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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2793-2798
GENE THERAPY
Improvement of erythropoiesis in  -thalassemic mice
by continuous erythropoietin delivery from muscle
Delphine Bohl,
Assumpció Bosch,
Ana Cardona,
Anna Salvetti, and
Jean Michel Heard
From the Laboratoire Rétrovirus et Transfert
Génétique and the Laboratoire de Technologie Cellulaire,
Institut Pasteur, Paris, France, and the Laboratoire de Thérapie
Génique, CHU-Hôtel-Dieu, Nantes, France.
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Abstract |
 -Thalassemias are highly prevalent genetic disorders that can
cause severe hemolytic anemia. The main pathophysiologic feature of
-thalassemia is the accumulation of unpaired  -globin chains in
erythrocyte precursors and red blood cells (RBCs). This
accumulation alters cell membrane function and results in
early cell destruction and ineffective erythropoiesis. Correction of
globin chain imbalance through the induction of fetal hemoglobin (HbF)
synthesis is a tentative therapeutic approach for this class of
diseases. In short-term in vitro or in vivo assays, recombinant human
erythropoietin increases the frequency of erythroid precursors
programmed to HbF in humans and to -minor globin in mice. In
contrast, long-term treatment of -thalassemic patients did not
induce HbF significantly. We took advantage of highly efficient
adeno-associated virus-mediated (AAV-mediated) gene
transfer into mouse muscle to induce a robust and sustained secretion
of mouse erythropoietin in -thalassemic mice, which represent a
suitable model for human -thalassemia intermedia. A 1-year follow-up
of 12 treated animals showed a stable correction of anemia associated
with improved RBC morphology, increased -minor globin synthesis, and
decreased amounts of -globin chains bound to erythrocyte membranes.
More effective erythropoiesis probably accounted for a reduction of
erythroid cell proliferation, as shown by decreased proportions of
circulating reticulocytes and by reduced iron 59 (59Fe)
incorporation into erythroid tissues. This study indicates that the
continuous delivery of high amounts of autologous erythropoietin induced a sustained stimulation of -minor globin synthesis and a
stable improvement of erythropoiesis in the -thalassemic mouse model.
(Blood. 2000;95:2793-2798)
© 2000 by The American Society of Hematology.
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Introduction |
Thalassemias are the most common monogenic diseases.
They are highly prevalent in the Mediterranean area, where Cooley's
anemia was first recognized, and in Southeast Asia, where there are
presumably more than 1 million severely affected individuals.
-Thalassemias are due to deficient -globin synthesis, which
causes globin chain imbalance in erythroid cells. Unmatched  -globin
chains liberate iron,1 bind to membranes, and alter
membrane lipids and proteins through oxidative mechanisms, thus causing
premature cell destruction.2 Intramedullary
hemolysis along with the decreased survival of adult erythrocytes in
the peripheral blood cause anemia, which in turn stimulates ineffective
dyserythropoiesis. Bone marrow expansion, bone complications, and iron
overload are the major causes of morbidity and early death in
-thalassemic patients.
Despite a marked increase in patient survival in recent years,
treatment of -thalassemia remains a challenging issue. Management relies on blood transfusions and iron chelation therapy. Both are
cumbersome and expensive and require sustained patient compliance. Patients with Cooley's anemia (thalassemia major, TM), the most severe
form of the disease, are strictly transfusion-dependent, while patients
with milder phenotypes (thalassemia intermedia, TI) do not need regular
transfusions. Although bone marrow transplantation can cure the
disease,3 it is often not an option.4
Reactivation of fetal hemoglobin (HbF synthesis) would be a reasonable
approach to improving globin chain balance in -thalassemia. Induction of HbF synthesis in adult cells has been observed under stress erythropoiesis conditions that trigger kinetics of rapid erythroid regeneration.5 In vitro, the formation of
HbF-containing cells is enhanced by erythropoietin (Epo) and by culture
conditions favoring the proliferation of less differentiated
progenitors.6,7 In vivo, expansion of HbF-programmed
progenitors and increased HbF synthesis were observed in baboons
receiving high doses of recombinant human Epo (rHuEpo)
during a period of several days.8 However, long-term trials
with rHuEpo gave ambiguous results in -thalassemic
patients.9-13 The treatment (perhaps with the exception of
combination therapy, which included hydroxyurea14) did not increase HbF significantly, although a clinical benefit was
nevertheless observed in some patients. High doses of rHuEpo were well
tolerated, but there are several potential drawbacks to its use. The
drug is expensive, which will limit the use of rHuEpo worldwide, and there is a theoretical risk of worsening bone marrow expansion and bone disease.
Efficient induction of fetal erythropoiesis in short-term in vitro
assays or by short-term in vivo stimulation with rHuEpo contrasts with
limited efficacy in long-term trials in -thalassemic patients. This
suggests that the stimulation of erythroid progenitors, which can be
programmed to HbF synthesis, could become inoperative over time. To
examine this point, we set up an experiment in which high amounts of
murine Epo were delivered during a period of several months to
-thalassemic mice. This was achieved by adeno-associated virus-mediated (AAV-mediated) gene transfer of the mouse Epo
complementary DNA (cDNA), as previously documented in normal
mice.15-19 Homozygous -thalassemic mice have a
microcytic, hypochromic anemia; highly dysmorphic red blood cells
(RBCs); and extensive intramedullar and intrasplenic destruction of
erythroid progenitors and erythroid hyperplasia.20 Similar
abnormalities in membrane functions have been documented in murine and
human -thalassemias.21 Although -minor globin
is expressed at a significant level in the adult mouse, expression is
much lower than in the fetus.22,23 As for primate HbF,
synthesis of -minor increases in response to in vitro culture of
erythroid progenitors,24 under stress erythropoiesis conditions,25 during hydroxyurea treatment,26
and transiently in mice injected with high doses of
rHuEpo.27 We show that a sustained secretion
of high amounts of Epo stimulated -minor chain
synthesis over several months in -thalassemic mice stably improved erythropoiesis.
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Materials and methods |
Vector preparation and administration
The AAV-CMV(cytomegalovirus)-Epo plasmid was obtained by inserting a
630-base pair (630-bp) DNA fragment containing the murine Epo coding
sequence between the human CMV immediate early promoter/enhancer and
the bovine growth hormone gene polyadenylation signal in
pSUB-201.28 A vector stock containing
1.2 × 1011 genomes/mL was generated by a triple
plasmid transfection of 293 cells, as previously
described.29 Infectious particles included
5.4 × 1010 Epo-encoding U/mL, less than
5 × 102 particle-forming U/mL replicative
adenovirus, and 5 × 104 Rep-transcomplementing
U/mL. The preparation was injected in ether-anesthetized C57Bl/6
Hbbth mice, 3-4 months old (bred from animals obtained at
the Jackson Laboratory, Ann Harbor, ME), either in both tibialis
anterior muscles (200 µL total, mice Nos. 1, 2, 3, 6, 11, and 12) or
in both tibialis anterior and both hind limb muscles (400 µL total, mice Nos. 4, 5, 7, 8, 9, and 10).
Hematology and Epo detection
Hemoglobin, hematocrit (Hct), and RBC counts were determined by
standard procedures. Reticulocytes were counted after staining with
brilliant cresyl blue. Serum Epo concentrations were measured by
radioimmunoassay (Biomérieux, Charbonnier les Bains, France).
Globin chains
Synthesis was analyzed by metabolic labeling. Blood cells were
washed and resuspended in methionine- and cystein-free
RPMI 1640 medium with 4% dialyzed fetal calf serum for 30 minutes at 37°C before a 20-minute labeling with 9.25 MBq (250 µCi) of 35S(sulfur 35)/methionine/cystein (Amersham Life
Science, Arlington Heights, IL) and a 1-hour chase. Protein extracts
(50 µg) were prepared in 0.5% Triton X-100, 20 mmol/L HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 150 mmol/L
sodium chloride (NaCl) and were analyzed on urea-Triton-polyacrylamide
gel electrophoresis. Gels were stained for 1 hour with Coomassie
Brilliant Blue, then fixed and dried for quantification on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Erythrocyte ghost
extracts were prepared as described.30
Iron 59 uptake
After a 2-hour fast, mice were injected in the tail vein with
0.028 MBq (0.75 µCi) of 59Fe (ferrous
sulfate in 0.1 mL 5% dextrose and water) (New England Nuclear, Boston,
MA). Plasma samples taken at regular intervals over 20-50 minutes were
counted in a Gamma Berthold scintillation spectrometer (Berthold,
Colombes, France). Mice were sacrificed 1 hour later, and spleens were
fixed with 4% paraformaldehyde overnight at 4°C. Radioactivity was
measured on 16-µm thick cryosections using a -imaging apparatus
(Biospace, Paris, France).
Statistics
Statistics were performed using SPSS 6.1 software
(SPSS, Chicago, IL). Significance level of linear
regression square coefficients (Rsq) was estimated by F tests.
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Results |
Transduction of skeletal muscles
A recombinant AAV vector (rAAV) containing the mouse Epo cDNA
inserted downstream of a human CMV promoter was constructed (rAAV-CMV-Epo, Figure 1A) and injected in
the leg muscles of 12 -thalassemic and 6 heterozygous C57BL/6 mice.
Animals received between 2.4 × 1010 and
4.8 × 1010 physical rAAV particles. Intense
polycythemia resulted in the death of treated heterozygous mice within
2 months following injection. -Thalassemia mice were monitored for
up to 1 year. With the exception of mice Nos. 9 and 12, which both died
of hemorrhage a few hours after a blood sampling, the entire injected
muscles were resected at the time of sacrifice, weighted, and used to
prepare high-molecular-weight DNA. Southern blot hybridization with an
Epo probe revealed double-stranded rAAV vector genomes representing an
average proportion of 2.8%-7% of diploid mouse genomes (Figure 1B). A
search for rAAV-CMV-Epo vector genomes by polymerase chain reaction
(PCR) was negative in the liver, spleen, lung, and kidney (data not
shown). Index values aimed at comparing the total amounts of transduced
tissue between mice were calculated by multiplying the mass of the
injected muscles by the proportion of vector genomes in these muscles. Data from mice Nos. 1, 2, 3, 4, 7, and 8, which were followed up for 10 to 12 months and fully investigated at the time of sacrifice, are shown
in Table 1.
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| Fig 1.
Quantification of rAAV-CMV-Epo vector genomes in the
muscles of injected -thalassemic mice.
(A) The vector diagram: ITR indicates AAV-2 inverted terminal repeats;
CMV, immediate early promoter/enhancer elements from CMV; mEpo, murine
erythropoietin; and bGH polyA, polyadenylation signal of the bovine
growth hormone gene. The BamH-I fragment used as a probe is indicated.
(B) Southern blot analysis of BamH-I-digested high-molecular-weight
DNA extracted from injected muscle. Analysis was performed after
injection at 2 months (mice Nos. 5 and 6), 5 months (mouse No. 11), 10 months (mice Nos. 3 and 10), and 1 year (mice Nos. 1, 2, 4, 7, and 8).
Reference copy numbers (ref. copy numbers) correspond to 1 copy (20 pg,
lane 1) or 0.1 copy (2 pg, lane 0.1) of the AAV-CMV-Epo plasmid DNA.
Endogenous Epo (Epo endo.) and vector signals are shown. Quantification
of vector genome copies was performed with a PhosphorImager (Molecular
Dynamics) according to the ref. copy number and Epo endo. signals.
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Table 1.
Data from untreated and treated -thalessmic mice
followed for 10-12 months and investigated at time of
sacrifice
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Systemic Epo delivery
Serum Epo concentrations were measured monthly (Figure
2A). Mean values were much higher in
untreated -thalassemic mice (150 ± 37 mU/mL) than in
heterozygous controls (39.2 ± 8.4 mU/mL). Three months after
vector injection, concentrations in treated -thalassemic mice ranged
between 150-1600 mU/mL. Mean serum Epo concentrations were calculated
over the last 6 months of life for mice Nos. 1, 2, 3, 4, 7, and 8 (Table 1). Values ranked with the indexes representing the amount
of transduced tissue (r = 0.943; P = .005,
Spearman rank correlation coefficient).
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| Fig 2.
Correction of hematologic values in treated
-thalassemic mice.
(A) Serum Epo concentration, (B) Hct, (C) Hb concentration, and (D) the
proportion of reticulocytes were measured monthly in treated
-thalassemic mice (continuous lines), 3 untreated -thalassemic
controls (dotted lines), and 3 heterozygous controls (dashed lines).
Values correspond to individually treated mice indicated by
numbers or are the mean ± SEM (standard error of the mean)
for controls.
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Correction of anemia
Hematocrit and Hb concentrations showed severe anemia in untreated
-thalassemic mice (Hct: 29.7% ± 1.3%; Hb:
86 ± 9.0 g/L [Hb: 8.6 ± 0.9 g/dL]; Figure 2B and 2C). Red
cell morphology showed marked anisocytosis and target cells (Figure
3). Hematocrits reached normal values
within 1 to 2 months after vector injection in all treated
-thalassemic mice. Later on, circulating RBCs either increased
further, which lead to intense polycythemia in mice Nos. 7 through 12 or stabilized to moderate polycythemia in mice Nos. 3 and 4, or
remained in the normal range in mice Nos. 1 and 2 (Figure 2B and 2C).
Red cell morphology was dramatically improved in all animals (Figure
3). However, faint staining of blood smears suggested persistent
hypochromia, which was confirmed by low mean corpuscular Hb content
(Table 1). Plasma iron concentration was measured at 8 months in mouse
No. 2 and found to be in the normal range (32.4 versus 34.2 ± 7
µmol/L in heterozygous controls). Mean Hct and Hb values calculated
over the last 6 months of life for mice Nos. 1, 2, 3, 4, 7, and 8 were
proportional to serum Epo concentrations (Table 1; Hct: Rsq = 0.726, P = .031; Hb: Rsq = 0.749, P = .026) and with
indexes of transduced tissue (Hct: Rsq = 0.740, P = .028;
Hb: Rsq = 0.692, P = .040). These data indicated that gene
transfer and high serum Epo concentrations were directly related to the
appearance of an effective erythropoiesis in treated mice.
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| Fig 3.
Improvement of peripheral red blood cell morphology in
treated -thalassemic mice.
May-Grünwald Giemsa staining of blood smears from an untreated
heterozygous mouse, from an untreated -thalassemic mouse, and from
the -thalassemic mouse No. 1 twelve months after intramuscular
injection of the rAAV-CMV-Epo vector.
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Improvement of globin chain imbalance
Globin chain synthesis was investigated by metabolic labeling of
blood cells followed by denaturing gel electrophoresis. Quantification of the -minor to -globin chain signal ratio showed significant improvement in mice Nos. 1, 2, 3, 4, 7, and 8 compared with 3 untreated
-thalassemic mice (Table 1; P = .005, Student t
test). However, the ratio never reached 1, indicating that unmatched -globin chains persisted in the erythrocytes of all treated mice.
Incorporation of unpaired -globin chains in erythrocyte membranes
causes membrane abnormalities which account for the early destruction
of erythroid precursors.31 We therefore analyzed the
-globin chain content in protein extracts from erythrocyte ghosts of
untreated and treated mice. Results showed a 2- to 3-fold reduction of
-globin chain in erythrocyte ghosts of treated -thalassemic mice
(Table 1). Nevertheless, membrane -globin chains were still much
more abundant than in nonthalassemic mice. Consistently with the
involvement of membrane -globin chains in cell destruction, reduction was more pronounced in mice with higher values of Hct (Rsq = 0.657, P = .050) and Hb (Rsq = 0.716,
P = .034). A significant relationship was also found between
the reduction of membrane -globin chains and serum Epo
concentrations (Rsq = 0.786, P = .019). In addition to the
dramatic changes observed in RBC morphology (Figure 3), these data
suggested that high serum Epo concentrations stimulated the production
of qualitatively improved RBC.
Erythroid hyperplasia and cell proliferation
A decreased proportion of reticulocytes in the blood of treated
-thalassemic mice suggested that erythroid cell proliferation was
reduced rather than accelerated in response to Epo secretion (Figure
2D). However, although the proportion of reticulocytes was reduced,
absolute reticulocyte counts remained elevated with respect to the
increased blood mass, even in normocythemic mice (10 and
9.8 × 105/µL in mice Nos. 1 and 2, respectively,
versus 3.1 ± 0.4 × 105/µL in
heterozygous controls). Together with the persistence of a splenomegaly
(Table 1), these data suggested that erythropoiesis was still very
active in treated mice.
To investigate erythroid proliferation more accurately, we examined the
uptake of 59Fe in erythroid tissues and its half-life in
the serum of treated mice and controls. The uptake of 59Fe
in tissues was assessed by scanning fixed erythroid tissue sections
with a -imager. Data from spleen sections are shown in Figure
4A. Similar data were obtained in the bone
marrow and the liver (data not shown). Imaging revealed an almost total
disappearance of actively regenerating foci in the spleen of treated
-thalassemic mice. Numbers of cpm/surface unit were higher in
untreated -thalassemic spleen than in heterozygotes, and they were
significantly reduced in treated mice (P < .0001,
Student t test). A significant relationship between decreased
uptake of 59Fe uptake in spleen sections and decreased
amounts of -globin chain content in erythrocyte membranes
(Rsq = 0.792, P = .018) indicated that the qualitative
improvement of RBCs was associated with a diminution of erythroid cell
proliferation.
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| Fig 4.
Erythroid cell proliferation measured by 59Fe
uptake assay.
Treated -thalassemic mice (continuous lines), untreated
-thalassemic controls (n = 3, dotted lines), and
heterozygous controls (n = 3, dashed lines) were injected with 0.028 MBq (0.75 µCi) of 59Fe in the tail vein. (A)
Radioactivity in spleen sections (-imager exposure, 755 minutes).
Values are the mean ± SEM of counted cpm/mm2 on 8 sections (treated mice), 29 sections (untreated controls), and 38 sections (heterozygote controls). Values scored in mice Nos. 4 and 7 were 551 ± 56 and 711 ± 67, respectively (not shown).
Gradation color indicates local radioactivity, according to the scale.
(B) Kinetics of the disappearance of radioactivity in plasma. Values
correspond to individually treated mice, indicated by number, or
are the mean ± SEM for controls.
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The half-life of 59Fe in the serum was shorter in untreated
-thalassemic mice than in heterozygous mice, accounting for
erythroid cell hyperproliferation (Figure 4B). Various kinetics were
observed in treated -thalassemic mice. In polycythemic mice Nos. 7 and 8, the disappearance of 59Fe from plasma was even more
rapid than in untreated -thalassemic mice. In contrast,
normocythemic mice Nos. 1 and 2 showed values almost equivalent to
heterozygotes. Mice Nos. 3 and 4, which had an intermediate phenotype,
showed intermediate values. Table 1 shows that the half-life of
59Fe in serum was inversely related to polycythemia (Hct:
Rsq = 0.956, P = .001; Hb: Rsq = 0.849,
P = .009) and spleen weight (Rsq = 0.915,
P = .003). These data indicated that the persistence of an
intense erythroid proliferation in treated mice was related to the
overstimulation of an effective erythropoiesis by Epo overdosage and to
the persistence of splenomegaly.
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Discussion |
AAV-derived vectors provide a simple method for introducing and
expressing cDNA into mouse skeletal muscle.15,17-19,32
Although double-stranded AAV genomes probably remain extrachromosomal
in mouse myofibers, thereby forming large circularized concatemers, their tight association with chromatin allows persistence and stable
expression during periods of several months.33 We took advantage of the unique properties of AAV vectors for introducing a
transcription unit in which the mouse Epo cDNA was strongly expressed
from human CMV enhancer and promoter elements into the skeletal muscles
of -thalassemic mice. A robust secretion of Epo was induced in all
animals and persisted at the same level during the 1-year observation
period, leading to high serum Epo concentrations. Increased serum Epo
concentrations had major consequences on erythropoiesis in
-thalassemic mice. These consequences included a qualitative
improvement of RBC phenotype, which allows for a stable correction of
anemia. Probably as a result of more effective erythropoiesis, cell
proliferation was reduced in erythroid tissues, as shown by the lower
proportion of reticulocytes in the circulating blood (albeit not
reaching normal mouse values) and by diminished 59Fe
incorporation in erythroid tissues, which was inversely proportional to
serum Epo concentrations. A reduced proportion of circulating reticulocytes was previously observed in mice transiently receiving high doses of rHuEpo,27 whereas contradictory data have
been reported from -thalassemic patients enrolled in rHuEpo
trials.9-13
The improved quality of erythroid cells was obvious on blood smears of
treated -thalassemic mice. Anisocytosis and poikilocytosis were much
less intense, and target cells were rare. However, some degree of
abnormality persisted, and hypochromia was not corrected. Morphological
changes were associated with modifications in RBC membrane proteins and
especially with decreased amounts of -globin chains associated with
erythrocyte membranes. A relationship between serum Epo concentrations
and the reduction of membrane-bound -globin chains as well as a
relationship between this latter parameter and the correction of anemia
could be demonstrated. Therefore more effective erythropoiesis in
treated mice was associated with an improved RBC phenotype. Previous
studies have pointed out the major role of membrane protein
abnormalities in ineffective erythropoiesis.2
The significant increase of -minor chain synthesis and the
subsequent reduction of unpaired -globin chains incorporated into
membranes probably played an important role in improving RBC phenotype.
This was likely a consequence of the action of Epo at the level of
primitive erythroid progenitors, as previously demonstrated for
primates.8 Indeed, as similarly observed for the synthesis
of HbF in the human erythroid lineage,34,35 -minor is
produced to higher levels in the progeny of primitive erythroid progenitors,24 which increases in frequency in vivo in
response to erythroid stress,25 hydroxyurea,26
and rHuEpo.27 In primates, the increased proportion of F
cells (cells containing HbF) under these conditions is thought to
result either from a premature commitment of primitive progenitors
recruited before completion of  gene inactivation or from a faster
cycling, which impairs completion of inactivation.8 In
mouse erythroid cell cultures36 and in vivo in
baboons,8 induction by Epo results from the expansion of
BFU-e, which has the potential to form -minor or fetal Hb, rather
than from late erythroid precursors, which are committed to -major
or HbA. In -thalassemic mice under strong Epo stimulation, the
preferential mobilization of BFU-e programmed for -minor synthesis
may account for the observed emergence of an effective erythropoiesis,
which slowed down erythroid cell proliferation, whereas the expansion
of late erythroid progenitors, which would aggravate dyserythropoiesis,
remained absent or limited. The expansion of a pool of primitive
erythroid progenitors has been previously reported in untreated
-thalassemic mice37 and in -thalassemic
patients.38 Interestingly, the possibility of inducing a
stable hypersecretion of autologous Epo over the long term in
-thalassemic mice indicated that effective erythropoiesis was
sustained in treated animals for at least 1 year.
If the model discussed above is correct, sustained improvement of
erythropoiesis suggests that a chronic expansion of the primitive BFU-e
compartment can be induced in mice continuously secreting high amounts
of Epo. Investigations of Epo-responding erythroid progenitors in
-thalassemic mice treated with the AAV-CMV-Epo vector are currently
being performed. In preliminary unpublished experiments, we used
different gene transfer methods to obtain sustained Epo secretion in
-thalassemic mice. Serum Epo concentration was significantly lower
than those reported in the present study using AAV-mediated gene
transfer. These attempts led to a transient and partial improvement of
erythropoiesis, which was observed in a minority of treated animals
only, without significant reduction of dyserythropoiesis. Similar
observations have been reported by Dalle et al39 using a
cell encapsulation method. This situation may be somehow reminiscent of
rHuEpo trials in human patients, which led to partial and inconsistent
improvements. It may be assumed that chronic expansion of the primitive
BFU-e compartment requires an intense and sustained stimulation in
order to be effective. The observed synergistic effects of rHuEpo and
hydroxyurea in -thalassemic patients support this
hypothesis.14
Although the reduction of erythroid cell proliferation was present in
all treated mice, 59Fe incorporation and half-life in serum
were comparable to those of normal mice only in normocythemic animals,
in which serum Epo concentrations remained between 250 and 350 mU/mL.
Higher concentrations resulted in the expansion of phenotypically
improved RBCs and in subsequent polycythemia (Figure
5). This result points out the need for a
regulated expression system which allows for control of Epo secretion
from engineered cells, such as that previously implemented in normal
mice and nonhuman primates.17-19 The reduction of erythroid
cell proliferation was also limited by splenomegaly, which persisted in
all treated animals 1 year after anemia had been corrected. The reasons
for the persistence of splenomegaly are unclear. Combination therapies
with hydroxyurea or splenectomy could be considered, with the aim of
facilitating control of erythroid cell proliferation in -thalassemic
mice.
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| Fig 5.
Schematic representation of the effects of serum Epo
concentration on the erythropoiesis of treated -thalassemic mice.
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The successful treatment of murine disease following intramuscular
injection of the AAV-CMV-Epo vector raises questions about the
relevance of this method for human -thalassemia. Gene therapy trials
with rAAV vectors are currently ongoing in hemophiliac patients.
Regulated expression systems may be considered for human application in
the near future. More concerns exist with respect to the clinical
benefits that may be expected from Epo therapy in human -thalassemia
because trials with rHuEpo produced ambiguous results.9-13
On the other hand, these trials indicate that high doses of rHuEpo did
not induce unwanted side effects. Cost, which is an obvious limitation
to treatments with rHuEpo, is potentially lower with a gene therapy
approach. Because rHuEpo seems at least to provide clinical benefit in
certain subpopulations of -thalassemic patients, Epo gene therapy
could be considered for long-term treatment in selected patients who
have demonstrated unambiguous responsiveness to rHuEpo during a
preliminary short-term trial.
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Acknowledgments |
We are grateful to Dr G. Millon for help in the 59Fe uptake
experiments, to Dr Y. Beuzard for helpful discussions during the origin
of this work, and to Drs G. Stamatoyannopoulos and F. Mavilio for
critical reading of the manuscript.
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Footnotes |
Submitted September 7, 1999; accepted January 4, 2000.
Supported by grant number 5902 from the Association Française
contre la Myopathie, Paris, France. D.B. is a fellow from Caisse Nationale d'Assurance Maternité (CANAM), Paris, France. A.B. is
a fellow from the European Commission (DG XII), Brussels, Belgium.
Reprints: Jean Michel Heard, Laboratoire Rétrovirus et
Transfert Génétique, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France; e-mail: jmheard{at}pasteur.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction