|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 465-474
Correction of Uroporphyrinogen Decarboxylase Deficiency
(Hepatoerythropoietic Porphyria) in Epstein-Barr Virus-Transformed
B-Cell Lines by Retrovirus-Mediated Gene Transfer: Fluorescence-Based
Selection of Transduced Cells
By
Antonio Fontanellas,
Frédéric Mazurier,
François Moreau-Gaudry,
Francis Belloc,
Cécile Ged, and
Hubert de
Verneuil
From the Laboratoire de Pathologie Moléculaire et
Thérapie Génique, FR 60 Biologie des Greffes,
Université Victor Segalen Bordeaux 2, Bordeaux, France; and the
Laboratoire d'Hémobiologie, Hôpital du
Haut-Lévêque, CHU de Bordeaux, Bordeaux, France.
 |
ABSTRACT |
Hepatoerythropoietic porphyria (HEP) is an inherited metabolic
disorder characterized by the accumulation of porphyrins resulting from
a deficiency in uroporphyrinogen decarboxylase (UROD). This autosomal
recessive disorder is severe, starting early in infancy with no
specific treatment. Gene therapy would represent a great therapeutic
improvement. Because hematopoietic cells are the target for somatic
gene therapy in this porphyria, Epstein-Barr virus-transformed B-cell
lines from patients with HEP provide a model system for the disease.
Thus, retrovirus-mediated expression of UROD was used to restore
enzymatic activity in B-cell lines from 3 HEP patients. The potential
of gene therapy for the metabolic correction of the disease was
demonstrated by a reduction of porphyrin accumulation to the normal
level in deficient transduced cells. Mixed culture experiments
demonstrated that there is no metabolic cross-correction of deficient
cells by normal cells. However, the observation of cellular expansion
in vitro and in vivo in immunodeficient mice suggested that genetically
corrected cells have a competitive advantage. Finally, to facilitate
future human gene therapy trials, we have developed a selection system
based on the expression of the therapeutic gene. Genetically corrected
cells are easily separated from deficient ones by the absence of
fluorescence when illuminated under UV light.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
PORPHYRIAS ARE INHERITED metabolic
diseases characterized by specific enzyme defects along the heme
biosynthetic pathway.1 Uroporphyrinogen decarboxylase
(UROD; EC. 4.1.1.37) is the fifth enzyme of this pathway and catalyzes
the sequential decarboxylation of uroporphyrinogen to yield
coproporphyrinogen.2 A deficiency of this enzyme is
responsible for porphyria cutanea tarda (PCT), the commonest form of
porphyria, and for hepatoerythropoietic porphyria (HEP). HEP, the
homozygous form of familial PCT, is clinically similar to congenital
erythropoietic porphyria (CEP) and is characterized by severe dermal
photosensitivity leading to scarring and mutilation of sun-exposed
areas of skin, sclerodermoid changes, hypertrichosis, erythrodontia,
anemia, and hepatosplenomegaly occuring very early in infancy or
childhood.1 The human enzyme has been purified to
homogeneity from erythrocytes3 and the human UROD gene has
been isolated and sequenced.4 The molecular lesions
responsible for HEP were investigated.4-6 The available treatments are mostly unsatisfactory and often limited to supportive care. The enzymatic deficiency is responsible for the accumulation of
large amounts of uroporphyrin and heptacarboxylic porphyrin and has
been found in all of the tissues investigated, especially bone marrow
and liver. This type of porphyria is a good candidate for somatic gene
therapy, because the genetic defect is well characterized at the
molecular level and inherited as a recessive trait. The ideal therapy
should concern two tissues: bone marrow and liver. One approach to
somatic gene therapy involves the use of recombinant retroviral vectors
to transduce cells ex vivo, followed by autologous transplantation of
genetically engineered cells.7 To facilitate the
development of ex vivo gene therapy, efficient selection procedures are
required for the isolation of genetically corrected cells before
autologous transplantation.
The aim of the present study is the transfer of functional UROD cDNA
into HEP-deficient cells by means of a retroviral vector. The
feasibility of the correction of the enzymatic defect is tested in
B-lymphocyte cell lines established from HEP patients. The efficiency
of gene transfer was analyzed in terms of retroviral integration,
enzymatic restoration, metabolic correction, and cellular expansion.
The ability of genetically corrected cells to transform the excess of
uroporphyrinogen produced by deficient cells was also analyzed.
Finally, a fluorescence-based selection procedure using flow cytometry
analysis and sorting was developed to isolate directly metabolically
corrected cells.
| |
MATERIALS AND METHODS |
Cell lines.
Epstein-Barr virus-transformed B lymphocytes were used because they can
be readily obtained from patients and because the cells are easy to
grow in culture. Peripheral blood lymphocytes (PBL) were isolated from
patients and controls. Cell lines (LB) were established from PBL after
Epstein-Barr virus infection at Laboratoire Genethon (Evry, France).
Deficient PBL were obtained from three HEP patients carrying two
different point mutations of the UROD gene. Two deficient (LBHEP) cell
lines, LB44 and LB45, harbored the G281E mutation (substitution of
glycine by glutamic acid at codon 281) leading to an unstable
protein.5 The third deficient cell line, LB86, had an
unpublished mutation F46L (substitution of phenylalanine by leucine at
codon 46). The impact of this mutation on UROD protein function is
under investigation. Normal (LBN) and deficient (LBHEP) lines are
maintained at 0.2 to 2 × 106 cells/mL in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS; Boehringer
Mannhein, Meylan, France), 100 U of penicillin per milliliter, and 1 mg/mL of streptomycin (GIBCO BRL, Grand Island, NY) at 37°C in 5%
CO2 atmosphere. The ecotropic Gp+env86 and the amphotropic
 CRIP packaging cell lines were maintained in Dulbecco's modified
Eagle's medium (DMEM), supplemented with 10% FCS, 100 U of penicillin
per milliliter, and 1 mg/mL of streptomycin at 37°C in 5%
CO2 atmosphere.
Construction of retroviral vector LUDSN and obtaining different
packaging cell lines.
The pLXSN vector8 has been widely used for gene transfer in
hematopoietic cells. The vector contains the NeoR gene that
allows the selection of transduced cells by G418 (Geneticin; GIBCO
BRL). NeoR gene is controlled by SV40 early promoter
sequences. The gene of interest is driven by the Moloney murine
leukemia virus (MoMLV)/Moloney murine sarcoma virus (MoMSV) long
terminal repeat (LTR). Normal full-length human UROD cDNA
(1.2 kb) was obtained from pG7UD vector.5 cDNA was cleaved
by EcoRI and Xho I and cloned into the pLXSN vector to
obtain the pLUDSN construct. The recombinant amphotropic particles were
obtained by a three cell-line transfection process (ping-pong
method).9 First, pLUDSN construct was introduced by DOTAP
transfection reagent (Boehringer Mannheim) into a first packaging cell
line CRIP. Second, filtered supernatants from virus-producing cells
were used to infect Gp+env86 cells in the presence of polybrene (Sigma,
St Louis, MO) during 6 hours for 3 consecutive days. Two days after the
last infection, cells were selected in the presence of 1 mg/mL G418
(Geneticin; GIBCO BRL) for 2 weeks. Resistant clones were isolated and
the clones producing the highest viral titer were selected using the
NIH3T3 cell line as a target. The Gp+env86/LUDSN8 clone was selected
for subsequent transduction experiments because of a high viral titer
(1.2 × 106 cfu/mL) and a UROD enzymatic activity
14-fold the normal value. Third, the CRIP packaging cell line
was transduced by filtered retroviral supernatant in the presence of
protamine sulfate (Sigma). The protocol of infection and selection was
as described for the Gp+env86 cells. G418-resistant clones
(CRIP/LUDSN) were isolated and screened for those producing the
highest titer using NIH3T3 cells as targets. Supernatants were tested
for the presence of replication competent virus.10
Transduction of target cells.
Retroviral supernatant was obtained from the producer cell lines
(CRIP/LUDSN7, 11, and 16 or CRIP/LXSN) maintained at 32° C in
5% CO2 atmosphere for 24 to 48 hours. Target cells were
transduced by retroviral supernatant infection under two different
conditions. For the first transduction protocol, 2 mL of the filtered
supernatant was added to 4 × 105 target cells (8:1
colony-forming units/cell ratio) in the presence of 8 µg/mL
polybrene. Cells and retroviral supernatant were incubated in a 6-well
plate and were centrifuged at 1,000g at 32°C for 60 minutes
followed by incubation for 6 hours at 32°C in a 5% CO2 incubator. Medium from producer cells was then replaced by RPMI 1640 medium supplemented with 20% FCS for 17 hours. Cells were transduced
for 3 consecutive days at 24-hour intervals. After the last infection,
LB were maintained without selection for 4 days to allow expansion and
NeoR expression. After expansion, LBHEP were subjected to
an initial period of selection in 0.2 mg/mL G418 for 4 days and then in
0.5 mg/mL for 1 week. Finally, LBHEP were maintained in medium
containing 1 mg/mL G418 for 2 to 3 weeks. In a second procedure, wells
(of a 6-well plate) were precoated with human recombinant CH296
fibronectin fragment (Retronectin, Takara; Boehringer Ingelheim, Gagny,
France) as recommended by the manufacturer. Two milliliters of filtered virus supernatant (2.4 × 106 viral particles) was
added for 20 minutes five times and then 4 × 105
normal or deficient LB cells were incubated in the wells for 6 hours at
37°C in 5% CO2 atmosphere for 3 consecutive days.
After each infection period, the medium was replaced by RPMI 1640 supplemented with 20% FCS. Selection was performed as described above.
Supernatants from these three clones were tested for the presence of
replication competent virus and were negative.
Southern blot analysis.
Southern blot analysis allows the quantification of integrated viral
copies in the target cell using known amounts of the vector as a
standard. High molecular weight DNA was extracted from LB cells by
proteinase K digestion and phenol/chlorophorm extraction. Ten
micrograms of genomic DNA was digested with Sac I; this enzyme
cut both LTR regions in our construct and produced a 3-kb band from the
LUDSN vector. The resulting fragments were separated on a 0.8% agarose
gel and transferred onto nylon membrane (Hybond N+; Amersham, Les Ulis,
France). Known amounts of plasmid DNA mixed with control LB DNA were
run on the same gel as standard. Filters were hybridized with a 1.2-kb
human UROD cDNA fragment that was labeled with 32P dCTP by
random priming (Multiprime DNA labeling system; Amersham Pharmacia,
Orsay, France). The vector copy number per cell was estimated by
comparison of the amount of signal generated by standards. The signal
obtained with 10 pg of plasmid DNA corresponds to the presence of one
copy of the transgene per cell when 10 µg of genomic DNA is loaded on
the gel.
UROD enzymatic activity and porphyrin level.
UROD activity and porphyrin level were determined in normal and LBHEP
cell lines to assess the phenotypic expression of the transferred gene.
Determination of UROD activity was performed according to de Verneuil
et al.3 UROD activity was expressed as picomoles of
coproporphyrinogen formed per hour per milligram of protein (units per milligram).
LB cell lines were seeded at 2 × 105 cells per
milliliter in the 6-well plate and maintained for 72 hours in RPMI 1640 medium containing 20% FCS. The medium was then kept, the cells were
washed with phosphate-buffered saline (PBS) and counted, and porphyrins were extracted from cell lysates and from the medium with 1 mol/L HClO4/CH3OH (1:1, vol/vol). Total porphyrin
level was quantitated by spectrofluorimetry (excitation, 405 nm;
emission, 595 nm). Uroporphyrin I solution (10 nmol/L) was used as a
standard. Porphyrin level was expressed as femtomoles of porphyrins per
103 cells. Individual carboxylated porphyrins were
identified by high performance liquid chromatography (HPLC)
assay.11
In vitro cell proliferation assays.
The MTS test12 (CellTiter 96rAQueous Solution
Cell Proliferation Assay; Promega Corp, Madison, WI) was used to
analyze the consequence of the metabolic deficiency on the cell
proliferation rate. LB cells were seeded at 2 × 105
cells per milliliter in the 6-well plate in RPMI 1640 medium containing
20% FCS. These cells were grown for 2 days before the MTS test. Viable
cells (104) were resuspended in 100 µL of fresh medium
per well of the 96-well plate reader. Assays were performed at 12, 24, 48, and 72 hours of culture at 37°C in a humidified 5%
CO2 atmosphere. Twenty microliters of the CellTiter
Solution Reagent was added directly to the wells and the plates were
incubated for 4 hours. Absorbance was measured at 490 nm.
The cellular expansion of LBN, LB44, LB44-LUDSN11, and a mixture of
LB44/LB44-LUDSN11 (80/20) was assessed at 0, 5, and 10 days by
measuring the proliferation index (number of cells at t = x days
divided by the number of cells at t = 0 day). Cells were suspended and
their viability was assessed with trypan blue. Viable cells were
counted in duplicate in a Malassez's count slide. Cell doubling time
was calculated on the first 5 days of culture. UROD enzymatic activity
was measured in the mixture at different times of culture.
In vivo cell proliferation in immunodeficient mice.
Injection of LB cells in immunodeficient mice results in the
development of a lymphoproliferative disorder characterized by B-cell
tumors.13 We used a double mutant, the
RAG2 // c mouse,
characterized by a complete absence of B and T lymphocytes and NK cells
in peripheral blood.14 Twenty-eight double homozygous mutant male mice between 8 to 10 weeks of age were randomly selected, divided into four groups, and inoculated subcutaneously with 2 × 106 LBN, LB44, or LB44-LUDSN11 cells or by the mixture of
LB44/LB44-LUDSN11 (80/20) LB cells as described above. Animals were
kept under sterile conditions in microisolators or air-filtered cages
and provided with autoclaved food and water. Most of the animals
developed subcutaneous tumors at the site of inoculation. Animals were
killed when tumors became clinically apparent (30 to 45 days after
injection), and tumors were resected for analysis of UROD enzymatic activity.
Fluorescence-based selection of retrovirally transduced cells.
Deficient and corrected HEP LB cell lines were subjected to preparative
fluorescence-activated cell sorting (FACS). Culture cells were seeded
at 2 × 105 cells/mL into T-75 culture flasks in RPMI
1640 medium supplemented with 7% FCS for 24 hours before flow
cytometry analysis. Accumulation of porphyrins in the deficient,
transduced, and control cells was monitored by fluorescence microscopy
using a 100 W mercury lamp and a near UV band filter. Cells were
harvested and concentrated at 106 cells/mL and analyzed by
flow cytometry using an Elite cytometer (Coulter, Coultronics,
Margency, France) at a rate of 1,500 events/s using saline as a sheath
fluid. Excitation was performed by an argon laser (Spectra Physics, Les
Ulis, France) tuned to emit 100 mW in UV light (340 to 360 nm).
Fluorescence was measured through a 675-nm band filter. For the cell
sorting experiments, the cells with either the lowest or the highest
fluorescence intensities were sorted in sterile tubes containing 0.5 mL
FCS. FACS efficiency was analyzed in terms of UROD enzymatic activity
and the percentage of transduced cells estimated by Southern blot.
We then tested a different strategy based on an increase in
uroporphyrin accumulation in untransduced deficient LB cells by exposure to  -aminolevulinic acid (ALA). Melatonin was also added to
the medium because of a protective role against oxidative damage induced by ALA.15 Culture cells were seeded at 2.5 × 105 cells/mL into T-75 culture flasks. ALA, melatonin, and
FeSO4 (Sigma, Saint-Quentin, France) were added to a final
concentration of 1 mmol/L, 2 mmol/L, and 50 µmol/L, respectively, in
RPMI 1640 medium containing 7% FCS at pH 7.4. After 12 hours, cells
were harvested, washed with PBS, and reseeded at the same dilution in
fresh RPMI 1640 medium supplemented with 7% FCS for 4 hours. Porphyrin
accumulation in cell lysates was quantitated by spectrofluorimetry, as
described above. The fluorescence of intact cells was estimated by
cytofluorimetry (Coulter Elite, Coultronics).
| |
RESULTS |
Transduction of target cells.
Three clones (CRIP/LUDSN7, CRIP/LUDSN11, and CRIP/LUDSN16)
characterized by a retroviral titer greater than 106 cfu/mL
were chosen to transduce the deficient LB45 cell line. UROD activity
was measured in the nontransduced and in the different transduced and
selected cell lines to find the best clone in terms of enzymatic
correction. Results are shown in Fig 1. The
number of integrated proviral copies was approximately one copy per
target cell in the three transduced and selected cell lines, as
estimated by Southern blot analysis (Fig 1A). CRIP/LUDSN11
clone, which demonstrated a 10-fold increase in enzymatic activity
compared with the deficient nontransduced cells (Fig 1B), was chosen
for the following experiments.
View larger version (55K):
[in this window]
[in a new window]
| Fig 1.
Identification of the LUDSN provirus and expression of
the transgene in HEP LB cells. Cells were infected with LUDSN7,
LUDSN11, and LUDSN16 by centrifugation in the retroviral supernatant at
1,000g and selected for 3 weeks with G418 (see Materials and
Methods). (A) Provirus integration into the genomic DNA of LB deficient
cells (Southern blot analysis). Ten micrograms of genomic DNA extracted
from transduced and selected LB45/LUDSN7, LB45/LUDSN11, and
LB45/LUDSN16 cell lines was digested with Sac I, blotted, and
probed with the 1.2-kb human UROD cDNA. Lane 0, 10 µg of DNA from
untransduced LB cells. Lanes 2 and 10 contained plasmid DNA (20 and 100 pg, respectively) mixed with 10 µg of noninfected DNA corresponding
to 2 and 10 copies per cell. The 3.0-kb band corresponds to the
integrated provirus and the 6.5-kb band corresponds to the endogenous
UROD gene. Note that the migration of the 3.0-kb band for LB45 samples
were artefactually sligthly different from the control plasmid DNA. (B)
UROD activity in transduced and deficient LB45 cell lines. Values are
the means ± SD of three to six determinations.** P < .01 v noninfected LB45 cells.
|
|
Results of UROD enzymatic activity in untransduced and transduced
normal, LB44, LB45, and LB86 cells are shown in
Table 1. UROD-specific activity of
unselected LB cells was higher when transduction was performed in
plates coated by Retronectin than when cells were centrifuged in the
retroviral supernatant. Consequently, all of the following experiments
were performed with selected LB cells transduced by Retronectin.
Deficient cell lines were transduced with LXSN (control virus) to
investigate a possible effect of the virus itself. The level of UROD
activity in LBHEP cell lines was not modified when compared with
selected LBHEP-LXSN cell lines (195 ± 76 U/mg for LB44-LXSN v 219 ± 123 U/mg for LB44; 251 ± 159 U/mg for LB45-LXSN
v 288 ± 108 U/mg for LB45; and 369 ± 195 U/mg for
LB86-LXSN v 396 ± 129 U/mg for LB86). After transduction of
the LBHEP cells by LUDSN11 and selection with G418, UROD activity was
increased to nearly normal levels (71%, 106%, and 89% for LB44,
LB45, and LB86 cells, respectively; Table 1). Transduced LB cells
expressed the same level of UROD activity for 2 months after the end of
selection, demonstrating that the UROD transgene introduced by the
retroviral vector was stably integrated and expressed (data not shown).
To evaluate the ability of the transgene to correct the metabolic
defect, the porphyrin level was measured in the cells and in the medium
for untransduced and transduced cells (Fig
2A). In the normal cells, a very low amount of porphyrins was found (0.12 ± 0.04 fmol per 103 cells). In deficient cells, a
significant increase in porphyrin level was found (from 4.5 to 24 fmol
per 103 cells) with the predominance of the highly
carboxylated forms, uroporphyrin, and heptacarboxylic porphyrin, which
are specific of the disease. Normalization of porphyrin level,
documented in Fig 2A, demonstrated the ability of the transduction to
correct the metabolic defect.
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
Porphyrin level in normal, deficient, and transduced
deficient LB cells. (A) LB cells were maintained in exponential phase
of culture and then placed in culture at 2 × 105
cells/mL. After 48 hours, porphyrins were measured ([ ] Cell
porphyrin level, [ ] Supernatant porphyrin level) as described in
Materials and Methods. Data are the mean ± SD of four to six
determinations. ** P < .01 v noninfected LBN cells.
(B) Porphyrin level in LBN cells, deficient LB45 cells, and a mixture
(50/50) of LB45 and LBN cells. Cells were placed in culture at 2 × 105 cells/mL. Porphyrin level was measured by
spectrofluorimetry immediatly ([ ] 0 hour) and after 24 (), 48 (), and 72 hours (), as described in Materials and Methods. Data
are the means ± SD of six independent experiments.
|
|
Absence of metabolic cross-correction between deficient and
transduced cells.
Porphyrin levels in normal, deficient, and a mixture (50/50) of normal
LB and LB45 cells at 0, 24, 48, and 72 hours are shown in Fig 2B. The
porphyrin level in the mixed cells was approximately half that in the
LB45 cell line. These in vitro results show that the UROD enzyme from
normal LB cells was not able to metabolize the excess of
uroporphyrinogens produced by the nontransduced deficient LB cells.
In vitro cell proliferation.
Two different tests were performed to analyze the influence of
metabolic correction on the cell proliferation rate. The MTS test was
performed to determine the number of metabolically active LB cells. For
these experiments, transduction with LXSN was used to rule out a
putative effect of the transduction itself. At 72 hours, cell numbers
in normal and LBHEP/LUDSN11 cells were significantly higher than in
noninfected and LB HEP/LXSN cells in the three LB HEP cell lines
investigated (Fig 3). The cell doubling
time was 34.5 hours for LB44 versus 19.4 hours for LB44-LUDSN11, 32.5 hours for LB45 versus 21 hours for LB45-LUDSN11, and 38.25 hours for
LB86 versus 21.1 hours for LB86-LUDSN11. As a reference, LBN doubling
time was 18.7 hours. The second test determined the proliferation index
by counting the cells at different time intervals
(Table 2). In these experimental
conditions, the proliferation index was 10.3 versus 21.1 after 10 days
of culture for the LB44 versus LB44-LUDSN11 cells, respectively,
corresponding to a doubling time of 46.7 hours versus 22.7 hours. As a
reference, the proliferation index was 21.9 for the LBN cells and the
doubling time was 21.9 hours.
View larger version (17K):
[in this window]
[in a new window]
| Fig 3.
Cell proliferation in normal, deficient, and deficient
transduced (LXSN or LUDSN11) and selected LB cell lines. Assays were
performed at different time intervals (0 to 72 hours) with the LB44
(A), LB45 (B), and LB86 (C) cell lines. For each cell line, results are
shown for nontransduced ( ), LXSN-transduced (), and
LUDSN-transduced cells ( ). As a reference, a normal LB cell line is
also shown ( ). Determination was performed by the CellTiter
96rAQueous Solution Cell Proliferation Assay (Promega Corp;
MTS test). Results of experiments for each cell line were the mean of
five determinations from three independent experiments. * P < .05, **P < .01 v the LBHEP/LUDSN11 cells.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Proliferation Index in LBN and LB44 Cells, in Transduced
and Selected LB44-LUDSN11 Cells, and in a Mixture (80/20) of LB44 and
Selected LB44-LUDSN11 Cells
|
|
Comparison of UROD activity in LB44/LB44-LUDSN11 mixed-cell experiments
between different time intervals indicated that there was a 1.3- and
2.3-fold enrichment of enzymatically corrected cells at days 5 and 10 of coculture, respectively (Fig 4).
View larger version (34K):
[in this window]
[in a new window]
| Fig 4.
Enrichment of enzymatically corrected cells in vitro and
in vivo in mixed-cell culture experiments evidenced by the increase in
enzymatic activity. LB44 (), LB44-LUDSN (), and a mixture of LB44
(80%) and LB44-LUDSN (20%) () was cultured in vitro for 5 and 10 days or injected into immunodeficient mice and analyzed after 30 to 45 days. LB cells or B-cell tumors were resected and UROD activity was
measured. * P < .05, **P < .01 v the
LB44/LB44-LUDSN11 cells at day 0.
|
|
In vivo cell proliferation in immunodeficient mice.
The subcutaneous injection of LB cells into immunodeficient mice
resulted in the development of a B-cell tumor (0.1 to 1.5 g in weight
for the tumor compared with a mean weight of 28 g for the whole mouse)
at the site of inoculation between 30 and 45 days after injection. Cell
proliferation was estimated in terms of increase in UROD enzymatic
activity in a mixture of deficient (80%) and transduced (20%) cells.
Control experiments with LB44 and LB44-LUDSN11 cells (Fig 4) showed
that UROD activity was not modified during engraftment. By contrast,
UROD activity in the mixture of deficient (80%) and transduced (20%)
cells was increased 1.9-fold compared with the initial UROD activity.
Mice injected with LB44-deficient cells evidenced higher plasma
porphyrin concentrations at the day of death (24.7 nmol/L; range, 7.4 to 73.8 nmol/L) than did noninjected control mice (4.6 nmol/L; range,
3.9 to 5.6 nmol/L) or injected mice with LBN (5.1 nmol/L; range, 2.9 to
5.9 nmol/L) or with LB44-LUDSN11 (7.2 nmol/L; range, 5.4 to 10.7 nmol/L).
Fluorescence-based selection of retrovirally transduced cells.
Deficient LB cells are characterized by an accumulation of porphyrins
resulting from the deficient UROD activity. In genetically modified
cells, the metabolic flow from uroporphyrinogen to protoporphyrin and
heme was restored and a decrease in porphyrin accumulation was observed
(Table 1). A mixture of deficient (80%) and corrected (20%) LBHEP
cells were cocultured and analyzed by flow cytometry (Fig 5). Two overlapping cell populations
were identified, which correspond to the enzymatically corrected (ie,
low fluorescent) and noncorrected (ie, high fluorescent) cells (Fig
5D). After sorting, the cell population with low fluorescence (Fig 5F)
had a normal UROD activity and normal porphyrin accumulation
(Table 3). Southern blot analysis
demonstrated that the low fluorescence fraction (Fig 5F) of cells had
one copy of retroviral vector per cell, the mixed cells had 0.2 copy
per cell, and the high fluorescent fraction (Fig 5E) had no band
corresponding to a retroviral vector sequence (data not shown).
View larger version (21K):
[in this window]
[in a new window]
| Fig 5.
Flow cytometry and FACS analyses of different populations
of LB cells. LBN (A), LB45 (B), selected LB45-LUDSN11 (C), and a
mixture of LB45/LB45-LUDSN11 (80/20) (D) cells were analyzed by flow
cytometry using a UV light excitation to characterize porphyrin
fluorescence in the cells. FACS analysis was then performed on mixed
cells. After sorting, the cell populations corresponding to high (E)
and low fluorescence (F) were reanalyzed.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3.
UROD Enzymatic Activity and Porphyrin Level in
Untransduced and Transduced HEP LB Cells Before and After FACS
Selection
|
|
A similar experiment was performed with another selected cell line,
LB45-LUDSN7. In these experiments, transduced cells had only 67% of
normal UROD activity (1,836 ± 460 U/mg in LB45-LUDSN7 v
2,728 ± 238 U/mg in LBN; P < .01) and the porphyrin level
was 0.6 ± 0.2 fmol/103 cells in LB45-LUDSN7 versus 0.12 ± 0.04 fmol/103 cells in LBN cells (P < .05).
The mixture of transduced (20%) and nontransduced cells (80%) showed
a single peak, ie, the two populations were not discriminated by flow
cytometry, in relation to the weak residual fluorescence of transduced
cells. Thus, we used a different strategy, based on an increase in
porphyrin accumulation in untransduced deficient LB cells by exposure
to ALA (Fig 6). After 12 hours of ALA
exposure, normal or genetically corrected cells were able to metabolize
porphyrinogens accumulated in the cell within 4 hours. By contrast,
deficient cells still presented a high accumulation of
porphyrin(ogen)s. The maximum difference of porphyrin accumulation,
determined by the LB45/LB45-LUDSN7 ratio, was observed 4 hours after
ALA incubation (39.1 ± 8 or 27.6 ± 2 when estimated in intact
cell by flow cytometry or in cell lysate, respectively, v 18.2 ± 5.1 before ALA exposure, P < .05). After ALA exposure,
flow cytometry analysis showed the mixture of LB45/LB45-LUDSN7 cells
(80/20) to have two nonoverlapping cell populations. Sorted cell
population with low fluorescence showed the same UROD activity (2,101 ± 310 U/mg) and porphyrin level (0.65 ± 0.31 fmol/103 cells) than the transduced cells (UROD activity,
1,836 ± 460 U/mg; porphyrin level, 0.6 ± 0.2 fmol/103 cells) with one copy of retroviral vector per cell
(data not shown).
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
In vitro cell porphyrin accumulation during and after ALA
exposure. LB45 (), LBN ( ), and LB45-LUDSN7 () cells were
cultured in RPMI 1640 and 7% FCS in the presence of 1 mmol/L ALA, 2 mmol/L melatonin, and 50 µmol/L FeSO4 for 12 hours as
indicated at the top of the figure. The medium was then removed, and
cells were washed with PBS and then resuspended with regular medium
(RPMI 1640 and 7% FCS). At regular time intervals, the porphyrin level
was measured in the cells.
|
|
In a separate experiment, cell survival was quantitated in untransduced
cells without (90% ± 1.4%) or with ALA exposure (73.9% ± 4.2%, P < .05) and also in transduced cells (91.8% ± 2.4% without ALA v 91.1% ± 2.1% after ALA exposure).
| |
DISCUSSION |
Porphyrias are inherited metabolic diseases characterized by specific
enzyme defects along the heme biosynthetic pathway.1 Therapy is often limited to supportive care that is only partially successful, especially in the severe forms, ie, CEP, HEP, some cases of
erythropoietic protoporphyria (EPP) inherited either as a dominant or a
recessive trait, and homozygous forms of acute hepatic porphyrias.
These are good candidates for gene therapy, because the genetic defect
is well characterized at the molecular level and inherited as a
recessive trait. We have previously documented sufficient gene transfer
rate and metabolic correction in different cells to indicate that CEP
is a good candidate for treatment by gene therapy in hematopoietic stem
cells (HSC).16,17 We also showed a persistance of the
expression of the transgene during erythroid differentiation by using
the in vitro model of K562 cells.18
In HEP, the UROD enzymatic defect is responsible for the accumulation
of large amounts of uroporphyrin in the different tissues investigated.
Both erythropoietic cells and liver are involved in the disease. The
available treatments are mostly unsatisfactory, and gene therapy by
retrovirus-mediated insertion of the normal UROD gene into
hematopoietic or/and hepatic cells seems to be the most appropriate
treatment in the future.19
We constructed a recombinant retroviral vector LUDSN to mediate
insertion and expression of human UROD cDNA in three lymphopoietic target cell populations. We documented sufficient metabolic correction of the porphyric phenotype in three different deficient LB cell lines
from HEP after gene transfer of the UROD cDNA. A complete correction of
enzymatic activity was found in the first two deficient lymphoblastoid
cell lines and only 71% of normal UROD activity in the third one. This
difference in expression is probably related to the site of insertion
of the provirus in the genome of the different cell lines.
Nevertheless, the porphyrin level was normal in all of the transduced
LB cells. Therefore, it may not be necessary to achieve a 100% normal
UROD activity to have a normal porphyrin level and a disappearence of
the photosensitivity for the patient.
One approach for the treatment by gene therapy of hematologic genetic
disease, such as HEP and other erythropoietic porphyrias, is to
introduce ex vivo a normal counterpart of the defective gene into HSC
from the affected patient and then to return them as an autologous bone
marrow transplant.7,19
Restoration of the functionality of all the HSC of a patient will
necessitate a number of consecutive autologous transplantations of
genetically modified cells. To avoid correcting all the HSC, we
wondered whether transformation of uroporphyrinogen from a deficient
cell by a UROD enzyme synthesized in a normal cell was possible.
Metabolic cross-correction is well-established for human lysosomal
storage diseases such as mucopolysaccharidosis type I (Hurler
syndrome),20 type II (Hunter syndrome),21 type
VII (Sly disease),22-24 or metachromatic
leukodystrophy.25 For adenosine deaminase (ADA) deficiency,
the toxic circulating substrate can be reduced by repeated
intramuscular injections of bovine ADA cross-linked to polyethylene
glycol26 or by inserting a normal ADA allele into
peripheral lymphocytes and circulating stem cells27-30 or
into epidermal keratinocytes.31 Our in vitro experiments with deficient cells showed a high level of porphyrins in the supernatant, implying that uroporphyrinogen or uroporphyrin are able to
cross the cell membrane of deficient cells, but these porphyrin(ogen)s
cannot be metabolized further because a cross-correction was not
observed. This is probably due to the oxidation of uroporphyrinogen into uroporphyrin that becomes a nonmetabolisable substrate and/or due
to the high affinity of porphyrins to serum proteins, such as albumin
or hemopexin.32 These results suggest that a high percentage of transduced HSC must be corrected for a benefit in gene
therapy trials involving HEP. However, in the case of a selective advantage of corrected cells, a benefit could be obtained even if a
small percentage of genetically corrected cells are reinfused to the
patient. A striking observation in this study was the growth inhibition
of LB cells in the presence of the metabolic defect. Transformation of
uroporphyrinogen by UROD enzyme encoded by the transgene reduced
porphyrin accumulation and increased the half-life of the cell. Growth
inhibition can be due to a heme defect and/or prolonged exposure to the
porphyrins. Heme synthesis is necesary for the electron-transport
chain, and a defective activity of one of the heme pathway enzymes
could affect cell proliferation. Koningsberger et al33
demonstrated that an elevated protoporphyrin level causes high
intracellular damage and inhibits HepG2 cell proliferation. How
protoporphyrin exerts its toxic dark effect has not been well
clarified. Nevertheless, it has been demonstrated that both
protoporphyrin and uroporphyrin, in the presence of H2O2, can potentiate the peroxidase-catalyzed
oxidation of NADPH.34,35
In our cell proliferation studies, the selective advantage in in vivo
experiments was lesser than in vitro. This could be due to much more
prolonged exposure to uroporphyrin in in vitro cultures. In vivo,
uroporphyrin excreted from LB cells is bound to albumin and hemopexin
and then excreted in the urine. Growth of transduced cells was higher
than in untransduced cells, suggesting a competitive advantage for
these corrected cells. Because bone marrow is the target organ for
somatic gene therapy of HEP and this is a proliferative organ,
corrected HSC could have a selective advantage.
To facilitate future ex vivo gene therapy in human beings, it could be
advantageous to design efficient selection procedures to increase the
frequency of genetically corrected patient cells before autologous
transplantation. The standard approach to selecting transduced cells is
the use of vectors encoding genes conferring cellular resistance to
potentially toxic drugs such as neomycin, hygromycin, puromycin, and
multidrug resistance (MDR-1).7 That study described a rapid
and efficient procedure for isolating retrovirally transduced HEP LB
cell lines. Because normal metabolic flow from uroporphyrinogen to
coproporphyrinogen was restored in transduced cells, the porphyrin
level (measured by fluorescence) returned to normal. The difference of
fluorescence between transduced and untransduced cells was detected by
flow cytometry analysis, and two populations could be isolated
differing in their fluorescence. Culture of the sorted LB cells with
low fluorescence led to the generation of a homogeneous cell population
that was metabolically corrected.
ALA is an immediate precursor in the biosynthetic pathway of heme.
Normally, the rate of synthesis of protoporphyrin is determined by the
rate of ALA synthesis, which, in turn, is regulated via a feedback
control mechanism dependent on the concentration of free
heme.1 The presence of exogeneous ALA bypasses the feedback control and thus may induce the intracellular accumulation of porphyrins. When porphyrin levels were not very different, ALA exposure
increased the difference in fluorescence between transduced and
untransduced cells, thus improving the discriminating power of the
method. In genetically modified cells, partially restored metabolic
flow from uroporphyrinogen to protoporphyrin led to a lesser
accumulation of porphyrins in the cells.
In the current study, LB cells from HEP patients were used as a model
to develop methods for the direct selection of genetically corrected
cells. Studies must now be performed on the HSC, the ultimate targets
for erythropoietic porphyria somatic gene therapy. Analogous selection
systems, based solely on the expression of a therapeutic gene, could be
used in other metabolic disorders, in which fluorescent substrates are
directly accessible.
Finally, HSC gene therapy in this porphyria is not the unique therapy
to be considered. Because there are two main target tissues, bone
marrow and liver, the ideal therapy should concern both tissues. We do
not know whether gene transfer in vivo in HSC could be sufficient to
metabolize the excess of uroporphyrinogen produced by the different
deficient tissues, especially the liver. Also, the decrease in
porphyrin synthesis in the bone marrow should be sufficient to limit
the excess of uroporphyrin accumulated in the body and then to suppress
the porphyric phenotype. The future availability of a mouse model of
the disease will permit ex vivo gene therapy experiments on
hematopoietic or/and hepatic cells to solve this problem.
| |
ACKNOWLEDGMENT |
The authors thank Prof R. Enriquez de Salamanca and Prof J.M. Mascaro
for providing patients specimens and Laboratoire Généthon (Evry, France) for transforming human lymphocytes.
| |
FOOTNOTES |
Submitted December 29, 1998; accepted March 21, 1999.
Supported by grants from Association Française Contre les
Myopathies, from Institut National de la Santé et de la Recherche Médicale (Grant No. CRI 9508), and from Conseil Régional
d'Aquitaine. A.F. was supported by a postdoctoral TMR Marie Curie
research fellowship.
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. section
1734 solely to indicate this fact.
Address reprint requests to Hubert de Verneuil, MD, PhD, Laboratoire de
Pathologie Moléculaire et Thérapie Génique,
Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,
33076 Bordeaux Cédex, France; e-mail: verneuil{at}u-bordeaux2.fr.
| |
REFERENCES |
1.
Kappas A, Sassa S, Galbraith RA, Nordmann Y:
The porphyrias, in
Scriver CR,
Beaudet AL,
Sly WS,
Valle D
(eds):
The Metabolic Basis of Inherited Disease, vol 2. New York, NY, Mc Graw Hill, 1995, p 2103.
2.
de Verneuil H, Grandchamp B, Nordmann Y:
Some kinetic properties of human red cell uroporphyrinogen decarboxylase.
Biochim Biophys Acta
611:174, 1980[Medline]
[Order article via Infotrieve]
3.
de Verneuil H, Sassa S, Kappas A:
Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. A single enzyme catalyzing the four sequential decarboxylations of uroporphyrinogens I and III.
J Biol Chem
4:2454, 1983
4.
Moran-Jimenez MJ, Ged C, Romana M, Enriquez De Salamanca R, Taieb A, Topi G, D'Alessandro L, de Verneuil H:
Uroporphyrinogen decarboxylase: Complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria.
Am J Hum Genet
58:712, 1996[Medline]
[Order article via Infotrieve]
5.
de Verneuil H, Grandchamp B, Beaumont C, Picat C, Nordmann Y:
Uroporphyrinogen decarboxylase structural mutant (Gly281  Glu) in a case of porphyria.
Science
234:732, 1986[Abstract/Free Full Text]
6.
Roberts AG, Elder GH, Enriquez de Salamanca R, Herrero C, Lecha M, Mascaro J:
A mutation (G281E) of the human uroporphyrinogen decarboxylase gene causes both hepatoerythropoietic porphyria and overt familial porphyria cutanea tarda: Biochemical and genetic studies on Spanish patients.
J Invest Dermatol
104:500, 1995[Medline]
[Order article via Infotrieve]
7.
Nolta A, Kohn D:
Haematopoietic stem cells for gene therapy, in
Potten CS
(ed):
Stem Cells. London, UK, Academic, 1997, p 447.
8.
Miller AD, Rosman GJ:
Improved retroviral vectors for gene transfer and expression.
Biotechniques
7:980, 1989[Medline]
[Order article via Infotrieve]
9.
Danos O, Mulligan RC:
Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.
Proc Natl Acad Sci USA
85:6460, 1988[Abstract/Free Full Text]
10.
Landau NR, Littman DR:
Packaging system for rapid production of murine leukemia virus vectors with a variable tropism.
J Virol
66:5110, 1992[Abstract/Free Full Text]
11.
Lim CK, Peters TJ:
Urine and faecal porphyrin profiles by reversed-phase high-performance liquid chromatography in the porphyrias.
Chim Acta
139:55, 1984
12.
Berridge MV, Tan AS:
Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.
Arch Biochem Biophys
303:474, 1993[Medline]
[Order article via Infotrieve]
13.
Cannon MJ, Pisa P, Fox RI, Cooper NR:
Epstein-Barr virus induces aggressive lymphoproliferative disorders of human B cell origin in SCID/hu chimeric mice.
J Clin Invest
85:1333, 1990
14.
Sharara LI, Andersson A, Guy-Grand D, Fisher A, Di Santo JP:
Deregulated TCR  T cell population provokes extramedullary hematopoiesis in mice deficient in the common chain.
Eur J Immunol
27:990, 1997[Medline]
[Order article via Infotrieve]
15.
Princ FG, Juknat AA, Maxit AG, Cardalda C, Batlle A:
Melatonin's antioxidant protection against delta-aminolevulinic acid-induced oxidative damage in rat cerebellum.
J Pineal Res
23:40, 1997[Medline]
[Order article via Infotrieve]
16.
Moreau-Gaudry F, Mazurier F, Bensidhoum M, Ged C, de Verneuil H:
Metabolic correction of congenital erythropoietic porphyria by retrovirus-mediated gene transfer into Epstein-Barr virus-transformed B-cell lines.
Blood
85:1449, 1995[Abstract/Free Full Text]
17.
Moreau-Gaudry F, Barbot C, Mazurier F, Mahon FX, Reiffers, Ged C, de Verneuil H:
Correction of the enzyme deficit of bone marrow cells in congenital erythropoietic porphyria by retroviral gene transfer.
Hematol Cell Ther
38:217, 1996[Medline]
[Order article via Infotrieve]
18.
Mazurier F, Moreau-Gaudry F, Salesse S, Barbot C, Ged C, Reiffers J, de Verneuil H:
Gene transfer of the uroporphyrinogen III synthase cDNA into haematopoietic progenitor cells in view of a future gene therapy in congenital erythropoietic porphyria.
J Inherit Metab Dis
20:247, 1997[Medline]
[Order article via Infotrieve]
19.
Moreau-Gaudry F, Ged C, de Verneuil H:
Gene therapy for erythropoietic porphyrias.
Gene Ther
3:843, 1996[Medline]
[Order article via Infotrieve]
20.
Stewart K, Brown OA, Morelli AE, Fairbairn LJ, Lashford LS, Cooper A, Hatton CE, Dexter TM, Castro MG, Lowenstein PR:
Uptake of alpha-(L)-iduronidase produced by retrovirally transduced fibroblasts into neuronal and glial cells in vitro.
Gene Ther
4:63, 1997[Medline]
[Order article via Infotrieve]
21.
Braun SE, Aronovich EL, Anderson RA, Crotty PL, McIvor RS, Whitley CB:
Metabolic correction and cross-correction of mucopolysaccharidosis type II (Hunter syndrome) by retroviral-mediated gene transfer and expression of human iduronate-2-sulfatase.
Proc Natl Acad Sci USA
90:11830, 1993[Abstract/Free Full Text]
22.
Taylor RM, Wolfe JH:
Cross-correction of beta-glucuronidase deficiency by retroviral vector-mediated gene transfer.
Exp Cell Res
214:606, 1994[Medline]
[Order article via Infotrieve]
23.
Taylor RM, Wolfe JH:
Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of beta-glucuronidase.
Nat Med
3:771, 1997[Medline]
[Order article via Infotrieve]
24.
Moullier P, Bohl D, Cardoso J, Heard JM, Danos O:
Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs.
Nat Med
1:353, 1995[Medline]
[Order article via Infotrieve]
25.
Sangalli A, Taveggia C, Salviati A, Wrabetz L, Bordignon C, Severini GM:
Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer arylsulfatase A to myelinating glia and deficient cells in vitro.
Hum Gene Ther
9:2111, 1998[Medline]
[Order article via Infotrieve]
26.
Hershfield MS:
PEG-ADA replacement therapy for adenosine deaminase deficiency: An update after 8.5 years.
Clin Immunol Immunopathol
76:S228, 1995[Medline]
[Order article via Infotrieve]
27.
Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF:
T lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years.
Science
270:475, 1995[Abstract/Free Full Text]
28.
Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG, Mavilio F:
Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients.
Science
270:470, 1995[Abstract/Free Full Text]
29.
Hoogerbrugge PM, von Beusechem VW, Kaptein LC, Einerhand MP, Valerio D:
Gene therapy for adenosine deaminase deficiency.
Br Med Bull
51:72, 1995[Abstract/Free Full Text]
30.
Kohn DB, Weinberg KI, Nolta JA, Heiss LN, Lenarsky C, Crooks GM, Hanley ME, Annett G, Brooks JS, el-Khoureiy A, Lawrence K, Wells S, Moen RC, Bastian J, Williams-Herman DE, Elder M, Wara D, Bowen T, Hershfield MS, Mullen CA, Blaese RM, Parkman R:
Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.
Nat Med
1:1017, 1995[Medline]
[Order article via Infotrieve]
31.
Fenjves ES, Schwartz PM, Blaese RM, Taichman LB:
Keratinocyte gene therapy for adenosine deaminase deficiency: A model approach for inherited metabolic disorders.
Hum Gene Ther
8:911, 1997[Medline]
[Order article via Infotrieve]
32.
Morgan WT, Smith A, Kostelo P:
The interaction of human serum albumin and hemopexin with porphyrins.
Biochim Biophys Acta
624:271, 1980[Medline]
[Order article via Infotrieve]
33.
Koningsberger JC, Rademakers LHPM, van Hattum J, Baart de la Faille H, Wiegman LJJM, Italiaander E, van Berge Henegouwen GP, Marx JJM:
Exogenous protoporphyrin inhibits Hep G2 cell proliferation, increases the intracellular hydrogen peroxide concentration and causes ultrastructural alterations.
J Hepatol
22:57, 1995[Medline]
[Order article via Infotrieve]
34.
Van Steveninck J, Boegheim JPJ, Dubbelman TMAR, Van der Zee J:
The mechanism of potentiation of horseradish peroxidase-catalysed of NADPH by porphyrins.
Biochem J
242:611, 1987[Medline]
[Order article via Infotrieve]
35.
Van Steveninck J, Boegheim JPJ, Dubbelman TMAR, Van der Zee J:
The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.
Biochem J
250:197, 1988[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
|
|
TOP
ABSTRACT
INTRODUCTION