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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1512-1517
RAPID COMMUNICATION
Control of Erythropoietin Delivery by Doxycycline in Mice After
Intramuscular Injection of Adeno-Associated Vector
By
Delphine Bohl,
Anna Salvetti,
Philippe Moullier, and
Jean Michel Heard
From Laboratoire Rétrovirus et Transfert Génétique,
CNRS URA 1157, Institut Pasteur, Paris; and Laboratoire de
Thérapie Génique, CHU-Hôtel-Dieu, Nantes, France.
 |
ABSTRACT |
We reported previously that controlled expression of a foreign gene
in response to tetracycline derivative can be accomplished in mice by
the autologous transplantation of retrovirus-modified muscle cells.
Although regulated systemic delivery of therapeutic proteins from
engineered tissues has potential clinical application, the
transplantation of muscle cells is not currently feasible in humans.
Several studies have shown that a single injection of adeno-associated
virus (AAV) vectors into mouse muscle results in long-term expression
of reporter genes as well as sustained delivery of proteins into the
serum. Because this method is potentially applicable clinically, we
constructed an AAV vector in which the expression of the mouse
erythropoietin (Epo) cDNA is modulated in response to doxycycline. The
vector was injected intramuscularly in normal mice. We observed that
hematocrit and serum Epo concentrations could be modulated over a
29-week period in response to the presence or absence of doxycycline in
the drinking water of these animals. Thus, a regulated gene expression
cassette can be incorporated into a single AAV vector, such that
intramuscular injection of the vector allows sustained and regulated
expression of a desired gene.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE CONTINUOUS SYSTEMIC delivery of
proteins from genetically modified tissues is a promising therapeutic
approach. Skeletal muscles are abundant, well vascularized, and have a
very slow turnover, and thus are attractive targets for gene transfer
designed to allow secretion of a protein into the blood. Feasibility
was shown in various animal models by the autotransplantation of
syngeneic myoblasts manipulated ex vivo with retrovirus
vectors.1-6 However, this method is too time-consuming,
costly, and potentially hazardous for consideration in humans. Direct
in vivo gene transfer could be preferrable. Adenovirus vectors have
been used in that manner. Long-term secretion of a protein into the
serum was observed, provided that the experiments were performed in
immuno-incompetent or immuno-suppressed animals, or that the vector
encoded a self protein, thus avoiding a strong immune
response.7-11 Plasmid DNA injection into mouse muscle can
also direct systemic secretion, although at a lower
level.12,13 Recently, several studies have suggested that
the intramuscular injection of adeno-associated virus (AAV) vectors in
mice results in stable expression of reporter genes,14-16
and sustained systemic delivery of erythropoietin
(Epo)17,18 or factor IX.19
The elements of the parental AAV-2 parvovirus genome retained in
AAV-derived (rAAV) vectors are limited to the inverted terminal repeats
(ITR), which are the only cis-acting sequences required for the
packaging and replication of recombinant genomes. In contrast with
adenovirus vectors, AAV vectors do not encode viral protein. Thus,
intramuscular injection into immuno-competent animals does not
stimulate immune response against the transgenic cells or the transgene
product.15 An additional potential advantage of rAAV
vectors is the absence of viral transcriptional control element, which
avoids interference with promoters aimed at directing tissue-specific or regulatable expression of the transgene. The packaging of rAAV vector genomes into viral particles currently relies on the transient expression of the rep and cap AAV genes together with
that of an adenovirus genome. Although the production methods are still empirical in the absence of a reliable packaging cell line, recent improvements have made high-titer vector stocks available, in which
contamination with wild-type AAV and adenovirus can be accurately determined and appears negligible.20
Hormones of therapeutic interest like growth hormone and Epo require a
tight adjustment of dose delivery to prevent adverse effects. Because
most of the physiological regulatory processes are difficult to
transfer to engineered cells, transgene expression must rely on
artificial regulatory systems. Artificial inducible expression systems
use transcriptional stimulation by chimeric transactivating factors,
the activity of which can be controlled by drugs such as tetracycline
derivatives,21 mifepristone,22 ecdysone,23 or rapamycine.24 Modulation of gene
expression in response to the controlling drug was documented in vitro
and in transgenic mice. Investigation in models relevant to gene
therapy is crucial, especially with regard to the potential
antigenicity of chimeric transcription factors. We previously observed
that retrovirus-engineered myoblasts expressing rtTA, the chimeric transactivator conferring doxycycline-inducible gene expression, can be
stably engrafted in mice, thus allowing the long-term control of Epo
secretion in vivo.5 However, the difficulties raised by the
transplantation of muscle cells in humans preclude clinical application
of this technique. We show here that comparable results can be achieved
using a single intramuscular injection of a rAAV vector containing the
components of the tetracycline-inducible regulatable system and a mouse
Epo cDNA. The efficacy and the simplicity of the method suggest that
this approach could be considered for future clinical applications.
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MATERIALS AND METHODS |
Vector construction.
A 630-bp DNA fragment containing the murine Epo coding sequence was
inserted between the tetO-CMV promoter and the 5 end of the SV40
polyadenylation signal of puHD10.3.25 An expression cassette for the reverse transactivator (rtTA) chimeric
protein21 was inserted 3 to the SV40 polyadenylation
signal in reverse orientation. This cassette contains a 1,858-bp
fragment of the MFG retroviral vector26 encompassing the
5 LTR and gag intronic sequences, followed by the
1,020-bp of the rtTA coding sequence. The construction was then moved
into the pSUB-201 AAV vector plasmid, giving rise to pAAV-ET, with a
total length of 5,017 bp.
rAAV production.
rAAV-ET was produced as described.27 Briefly, 293 cells
were cotransfected with pAAV-ET and pspRC transcomplementing rep-cap plasmid DNAs, then infected 6 hours later with wild-type adenovirus 5 (multiplicity of infection [MOI] = 10). Cell and
adenovirus stocks were free of AAV2 sequences detectable by
PCR.27 Vector particles were purified on CsCl gradients
from cell extracts collected at day 3 and dialyzed against
phosphate-buffered saline (PBS). Quantification of AAV particles
containing the rAAV-ET genome by dot blot and hybridization with an
Epo-specific probe measured 2.5 × 1011 rAAV-ET
genomes/mL. Infectious virus particles (i.p.) were quantified by a
modified replication center assay. HeLa32 cells, which constitutively express the AAV Rep and Cap proteins,27 were exposed to
serial dilutions of rAAV-ET and cells in which the vector replicated were revealed by hybridization with an Epo-specific probe. After addition of transcomplementing adenovirus (MOI: 10), rAAV-ET vector replication indicated 2.1 × 1010 i.p./mL. The
detection of 2.3 × 104 rAAV-ET vector replication
foci per milliliter on HeLa32 cells without addition of adenovirus
indicated that the preparation contained contaminating adenovirus 5. The replication of rAAV-ET in HeLa cells infected with adenovirus 5 (MOI: 10) indicated that AAV particles transcomplementing for Rep
contaminated the vector stock in a proportion of 0.005%.
Human myotube cultures.
Cell isolation and culture protocols were as described,28
in accordance with the French legislation. Differentiation of human
myoblasts was induced by seeding cells in 24-well plates at a density
of 3.5 × 104 cells/well and grown to confluence.
Incubation for 48 hours in serum-free Dulbecco's Modification of
Eagle's Medium (DMEM) with insulin (10 µg/mL) and
transferin (100 µg/mL) induced myotube formation.
Animal experiments.
One hundred microliters of the rAAV-ET preparation was injected in the
tibialis anterior of ether-anesthetized 6-week-old male C3H mice from
IFFA-CREDO (Orléans, France). Doxycycline-HCl (Sigma,
Saint-Quentin Fallavier, France) was dissolved in the drinking water to a final concentration of 200 µg/mL with 5%
sucrose. No obvious side effect was observed in animals. Blood samples were obtained by retro-orbital puncture of ether-anesthetized animals.
Epo detection.
Epo concentration in culture supernatants was measured on
Epo-dependent cells, using recombinant human Epo as a reference, as described previously.29 Serum Epo concentrations were
measured by a radioimmunoassay (BioMérieux, Charbonnier les
Bains, France).
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RESULTS |
rAAV-ET vector design.
The rAAV-ET vector contains two transcriptional units oriented in
opposite directions, with a central bidirectional SV40 polyadenylation site (Fig 1A). Sequences encoding the
chimeric transcription factor rtTA, which confers doxycycline-inducible
expression,21 were inserted downstream of a retrovirus LTR
promoter. This promoter has been previously used for long-term gene
expression in mouse skeletal muscle.30 A minimal human CMV
promoter flanked with tetracycline operator motifs (tetO-CMV promoter),
to which the rtTA protein can bind, controlled the transcription of the
mouse Epo cDNA.25
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| Fig 1.
Transduction and expression of rAAV-ET in skeletal muscle
cells. (A) Structure of the rAAV-ET vector. ITR: AAV-2 inverted
terminal repeats; tetO-CMV: tetracycline-inducible promoter including
seven repeats of the tetracycline operator inserted upstream of the CMV
minimal promoter; mEpo: murine erythropoietin cDNA; (A)n: SV4O
bidirectional polyadenylation signal; rtTA: coding sequences for the
tetracycline reverse transactivator; LTR: long terminal repeat of the
MFG retrovirus construct. The BamHI fragment used as an
Epo-specific probe is indicated. (B) Northern blot analysis of total
RNAs extracted 7 days after exposure of human primary myotubes to
rAAV-ET (2 × 109 vector genomes/culture well). Cells were
incubated in the presence (+) or in the absence ( ) of doxycycline
(1 µg/mL). (c) Control myotubes not exposed to rAAV-ET. Hybridization
was performed with the combination of an Epo-specific and a
rtTA-specific 32P-labeled probe. Migration of 18S and 28S
rRNAs is indicated. Amounts of RNAs loaded on each lane can be
appreciated on the ethidium bromide staining of 28 S rRNA (28S).
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In vitro induction.
AAV-mediated gene transfer and doxycycline-inducible Epo secretion were
investigated in cultured human primary myotubes, 7 days after exposure
to the vector. Epo mRNAs were detected by Northern blot in transduced
cells not exposed to doxycycline, indicating basal transcription from
the tetO-CMV promoter (Fig 1B). Addition of doxycycline (1 µg/mL)
resulted in a 12-fold increase of Epo mRNAs amounts. Basal Epo
secretion was also detected in culture supernatant (49.3 ± 20.4 Epo
mU/24 h/mg protein). Secretion increased ninefold (434 ± 53 Epo
mU/24 h/mg protein) when doxycycline was added. Thus, the transduction
of human primary myotubes with rAAV-ET conferred inducible expression
of murine Epo.
Control of Epo secretion in vivo.
Hematocrit and serum Epo concentration were measured over a period of
29 weeks in mice injected intramuscularly with the rAAV-ET vector
(Fig 2). Low-dose animals (LD, n = 6)
received 2.5 × 1010 rAAV-ET genomes through a
100-µL injection in a single tibialis anterior; high-dose animals
(HD; n = 8) received 5 × 1010 rAAV-ET genomes through
a 100-µL injection in each tibialis anterior.
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| Fig 2.
Long-term monitoring of hematocrit and serum Epo
concentration in individual mice injected with rAAV-ET. LD, mice
injected with 2.5 × 1010 rAAV-ET genomes; HD, mice
injected with 5 × 1010 rAAV-ET genomes. Hematocrit was
measured weekly by collecting 40 µL of blood via retro-orbital
puncture. Doxycycline (200 µg/mL) was present (red lines) or not
(blue lines) in the drinking water. Serum Epo concentration values (in
Epo mU/mL) are shown within circles on hematocrit curves at the
timepoint they were determined. Hematocrit and serum Epo concentration
measured before rAAV-ET injection were: 45.2% ± 1.2% and 20.8 ± 5.4 Epo mU/mL, respectively (n = 14). Dotted lines indicate
hematocrit values in animals not injected with rAAV-ET (n = 6).
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To assess basal Epo secretion level, five animals injected with the
rAAV-ET vector were not treated with doxycycline. Hematocrit slowly
increased in the two untreated LD mice, reaching a plateau at 53.9% ± 4.1%, with serum Epo concentration sligthly over normal. Basal
hematocrit (63.1% ± 3%) and serum Epo were higher in the three
untreated HD mice. These results showed that in vivo basal Epo
secretion levels depended on the amount of injected vector.
Doxycycline (200 µg/mL) was added to the drinking water of naive (n = 6) and rAAV-ET-injected mice (n = 9). The treatment was initiated 2 weeks after rAAV-ET injection. Whereas hematocrit was not modified in
naive mice, a rapid increase was observed during the first 6 weeks of
treatment in rAAV-ET-injected animals, reaching 75.7% ± 2.3% in
4 LD mice and 76.9% ± 3.1% in 5 HD mice, after which the increase
was slower. Two LD mice showed a plateau at 80% to 85% until
sacrifice, whereas 3 HD mice died between weeks 21 and 23, presumably
as a consequence of polycythemia. Although hematocrit and serum Epo
values were not strictly related (r = .81 for LD mice and
r = .86 for HD mice), serum Epo concentrations were
consistently increased in polycythemic animals, with higher levels in
HD (320 to 1,050 Epo mU/mL) than in LD (140 to 430 Epo mU/mL) mice
(P = .032, F-test) (Fig 2). These results indicate that the
intramuscular injection of rAAV-ET induced robust and sustained Epo
secretion in doxycycline-treated mice.
Control of secretion levels supposes that a phenotypic reversion is
observed when stimulation is arrested. Doxycycline was withdrawn from
water given to polycythemic animals at week 8 (HD, n = 2; LD, n = 2)
and at week 25 (LD, n = 2). Measurement of serum Epo concentration 3 and 4 weeks later, respectively, showed values equivalent to that of
control mice, indicating that the stimulation of Epo secretion by
doxycycline was reversible. Hematocrits decreased more slowly, as
expected considering that the half-life of mouse erythrocytes is 24 days.
To ensure that responsiveness to doxycycline was maintained overtime,
the drug was administered again at week 19 to LD and HD animals which
had recovered background hematocrit level after doxycycline was
stopped, and at week 25 to LD animals which had not been stimulated
previously. Serum Epo levels measured 3 and 4 weeks later showed
concentrations equivalent to those of animals continuously treated with
the drug, indicating that the tetracycline regulatory system was fully
functional several months after gene transfer. Hematocrit values
increased accordingly (Fig 2). Interestingly, doxycycline stimulation
induced polycythemia more rapidly when administered at late times than
immediately after AAV injection.
The phenotypic changes induced by rAAV-ET transduction must be
interpreted with regard to the number of vector genomes which persisted
in injected muscles. High-molecular-weight DNA was extracted from
entire injected and control muscles of animals killed at 29 weeks and
vector sequences were detected by hybridization of Southern blots with
an Epo-specific probe (Fig 3). As expected, considering the delay after injection, vector signal was not detected in undigested DNA. BamHI, which generates vector internal
fragments, allowed quantification of vector copy numbers per cell. An
average of 0.043 ± 0.018 copies (n = 13) of double-stranded vector
genome per diploid cell genome was detected in injected muscles. Weak signals prevented detection of vector integration or concatemer formation. A search for rAAV-ET vector genome was performed by polymerase chain reaction (PCR) in various tissues. Whereas injected muscles generated a specific signal, DNA samples from liver, lung, spleen, and brain were negative (not shown).
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| Fig 3.
Southern blot analysis of high-molecular-weight DNA
extracted from muscles. Entire tibialis anterior muscles were resected
from animals killed 29 weeks after vector injection and used for
high-molecular-weight DNA extraction. HD mice, animals injected with
2.5 × 1010 rAAV-ET genomes in each tibialis anterior; LD
mice, animals injected with 2.5 × 1010 rAAV-ET genomes in
a single tibialis anterior; i.m., rAAV-ET injected muscle; n.i.m.,
controlateral muscle not injected with the vector. Undigested () and
BamHI digested (+) DNA was hybridized with a
32P-labeled Epo-specific probe. Reference copy numbers
(ref. copy number) correspond to high-molecular-weight DNA extracted
from normal C3H mouse muscle and run with plasmid DNAs corresponding to
1 copy (20 pg, lane 1) or 0.1 copy (2 pg, lane 0.1) of pAAV-ET DNA. (C)
DNA extracted from a muscle of a noninjected mouse. Signals
corresponding to the endogenous Epo gene (Epo endo) and to the internal
BamHI vector fragment are indicated. Precise quantification of
vector signals relative to ref. copy number signals and Epo endo
signals was done on a phosphorimager. Molecular weight markers are in
kilobases.
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DISCUSSION |
This study documents the sustained responsiveness of gene expression in
response to doxycycline in mice which have received a single
intramuscular injection of a recombinant AAV vector. Immune tolerance
for genetically modified cells and therapeutic proteins is critical for
the success of a gene therapy approach. The persistence of genetically
modified myofibers several months after injection was attested by the
detection of vector genomes and by the induction of Epo secretion in
response to the administration of doxycycline to recipient animals. The
absence of an immune reaction directed against the rtTA protein, which
contains xenogeneic epitopes, was confirmed by the histological
normality of recipient muscles examined after sacrifice, as well as by
the absence of antibody directed against rtTA (not shown). In agreement
with our previous observation,5 these data confirmed that
the components of the tetracycline regulatory system can be stably
expressed in mouse muscles. Consistent with a previous report
documenting the lack of an immune response against foreign proteins
expressed from AAV vectors,15 immune tolerance toward the
rtTA protein was not broken by using rAAV as a gene transfer vehicle.
In muscles injected with 2.5 × 1010 rAAV-ET
particles, stably transduced vector genomes were associated with mouse
high-molecular-weight DNA. They represented approximatively 5% of
diploid mouse genomes. This proportion is consistent with data from
previous studies in which equivalent18 or higher
numbers19 of rAAV genome were injected into mouse muscles.
We independently observed that the proportion of stably transduced
myofibers is directly related to the number of injected vector genomes
(D.B., unpublished data). Analysis performed in previous
studies showed that persisting rAAV genomes are organized as tandem
oligomers and interlocked circles closely associated with
high-molecular-weight mouse DNA. Whether these genomes are integrated
in the host DNA or remain as large episomes has not been clearly
elucidated.14,15,18,19
Northern blot analysis of human primary myoblasts transduced in vitro
showed basal activity of the tetO-CMV promoter in the absence of the
inducer. The presence of retroviral LTR enhancer sequences controlling
the expression of rtTA on the same recombinant AAV genome as the
inducible tetO-CMV promoter probably results in a nonspecific
transactivation. Basal promoter activity account for lower induction
ratio than previously observed with this system.5 In mice
injected with 2.5 × 1010 rAAV-ET genomes
and not treated with doxycycline, basal Epo secretion was responsible
for a moderate increase of serum Epo levels and hematocrit values,
which were only slightly above normal. However, even minimal basal
secretion could be deleterious in a situation where the transgene
product is immunogenic.
After the addition of doxycycline in the drinking water, serum Epo
levels were roughly 10-fold higher than basal concentrations. It is
noticeable that this increase was equivalent to that observed after the
addition of doxycycline to primary human myotubes maintained in tissue
culture. Values of 200 to 1,000 Epo mU/mL of serum were measured, which
correspond approximately to 2 to 10 ng/mL of circulating Epo. Secreted
Epo amounts appear slightly higher than in previously published works
in which muscles had been transduced with a comparable efficiency, but
using AAV vectors expressing Epo cDNA constitutively from CMV promoter
and enhancer elements.17,18 This suggests very efficient
expression from the inducible tetO-CMV promoter in the presence of
doxycycline. Hematocrits increased rapidly during the first weeks of
doxycycline treatment, then more slowly. Interestingly, the initial
increase was more rapid when the treatment was started at late times
after gene transfer rather than at week 2. A possible explanation is
that the conversion of AAV genomes to transcriptionally active
double-stranded DNA was still building up at early time points, whereas
it was fully achieved several weeks after gene transfer.19
Two weeks after the doxycycline stimulation was arrested, serum Epo
levels equivalent to those of animals never treated with the drug were
measured. Presumably, delays needed for returning to the basal
situation are actually much shorter, as suggested by our previous study
with retrovirus-engineered myofibers.5 Normalization of
hematocrits required several weeks, as expected considering the
half-life of mouse erythrocytes.
The feasibility of a long-term control of the systemic delivery of Epo
after a single intramuscular injection may have implications for the
development of gene therapy protocols for patients with  -thalassemia. In the -thalassemic mouse model, the
injection of high doses of recombinant human (rHu) Epo allowed a
partial and transient correction of the disease,31 whereas
the engraftment of Epo-secreting hematopoietic cells fully corrected
the -thalassemic phenotype and induced a lethal
polycythemia.32 Normalization of the / -globin chain
ratio and unpaired -globin chain level was obtained with serum Epo
concentrations in the range of 200 mU/mL, suggesting that therapeutic
levels could be easily attained by the intramuscular injection of
rAAV-ET, which would also allow to avoid fatal polycythemia. A partial
correction of hemolysis and anemia was reported after the
administration of rHuEpo in patients with intermediate
-thalassemia.33-35 Although rHuEpo amounts needed for a
complete correction of the -thalassemic syndrome in human patients
have not been determined, they are presumed to be higher than 3,000 units/kg/wk. The high frequency of the disease and the necessity of a
life-long treatment with such doses of rHuEpo would result in
unaffordable costs. The systemic delivery of potentially very high
amounts of Epo and the possibility of preventing polycythemia by a
tight control of gene expression by doxycycline may allow consideration
of rAAV-mediated gene transfer into muscles for future human trials.
Assessment of this approach in the -thalassemic mouse model will be
crucial in that respect.
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FOOTNOTES |
Submitted April 9, 1998;
accepted June 14, 1998.
Supported by grants from the association Vaincre les Maladies
Lysosomales, the Association Française contre les Myopathies, and
the French Ministère de l'Education Nationale et de la
Recherche.
Address reprint requests to Jean Michel Heard, MD, 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. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We are grateful to Dr H. Bujard for the gift of puHD10.3 and
pUHD172.1-neo plasmids; to Dr V. Mouly for providing us with primary
human myoblast cultures, and to S. Orève for excellent technical
assitance in the preparation of rAAV-ET stocks.
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Abstract
Introduction
Abstract