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HUMAN GENE THERAPY 13:947–957 (May 20, 2002)
© Mary Ann Liebert, Inc.
Effective Retrovirus-Mediated Gene Transfer in Normal and
Mutant Human Melanocytes
MARIA VITTORIA SCHIAFFINO,
1,2
ELENA DELLAMBRA,
3
KATIA CORTESE,
4
CINZIA BASCHIROTTO,
1
SERGIO BONDANZA,
3
MAURIZIO CLEMENTI,
5
PAOLO NUCCI,
6
ANDREA BALLABIO,
1,7,8
CARLO TACCHETTI,
4
and MICHELE DE LUCA
3
ABSTRACT
Melanocytes represent the second most important cell type in the skin and are primarily responsible for the
pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired
disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and in-
creased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of
genetically manipulating human melanocytes has been hampered so far by a number of limitations, includ-
ing their resistance to retroviral infection. To address the problem of human melanocyte transduction, we
generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pig-
mentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the pres-
ence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient
melanocytes displayed a significant impairment in replication ability and showed complete absence of en-
dogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure.
To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited
a melanocyte–keratinocyte coculturing approach. Despite their lower replication rate with respect to wild-
type cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found
to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the
feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, hu-
man melanocytes with nonmitogenic genes.
947
OVERVIEW SUMMARY
The skin represents one of the most attractive target tissues
for ex vivo gene therapy, not only for its accessibility, but also
for the availability of advanced skin culture and surgery tech-
niques. Moreover, we previously showed that the main cell
type in the skin, the keratinocyte and its precursor stem cells,
can be efficiently and stably transduced by retroviral vectors
and selected in vitro. However, no effective gene transfer pro-
cedure has been developed yet for the melanocytes, despite
the fact that these cells represent the second physiologically
most important cell type in the skin. We now report the set-
ting up of an improved gene transfer method for normal and
mutant human melanocytes, allowing the expression of non-
mitogenic recombinant proteins in high amounts and in a
uniform and a stable manner. Our findings indicate the fea-
sibility of an ex vivo gene therapy approach for the treatment
of inherited and acquired pigmentation disorders.
1
TIGEM, Telethon Institute of Genetics and Medicine, 20132 Milan, Italy.
2
Present address: DIBIT, Scientific Institute San Raffaele, 20132 Milan, Italy.
3
Laboratory of Tissue Engineering, IDI IRCCS, Istituto Dermopatico dell’Immacolata, 00040 Pomezia (Rome), Italy.
4
Department of Experimental Medicine, Anatomy Section, University of Genoa, 16132 Genoa, Italy.
5
Medical Genetics, Department of Pediatrics, University of Padua, 35128 Padua, Italy.
6
Department of Ophthalmology, San Raffaele Hospital, 20132 Milan, Italy.
7
San Raffaele Faculty of Medicine, 20132 Milan, Italy.
8
Present address: TIGEM, Telethon Institute of Genetics and Medicine, and Second University of Naples, 80131 Naples, Italy.
INTRODUCTION
I
N MAMMALS, pigmentation of the skin, hair, and eyes results
from the presence and distribution of melanins. These black-
brown and yellow-red pigments are exclusively synthesized by
a relatively small subpopulation of highly specialized pigment
cells, including the melanocytes of the skin, eyes, and other tis-
sues, and the retinal pigment epithelium (RPE) (for review see
Quevedo
et al
., 1987; Hearing and Tsukamoto, 1991; King
et
al
., 1995). The melanogenic function of the pigment cells may
be affected in a number of inherited and acquired disorders, re-
sulting in clinical conditions characterized by hyperpigmenta-
tion or hypopigmentation. These can generate serious aesthetic
problems: the cosmetic disfigurement caused, for example, by
vitiligo can generate profound psychological and psychosocial
effects in the affected patients, including depression and social
rejection. Moreover, melanins act as a weak sunscreen, repre-
senting an important defense against ultraviolet radiation.
Therefore, melanocyte malfunctioning can expose the skin to
the damaging effects of sunlight and increase the risk of ma-
lignant melanoma (Sober
et al
., 1991).
The genetic disorders of pigmentation consist of a vast and
heterogeneous collection of pathological conditions. Among
these, albinism represents a group of inherited abnormalities
characterized by primary and specific involvement of the pig-
ment cells of the skin and eyes, which show defective melanin
synthesis and/or distribution. Patients affected with albinism ex-
hibit variable hypopigmentation of the skin and severe devel-
opmental defects of the optic system. The most common forms
of albinism, that is, oculocutaneous albinism (OCA) types 1 and
2, result from alterations of the melanin biosynthetic pathway
with absence or major reduction of melanin (King
et al
., 1995).
In contrast, ocular albinism type 1 (OA1; MIM [Mendelian In-
heritance in Man (McKusick, 1998)] 300500), representing the
most common form of ocular albinism with an estimated preva-
lence of 1:50,000, is thought to arise from abnormal biogene-
sis of melanosomes, that is, the specialized subcellular or-
ganelles of the pigment cells devoted to the synthesis, storage,
and transport of melanins (O’Donnell
et al
., 1976; Garner and
Jay, 1980; Wong
et al
., 1983; Incerti
et al
., 2000).
Ocular albinism is transmitted as an X-linked trait, with car-
rier females showing only minor ocular and skin abnormalities.
Conversely, affected males exhibit the optic changes typical of
all forms of albinism (severe reduction of visual acuity due to
foveal hypoplasia, nystagmus, strabismus, photophobia, iris
translucency, hypopigmentation of the retina, and misrouting
of the optic tracts resulting in loss of stereoscopic vision) and
the presence of giant melanosomes (macromelanosomes) in
skin melanocytes and RPE (O’Donnell
et al
., 1976; King
et al
.,
1995). Progress has been achieved in understanding the mo-
lecular bases for ocular albinism. Indeed, we previously iden-
tified the gene responsible for this disorder and characterized
its protein product as a pigment cell-specific glycoprotein lo-
calized to the melanosomal membrane and displaying features
of G protein-coupled receptors (GPCRs) (Bassi
et al
., 1995;
Schiaffino
et al
., 1996, 1999).
As a first step toward the development of gene therapy ap-
proaches aimed at the correction of pigmentation disorders, we
address here the problem of setting up an effective gene trans-
fer protocol for transducing with nonmitogenic genes not only
normal, but also mutant, melanocytes. For this purpose, we iso-
lated and cultured
in vitro
skin melanocytes obtained from a
patient affected with ocular albinism and showing complete ab-
sence of endogenous OA1 protein. Using OA1 as a physiolog-
ical marker protein, we exploited these cells as a model to de-
velop an extremely efficient gene transfer procedure mediated
by retroviral vectors.
MATERIALS AND METHODS
Microsatellite and sequence analyses of the OA1 gene
Microsatellite markers DXS1223 and DXS7108 were previ-
ously described (Ferrero
et al
., 1995). In addition, we used a
newly identified, highly polymorphic, 19-CA dinucleotide re-
peat (named OA1-CA2) located approximately 1 kb upstream
of exon 1. Oligonucleotide primers flanking the repeat sequence
were OA1-CA2/F (59-TCTTGTGTTGTACTTATGCTGAG)
and OA1-CA2/R (59-GATTACACCACTGCACTCCAG). Be-
cause of the presence of an
Alu
sequence in the region of OA1-
CA2/R, labeling was performed on the F primer only. Poly-
merase chain reaction (PCR) conditions were 30 cycles of 94°C
for 50 sec, 58°C for 50 sec, and 72°C for 40 sec to obtain an
amplification product of 164 bp (with 19 repeats). Exons of the
OA1
gene were amplified and subjected to direct sequencing
analysis as described (Schiaffino
et al
., 1995). Blood and skin
biopsy samples were obtained after all members of the OA1
family had given informed consent.
Melanocyte isolation, culturing, and transduction
Human keratinocytes and melanocytes were isolated from
in
vitro
-reconstituted epidermal sheets and cultivated as described
(De Luca
et al
., 1988; Schiaffino
et al
., 1996). A 2-cm
2
shave
biopsy from the abdominal skin of patient II-3 was used as start-
ing material to isolate the
OA1
-mutant melanocytes. After pu-
rification, melanocytes were cultivated in melanocyte growth
medium: Dulbecco’s modified Eagle’s medium (DMEM) and
Ham’s F12 medium (2:1 mixture), containing fetal calf serum
(FCS, 5%), insulin (5 mg/ml), adenine (0.18 m
M
), hydrocorti-
sone (0.4 mg/ml), triiodothyronine (2 n
M
), epidermal growth
factor (EGF, 10 ng/ml), basic fibroblast growth factor (bFGF,
1 ng/ml), cholera toxin (CT, 0.1 n
M
), phorbol 12-myristate
13-acetate (PMA, 10 ng/ml), glutamine (4 m
M
), and peni-
cillin–streptomycin (50 IU/ml).
In some experiments, aimed at testing the possibility that
macromelanosome formation could be impaired by growth fac-
tors present in the medium (particularly those known to act
along typical GPCR-mediated pathways) or by absence of the
keratinocyte environment, the patient melanocytes were also
cultured (1) in the absence of CT, which irreversibly activates
Gs protein a chain; (2) in the absence of PMA, which acti-
vates protein kinase C (PKC); (3) in the absence of both CT
and TPA; (4) in the presence of keratinocytes with or without
cholera toxin, or by substituting cholera toxin with the a chain
of melanocyte-stimulating hormone (a-MSH), for up to 2
weeks.
The ages of the donors of human melanocyte strains utilized
in this study were as follows: MK69, 16 years old; MK57, 10
years old; MK106, 40 years old; patient II-3, 5 years old. The
doubling time of the isolated melanocyte cultures was as fol-
lows: normal melanocyte strains MK69, MK57 and MK106,
SCHIAFFINO ET AL.
948
2–4 days; patient melanocytes (either untreated or transduced),
6 days (the transduced patient melanocytes displayed a short-
ening of the doubling time from 6 to 4 days during the initial
passages after infection; however, by the time they were har-
vested for analysis, their replication rate had lowered again to
6 days). Melanosome counts were performed on isolated me-
lanocyte cultures between passages 8 and 17 (MK69, passage
15; MK57, passage 8; MK106 wt, passage 5; MK106 trans-
duced, passage 10; patient untreated, passage 15; patient trans-
duced, passage 17).
The LBSN retroviral vector, carrying the cDNA encoding b-
galactosidase (b-Gal), was previously described (Mathor
et al
.,
1996). The LOA1SN retroviral vector, carrying the OA1 cDNA,
was obtained by cloning the OA1 cDNA into the
Eco
RI and
Xho
I sites of the LXSN retroviral vector (Miller and Rosman,
1989). Preparation of high-titer retroviral supernatants and pro-
ducer cell lines was performed essentially as described (Mathor
et al
., 1996). For retroviral infection, human melanocytes from
normal donors and from the OA1 patient (2 3 10
4
/cm
2
) were
seeded onto a feeder layer composed of lethally irradiated (6000
rads) keratinocytes (8 3 10
4
/cm
2
) and producer Am12/LBSN
or Am12/LOA1SN cell lines (4 3 10
4
/cm
2
) in melanocyte
growth medium, containing PMA (10 ng/ml). After 5 days of
cocultivation, melanocytes were passaged and G418 (7.5
mg/ml) was added for 15 days to select the transduced cells.
Selected cells were used for further molecular analysis.
In the melanocyte–keratinocyte coculture assays, transduced
melanocytes (5 3 10
2
/cm
2
) and normal human keratinocytes
(5 3 10
3
/cm
2
) were cocultivated onto a feeder layer of lethally
irradiated 3T3-J2 cells in keratinocyte growth medium: DMEM
and Ham’s F12 medium (2:1 mixture), containing fetal calf
serum (FCS, 10%), insulin (5 mg/ml), adenine (0.18 m
M
), hy-
drocortisone (0.4 mg/ml), triiodothyronine (2 n
M
), epidermal
growth factor (EGF, 10 ng/ml), CT (0.1 n
M
), glutamine (4 m
M
)
and penicillin–streptomycin (50 IU/ml). To determine the lo-
cation and the distribution of melanocytes within the reconsti-
tuted epidermal sheet, confluent cultured epithelia were de-
tached from the culture vessel with the neutral protease Dispase
II, stained with b -galactosidase or dihydroxyphenylalanine
(DOPA), and subjected to histological analysis (De Luca
et al
.,
1988).
Northern, Western, and immunofluorescence analyses
Northern blot analysis was performed as described, using the
OA1 cDNA as a probe (Bassi
et al
., 1995). Western and im-
munofluorescence analyses were performed essentially as de-
scribed (Schiaffino
et al
., 1996, 1999; d’Addio
et al
., 2000).
Protein extracts from normal, patient, and transduced
melanocytes were separated on a sodium dodecyl sulfate
(SDS)–7.5% polyacrylamide gel and transferred to a polyvinyli-
dene difluoride (PVDF) membrane sheet (Hybond-P; Amer-
sham Pharmacia Biotech, Piscataway, NJ), using the Mini-
PROTEAN and the Mini Trans-Blot apparatus (Bio-Rad,
Hercules, CA). Visualization of antibody binding was carried
out with Enhanced ChemiLuminescence Plus (Amersham, Ar-
lington Heights, IL) according to the manufacturer’s instruc-
tions. For immunofluorescence colocalization studies,
melanocytes were cultured on plastic coverslips and fixed in
methanol at 220°C. Affinity-purified anti-OA1 antibody W7
was previously described (Schiaffino
et al
., 1996) and used at
0.5 and at 1.5 mg/ml for Western and immunofluorescence
analyses, respectively. The monoclonal antibody (MAb)
HMB45 against Pmel17 (Dako, Glostrup, Denmark) was used
at a 1:80 dilution for immunofluorescence. Fluorescein isoth-
iocyanate (FITC)-conjugated goat anti-mouse and tetramethyl
rhodamine isothiocyanate (TRITC)-conjugated pig anti-rabbit
immunoglobulins (Dako) were used as secondary antibodies.
Electron microscopy analysis and morphometry
For ultrastructural analysis, normal and OA1 patient
melanocytes were fixed with 2.5% glutaraldehyde in cacody-
late buffer, postfixed in osmium tetroxide, dehydrated through
a graded ethanol series, and embedded in LX112 (Polysciences,
Warrington, PA). Ultrathin sections were stained with uranyl
acetate and lead citrate, and analyzed with EM10C or EM902A
electron microscopes (Zeiss, Thornwood, NY). Melanosome
counts were performed on an average of 10 and 20 cells for
normal and OA1 patient melanocytes, respectively. Counting
included all visible melanosomes, excluding those organized as
aggregates surrounded by a single membrane.
RESULTS
Affected individuals of the OA1 family carry a
frameshift within the OA1 gene
The pedigree of the OA1 family analyzed in this study is
shown in Fig. 1. Complete ocular examination of all family
members was performed. Individual II-1 was examined at the
age of 24 years. Visual acuity for distance, with correction, was
20/100 (20/60 for near vision). The patient showed nystagmus
and iris translucency at slit lamp examination. Fundus obser-
vation disclosed prominent choroidal vessels, indicating hy-
popigmentation of the RPE, and absence of the foveal reflex.
Histological and ultrastructural examinations of a skin bioptic
sample from the patient revealed the presence of typical
macromelanosomes. Individual II-3 was examined at the age of
5 years. Visual acuity for distance, with correction, was 20/200
(20/80 for near vision). The patient also showed nystagmus, iris
translucency, and fundus hypopigmentation, with marked at-
tenuation of the foveal reflex.
Individual I-2 displayed visual acuity of 20/20 and a pattern
of fundus depigmentation consistent with a carrier status for oc-
ular albinism. Ocular examinations of individuals I-1 and II-2
were unremarkable. To confirm the diagnosis of ocular albinism
type 1 at the molecular level, we performed both linkage and
mutation analyses, using DNA samples obtained from fresh
blood of all family members. Linkage analysis was performed
with microsatellite markers located adjacent or internal to the
OA1
gene and showed cosegregation of the disease with the
OA1
locus (Fig. 1). Finally, the direct sequencing of PCR-am-
plified
OA1
exons revealed the presence of a four-nucleotide
deletion at the end of exon 1 in the affected members of the
family, that is, II-1 and II-3 (252del4; Fig. 2A, PT).
OA1 protein is undetectable in OA1
patient melanocytes
The
OA1
gene is expressed, at high levels, only in
melanocytes and RPE, consistent with the clinical phenotype
GENE TRANSFER IN HUMAN MELANOCYTES
949
of the disease. Therefore, to determine the effect of the 252del4
mutation on the OA1 mRNA and protein we obtained a skin
biopsy from patient II-3 and generated a melanocyte culture.
Although the patient melanocytes displayed a lower replication
rate with respect to wild-type cells (the mean population dou-
bling time was 6 days instead of 2–4 days, respectively), they
were isolated and successfully passaged more than 20 times.
The patient melanocytes were initially subjected to Northern
blot analysis with the OA1 cDNA, revealing that the size and
abundance of the OA1 mRNA are normal in these cells (Fig.
2B, PT).
As illustrated in Fig. 3A, the 252del4 mutation results in a
frameshift that could theoretically give rise to two truncated
protein products: a short peptide corresponding to the N termi-
nus and the first transmembrane domain of OA1 up to residue
65, plus 19 unrelated amino acids; and an N terminus-truncated
form of OA1, lacking the first 84 residues and starting from the
second in-frame ATG.
In vitro
studies, performed with the anti-
OA1 antibody (directed against the C terminus of OA1) and a
recombinant construct missing the first ATG, showed that the
N terminus-truncated mutant could actually be produced in
transfected HeLa cells. However, the truncated protein was not
correctly sorted to the lysosomes, displaying a reticular pattern
consistent with retention in the endoplasmic reticulum (ER)
(data not shown).
To test for the presence of a truncated form of OA1 in the
patient melanocytes, we performed Western and immunofluo-
rescence analyses with the anti-OA1 antibody (Fig. 3B, WT and
PT, and not shown). We did not obtain any specific signal by
either method, suggesting that, if alternative start codons down-
stream of the mutation are being used, they produce unstable
proteins not detectable by Western blot and immunofluores-
cence. This interpretation is in agreement with our data indi-
cating that, when expressed at physiological levels, mutant OA1
proteins retained in the ER are rapidly degraded (d’Addio
et
al
., 2000). Conversely, because antibodies against the N termi-
nus of OA1 are not available, we were unable to evaluate the
expression and stability of the theoretical C terminus-truncated
mutant of OA1.
OA1 patient melanocytes display an excess
of mature melanosomes
By light microscopy analysis, the patient melanocytes dis-
played a normal bipolar morphology and an overall appearance
similar to wild-type cells. To look for the presence of subcel-
lular abnormalities and particularly of macromelanosomes, we
performed extensive ultrastructural examination studies. Sur-
prisingly, the cultured patient melanocytes did not show any
structural abnormalities of melanosomes with respect to wild-
type cells, despite the huge number of samples analyzed and
despite the several culture conditions utilized (see Materials and
Methods). Indeed, both normal and patient melanocytes showed
the presence of normal melanosomes at all stages of matura-
tion, and occasionally of melanosomal aggregates. This incon-
sistency between
in vivo
versus cultivated melanocytes cannot
be attributed to patient-based variability. Indeed, we also ana-
lyzed a melanocyte culture obtained from the affected brother
of patient II-3, that is, patient II-1 (the poor viability of this lat-
ter culture did not allow us to use it for further investigation
and transduction). We found that, although examination of a
skin biopsy from patient II-1 revealed the presence of typical
macromelanosomes (see the description of the OA1 family), no
structural abnormalities of melanosomes were observed in the
cultured melanocytes obtained from this same patient.
Nevertheless, a major difference between normal and patient
melanocytes emerged by considering the number of melanosomes
and their maturation stage. Comparing the ratios between mature
SCHIAFFINO ET AL.
950
FIG. 1. Pedigree and microsatellite analysis of the OA1 fam-
ily. DXS 1223 and DXS 7108 are microsatellite markers flank-
ing the
OA1
locus on the telomeric side and on the centromeric
side, respectively. OA1-CA2 represents a novel marker located
within the
OA1
gene (see Materials and Methods for details).
The analysis shows cosegregation of the disease with the
OA1
locus.
FIG. 2. Genomic sequence of the
OA1
gene (A) and North-
ern blot analysis of the
OA1
transcript (B) in a normal indi-
vidual (WT) and in patient II-3 (PT). (A) The patient carries a
deletion of four bases within the coding region of exon 1 of the
OA1
gene (252del4). (B) Northern analysis, performed using 4
mg of total RNA from wild-type melanocytes (WT) and 2 mg
of total RNA from patient melanocytes (PT), reveals that the
OA1 mRNA expressed by patient melanocytes is similar in size
and abundance to that expressed by wild-type cells.
(stage IV, fully pigmented) versus immature (stage II–III, non-
pigmented or partially pigmented) melanosomes, we found a
higher prevalence of mature melanosomes in the patient
melanocytes with respect to wild-type cells. Table 1 shows the
melanosome counts performed in three independent normal me-
lanocyte strains (MK69, MK57, and MK106) and in the patient
melanocytes. Normal melanocytes, obtained from white subjects
with type III–IV skin complexions (as the patient), displayed a
mature/immature melanosome ratio ranging between 0.1 and 0.8,
with an average of 0.46 (Table 1, bottom row, average of wild
type). No major variations were observed in cultures of different
passage number or obtained from donors of different ages (see
Materials and Methods). In contrast, in the patient melanocytes
the mature melanosomes represented the vast majority of the or-
ganelles, with a mean mature/immature melanosome ratio of 6.99
(Table 1, bottom row, patient untreated).
Normal and mutant melanocytes can be
efficiently and stably transduced
Transduction of normal human melanocytes
. Many pigmen-
tation disorders, including albinism and xeroderma pigmento-
sum, result from the loss-of-function of the involved genes and
might therefore be considered as candidates for classic re-
placement gene therapy approaches. However, a major prereq-
uisite to the genetic correction of pigmentation disorders is rep-
resented by the availability of effective gene transfer procedures
for normal and mutant human pigment cells. To set up an effi-
cient transduction protocol for human melanocytes, we initially
utilized wild-type cells and infected them with a Moloney
murine leukemia virus-derived retroviral vector carrying the
bacterial b-galactosidase cDNA (LBSN) (Mathor
et al
., 1996).
In the search for an effective transduction strategy, we rea-
soned that when grown in close contact with keratinocytes, hu-
man melanocytes proliferate at substantially higher rates (the
mean population doubling time becomes 24 hr with respect to
the 2–4 days required by isolated melanocytes), undergo proper
morphological and functional (melanin synthesis) differentia-
tion, and physiologically migrate within the basal layer of the
cultured epidermis (De Luca
et al
., 1988). The ability of ker-
atinocytes to promote melanocyte growth and differentiation
could facilitate melanocyte transduction as well. Therefore, we
cocultured normal human melanocytes with lethally irradiated
keratinocytes and infected them with the retroviral vector
LBSN. The highest transduction efficiency (40–50%) was ob-
tained when melanocytes were seeded for at least 5 days on a
feeder layer composed of lethally irradiated keratinocytes and
producer GP1envAm12 cells (2:1 mixture; see Materials and
Methods for details).
After infection, melanocytes were passaged and geneticin
was added for 15 days to select the transduced cells. By this
treatment, the transduction efficiency reached 95–100% (Fig.
4A) and this value was maintained during the following culti-
vation without geneticin, demonstrating that normal neonatal
human melanocytes can be efficiently and stably transduced
in
vitro
with a nonmitogenic marker gene. Finally, to evaluate
whether the transduced melanocytes maintained the proper or-
ganization in the reconstituted epithelial sheet, we cocultured
human keratinocytes with LBSN-transduced melanocytes. We
found that the transduced melanocytes were associated with ke-
ratinocyte colonies like wild-type cells (Fig. 4B) and, after
colony fusion and epidermal sheet reconstitution, were physi-
ologically organized in the basal layer and maintained a nor-
mal melanocyte/keratinocyte ratio (Fig. 4C).
Transduction of OA1 patient melanocytes
. To transduce the
patient melanocytes, we took advantage of the same infection
protocol as described above, with the exception that in this case
GENE TRANSFER IN HUMAN MELANOCYTES
951
FIG. 3. Theoretical and biochemical consequences of the
252del4 mutation on the OA1 protein. (A) Schematic repre-
sentation of the truncated OA1 proteins that could theoretically
be translated by the patient mRNA starting from the first (phys-
iological) and second in-frame ATGs. The predicted heptahe-
lical topology of the wild-type OA1 protein is used as frame-
work. Vertical rectangles (I to VII), transmembrane a helices;
e
1–3
and i
1–3
, hydrophilic lumenal and intracellular loops; N and
C, N terminus and C terminus, respectively; solid circles,
residues encoded by the mutant mRNA following the
frameshift. (B) Western blot analysis of normal melanocytes
(WT), and of patient melanocytes before (PT) and after (PT-
LOA1SN) infection with the LOA1SN retroviral vector. Ar-
rows, 60-kDa glycosylated form and 45- to 48-kDa unglyco-
sylated doublet of the OA1 protein; asterisk, unrelated protein
cross-reacting with the anti-OA1 antibody. A comparable
amount of OA1 protein is detected in 25 mg and in 0.6 mg of
extract from wild-type melanocytes and from LOA1SN-trans-
duced patient melanocytes, respectively, indicating that the lat-
ter express approximately 40 times more OA1 protein than the
former.
SCHIAFFINO ET AL.
952
TABLE 1. MELANOSOM E COUNTS IN NORMAL, PATIENT, AND TRANSDUCED MELANOCYTES
Melanocyte culture
a
Average
MK69 MK57 MK106 MK106 MK106 of wild Patient Patient
(wt) (wt) (wt) LXSN LOA1SN type untreated LOA1SN
Melanosomes per cell
b
125 204 182 152 178 171 354 309
Mature melanosomes
c
012 068 081 064 065 054 304 191
Immature melanosomes
d
113 136 101 088 113 117 050 118
Mature versus immature 0.1 6 0.09 0.57 6 0.25 0.8 6 0.48 0.73 6 0.24 0.63 6 0.38 0.46 6 0.42 6.99 6 3.67 1.64 6 0.60
melanosomes
e
a
MK69, MK57, and MK106, three independent normal human melanocyte strains; MK106-LXSN, normal melanocytes trans-
duced with the empty retroviral vector LXSN; MK106 LOA1SN and patient LOA1SN, normal and patient melanocytes trans-
duced with the retroviral vector LOA1SN, respectively.
b
The number of melanosomes per cell was calculated as the arithmetic average of the counts obtained in 10–20 independent
sections from different cells of the same melanocyte culture.
c
Mature melanosomes: stage IV, fully pigmented melanosomes (mean number per cell).
d
Immature melanosomes: stage II–III, nonpigmented or partially pigmented melanosomes (mean number per cell).
e
The mature/immature melanosome ratio, represented in the figure as a histogram, was calculated as the arithmetic average of
the ratios obtained in each independent section analyzed, with the standard deviation.
FIG. 4. Transduction of normal human melanocytes with the LBSN retroviral vector. (A) b-Gal staining of the melanocyte cul-
ture after transduction and G418 selection. All visible cells appear to express the b-Gal enzyme. (B) LBSN-transduced melanocytes
associated with a keratinocyte colony. (C) After colony fusion and epidermal sheet reconstitution, the LBSN-transduced
melanocytes maintain a normal melanocyte/keratinocyte ratio, as previously observed with wild-type melanocytes. Original mag-
nification: 3400.
FIG. 5. Immunofluorescence analysis of patient melanocytes transduced with the LOA1SN retroviral vector. The recombinant
OA1 protein is detected by anti-OA1 antibody (OA1), whereas the melanosomal marker Pmel-17/gp100 is visualized by MAb
HMB45 (gp100). (A) Comparison between staining for OA1 and Pmel-17/gp100 in a representative field at lower original mag-
nification (3400), showing that virtually all G418-selected melanocytes are expressing the recombinant OA1 protein. Note that
in contrast to OA1, which is found only at an intracellular level, Pmel-17/gp100 is also detected at the plasma membrane, as pre-
viously reported. P.O., Phase optics. (B) Two examples of colocalization between OA1 and Pmel-17/gp100 at higher magnifi-
cation (31000), indicating that the recombinant OA1 protein is sorted to the melanosomes as the endogenous protein in wild-
type cells. The strong perinuclear staining obtained with the anti-OA1 antibody is probably due to accumulation of highly
overexpressed protein in the Golgi region (the recombinant OA1 in the transduced melanocytes is expressed about 40 times more
that the endogenous protein in normal melanocytes; see Fig. 3B). m., Merge.
FIG. 4
FIG. 5
the retroviral vector LOA1SN, carrying the OA1 cDNA, was
used. Because endogenous OA1 protein was undetectable in
the patient melanocytes, we could use the anti-OA1 antibody
to verify the reconstitution of OA1 expression, physiological
processing, and subcellular distribution in the transduced
cells. As shown in Fig. 3B, Western blot analysis of extracts
from the patient melanocytes after transduction with the
LOA1SN retroviral vector revealed the reappearance of both
the 60-kDa fully glycosylated form and the 45- to 48-kDa
unglycosylated polypeptides of the OA1 protein [Fig. 3B,
PT(LOA1SN)]. Serial dilutions of the LOA1SN-transduced
melanocyte extracts showed that these cells express OA1 at
a much higher level than wild-type cells, possibly because of
the strength of the long terminal repeat (LTR) promoter com-
pared with the OA1 promoter, and/or to multiple proviral in-
tegrations (Fig. 3B).
Moreover, immunofluorescence analysis using bright-field
and melanocyte markers for comparison revealed that trans-
duction efficiency was about 80–90% before the selection and
reached 100% after the addition of geneticin (not shown and
Fig. 5A). The recombinant OA1 protein was found to be sta-
bly expressed at high levels for several passages after infection
(at least six passages) and to partially colocalize with the
melanosomal marker gp100/Pmel-17 (Fig. 5B), thus displaying
a distribution pattern similar to that of the endogenous protein
in wild-type melanocytes. Finally, transduction of the patient
melanocytes with the LOA1SN retroviral vector was sufficient
to substantially revert the melanosomal maturation phenotype,
characterized by predominance of mature melanosomes, re-
ducing the mature/immature melanosome ratio toward normal
values (Table 1, from 6.99 in patient untreated to 1.64 in pa-
tient LOA1SN). Instead, no significant modification of the ma-
ture/immature melanosome ratio, or of melanosome morphol-
ogy, was observed in normal melanocytes transduced with the
empty LXSN vector or with the LOA1SN vector (Table 1,
MK106 LXSN and MK106 LOA1SN, respectively).
The LOA1SN retroviral vector appeared to confer a moder-
ate growth advantage to the patient melanocytes. During the
initial passages after infection, the LOA1SN-transduced patient
melanocytes displayed a shortening of doubling time (from 6
to 4 days in untreated and transduced cells, respectively), which
possibly facilitated cell recovery and selection. However, such
advantage was apparently lost after a few passages, so that, by
the time of harvesting for analysis, the replication rate of the
transduced melanocytes had lowered again to 6 days. Instead,
we could not obtain successful transduction of the patient
melanocytes with the empty LXSN retroviral vector, probably
because of the impaired replication ability of these cells. Nev-
ertheless, the inability of both the LOA1SN and LXSN vectors
to influence melanosome maturation in normal melanocytes
supports the specificity of the phenotype rescue obtained with
the LOA1SN vector in the patient melanocytes.
DISCUSSION
Transduction of normal and mutant
human melanocytes
The skin certainly represents one of the most attractive tar-
get tissues for gene therapy. Over the last 50 years, major ad-
vances have been achieved in the development of skin culture
and surgery techniques aimed at the repair of massive full-thick-
ness burns. Large sheets of stratified squamous epithelium can
be obtained in a limited amount of time from small bioptic sam-
ples and are routinely utilized for autologous grafting in pa-
tients suffering from large skin and mucosal defects (Gallico
et
al
., 1984; Pellegrini
et al
., 1997, 1999; Rama
et al
., 2001).
Moreover, we have successfully utilized cultured epidermal au-
tograft, bearing a controlled and physiological melanocyte/ke-
ratinocyte ratio, for the treatment of “stable” vitiligo (Guerra
et
al
., 2000). Finally, we have previously shown that the main cell
type in the skin, the keratinocyte and its precursor stem cells,
can be efficiently and stably transduced by retroviral vectors
and selected
in vitro
(Mathor
et al
., 1996; Dellambra
et al
.,
1998). These findings demonstrate that, at least in principle,
ex
vivo
gene therapy approaches using the skin as the target tis-
sue may be feasible for the correction of cutaneous as well as
noncutaneous diseases.
The melanocytes represent the second physiologically most
important cell type in the skin (with a mean rate of 1:35 with
respect to keratinocytes). They have protective and aesthetic
functions, but can also generate a common and extremely se-
vere type of cancer, malignant melanoma. Therefore, the trans-
duction of melanocytes represents a key step for the develop-
ment of therapeutic approaches aimed at the correction of skin
pigmentation disorders, involving melanocytes alone, such as
oculocutaneous albinism, or both melanocytes and ker-
atinocytes, such as xeroderma pigmentosum (XP). Indeed, XP
is an extremely severe genetic disorder characterized by muta-
tion of the genes involved in excision repair of damaged DNA.
Patients with this disorder show hypersensitivity to ultraviolet
rays, with an incidence of squamous and basal cell carcinomas
and melanomas over 2000 times greater than in the normal pop-
ulation. Therefore, both keratinocyte and melanocyte gene cor-
rection would be required for the treatment of this disorder (Car-
reau
et al
., 1995; Zeng
et al
., 1997).
Gene transfer or transgenesis approaches have been widely
utilized in mice and other rodents to perform biological stud-
ies on melanocyte function and development (Kucera
et al
.,
1996; Dunn
et al
., 2000), and to correct various types of ge-
netic or acquired pigmentation disorders, including the melan-
ocyte-derived cancer malignant melanoma (Hirschowitz
et al
.,
1998). Rescue of the hypopigmented phenotype in different
kinds of albinism has been accomplished by several approaches.
Melanocyte cultures, generated from pink-eyed dilution and
brown mice, have been transduced with the corresponding
genes by using LipofectAMINE reagents and retroviruses, re-
spectively (Bennett
et al
., 1990; Sviderskaya
et al
., 1997). Mu-
tations of the tyrosinase gene have been complemented in al-
bino mouse melanocytes and skin by RNA–DNA
oligonucleotide strategies (Igoucheva and Yoon, 2000), in al-
bino mice by cell-type-directed gene targeting
in utero
(Dunn
et al
., 2001), and in albino rabbits by yeast artificial chromo-
some transgenesis (Brem
et al
., 1996).
However, the possibility of using genetically modified hu-
man melanocytes to correct human pigmentation disorders has
been hampered so far by a number of limitations, including the
resistance of these cells to retroviral infection compared with
keratinocytes, possibly depending on their lower replication
abilities. Indeed, it was previously shown that normal human
melanocytes can be transduced with retroviral vectors, although
SCHIAFFINO ET AL.
954
with low efficiency (Coleman and Lugo, 1998; Hamoen
et al
.,
2001). Coleman and Lugo (1998) transduced human
melanocytes with a bFGF-retrovirus or an empty retrovirus, re-
porting yields of 5–10 or 20–60 G418-resistant colonies per in-
fection, respectively. Hamoen
et al
. (2001) transduced human
melanocytes with a hepatocyte growth factor (HGF)-retrovirus,
achieving an efficiency of 6%. Higher transduction efficiencies
were obtained with adenoviral vectors; however, the episomal
nature of adenoviral replication makes such vectors unsuitable
for the gene therapy of a highly self-renewing tissue like the
skin (Nesbit
et al
., 1999). Thus, the possibility of efficiently
and stably transducing human pigment cells, particularly if car-
rying mutations that may affect their viability and growth ca-
pacity and using nonmitogenic genes, remains to be established.
We obtained a melanocyte culture from a 5-year-old indi-
vidual affected with ocular albinism type 1. The patient
melanocytes did not express any detectable OA1 protein and,
as often observed with mutant cells, displayed a significantly
impaired replication ability compared with wild-type cells. Thus
they represented a suitable model for setting up an efficient
gene transfer procedure. For this purpose, we took advantage
of a melanocyte–keratinocyte coculturing approach and trans-
duced the patient melanocytes with the retroviral vector
LOA1SN, carrying the OA1 cDNA. Consequently, we could
evaluate both the efficiency of the transduction protocol and re-
constitution of the physiological processing, targeting, and ac-
tivity of the melanocyte-specific protein OA1 in human pig-
ment cells.
Our results showed that the LOA1SN-transduced patient
melanocytes were able to express large amounts of recombi-
nant OA1 protein and to sustain its correct processing and tar-
geting to the melanosomes. Moreover, the transduced cells dis-
played a substantial recovery from their aberrant predominance
of mature melanosomes, regaining a normal mature/immature
melanosome ratio. The recombinant OA1 protein was found to
be expressed at high levels in 80–90% of the infected
melanocytes before selection and in virtually 100% of the cells
after selection with G418. OA1 expression was maintained for
several passages after infection, indicating that the transgene is
stably integrated in the melanocyte genome and remains func-
tional during the subsequent doublings of the cells. Although
we obtained only a moderate and transient increase in the repli-
cation rate of LOA1SN-transduced patient melanocytes, the
presence of a more powerful and effective growth advantage in
other systems would probably increase the recovery and facil-
itate the selection of the genetically corrected melanocytes.
In summary, we have developed an efficient gene transfer
procedure for human melanocytes, allowing the expression of
nonmitogenic recombinant proteins in high amounts, and in a
uniform and stable manner. We showed that pure cultures of
normal or mutant melanocytes can be transduced, selected, and
eventually used together with keratinocytes for the generation
of genetically modified epidermal sheets suitable for grafting
onto patients. Our results demonstrate the feasibility of an
ex
vivo
gene therapy approach for the correction of inherited and
acquired disorders involving pigment cells of the skin.
The melanosomal phenotype in ocular albinism type 1
The culture conditions used for growing melanocytes
in vitro
are different with respect to those used for intact skin. As a con-
sequence, normal human melanocytes in culture can exhibit var-
ious types of melanosomal abnormalities (Breathnach
et al
.,
1988). Nevertheless, a number of melanocyte strains and lines
have been obtained previously from mice (and humans) affected
with various forms of albinism, with preservation of the original
phenotypes (Park
et al
., 1993; Zhao
et al
., 1994; Bennett and
Sviderskaya, 1996). Although somewhat unexpected, the lack of
typical macromelanosomes in the OA1 patient melanocytes ap-
pears consistent with the variable expressivity (within the same
cell and in different cell types and patients) and with the non-
specificity displayed
in vivo
by this particular melanosomal phe-
notype (O’Donnell
et al
., 1976; Garner and Jay, 1980; Wong
et
al
., 1983; King
et al
., 1995; Schnur
et al
., 1998). Thus, the de-
ficiency of OA1 appears neither sufficient nor necessary by it-
self to determine the manifestation of the macromelanosomal
phenotype, which instead might depend on multiple factors giv-
ing rise to (or preventing) this abnormality in different patho-
logical conditions. On the other hand, the role of macrome-
lanosomes in the pathogenesis of ocular albinism remains to be
established (Incerti
et al
., 2000).
Despite the absence of macromelanosomes, we noticed a sur-
prisingly high prevalence of mature melanosomes in the OA1
patient melanocytes with respect to wild-type cells and a sig-
nificant rescue of this abnormality after transduction with the
LOA1SN retroviral vector (Table 1). These results suggest that
the dysregulation of melanosome biogenesis caused by the de-
ficiency of OA1 might manifest with alternative phenotypes,
presence of macromelanosomes or prevalence of mature
melanosomes,
in vivo
and
in vitro
, respectively. Consistent with
our previous studies on the
Oa1
knockout (Incerti
et al
., 2000),
these findings further support the idea that OA1 could act as a
negative regulator of melanosome maturation, by preventing
melanosome overgrowth and/or uncontrolled melanin deposi-
tion.
ACKNOWLEDGMENTS
We thank Dr. V. Marigo for critical reading of the manu-
script. This work was supported by generous donations from
Vision of Children Foundation-San Diego (to M.V.S. and A.B.),
Telethon-Italy (Telethon grants F.3 to M.V.S., A.106 and B.53
to M.D.L., and E0942 to C.T.), CNR (target project “Biotech-
nology” to C.T.), and MURST (to C.T.). We also warmly thank
all members of the OA1 family involved in this study, as with-
out their collaboration this work would not have been possible.
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Address reprint requests to:
Dr. M. Vittoria Schiaffino
DIBIT, Scientific Institute San Raffaele
Via Olgettina 58
20132 Milan, Italy
E-mail:
schiaffino.mariavittoria@hsr.it
Received for publication July 5, 2001; accepted after revision
April 9, 2002.
Published online: April 23, 2002.
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