Federal Register / Vol. 46, No. 233 / Friday, December 4, 1981 / Notices 
59381 ; 
harmful effect of eukaryotic DNA for a 
prokaryote. Therefore, experiments 
which do not include the intentional 
expression of eukaryotic DNA in 
prokaryotes, or prokaryotic information 
in eukaryotes, should be considered 
unlikely to express a product: any harm 
would have to be due to the DNA itself, 
and any competitive advantage for 
survival would likewise have to be due 
to the DNA itself. We will consider 
below those cases where the 
recombinant is designed to express a 
foreign product. 
b. Small pieces in iarge organisms. 
Many recombinant DNA experiments 
involve the introduction of pieces of 
relatively small foreign DNA into a host. 
Thus, for many experiments in E. coii, 
pieces which represent no more than 
0. 5-1% of the recipient’s genetic 
information are added as recombinant 
DNA. In most cases, this kind of new 
information, unless carefully integrated 
with information already present, will 
not significantly alter the ecology of the 
organism (Ayala, 1977). Carried further, 
this argument may suggest that new 
information introduced into a pathogen 
is not likely to significantly change its 
mechanism of pathogenicity. 
Information which must be processed to 
serve as part of a more complex 
biochemical pathway, as is often the 
case for the surface components which 
play an important role in bacterial 
pathogenesis, will be even more difficult 
to alter via recombinant DNA. 
For more complex cells, the added 
DNA will represent a small part of the 
recipient’s genome. This argument 
clearly does not hold as well for viral 
recombinants, where the added 
recombinant DNA information may be 
equal in quantity to what is already 
present. In addition, it may not apply if 
one is speaking of planned changes, 
such as introducing antibiotic resistance 
genes or causing overproduction or 
change in some normal cell component; 
1. e., increasing toxin synthesis, or 
altering the characteristics of a surface 
component of a bacterial cell such that 
the antigenicity of the cell will be 
changed. 
c. Specific Cases. 
i. Expression of Active Peptides: 
Hormones, Toxins. One major class of 
concerns in the discussion of the risks of 
recombinant DNA is based on the 
assumption that bacteria could be 
programmed to express, in large 
quantities, active proteins which would 
cause, either by themselves or through 
their antigenicity, some untoward 
reaction in their host. This issue was 
specifically addressed for E. coil hosts 
at a meeting sponsored by NIAID in 
Pasadena, California in April 1980. Since 
for the sake of discussion the 
participants assumed the “worst case” 
of transfer of the recombinant DNA to 
an established colonizing organism, 
some of the conclusions from that 
meeting can be generalized to the case 
of cloning in the prokaryotes which 
colonize the intestinal tract. Reports 
from that meeting have been published 
in the Recombinant DNA Technicai 
Buiietin (Volumes 3 and 4, 1980-1981) 
and are summarized here. 
If one assumes transfer and 
maintenance of a recombinant plasmid 
in essentially all E. coii of the intestinal 
tract one can calculate that the bacteria, 
at their maximum synthetic capacity, 
will produce in the range of 10® 
molecules/cell/generation, or about 50 
micrograms a day of a product of the 
size of insulin. Given this number as an 
upper limit, one can calculate for 
various active peptides the maximum 
dosage the hose will receive and the 
effect on the host from these dosages. 
Such calculations suggest that most 
hormones with activities similar to 
insulin will not be expected to have 
much, if any, effect on a mammalian 
host, even if they are exported from the 
bacterial factory and absorbed from the 
intestine in active form. Much more 
active peptides or proteins, however, 
might be of some concern under such 
circumstances. If, in addition, one 
imagined as the host anaerobic bacteria 
as well as or instead of aerobes, the 
body dose of an expressed protein might 
be significantly greater. 
Fifty micrograms per day, on the other 
hand, of the most active toxins (e.g., the 
botulinum toxins, Shigeiia dysenteriae 
neurotoxin, tetanus toxin, diphtheria 
toxin) is well above the intravenous 
lethal dose for an average man. Several 
other toxins, including three of plant 
origin (abrin, ricin and modeccin), may 
be lethal to man within the range of 10 
to 100 micrograms. The effects of the 
cytotoxic toxins, intraintestinally 
produced, on the lining of the lower GI 
tract are generally unknown. They 
presumably might (1) damage the 
colonic or intestinal lining directly, (2) 
pass through the lumen and cause 
damage. elsewhere, or (3) pass into the 
bloodstream subsequent to lumen 
damage. Little is known of the 
pharmacokinetics of toxins in the body. 
Thus, these arguments suggest that for 
cloning of foreign proteins, only the 
most active kinds of peptides, at 
maximal levels of expression, would be 
likely to have some effect, if one ignores 
the problems of dissemination and 
establishment. 
ii. Expression of Cross-Reacting 
Antibodies. The implication of inserting 
eukaryotic genetic material coding for 
human “self antigens into prokaryotic 
microbe vectors that parasitize humans 
was also specifically addressed by the 
NIAID meeting in Pasadena, California 
(Workshop on Recombinant DNA Risk 
Assessment). When considered within 
the framework of microbial parasitism 
in its broadest perspective and against 
contemporary concepts of immunologic 
tolerance to “self’ constituents and host 
autoreactive immune responses, the 
injurious potential of autoreactive 
immune responses elicited by “cross- 
reacting” infecting host-vector microbes 
would appear to be extraordinarily low. 
This conclusion is supported by the 
following observations: 
• Literally hundreds of cross-reactivities 
exist between human proteins and 
bacterial and viral surface proteins. 
Most of these cross-reacting proteins 
have not been implicated in 
autoimmune injury. 
• Abundant data exists indicating that 
some cross-reacting systems do lead 
to production of autoantibodies. Most 
of these autoantibodies are low 
affinity binding antibodies which do 
not lead to tissue injury and clinically 
manifest disease. Whether low 
affinity or high affinity antibodies will 
be induced by cross-reacting systems 
displaying human antigens is 
unknown. 
• Some type of regulatory restraint 
exists which allows “self’ to be 
recognized by host immunocompetent 
cells but prohibits such cells from 
launching an “anti-self’ immunologic 
attack (Paterson, 1981). 
iii. Animai Virus Cioning. 
Recombinant DNA experiments 
involving the use of animal virus 
genomes fall into several possible 
categories: 
(1) Cloning of entire or partial 
genomes in prokaryotes, 
(2) Cloning of animal virus genomes in 
eukaryotic cells under conditions where 
the genome may replicate either 
autonomously or in concert with the 
host genome, but cannot produce an 
infectious particle (defective virus or 
non-permissive infection), 
(3) Cloning of an animal virus genome 
in eukaryotic cells under conditions 
where the genome may not only be 
replicated but also may be matured into 
an infectious virus. 
In all these cases, the animal virus 
may be considered as either "recipient” 
or “donor”: the difference is frequently 
semantic only. General considerations 
will be reviewed here. A more technical 
analysis of the issues to each class of 
animal virus are considered in the report J 
of the Ascot Meeting (Federal Register, 
July 28, 1978). 
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