The definition of gene therapy can be expanded to include both the delivery of non-coding nu-cleic acids eg, oligonucleotides, which have the ability to modify gene expression in the reci
Trang 1Gene Therapy for the Treatment
of Musculoskeletal Diseases
Christopher H Evans, PhD, Steven C Ghivizzani, PhD, James H Herndon, MD, and Paul D Robbins, PhD
Abstract
Gene therapy involves the transfer of
genes to patients for therapeutic
pur-poses.1This approach is intuitively
obvious for the treatment of
mende-lian disorders, but it also has wide
ap-plication for diseases that lack a
strong or simple genetic basis In such
instances, gene transfer is used as a
biologic delivery system for
thera-peutic RNAs or proteins encoded by
the transgene The definition of gene
therapy can be expanded to include
both the delivery of non-coding
nu-cleic acids (eg, oligonucleotides),
which have the ability to modify gene
expression in the recipient cells, and
the in situ repair of mutations
through gene correction.2
At a minimum, a successful gene
therapy protocol must answer the fol-lowing questions: (1) Which gene or genes should be transferred? (2) Where should the therapeutic genes
be transferred? (3) How can the trans-genes be transferred to the target cells? (4) How should the level and duration of transgene expression be regulated? (5) How can safety be en-sured?
Gene Transfer
Vectors, which can be viral or nonvi-ral, are vehicles that deliver genetic material into a living cell To create vectors, wild-type viruses are genet-ically altered to eliminate virulence
and, in most cases, their ability to rep-licate, while retaining infectivity Vi-ral vectors being used in human clin-ical trials include oncoretrovirus (ie, retrovirus), adenovirus, adeno-associated virus (AAV), and herpes simplex virus Lentivirus, another type of retrovirus, is also undergoing rapid development The key charac-teristics of any viral vector include its host range, ability to infect nondivid-ing cells, packagnondivid-ing capacity, immu-nogenicity, titer, ease of manufacture, and safety, as well as whether it in-tegrates into the host genomic DNA.3 Gene transfer using a viral vector is known as transduction Nonviral vectors may be as
sim-Dr Evans is The Robert Lovett Professor of Or-thopaedic Surgery, Center for Molecular Ortho-paedics, Department of Orthopaedic Surgery, Har-vard Medical School, Boston, MA Dr Ghivizzani
is Associate Professor, Department of Orthopaedic Surgery, University of Florida College of Medi-cine, Gainesville, FL Dr Herndon is The Wil-liam Harris Professor of Orthopaedic Surgery, Center for Molecular Orthopaedics, Department
of Orthopaedic Surgery, Harvard Medical School.
Dr Robbins is Professor, Department of Molec-ular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA Reprint requests: Dr Evans, Center for Molec-ular Orthopaedics, BLI-152, 221 Longwood Av-enue, Boston, MA 02115.
Copyright 2005 by the American Academy of Orthopaedic Surgeons.
Research into the orthopaedic applications of gene therapy has resulted in progress
toward managing chronic and acute genetic and nongenetic disorders Gene
ther-apy for arthritis, the original focus of research, has progressed to the initiation of
several phase I clinical trials Preliminary findings support the application of gene
therapy in the treatment of additional chronic conditions, including osteoporosis and
aseptic loosening, as well as musculoskeletal tumors The most rapid progress is
likely to be in tissue repair because it requires neither long-term transgene
expres-sion nor closely regulated levels of transgene expresexpres-sion Moreover, healing
prob-ably can be achieved with existing technology In preclinical studies, genetically
mod-ulated stimulation of bone healing has shown impressive results in repairing segmental
defects in the long bones and cranium and in improving the success of spinal
fu-sions An increasing amount of evidence indicates that gene transfer can aid the
repair of articular cartilage, menisci, intervertebral disks, ligaments, and tendons.
These developments have the potential to transform many areas of musculoskeletal
care, leading to treatments that are less invasive, more effective, and less expensive
than existing modalities.
J Am Acad Orthop Surg 2005;13:230-242
Trang 2ple as naked, plasmid DNA
Trans-fer efficiency can be increased by
com-bining the DNA with natural or
synthetic polymers or by applying
biophysical methods, such as
elec-troporation Nonviral gene transfer,
known as transfection, is less
expen-sive, safer, and simpler than
transduc-tion, but it is considerably less
effi-cient.4
Regardless of the vector, genes
may be transferred to their targets by
in vivo or ex vivo strategies For in
vivo delivery, vector is introduced
di-rectly into the recipient During ex
vivo delivery, cells are recovered,
ge-netically manipulated outside the
body, then returned to the recipient
Of the two, in vivo gene transfer is
less expensive and technically
sim-pler, but its use raises safety concerns
because infectious or transfecting
agents are introduced directly into the
body Moreover, many of these
agents, particularly viral vectors, are
antigenic, which may provoke
im-mune problems and prevent repeat
dosing The major limitation of in
vivo gene transfer is the inability of
the vector selectively to target cells
as-sociated with the tissue of interest
Ex vivo gene transfer is considered
safer because transduced or
transfect-ed cells—not vectors—are introductransfect-ed
into the body Moreover, the
geneti-cally modified cells can be
exhaus-tively tested before reimplantation
Ex vivo transfer also facilitates the use
of oncoretroviral vectors, which
transduce only dividing cells, because
many cells with low mitotic indices
in vivo replicate readily in culture Ex vivo gene transfer also helps address certain immune problems because vectors can be chosen that express no viral proteins in transduced cells
Thus, the cells returned to the patient synthesize no foreign antigens,
there-by enabling both long-term gene ex-pression and repeat dosing Finally,
ex vivo methods enable more
specif-ic targeting and, therefore, better con-trol of the transduced cells
The primary disadvantage of ex vivo delivery is the expense and com-plexity of harvesting cells and main-taining them in cell culture before transducing, testing, and returning them Patients are exposed to the ad-ditional procedures involved in cell harvesting, and cell transplantation brings its own set of issues that are absent from in vivo delivery proto-cols Approaches for obviating the disadvantages of ex vivo gene trans-fer are being explored
Preliminary evidence indicates that certain cells may be successfully allografted, thus acting as so-called universal donors For example, der-mal fibroblasts expanded from a sin-gle donor provide all the cells used
in living artificial skin grafts The do-nor cells persist in the recipient’s skin for extended periods, possibly be-cause of the dense, collagenous, ex-tracellular matrix that surrounds them Similarly, mesenchymal stem cells may possess immunosuppres-sive properties, thereby enabling their survival in allogeneic hosts.5If allo-geneic cells can indeed be used in this
manner, batches of transduced, screened, and standardized universal donor cells could be established and injected into recipients on demand, increasing the ease of ex vivo gene de-livery
Ex vivo gene delivery also may be expedited by using cells that can be recovered, transduced, and returned
to the patient in one sitting Blood and bone marrow lend themselves to these abbreviated ex vivo delivery strategies.6-8
Duration and Regulation of Transgene Expression
The optimal duration and level of transgene expression is specific to each application For example, some cancer applications may require a very large burst of transgene expres-sion for a limited period to kill tumor cells without causing subsequent damage to uninvolved tissues In con-trast, successful treatment of many monogenic diseases (eg, hemophilia) requires prolonged expression at low
to moderate levels Modest levels of transgene expression for limited pe-riods may be appropriate for tissue repair (eg, cartilage or bone healing) Episodic conditions, such as rheuma-toid arthritis (RA), which is charac-terized by flares and remissions, might require persistent carriage of the transgene, with levels of expres-sion increased or reduced to match disease activity
Transient transgene expression is
Dr Evans or the department with which he is affiliated has received research or institutional support from NIH–National Institute for Arthritis Mus-culoskeletal and Skin Diseases; National Institute for Diabetes, Digestive and Kidney Diseases; the Orthopaedic Trauma Association; Orthogen; Valentis; Osiris; and TissueGene Dr Evans or the department with which he is affiliated has received royalties from Valentis and TissueGene Dr Evans or the department with which he is affiliated has stock or stock options held in Valentis, GenVec, and Orthogen Dr Evans or the department with which he
is affiliated serves as a consultant to or is an employee of Valentis and TissueGene Dr Evans is on the Scientific Advisory Board of TissueGene and Orthogen Dr Ghivizzani or the department with which he is affiliated has received research or institutional support from NIH–National Institute for Arthritis, Musculoskeletal and Skin Diseases Dr Herndon or the department with which he is affiliated has stock or stock options held in Valentis Dr Robbins or the department with which he is affiliated has received research or institutional support from Valentis and TissueGene Dr Robbins or the department with which he is affiliated has received nonincome support (such as equipment or services), commercially derived honoraria, or other non-research–related funding (such as paid travel) from TissueGene and Orthogen Dr Robbins or the department with which he is affiliated has received royalties from TissueGene and Valentis Dr Robbins or the department with which he is affiliated has stock or stock options held in Valentis Dr Robbins
or the department with which he is affiliated serves as a consultant to or is an employee of TissueGene and Orthogen.
Trang 3more easily achieved than long-term
expression When prolonged
trans-gene expression is required, it is
nec-essary to introduce the genes into
either long-lived cells or, if an
inte-grating vector is used, cells whose
progeny can continue to express the
transgene Skeletal muscle as well as
brain and liver cells are examples of
nondividing cells that could provide
extended periods of transgene
ex-pression Stem cells are alternative
targets because their capacity for
self-renewal could ensure carriage of the
transgene for extended periods,
pos-sibly for life Moreover, progeny cells
could carry the transgene as they
dif-ferentiate into mature cells, which
otherwise might be difficult to target
It has been difficult to transduce and
maintain transgene expression
with-in hematopoietic9 and
mesenchy-mal10stem cells, but these technical
limitations may soon be overcome
The immune system acts as a
bar-rier to long-term gene expression
when the vector used for gene
trans-fer contrans-fers antigenicity on the
re-cipient cells For example, cells
transduced with first-generation
ad-enoviruses express certain residual
adenoviral proteins.11These proteins
are highly antigenic, and cells
ex-pressing them are killed by the
im-mune system This difficult problem
has been put to rest only with the
ad-vent of “gutted” adenoviruses from
which all viral coding sequences have
been eliminated
Retroviral and AAV vectors do not
express viral proteins in transduced
cells Nonviral vectors also avoid the
expression of viral proteins
Howev-er, they may nevertheless activate the
immune system because plasmid
DNA used by most nonviral systems
is grown in bacteria that, unlike
eu-karyotic cells, do not methylate
cy-tosine residues in DNA The
un-methylated dinucleotide sequence
cytosine-guanosine (CpG) strongly
activates cell-mediated immunity.12
In the absence of problems with
cell turnover or the immune system,
long-term gene expression also can be curtailed at the level of the promoter
The strong viral promoters often fa-vored for gene therapy experiments may become turned off (silenced) in certain eukaryotic cells As a result, there is interest in using constitutive eukaryotic promoters In general, this strategy can be successful, but in many cases, the level of transgene ex-pression is considerably lower than that achieved with viral promoters
Manipulation of gene expression
at the level of the promoter currently offers the best prospect of achieving regulated gene expression; there are two general approaches to regulating transgene expression in this way One method consists of using an exoge-nous molecule to control the level of gene expression Systems responsive
to agents such as tetracycline, rapa-mycin, and RU486 are available.13An alternative strategy makes use of in-trinsic regulation, taking advantage
of the natural responsiveness of many promoters to endogenous stimuli, such as inflammation.14These types
of inducible systems are attractive be-cause they are self-regulating How-ever, they raise safety concerns be-cause there is no easy way to control them, should that become medically necessary
Safety
Several safety concerns are
associat-ed with gene therapy, some more psy-chological than actual Recombinant viruses used for gene transfer are de-rived from wild-type viruses that cause disease, thus raising tangible concerns regarding the use of viral vectors For example, lentiviral vec-tors are derived from HIV; oncoret-roviral vectors are commonly derived from the Moloney murine leukemia virus; wild-type adenoviruses cause colds and flu; and herpes simplex vi-rus causes conditions such as cold sores and herpes In contrast, AAV causes no known human disease
The viruses used for gene transfer have been altered and in principle are
no longer virulent Theoretically, how-ever, during the production of large batches of virus for clinical use or dur-ing transduction of the target cells, vi-ral vectors may undergo genetic re-arrangements that restore virulence There is particular concern regarding the possible generation of replication-competent viruses, which not only would spread within the recipient but also could permit horizontal transfer
to other individuals, with unknown consequences The presence of replication-competent virus also in-creases the likelihood of germ-line gene transfer, another matter of concern Considerable effort has been ex-pended in developing very sensitive assays for replication-competent vi-ruses; in fact, this is mandatory in hu-man clinical studies With
lentivirus-es, using vectors derived from equine
or feline sources rather than from HIV may be safer Although these nonhu-man lentiviruses do not normally cause disease in humans, the prop-erties of recombinant viruses engi-neered in the laboratory to transduce human cells may be different Ironically, the first documented death as a result of gene transfer oc-curred with adenovirus, a vector con-sidered to be safe because it is non-integrating and, in its wild-type state,
is associated with only mild respira-tory infections.15A large adenoviral load of approximately 1014particles was infused into the hepatic portal vein
of a patient, which led to an uncon-trollable, systemic inflammatory re-action and death from respiratory fail-ure The exact mechanism remains unclear, but a hypersensitivity reac-tion could occur with a high antigenic load Moreover, infection of cells with adenoviruses activates the intracellular signaling machinery (mitogen-activated protein kinases and the transcription factor NFκB), which are involved with the induction of inflammatory cyto-kines that could trigger a massive, sys-temic inflammatory response
Trang 4Gener-alized reactions should not occur when
smaller doses of adenovirus are locally
applied
Insertional mutagenesis has
al-ways been a theoretic possibility with
retroviral vectors, but until recently,
it had never been observed despite
the widespread use of retroviral
vec-tors in human trials However, in
1999, a lymphoproliferative disorder
resembling leukemia occurred in a
child treated for X-linked severe
com-bined immunodeficiency disease
with retroviral gene transfer.16 Two
more children in the same study also
developed leukemia, which resulted
from insertion of the retrovirus near
a known oncogene Two of these three
children subsequently died from the
secondary leukemia Several
circum-stances conspired to make this
clin-ical trial singularly vulnerable to this
type of adverse event: the subjects
lacked adaptive immunity, the
retro-virus was targeted to hematopoietic
stem cells, the transgene encoded one
chain of a receptor common to
sev-eral different growth factors, and the
genetically modified cells had a
se-lective, in vivo growth advantage
over unmodified cells Ironically, the
protocol that produced the leukemia
successfully treated the genetic
dis-ease Because childhood leukemia can
be successfully treated in most cases
and because X-linked severe
com-bined immunodeficiency disease is
lethal, permission has been given to
treat additional patients with
retro-viral gene transfer However, the
ep-isode has renewed concerns about
insertional mutagenesis, and the use
of retroviral vectors for
non–life-threatening diseases has been subject
to renewed questioning
Although nonviral vectors involve
fewer safety concerns than their
vi-ral counterparts, they are not devoid
of potential side effects For example,
DNA is inflammatory, and
unmeth-ylated CpG dinucleotide sequences
present in plasmids generated in
bac-teria stimulate cell-mediated
immu-nity The inefficiencies of nonviral
gene delivery often require the ad-ministration of very large amounts of DNA, thus increasing the chances of unwanted side effects
Despite the disproportionate amount of negative publicity
attract-ed by these events, there have been only scattered reports of nonfatal side effects and three deaths among more than 4,000 individuals treated
Musculoskeletal Applications of Gene Therapy
Interest in gene therapy for muscu-loskeletal applications began with re-search focused on gene delivery to synovium to treat arthritis.17
Howev-er, the rich potential of gene therapy for other musculoskeletal indications was quickly appreciated and, by the time the first review was published
in 1995,1most major applications of the technology had been foreseen To facilitate communication and collab-oration between the growing num-bers of investigators in this area, sev-eral workshops on orthopaedic gene therapy have been held.18-20
Although gene therapy was con-ceived of as a method for treating mendelian diseases, much attention
is devoted to its use in nongenetic dis-orders It is useful to divide the field
of orthopaedic gene therapy into four main areas based on the genetics and chronicity of the target diseases be-cause each entails different gene ther-apy approaches (Fig 1) Of the four areas illustrated, gene therapy for or-thopaedic tumors has received very little experimental attention
Mendelian Diseases
Considerable progress has been made in identifying the mutations
Figure 1 Categories of orthopaedic disease amenable to gene therapy CACP = campodactyly-arthropathy-coxa vara-pericarditis.
Trang 5that lead to mendelian disorders of
the musculoskeletal system.21 With
completion of the Human Genome
Project and rapid advances in
tech-nology, there is a reasonable prospect
of determining the molecular basis for
all of them within the next decade
Despite such progress, these
diseas-es prdiseas-esent considerable challengdiseas-es to
gene therapy Many of them are rare,
dominant negative disorders, which
require suppression of mutant gene
expression Another problem is the
developmental nature of many of
these diseases Thus, gene therapy
may need to be administered at a very
early developmental age, possibly in
utero, before the musculoskeletal
sys-tem becomes fully developed and
dif-ficult to alter
As well as challenging the limits
of gene therapy, such constraints also
require sophisticated early diagnosis
Even when postnatal gene therapy is
a reasonable option, the abundant
ex-tracellular matrix present in many
musculoskeletal tissues renders gene
delivery inefficient Finally, effective
gene therapy of most genetic
disor-ders probably requires transgene
ex-pression for life and, in the case of
dominant negative mutations,
equal-ly long suppression of mutant alleles
Nevertheless, for most genetic
diseas-es, the choice of transgene is obvious
and, in many cases, the level of
trans-gene expression does not need to be
finely regulated A gene therapy
ap-proach may be optimal because these
diseases currently are often difficult
to treat and impossible to cure
Osteogenesis Imperfecta
Osteogenesis imperfecta (OI) is
caused by mutations in the genes
en-coding the alpha chains of type I
col-lagen Type III OI is recessive; types
I, II, and IV are dominant In tissue
culture, antisense RNA both inhibits
expression of the mutated gene and
reduces expression of the
unmutat-ed gene Ribozymes and small,
inter-fering RNAs, however, achieve
sub-stantial suppression of the mutant
allele without influencing expression
of the wild-type allele.22,23
The oim mouse, which lacks the
alpha-2 chain of type I collagen, serves as a useful experimental
mod-el for recessive forms of human OI
Niyibizi et al24corrected the molec-ular defect in vitro by introducing a cDNA encoding the wild-type alpha-2 chain into fibroblasts derived
from the oim mouse They also
cor-rected the molecular defect in vivo in
a small patch of skin injected with an adenovirus vector carrying the wild-type gene The current challenge is to develop techniques that permit the introduction of the therapeutic gene into a sufficient proportion of osteo-blasts to correct the disease and to maintain expression of the gene for the life of the animal Ex vivo strat-egies using stem cells (eg, mesenchy-mal stem cells) seem to be promising for correcting genetic defects, not only
in bones but also in other collagenous tissues affected by the disease
Lysosomal Storage Disorders
Several lysosomal storage
diseas-es have important orthopaedic se-quelae, and they appeal to gene ther-apists for several reasons The genes whose mutations cause the diseases are cloned and well characterized, the diseases are recessive, and treatment with recombinant protein or by bone marrow transplant typically is suc-cessful In addition, the level of gene expression does not need to be
tight-ly regulated and, in many cases, the therapeutic gene may be expressed in any convenient tissue with access to the systemic circulation.25
Gaucher’s disease is caused by mutations in the gene encoding the enzyme glucocerebrosidase In a phase I clinical trial in which gene therapy was used to treat Gaucher’s disease, a retrovirus was used to transfer the glucocerebrosidase cDNA via ex vivo delivery into he-matopoietic stem cells.26Four patients were treated, and the trial is now closed
The mucopolysaccharidoses (MPSs),
a group of lysosomal storage disor-ders in which various enzymes nec-essary for the breakdown of glycosami-noglycans are missing, may have associated skeletal abnormalities (ie, Hunter’s and Hurler’s syndromes) Currently in progress is a phase I pro-tocol for subjects with a mild form of Hunter’s syndrome (MPS II), in which the enzyme iduronate-2-sulfatase is defective
Fibrodysplasia Ossificans Progressiva
Fibrodysplasia ossificans progres-siva is characterized by the exagger-ated deposition of ectopic bone fol-lowing even mild trauma, and afflicted individuals are said to
devel-op a second skeleton Although the molecular basis for the disease is un-known, it is thought to reflect muta-tions that disturb bone morphoge-netic protein (BMP)-4 synthesis or signaling In an interesting approach
to the therapy of a genetic disease whose molecular lesion is unknown,
investigators are evaluating the nog-gin gene, whose product
antagoniz-es BMP-4–induced heterotopic ossi-fication.27
Chronic Nonmendelian Diseases
The goal of gene therapy in man-aging the chronic nonmendelian dis-eases is not to compensate for a ge-netic abnormality in the patient but
to use gene transfer as a biologic de-livery method for therapeutic gene products In the absence of a clear ge-netic basis for the disease, the choice
of therapeutic transgene is not always obvious, and its selection relies on an understanding of the etiology and pathogenesis of the disorder in ques-tion Achieving long-term transgene expression is a major challenge; even developing convenient methods of re-administration may be problematic Nevertheless, continued research is necessary because many of the target diseases are common, are poorly treated by existing modalities, and are increasing in incidence as the
Trang 6popu-lation ages Most progress has been
made in the treatment of arthritis, the
first musculoskeletal disorder
target-ed for gene therapy
Rheumatoid Arthritis
Although RA is an autoimmune
condition with significant pathology
involving the joints, there are
impor-tant extra-articular and systemic
manifestations of the disease
Accord-ingly, attempts to treat RA with gene
therapy in animal models have
con-sisted of local gene delivery to joints,
systemic delivery to various organs,
and delivery to lymphocytes and
antigen-presenting cells, which have
the ability to migrate between
differ-ent lymphoid tissues.28Genes
encod-ing a variety of type 2 cytokines
(par-ticularly interleukins [ILs]-4, -10, and
-13), antagonists of IL-1 and tumor
necrosis factor, and antiangiogenic
proteins, have shown efficacy in
an-imal models Rather than attempting
to modulate the natural disease
cess, other investigators have
pro-duced genetic synovectomies by
in-jecting joints with genes whose
products cause apoptosis within the
synovium The advantage of this
ap-proach is that it circumvents the need
for long-term gene expression The
disadvantage is that the clinical
re-sults may be no better than those
achieved by conventional
synovecto-my Preclinical studies have
estab-lished a convincing proof of
princi-ple that justifies and has propelled the
development of the four human gene
therapy protocols for RA.29
The first clinical protocol30
select-ed an IL-1 blocker, the IL-1 receptor
antagonist (IL-1Ra),31 as the
trans-gene Using ex vivo delivery, a
retro-virus was used to transfer the IL-1Ra
cDNA to autologous synovial
fibro-blasts obtained from nine
postmeno-pausal women with advanced RA
Control cells were not genetically
modified In a double-blind fashion,
genetically modified and control cells
were delivered by intra-articular
in-jection to the 2nd-5th
metacarpopha-langeal (MCP) joints of one hand of each subject One week later, these MCP joints were recovered and amined for evidence of transgene
ex-pression (Fig 2) This study was not designed to determine efficacy; how-ever, it confirmed that it is indeed pos-sible to transfer genes to human joints
Figure 2 The sequence of events in a phase I clinical trial of gene therapy in nine postmeno-pausal women with advanced RA who failed pharmacologic control and required multiple joint surgeries, including replacement of MCP joints 2 through 5 on one hand Monolayers
of autologous synovial fibroblasts were expanded in culture (1) and divided into two
pop-ulations, one of which was transduced with a retrovirus carrying human IL-1Ra transgene
(2) After safety testing (3), in a double-blinded fashion, two of the recipients’ MCP joints 2-4 were injected with genetically modified cells, while the other two were injected with nạve control cells (4) Seven days later, the four MCP joints were surgically replaced (5), and
re-covered tissues were analyzed for expression of the transferred IL-1Ra gene (6) (Adapted
with permission from Evans CH, Ghivizzani SC, Robbins PD: Blocking cytokines with genes.
J Leukoc Biol 1998;64:55-61.)
Trang 7and to successfully express them
intra-articularly (Fig 3) in a manner
that is safe and acceptable to
pa-tients.32
A similar phase I protocol using ex
vivo, retroviral transfer of human
IL-1Ra cDNA to MCP joints is underway
in Germany.29However, in that study,
there is a gap of 1 month between the
introduction and surgical removal of
the transgene So far, four
individu-als have been treated, with results
sim-ilar to those in the United States trial
A phase I protocol involving the
direct, intra-articular injection of a
re-combinant AAV vector began last
year This vector carries a cDNA
en-coding a fusion protein composed of
two tumor necrosis factor–soluble
re-ceptors combined on an
immuno-globulin molecule In essence, this is
a gene that encodes the
anti-rheumatic drug etanercept
The only clinical trial of gene
ther-apy in RA using nonviral gene
deliv-ery employs the genetic
synovecto-my approach Joints are injected with
DNA encoding herpes simplex
thy-midine kinase Cells expressing this
gene become susceptible to
ganciclo-vir and, because of a pronounced
by-stander effect, there is widespread
death of cells within the synovium.33
This approach obviates the necessity
of long-term gene expression;
more-over, readministration of the gene
upon recurrence of symptoms should
be possible It remains to be seen how
the clinical results compare with those
of conventional synovectomy
Osteoarthritis
IL-1 also may be an important
me-diator in osteoarthritis (OA) Three
studies confirm the promise of IL-1Ra
gene therapy in treating OA.34The first
showed that retroviral, ex vivo
deliv-ery of human IL-1Ra cDNAto the knee
joints of dogs after transection of the
anterior cruciate ligament slowed
car-tilage loss.35In a subsequent study,
plasmid DNA encoding canine IL-1Ra
delivered nonvirally to the knee joints
of rabbits suppressed development of
surgically induced OA.36 Convincing data were reported from a series of experiments in which equine IL-1Ra cDNA was delivered
to the joints of horses by direct, in vivo, adenoviral delivery.37Intra-articular expression of equine IL-1Ra
inhibit-ed the development of experimental
OA induced by the surgical creation
of osteochondral fragments In addi-tion to strongly protecting the artic-ular cartilage, this therapy reduced the lameness index of the horses, dem-onstrating improvement in both clin-ical and laboratory parameters
Given the late stage at which hu-man OA is typically diagnosed, ar-resting the progress of the disease with an anti-inflammatory and
chon-droprotective gene, such as IL-1Ra,
may be insufficient More often, it may be necessary to restore damaged cartilage, possibly using gene
thera-py approaches Such complications could be avoided if earlier diagnosis were possible and if gene transfer
could be given prophylactically after injuries known to predispose to OA, such as rupture of the anterior cru-ciate ligament
In several ways, OA is well suited
to local, intra-articular gene therapy Unlike RA, it is not a systemic con-dition; rather, it is restricted to a small number of accessible joints with lim-ited extra-articular manifestations of disease Moreover, there are few ef-fective pharmacologic treatments A phase I human protocol for gene transfer in subjects with OA is under-going the review process
Aseptic Loosening
Proteins that maintain or restore bone mass around prosthetic joint ar-throplasties or inhibit cellular reac-tions to wear debris may prevent or reverse aseptic loosening Delivery of genes encoding such proteins has shown promise in relevant animal models Using a murine air pouch model, Yang et al38 showed that in
Figure 3 Expression of IL-1Ra transgene in human rheumatoid synovium following ex vivo
gene transfer The human IL-1Ra cDNA was transferred to human rheumatoid MCP joints
by the protocol described in Figure 2 Genetically modified synovia were recovered at the time of joint arthroplasty, and expression of the transgene was detected by in situ hybrid-ization The image is pseudocolored to show mRNA (green) and synovium (red) Arrows indicate areas of particularly high transgene expression (Courtesy of Simon C Watkins, PhD, Pittsburgh, PA.)
Trang 8vivo delivery of cDNAs encoding
IL-1Ra and IL-10 strongly reduced the
inflammatory cellular reaction to
par-ticles of ultra-high-molecular-weight
polyethylene or
polymethylmeth-acrylate In another study, when
frag-ments of bone were introduced into
the air pouch along with the wear
de-bris, transfer of the osteoprotegerin
(OPG) cDNA inhibited loss of bone
matrix.39
In a complementary series of
stud-ies, titanium particles were
implant-ed onto the calvarium in a murine
model.40Using adenovirus and AAV
vectors, the investigators found that
genes encoding a bivalent soluble
tu-mor necrosis factor receptor, OPG, or
IL-10 were able to inhibit bone
resorp-tion in response to the particles.41
Pro-tection occurred whether the vector
was delivered locally to the calvarial
surface or systemically via
intramus-cular injection
Osteoporosis
Genes whose products retard bone
loss or promote bone formation have
potential for managing osteoporosis
In a murine ovariectomy model of
os-teoporosis, the injection of
adenovi-rus carrying human IL-1Ra cDNA
transduced cells in marrow and the
surrounding bone, leading to a
dra-matic reduction in bone loss.42
Al-though gene expression persisted for
only 2 to 3 weeks, the protective
ef-fects of gene transfer lasted for at least
5 weeks In similar experiments,
ad-enovirus carrying OPG cDNA was
in-jected intravenously,43 a route of
application that predominantly
trans-duces the liver It led to high
circu-lating levels of OPG, which produced
a prolonged anti-osteoporotic effect
Of particular note was the remarkable
duration of OPG gene expression
achieved in this study, which may
re-flect the ability of OPG to interfere
with immune responses involved in
the clearance of adenovirally
infect-ed cells Similar data were
subse-quently obtained using an AAV
vec-tor.44
Tissue Repair
There are several advantages to us-ing gene therapy to heal musculo-skeletal tissues: long-term transgene expression is neither necessary nor desirable; in most cases, the level of transgene expression need not be un-realistically high or closely regulated;
and it may be possible to achieve clin-ical success using existing
technolo-gy Moreover, there is a need for bet-ter ways to heal injuries to bone and soft tissues Many of these injuries oc-cur in younger individuals as a result
of sporting activities; when unsatis-factorily repaired, such injuries have
a major accumulated impact on qual-ity of life The ultimate role of gene transfer strategies in musculoskeletal tissue repair will depend on the suc-cess of competing technologies, par-ticularly those based on the use of re-combinant growth factors and tissue engineering
Bone Healing
The ability of gene transfer to in-duce bone formation has been con-firmed by multiple independent lab-oratories using both ex vivo and in vivo strategies.45,46In evaluating heal-ing, the model of choice has been a defect of critical size surgically cre-ated in the long bones or crania of ex-perimental animals In the ex vivo ap-proach, adenovirus was used to deliver BMP-2 cDNA to bone marrow stromal cells in cell culture.47The ge-netically modified cells were seeded onto a collagenous scaffold and in-serted into defects of critical size in the femurs of rats Unlike control de-fects, the genetically treated femurs healed within a few weeks By his-tologic criteria, healing achieved by
BMP-2 gene transfer was superior to
that achieved with recombinant BMP-2 protein
One advantage of using marrow stromal cells is their ability not only
to express the BMP-2 transgene but
also to respond to it and form bone
Subsequent investigators have con-firmed this approach, using
osteopro-genitor cells derived from perios-teum, muscle, and fat.46Success also has been reported with cells (eg, skin fibroblasts) with no obvious osteo-genic potential Other transgenes,
such as BMP-4, also are effective in
these models, and it is assumed that additional genes encoding
osteogen-ic proteins, such as BMP-6, -7, and -9, also will be successful
Because of the cost and complex-ity of ex vivo delivery methods, there have been attempts to heal osseous defects by in vivo delivery of genes
to the lesion One approach involves the use of matrices impregnated with DNA, known as gene-activated ma-trices (GAMs) Fang et al48 healed segmental defects in rat bone by in-serting GAMs containing a cDNA en-coding the first 34 amino acids of par-athyroid hormone (PTH 1-34) and BMP-4 This group later confirmed stimulated bone formation in large osseous defects in dogs using GAMs carrying PTH 1-34 cDNA.49
An alternative in vivo strategy in-volves the direct, intralesional injec-tion of vectors, such as adenovirus-carrying osteoinductive genes, in the absence of a matrix or scaffold Baltzer
et al50demonstrated the feasibility of this in a rabbit segmental defect
mod-el (Fig 4) Those findings have been reproduced in rats.46Safety is of
great-er concgreat-ern when using in vivo gene delivery of adenovirus However, in the studies of Baltzer et al,51transgene expression was almost entirely re-stricted to the site of administration, with only slight and temporary ex-pression in the liver No exex-pression occurred in other organs that were ex-amined It is not known whether there will be immunologic constraints
to the application or reapplication of adenoviral vectors in the healing of human bones
Although the data from the afore-mentioned studies are impressive, it remains to be seen whether the osteo-genic response in humans,
especial-ly those who are older, diabetic, or traumatized, or who smoke, will be
Trang 9as vigorous as that of the young,
oth-erwise healthy rats and rabbits
stud-ied
Spine Fusion
Gene transfer strategies are being
developed to improve the outcome of
spinal fusions using the osteogenic
factor LIM mineralization protein-1
(LMP-1).52Because it is an
intracellu-lar protein, its delivery by gene
trans-fer is particularly appropriate One of
the oddities about LMP-1 is its
re-markable potency In fact,
investiga-tors have the problem of needing to
prevent excessively high levels of
gene expression because under such circumstances, the efficiency of bone formation is reduced
Limited transgene expression has been achieved with plasmid DNA and by transducing cells with adeno-virus vectors for only a short period
The latest version of the application consisted of an abbreviated ex vivo gene delivery approach in which buffy coat cells were isolated from au-tologous blood intraoperatively,
brief-ly incubated with the adenoviral vec-tor, placed on a collagen-ceramic composite carrier, and immediately inserted into the fusion site In
rab-bits with a single-level arthrodesis of the lumbar spine, this procedure re-sulted in full spinal fusion within 4 weeks; none of the control rabbits un-derwent spinal fusion.8Genes encod-ing additional osteogenic genes, such
as BMP-2, also show preliminary
promise in experimental spinal fusion studies.53
Articular Cartilage and Meniscus
Several approaches to repairing cartilage using gene transfer are be-ing evaluated.54One approach is to use technologies developed for man-aging arthritis and delivered to
syn-Figure 4 Healing of an osseous defect of critical size (1.3 cm) by in vivo gene transfer in the femurs of rabbits An adenovirus was used
to deliver human BMP-2 cDNA to the defects shown in panels A-D Control defects (E-H) received a luciferase gene Plain radiographs were taken immediately after surgery (A and E) and at 5 weeks (B and F), 7 weeks (C and G), and 12 weeks (D and H) postoperatively
(Re-produced with permission from Baltzer AW, Lattermann C, Whalen JD, et al: Genetic enhancement of fracture repair: Healing of an
ex-perimental segmental defect by adenoviral transfer of the BMP-2 gene Gene Ther 2000;7:734-739.)
Trang 10ovium growth factor genes whose
se-creted products diffuse to areas of
damaged cartilage Other methods
in-volve ex vivo gene delivery using
chondrocytes or chondroprogenitor
cells as vehicles and in vivo delivery
using vectors associated with
matri-ces or autologous blood and bone
marrow clots
Delivery of transgenes encoding
insulin-like growth factor-1 (IGF-1) or
BMP-2 to synovium increases matrix
synthesis by chondrocytes in the
ad-jacent cartilage.55However, delivery
of a transforming growth factor-β
(TGF-β) gene in this way is
deleteri-ous, causing massive fibrosis,
ectop-ic cartilage formation, and
osteo-phytes.56Moreover, this approach to
gene therapy does not, by itself,
in-crease the cellularity of the lesion
However, it might be a useful adjunct
to cell-based repair methods or
mi-crofracture
Using marker genes, it has been
es-tablished that genetically modified
chondrocytes and periosteal cells can
be implanted into cartilaginous
le-sions, where they continue to express
the transgene for up to several weeks
Transfer of cDNAs encoding BMP-2,
BMP-7, IGF-1, or TGF-β
dramatical-ly increases matrix synthesis by
cul-tures of chondrocytes, even in the
presence of IL-1.57In an equine
mod-el of cartilage repair by chondrocyte
transplantation, the introduction of a
BMP-7 cDNA into the transplanted
chondrocytes accelerated repair.58
BMP-7 also promotes the
chondro-genic differentiation of precursor cells
derived from periosteum The
im-plantation of periosteal cells
trans-duced with BMP-7 or sonic hedgehog
cDNAs enhances repair of
osteochon-dral defects in rabbits.59
To avoid the complexities of ex
vivo gene delivery, there have been
attempts to deliver genes directly to
full-thickness lesions in cartilage In
one approach, adenoviral vectors
are associated with a
collagen-gly-cosaminoglycan matrix that is
in-serted into the defect Alternatively,
the vectors are mixed with autolo-gous blood or bone marrow during clotting The resulting “gene plug”
can be press-fit into lesions in artic-ular cartilage.6
As an alternative to implanting vectors or genetically modified cells into lesions, the genetically modified cells can be allowed to develop into mature tissue before implantation
This approach combines gene
thera-py with tissue engineering Prelimi-nary success has been reported with chondrocytes that have been trans-fected with IGF-1 cDNA, seeded onto scaffolds, and incubated in a bioreac-tor.60
Many of the principles for repair-ing articular cartilage can be
extend-ed to the repair of meniscal lesions
Marker genes have been
successful-ly expressed in experimental menis-cal lesions by ex vivo and gene plug approaches.7Using a tissue engineer-ing approach, genetically modified meniscal cells have been seeded onto
a matrix and implanted into nude mice, where the cells develop into me-niscal tissue.61
Intervertebral Disk
Using strategies similar to those employed in the repair of articular cartilage, investigators are develop-ing methods of introducdevelop-ing genes into cells of intervertebral disks to prevent or reverse disk degenera-tion.62 One interesting and unex-pected finding is the remarkably prolonged duration of transgene ex-pression that follows the intradiskal injection of recombinant adenoviral vectors This duration appears to re-flect the immunologically protected environment of the disk and the nondividing state of its cells It should be a major asset to the fur-ther development of this approach
to therapy Introduction of growth factor genes into disk cells elevates the synthesis of matrix macromole-cules, but whether this protects or heals disks in vivo has not yet been evaluated in animal models
Ligament and Tendon
Cells recovered from ligaments and tendons are readily transduced
by a variety of viral and nonviral vec-tors, and gene transfer can be accom-plished by ex vivo and in vivo strat-egies.63Delivery of cDNAs encoding growth factors promotes cell division and the deposition of extracellular matrix in vitro,64 but it is not yet known whether this accelerates
heal-ing in vivo.
BMP-12 and -13 proteins are of particular interest because they pro-mote the differentiation of mesenchy-mal stem cells into tissue with the ap-pearance of ligament and tendon Intramuscular injection of an adeno-virus encoding BMP-12 leads to the formation of ectopic ligamentous tis-sue When this vector is injected into chicken tendon cells, there is an in-crease in the synthesis of type I col-lagen In a complete tendon
lacera-tion chicken model, BMP-12 gene
transfer doubled the tensile strength and stiffness of the repaired ten-dons.65
Another strategy for improving the healing of ligaments and tendons
is to reduce the synthesis of decorin This small proteoglycan is an attrac-tive target because it limits the diam-eter of collagen fibrils and also acts
as an antagonist of TGF-β Blocking decorin production has been evalu-ated as a means to improve healing
of the medial collateral ligament in a rabbit model Inhibiting decorin ex-pression with antisense RNA in-creased the average diameter of the collagen fibers within the repair tis-sue and improved the mechanical properties of the ligament.66
Summary
Gene therapy offers a broad range of potential applications for treating musculoskeletal conditions in all specialty areas.67In particular, gene transfer offers novel therapeutic ap-proaches to all six focus areas