be beneficial. Furthermore, a mild over- 
production of enzyme should not be 
harmful to the cell. In addition, in all 
three the gene has been cloned and a 
complementary DNA is available. 
Since combined immunodeficiency 
due to a defect in the ADA gene in T 
lymphocytes can be corrected by infu- 
sion of normal bone marrow cells from a 
histocompaiible donor, selective replica- 
tion of the normal T cells appears to take 
place (61. This observation offers hope 
that defective bone marrow can be re- 
moved from a patient, the normal ADA 
gene inserted into a number of cells 
through gene therapy, and the treated 
marrow rcimplanted into the patient 
where it may have a selective growth 
advantage. There is also evidence that 
marrow cells containing H PRT ( FI PRT ' f 
may have a selective advantage (in both 
mice and humans) over cells that do not 
(FIPRT ) (7). If selective growth occurs, 
no ablation of the patient's own marrow 
would be necessary. If. however, cor- 
rected stem cells have no growth advan- 
tage over endogenous ones, then partial 
or complete marrow destruction (either 
by irradiation or by other means) may be 
required in order to allow- the corrected 
marrow cells an environment favorable 
for expansion. 
Lillies. The ethics of gene therapy in 
humans has been discussed for many 
years (If) and is being widely debated at 
present (9). Essentially all observers 
have stated that they believe that it 
would be ethical to insert genetic materi- 
al into a human being for the sole pur- 
pose of medically correcting a severe 
genetic defect in that patient — that is. 
somatic cell gene therapy. Attempts to 
correct germ cells (that is, to permit the 
new gene so be passed on to the patient's 
children) or to enhance or improve a 
"normal'’ person by gene manipulation 
do not have societal acceptance at this 
time (9). Flowcver. somatic cell gene 
therapy for a patient suffering a serious 
genetic disorder would be ethically ac- 
ceptable if carried out under the same 
strict criteria that cover other new and 
experimental medical procedures (10). 
The techniques that are now being devel- 
oped for human application are for so- 
matic cell, not germ line, gene therapy. 
The question examined here is: What 
criteria should be used in evaluating gene 
therapy protocols? Three general re- 
quirements. first presented in 1980 (10). 
are that it should be shown in animal 
studies that (i) the new' gene can be put 
into the correct target cells and will 
remain there long enough to be effective: 
(ii) the new gene will be expressed in the 
cell at an appropriate level: and (iii) the 
■mi: 
new gene will not harm the cell or. by 
extension, the animal. These three requi- 
sites. summarized as delivery, expres- 
sion. and safety, will each be examined 
in turn. 
Delivery 
At present, the only human tissues 
that can be used for gene transfer are 
bone marrow and skin cells. No other 
cells can be extracted from the body, 
grown in culture to allow manipulation, 
and then successfully rcimplanted into 
the patient from whom the tissue was 
taken. In the future, as more is learned 
on how to package the injected DNA and 
to make it tissue- or even cell type- 
specific. the intravenous route would be 
the simplest and most desirable. At- 
tempting to give a foreign gene by injec- 
tion directly into the bloodstream is not 
advisable w'ith our present state of knowl- 
edge. since the procedure would be enor- 
mously inefficient and there would be little 
control over the DNA's fate (II). 
Studies are considerably more ad- 
vanced with bone marrow than skin cells 
as a recipient tissue for gene transfer. 
Bone marrow consists of a heteroge- 
neous population of cells, most of which 
are committed to differentiation into 
erythrocytes, lymphocytes, megakaryo- 
cytes. and so on. Only a small proportion 
(0.1 to 0.5 percent) of nucleated bone 
marrow cells arc stem cells (that is. cells 
that have not yet differentiated into spe- 
cific cell types and which divide as need- 
ed to maintain the marrow population). 
In gene therapy, stem cells would be the 
primary target. Because they are low in 
number and are not recognizable, a de- 
livery system for transferring a gene into 
stem cells must be efficient. 
Techniques for transferring cloned 
genes into cells can be grouped in four 
categories: (i) viral, both RNA viruses 
(or retroviruses) and DNA viruses (for 
example. SV40. adenovirus, and bovine 
papilloma): (ii) chemical, such as calci- 
um phosphate-mediated DNA uptake: 
(iii) fusion, that is. fusion of DN A-loaded 
membranous vesicles, such as lipo- 
somes, red blood cell ghosts, or proto- 
plasts. to cells: and (iv) physical, that is. 
microinjection or electroporation. Each 
technique is valuable for certain types of 
experiments, but none can yet be used to 
insert a gene into a specific chromosomal 
site in a target cell. Fusion techniques 
are the least well characterized and will 
not be discussed. As noted, retroviral- 
based vectors appear to be the most 
promising approach at present for use in 
humans. 
Viral Techniques 
RNA viruses (retroviruses). There are 
a number of advantages of vectors de- 
rived from retroviruses as a gene deliv- 
ery system. First, up to 100 percent of 
cells can be infected and can express the 
integrated viral (and exogenous) genes; 
this is in contrast to chemical methods 
where, although most cells take in the 
DNA. as shown by positive assays after 
48 hours, only one cell in 10* to I0 7 
stably expresses the exogenous gene. 
Second, as many cells as desired can be 
infected simultaneously: I0 h to I0 7 is a 
convenient number for a simple proto- 
col. Third, under appropriate conditions 
the DNA can integrate as a single copy at 
a single, albeit random, site, whereas the 
chemical and physical techniques often 
result in the insertion of multiple copies 
of the transferred gene, all linked head- 
to-tail in tandem repeats. Fourth, al- 
though integration is random with re- 
spect to the host genome, it is precise 
with respect to the viral genome — that is. 
the structure of the integrated DNA is 
known. Fifth, the infection and long- 
term harboring of the retroviral vector 
usually docs not harm cells. Finally, a 
wide and controllable host range is avail- 
able. A number of retroviral vector sys- 
tems have been developed. Here we 
concentrate on vectors based on Ma- 
loney murine leukemia virus (MoMLV). 
I) Life cycle and structure. The details 
of the life cycle of retroviruses have been 
reviewed recently (12). In brief, the ret- 
rovirus. composed of an RNA-protcin 
core and a glycoprotein envelope, enters 
a cell where the RNA acts as a template 
for the reverse transcription of the genet- 
ic information into a double strand of 
DNA. This DNA can precisely integrate 
as a single copy, called a provirus, at a 
random location in the genome of the 
host. 
Although much has been learned 
about the regulatory features of retrovi- 
ruses. uncertainties remain. Those fea- 
tures of the proviral structure that are 
thought to be necessary for transcription 
and transmission of the viral genome are 
(see Fig. I): a long terminal repeat (LTR) 
sequence on each end. containing regula- 
tory signals for initiating and terminating 
transcription, sequences required for re- 
verse transcription and others for provi- 
ral integration: short sequences (called 
here, for short, r~ and r') immediately 
adjacent to each LTR and necessary for 
reverse transcription: the packaging se- 
quence called J; in MoMLV. necessary 
for the viral RNA to be packaged into an 
infectious viral particle: and the donor 
(D) and acceptor (A) splice sites. 
st ii-nci:. vot. ::r. 
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Recombinant DNA Research, Volume 12 
