Human Disease Gene Identification and Correction 
C. Thomas Caskey, M.D. — Investigator 
Dr. Caskey is also Professor of Molecular Genetics, Biochemistry, Medicine, and Cell Biology at Baylor 
College of Medicine. He received his M.D. degree at Duke University. His internship and residency training 
were in internal medicine, also at Duke; his postdoctoral training was at the NIH under the supervision 
of Marshall Nirenberg. Dr. Caskey is a past president of the American Society for Human Genetics and 
was recently named Distinguished Service Professor by the Board of Trustees of Baylor. 
MOLECULAR genetics offers unprecedented 
opportunities for correction of single-gene 
defects, the development of simple DNA-based 
diagnostics, and the discovery of disease genes. 
This laboratory has made significant progress in 
the development of gene replacement therapies 
for three diseases. The isolation of the fragile X 
locus has been achieved as a collaborative effort 
with two laboratories. DNA-based methods for 
diagnosis of affected males and carrier females 
are now fully developed for four diseases in- 
herited through the X chromosome. 
Genetic Correction of Inherited Disease 
Human genes can now be cloned, placed into 
defective viruses used as vectors, and transferred 
into other cultured cells, embryonic cells, and 
mice. These encouraging developments increase 
the likelihood of successful gene replacement 
therapy. Our laboratory is developing technology 
toward that objective for three heritable diseases. 
Each disease offers different technical and strate- 
gic challenges. 
Adenosine deaminase (ADA) deficiency is 
an inherited autosomal recessive disease. Bone 
marrow transplantation provides a once-per-life- 
time cure, but carries the potential complica- 
tions of graft-versus-host reaction. PEG-ADA ad- 
ministration (ADA attached to polyethylene 
glycol) on a continuing basis has provided im- 
provement in immunologic function of patients, 
but not a cure. We reported previously our high- 
efficiency retroviral delivery of a human ADA 
minigene to mouse stem cells that, when re- 
turned to a lethally irradiated mouse, resulted in 
99 percent rescue and high-level, long-term ex- 
pression of the gene. Thus stem cell infection has 
been achieved in mice, and recent human experi- 
ments are equally encouraging. 
Both "safe" virus and helper cell lines were 
used to deliver the human ADA gene to normal 
and ADA-deficient long-term bone marrow cul- 
tures. The infection rate exceeds 90 percent, and 
the level of ADA expression is equal to normal 
myeloblastic progenitors. This success is a conse- 
quence of precisely defined conditions of stem 
cell stimulation and growth. The parameters in- 
clude cocultivation of human bone marrow cul- 
tures with viral producer lines and selected re- 
combinant interleukin cell-stimulatory reagents. 
Presently the research is focused on the use of 
CD34 monoclonal selected cells. These have 
stem cell properties and represent about lO""*- 
10~' of human bone marrow cells. If transfer can 
be mediated by these selected cells, the current 
large cell-culture requirements for the human 
experiment are reduced to a trivial number of 
cells. Clinical protocols are proposed for 1991. 
Ornithine transcarbamylase (OTC) defi- 
ciency, the most common urea cycle defect in 
humans, is inherited in an X-linked recessive 
manner. The coma, seizures, and retardation are 
the result of hyperammonemia secondary to the 
enzyme deficiency. Liver transplantation pro- 
vides a once-per-lifetime cure, although again 
with the potential complications of graft-versus- 
host reaction. This condition is poorly managed 
through dietary protein restriction and medical 
therapy. Two mouse models, sparse fur and ash, 
are available for developing gene correction tech- 
nology. Using a human OTC minigene under reg- 
ulation of a small bowel liver-specific promoter, 
the sparse fur mouse has been totally corrected 
of the deficiency, including both coat features 
and metabolic defects. 
Surprisingly, the correcting enzyme was ex- 
pressed in small bowel (an ectopic tissue) and 
not in liver. This observation has directed our ef- 
forts for human correction to two target organs: 
liver and small bowel. Toward that objective, two 
viral vectors are now available and are found to 
transfer human OTC successfully to cells in cul- 
ture. For liver delivery, we use a retroviral vector 
with tissue-specific promoter. For small bowel 
delivery, we use a defective adenoassociated 
virus. Each has biosafety features and delivers 
genes efficiently. Our studies now focus on the 
correction of the ash mouse mutant, which has 
profound OTC deficiency. All human studies 
currently involve liver cells in culture. 
Duchenne muscular dystrophy is a severe 
disorder also inherited in an X-linked recessive 
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