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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2536-2542
GENE THERAPY
From the Departments of Pediatrics and Pathology, University of
Pennsylvania Medical Center and The Children's Hospital of
Philadelphia, Philadelphia, PA; Avigen Inc, Alameda, CA; and Institute
for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA.
Hemophilia B is caused by the absence of functional coagulation
factor IX (F.IX) and represents an important model for treatment of
genetic diseases by gene therapy. Recent studies have shown that
intramuscular injection of an adeno-associated viral (AAV) vector into
mice and hemophilia B dogs results in vector dose-dependent, long-term
expression of biologically active F.IX at therapeutic levels. In this
study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular
injection of mice using a 2- to 4-fold lower vector dose
(1 × 1011 vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished
through the use of an improved expression cassette that uses the
cytomegalovirus (CMV) immediate early enhancer/promoter in combination
with a 1.2-kilobase portion of human skeletal actin promoter. These
results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone
was 120 to 200 ng/mL in mice injected with 1 × 1011
vector genomes. Muscle-specific promoters performed poorly for F.IX
transgene expression in vitro and in vivo. However, the incorporation of a sequence from the
The severe bleeding disorder hemophilia B is caused by
an absence of functional coagulation factor IX (F.IX). The X-linked disease affects 1 in 30 000 males in the United States. Current treatment for hemophilia is based on intravenous infusion of
plasma-derived or recombinant clotting factor concentrates. Treatment
can be episode-based (in response to a bleeding episode) or
prophylactic (2-3 infusions per week). The latter is expensive and not
without complications and is therefore not widely adopted in the United States. However, 30 years of experience with prophylactic treatment as
pioneered in Sweden has shown that a continuous supply of clotting factor levels above 1% of normal (greater than 50 ng F.IX/mL plasma) results in prevention of most joint damage that constitutes the major
morbidity of the disease. Furthermore, more life-threatening bleeding
episodes, such as intracranial or retroperitoneal bleeds, are also
prevented if a continuous level of factor can be
maintained.1,2 These observations, as well as the
availability of animal models, have contributed to the development of
hemophilia into an important model for treatment of genetic diseases by
gene therapy. The goal of a gene-based treatment is to achieve
long-term expression of F.IX in the circulation of a severe hemophiliac
in order to maintain a continuous supply of F.IX.
Progress toward gene therapy has been substantial over the past 2 years
due to successful studies in a large animal model of hemophilia B using
liver or muscle as a target organ for F.IX gene transfer with
adeno-associated viral (AAV) vectors.3,4 In a recently
developed approach, we have taken advantage of the observation that AAV
vectors efficiently transfer genes to muscle fibers in
vivo.5-7 AAV vectors are single-stranded,
replication-deficient DNA viruses that can be produced in a helper
virus-free system.8 Recombinant AAV contains an expression
cassette flanked by viral inverted terminal repeats and is devoid of
any sequences encoding viral gene products. The viral packaging limit
is approximately 5 kilobases (kb). Gene transfer to nondividing cells
such as muscle fibers is stable and often not associated with cellular
immune responses against transduced fibers (as commonly observed with, for example, adenoviral vectors). Although F.IX is normally made in the
liver, muscle cells are capable of synthesizing biologically active
F.IX.9,10 In previous studies, we achieved sustained expression of therapeutic levels of F.IX in the circulation of experimental animals by intramuscular administration of AAV vectors for
expression of human F.IX in immunodeficient mice or canine F.IX in
hemophilia B dogs (up to 7% of normal human levels in mice and up to
1.4% in dogs).3,11 While these levels of expression are
likely to have a therapeutic effect in a patient with severe hemophilia
B, further improvements in expression are desirable. On a
transcriptional level, this could be achieved by an improved expression
cassette resulting in increased expression per delivered vector
particle. Equally important, the ability to direct expression of a set
amount of F.IX using a lower total dose of vector is an important
therapeutic goal, because biodistribution to sites outside muscle (eg,
to brain or gonads, an undesirable outcome) is a direct function of
dose (in vector genomes) administered. Thus, the use of an improved
vector to achieve higher levels of F.IX expression with lower total
doses of vector will result in an added measure of safety for this gene
therapy approach. In addition, optimization of gene expression on a
transcriptional level will benefit not only a protocol for F.IX, but
other gene therapy strategies that might use muscle-derived expression
of the transgene product (eg, growth hormone, dystrophin, leptin, erythropoietin, insulin-like growth factor-1) as well.
In our initial studies in mice and dogs, we have used the
cytomegalovirus (CMV) immediate early (IE) enhancer/promoter to drive
sustained high levels of F.IX expression in muscle (for at least 2 years postvector administration). In this study, we demonstrate that
expression of muscle-derived human F.IX can be enhanced by a
combination of human Construction of plasmids encoding AAV vectors
Production of AAV vectors
Transduction of myotubes in vitro Murine C2C12 myoblasts were grown in DMEM medium containing 10% fetal bovine serum and antibiotics. Cells were plated in 6-well plates, allowed to reach confluence, and then differentiated to myotubes by exchanging media to DMEM containing 2% horse serum (differentiation media). Cells were incubated for 5 to 6 days in differentiation medium prior to transduction with AAV vector. Immediately prior to addition of vector, fresh differentiation media was added to the cells, and vector was added at multiplicities of infection (MOIs) ranging from 104 to 105 vector genomes/cell. The media was replaced on the following day. All transduction experiments were carried out in duplicate. MOIs were calculated for number of myoblasts used per well for formation of myotubes (2 × 106 myoblasts per confluent 6-well). Supernatants (24-hour) were collected 7 to12 days posttransduction, and myotubes were subsequently harvested for RNA isolation. Concentrations of human F.IX in supernatants were measured by enzyme-linked immunosorbent assay (ELISA) as described.22 Human myotubes derived from primary human myoblasts were a gift from Dr Heiman-Petterson, Hahneman University, Philadelphia, PA. These cells were maintained and transduced as described for differentiated C2C12 myotubes, except that vector was added in OptiMEM medium (Gibco/BRL, Gaithersburg, MD), and the differentiation medium contained 10% horse serum.5 Transduction experiments in other cell types (HEK-293 cells and human hepatoma [HepG2] cells) were carried out in 24-well plates as described.13 These cells were transduced at 50% confluence in DMEM/2% fetal bovine serum medium. Supernatants (24-hour) were harvested 96 hours posttransduction.Analysis of F.IX mRNA in transduced myotubes Total RNA was isolated from transduced myotubes using the RNAzol kit (Tel Test, Friendswood, TX). Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on 1 µg of total RNA using an RT-PCR kit purchased from Applied Biosystems (Foster City, CA) and using primers specific for messenger RNAs (mRNAs) from AAV-hF.IX vectors. PCR products were separated by agarose gel electrophoresis. Duplicate Northern blots were performed by separation of 10 µg of total RNA using agarose gel electrophoresis under denaturing conditions. Following transfer to nylon membranes, 1 blot was hybridized with a radioactively labeled probe specific for the coding region of the human F.IX cDNA and the other to a -actin probe.
Phosphoimage analysis of the blots was performed using a Storm 860 phosphoimager (Molecular Devices, Sunnyvale, CA) and ImageQuaNT
software (Molecular Devices). The -actin signal was used to
normalize the amount of RNA loaded in each lane, and the relative
amounts of human F.IX transcript observed in the various RNA samples
was calculated.
Animal experiments Immunodeficient Rag-1 mice (4-6 weeks old, 20-25 g) were injected intramuscularly into 4 sites of the hind limbs with 1 × 1011 vector genomes of recombinant AAV (n = 4 for each vector) as described previously.11 Rag-1 mice lack recombinase activating gene 1 and are therefore devoid of mature B and T cells and circulating immunoglobulins.23 Quadriceps and tibialis anterior muscles were exposed by a 1-cm incision through the skin while the animals were under general anesthesia from an intraperitoneal injection of ketamine/xylazine. Vector was diluted in 1 × phosphate-buffered saline and slowly injected with a Hamilton syringe (50 µL of vector suspension per quadriceps and 25 µL per tibialis anterior). Incisions were closed with suture. Mice were bled retro-orbitally on a biweekly schedule for 10 to 12 weeks, and F.IX levels in plasma samples were determined by ELISA specific for human F.IX as described.22
A series of AAV vectors for expression of the human F.IX cDNA was
constructed and produced in high titer (greater than or equal to
1012 vector genomes/mL) in HEK-293 cells using an
adenovirus-free production system that is based on the
triple-transfection method (see "Materials and methods"). These
vectors are derived from constructs that were successfully used for
expression of therapeutic levels of human or canine F.IX in mice and
hemophilic dogs.3,11 All expression cassettes were designed
not to exceed the packaging limit of AAV (approximately 5 kb). While
all vectors contain the human F.IX cDNA, they differ in
enhancer/promoter, intron, and polyadenylation sequences (Table
1). When tested for expression of muscle
cell-derived F.IX in murine C2C12 myotubes, all constructs showed
detectable expression and correct splicing when analyzed by RT-PCR
(data not shown) but differed substantially with regard to transcript
levels and secretion of F.IX into culture media (see below). For in
vivo experiments, immunodeficient Rag-1 mice were chosen because
intramuscular administration of vector results in antibody formation
against the nonspecies-specific transgene product (human F.IX) in
immunocompetent mice.11 We have previously shown that F.IX
expression in Rag-1 mice is vector dose-dependent, and we therefore
chose an intermediate dose of 1 × 1011 vector genomes/mouse for intramuscular administration of recombinant AAV
vectors (based on experiments with 3 × 109 to
4 × 1011 AAV-CMV-hF.IX-3 vector
genomes/mouse24).
Expression from the CMV promoter is sufficient to achieve therapeutic levels In our published experiments in mice and dogs, we have used 2 similar expression cassettes. Both use the CMV IE enhancer/promoter but differ in intron and polyadenylation sequences and in the amount of 3'-UT region of the F.IX cDNA. Hence, we decided to compare these vectors (AAV-CMV-hF.IX-2 and -3 as outlined in Table 1) for expression of human F.IX in vitro using murine C2C12 myotubes and in vivo using Rag-1 mice. Based on other investigators' observations on transgene expression, we additionally constructed vectors AAV-CMV-hF.IX-3-bGH and AAV-CMV-hF.IX-8. The first vector contains the bovine growth hormone polyadenylation signal, which has been shown to improve expression in the context of a plasmid DNA vector when substituted for the SV40 polyA (which is used in AAV-CMV-hF.IX-3).25 The second vector contains the entire human F.IX 3'-UT region and polyadenylation signal of the human F.IX message, which was found to increase expression of human F.IX in mice after hepatic gene transfer with an adenoviral vector.26
Muscle-specific promoters fail to produce therapeutic levels of
expression
Improved expression of muscle-derived F.IX by addition
of skeletal actin regulatory sequences to the CMV enhancer/promoter
Previously published studies have established the feasibility of
muscle-directed gene transfer for treatment of hemophilia B. Vector
dose-dependent sustained expression of therapeutic F.IX levels was
demonstrated in a large animal model using intramuscular injection of
an AAV vector.3 Injection of up to
8.5 × 1012 vector genomes of recombinant AAV per
kilogram did not result in detectable toxicity in the hemophilia B dogs
used in that study. However, levels of systemic F.IX expression per
delivered vector particle were clearly less than published for
liver-directed approaches.13,27 Therefore, improvement of
expression levels remains an important issue. In this investigation, we
have shown that addition of a muscle-specific element of the skeletal
actin promoter region to our CMV-driven expression cassette improved
systemic muscle-derived F.IX expression substantially. Systemic levels
of 6% of the normal human F.IX concentration were achieved with a 2- to 4-fold lower vector dose per animal than described previously with a
vector containing the CMV enhancer/promoter alone.11,24
These results further underline the feasibility of muscle as a target
organ for systemic delivery of F.IX or other secreted proteins and give more insight into the use of the CMV enhancer/promoter sequence for
transgene expression. Moreover, the potential use of this expression
cassette in a clinical trial might additionally improve safety of the
protocol because therapeutic levels of expression could be reached with
a lower vector dose per kilogram.
The authors thank Dr Heiman-Patterson for human myotubes, Dr
Hardeman for the HSA DNA, Dr Ordahl for the troponin DNA, and J. H. Liu
and N. Chung for technical assistance.
Submitted September 27, 1999; accepted December 21, 1999.
Supported by National Institutes of Health grants R01 HL53668 and P50
HL54500 to K.A.H, grant F32 HL09397 to J.N.H., a K. Dormandy Trust
grant to P.A.F., and Avigen, Inc, a company in which K.A.H.,
L.B.C., and C.S. hold equity.
Reprints: Katherine A. High, The Children's Hospital of
Philadelphia, Abramson Research Center, Room 310, 34th St and Civic
Center Blvd, Philadelphia, PA 19104; e-mail: high{at}emailchop.edu.
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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Transforming growth factor- |
Top
Abstract
Introduction
-skeletal actin promoter containing at least
1 muscle-specific enhancer and 1 enhancer-like element further improved
muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of
F.IX expression with lower vector doses, thus enhancing efficacy and
safety of the protocol.
(Blood. 2000;95:2536-2542)
-skeletal actin and CMV enhancer/promoter sequences driving transgene expression.
1281 to
overtreatment or optimal model?
Haemophilia.
1998;4:409-412