range) env gene tor the MoMI.V env 
gene, whieh produces a particle coal 
with a narrow host range. This helper 
vir.il construct is called pSAM. Miller cl 
til. I/V) huill a retroviral vector contain- 
ing a full-length complementary DNA for 
the human enzyme HPRT. T his vector, 
called pLPL. was cotransfected along 
with the vli helper. pSAM. into HPRT 
BALB/3T3 cells. One clone Ic7cl) was 
obtained that produced high levels of 
viral particles containing the HPRT vec- 
tor. Injection of these cells into Icthally 
irradiated mice resulted in animals that 
continued to produce HPRT-vcctor par- 
ticles for at least 6 months (/V). The 
infectious particles resulted from the 
presence of low levels <<().! percent of 
the HPR T- vector virus) of packagcahlc 
helper virus along with the injection of 
MoMLV as additional helper which led 
to multiple rounds of replication in the 
host. In addition, human HPRT enzyme 
was detected in spleen cells. 
4) Shortcomings of retroviral delivery 
systems. The evidence indicates that ret- 
roviruses can be used as a reasonably 
efficient delivery system. A gene therapy 
procedure, however, also requires a reli- 
able system. In most of the work report- 
ed to date, a number of cells are found to 
contain altered proviral DNA. The big- 
gest problem appears to be that retrovi- 
ruses have a strong propensity for delet- 
ing sequences during virus replication 
t/Vu). Many vectors have been ineffec- 
tive because the foreign DNA is partially 
or totally removed from the construct or 
is rearranged. For example. Joyner and 
Bernstein (2//) have used the Friend 
spleen focus-forming virus as a potential 
vector system for hematopoietic cells. 
Constructs containing a thymidine ki- 
nase (TK) gene in the v«V region and an 
intact env gene tgp55) were used, along 
with MoMLV as helper, to obtain viral 
particles. The particles were injected 
into Icthally irradiated mice and also 
layered onto rat TK (LTA) cells. 
Southern blot analysis of the integrated 
proviral DNA in erythrolcukemic 
spleens demonstrated vector constructs 
with intact gp55 genes but deleted TK 
sequences, whereas TK' LTA clones 
possessed intact TK genes but deleted or 
rearranged gp55 sequences. In other 
words, in no case could a provirus be 
found that still contained both the TK 
and gp55 genes. Even the successful 
MSV DHFR-NEO vector, which pro- 
duces neo' expression in mice, has lost a 
portion of its DHFR gene during produc- 
tion of the viral particles 1/6). Several 
approaches are being tried to circumvent 
this problem of instability (/V«). 
5) Properties still needed for an opti- 
4IU 
mal delivery system. An ideal delivery 
system not only would be stable but also 
would be tissue-specific. When a genetic 
disorder is in the hematopoietic system, 
then the isolated bone marrow can be 
treated. But no other tissue, except skin 
cells, can be removed, treated, and re- 
placed at present. Since many viruses 
are known to infect only specific tissues 
(that is. to bind to receptors that are 
present only on certain cell types), a 
retroviral particle containing a coat gly- 
coprotein that recognizes only human 
hematopoietic stem cells would permit 
the retroviral vector to be given intrave- 
nously with little danger that cells other 
than (hose in the marrow would be in- 
fected. Such specificity could permit the 
liver and brain, for example, to be treat- 
ed individually. In addition, the danger 
of inadvertently infecting germ cells 
could be eliminated. One problem, how- 
ever. is that cell replication appears to be 
necessary for integration. It would not 
be possible to infect nondividing brain 
cells, for example, as far as we now’ 
know. 
The optimal system not only would 
deliver the vector specifically into the 
cell type of choice but would also direct 
the vector to a predetermined chromo- 
somal site. Specific insertion into a se- 
lected site of a chromosome by means of 
homologous recombination can be readi- 
ly achieved in lower organisms but ap- 
pears to be a formidable task in mam- 
mals. whether retroviral vectors or plas- 
mid-based vectors arc used. Present evi- 
dence suggests that homologous site- 
specific integration occurs at a very low 
level, when it occurs at all. in mammals 
121 ). 
DNA druses. Viruses, such as SV40. 
with DNA as the nucleic acid in their 
core have been employed for several 
years as gene transfer vectors (22). A 
conditionally nonreplicating adenoviral 
vector has recently been developed that 
will efficiently infect animal and human 
cells (including hematopoietic cells) with 
the result that one or a few copies of the 
recombinant virus are integrated into the 
host cell's genome {23). Whether adeno- 
viral vectors will be as efficient as retro- 
viral vectors, or will offer other advan- 
tages as a gene transfer delivery system, 
remains to be determined. One subcate- 
gory of DNA viruses should be men- 
tioned: bovine papilloma virus (BPV) 
(24). This viral DNA replicates extra- 
chromosomally so that BPV-based vec- 
tors may prove to be useful for maintain- 
ing genes in cells in a nonintegrated 
manner. Transfection of hematopoietic 
cells with BPV- vectors has not vet been 
reported. 
Chemical Techniques 
The other procedure under active con- 
sideration for insertion of genes into hu- 
man bone marrow cells is calcium phos- 
phate-mediated DNA uptake. The origi- 
nal procedure of Graham and van der Eb 
(23) was modified by Wigler cl til. (26) in 
order to insert into the genome of mam- 
malian cells growing in culture a frag- 
ment of DNA carrying one or more 
genes. A number of genes have been 
used including the herpes simplex TK 
gene complementing TK cells, the 
DHFR gene protecting against the drug 
methotrexate, and the nco r gene protect- 
ing against the antibiotic G4I8. 
Procedure. Transfection is carried out 
by pipetting a suspension of DNA. com- 
plcxcd into small precipitates with calci- 
um phosphate, onto a monolayer of cells 
growing in a tissue culture dish (26). A 
number of techniques are used to in- 
crease the efficiency of transfection in 
different cell types: for example, diethyl- 
aminocthyl dextran can be employed in- 
stead of calcium phosphate or the cells 
can be shocked with glycerol after 2 
hours of incubation (27). The efficiency 
of the process varies with the cell line. 
Under optimal conditions and very re- 
ceptive cells (for example, mouse L 
cells), one cell in I0 : to 10’ can be 
obtained that has integrated and ex- 
pressed the exogenous DNA. Because 
the usual efficiency is 10"' to I0' 7 . a 
procedure is required to detect the occa- 
sional transfected cell. In other words, a 
gene must be present that can protect the 
cell from a lethal selective agent that is 
added to the incubation medium or that 
complements a genetic defect (HPRT or 
TK. for example). The transfected cell 
will survive while all others arc killed. 
Attempts to obtain transfected cells 
without selective pressure have general- 
ly been unsuccessful. 
Transfection appears to work poorly 
in suspension cells, namely bone marrow 
cells. Efficiencies can only be estimated, 
but the value is probably one cell in I O'* 
or 10 7 . Using the powerful selection sys- 
tem offered by the mutant DHFR gene 
(isolated from 3T6-R400 cells) that pro- 
vides exceptional resistance to metho- 
trexate. Carr ct til. (2/0 reported that the 
calcium phosphate transfer technique 
can be successfully employed to obtain 
mouse bone marrow- cells that contain a 
functional exogenous DHFR gene. The 
permanently transfected cells can par- 
tially repopulate a lethally irradiated 
mouse. These results support the studies 
of Cline ei til. (2V) who reported success- 
ful transfer of a functional DHFR gene 
into the bone marrow of mice. However. 
sc ii nc k. vo!.. ;:c. 
Recombinant DNA Research, Volume 12 
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