From Molecular Biology to Therapy 
of Human Disease 
Fred D. Ledley, M.D. — Assistant Investigator 
Dr. Ledley is also Associate Professor of Cell Biology and Pediatrics at Baylor College of Medicine. He 
received his B.S. degree in physical sciences from the University of Maryland, College Park, and his M.D. 
degree from Georgetown University. He trained in pediatrics and medical genetics at the Children 's 
Hospital, Boston, and Harvard Medical School. His postdoctoral research was conducted with David 
Baltimore at the Massachusetts Institute of Technology and with Savio Woo at Baylor College of Medicine. 
SINCE its inception this laboratory has focused 
on genetic deficiency of the enzyme methyl- 
malonyl CoA mutase (MCM) as a model for molec- 
ular genetic investigations of human disease. We 
have cloned and sequenced human and murine 
cDNAs for MCM, have mapped and cloned the 
genomic locus in mice and humans, and have de- 
scribed a series of mutations causing interesting 
and informative phenotypes of this disorder. 
While continuing these studies, we have begun 
to address the technologies that will be essential 
in moving toward somatic gene therapy for MCM 
deficiency. We have focused our efforts on un- 
derstanding the consequences of metabolic engi- 
neering of MCM activity in human cells, on devel- 
oping methods for efficient manipulation and 
transduction of human hepatocytes, and on as- 
sessing methods for hepatocellular transplanta- 
tion in large animals and humans. 
Retroviral-mediated Correction 
of MCM in Vitro 
We constructed a high-titer, amphotropic re- 
troviral vector containing the full-length normal 
human MCM gene. MCM-deficient fibroblasts 
were transduced with this vector, and the effi- 
ciency of transduction was assessed by semiquan- 
titative identification of the recombinant pro- 
virus. The restoration of MCM activity was 
measured by the ability of cells to metabolize 
['^C]-propionate. The metabolic capacity of fibro- 
blasts was restored to normal levels by transduc- 
tion, even though only 1 0-30 percent of the cells 
were effectively transduced. No further increase 
in metabolic capacity was evident when MCM 
apoenzyme activity was increased above normal 
levels by varying transduction or transfection 
conditions. Transduction of normal fibroblasts or 
hepatoma cells increased MCM apoenzyme activ- 
ity, but not the capacity for propionate metabo- 
lism, suggesting that other steps on these path- 
ways are normally limiting. 
Metabolic cooperation between cells was 
shown to increase the flux of propionate through 
subpopulations of metabolically competent 
cells. The capacity for propionate metabolism of 
hepatic cells was also found to be more than 10- 
fold greater than the maximal capacity of fibro- 
blasts, suggesting that the phenotypic impact of 
metabolic engineering in hepatic cells would be 
greater than in other potential targets for gene 
therapy. 
Transduction of Primary Human 
Hepatocytes 
One strategy for targeting gene therapy to the 
liver involves harvesting and culturing hepato- 
cytes from patients, transducing these cells ex 
vivo with retroviral vectors, and returning them 
to patients by hepatocellular transplantation. We 
demonstrated the feasibility of hepatocellular 
harvest from human liver segments preserved in 
UW (University of Wisconsin) solution, demon- 
strated proliferation of hepatocytes and preserva- 
tion of differentiated hepatocellular functions in 
cells cultured in hormonally defined media, and 
established methods for transducing human cells 
with recombinant retroviral vectors. 
The efficiency of transduction of human cells 
with amphotropic vectors (1-10 percent) was 
significantly less than that observed in various an- 
imal models. Vectors containing MCM were 
shown to constitute expression of recombinant 
mRNA from the 5' long terminal repeat (5'LTR) 
promoter. Higher-efficiency transduction was 
obtained using retroviral vectors with xenotropic 
or gibbon ape enf determinants. Ongoing studies 
are aimed at optimizing conditions for hepato- 
cyte cultivation and transduction. 
Hepatocellular Transplantation 
One of the major factors limiting the applica- 
tion of ex vivo strategies for hepatic gene therapy 
is that hepatocellular transplantation has never 
been attempted in clinical practice. We have as- 
sessed the feasibility in large animal models, us- 
ing a novel method for tracking transplanted 
cells. The cells are stained with a fluorescent dye, 
Dil. This dye is not metabolized or exchanged 
between cells, and engraftment can be identified 
in recipient tissues by fluorescent microscopy or 
flow cytometry. 
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