasitic
microbes -- the person suffering from an enteric infection who fails to wash his hands, or
the influenza victim who insists on going to work is behaving unethically, and to the
peril of his fellows. But we would scarcely invoke serious regulatory sanctions in
preference to public education except where there is an unusual public risk, and some
evidence that an enforced quarantine was likely to yield a positive gain.

While a newly awakened scientific conscience is the main source of public
discussion of the hazards of microbiological research, some of it also appears to be
directed to the frustration of its benefits. For some such critics, any measure that might
prolong lives (especially of our own citizens) is merely worsening the population
problem and the excessive consumption of limited world resources. Others are sure that

any technological advance in a  free-enterprise society will inevitably benefit only the
already powerful and wealthy. Still others harbor deep-seated resentments against the
professional and economic dominance of the medical profession. These motifs do not alter by
one whit the actual substance of concerns about the hazards of research; we must
understand their prevalence to foresee the ways in which the issue may be exploited for
reasons quite far from scientific conscience.

Senator Kennedy has remarked that society must give its informed consent to
technological innovation. The power of the purse is enough to enforce that doctrine, nor can
there be any quarrel with it on ethical grounds. The relevant information surely includes
the hazards of saying no to prospects of significant medical advances. The particular
field of DNA- splicing research, far from being an idle scientific toy, or the basis of
expensive and specialized aid to a few lives, promises some of the most pervasive benefits for
the public health since the discovery and promulgation of the antibiotics.