Dept. of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
Duchenne muscular dystrophy (DMD) is a lethal muscle disease typically caused by
frame-shifting mutations in the DMD gene that impede the synthesis of the
dystrophin protein. In the milder allelic Becker muscular dystrophy (BMD), the
mutations do not disturb the reading frame and a still partly functional protein
is expressed. In this study, we explored a therapeutic strategy aimed at
restoration of the reading frame in DMD patients through targeted modulation of
dystrophin pre-mRNA splicing. Considering that exon 45 is the single most
frequently deleted exon in DMD, whereas exon 45+46 deletions only cause a milder
form of BMD, we designed a system to induce exon 46 skipping. Following
transfection of antisense oligoribonucleotides (AONs) complementary to a
putative splicing regulatory sequence within exon 46, we induced the skipping of
this exon from the transcript in cultured myotubes of both mouse and human
origin. Immunohistochemical and RT-PCR analysis of transfected myotubes from two
unrelated DMD patients carrying an exon 45 deletion, showed that the skipping of
exon 46 to a level of 20% allowed the synthesis of normal amounts of properly
localized dystrophin. Our results demonstrate the therapeutic potential of
antisense oligonucleotides to restore dystrophin expression from the endogenous
gene in DMD patients. This gene therapy strategy should be applicable to a
variety of DMD mutations, as well as other genetic diseases.
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh
Recently, gene delivery in vivo using electroporation has became an area of
greater research interest. The prospect of utilizing electroporation for gene
transfer in vivo is evident based on the success of delivering genes in vitro
and effective delivery of drugs in electrochemotherapy studies. Although the
detailed molecular mechanism of electroporation is still not completely
understood, there is agreement in the literature that the field strength induces
some sort of metastable structure defect in the membrane which serves as a
pathway from the extracellular space to the cell interior. Electroporation is a
threshold phenomenon. The field strength necessary for gene delivery must exceed
a threshold value. Moderate increases in the applied field strength resulting
delivery; however, large increases of the applied field from this threshold
result in tissue damage. Here, we report a novel design of an injectable
electrode, which utilizes comparatively much lower electric field strength to
achieve high transfection efficiency. The hypothesis of this design was based on
two electroporation parameters we defined. One is the Effective Threshold Value
(ETV), which is required for the electrotransfer and is defined by the cell and
its environment. Another one is the Apparent Threshold Value (ATV), which is
defined by the electroporater. ATV is usually much greater than ETV.
Theoretically, the field strength is reversely proportional to the distance from
its source. Therefore, when the field strength is delivered from the
conventional electrodes to the injection site of DNA, the field strength is
dissipated due to the distance between the injection site and the electrode. To
compensate for the loss in the field strength, higher ATV has to be applied
which results in toxicity to the tissue. A logical approach to decrease ATV is
to directly deliver the field strength to the injection site. In this case, ATV
is approximately equal to ETV such that gene transfer can be achieved at lower
field strength. This hypothesis was supported by our data. The maximal level of
gene transfer was achieved at as low as 50 V/cm of field using an injectable
electrode, whereas 200 V/cm was required by using a conventional electrode to
achieve the similar level of gene transfer. Histochmical analysis showed that no
muscle damage was observed using the new electrode. However, severe damage was
induced by using the conventional electrode. We conclude that the new injectable
electrode represents a significant improvement in elctroporative gene transfer
to tissues.
Supported by NIH grants CA74918, DK54225, AR45925, DK44935 and by Muscular
Dystrophy Association of America.
Center
for Pharmacogenetics
School
of Pharmacy
University
of Pittsburgh
Gene delivery to skeletal muscle is a promising method for the treatment of
muscular diseases as well as for the production of therapeutic proteins.
Electroporation has been proved to be an efficient method for gene delivery to
muscle. In this study, we report a significant enhancement of
electroporation-mediated gene expression in skeletal muscle by injecting plasmid
DNA mixed with hyaluronidase before the application of electroporation.
Luciferase gene expression was about 10 fold higher in mice injected with DNA
and hyaluronidase, compared with mice injected with DNA alone. Hyaluronic acid
showed no effect on promoting gene expression. Moreover, the enhancement of gene
expression required enzymatic activity, because co-injection of DNA with
denatured hyaluronidase or bovine serum albumin had no effect on gene expression.
More importantly, the area of the muscle that expressed transgene product was
increased very significantly in mice treated with hyaluronidase. More than 40%
of muscle fibers expressed b-galactosidase when mice were co-injected with a
plasmid containing LacZ gene and hyaluronidase. On the other hand, less than 5%
of muscle fibers expressed b-galactosidase in mice injected with plasmid alone.
Supported by NIH grant AR45925 and by Muscular Dystrophy Association of America.
Harvard
Medical School, Boston, MA 02114
Duke
University Medical Center, Durham, NC 27710
University
of Miami School of Medicine, Miami, FL 33136
To understand the functions of specific proteins in vestibular sensory hair
cells, it is necessary to add or inactivate those proteins in a system where
their physiological effects can be studied both in vitro and in vivo.
The loss of vestibular hair cells, or the loss of their capacity to transduce
vestibular stimuli, is the cause of many balance disorders. In order to develop
viral vectors that could allow an understanding of specific protein functions,
and for the potential treatment of balance disorders, recombinant adenovirus
vectors were used to infect vestibular hair cells of the guinea pig inner ear in
vivo, and cultured mouse vestibular endorgans. Recombinant adenovirus vectors
[E1-], [E1-, E2b-] containing either green fluorescent protein (GFP) or lacZ as
a reporter gene were used in this study. [E1-] adenovirus vectors (105-108 pfu/ml)
were found to be toxic to cultured vestibular hair cells, such that by 48 hrs
after infection, there were no intact stereocilia bundles. On the other hand,
[E1-, E2b-] adneovirus vectors (108 pfu/ml) were not toxic to cultured
vestibular hair cell stereocilia up to 2 weeks post-infection. The [E1-, E2b-]
adenovirus vector (5 x 108 pfu/infused ear) was also capable of driving
transgene expression to vestibular hair cells in vivo.
The development of a viral vector that infects hair cells of the vestibular
system in vivo and in vitro without ototoxicity, is crucial for gene replacement
therapy to be effectively used to treat balance disorders.
This work was supported by grants from the Public Health Service (DC03086,
DK52925, DC00304), Muscular Dystrophy Association, USA, and funds from Glaser
Foundation and the University of Miami's Chandler Chair.
INSERM U491, Fac de Medecine, 13385 Marseille Cedex5 France
We have constructed 4 mouse lines by introducing a human YAC containing the
human gene PMP22 into the murine oocyte (Huxley et al, 1996; 1998). This gene
has been involved in the Charcot-Marie-Tooth type 1A disorder, the most frequent
inherited peripheral neuropathy (1/3000). Patients suffering from this disease
present a duplication (1.5 Mb) of the proximal short arm of chromosome 17, which
includes the PMP22 gene. Two transgenic lines exhibit a peripheral neuropathy
transmitted as an autosomal dominant trait, and clinical signs of transgenic
mice are very similar to the human disorder. In a further step, we have explore
the model, and these data clearly demonstrate the following points: - The
neuropathic phenotype linked to PMP22 overexpression, appears by a non linear
mechanism. - The peripheral neuropathy is not a demyelinating neuropathy, but a
dysmyelinating. – Disorders linked to PMP22 overexpression are in fact
pathologies of the differentiation of Schwann cells.
In a last step, we used these CMT mice, as a preclinical model, in order to test
therapeutical approaches (we may note that it is a human YAC we have introduced
in the mouse). In a first attempt, we have tested a battery of molecules
susceptible to act on peripheral nerve and myelination. We tested transgenic
mice from the same litter either with an active molecule or a placebo. We choose,
as inclusion criteria, only males (more severely affected than females) of an
age of two month (clinical signs appear about one month after birth in this line).
Muscular capacity has been evaluated using a rotarod test, and at the end of the
test, by measuring the percentage of nerve fibers with myelin on sciatic
histological slices. Our two first set of experiments was very promising, as one
molecule is reverting the phenotype with a great efficiency, allowing us to
envisage the first clinical trials on CMT1A patients.
Children’s
Institute for Surgical Science, Children’s Hospital of Philadelphia,
Philadelphia, PA 19104
Institute
for Human Gene Therapy, Philadelphia, PA 19104
The
Salk Institute, La Jolla, CA 92037
In utero gene therapy is a promising technique to allow long-term correction of
congenital genetic diseases. The early gestational fetal environment may provide
unique biological and practical advantages for gene therapy in that actively
proliferating stem cell compartments are accessible. Given the potential of
lentiviral vectors to transduce stem cells, we sought to define the biology of
these vectors following in utero administration in pre-immune mouse fetuses.
Balb/c fetuses at 14-15 days’ gestation were injected with lentiviral vectors
carrying the transgene lacZ under the control of the human CMV promoter in one
of three routes: intraperitoneal (IP), intrahepatic (IH), or intramuscular (IM).
The lentiviral vectors were pseudotyped with either VSV-G, Mokola, or Ebola
virus proteins at a titer of 1e9 or 1e10 infectious units (IU)/ml (VSV-G), 2e8
IU/ml (Mokola) or 3e8 IU/ml (Ebola). 5 ul of virus was introduced directly into
each fetus using a pulled glass micropipette. The pups were harvested at time
points between 5 days to 19 weeks following injection. Detailed histologic
assessment was performed by analyzing 1-2 um plastic sections of X-gal stained
tissues. Mortality following IH injection was compared to control pups injected
with PBS alone.
Survival to birth following direct intrahepatic injection was 53% with PBS, 42%
with VSV-G, 56% with Ebola, and 50% with Mokola pseudotyped virus. IH injected
animals showed gene expression in liver hepatocytes and peri-ductular cells,
with the comparative efficiency of each pseudotype being Mokola>VSV-G>Ebola.
IM injection resulted in transduction of myocytes, satellite cells,
neuromuscular spindles, and perivascular cells, with greatest efficiency from
the Mokola-pseudotyped vector. Systemic spread of the virus was documented by
transduction of cardiomyocytes after IP, IH, and IM injection with the
VSV-G-pseudotyped vector and after IH and IM injection with the
Mokola-pseudotyped vector. Expression has persisted in liver, heart, and muscle
up to the time of last analysis at 19 weeks following injection without evidence
of an inflammatory infiltrate in the transduced tissues.
Long-term gene expression in multiple tissues can be achieved after in utero
administration of lentiviral vectors with minimal apparent toxicity. The
efficiency and distribution of transduction after in utero administration was
highly dependent upon the route of administration and the pseudotype of vector
used. Our findings of long term transduction of skeletal myocytes, satellite
cells, cardiomyocytes, and hepatocytes after in utero administration suggests a
novel strategy for the treatment of congenital muscular dystrophies,
coagulopathies, and inborn errors of metabolism.
Department
of Neurology, University of Washington, School of Medicine, Seattle, WA
98115-7720
Cellular
and Molecular Biology Program, University of Michigan Medical School, Ann Arbor,
MI 48109
Department
of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109
The goal of this study was to evaluate the correction of muscular dystrophy in a
mouse model (the mdx mouse) using human dystrophin transgenes (micro-dystrophins)
that contain a series of large deletions sufficient enough to allow packaging
into the small (~5 kb cloning capacity) gene therapy vector, adeno-associated
virus (AAV). AAV is the most efficient skeletal muscle delivery vector that does
not generate a significant immune response, yet it is incapable of accommodating
a full-length dystrophin cDNA (14 kb). The creation of functional
micro-dystrophin expression vectors of ~4.6 kb in size is therefore essential to
develop a successful AAV-mediated gene therapy strategy for Duchenne muscular
dystrophy (DMD).
DMD is an X-linked, recessive disorder caused by defects in the dystrophin gene.
It is the most common form of muscular dystrophy and is characterized by
progressive muscular degeneration and weakness, leading to death in the early
twenties. The full-length dystrophin gene product is a 427 kDa, multi-domain
protein that is localized to the subsarcolemmal membrane and is believed to
provide a critical link between the actin cytoskeleton and the extracellular
matrix via interactions with the dystrophin associated protein complex, or DAP.
The binding motifs of the dystrophin molecule that are necessary for assembly of
the DAP complex and subsequent stabilization of the muscle membrane are located
near the N and C termini, while the majority of the protein lies in the central
rod domain, which is comprised of 24 spectrin-like, triple helical repeats
spaced by four non-repeat hinge regions. Natural in-frame deletions in this
region often result in a less severe form of muscular dystrophy, called Becker
muscular dystrophy.
A recent paper in PNAS vol. 97 (Wang et al., 2000) demonstrated that
dystrophin molecules with large C terminal and rod domain deletions (maintaining
5-6 repeats) can be delivered by AAV and act to prevent the development of
muscular dystrophy in young mdx mice. Although this work suggested that
protection can be achieved in limb muscle by a micro-dystrophin molecule, it
left unanswered whether these dystrophins possessed the functional capacity to
protect against the harsh forces associated with damaging eccentric contractions,
and whether this protection would extend to the mdx diaphragm, which is
the most severely affected muscle in mice and humans.
We generated transgenic mice expressing several different micro-dystrophin
clones containing only four repeats, and analyzed their ability to correct
muscular dystrophy by both morphological and physiological assays. Two of three
transgenes significantly reduced the amount of dystrophy observed in the mdx
limb and diaphragm muscles. The best transgene produced muscle with wild-type
levels of central nuclei, Evans Blue dye exclusion, resistance to
contraction-induced injury, and the ability to run distances on a treadmill. In
addition, no areas of fibrosis nor monocyte infiltration were observed. We are
currently evaluating these micro-dystrophin clones in adult animals using AAV
vectors.
Human
Genetics Laboratory, Laval University Hospital Center, Sainte-Foy, Canada.
Institut
fur Zellbiologie, Universitat Witten/Herdecke, Stockumer Strasse 10, D-58448
Witten, Germany.
Many human gene therapies require the use of non-immunogenic vectors for stable
gene expression. Stable gene expression can be mediated by an integrated or
episomal DNA sequence. Episomal vectors offer the advantage of not modifying the
chromosomes, but most episomal vectors require the expression of viral genes. A
plasmid with a Scaffold/Matrix-Attached Region from the human interferon-
gene and a SV40 replication origin has previously been described as mitotically
stable in chinese hamster ovary cells. We introduced the EGFP gene into this
plasmid and transfected it into primary human myoblasts. This plasmid replicated
episomally for more than two months under selective pressure. Intact episomal
plasmid DNA could be recovered and used to transform E. coli cells. This plasmid
is thus very interesting because it permits efficient episomal replication in
primary human cells without requiring the expression of any viral gene. This
plasmid would be particularly usefull in our field of autologous myoblast
transplantation for Duchenne muscular dystrophy since human myoblasts are
difficult to transfect with large plasmids (containing the dystrophin cDNA) and
the stability of the DNA introduced into a few cells is desired. Research is
underway to characterize the segregation efficiency of this plasmid, experiment
alternate replication origins and introduce the dystrophin cDNA.
Department of Molecular Genetics and Biochemistry, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA15261
Duchenne Muscular dystrophy (DMD) is one of the most common, disabling and
lethal genetic diseases afflicting one of every 3500 boys. The patients suffer
from progressive muscle degeneration and weakness, become wheelchair-bound in
their early teens and die prematurely in their late teens and early twenties. No
effective treatment is currently available for DMD. The etiological cause of the
disease is the mutation in the dystrophin gene. The lack of a functional
dystrophin protein in the striatal muscle causes instability of the muscle cell
membrane and results in persistent damage by muscle contraction. Because DMD is
X-linked and genetically recessive, it has been considered as a good candidate
for gene replacement therapy. Adeno-associated viral (AAV) vector has proven to
be the most promising vector system, which renders efficient and stable gene
delivery in muscle tissues without triggering cellular immune responses to the
transgene products. Previously, a major hurdle preventing the use of AAV vectors
for DMD gene therapy is the small capacity of AAV (5 kb) and the immense size of
the dystrophin gene, which spans over 3 million basepairs on the X-chromosome
and has a cDNA of 14 kb. Recently, we have overcome this hurdle by selectively
truncating the non-essential regions of the human dystrophin and created novel
minidystrophin genes that can be packaged into AAV vectors. Injection of
AAV-mini-dystrophin vectors into the hindleg muscles of mdx mice, a DMD animal
model, effectively restored the missing dystrophin protein along with the
dystrophin associated protein complex onto the muscle cell membrane. Moreover,
AAV vector treatment alleviated the muscle histopathological conditions and
protected the muscle cell membrane integrity for over a year.
In this report, we show that AAV-mini-dystrophin vector treatment in the mdx
mouse muscle is also able to improve the contractile forces. To test the effect
of muscle contractile force recovery, adult male mdx mice at the age of 1.5 to
2.5 months were injected with AAV-mini-dystrophin on the tibialis anterior (TA)
muscle of the left legs, while the right legs remained untreated as the internal
control. At the time of vector treatment, the muscle tissues already went
through massive waves of degeneration and regeneration, and exhibited overt
signs of pathology. At 7 months after vector injection, the AAV treated and
untreated muscles were isolated for in vitro contractile force tests. The
experimental results showed that 1) the specific peak force was slightly higher
after vector treatment (P=4.08±0.53N/cm2, n=8) when compared to the untreated
control muscle (P=3.75±0.65N/cm2, n=8); 2) the specific tetanic force was 30%
higher in the vector treated muscles (P=13.05±2.00N/cm2, n=8) than the
untreated control muscles (P=10.30±1.56N/cm2, n=8). 3) during the eccentric
lengthening tests, the vector treated muscles retained significantly higher
contractile forces than the untreated muscles. Immunofluorescent staining of
minidystrophin in the AAV vector treated muscles after in vitro force
measurement showed 20% to 50% of the myofibers positive for dystrophin
expression. Measurement of the age-matched normal male mouse TA muscle
contractile forces is in progress. In summary, this study demonstrated that the
mini-dystrophin can recover the contractile force deficits in the adult
dystrophic muscles of the mdx mice, therefore, further supporting the
therapeutic functionality of the minigenes delivered by AAV vectors.
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada
Production of Adeno-associated Virus Vectors by Cre-loxP Recombination
System*
Qingshan Teng, Josephine Nalbantoglu, George Karpati, Paul C. Holland
Department of Neurology and Neurosurgery, Montreal Neurological Institute,
McGill University, Montreal, Quebec, Canada
Adeno-associated viruses are attractive vectors for the delivery of foreign
genes into mammalian cells for gene therapy. The production of AAV vectors
usually involves transfection of 293A cells with an AAV plasmid which contains a
foreign gene expression cassette flanked by AAV terminal repeat sequences, the
AAV helper functions rep and cap, and adenovirus genes E1A, E1B, E2A, E4 and VA
RNA. We describe a novel approach to produce AAV vectors by using the
prokaryotic Cre-loxP recombination system. Cells that stably express the
Cre recombinase (293Cre4) were cotransfected with AAV vector plasmids and AAV
helper plasmids containing rep and cap, followed by infection with AdLC8cluc
helper viruses. The AdLC8cluc helper viruses have their packaging signals
flanked by loxP sequences, rendering them unpackageable in 293Cre4 cells.
However, they retain the ability to replicate and provide all the functions
necessary for packaging of AAV vectors. We tested this strategy to produce
AAV-LacZ, a recombinant AAV containing the reporter gene E.coli LacZ. 293Cre4
cells were cotransfected with the AAV vector AAV-LacZ and AAV helper plasmid
ACG2, followed by infection with AdLC8cluc helper viruses. The cells were
harvested 72 hours post-infection. AAV-LacZ virus was titrated on 293A cells by
staining for the LacZ product, beta-galactosidase. AAV vectors produced by this
scheme yielded 6.0 x 107 blue cell forming units/1.2 x 106
cells. Contamination with adenovirus was estimated less than one in 104
AAV functional particles. The use of such packaging deficient helper adenovirus
may circumvent some of the problems associated with the large-scale production
of recombinant AAV.
*Supported by Muscular Dystrophy Association (USA).
Department
of Physiology, University of Michigan, Ann Arbor
Department
of Neurology, University of Washington, Seattle.
Cellular
and Molecular Biology Program, University of Michigan, Ann Arbor
Department
of Human Genetics, University of Michigan, Ann Arbor
Duchenne muscular dystrophy (DMD), a degenerative lethal muscle disorder that
affects one in 3500 newborn males, is caused by mutations in the dystrophin
gene. Adenoviral vectors are promising tools that may be used to deliver the
dystrophin gene to affected muscle to alleviate the symptoms of DMD. First
generation adenoviral vectors, however, elicit a potent immune response that
prevents long term transgene expression, and are limited in cloning capacity.
Therefore, we have constructed "gutted" adenoviral vectors that are
devoid of all viral genes and contain a full-length human (HDys) or mouse (MDys)
dystrophin cDNA driven by a muscle specific promoter. Preliminary five day
experiments in six week old mdxmouse tibilais anterior (TA) muscles
injected with either viral construct showed high levels of dystrophin protein
expression and exclusion of the vital dye Evans Blue, demonstrating improved
muscle sarcolemma integrity in the presence of dystrophin. Injections into adult
(11-13 mo.) mdxmouse TA muscles were performed and a novel eccentric (lengthening)
contraction protocol was used to stringently test for restored muscle function.
This protocol demonstrates marked differences between mdxand wild type TA
muscle susceptibility to contraction-induced injury; after one injury-inducing
lengthening contraction, wild type TA muscle force is reduced by 10%, while mdxmuscles
demonstrate a 72% force loss. After five days, HDys injected muscles were
significantly protected from injury and showed high levels of dystrophin
expression in contrast to the sham injected contralateral control muscles.
However, 25 days after injection, significant decreases in force generating
capacity were detected. This loss of force generation was similar to that
observed after injection of a first generation virus containing a ß-galactosidase
transgene (CNß), suggesting that an immune response against human dystrophin
protein was elicited. MDys injected muscles demonstrated high levels of
dystrophin protein expression and no functional defects after 25 days. In
addition, MDys injected muscles were able to produce 62% of wild type force
levels after one lengthening contraction that, in contrast, reduced mdxand
sham injected control muscles to 38 and 29% of wild type force, respectively.
FACS sorting of injected muscles revealed CD4+ and CD8+ cells present in all
virus-injected muscles. MDys injected muscles showed the least amount of
infiltration, HDys injected muscles showed approximately 1/3 more immune cells
than MDys injected muscles, and CNß injected muscles displayed the highest
amount of immune cell infiltration. Collectively, these data demonstrate 1)
efficient gutted adenoviral vectors are successful in transducing dystrophin in
TA muscles of one year old mdxmice, 2) evidence for an immune response
against the human dystrophin protein that causes atrophy and loss of function,
and 3) a partial functional correction of adult mdxmouse muscle after
delivery of full-length mouse dystrophin with a gutted adenoviral vector.
1 Department of Molecular genetics and Biochemistry
2 Department of Orthopedic Surgery, and 3 Department of Surgery
University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261
Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the
d-sacroglycan (SG) gene. Previously, by directly injecting an adeno-associated
virus (AAV) vector carrying human d-sacroglycan gene into the skeletal muscle,
we have shown successful genetic and biochemical rescue and muscle force
recovery in the Bio.14 hamster, a homologous animal model for LGMD 2F(J. Li et
al, Gene Therapy 6:74; X. Xiao et al, J.Virology, 74:1438). Like the human
patients, the Bio14.6 hamsters also suffer from cardiac myopathy and die
prematurely. Hence, the animal has also been widely used as a model of
congestive heart failure. In this report, using a heterotopic heart transplant
model, we show high-efficiency and long-term intra-cardiac gene transfer by AAV
vectors carrying either the reporter Lac-Z gene or the therapeutic d-sarcoglycan
gene.
In this study, the 2-month old dystrophic Bio14.6 hamsters and healthy control
F1B hamsters were subjected to the heterotopic heart transplantation with the
syngeneic donor hearts, which had a single perfusion ex vivo with the AAV
vectors via the aorta. In detail, the vectors were delivered into hamster
coronary circulation by perfusion through aorta after the heart was bathed in
cold Ringer’s buffer for 30 minutes before transplanted into recipient
hamsters. For each donor heart, 0.5 ml (5x1012 viral particles/ml) of AAV-LacZ
or AAV-SG was used for the ex vivo perfusion. To examine the gene transfer
efficiency, the recipient hamsters were sacrificed at four-month and one-year
after heart transplantation. Cryo-thin-sections (10 mm) of the heart tissue were
subjected to immunofluorescent staining for d-SG expression and X-gal staining
for Lac-Z expression. Our experiments demonstrated 1) highly-efficient AAV-LacZ
vector transduction in over 90% of the cardiac muscle tissues for one year in
the F1B normal hamsters; 2) highly efficient AAV-SG vector transduction in 80%
to 90% of the cardiac muscle in the Bio 14.6 hamster heart for four month (the
duration of the experiment). 3) no immune rejection of the transplanted hearts
that expressed either E. coli LacZ or the human SG proteins. Our studies suggest
that the Bio 14 hamster is an excellent LGMD animal model for gene therapy, and
that AAV is the vector of choice for the long-term intra-cardiac gene therapy
strategy. Further studies are needed to evaluate the physiology and
histopathology improvement after AAV vector-mediated gene therapy in the
cardiomyopathic hamster hearts.
Montreal Neurological Institute, McGill University, Montreal, QC, Canada
Duchenne muscular dystrophy (DMD) is a relatively common inherited neuromuscular
disease caused by deficiency of dystrophin. A potential and useful treatment of
DMD by gene therapy will require the efficient and prolonged expression of
dystrophin in most muscles of the body. Helper-dependent adenovirus (HDAd), also
known as gutted adenovirus (Ad), are deleted of most viral genes and have
permitted the safe an long term expression of various transgenes in several
tissues. We have previously constructed a HDAd encoding the full-length
dystrophin regulated by the cytomegalovirus (CMV) promoter (HDAdCMVDysFl), and
have observed a weak dystrophin expression after gene transfer with this vector
in muscle. In the present study, in an attempt to increase dystrophin expression,
we have constructed a HDAd encoding a single copy of dystrophin regulated by the
strong hybrid CMV enhancer/-actin
promoter (CB) (HDAdCBDys) and a HDAd encoding two copies of dystrophin regulated
by the CB promoter (HDAdCBDysDys). To evaluate dystrophin expression from these
vectors, the tibialis anterior muscle of neonatal mdx mice (the mouse model for
DMD) was injected with HDAdCMVDysFl, HDAdCBDys or HDAdCBDysDys at a titer
ranging from 1.0 to 2.0 X 1012 particles/ml, and the number of
dystrophin positive fibers was determined by immunohistochemistry. At the
earliest time point investigated (10 days post-injection), the average number of
dystrophin positive fibers was higher after gene transfer with HDAdCBDysDys (mean
= 240) compared to HDAdCBDys (mean = 70) and to HDAdCMVDysFl (mean = 20). At 30
days post-injection, the number of dystrophin positive fibers remained the same
after gene transfer with HDAdCBDys and HDAdCBDysDys, but in some muscles, nearly
100% of fibers were dystrophin positive. In conclusion, in the backbone of a
HDAd the CB promoter confers a much stronger dystrophin expression than the CMV
promoter, which persists unabated for at least 30 days without any evidence of
inflammation. The use of the double dystrophin expression cassette in some
muscles dramatically increased the amount of dystrophin produced by the HDAd.
Montreal
Neurological Institute, McGill University, Montreal, QC, Canada
Department
of Genetics, Duke University Medical Center, Durham, NC, USA
Respiratory
Division, McGill University Health Center and Meakins-Christie Laboratories,
McGill University, Montreal, QC, Canada
Biotechnology
Research Institute, the National Reasearch Council of Canada, Montreal, QC,
Canada
Helper-dependent adenovirus (HDAd) vectors lack all or most of adenoviral (Ad)
genes. They hold great promise for gene therapy of diseases such as Duchenne
muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted
Ad (first generation Ad or FGAd) and can carry the full-length dystrophin cDNA
(12 kb). We have previously observed that the transgene expression was less
efficient in cell culture and in the muscle in vivo after gene transfer with a
HDAd encoding the full-length dystrophin cDNA (HDAdCMVDysFl) compared to a FGAd
encoding the mini-dystrophin cDNA (FGAdCMV-dys). The two dystrophin transgenes
were regulated by the same cytomegalovirus (CMV) promoter. We have also
demonstrated that Ad gene products provided in trans by a FGAd could increase
the amount of dystrophin produced by HDAdCMVDysFl in cell culture as well as in
muscle. In the present study, we have tried to characterize the Ad gene products
responsible for this enhancement. Cultures of HeLa cells and C2C12 myotubes were
infected with HDAdCMVDysFl alone, or mixed with an E1/E4-deleted Ad (Ad5/E1(-gal)E4),
or with a HDAd encoding the complete E4 region (HDAdMCKDysE4), or with an
E1/E3-deleted Ad lacking the expression of the terminal protein and polymerase (AdLacZpp).
The tibialis anterior muscle of neonatal mdx mice (the mouse model for DMD) was
also injected with the same vector preparations. Dystrophin expression was
monitored by western blot analysis at 4 days post-infection (cell culture) and
by immunohistochemistry at 10 days post-injection (mdx muscle). No dystrophin
enhancement occurred in the presence of (Ad5/E1(-gal)E4)
indicating that E4 was required to increase dystrophin expression from
HDAdCMVDysFl in vitro and in vivo. Enhanced dystrophin expression was observed
in cell culture but not in mdx muscle in the presence of HDAdMCKDysE4 and AdLacZpp.
Thus, E4 is sufficient to increase dystrophin expression of HDAdCMVDysFl in
vitro but not in the muscle in vivo where additional gene products encoded by E2
are needed. The inclusion of E4 and E2 in the backbone of a HDAd may be required
to produce sufficient dystrophin to mitigate the pathology of DMD after gene
transfer with a HDAd.
Departments
of Molecular Genetics & Biochemistry, and Human Genetics, University of
Pittsburgh, Pittsburgh, PA
Department
of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA
Department
of Molecular Genetics & Biochemistry, University of Pittsburgh, Pittsburgh,
PA
Duchenne Muscular Dystrophy (DMD) is a genetic muscular disease caused by loss
of dystrophin protein, an important component of the cytoskeleton of myofibers.
Dystrophin deficiency results in loss of the connection between the sarcolemmal
cytoskeleton and the extracellular matrix, leading to progressive muscle
weakness and death in young ages in DMD patients. Transplantation of myoblasts
has been shown to be effective in restoring normal dystrophin expression in
dystrophic muscle. Although primary muscle-derived stem cells isolated from
hindlimb muscle of mdx mice have displayed a better survival rate than primary
myoblasts after transplantation, all cell populations that we have tested showed
significant loss 5 days post-transplantation. However, when we genetically
engineered primary muscle-derived cells to express IL-Ra, an anti-inflammatory
protein, the early loss of the injected cells was significantly reduced 48 hr
after injection. To improve the in vivo survival of transplanted myoblasts, we
have compared genes encoding immunosuppressive and anti-inflammatory proteins.
For this study, primary muscle-derived stem cells (PP6) isolated from hindlimb
muscle of mdx mice were used. Retroviral vectors have been constructed with
immunosuppressive genes including TGF-ß, vIL-10, IL-Ira, and soluble IL-1
receptor Type II-Ig (sIL-IRII-Ig). Stable selected populations of primary
myoblasts expressing the different immunosuppressive gene products were
generated by retroviral infection. The cells were transduced with adenovirus
carrying the LacZ reporter gene 24 hr. before the transplantation into the
gastrocnemius muscle of mdx mice. The animals were sacrificed on day 5 and 21
post-transplantation and the hindlimb muscles removed, sectioned and stained for
LacZ expression. The LacZ reporter-gene expression was detected in all groups
including the control group on day 5. However, a significant loss of LacZ
expression was observed on day 21. We could not detect any LacZ-positive
myofibers in the non-modified control group. In contrast, we have observed
varying levels of LacZ expression in the other groups on day 21. In particular,
36 % and 35% of the myoblasts producing sIL-IRII-Ig and vIL-10 respectively
survived and continued to express LacZ for 21 days compared to the number of
LacZ-positive myofibers on day 5 of the same group. Ratios of 12% and 1.5% were
observed for the genetically engineered myoblasts producing IL-IRa and TGF-ß
respectively. Comparing the various immunosuppressive genes, sIL-IRII-Ig and
vIL-10 are the most promising to facilitate prolonged survival of myoblasts
following intra-muscular transplantation. This approach may be help in
developing therapeutic strategies in DMD.
University of Pittsburgh, Dpt. Neurology, 546 S-BST Pittsburgh, PA15213
Adenoviruses have a number of features that suit them particularly well as gene
therapy vectors for skeletal muscle. In spite of these advantages, adenoviral
vector-mediated gene transfer to muscle still faces serious preclinical,
technical hurdles. Muscle tissue is widespread, comprising roughly 40% of the
body's mass. Therefore, if a therapy is to be effective against an inherited
muscle disease such as Duchenne Muscular Dystrophy that affects widely
distributed muscles, delivery of the therapy must take advantage of the vascular
system. Since the native attachment receptor for adenovirus,
Coxsackievirus-Adenovirus receptor, is expressed at a low level in mature muscle
and at a high level in other tissues such as liver, systemic vascular delivery
of adenovirus to muscle will likely require targeting to a muscle-specific
molecule. We therefore seek to identify and characterize muscle-specific ligands
that could be used for targeting adenoviral vectors to fetal and adult muscle.
To generate myogenic reagents that will be useful for the development of muscle
targeting, we are characterizing myoblasts derived from both fetal and adult
murine muscle. We have successfully isolated myoblasts from fetal (embryonic day
16) and post-natal muscle, demonstrating proliferation as mononuclear myoblasts,
fusion in vitro to form multinuclear myotubes, and expression of myogenic
markers. To characterize adenoviral vector-mediated gene delivery, we infected
both fetal and post-natal myoblasts with a first generation adenovirus (E1, E3
deleted) carrying the lacZ reporter gene driven by the CMV promoter over a range
of multiplicities of infection. We have found that reporter gene expression is
approximately 20-fold higher in fetal myoblasts as compared with post-natal
myoblasts.
Successful targeting of adenoviral vectors to either fetal or post-natal muscle
will require both an understanding of mechanisms of un-targeted transduction and
the identification of candidate muscle-specific molecules. Our studies will
compare fetal and post-natal myogenic cells for the expression of cell-surface
molecules, including CAR, integrins and muscle-specific molecules, and markers
of myogenic differentiation. These myogenic cells can also be used for the
investigation of adenoviral vector targeting to known muscle-specific cell
surface molecules and for the identification of novel targeting ligands.
Department of Child Development, Kumamoto University School of Medicine.
Intro: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle disease
caused by the deficiency of dystrophin. Various approaches, including cell
transplantation and gene therapy, have been carried out in order to restore the
missing dystrophin gene in DMD patients. Although gene therapy for DMD has shown
some promise, viral vectors are limited to the injected area and we believe that
DMD is a systemic disease. Conversely, muscle and/or bone marrow derived stem
cell transplantation to dystrophic muscle by systemic, intravenous injections
have succeeded for the delivery of dystrophin (1, 2). Furthermore, embryonic
stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos are
totipotent cells and have the capacity to differentiate into all tissues and
cell types. ES cells induced to differentiate in vitro gave rise to many cell
types including hematopoietic precursors, cardiac and skeletal muscle,
endothelium, and neural cells. Previously, it was impossible to limit
differentiation of the ES cells to the muscle lineage. However, we were capable
of inducing differentiation of these ES cells to muscle cells only by
transducing them with an Adenovirus encoding for MyoD, a transcription factor
used for muscle cell differentiation (3).
Methods: Following the removal of feeder cells (primary embryo fibroblasts), the
ES cells were infected in vitro with Adenovirus (AdMyoD) at a multiplicity of
infection (MOI) of 50 and cultured in ES medium containing Leukemia inhibitory
factor (LIF) and 15% FCS. One day following the AdMyoD infection, LIF was
removed from the culture medium and the concentration of FSC was changed to 2%
in order to induce differentiation of cells into myotubes. At 7 days
post-infection, the differentiated ES cells were assessed for desmin expression
by immunofluorescence using a monoclonal mouse anti-desmin antibody (1:200)
followed by a FITC-goat anti-mouse IgG (1:50).
Results: Some of the ES cells began to fuse and form myotubes at 3 days
post-infection. Many myotube-like cells were detected at 7 days post-infection.
The immunofluorescent staining showed that almost all fused (myotubes) and
non-fused cells were desmin positive.
Discussion: To achieve success for DMD therapy, differentiation of ES cells must
be limited to the muscle lineage. Our results indicated that ES cells are easily
transformed to muscle cells upon stimulation with MyoD. This data suggests that
further development in ES cell technology could be useful for clinic treatments
of DMD.
1.Gussoni, E., Y. Soneoka, C. D. Strickland et al. Dystrophin expression in the
mdx mouse restored by stem cell transplantation. Nature. 401 390-394 1999
2.Ferrari, G., G.C.D. Angelis, M. Coletta, et al. Muscle Regeneration by Bone
Marrow-Derived Myogenic Progenitors Science 279 1528-1530 1998
3.D. F. Pinney, Pearson-W SH, S. F.Konieczny, et al. Myogenic lineage
determination and differentiation: evidence for a regulatory gene pathway.
Cell.53 781- 93 1988.
Faculty of Medicine, University of Geneva, Switzerland
Lentiviral vectors can transduce cells irrespective of their proliferating
status. We have taken advantage of this property to immortalize conditionally
human muscle satellite cells through the use of LoxP-lentivectors expressing
growth promoting and antisenescence genes. Primary myoblasts from control
individuals and from patients suffering from Duchenne or Steinert muscular
dystrophies could be immortalized with gene cocktails that included the SV40
large T antigen (TAg) or the INK4a locus antagonist bmi-1, with or without
telomerase (hTERT). The resulting cell lines were not tumorigenic in nude mice,
but grew continuously in culture. TAg-expressing clones showed a great degree of
genetic instability and exhibited aberrant morphological features. In contrast,
myoblasts immortalized with bmi-1 retained a normal karyotype, grew in "proliferation"
but not in "differentiation" medium, and conserved more faithfully the
phenotypic characteristics of primary myoblasts. However, only rare clones
demonstrated a clear fusogenic potential, even once arrested by Cre-mediated
excision of the transgenes. These results further validate the use of lentiviral
vectors for the conditional immortalization of human primary cells, and also
demonstrate that the composition of the immortalization cocktail greatly
influences the potential utility of the resulting cells.
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry
Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder caused by a
defect in the DMD gene. Utrophin is an autosomal homologue of the DMD
gene product, dystrophin, and over-expression of utrophin is expected to
compensate the defect of dystrophin.
We have previously reported that utrophin was up-regulated at the sarcolemma of
dystrophin-deficient mdx mice, when a -galactosidase-expressing
adenovirus vector, AxCALacZ was injected into the skeletal muscle. Moreover,
up-regulated utrophin mitigated dystrophic phenotypes. Since this
over-expression was inhibited by immunosuppression with FK-506 or anti-CD4
antibody treatment, we concluded that the immune response against AxCALacZ
resulted in the up-regulation of utrophin (Yamamoto et al., Hum Gene Ther
11, 669-680, 2000).
To apply over-expression of utrophin to treatment of DMD, we need to know the
molecular mechanism of the up-regulation. We hypothesize that some cytokines
would be produced by infiltrating cells and up-regulate the expression of
utrophin in muscle fibers. To clarify which cytokine is involved in the
up-regulation of utrophin, we daily injected candidates of cytokines into
tibialis anterior muscles of 2-week-old mdx or normal C57Bl/10 mice for
five days. Two, five and eight days after the final injection, we evaluated
utrophin expression by immunohistochemistry.
The experiment with anti-CD4+ treatment suggests that CD4+
T cells were mainly involved in the up-regulation of utrophin. Hence, we firstly
focused on cytokines involved in differentiation or maturation of CD4+
T cells. Interferon-
(IFN-)
and Interleukin-12 (IL-12) play an important role in differentiation of CD4+
cells into Th1 subset. We injected IFN-
at a dose of 400 U/day or 1000 U/day, and IL-12 at a dose of 80 ng/day or 800 ng/day,
however these cytokines showed no effects on utrophin expression. IL-4 is known
to be involved in differentiation of CD4+ cells into Th2 subset.
Therefore, we next tried IL-4 at a dose of 80 ng/day or 800 ng/day, but it also
failed to up-regulate utrophin.
IL-6 is expressed in tissues infected with adenovirus vector. IL-6 is also
detected in the muscle of patients with inflammatory myopathies or in
regenerating muscle, where utrophin is up-regulated. Therefore we injected IL-6
at a dose of 800 ng/day and found that utrophin was widely expressed at the
sarcolemma of mdx muscles. Importantly, the expression of utrophin was
observed in mature myofibers as well as regenerating myofibers. In mdx
muscles injected with IL-6 at a dose of 80 ng/day utrophin expression was weaker
and partial. We also injected IL-6 into the muscles of normal C57Bl/10 mice at a
dose of 800 ng/day, but utrophin was not expressed at the sarcolemma.
These results strongly suggest that IL-6 is involved in over-expression of
utrophin in the AxCALacZ injected mdx muscle. In contrast, the
up-regulation of utrophin did not occur in muscles of normal mice. This implies
that other prerequisite factors which particularly express in mdx muscle
are also involved in the utrophin up-regulation.
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