Molecular Studies of Human Genetic Disease 
inhalation rather than injection. We are preparing 
to evaluate various methods for pulmonary gene 
therapy. Liposomes containing DNA, retroviral 
vectors, and adenoviral vectors will be used for 
direct instillation into the lungs or for various 
aerosol deliveries, beginning with normal ro- 
dents and perhaps progressing to primates. 
Leukocyte and Endothelial Cell 
Adhesion Molecules 
Leukocytes (white blood cells) are involved in 
defense against infection and in most inflamma- 
tory responses. Through complex mechanisms, 
the various forms of leukocytes roll along the 
blood vessel walls and eventually migrate 
through the endothelial layer. This ability is me- 
diated to a large extent by carefully regulated ad- 
hesion molecules on the surface of leukocytes 
and endothelial cells. 
We are attempting to understand the role of 
these proteins in normal biology and in multifac- 
torial genetic diseases. This involves preparing 
mutant mice and analyzing naturally occurring 
genetic variation in humans. It is quite possible 
that variation in these genes could be an impor- 
tant factor in the predisposition to multifactorial 
disorders such as atherosclerosis, autoimmune 
disease, and a wide range of inflammatory pro- 
cesses, including arthritis and diabetes mellitus. 
We are focusing initially on CD 18, which is a 
subunit of leukocyte integrin, and on intracellu- 
lar adhesion molecule- 1 (ICAM-1), a member of 
the immunoglobulin gene family. CD 18 is found 
on the surface on many leukocytes, and its adhe- 
siveness can be sharply increased through an acti- 
vation process. ICAM-1 is found on many cells, 
particularly endothelial cells, where it can bind 
the CD18-containing integrins and mediate firm 
cell attachment and migration of leukocytes out 
of the bloodstream. 
Individual genes can be altered in mice by the 
technique of homologous recombination (ex- 
change of DNA between related molecules) . Us- 
ing this methodology in ES cells, a mutation was 
introduced into the mouse CD 18 gene, and the 
alteration was transmitted through the germline 
so that the altered animals could be bred. The 
mutant animals have a partial rather than com- 
plete impairment of the CD 18 gene's function. 
Leukocyte function shows significant alteration 
in these animals, and they will be studied for any 
differences in inflammatory responses or suscep- 
tibility to atherosclerosis. 
A mutation has also been introduced into the 
ICAM-1 gene, and mice carrying this mutation in 
the majority of their cells are being bred to obtain 
transmission of the defect to the germline. Eff^orts 
have also been initiated to prepare a mutation in 
the P-selectin gene, whose product is expressed 
on platelets and endothelial cells. Adhesion pro- 
teins of the selectin family bind to sugar mole- 
cules, rather than to proteins, on other cells. The 
work to obtain mouse mutations in leukocyte and 
endothelial cell adhesion molecules is supported 
in part by a grant from the National Institutes of 
Health. 
A defect in CD 18 causes a rare human genetic 
disease. In its severe form, this condition, known 
as leukocyte adhesion deficiency (LAD), leads to 
fatal susceptibility to infections. LAD is an attrac- 
tive model for development of somatic gene ther- 
apy for bone marrow-derived cells. A recombi- 
nant retrovirus encoding the human CD 18 
protein has been prepared and has been used to 
transfer the gene into mouse and human cells. 
Bone marrow transplantation was performed in 
mice, and the infected cells express human CD 18 
when reimplanted into the mouse. Cultured cells 
from LAD patients were infected with retrovirus, 
and the adhesion properties of the cells were re- 
stored to normal. Bone marrow cells from af- 
fected patients are being cultured in the labora- 
tory and infected with the retrovirus in 
preparation for performing human gene therapy. 
Genetic variation in the adhesion molecules of 
human leukocytes and endothelial cells is being 
identified. In the ICAM-1 gene, for example, 
there is an amino acid difference that is prevalent 
in the population. It is of interest to determine 
whether different forms of the ICAM-1 gene in 
the human population are associated with differ- 
ent susceptibilities to inflammatory diseases and 
atherosclerosis. Some of the long-term goals of 
this project are to identify naturally occurring ge- 
netic variations that alter susceptibility to com- 
mon disease processes. In addition, the mutant 
mice can be used to determine if decreased func- 
tion of some of these cell adhesion molecules re- 
duces chronic inflammatory disease processes 
(e.g., autoimmune disease, arthritis, multiple 
sclerosis, diabetes mellitus). If reduced gene 
function were to lessen the disease processes in 
mouse models, this would provide evidence that 
the function of these proteins might be blocked 
by drugs as a means of slowing the analogous pro- 
cesses in humans. 
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