TECHNIQUES FOR GENE THERAPY 
John W. Belmont, M.D., Ph.D., Assistant Investigator 
I. Transfer of Human Adenosine Deaminase into 
Murine Hematopoietic Cells. 
Dr. Belmont's laboratory has chosen the human 
adenosine deaminase (ADA) gene as a convenient 
biochemical marker for gene transfer into murine 
bone marrow cells. One form of human severe 
combined immune deficiency is caused by ADA defi- 
ciency. This is a rare autosomal recessive disorder, 
but it appears to be a good model for gene therapy 
research, because the molecular pathophysiology is 
relatively well characterized, and the disease is suf- 
ficiently severe to warrant investigation of experi- 
mental therapies. Extensive characterization in the 
murine model system should allow a smooth transi- 
tion of promising gene transfer vectors into large 
animal and human experiments. 
A promising basic vector had previously been 
identified that efficiently transduced human ADA 
into mouse hematopoietic progenitors. That vector 
utilizes the Moloney murine leukemia virus long 
terminal repeat (LTR) sequences to provide the 
promoter and enhancer functions for expression. 
Initial experiments indicated that this vector could 
be used to infect pluripotent hematopoietic stem 
cells and allow expression of the human ADA en- 
zyme in their differentiated progeny of all lineages. 
This vector has been reevaluated in transplant ex- 
periments using virus produced from a new packag- 
ing cell line, GP+E86 (provided by Dr. A. Banks). 
This cell line has multiple modifications in the 
packaging elements and substantially reduces the 
risk of generating replication-competent recombi- 
nant virus, which had complicated the previous 
studies. Several aspects of the bone marrow infec- 
tion protocol were systematically investigated, in- 
cluding the possible contribution of costimulation 
of the marrow cells by hematopoietic growth fac- 
tors during the period of infection. All of the 37 
transplanted animals expressed human ADA in 
their peripheral red blood cells for up to 9 weeks 
after the transplant. When the hematolymphoid tis- 
sues were surveyed by Western analysis and ADA- 
specific polymerase chain reaction (PCR), 68% of 
the animals were positive at 6 months post-trans- 
plant. The percentage of cells bearing the provirus 
was not uniform in the various tissues examined. 
This suggested variable infection of the repopulat- 
ing cells in the different lineages. The addition of a 
cocktail of growth factors [including interleukin-3 
(IL-3), IL-la, and IL-6] improved the percentage 
representation of infected cells in the myeloid lin- 
eage. This result is consistent with the possibility 
that stimulation of the cells during infection may 
improve the infection efficiency of the most primi- 
tive repopulating cells but may also affect their sub- 
sequent distribution in the transplanted animal. 
The role of hematopoietic growth factors in the 
growth of pluripotent hematopoietic stem cells is 
being investigated using purified recombinant pro- 
teins. A new competitive repopulation assay has 
been developed to aid in the quantitative analysis 
of growth factor activities. Two families of inbred 
FVB strain transgenic mice are being used as do- 
nors into parental strain recipient mice. The cells 
from the transgenic strains are readily distinguish- 
able by Southern analysis. By stimulating cells of 
one strain during the infection and mixing the cells 
from each strain prior to infusion into the trans- 
plant recipients, it should be possible to analyze 
the growth factor effects on the long-term repop- 
ulating cells. 
Initial experiments examining spleen colony- 
forming unit (CFU-S) progenitors indicate that IL-3, 
granulocyte colony-stimulating factor (G-CSF), IL-6, 
IL-7, and leukemia inhibitory factor (LIF) all in- 
crease retrovirus infection efficiency. The effect of 
IL-6 occurs in the absence of a measurable increase 
in CFU-S number and is consistent with the hy- 
pothesis that this factor influences the latency of G^ 
to G^ transition in stem cells. LIF appears to in- 
crease CFU-S growth in vitro by an effect on a pre- 
cursor cell. This factor also stimulates the growth of 
embryonic stem cells, which raises the possibility 
that the factor directly affects the pluripotent stem 
cells. A technique for in vitro propagation of hema- 
topoietic stem cells would open the way for a vari- 
ety of gene modification strategies. 
Several new vectors bearing immunoglobulin 
and T cell receptor (TCR) enhancer sequences have 
been tested. In tissue culture cell lines, no increase 
in expression was noted in the appropriate lym- 
phoid cell types. A similar result has been observed 
in vivo after bone marrow transplant. The explana- 
tion for the failure of the modifications to affect 
specificity of expression is being investigated. 
Continued 
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