Proc. Natl. Acad. Sci.
USA
Vol. 92, pp. 1674-1678, February 1995
Medical Sciences
Retroviral-mediated gene transfer corrects very-long-chain fatty
acid metabolism in adrenoleukodystrophy fibroblasts
(peroxisomes/retroviral vector/gene therapy)
NATHALIE CARTIER*, JACQUELINE LoPEZ*, PHILIPPE MOULLIERt, FRANcis RocCHICCIOLI*,
MARIE-ODILE ROLLANDt, PAULA JORGE*§, JEAN MOSSER%, JEAN-LOUIS MANDELI,
PIERRE-FRANCOIS BOUGNEiRES*,OLIVIER DANOSt, AND PATRICK AUBOURG*
*Institut National de la Sante et de la Recherche Medicale U342, Universite Rene Descartes, H6pital Saint-Vincent de Paul, 82 avenue Denfert Rochereau, 75014
Paris, France; tLaboratoire Retrovirus et Transfert Genetique, Centre National de la Recherche Scientifique Unite de Recherche Associee 1157, Institut Pasteur,
25 rue du Dr. Roux, 75724 Paris 15 France; tBiochemistry Department, H6pital Debrousse, 29 rue Soeur Bouvier 69322 Lyon Cedex 05, France; and 'Institut
National de la Sante et de la Recherche Medicale U184, Laboratoire de Genetique Moleculaire des Eucaryotes, Faculte de Medecine et Centre Hospitalier
Regional Universitaire, 11 rue Humann, 67085 Strasbourg Cedex, France
Communicated by N. Edward Tolbert, Michigan State University, East Lansing, MI, November 16, 1994 (received for review July 15, 1994)
ABSTRACT Adrenoleukodystrophy (ALD), a lethal de-
myelinating disease of the brain, is caused by mutations of a
gene encoding an ATP-binding transporter, called ALDP,
localized in the peroxisomal membrane. It is associated with
a defective oxidation of very-long-chain fatty acids, leading to
their accumulation in many tissues. This study reports that
the retroviral-mediated transfer of the ALD cDNA restored
very-long-chain fatty acid oxidation in ALD fibroblasts in
vitro following abundant expression and appropriate target-
ing of the vector-encoded ALDP in peroxisomes. The same
method may be used in hematopoietic cells as a further step
of a gene therapy approach of ALD.
X chromosome-linked adrenoleukodystrophy (ALD) is a dev-
astating neurologic disorder that affects 1/15,000 males with
different clinical phenotypes within the same kindred. The
cerebral lesions occur in young boys between the age of 5 and
12 years and are characterized by progressive demyelination
leading to rapid deterioration and death within 3-5 years of
diagnosis (1).
The main biochemical defect of ALD is an impaired oxi-
dation of saturated very-long-chain fatty acids (VLCFAs) and
their accumulation in cerebral white matter, adrenal glands,
fibroblasts, and plasma (1, 2). In ALD fibroblasts, VLCFAs
are normally transported across the peroxisomal membrane
(3), but a functional defect of VLCFA-CoA synthetase impairs
their (3-oxidation (4-6).
The recent cloning and demonstration of deletions and
mutations of theALD gene (7-10) revealed that the metabolic
abnormality is due to defects of a 75-kDa peroxisomal integral
membrane protein (11), a member of the ATP-binding cas-
sette (ABC) transporter family (12). The function of ALD
protein (ALDP) is unknown, but it is necessary for the activity
of the VLCFA-CoA synthetase, also localized in the peroxi-
somal membrane (13).
Various therapeutic approaches including a lipid diet com-
bining the administration of glycerol trioleate and trierucate
(Lorenzo's oil) (14-16) failed to modify the evolution of the
cerebral lesions of ALD. Four years ago, we reported that bone
marrow transplantation (BMT) can reverse demyelination, if
performed at a relatively early stage of its development (17).
Since allogenic BMT is often limited by the lack of histocom-
patible donors and has serious secondary effects, we consid-
ered transplantation of the patient's own bone marrow cells
after transfer of the normal ALD gene as an alternative strat-
egy.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
1674
To initiate this gene therapy approach, we first studied if the
retroviral-mediated transfer of the ALD cDNA into ALD skin
fibroblasts could correct their abnormal phenotype in vitro.
METHODOLOGY
Cell Lines and Cell Culture. Patients JL and SM have
X-linked ALD but with a different clinical phenotype. Patient
JL has the severe childhood cerebral form of ALD, while
patient SM has the less severe adult adrenomyeloneuropathy
form. Their cultured skin fibroblasts had increased content
and defective oxidation of VLCFA as reported in ALD (1).
Cells were maintained in Dulbecco's modified Eagle's medium
(DMEM; ATGC), supplemented with 10% (vol/vol) inacti-
vated fetal calf serum.
JL and SM fibroblasts were immortalized with a retroviral
construct that encodes for simian virus 40 (SV40) large tumor
antigen and the selectable marker gene for neomycine phos-
photransferase. The retroviral producer cells (SV40 pop) were
derived from the pZipNeoSV(X) vector system (18). SV40-
immortalized ALD fibroblasts and the murine retroviral pack-
aging cell line (TCRIP) were cultured in DMEM with 10%
newborn calf serum. All media were supplemented with 2 mM
glutamine, penicillin (50 units/ml), and streptomycin sulfate
(50 mg/ml).
Construction and Packaging of the Retroviral Vector M48-
ALD. A full-length ALDP cDNA (H83, nt 333-2781 of the
published sequence; ref. 7) was obtained by ligation of two
previously characterized partial cDNAs (H3 and H8) and
direct cloning in the EcoRI site of pBluescript KS(+) (Strat-
agene). To construct the retroviral ALDP vector (see Fig. 1A),
a 2.43-kb Spe I-EcoRI fragment was isolated from the full-
length human ALDP cDNA, Bcl I linkered, and inserted in the
sense orientation into the unique BamHI restriction site of the
M48 retroviral vector, a Moloney-based retroviral vector (19).
In this M48-ALD construct, expression of ALD cDNA is
under the control of the mouse phosphoglycerate kinase
(PGK) gene promoter.
The GLU-3 control vector (GLU-3) was obtained by insert-
ing the human ,B-glucuronidase gene in the same M48 vector
(20). The PGK-neo vector containing the neomycin-resistance
gene from TnS under the control of the PGK promoter was
used for G418 selection (21).
M48-ALD and PGK-neo vectors were cotransfected into the
amphotropic packaging TCRIP cell line (22) using DNA/
Abbreviations: ALD, adrenoleukodystrophy; ALDP, adrenoleu-
kodystrophy protein; VLCFA, very-long-chain fatty acid; PGK, phos-
phoglycerate kinase; BMT, bone marrow transplantation; mAb,
monoclonal antibody.
§Present address: Instituto Genetica Medica Jacinto Magalhaes, Porto,
Portugal.
 Medical Sciences: Cartier et al.
calcium phosphate coprecipitation. Transfected cells were se-
lected in a medium containing G418 (1 mg of active base per
ml; GIBCO/BRL). Clones were isolated by ring cloning and
expanded to cell lines. M48-ALD recombinant clones were
selected on the basis of positive immunofluorescence assay
with anti-ALDP antibodies. The absence of helper virus was
assayed by a virus mobilization assay (23).
Retroviral Gene Transfer. ALD skin fibroblasts were
seeded at 5% confluency, 16 h prior to infection. Cells were
incubated for 8 h with 10 ml of the fresh filtered M48-ALD or
GLU-3 supernatant in the presence of Polybrene (8 ,ug/ml,
Sigma). Infection was performed twice on two consecutive
days. We selected the highest M48-ALD-virus-producer
clones on the basis of the number of integrated copies of intact
M48-ALD provirus, after infection of SV40-immortalized
ALD fibroblasts. As determined by Southern blotting, infec-
tion resulted in the integration of approximately one provirus
copy per cell genome.
Southern Blot Analysis. The copy number of integrated
provirus was determined by Southern blot analysis. Genomic
DNA was isolated and digested with Kpn I restriction enzyme.
Gel electrophoresis, Southern blotting, probing, and autora-
diography were performed as described (7). Probe Ex13 is a
partial ALD cDNA (nt 1221-nt 1829) (7).
Monoclonal Antibodies (mAbs) and Polyclonal Antibodies.
The production and characteristics of the anti-human ALDP
mAbs antibodies (1D6 and 2B4 mAbs produced in mouse) used
in these investigations are described elsewhere (11). These two
mAbs do not cross-react with the mouse ALDP. Rat catalase
antibodies produced by immunizing rabbits are generous gifts
from R. J. A. Wanders (University Hospital, Amsterdam).
Immunoblotting and Immunocytofluorescence Analysis.
Whole cell extraction, SDS/PAGE, and Western blotting were
performed as described (11).
Human skin fibroblasts (50 x 103 per ml) were cultured for 16
h in the wall of a chamber slide (Nunc), fixed in 2% formaldehyde
for 4 min, and permeabilized in phosphate-buffered saline
(PBS)/0.1% Triton X-100. Cells were then incubated at room
temperature with mnAb 1D6 or 2B4 in PBS/0.1% Triton X-100
with 500 ,ug of goat IgG per ml. Biotinylated anti-mouse anti-
bodies were added at 10 ,ug/ml (Vector Laboratories) and spe-
cific labeling of ALDP was revealed by using Cy3-streptavidin
(Jackson ImmunoResearch).
For colocalization experiments, cells were first treated with
mAb 1D6 followed by incubation with anti-mouse antibodies
directly conjugated to Cy3 (Jackson ImmunoResearch). Rat
catalase antiserum was added and cells were further incubated
with anti-rabbit antibodies directly conjugated to fluorescein-
avidin D (Jackson ImmunoResearch). The microscope filters
used to study protein expression revealed by Cy3 or fluorescein
isothiocyanate had excitation/absorption length-wave channel
specificity for each of these fluorochromes.
Three sets of experiments were performed to exclude non-
specific cross-reactions between secondary and primary anti-
bodies. Cells were incubated with (i) mAb 1D6 and biotiny-
lated anti-rabbit antibodies labeled with fluorescein-avidin D,
(ii) catalase antiserum and anti-mouse antibodies labeled with
Cy3-streptavidin, or (iii) biotinylated anti-isotype (IgGl)
mouse antibody and Cy3-streptavidin.
Determination of VLCFA Levels and Peroxisomal f8-Oxi-
dation Activity. VLCFA concentrations were measured in
cultured skin fibroblasts by gas/liquid chromatography/mass
spectrometry (24). The capacity of the fibroblasts to oxidize
[1-14C]lignoceric acid to acetate was measured as described
(25).
RESULTS
Retroviral Transduction of ALD cDNA in Skin Fibroblasts
from ALD Patients. To evaluate if gene transfer could correct
the metabolic defect in ALD fibroblasts, we constructed a
Proc. NatL Acad. Sci USA 92 (1995) 1675
A A
LTR I-PGK G
ALD _LTR
Kpn I Kpn I
B 1 2 3 4 5
kb
40 .340. - 15
OW :** 4 _ 11
As - 5.8
,q,w
FIG. 1. (A) Schematic diagram of retrovirus M48-ALD construct.
The vector was assembled by reconstruction of the ALD coding
sequence in the Moloney murine leukemia virus-derived vector M48.
In this construct, expression of ALD cDNA is under the control of the
mouse PGK gene promoter. Locations of the Kpn I sites used for
Southern blot analysis are indicated. LTR, long terminal repeat. (B)
Integration of intact M48-ALD provirus in ALD fibroblasts. Kpn
I-digested genomic DNA from normal and ALD skin fibroblasts and
plasmid vector were probed to Ex13 cDNA. Lane 1, 0.2 copy per cell
genome of M48-ALD plasmid; lane 2, normal fibroblasts; lane 3,
nontransduced ALD fibroblasts; lane 4, ALD fibroblasts transduced
with the control GLU3-vector; lane 5, ALD fibroblasts transduced
with M48-ALD vector. A 5.8-kb band comigrating with the proviral
band (lane 1) was present in ALD fibroblasts (patient SM) transduced
with the M48-ALD vector (lane 5). In addition, Ex13 probing showed
two endogenous DNA fragments (11 and 15 kb) in normal and ALD
fibroblasts.
recombinant retroviral vector, M48-ALD (Fig. 1A), carrying
the human ALDP cDNA under the control of the mouse PGK
promoter. This construct was cotransfected with a PGK-neo
plasmid confering G418 resistance into the packaging TCRIP
cells, allowing the isolation of several amphotropic virus (M48-
ALD)-producer cell lines.
ALD skin fibroblasts were then transduced with superna-
tant from M48-ALD-producer TCRIP cells or supernatant
from the control GLU3-TCRIP cells. Based on immunocyt-
ofluorescence studies, 75% ± 6% of JL fibroblasts and 66% +
4% of SM fibroblasts express recombinant ALDP after two
kDa 1 2 3 4 5 6 7
756
ALDP
Catalase
62 -
FIG. 2. Immunoblotting of ALDP in ALD fibroblasts before and
after correction with the M48-ALD vector. One hundred micrograms
of whole cell extracts from skin fibroblasts were separated by 8%
SDS/PAGE and immunoblotted with anti-ALDP (Upper) and anti-
catalase (Lower) antibodies. Lane 1, normal fibroblasts; lane 2, SM
fibroblasts before transduction; lane 3, SM fibroblasts cultured for 2
weeks after transduction; lanes 4 and 6, JL fibroblasts before trans-
duction; lane 5, JL fibroblasts cultured for 2 weeks after transduction;
lane 7, JL fibroblasts cultured for 2 months after transduction. Based
on catalase band intensity, the protein amount is lower in lane 7.
1676 Medical Sciences: Cartier et al. Proc. Natl Acad ScL USA 92 (1995)

FIG. 3. (Legend appears at the bottom of the opposite page).

 Medical Sciences: Cartier et al. Proc. Natl Acad Sci USA 92 (1995) 1677
cycles of infection. Intact M48-ALD proviral integration was
demonstrated by Southern blot analysis in ALD skin fibro-
blasts (Fig. 1B).
In Vitro Expression of ALDP in Vector-Treated ALD Fibro-
blasts. To determine the effect of retroviral transduction on xc
the expression of recombinant ALDP, transduced ALD fibro- X >80
blasts were cultured to confluency in fresh medium and then
studied using immunoblotting and indirect immunocytofluo-
rescence. a0~E
0
Immunoblotting of normal fibroblasts with mAbs 1D6 and
2B4 revealed a specific 75-kDa ALD protein (ALDP) (Fig. 2, -40
lane 1 and ref. 11). This 75-kDa band was absent in fibroblasts
from patient JL (Fig. 2, lanes 4 and 6) and present in low
amount in fibroblasts from patient SM (Fig. 2, lane 2). After
transduction with the M48-ALD vector, a 75-kDa ALDP band
was present in fibroblasts from both ALD patients (Fig. 2, 0.3 B
lanes 3 and 5). Based on band intensity scanning, the expres-
sion of ALDP increased to 5-fold the normal value in these
fibroblasts. The 75-kDa band was not detectable in ALD
fibroblasts transduced with the GLU-3 control vector (not a
0
shown). CL
Using indirect immunocytofluorescence, mAbs 1D6 and
2B4 failed to reveal any punctate fluorescent signal in fibro-
blasts from patient JL (Fig. 3A). In agreement with immuno- OE
Zw..
blotting experiments, these data demonstrate a complete ab- o 0.1
sence of ALDP in this cell line. 0~ ~
-
~ 01
On the other hand, a punctate cytoplasmic fluorescent US~J S
staining was observed in the fibroblasts from patient SM, who
had also ALD (Fig. 3C). In agreement with the immunoblot-
ting data, the punctate pattern was less intense than in normal -1 2 -12
fibroblasts (11), suggesting that the mutated ALDP was syn-
thesized in lower amounts or unstable in this cell line. Normal Patient Patient
After transduction with the M48-ALD vector, a marked JL SM
punctate fluorescent staining reappeared in the fibroblasts FIG. 4. Lignoceric acid oxidation (A) and hexacosanoic acid con-
from patient JL (Fig. 3B) and the punctate pattern increased tent (B) in fibroblasts from ALD patients before and after correction
noticeably in fibroblasts from patient SM (Fig. 3C). These with the M48-ALD vector. -, Nontransduced fibroblasts; 1, trans-
modifications did not occur in the cells transduced with the duced fibroblasts cultured for 10 days; 2, transduced fibroblasts cul-
control GLU-3 vector (not shown). No cytosolic aggregates tured for 2 months. Hexacosanoic acid values of ALD fibroblasts are
were observed in the two transduced ALD cell lines, indicating mean ± SD of triplicate samples. Lignoceric acid oxidation values of
ALD fibroblasts are mean ± SD of duplicate samples. Values of
that most, if not all, recombinant ALDP had been correctly hexacosanoic acid content and lignoceric acid oxidation in normal
incorporated into peroxisomal membranes. This supports the fibroblasts are from refs. 24 and 25.
idea that ALD fibroblasts have an import system capable of
targeting large amounts of ALDP to peroxisomes. Long-Term Expression of ALDP in Vector-Treated ALD
Colocalization studies confirmed that the location of re- Fibroblasts. The intensity of the 75-kDa band detected by
combinant ALDP was peroxisomal. Fibroblasts from patient immunoblotting did not change significantly in ALD skin
JL transduced with the M48-ALD vector were first incubated fibroblasts transduced with the M48-ALD vector and cultured
with anti-ALDP mAbs labeled to Cy3 and then incubated with for 2 months (corresponding to five passages) (Fig. 2, lanes 5
peroxisomal catalase labeled to fluorescein. They showed a and 7). Using immunofluorescence studies, we observed no
red punctate signal corresponding to ALDP when a filter with decrease with time (2 months) of the percentage of JL and SM
channel specificity for Cy3 fluorescence was used (Fig. 3E) and fibroblasts expressing recombinant ALDP.
a green punctate signal when a filter with channel specificity Correction of VLCFA Metabolism in Vector-Treated ALD
for fluorescein fluorescence was used (Fig. 3F). Using a filter Fibroblasts. The 83-oxidation of lignoceric acid (C24:0) was
with broader fluorescence specificity, a yellow-brown color decreased to 27% and 35% of control values in fibroblasts
corresponding to a match between the green and the red from patients JL and SM (Fig. 4A). After transduction with the
punctate signals (Fig. 3G) was observed, indicating that re- M48-ALD vector, these fibroblasts degraded lignoceric acid at
combinant ALDP and the endogenous peroxisomal catalase normal rates (Fig. 4A). The (3-oxidation of lignoceric acid in
colocalized and therefore that recombinant ALDP was per- the transduced ALD fibroblasts cultured for 2 months re-
oxisomal. The secondary antibodies did not cross-react with mained normal (Fig. 4A).
the primary antibodies in the single or colocalization experi- To evaluate further the correction of phenotype, VLCFA
ments. concentrations were measured in ALD fibroblasts before and

FIG. 3. Expression and localization of ALDP in ALD fibroblasts before and after correction with the M48-ALD vector. Using anti-ALDP
antibodies (1D6 mAb), immunocytofluorescence studies were performed before (A and C) and after (B and D) transduction with the M48-ALD
vector. (A and B) Fibroblasts from ALD patient JL. (C and D) Fibroblasts from ALD patient SM. (E-G) Colocalization of ALDP and peroxisomal
catalase in fibroblasts from patient JL after transduction with the M48-ALD vector. The images were taken from an identical field. (E) A filter
with channel specificity for Cy3 detects a red punctate pattern corresponding to the presence of recombinant ALDP. (F) A filter with channel
specificity for fluorescein detects a green punctate pattern corresponding to the endogenous presence of peroxisomal catalase. (G) A filter with
a broad fluorescence channel specificity detects on the same field a yellow-brown punctate pattern indicating that recombinant ALDP and
endogenous catalase colocalized. (A-D, X800; E-G, X400.)
1678 Medical Sciences: Cartier et al.
after transduction with M48-ALD vector. Fig. 4B shows that
VLCFA content of nontransduced fibroblasts from patients
SM and JL was 3.5- to 4.0-fold that of normal fibroblasts.
Transduction with the M48-ALD vector normalized the C26:0
(hexacosanoic acid) concentration in fibroblasts (Fig. 4B) and
C26:0/C22:0 and C24:0/C22:0 ratios. No change occurred in
ALD fibroblasts transduced with the GLU-3 control vector
(not shown). The levels of VLCFA in the transduced ALD
fibroblasts cultured for 2 months remained normal (Fig. 4B).
DISCUSSION
The demyelinating lesions in the brain of ALD are diffuse (1).
They often involve occipital and/or frontal lobes and internal
capsules (1). It is likely that the demyelination observed in
ALD is related to the accumulation of VLCFA in the various
lipid fractions of myelin, leading to alteration in their physical
properties and destabilisation of myelin membrane (1). To
deliver foreign genes to different brain regions, the stereotac-
tical injection of replication-defective adenovirus (28-30) or
herpes virus (31, 32) vectors has been proposed. This ap-
proach, however, is unsuitable in ALD since it would require
numerous injections at different sites of the brain.
In the brain, up to 20% of glial cells are microglia cells (33).
Although the turnover of resident microglia cells is still a
controversial issue, several lines of evidence suggest that
monocytes derived from peripheral hematopoietic cells can
cross the blood-brain barrier, enter the brain parenchyma, and
differentiate as microglia cells (33, 34). Amelioration of sev-
eral neurodegenerative disorders after BMT can be explained
by the replacement of abnormal microglia cells by new hema-
topoietic cells (35). In ALD, allogenic BMT can reverse or stop
demyelination when performed in children with moderate and
slowly progressive demyelinating lesions (17). This procedure,
however, requires a perfectly matched histocompatible donor
and is associated with significant risks, including severe graft-
versus-host-disease. To circumvent these problems, we con-
sidered that autologous BMT after the insertion of the normal
ALD gene could be an alternative approach. As a first step, we
tested if retroviral-mediated transfer of the ALD cDNA could
correct the phenotype of ALD fibroblasts in vitro.
We constructed a recombinant retroviral vector, M48-ALD,
to mediate insertion and expression of human ALD cDNA and
tested this construct in skin fibroblasts from two unrelated
ALD patients. Before transduction with the M48-ALD vector,
ALDP was absent in one mutated cell line and present but
defective in the second. After transduction with the M48-
ALD, recombinant ALDP, expressed abundantly, was nor-
mally incorporated into peroxisomes in the fibroblasts from
both patients. The cellular distribution of recombinant ALDP
was punctate, as in normal fibroblasts, and colocalized with the
peroxisomal catalase. Although ALDP was overexpressed, no
ALDP aggregates were observed in the cytosol. These results
support the observation that, most if not all, recombinant
ALDP was correctly targeted to peroxisomes.
The oxidation of lignoceric acid, a substrate for peroxisomal
,B-oxidation, is reduced to 20-35% of normal in ALD fibro-
blasts (1, 2). It was completely normalized by gene transfer.
Consequently, VLCFA content returned to normal in the
patients' fibroblast cell lines in which they had previously
accumulated. These results indicate that the recombinant
ALD localized in the peroxisomal membrane was functional
and allowed a complete restoration of the VLCFA-CoA syn-
thetase activity, deficient in ALD. Stable expression of ALDP
and prolonged metabolic correction were achieved for 2
months.
Information from other genetic disorders is encouraging to
our retroviral gene therapy approach. Retroviral vectors can
efficiently transfer and express physiological amounts of glu-
cocerebrosidase into long-lived bone marrow hematopoietic
cells from patients with Gaucher disease (36). Furthermore,
Proc. NatL Acad ScL USA 92 (1995)
murine bone marrow cells expressing human glucocerebrosi-
dase from a retroviral vector can repopulate efficiently central
nervous system microglia cells (26). The gene-transfer effi-
ciency and the longevity of transduced cells in large animal
need to be improved but significant progress has also been
made in this field (27). Our results provide a basis for studying
in vitro the ALD gene transfer and expression in hematopoietic
stem cells from normal humans, ALD patients, and animals.
The capacity of transduced cells to replace abnormal microglia
cells may be studied in a "knock-out" murine ALD model and
in large animals as a preliminary attempt before clinical trials
could be eventually envisaged in patients.
We thank Anne-Marie Reppelin for technical assistance and Anna
Salvetti for helpful advice. This work was supported by the Association
Frangaise contre les Myopathies, the Fondation Recherche et Partage,
La Fondation de France, the Institut Scientifique Roussel, and the
European Leukodystrophy Association.
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