Molecular Genetic Investigation and Therapy for 
Inborn Errors of Metabolism 
Fred D. Ledley, M.D. — Assistant Investigator 
Dr. Ledley is also Associate Professor of Cell Biology and Pediatrics at Baylor College of Medicine. He re- 
ceived 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 Hos- 
pital, 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. 
OUR laboratory has focused on molecular ge- 
netic investigations of the enzyme methyl- 
malonyl CoA mutase (MCM) and its deficiency 
state in humans, methylmalonic aciduria. This 
enzyme is required for the metabolism of propio- 
nate, which can be produced by catabolism of 
certain amino acids or absorbed from fermenta- 
tion products in the intestines. 
We have cloned genes for human and mouse 
MCM, identified mutations that give rise to nota- 
ble phenotypes of methylmalonic aciduria, and 
demonstrated correction of the metabolic defect 
in cultured cells from methylmalonic aciduria pa- 
tients by gene transfer of a normal MCM gene. 
This last experiment is prescient of somatic gene 
therapy in which patients with inborn errors of 
metabolism might be treated by introducing a 
normal gene into somatic cells to perform the 
function of the inherited mutant genes. 
Successful somatic gene therapy involves more 
than introducing a normal gene into genetically 
defective cells. It will be necessary to achieve 
proper compartmentalization and regulation of 
the recombinant enzyme, substrates, cofactors, 
and reaction products; to determine which or- 
gans and somatic cells represent the best possible 
targets for gene transfer, how many cells need to 
be transformed, and how the metabolic capacity 
and phenotypic effect of reconstituted cells may 
be maximized; and to address a variety of clinical 
issues to ensure that clinical trials embody the 
greatest potential benefit and entail the least pos- 
sible risk. It is also necessary to adhere to the 
regulatory and review process that has been es- 
tablished to monitor human gene therapy. 
Our studies are directed at attaining an under- 
standing of the structure and function of MCM; of 
its role in homeostasis, nutrition, and pathology; 
and of the multifaceted issues required to pro- 
pose gene therapy for methylmalonic aciduria. In 
the past year this has involved basic research con- 
cerning MCM and gene transfer as well as the es- 
tablishment of a clinical foundation for gene ther- 
apy trials. 
Role of Methylmalonyl CoA Mutase in 
Health and Disease 
The clinical expression of MCM deficiency 
presents several enigmas. Why is this disorder 
lethal? Why are the symptoms intermittent? Why 
are some individuals with persistent methylma- 
lonic aciduria clinically normal? We have de- 
scribed a variety of mutations in the gene for 
MCM. Some give rise to completely inactive en- 
zymes, others to enzymes that show limited activ- 
ity in the presence of vastly excessive cofactor, 
and an interesting enzyme that is nonfunctional 
in most cells but active in heterologous combina- 
tion with some other mutant enzymes. We will 
study whether the residual activity expressed by 
mutant genes correlates with the different pheno- 
typic forms of methylmalonic aciduria or 
whether other factors are involved in this 
pleomorphism. 
We are studying propionate metabolism in 
various normal and genetically defective cells to 
investigate the role of MCM in homeostasis. Our 
studies suggest that under normal conditions the 
rate of propionate metabolism is not limited by 
the amount of the enzyme. Barely detectable lev- 
els of MCM can sustain maximal levels of propio- 
nate metabolism, and the addition of more en- 
zyme to normal cells has no effect on this 
metabolic activity. 
Interestingly, the level of propionate metabo- 
lism in cultures containing few cells with recon- 
stituted enzyme is similar to that of a population 
of completely normal cells, indicating exchange 
of intermediate metabolites between cells. The 
exchange appears to require cell contact; it does 
not occur between cells separated by a fluid 
phase. This suggests that gene therapy targeting a 
small population of cells may have a significant 
phenotypic impact, though therapy may have to 
be targeted to organs damaged by the accumula- 
tion of organic acids, particularly the liver, rather 
than more accessible sites such as bone marrow. 
We have also observed that the ability of a colonic 
epithelial cell line (CACO-2) to metabolize propio- 
nate increases dramatically as the cell differentiates 
in culture. This suggests that propionate metabolism 
may be a differentiated function of intestinal epithe- 
lium. There are several reasons to think that the in- 
testine may play a critical role in propionate homeo- 
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