plementation with the mutant allele R93H. Four 
mutations were identified that clustered at the 3' 
end of the gene, close to another mutation with 
this characteristic phenotype that was identified 
previously. 
Alleles with each of these mutations were shown 
to express an intermediate, cobalamin-responsive 
phenotype after gene transfer into cells that do not 
express MCM. Each complemented with clones hav- 
ing R93H in cotransformation experiments, and 
each expressed a mutant phenotype when overex- 
pressed in Saccharomyces cereviseae. Several lines 
of evidence from the laboratory's studies and 
from studies on Propionibacterium shermanii 
MCM point to these mutations occurring within the 
cobalamin-binding domain of the protein. Studies 
are under way to confirm the assignment of this do- 
main by direct binding studies and to identify the 
nature of the defect expressed by R93H. 
Toward Gene Therapy for MCM Deficiency 
Genetic MCM deficiency represents an attractive 
candidate for hepatic gene therapy. Two models 
have been developed. 
One model involves the transduction of primary 
cells with retroviruses capable of permanently 
transforming cells with a normal MCM-containing 
provirus. High-titer retroviral vectors have been es- 
tablished that are capable of complementing the bio- 
chemical defect in mutant fibroblasts and express- 
ing the recombinant gene product in primary 
human hepatocytes. Other studies in the laboratory 
have demonstrated that human hepatocytes can be 
effectively harvested, cultivated, and transplanted 
into immunodeficient animals, where they will en- 
graft within the liver. The transduction of human 
hepatocytes with amphotropic retrovirus was found 
to be significantly worse than in many common ani- 
mal models. Greater efficiency of transduction is 
obtained with xenotropic vectors that recognize a 
distinct receptor on human cells. Coupled with an 
ongoing clinical trial of hepatocellular transplanta- 
tion with retroviral-marked cells, these experiments 
may pave the way for considering an ex vivo scheme 
for somatic gene therapy. 
An alternative scheme for gene therapy involves 
the targeting of DNA vectors to the liver with pro- 
tein-DNA complexes. In collaboration with Dr. 
George Wu (University of Connecticut), an MCM 
expression vector was coupled with asialorosomu- 
coid via polylysine and injected into the tail vein of 
mice. Studies demonstrated that 1) DNA was cleared 
from the blood in 10-20 minutes, 2) >90% of DNA 
was taken up by the liver, 3) DNA was rapidly de- 
graded by the liver and largely eliminated by 24 
hours, 4) MCM mRNA was expressed in the liver 
8-24 hours after administration, and 5) MCM en- 
zyme activity was increased 25-50% over baseline 
(normal) between 24 and 48 hours after administra- 
tion. This approach to therapy may be highly effi- 
cacious in MCM deficiency, which is characterized 
by an acute episode of dyshomeostasis punctuating 
relative stability on dietary and vitamin therapy. 
Targeting to the Thyroid, a Potential 
for Somatic Gene Therapy 
This laboratory has begun to explore methods for 
gene delivery to the thyroid, which represents a 
novel target for somatic gene therapy. The thyroid 
would be a particularly attractive target because it 
can be surgically manipulated using minimally inva- 
sive procedures, because thyroid follicular cells 
have an extremely large capacity for protein synthe- 
sis and are susceptible to extensive temporal regula- 
tion, and because the thyroid has a high rate of con- 
stitutive blood flow. The thyroid may be used as a 
target for treating monogenic forms of congenital 
hypothyroidism (cretinism), acquired thyroid dis- 
ease, and disorders requiring constitution of hor- 
mones or serum proteins in the blood. 
Two methods for gene delivery to the thyroid 
have been established. The first involves a classic ex 
vivo scheme. In dogs, thyroid follicular cells have 
been harvested, cultivated in a serum-free media, 
transduced with retroviral vectors, and transplanted 
into autologous animals. Studies have shown that 
follicular cells preserve differentiated thyroid func- 
tions, can be selected for G418 resistance after 
transduction with NEO-R-containing retroviruses, 
and express the recombinant gene product in vivo. 
The thyroid can also be transiently transformed by 
direct injection of DNA vectors into the body of the 
gland. In rabbits, injection of vectors expressing 
chloramphenicol acetyltransferase leads to high lev- 
els of gene expression for 3-5 days. Studies with 
(8-galactosidase indicate that thyroid follicular cells 
are expressing the recombinant gene. The thyroid 
thus shares with muscle the unusual property of ef- 
fectively taking up and expressing recombinant 
genes after injection of naked DNA. Thus gene ther- 
apy directed at these organs may be considered, us- 
ing genes as medicines that will resemble conven- 
tional pharmaceuticals in their mode of delivery, 
metabolism, and pharmacology. 
Direct Gene Delivery to Joints: 
Potential Gene Therapy for Arthritis 
Studies in Dr. Ledley's laboratory have also been 
directed at assessing methods for transferring genes 
into the synovium of joints as a means of treating 
220 
