only four genomic promoters have been 
reported to show significant activity: me- 
tnllothioncin (2). transferrin I.U). immu- 
noglobulin (,f5l. and elastase (40). How- 
ever. essentially any complementary 
DNA can be attached to an active pro- 
moter. such as metallothionein. and the 
coding sequence will usually he ex- 
pressed in a transgenic mouse under the 
control of that promoter. 
Why are most promoters inactive after 
microinjection into mouse oocytes? At 
least one promoter has been examined in 
this regard: mouse (i" 1 '*' globin. The se- 
quences are found to be heavily methyl- 
ated in mouse tissues where they tire 
inactive but relatively unmethylated in 
tissue culture cells where they are active 
(V/). Therefore, the mouse zygote ap- 
pears to respond to this foreign DNA by- 
covering it with methyl groups which 
remain on the DNA throughout the life- 
time of the animal. Attempts to decrease 
the methvlation of the genomic DNA by 
treating adult mice carrying an exoge- 
nous (J-globin promoter with the hypo- 
methylating drug 5-azacytidinc have 
been essentially unsuccessful (■//). The 
metallothionein promoter, however, 
even if methylated, can remain active 
(42). Why some promoters are inactivat- 
ed by methylalion. or other mechanisms, 
while others are not is not know n. 
Expression from retroviral vectors. If 
a retroviral vector is used for gene trans- 
fer. the transcriptional signals in the re- 
trovirus's own LTK's can be used (Fig. 
I). Expression of exogenous genes car- 
ried by retroviral vectors into bone mar- 
row- cells has been reported by three 
laboratories. The two studies in which a 
neo' gene was expressed in mouse bone 
marrow were described above ( 13. 16). 
The most extensive data, however, are 
from Willis et al. (4.t). A homozygous 
Lesch-Nyhan (l.N) lymphoblast cell line 
was used to determine whether an 
HPKT human hematopoietic cell could 
be corrected by a retroviral vector con- 
taining a functional HPK T gene. The LN 
cells have all the characteristics of a cell 
line totally defective in HPRT. specifi- 
cally a disruption in their inosinate cycle 
that leads to a high purine production 
and a number of other metabolic abnor- 
malties (44). LN cells infected with viral 
particles containing the HPRT vector 
could be rescued in selective medium. 
Seventeen HPRT' clones were isolated 
and studied. These cell lines had HPRT 
levels ranging from 4 to 23 percent of the 
normal level, and the abnormalities asso- 
ciated with a deranged inosinate cycle 
were partially to nearly completely cor- 
rected (4.f). In a corollary study, viral 
particles containing the HPRT-vector 
4IK> 
were used to infect mouse bone marrow 
cells that were then injected into lethally 
irradiated mice (/V). Both human HPRT 
proteins and chronic production of 
HPRT- vector particles were detected in 
the hematopoietic tissue of the mice. 
A problem must still be overcome, 
however. Even though expression of 
HI'RT and neo' genes has been obtained 
in the hematopoietic tissue of irradiated 
mice, the efficiency of the combined 
delivery-expression system is poor. If 15 
percent of stem cells can be infected and 
if 4 to 23 percent of normal expression 
can be obtained in them, can sufficient 
enzyme be synthesized to be of benefit 
to a patient? The issue, once again, is 
whether or not the treated cells w ill have 
a selective growth advantage in the pa- 
tient's body. If they do not. then, either 
the patient's own bone marrow must be 
partially or totally eliminated before re- 
implantation of the treated cells or the 
gene therapy protocol must demonstrate 
at least some expression in nonirradiated 
animals. It must be recognized, howev- 
er. that, in the absence of a true animal 
model for a given genetic disease, it 
might be difficult or impossible to dem- 
onstrate selective growth advantage ex- 
cept in human patients. 
Use of enhancers to increase expres- 
sion. How can the level of expression be 
increased and properly regulated? One 
key element may be the enhancers. 
These are DNA sequences usually 50 to 
150 base pairs in length that increase the 
expression of the adjacent gene 10 to 
1000 times (45). A retrovirus has its own 
enhancer immediately upstream from its 
promoter in the LTR (Fig. I). Enhancers 
are known to be species-specific (46). A 
primate enhancer (for example, the 72 
base pair repeat from SV40) is several 
times more active in primate tissue cul- 
ture cells than in rodent cells. Likewise, 
a mouse enhancer (for example, the 73 
base pair repeat from MSV) is more 
active in rodent cells than in primate 
cells. The promoter acted upon does not 
influence the species specificity (a 
mouse (3-globin promoter and a primate 
SV40 promoter are both activated more 
by a primate enhancer in primate cells 
than in rodent cells), although different 
promoters can be enhanced to different 
extents (47). Retroviral vectors designed 
for therapeutic application in humans 
may need primate, or even human, en- 
hancing sequences rather than the mouse 
ones that are now used. 
Some enhancers may even be tissue- 
specific (4#). With a tissue-specific en- 
hancer it may not be necessary to devel- 
op a cell-specific delivery system. The 
DNA cogld be integrated into all cells 
but only be expressed significantly in 
(hat tissue in which the enhancer is ac- 
tive. Even more precision may he 
achieved if one could place a tissue- 
specific coat on a retroviral particle that 
would direct the virus into the target cell, 
along with a tissue-specific (and possibly 
even a devclopmental-lime-period-spe- 
cific) enhancer in the construct itself. 
Systems like globin undoubtedly have 
other regulatory regions in addition to 
enhancers which recognize cellular fac- 
tors that are involved in control. Much 
information still needs to be learned 
about the regulatory signals in these mul- 
tigene families. 
Expression from plasmid-haseJ 
expression vectors. If a chemical gene 
transfer technique is used as a delivery 
system, then the gene must be inserted 
into an appropriate expression vector. 
An expression vector is a plasmid (usual- 
ly pBR322l in which the complementary 
DNA (or genomic gene) of interest is 
inserted together with regulatory signals. 
A typical expression vector would be 
composed of a promoter (for example, 
from the mouse metallothionein gene), 
the complementary DNA of choice, a 
splice site and polyadenylation site (nec- 
essary for correct processing of the tran- 
scribed RNA). and an enhancer. 
Plasmid-based expression vectors con- 
taining an enhancer have not yet been 
used to transfect bone marrow cells. 
Therefore, how effective expression might 
be is unknown. The inefficiency of the 
presently available delivery systems for 
these vectors was discussed above. 
One additional complication is that 
calcium phosphate-directed transfec- 
tion. as well as microinjection, does not 
usually result in the integration of a 
single copy of the expression vector. The 
plasmid DNA vector appears to be ligat- 
ed or replicated, or both, inside the cell 
to form a long hcad-(o-tail structure 
called a tandem repeat (49). This tandem 
repeat, which can be a few or up to 
hundreds of copies in length, is randomly 
inserted usually in one site in the 
genome. The tandem repeals may pro- 
duce problems for genes requiring intri- 
cate regulation because of the uncertain- 
ty as to how many of the copies are 
active. 
Regulation hy genomic control sig- 
nals. Can cither plasmid-based expres- 
sion vectors or retroviral vectors be used 
to transfer genes that are controlled by 
the gene's own genomic regulatory se- 
quences? Plasmid-based expression vec- 
tors in transgenic mice do respond to 
normal physiological control signals in 
some cases. Mctallothionein-promoted 
genes express primarily in the liver, the 
sctrNtT.. vot.. ::r. 
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Recombinant DNA Research, Volume 12 
