normal tissue for mctallothioncin synthe- 
sis. and can be induced by cadmium, as 
occurs in vivo for the endogenous gene: 
however, they do not respond to ste- 
roids. which arc another physiologic in- 
ducer in vivo (.50). An immunoglobulin 
gene is expressed in the spleen, the cor- 
rect in vivo tissue, and not in liver (,f.5|. 
A mouse-human (i-globin fusion gene 
expresses in hematopoietic tissue (.5/1. 
In tissue culture cells, a number of 
plasmid-based expression vectors have 
demonstrated at least a degree of normal 
regulation. For example, the human |i- 
globin gene with approximately I kilo- 
base of genomic 5' Hanking sequence can 
be induced (along with endogenous 
mouse globin) in a transfected MEL cell 
(J2). The level of expression is not as 
high as that of the normal endogenous (i- 
globin gene, suggesting that other regula- 
tory signals arc needed. However, trans- 
fection of MEL cells with cosmids carry- 
ing 30 to 40 kilobases of human genomic 
DNA containing (he human (3-globin 
gene docs not result in higher expression 
of human P globin messenger RNA (.5./). 
Miller el al. (.54) obtained encouraging 
results when they placed a rat growth 
hormone complementary DNA together 
with 237 bases of genomic 5’ Hanking 
sequence into the env region of the 
HPRT-vector already described above. 
This growth hormone gene was regulated 
in rescued HPRT fibroblast cells by its 
own genomic promoter and regulatory 
sequences as shown by (i) stimulation by 
glucocorticoid and thyroid hormones, 
which are normal in vivo regulators, and 
(ii) equal activity whether the fragment 
was placed in the same direction or op- 
posite to the vector's LTR's (.54). 
Expression of (he vector in an animal has 
not yet been studied. 
These data provide hope that vectors 
can be built with all the genomic regula- 
tory signals necessary to produce cor- 
rectly controlled expression in target 
cells. In the future, one might use only 
selected portions of a retrovirus in order 
to construct a delivery and integration 
system that would place one copy of the 
vector DNA into the target cell's 
genome. Expression would be controlled 
by the exogenous gene's own genomic 
regulatory signals. One possible problem 
is size: it appears that MoMLV con- 
structs must not be over 9 to 12 kilobases 
in order to be packaged. Since 2 or 3 
kilobases are necessary for essential 
function, only 6 to 9 kilobases are avail- 
able for insert. This amount may be 
adequate, but further studies are needed 
to determine the answer (.5.5). 
Importance of chromosomal loc ation. 
A major question that remains is: How 
XXTOIU K IVX4 
important is chromosome location? Inte- 
gration of a proviral structure can in 
some cases activate a downstream gene, 
as can occur with oncogenes. This prob- 
lem could be eliminated by deleting the 
enhancer and promoter regions from the 
3’ (right-hand) LTR in the retroviral vec- 
tor. One round of reverse transcription 
could (hen occur which would result in 
double-stranded retroviral DNA with 
both LTR's defective. The retroviral 
vector DNA would then integrate with 
no transcription initiation signals. There- 
fore. expression would have to be con- 
trolled by exogenous signals in the in- 
serted gene, and no downstream activa- 
tion of other genes could take place. 
Certainly an integration site that dis- 
rupts an important gene or regulatory 
sequence would normally be detrimen- 
tal. How often this would occur must be 
determined by experiment. It is probable 
though that in most such cases the inser- 
tional event would dimmish the fitness of 
the recipient cell so that it would be 
outgrown by normal cells. 
Are there only certain active chroma- 
tin regions that can allow expression of a 
gene? Or could an expression vector 
take its own "active domain" with it so 
that essentially any location would be 
acceptable? The answers to these ques- 
tions arc still not known. 
Safety 
The third and final criterion for evalu- 
ating a human gene therapy protocol is 
that the delivery-expression system be 
safe. 
Retroviral vectors. Although retrovir- 
uses have many advantages for gene 
transfer, they also have disadvantages. 
One problem is that they can rearrange 
their own structure as well as exchange 
sequences with other retroviruses. In the 
future it might be possible to modify 
retroviral vectors in such a way that they 
become less unstable. At present, how- 
ever. there is the possibility that a retro- 
viral vector might recombine with an 
endogenous viral sequence (,5b) to pro- 
duce packageablc. infectious recombi- 
nant virus. Properties that such a recom- 
binant would have arc unknow-n. but ihe 
potential homology between retroviral 
vectors and as-yet unknown primate 
cancer retroviruses or human T-ccll leu- 
kemia viruses might be sufficiently close 
so that possible recombinants should be 
sought. There is. however, a built-in 
safety feature with the mouse retroviral 
vectors now in use. These mouse struc- 
tures have a very different sequence 
from known primate retroviruses, and 
there appears to be little or no homology 
between the two (.57). Therefore, a po- 
tentially "safe" proviral vector con- 
struct might he one composed of mouse 
LTR's. with their enhancer and promot- 
er regions deleted, and a human gene 
controlled by the appropriate human ge- 
nomic regulatory signals. 
With the present constructs, three 
types of experiments ought to be carried 
out before any retrovirus-treated bone 
marrow is injected into a patient. These 
protocols, designed to test the safety of 
the delivery-expression system, are nec- 
essary since once treated bone marrow is 
reinserted into a patient, it and all retro- 
viruses that it contains arc irretrievable. 
First, studies in vitro with human bone 
marrow arc needed. Marrow cultures 
infected with the therapeutic vector 
should be tested for a period of lime for 
the production of recombinant viruses. 
Any infectious virus isolated should be 
studied for possible pathogenicity. 
Second, studies in vivo with mice are 
needed. Since many retroviral vectors 
arc constructed from mouse retrovir- 
uses. and expression studied in mouse 
bone marrow transplanted into Icthally 
irradiated (or nonirradiated) mice, these 
animals should be followed to determine 
if genomic rearrangement or the site of 
chromosomal integration has resulted in 
any pathologic manifestations or the pro- 
duction of any infectious viruses. 
Third, studies in vivo with primates 
arc needed. A protocol similar to the one 
planned for human application should be 
carried out in primates, not just mice, 
because the endogenous proviral se- 
quences in primate, including human. 
DNA are different from those in mouse 
DNA. Therefore, the nature of any viral 
recombinants would be different. Treat- 
ed bone marrow should be reimplanted 
into primates, the successful transfer of 
intact vector DNA into hematopoietic 
cells demonstrated, the expression of at 
least small amounts of gene product veri- 
fied. and the existence of infectious 
recombinant viruses sought and. if 
found, analyzed. 
Plasmid-hased expression vectors. 
The calcium-phosphate procedure for 
transferring a plasmid-based expression 
vector into human bone marrow has not 
yet been demonstrated to be an effective 
delivery system. However, the proce- 
dure itself docs not appear to represent a 
significant risk of harm. In theory, of 
course, a stem cell could be altered to 
make it carcinogenic so that it would still 
be necessary to follow treated mice over 
time to determine the likelihood of pa- 
thology. Primate studies, however, 
would appear not to be necessary. 
407 t 
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Recombinant DNA Research, Volume 12 
