Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 888-893
Stomach Implant for Long-Term Erythropoietin Expression in Rats
By
Daniel V. Lejnieks,
N. Ramesh,
Stella Lau, and
William R.A. Osborne
From the Department of Pediatrics, University of Washington, Seattle,
WA.
 |
ABSTRACT |
To approach the goal of consistent long-term erythropoietin (Epo)
expression in vivo, we developed an implantation procedure in which
transduced autologous vascular smooth muscle was introduced into rats
in a chamber created from a polytetrafluoroethylene (PTFE) ring placed
under the serosa of the stomach. The implant became vascularized and
permitted the long-term survival of smooth muscle cells expressing Epo.
Hematocrits of treated animals increased rapidly and monitored over 12 months gave a mean value of 56.0 ± 4.0% (P < .001; n = 9), increased from a presurgery mean of 42.3 ± 1.6%. Hemoglobin
levels rose from a presurgery mean of 15.2 ± 0.4 g/dL and for 12 months were significantly elevated with a mean value of 19.5 ± 1.3 g/dL (P < .001; n = 9). The hematocrit and
hemoglobin levels of control animals receiving human adenosine deaminase (ADA)-expressing cells were not significantly
different from baseline (P > .05; n = 5). In response to
tissue oxygenation, kidney, and (to a lesser extent) liver are specific
organs that synthesize Epo. Treated animals showed downregulation of
endogenous Epo mRNA in kidney over a 12-month period. The PTFE implant
provides sustained gene delivery, is safe, and is minimally invasive.
It allows easy engraftment of transduced cells and may be applied generally to the systemic delivery of therapeutic proteins such as
hormones and clotting factors.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ERYTHROPOIETIN (Epo) is a 30-kD
glycoprotein hormone that is the regulator of red cell production and
maintenance in mammals.1,2 Understanding of the molecular
mechanisms of its action was significantly advanced by the cloning of
human Epo cDNA.3,4 Epo from many mammals has now been
cloned, and a high degree of sequence similarity and genetic structure
has been found.1,5 The availability of recombinant human
Epo provided a major advance in the treatment of renal failure patients
receiving dialysis.6 The attendant dangers of transfusion
therapy were eliminated and the quality of life of these patients has
significantly increased.2 The administration of recombinant
Epo is now widely used for long-term treatment of anemia associated
with chronic renal failure, cancer chemotherapy, and human
immunodeficiency virus infections.2 Delivery of this
hormone by gene therapy rather than by repeated injections would
provide substantial clinical and economic benefits and would serve as a
model for the expression of other therapeutic proteins.
In adults, Epo is produced primarily in the kidney with the liver as a
secondary source.7,8 Tissue oxygen tension regulates the
overall level of Epo and red cell production, primarily through the
rates of gene transcription in the kidney.1 The
hypoxia-responsive cis elements of the Epo gene have been found to be
localized to the 3
untranslated region.9-15 Deletion
analysis has shown that a 24-bp portion of the 3 flanking
sequence of the Epo gene was sufficient to give a transcriptional
response to hypoxia.12 The oxygen-sensing mechanism
involved in the induction of Epo synthesis includes a 120-kD
hypoxia-inducible factor 1 (HIF-1) and other transacting
factors.16 Most interestingly, the oxygen-sensing system
initially identified in Epo-producing cells was found to be present in
a wide variety of cell types.11,13,14 Unfortunately these
regulatory DNA sequences are too large to be suitable for insertion in
a retroviral vector.
Long-term in vivo gene expression requires both target cells and gene
delivery vectors that permit continuous vector-encoded activity. Of the
three common virus-based methods of gene transfer, retroviral vectors
are probably the most useful for ex vivo gene transfer.17-20 Adeno-associated virus (AAV) vectors have
many attractive features, such as safety and ability to transduce
nonproliferating cells,21-25 but they do not possess
advantages over retroviruses for ex vivo gene transduction.
Replication-defective retroviral vectors can be made with high titers
and will infect a wide variety of cell types. Infection results in
stable proviral integration into the host chromosome providing gene
expression for the lifetime of the cell and its
progeny17-20 Therapeutic genes can be expressed at high
level from the viral long terminal repeat (LTR) promoter/enhancer or
strong internal promoters. Recently, the incorporation of internal ribosome entry sites from picornaviruses into retroviral vectors has
allowed the generation of bicistronic vectors and subsequent advantages
in linked-gene selection.26-28
Nonhematopoietic cells studied as vehicles for gene therapy include
skin fibroblasts, myoblasts, and vascular smooth muscle cells. Skin
fibroblasts are easily obtained, cultured, and transduced but have a
major disadvantage of inactivating vector sequences after
transplantation.29,30 Myoblasts represent a promising target cell type for gene therapy. Transduced skeletal myoblasts have
been used to deliver Epo in mice,31-33 and transplantation of retrovirally transduced skeletal muscle myoblasts has been successfully achieved in dogs with alpha-L-iduronidase
deficiency.34 Intramuscular injection of plasmid DNA has
produced systemic expression of Epo in mice.35
Smooth muscle cells are present within the vasculature as a
multilayered mass of long-lived cells in proximity to the circulation and have been investigated as targets for gene
therapy.36-42 We have shown that transduced vascular smooth
muscle cells seeded into carotid arteries in the rat will provide
sustained expression of both marker and therapeutic
genes.36,38,42 However, while showing the potential of
smooth muscle cells to provide long-term gene expression of
therapeutic proteins, this procedure may not be applied to patients as
it requires arterial injury to achieve cell
engraftment.36,38,41 As an alternative site for smooth muscle cell implantation, we recognized that the tissue plane between
the tunica muscularis and tunica serosa might provide a niche for
retention of transduced smooth muscle cells. This tissue is composed of
smooth muscle cells, is well vascularized, and is able to provide
nutrition for implanted cells. Smooth muscle cells are present at this
site, and it is important to the survival of transplanted cells that
they are a normal constituent of the targeted area. To examine the
potential of this method of cell implantation to provide long-term gene
expression we studied the effect of Epo secretion on hematopoiesis in
rats.
| |
MATERIALS AND METHODS |
Construction of retroviral vectors.
The retroviral vector LrEpSN was made by inserting an
EcoRI-BamHI fragment of the rat Epo cDNA into
LXSN.38,43 A plasmid containing the rat Epo gene was kindly
provided by Drs J.-P.R. Boissel and H.F. Bunn, Boston, MA.5
The amphotropic retroviruses and the control retroviral vector LASN,
encoding human adenosine deaminase (ADA), were generated as described
earlier.44
Cell culture.
Rat smooth muscle cell cultures were prepared by enzymatic
digestion of the aorta from a male Fisher 344 rat, and the cells were
characterized by positive staining for muscle cell-specific actins with
HHF35 antibody37 and staining negative for von Willebrand factor,37 an endothelial cell specific marker. Ecotropic
PE501 and amphotropic PA317 retrovirus packaging cell
lines,43,45 NIH 3T3 thymidine kinase negative
cells,45 and primary cultures of rat smooth muscle cells
were grown in Dulbecco/Vogt modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum in humidified 5%
CO2 at 37°C. Early passage smooth muscle cells were
exposed to 16-hour virus harvests from PA317-LrEpSN or PA317-LASN amphotropic virus-producing cell lines for a period of 24 hours in the
presence of polybrene (4 µg/mL). Infected cells were selected in
medium containing G418 at 1 mg/mL. Vascular smooth muscle cells infected with LrEpSN and selected in 1 mg/mL G-418 antibiotic secreted
6.7 mU/24 h per 105 cells of Epo.38 In
experiments to determine cell distribution in polytetrafluoroethylene
(PTFE) implants, LrEpSN-transduced cells were labeled with the
fluorescent marker
1,1-dioctadecyl-3,3,33-tetramethylindocarbocyanine perchlorate (DiI).36
Epo mRNA analysis.
Total RNA was isolated from rat kidney by homogenization in the
presence of RNAzol. Using SuperScript reverse transcriptase (GIBCO-BRL, Gaithersburg, MD), 1 µg of total RNA was
reverse-transcribed in the presence of random hexamer primers.
Polymerase chain reaction (PCR; 30 cycles) was performed with rat
Epo-specific primers (5 AGG CGC GGA GAT GGG GGT GC 3 and
5 CCC CGG AGG AAG TTG GAG TAG 3) to give a 540-bp
amplified fragment. An aliquot of the amplified reaction mixture was
electrophoresed in a 2% agarose gel and, after Southern transfer, the
membrane was hybridized with a 32P-labeled 500-bp Epo cDNA
probe. As a control for RNA extraction, integrity, reverse
transcription, and amplification, 
-actin-specific primers (5
GTG GGG CGC CCC AGG CAC CA 3 and 5 CTC CTT AAT GTC ACG
CAC GAT TTC 3) were used to amplify a 500-bp fragment from the
same cDNA preparation. Equal amplification of the samples was confirmed
by both ethidium bromide staining and subjecting diluted aliquots of
each sample to agarose electrophoresis, Southern transfer, and
hybridization of the transferred DNA to a -actin-specific 32P-cDNA probe.
Smooth muscle cell implantation.
Rats were anesthetized by intraperitoneal (IP) injection with 44 mg/kg
ketamine, 5 mg/kg xylazine, and 0.5 mg/kg acepromazine. All rats
received 0.04 mg dexamethasone IP just before surgery. An area from
thoracic inlet to the pubis was prepared for surgery, and a 3-cm
midline abdominal incision was made from the xyphoid to the umbilicus.
The stomach was temporarily exteriorized and held in place with a
mosquito hemostat. A 0.5-cm superficial incision was made in the
capsule on the cranial face of the body of the stomach, and a small
pocket approximately 0.6 cm in diameter was created under the capsule
using blunt dissection. A small PTFE ring (inner diameter 4 mm, outer
diameter 6 mm) was inserted into the pocket and sutured in place using
5-0 maxon on a taper needle in a simple continuous pattern. The suture
material was drawn tightly to constrict the ring to a final inner
diameter of 2 to 3 mm before finishing the knot. The fibrous tunic
directly overlying the ring was cryofrozen using a steel probe, and the
ring was mechanically elevated to prevent the freezing of the
underlying muscular layer to minimize tissue damage. The ring was
rinsed with 0.9% saline to remove any blood clots formed during
surgery. Cell aliquots of 1 × 106 cells/50 mL media
were then introduced into the center of the ring through a 24-g
intravenous (IV) catheter. Animals received two rings each containing 1 × 106 transduced vascular smooth muscle cells
expressing either Epo or human ADA.
Blood analysis.
Anticoagulated blood samples (300 µL) were obtained from the tail
vein and reticulocyte count determined by vital staining with brilliant
cresyl blue and cell counting by standard techniques. Hematocrit,
hemoglobin, platelet, and white blood cell (WBC) number were measured
using a Coulter counter (Coulter Immunology, Hialeah, FL).
| |
RESULTS |
To achieve cell implantation we positioned PTFE rings under the serosal
plane of the rat stomach to create an area above the muscle layer
enclosed by the serosa membrane. The hematocrits of animals implanted
with Epo-secreting transduced cells increased steadily over 50 days
from a mean of 42.3 ± 1.6% to a maximum of 67.5 ± 3.1% and
remained elevated with a mean value of 56.0 ± 4.0% (P < .001) over the 12-month observation period
(Fig 1 and Table 1).
Hemoglobin levels rose from a presurgery mean of 15.2 ± 0.4 g/dL to
a maximum of 22.6 ± 1 g/dL at around 7 weeks and at 12 months were
significantly elevated with a mean value of 19.5 ± 1.3 g/dL (P
< .001). The hematocrit and hemoglobin levels of control animals
treated with transduced cells expressing human ADA were not
significantly different from baseline during the experiment (P
>.05; Table 1). Greater than 95% of the treated animals showed
significant increases in red cell production, indicating this procedure
is very reproducible. The control hematological levels were in
agreement with normal rat blood values.46 WBC and platelet
counts remained within the normal range during the course of the
experiment in both the treated and control groups (Table 1). This was
expected because previous studies have documented the selective effect
of sustained Epo delivery on hematopoiesis with no significant changes
in leukocyte or megakaryocyte production.6,38,47,48 Reticulocytes were elevated in treated rats and not in controls. The
mean value presurgery was 1.9%, with a range of 0.5% to 3.5%, and
the postsurgery mean was 3.7%, with a range of 1.3% to 10.3%. The
reticulocyte counts peaked between 10 to 30 days, in contrast to
implantation of transduced smooth muscle cells on the carotid artery,
which gave peak levels at about 8 to 14 days.38 The single
administration of dexamethasone at the time of surgery increased the
red cell production of treated animals to implantation of Epo-secreting
cells. At 2 months animals receiving dexamethasone had a mean
hematocrit of 59% compared with a mean hematocrit of 51% recorded
from untreated animals (data not shown).
View larger version (22K):
[in this window]
[in a new window]
| Fig 1.
Effect of seeding of transduced vascular smooth muscle
cells on hematocrit (Hct). Closed symbols represent animals seeded with
LrEpSN-transduced cells, and open symbols are control rats receiving
LASN-transduced cells.
|
|
A rat showing an elevated hematocrit of 63% at 12 months after surgery
was sacrificed, and the PTFE implant was removed, fixed, and stained
with hematoxylin and eosin. A photomicrograph of a cross-section showed tissue within and around the PTFE graft that was
fully integrated and well vascularized (Fig
2). This suggests that the PTFE structure is well tolerated between the
muscle and serosal layers.
View larger version (107K):
[in this window]
[in a new window]
| Fig 2.
Histological cross-sections of stomach PTFE implants
containing transduced smooth muscle cells. Tissues in panels 1 and 2 were obtained at 12 months postsurgery from a rat that had a hematocrit of 62%, fixed in formalin, and stained with H&E. PTFE implants containing Epo-secreting vascular smooth muscle cells unlabeled (panel
3) or marked with DiI (panel 4) were removed 2 months postsurgery from
a rat with a hematocrit of 67%, frozen, sectioned, and photographed using a Nikon Microphot FXA equipped with a rhodamine filter. PTFE
material is denoted P, and mucosal tissue as M. (Panels 1, 2, and 4:
×40 original magnification; panel 3: ×100 original
magnification.
|
|
To show that transduced vascular smooth muscle cells expressing Epo are
contained within the PTFE ring structure, Epo-secreting cells were
labeled with DiI, a fluorescent dye, before implantation. Unlabeled
Epo-secreting cells were implanted in control rats. At 2 months after
surgery a rat with a hematocrit of 68% was sacrificed and tissue
cross-sections photographed (Fig 2). A large mass of dye-marked
fluorescing cells was evident in the area encompassed by the PTFE ring,
and fluorescent cells were not visible in adjacent areas or in the
control sections (Fig 2). These data show that transduced vascular
smooth muscle cells remain in the PTFE-enclosed area and provide
therapeutic Epo expression for at least 12 months.
To determine if vector-encoded Epo expression resulted in
downregulation of endogenous Epo,8 test rats were
sacrificed when elevated hematocrit and hemoglobin levels were
established. Control rats that received LASN-transduced cells were
sacrificed at similar time points. Because Northern analysis is not
sensitive enough to detect endogenous Epo message, RNA obtained from
kidney was subjected to reverse transcription (RT)-PCR using rat
Epo-specific probes.5 Endogenous Epo mRNA in kidneys of
rats seeded with LrEpSN-transduced cells and analyzed at 11 and 12 months was greatly reduced in comparison to a control kidney,
suggesting significant downregulation of endogenous Epo production
(Fig 3). Epo mRNA in liver was not
detectable by this RT-PCR method (data not shown). Southern band
intensities from RT-PCR amplification of actin mRNA isolated from test
and control tissues were similar, indicating equivalence in RNA
isolation and amplification (Fig 3).
View larger version (48K):
[in this window]
[in a new window]
| Fig 3.
Epo mRNA analysis. Total RNA was isolated from kidneys of
rats receiving Epo expressing LrEpSN-transduced cells and control rats
receiving LASN-transduced cells expressing human ADA. RT-PCR was
performed with rat Epo-specific primers to give a 540-bp amplified segment that was subjected to electrophoresis and hybridized with a
32P-labeled Epo probe. As a control RT-PCR was performed
using -actin-specific primers to amplify a 500-bp fragment from the
same cDNA preparation. Diluted aliquots of each sample were subjected
to agarose electrophoresis, Southern transfer, and hybridization of the
transferred DNA to a -actin-specific 32P-cDNA probe.
|
|
| |
DISCUSSION |
We have described a novel site to implant transduced cells for the
systemic delivery of proteins. The time from surgery to maximum
hematocrit in this study was about 50 days, nearly twice as long as the
3-week induction period we observed using Epo-secreting smooth muscle
cells seeded onto denuded rat carotid arteries.38 This
difference in achieving maximal hematocrit may be due to arterial
seeding providing immediate systemic secretion of Epo, whereas a
stomach implant requires time to establish vascularization and a
pathway for hormone delivery to the bone marrow. The normal kinetics of
red cell production involve a 2- to 3-week period from Epo secretion to
mature red cell formation from erythroid precursors.2
The dye-labeling experiment showed that placement of cells within the
PTFE ring initiated the creation of a structure that served both to
retain and nurture implanted cells that were able to provide sustained,
high-level Epo expression. Extrapolation of the increases in red cell
production obtained over 12 months suggests that the cell implant would
function to deliver potentially therapeutic Epo levels for greater than
3 years, longer than the life time of the rat.
The protocol used in these experiments involved the single
administration of dexamethasone at the time of surgery. This was based
on initial experiments showing that dexamethasone, a powerful synthetic
glucocorticoid, enhanced the response of treated animals to
implantation of Epo-secreting cells. The viral LTR promoter, which
drives Epo cDNA expression, contains a known steroid-responsive element, but the short serum half-life of dexamethasone (1.8 to 4.7 hours) makes an effect mediated through this promoter element unlikely
to be sustained for 12 months.49 Dexamethasone has been
used clinically to decrease surgical inflammation and edema and to
decrease immune-mediated tissue destruction, making preservation of the
transplanted cells by dexamethasone treatment the most probable
explanation for increased cell survival and
hematocrit.50,51
The analysis of kidney Epo mRNA indicated that the systemic delivery of
hormone from the stomach implant caused downregulation of endogenous
Epo production, and this was sustained for at least 12 months. These
data provide evidence of long-term high-level Epo expression from
transplanted cells and, furthermore, indicate that the elevated red
cell production we observed was mediated by implanted transduced cells
with a minimal contribution from endogenous Epo. Prolonged red cell
production in excess of 60% from genetically modified cells is a
significant result from this cell transduction and implantation
protocol. In these experiments we used the strong viral LTR promoter to
achieve unregulated Epo expression. Although multiple cis
genetic elements have been identified that induce hypoxia-mediated Epo
expression in kidney and liver, they have yet to be defined in a size
suitable for insertion in a retroviral vector9,10,52 and
may not function in vascular smooth muscle cells.
The expression of bacterial neomycin phosphotransferase from the virus
we used (LrEpSN) did not appear to cause an immune-mediated loss of
transplanted cells. This is supported by the longevity of cell survival
and the sustained red cell overproduction. The elimination of
autologous transduced cells by an immune-mediated mechanism to foreign
transgenes has been reported in rats receiving glioma
cells53 and human T-cell transplantations.54
The smooth muscle cells we targeted for gene expression and
implantation are not usually involved in antigen processing and
presentation, and the neo gene is expressed in the cytosol and may not
be secreted, providing an explanation for the apparent lack of
immune-mediated cell loss.
The persistence for at least 12 months of transduced smooth muscle
cells contained within a PTFE gastric ring suggests this approach may
be useful for human gene therapy. Vascular smooth muscle cells can be
obtained from a peripheral vein and can be cultured, transduced, and
selected with high efficiency. This method may be applied to patients
by using laparoscopic surgery to implant PTFE structures to contain
therapeutically relevant cell numbers. We estimate that 108
transduced vascular smooth muscle cells would provide a therapeutic supply of Epo to an 80-kg patient,38 and implantation of
this cell number is achievable in a PTFE graft. In the absence of a hypoxia-regulated Epo promoter of a size to permit inclusion in a
virus, retroviral-mediated red cell production will be controlled by
the number of cells implanted. As we know the level of transduced Epo
gene expression we can achieve a desired hematocrit by manipulating the
number of cells implanted. The use of laparoscopes is now widespread
and affords a minimally invasive method to access and perform surgery
on the stomach and intestine. This study and others36,38,42 have shown that retroviral vectors are not subject to vector
inactivation in smooth muscle cells. A patient's cells can be stored
frozen for an indefinite period enabling this process to be repeated if
desired. Cells can be modified to express and systemically deliver
therapeutic proteins such as granulocyte colony-stimulating factor,
insulin, clotting factors, and enzymes such as glucocerebrosidase for
the treatment of Gaucher's disease. Furthermore, this method is
inherently safe because transplanted cells remain within the PTFE
structure and can therefore be removed if necessary.
| |
FOOTNOTES |
Submitted December 8, 1997;
accepted March 27, 1998.
Supported by Grant Nos. DK 43727, DK 47754, and DK 50686 from the
National Institutes of Health.
Address reprint requests to William R.A. Osborne, PhD, Department of
Pediatrics, MS 356320, University of Washington, Seattle, WA 98195.
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.
| |
ACKNOWLEDGMENT |
We thank Drs D.C. Dale, S.C. Barry, D. Liggitt, and A.M. Gown for much
helpful advice, and Drs J.-P.R. Boissel and H.F. Bunn for kindly
supplying the rat Epo cDNA.
| |
REFERENCES |
1.
Koury MJ,
Bondurant MC:
The molecular mechanism of erythropoietin action.
Eur J Biochem
210:649,
1992[Medline]
[Order article via Infotrieve]
2.
Spivak JL:
Hematology/Oncology Clinics of North America. Erythropoietin: Basic and clinical aspects.
Hematol Oncol Clin North Am
8:1043,
1994
3.
Lin F-K,
Suggs S,
Lin C-H,
Browne JK,
Smalling R,
Egrie JC,
Chen KK,
Fox GM,
Martin F,
Stabinsky Z,
Bradrawi SM,
Lai P-H,
Goldwasser E:
Cloning and expression of the human erythropoietin gene.
Proc Natl Acad Sci USA
82:7580,
1985[Abstract/Free Full Text]
4.
Jacobs K,
Shoemaker CB,
Rudersdorf R,
Neill SD,
Kaufman RJ,
Mufson A,
Seehra J,
Jones SS,
Hewick R,
Fritsch EF,
Kawakita M,
Shimizu T,
Miyake T:
Isolation and characterization of genomic and cDNA clones of human erythropoietin.
Nature
313:806,
1985[Medline]
[Order article via Infotrieve]
5.
Wen D,
Boissel J-PR,
Tracy TE,
Gruninger RH,
Mulcahy LS,
Czelusniak J,
Goodman M,
Bunn HF:
Erythropoietin structure-function relationships: High degree of sequence homology among mammals.
Blood
82:1507,
1993[Abstract/Free Full Text]
6.
Eschbach JW,
Egrie JC,
Downing MR,
Brown JK,
Adamson JW:
Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial.
N Engl J Med
316:73,
1987[Abstract]
7.
Koury ST,
Bondurant MC,
Koury MJ,
Semenza GL:
Localization of cells producing erythropoietin in murine liver by in situ hybridization.
Blood
77:2497,
1991[Abstract/Free Full Text]
8.
Schuster SJ,
Koury ST,
Bohrer M,
Salceda S,
Caro J:
Cellular sites of extrarenal erythropoietin production in anaemic rats.
Br J Haematol
81:153,
1992[Medline]
[Order article via Infotrieve]
9.
Beck I,
Weinmann R,
Caro J:
Characterization of hypoxia-response enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells.
Blood
82:704,
1993[Abstract/Free Full Text]
10.
Blanchard KL,
Acquaviva AM,
Galson DL,
Bunn HF:
Hypoxia induction of the human erythropoietin gene: Cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.
Mol Cell Biol
12:5373,
1992[Abstract/Free Full Text]
11.
Firth JD,
Ebert BL,
Pugh SW,
Ratcliffe PJ:
Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A gene: Similarities with the erythropoietin 3 enhancer.
Proc Natl Acad Sci USA
91:6496,
1994[Abstract/Free Full Text]
12.
Madan A,
Curtin PT:
A 24-base-pair sequence 3 to the human erythropoietin gene contains a hypoxia-responsive transcriptional enhancer.
Proc Natl Acad Sci USA
90:3928,
1993[Abstract/Free Full Text]
13.
Maxwell PH,
Pugh CW,
Ratcliffe PJ:
Inducible operation of the erythropoeitin 3' enhancer in multiple cell lines: Evidence for a widespread oxygen-sensing mechanism.
Proc Natl Acad Sci USA
90:2423,
1993[Abstract/Free Full Text]
14.
Semenza GL,
Roth PH,
Fang HM,
Wang GL:
Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.
J Biol Chem
269:23757,
1994[Abstract/Free Full Text]
15.
Wang GL,
Semenza GL:
General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.
Proc Natl Acad Sci USA
90:4304,
1993[Abstract/Free Full Text]
16.
Wang GL,
Semenza GL:
Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.
J Biol Chem
268:21513,
1993[Abstract/Free Full Text]
17.
Miller AD:
Human gene therapy comes of age.
Nature
357:455,
1992[Medline]
[Order article via Infotrieve]
18.
Morgan RA,
Anderson WF:
Human gene therapy.
Annu Rev Biochem
62:191,
1993[Medline]
[Order article via Infotrieve]
19.
Mulligan RC:
The basic science of gene therapy.
Science
260:926,
1993[Abstract/Free Full Text]
20.
Kohn DB:
The current status of gene therapy using hematopoietic stem cells.
Curr Opin Pediatr
7:56,
1995[Medline]
[Order article via Infotrieve]
21.
Muzyczka N:
Use of adeno-associated virus as a general transduction vector for mammalian cells
, in Muzyczka N
(ed):
Current Topics in Microbiology and Immunology. Viral Expression Vectors (vol 158).
New York, NY, Springer-Verlag
, 1992
, p 97
22.
Samulski RJ:
Adeno-asssociated virus: Integration at a specific chromosomal locus.
Curr Opin Genet Dev
3:74,
1993[Medline]
[Order article via Infotrieve]
23.
Kessler PD,
Podsaloff GM,
Chen X,
McQuiston SA,
Colosi PC,
Matelis LA,
Kurtzman GJ,
Byrne BJ:
Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.
Proc Natl Acad Sci USA
93:14082,
1996[Abstract/Free Full Text]
24.
Kaplitt MG,
Leone P,
Samulski RJ,
Xiao X,
Pfaff DW,
O'Malley KL,
During MJ:
Long-term gene expression and phenotypic correction using adeno-associated virus vectors in mammalian brain.
Nat Genet
8:148,
1994[Medline]
[Order article via Infotrieve]
25.
Snyder RO,
Miao CH,
Patijn GA,
Spratt SK,
Danos O,
Nagy D,
Gown AM,
Winther B,
Meuse L,
Cohen LK,
Thompson AR,
Kay MA:
Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.
Nat Genet
16:270,
1997[Medline]
[Order article via Infotrieve]
26.
Adam MA,
Ramesh N,
Miller AD,
Osborne WRA:
Internal initiation of translation in retroviral vectors carrying picornavirus 5 nontranslated regions.
J Virol
65:4985,
1991[Abstract/Free Full Text]
27.
Morgan RA,
Couture L,
Elroy-Stein O,
Ragheb J,
Moss B,
Anderson WA:
Retroviral vectors containing putative internal ribosome entry sites: Development of a polycistronic gene transfer system and applications to human gene therapy.
Nucleic Acids Res
20:1293,
1992[Abstract/Free Full Text]
28.
Ramesh N,
Kim S-T,
Wei MQ,
Khalighi M,
Osborne WRA:
High-titer bicistronic vectors employing foot-and-mouth disease virus internal ribosome entry site.
Nucleic Acids Res
24:2697,
1996[Abstract/Free Full Text]
29.
Ramesh N,
Lau S,
Palmer TD,
Storb R,
Osborne WRA:
High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation.
Hum Gene Ther
4:3,
1993[Medline]
[Order article via Infotrieve]
30.
Palmer TD,
Rosman GJ,
Osborne WRA,
Miller AD:
Genetically-modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.
Proc Natl Acad Sci USA
88:1330,
1991[Abstract/Free Full Text]
31. Barr E, Tripathy S, Leiden JM: Genetically modified myoblasts
for the treatment of erythropoietin-responsive anemias. J Cell Biochem
18:DZ012, 1994 (suppl 18A)
32.
Hamamori Y,
Samal B,
Tian J,
Kedes L:
Myoblast transfer of human erythropoietin gene in a mouse model of renal failure.
J Clin Invest
95:1808,
1995
33.
Naffakh N,
Pinset C,
Montarras D,
Paulin D,
Danos O,
Heard JM:
Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.
Hum Gene Ther
7:11,
1996[Medline]
[Order article via Infotrieve]
34.
Shull RM,
Lu X,
McEntee MF,
Bright RM,
Pepper KA,
Kohn DB:
Myoblast gene therapy in canine mucopolysaccharidosis I: Abrogation by an immune response to alpha-L-iduronidase.
Hum Gene Ther
7:1595,
1996[Medline]
[Order article via Infotrieve]
35.
Tripathy SK,
Svensson EC,
Black BH,
Goldwasser G,
Margalith M,
Hobart PM,
Leiden JM:
Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector.
Proc Natl Acad Sci USA
93:10876,
1996[Abstract/Free Full Text]
36.
Clowes MM,
Lynch CM,
Miller AD,
Miller DG,
Osborne WRA,
Clowes AW:
Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.
J Clin Invest
93:644,
1994
37.
Geary RL,
Clowes AW,
Lau S,
Vergel S,
Dale DC,
Osborne WRA:
Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally-transduced smooth muscle cells: A model for local and systemic gene therapy.
Hum Gene Ther
5:1213,
1994
38.
Osborne WRA,
Ramesh N,
Lau S,
Clowes MM,
Dale DC,
Clowes AW:
Gene therapy for long-term expression of erythropoietin in rats.
Proc Natl Acad Sci USA
92:8055,
1995[Abstract/Free Full Text]
39.
Ohno T,
Gordon D,
San H,
Pompili MJ,
Nabel GJ,
Nabel EG:
Gene therapy for vascular smooth muscle cell proliferation after arterial injury.
Science
265:781,
1994[Abstract/Free Full Text]
40.
Plautz G,
Nabel EG,
Nabel GJ:
Introduction of vascular smooth muscle cells expressing recombinant genes in vivo.
Circulation
83:578,
1991[Abstract/Free Full Text]
41.
Lejnieks DV,
Han SW,
Ramesh N,
Lau S,
Osborne WRA:
Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats.
Hum Gene Ther
7:1431,
1996[Medline]
[Order article via Infotrieve]
42.
Lynch CM,
Clowes MM,
Osborne WRA,
Clowes AW,
Miller AD:
Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: A model for gene therapy.
Proc Natl Acad Sci USA
89:1138,
1992[Abstract/Free Full Text]
43.
Miller AD,
Rosman GJ:
Improved retroviral vectors for gene transfer and expression.
Biotechniques
7:980,
1989[Medline]
[Order article via Infotrieve]
44.
Hock RA,
Miller AD,
Osborne WRA:
Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines.
Blood
74:876,
1989[Abstract/Free Full Text]
45.
Miller AD,
Buttimore C:
Redesign of retrovirus packaging cell lines to avoid recombination to helper virus production.
Mol Cell Biol
6:2895,
1986[Abstract/Free Full Text]
46. Schalm OW, Jain NC, Carroll EJ: Veterinary Hematology (ed 3).
Philadelphia, PA, Lea and Febiger, 1975
47.
Semenza GL,
Nejfelt MK,
Chi SM,
Antonarakis SE:
Hypoxia-inducible nuclear factors bind to an enhancer element located 3 to the human erythropoietin gene.
Proc Natl Acad Sci USA
88:5680,
1991[Abstract/Free Full Text]
48.
Spivak JL,
Pham T,
Isaacs M,
Hankins WD:
Erythropoietin is both a mitogen and a survival factor.
Blood
77:1228,
1991[Abstract/Free Full Text]
49.
Axelrod L:
Corticosteroid therapy
, in Becker EL
(ed):
Principles and Practice of Endocrinology and Metabolism.
Philadelphia, PA, Lippincott
, 1995
, p 695
50.
Fletcher DS,
Osinga D,
Bonney RJ:
Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse Arthus reaction.
Biochem Pharmacol
35:2601,
1986[Medline]
[Order article via Infotrieve]
51.
Weber CR,
Griffin JM:
Evaluation of dexamethasone for reducing postoperative edema and inflammatory response after orthognathic surgery.
J Oral Maxillofac Surg
52:35,
1994[Medline]
[Order article via Infotrieve]
52.
Madan A,
Lin C,
Hatch SL,
Curtin PT:
Regulated basal, inducible, and tissue-specific human erythropoietin gene expression in transgenic mice requires multiple cis DNA sequences.
Blood
85:2735,
1995[Abstract/Free Full Text]
53.
Tapscott SJ,
Miller AD,
Olsen JM,
Berger MS,
Groudine M,
Spence AM:
Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes does not require drug selection.
Proc Natl Acad Sci USA
91:8185,
1994[Abstract/Free Full Text]
54.
Riddel SR,
Elliott M,
Lewinsohn DA,
Gilbert MJ,
Wilson L,
Manley SA,
Lupton SD,
Overell RW,
Reynolds TC,
Corey L,
Greenberg PD:
T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV patients.
Nat Med
2:216,
1996[Medline]
[Order article via Infotrieve]
Abstract
Introduction
Abstract
