LTR 0 A LTR 
Kip. I. Simplified \truclurc of MoMI.V retroviral provirus l)NA. Abbreviations E. enhancer: 
P. promoter; I. initiation (('apt cite for viral KNA synthesis: r . replication initiation site for 
minus UNA strand (transfer KNA hindinp site): I). donor splice site: 4. packaging sequence: A. 
the major acceptor splice site: r’. replication munition site for plus l)N A strand (purine-rich 
silci. T. terminal IpolytAl additionl site for viral KNA synthesis; t.TK. lonp terminal repeat: 
UJ. K. and l).' arv portions of the I.TK: j •«»*. group-specific (that is. viral core) antipens: pi.', 
p 1 2. p.Vl. and p Ml; pul. KNA-dcpendent UNA polymerase (reverse tninscriptasel: and rue. 
envelope proteins: gp7tl. pl.'E. and K. (Not drawn to scale) 
Retroviral KNA is synthesized from 
the proviral DNA by Ihe host cell’s own 
RNA polymerjsc. A portion of this RNA 
is used in the cell’s translational machin- 
ery to synthesize the viral proteins that 
go into the final viral panicles along with 
the genomic RNA. These viral panicles 
bud ofT from the cell and can then infect 
other cells. 
From experimental studies as well as 
the existence of a number of naturally 
occurring defective viruses, it is known 
that almost all of the regions coding for 
viral proteins pol. and ntr in Fig. 
I ) can be deleted and some or all of these 
sequences replaced with other DNA. 
Once the viral genes are deleted, the 
retroviral vector becomes defective. In 
order to obtain infectious viral panicles, 
a cell harbonng a defective provirus 
must be infected with a "helper" virus, 
which carries all the viral functions need- 
ed — that is. the genes for /><*/. and 
ntr. 
2) Use as gene delivery system. The 
proviral DNA for the desired retrovirus 
Icommonly cither MoMLV or murine 
sarcoma virus (MSV)| is isolated and 
insened into a convenient plasmid. The 
viral genes can then be replaced with the 
exogenous genes of choice by standard 
recombinant DNA techniques. This con- 
struct is used to transfect tissue culture 
cells (for example. NIH 3T3 cells t by a 
convenient gene transfer procedure (for 
example, calcium phosphate). After in- 
fecting the cells with a helper virus (such 
as intact MoMLV). infectious viral parti- 
cles. possessing both the retroviral vec- 
tor and the helper virus, bud oirfrom the 
cells into the surrounding medium. This 
panicle-containing supernatant is col- 
lected and used to infect bone marrow 
cells in culture or. more simply, freshly 
extracted bone marrow is incubated di- 
rectly with (he cells budding the viral 
panicles. The marrow cells are removed 
and injected intravenously into a mouse 
whose bone marrow has been killed by 
x-rays (lethally irradiated). The animal is 
then studied to determine if the trans- 
ferred marrow cells express the desired 
gene from the vector. 
3) Successful gene transfer into adult 
mice. Joyner el til. ( IJ) have successfully 
used this procedure to transfer a func- 
tional gene for neomycin resistance 
{ntrt>' ) into mouse hematopoietic progen- 
itor cells by use of a MoMLV retroviral 
vector. The presence and expression of 
this gene in granulocytic progenitor cells 
rendered these cells resistant to the neo- 
mycin-like antibiotic G4I8 as determined 
by in vitro colony assays. Treated cells 
were injected into lethally irradiated 
mice: Southern blot analysis and colony 
» (N'tltlll K tnu 
assays showed that the net/ gene is 
present and functional in the spleens of 
(he recipient animals |/.f). 
An improvement on this procedure 
would be to treat bone marrow cells with 
a retroviral panicle that could deliver the 
vector but which would nut itself pro- 
duce a spreading infection. Mann el til. 
( 14) have developed a technique for ac- 
complishing this goal. The regulatory 
signal <1 » ( Fig. I ) contains a sequence, the 
exact size and structure of which arc not 
yet known (/.'). that must be present in 
the viral KNA for it to be packaged into a 
viral panicle. A helper virus was con- 
structed with this sequence deleted (tfc I 
by making use of convenient restriction 
endonuclease sites (Bal I and Pst 1 1 
flanking the sequence in MoMLV. The 
tl) helper is able to produce all the viral 
proteins required to make a particle, but 
the panicle does not package its own 
KNA. Since the retroviral vector has a <k 
sequence, it is packaged Consequently, 
the panicle can just infect once: it is only 
a delivery system for the vector, not an 
infectious agent. 
In order to use the d> helper virus 
conveniently, a line of NIH 3T3 cells 
was established with the helper proviral 
DNA permanently integrated (/*/): 
helper viral RNA is produced constitu- 
livcly. The transfection of this cell line 
(called tl»-2) with the retroviral vector 
DNA results 48 hours later in a superna- 
tant that contains viral panicles with 
only the vector. 
Williams el til. t/6) have used the ti»-2 
cell line to place a functioning net/ gene 
into (he hematopoietic cells of adult 
mice. Freshly extracted murine bone 
marrow was layered onto tit-2 cells pro- 
ducing a retroviral vector called MSV 
DHFR-NEO. which contains genes for 
dihydrofolate reductase (DHFR) and 
nco’ in an MSV backbone. The marrow 
and ti<-2 cells were incubated for 48 hours 
under standard incubation conditions: 
similar results were obtained when bone 
marrow cells with the ti>-2 MSV DHFR- 
N EO cell layer were incubated for 6 days 
under Dexter-type conditions 117). The 
viral panicles that budded off into the 
supernatant contained the MSV DHFR- 
NEO vector but. to the extent that could 
be determined, no helper viral RNA. 
Ten days after lethally irradiated mice 
were injected with the treated bone mar- 
row cells, analysis of the regenerating 
spleens showed that the mice carried the 
MSV DHFR-NEO proviral DNA in their 
hematopoietic cells. Individual spleen 
colonics, each arising from a single stem 
cell, were generated by injecting an esti- 
mated one to ten stem cells into another 
group of lethally irradiated mice. Cells 
from individual colonies were able to 
produce spleen colonies in a secondary 
group of lethally irradiated animals. 
These mice also were shown to carry 
MSV DHFR-NEO DNA in their total 
spleen DNA and. in each case, to have 
the same integration site restriction pat- 
tern as the colony from the primary 
mouse. These data show that (he deliv- 
ery system is effective, at least for mouse 
bone marrow cells. Preliminary evidence 
indicates that the neu' gene is expressed 
</A). 
Southern blot analysis of total spleen 
DNA with a number of restriction en- 
zymes revealed in some cases a small 
number of proviral integration sites. This 
result suggests that only a few infected 
stem cells were proliferating to repopu- 
late the irradiated spleen. Secondary 
transfers of individual colonies showed 
that only 7 of 48 colonies (15 percent) 
contained the net/ gene. This is a lower 
limit since an occasional colony might 
have been formed from endogenous stem 
cells that survived the irradiation. These 
data suggest that the present bone mar- 
row procedure might still be made more 
efficient as a delivery system. 
A similar retroviral vector system 
based primarily on MoMLV has been 
developed by Verma and his co-workers 
(/#). In their vk helper virus they substi- 
tuted an amphotrophic (that is. wide host 
ant 
Recombinant DNA Research, Volume 12 
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