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Gene 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.
1
This approach is intuitively
obvious for the treatment of mende-
lian disorders, butit 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, packaging 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 Sur gery, 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 expression. 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
Orthopaedic Research Society Special Article
230 Journal of the American Academy of Orthopaedic Surgeons
ple 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 intr oduced 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 introduced
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 r ecipient’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.
5
If 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.
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 231
more 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 hematopoietic
9
and mesenchy-
mal
10
stem 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 confers antigenicity on the re-
cipient cells. For example, cells

transduced with first-generation ad-
enoviruses express certain residual
adenoviral proteins.
11
These 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.
13
An
alternative strategy makes use of in-
trinsic regulation, taking advantage
of the natural responsiveness of many
promoters to endogenous stimuli,
such as inflammation.
14
These types

of inducible systems ar e 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 r ecipient 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 fr om 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.
15
A large adenoviral
load of approximately 10
14
particles
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 NF κB), which are involved with
the induction of inflammatory cyto-
kines that could trigger a massive, sys-
temic inflammatory response. Gener-
Gene Therapy for the Treatment of Musculoskeletal Diseases
232 Journal of the American Academy of Orthopaedic Surgeons
alized reactions should not occur when
smaller doses of adenovirus ar e locally
applied.
Insertional mutagenesis has al-
ways been a theor etic 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. Tw o 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,
1
most 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 differ ent 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.
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 233
that 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 present considerable challenges 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 al
24
corrected 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.
26
Four 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 pr ogres-
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 popu-
Gene Therapy for the Treatment of Musculoskeletal Diseases

234 Journal of the American Academy of Orthopaedic Surgeons
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.
28
Genes 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 pro-
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 protocol
30
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 contr ol 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 ex-
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 naïve
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.)
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 235
and 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 cDNAto MCP joints is underway

in Germany.
29
However, 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 r esults 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 r emains 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.
34
The first
showed that retroviral, ex vivo deliv-
ery of human IL-1Ra cDNA to the knee
joints of dogs after transection of the
anterior cr uciate ligament slowed car-
tilage loss.
35
In a subsequent study,
plasmid DNAencoding 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.

37
Intra-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 r educed 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 r estore 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, i t i s 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 al
38
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.)
Gene Therapy for the Treatment of Musculoskeletal Diseases
236 Journal of the American Academy of Orthopaedic Surgeons
vivo delivery of cDNAs encoding IL-
1Ra and IL-10 strongly reduced the

inflammatory cellular r eaction 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.
40
Using 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 pr oducts 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 cDNAwas in-
jected intravenously,
43
a route of
application that pr edominantly 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.
45,46
In 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 cDNAto bone marrow
stromal cells in cell culture.
47
The 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 appr oach, using osteopro-
genitor cells derived from perios-
teum, muscle, and fat.

46
Success 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, ther e
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 al
48
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 al
50
demonstrated the feasibility of
this in a rabbit segmental defect mod-
el (Fig. 4). Those findings have been
repr oduced in rats.
46
Safety is of great-
er concern when using in vivo gene
delivery of adenovirus. However, in
the studies of Baltzer et al,
51
transgene
expression was almost entirely re-
stricted to the site of administration,
with only slight and temporary ex-
pression in the liver. No expression
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
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 237
as vigorous as that of the young, oth-
erwise healthy rats and rabbits stud-
ied.
Spine Fusion
Gene transfer strategies are being
developed to impr ove the outcome of
spinal fusions using the osteogenic
factor LIM mineralization protein-1
(LMP-1).
52
Because it is an intracellu-
lar protein, its delivery by gene trans-
fer is particularly appr opriate. 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 contr ol rabbits un-
derwent spinal fusion.
8
Genes 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.
54
One 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.)
Gene Therapy for the Treatment of Musculoskeletal Diseases
238 Journal of the American Academy of Orthopaedic Surgeons
ovium 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.
55
However, 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.
56
Moreover, this approach to
gene therapy does not, by itself, in-
crease the cellularity of the lesion.

However, i t 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.
57
In 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.
7
Using 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 introducing 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.
63
Delivery 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 differ entiation 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.
67
In particular, gene
transfer offers novel therapeutic ap-
proaches to all six focus areas iden-
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 239
tified in the Unified Research
Agenda of the American Academy
of Orthopaedic Surgeons: OA, spi-
nal disorders, osteoporosis, limb
trauma, soft-tissue injury, and child-
hood disorders. In the last 15 years,
the field of musculoskeletal gene
therapy has developed rapidly and
has emerged as a thriving area of re-
search in its own right. Particularly
impressive progress has been made
in the field of arthritis, where a
phase I study has been completed,
four phase I studies are in progress,
and a phase II study is being
planned. The use of gene transfer to
enhance the repair of musculoskel-
etal tissues is also developing rap-
idly, especially in bone healing,
which is responsive to genetic en-
hancement. Impressive proof of
principle has been reported for the

healing of segmental defects and
spine fusions.
Orthopaedic surgery has much to
offer the field of gene therapy, partic-
ularly by providing opportunities in
which gene therapy has a good
chance of early success in clinical
studies using existing technolo-
gies.
68,69
The volume of orthopaedic
cases and the pressing need for
improved treatment modalities in
certain areas may provide gene ther-
apy with some of its earliest clinical
successes and its largest patient
base.
The degree to which these predic-
tions will be fulfilled depends on a
variety of circumstances, not all of
which are internal to the field of mus-
culoskeletal gene therapy. Cost and
safety are obvious factors, as is the
success of competing, nongenetic
therapies. Some gene therapy appli-
cations may reduce the need for com-
plex surgery, with obvious cost sav-
ings. A critical mass of accomplished
investigators engaged in fruitful, col-
laborative research has been attract-

ed to the field of gene therapy. How-
ever, the limited availability of vectors
has become an impediment to inves-
tigations, and it has been proposed
that the National Institute of Arthri-
tis and Musculoskeletal and Skin Dis-
eases consider funding the establish-
ment of national vector production
cores for investigators who wish to
engage in musculoskeletal gene ther-
apy research.
19
The economics of gene therapy
and its application to large numbers
of patients make in vivo delivery
particularly favorable because such
transfer avoids the expense and
complexity of having to culture each
patient’s cells individually, trans-
duce them, and then return them,
especially if this can be accom-
plished without the need for artifi-
cial scaffolds.
70
Although in vivo
gene therapy raises greater safety
concerns, these should not be exag-
gerated. Safety issues can be com-
prehensively evaluated in preclini-
cal studies and well-designed

clinical protocols.
References
1. Evans CH, Robbins PD: Possible ortho-
paedic applications of gene therapy.
J Bone Joint Surg Am 1995;77:1103-1114.
2. Culver KW, Hsieh WT, Huyen Y, et al:
Correction of chromosomal point mu-
tations in human cells with bifunctional
oligonucleotides. Nat Biotechnol 1999;
17:989-993.
3. Thomas CE, Ehrhardt A, Kay MA:
Progress and problems with the use of
viral vectors for gene therapy. Nat Rev
Genet 2003;4:346-358.
4. Davis ME: Non-viral gene delivery sys-
tems. Curr Opin Biotechnol 2002;13:128-
131.
5. Devine SM, Peter S, Martin BJ, Barry F,
McIntosh KR: Mesenchymal stem cells:
Stealth and suppression. Cancer J 2001;
7(suppl 2):S76-S82.
6. Pascher A, Palmer GD, Steinert A, et al:
Gene delivery to cartilage defects using
coagulated bone marrow aspirate. Gene
Ther 2004;11:133-141.
7. Goto H, Shuler FD, Lamsam C, et al:
Transfer of lacZ marker gene to the me-
niscus. J Bone Joint Surg Am 1999;81:918-
925.
8. Viggeswarapu M, Boden SD, Liu Y, et

al: Adenoviral delivery of LIM miner-
alization protein-1 induces new-bone
formation in vitro and in vivo. J Bone
Joint Surg Am 2001;83:364-376.
9. Kohn DB: Gene therapy using hemato-
poietic stem cells. Curr Opin Mol Ther
1999;1:437-442.
10. Van Damme A, Vanden Driessche T,
Collen D, Chuah MK: Bone marrow
stromal cells as targets for gene therapy.
Curr Gene Ther 2002;2:195-209.
11. Lowenstein PR (ed): Immune responses
to viral vectors for gene therapy. Gene
Ther 2004;10:933-998.
12. Sato Y, Roman M, Tighe H, et al: Immu-
nostimulatory DNA sequences neces-
sary for effective intradermal gene im-
munization. Science 1996;273:352-354.
13. Zoltick PW, Wilson JM: Regulated gene
expression in gene therapy. AnnNY
Acad Sci 2001;953:53-63.
14. Varley AW, Munford RS: Physiological-
ly responsive gene therapy. Mol Med To-
day 1998;4:445-451.
15. Carmen IH: A death in the laboratory:
The politics of the Gelsinger aftermath.
Mol Ther 2001;3:425-428.
16. Kaiser J: Gene therapy: Seeking the
cause of induced leukemias in X-SCID
trial. Science 2003;299:495.

17. Bandara G, Robbins PD, Georgescu HI,
Mueller GM, Glorioso JC, Evans CH:
Gene transfer to synoviocytes: Pros-
pects for gene treatment of arthritis.
DNA Cell Biol 1992;11:227-231.
18. Evans CH, Scully SP (eds): Orthopaedic
gene therapy. Clin Orthop 2000;379:S2-
S316.
19. Rosier R, Evans CH (eds): Molecular Bi-
ology in Orthopaedics. Rosemont, IL:
American Academy of Orthopaedic
Surgeons, 2003.
20. Evans CH (ed): Gene therapy for ortho-
paedics. Gene Ther 2004;11:343-426.
21. Warman ML: Human genetic insights
into skeletal development, growth, and
homeostasis. Clin Orthop 2000;379:S40-
S54.
22. Dawson PA, Marini JC: Hammerhead
ribozymes selectively suppress mutant
type I collagen mRNA in osteogenesis
imperfecta fibroblasts. Nucleic Acids Res
2000;28:4013-4020.
23. Millington-Ward S, McMahon HP,
Allen D, et al: RNAi of COL1A1 in mes-
enchymal progenitor cells. Eur J Hum
Gene Therapy for the Treatment of Musculoskeletal Diseases
240 Journal of the American Academy of Orthopaedic Surgeons
Genet 2004;12:864-866.
24. Niyibizi C, Smith P, Mi Z, Phillips CL,

Robbins P: Transfer of proalpha2(I)
cDNA into cells of a murine model of
human Osteogenesis Imperfecta re-
stores synthesis of type I collagen com-
prised of alpha1(I) and alpha2(I) het-
erotrimers in vitro and in vivo. J Cell
Biochem 2001;83:84-91.
25. Cabrera-Salazar MA, Novelli E, Bar-
ranger JA: Gene therapy for the lyso-
somal storage disorders. Curr Opin Mol
Ther 2002;4:349-358.
26. Barranger JA, Rice EO, Dunigan J, et al:
Gaucher’s disease: Studies of gene
transfer to haematopoietic cells.
Baillieres Clin Haematol 1997;10:765-778.
27. Kaplan FS, Ahn J, Serrano de la Peña L,
Glaser DL, Shore EM: Fibrodysplasia
ossificans progressiva: Deciphering the
molecular pathways of ectopic skeleto-
genesis, in Rosier RN, Evans CH (eds):
Molecular Biology in Orthopaedics. Rose-
mont, IL: American Academy of Ortho-
paedic Surgeons, 2003, pp 95-105.
28. Evans CH, Ghivizzani SC, Kang R, et al:
Gene therapy for rheumatic diseases.
Arthritis Rheum 1999;42:1-16.
29. Evans CH, Ghivizzani SC, Herndon JH,
et al: Clinical trials in the gene therapy
of arthritis. Clin Orthop 2000;379:S300-
S307.

30. Evans CH, Robbins PD, Ghivizzani SC,
et al: Clinical trial to assess the safety,
feasibility, and efficacy of transferring a
potentially anti-arthritic cytokine gene
to human joints with rheumatoid ar-
thritis. Hum Gene Ther 1996;7:1261-1280.
31. Arend WP, Evans CH: Interleukin-1 re-
ceptor antagonist, in Thomson AW,
Lotz MT (eds): The Cytokine Handbook,
ed 4. London, England: Elsevier Science
Ltd, 2003, vol 2, pp 669-708.
32. Evans CH, Robbins PD, Ghivizzani SC,
et al: Gene transfer to human joints:
Progress toward a gene therapy of ar-
thritis. Proc Natl Acad Sci U S A 2005;
102:8698-8703.
33. Sant SM, Suarez TM, Moalli MR, et al:
Molecular lysis of synovial lining cells
by in vivo herpes simplex virus-
thymidine kinase gene transfer. Hum
Gene Ther 1998;9:2735-2743.
34. Evans CH: Gene therapies for osteoar-
thritis. Curr Rheumatol Rep 2004;6:31-40.
35. Pelletier JP, Caron JP, Evans C, et al: In
vivo suppression of early experimental
osteoarthritis by interleukin-1 receptor
antagonist using gene therapy. Arthritis
Rheum 1997;40:1012-1019.
36. Fernandes J, Tardif G, Martel-Pelletier J,
et al: In vivo transfer of interleukin-1 re-

ceptor antagonist gene in osteoarthritic
rabbit knee joints: Prevention of os-
teoarthritis progression. Am J Pathol
1999;154:1159-1169.
37. Frisbie DD, Ghivizzani SC, Robbins
PD, Evans CH, McIlwraith CW: Treat-
ment of experimental equine osteoar-
thritis by in vivo delivery of the equine
interleukin-1 receptor antagonist gene.
Gene Ther 2002;9:12-20.
38. Yang S, Wu B, Mayton L, Evans CH,
Robbins PD, Wooley PH: IL-1Ra and
vIL-10 gene transfer using retroviral
vectors ameliorates particle-associated
inflammation in the murine air pouch
model. Inflamm Res 2002;51:342-350.
39. Yang SY, Mayton L, Wu B, Goater JJ,
Schwarz EM, Wooley PH: Adeno-
associated virus-mediated osteoprote-
gerin gene transfer protects against
particulate polyethylene-induced os-
teolysis in a murine model. Arthritis
Rheum 2002;46:2514-2523.
40. Ulrich-Vinther M, Carmody EE, Goater
JJ, S balle K, O’Keefe RJ, Schwarz EM:
Recombinant adeno-associated virus-
mediated osteoprotegerin gene therapy
inhibits wear debris-induced osteoly-
sis. J Bone Joint Surg Am 2002;84:1405-
1412.

41. Carmody EE, Schwarz EM, Puzas JE, Ros-
ier RN, O’Keefe RJ: Viral interleukin-10
gene inhibition of inflammation, osteo-
clastogenesis, and bone resorption in re-
sponse to titanium particles. Arthritis
Rheum 2002;46:1298-1308.
42. Baltzer AW, Whalen JD, Wooley P, et al:
Gene therapy for osteoporosis: Evalua-
tion in a murine ovariectomy model.
Gene Ther 2001;8:1770-1776.
43. Bolon B, Carter C, Daris M, et al: Ade-
noviral delivery of osteoprotegerin
ameliorates bone resorption in a mouse
ovariectomy model of osteoporosis.
Mol Ther 2001;3:197-205.
44. Kostenuik PJ, Bolon B, Morony S, et al:
Gene therapy with human recombinant
osteoprotegerin reverses established
osteopenia in ovariectomized mice.
Bone 2004;34:656-664.
45. Lieberman JR, Ghivizzani SC, Evans
CH: Gene transfer approaches to the
healing of bone and cartilage. Mol Ther
2002;6:141-147.
46. Betz O, Vrahas M, Baltzer A, Lieberman
JR, Robbins PD, Evans CH: Gene trans-
fer approaches to enhancing bone heal-
ing, in Lieberman JR, Friedlaender GE
(eds): Bone Regeneration and Repair: Biol-
ogy and Clinical Applications. Totowa,

NJ: Humana Press, 2005, pp 157-168.
47. Lieberman JR, Daluiski A, Stevenson S,
et al: The effect of regional gene therapy
with bone morphogenetic protein-2-
producing bone-marrow cells on the re-
pair of segmental femoral defects in
rats. J Bone Joint Surg Am 1999;81:905-
917.
48. Fang J, Zhu YY, Smiley E, et al: Stimu-
lation of new bone formation by direct
transfer of osteogenic plasmid genes.
Proc Natl Acad SciUSA1996;93:5753-
5758.
49. Bonadio J, Smiley E, Patil P, Goldstein
S: Localized, direct plasmid gene deliv-
ery in vivo: Prolonged therapy results
in reproducible tissue regeneration. Nat
Med 1999;5:753-759.
50. Baltzer AW, Lattermann C, Whalen JD,
et al: Genetic enhancement of fracture
repair: Healing of an experimental seg-
mental defect by adenoviral transfer of
the BMP-2 gene. Gene Ther 2000;7:734-
739.
51. Baltzer AW, Lattermann C, Whalen JD,
Braunstein S, Robbins PD, Evans CH: A
gene therapy approach to accelerating
bone healing: Evaluation of gene ex-
pression in a New Zealand white rab-
bit model. Knee Surg Sports Traumatol

Arthrosc 1999;7:197-202.
52. Boden SD, Hair GA, Viggeswarapu M,
Liu Y, Titus L: Gene therapy for spine
fusion. Clin Orthop 2000;379:S225-S233.
53. Wang JC, Kanim LE, Yoo S, Campbell
PA, Berk AJ, Lieberman JR: Effect of re-
gional gene therapy with bone mor-
phogenetic protein-2-producing bone
marrow cells on spinal fusion in rats.
J Bone Joint Surg Am 2003;85:905-911.
54. Palmer G, Pascher A, Gouze E, et al: De-
velopment of gene-based therapies for
cartilage repair. Crit Rev Eukaryot Gene
Expr 2002;12:259-273.
55. Mi Z, Ghivizzani SC, Lechman ER, et al:
Adenovirus-mediated gene transfer of
insulin-like growth factor 1 stimulates
proteoglycan synthesis in rabbit joints.
Arthritis Rheum 2000;43:2563-2570.
56. Bakker AC, van de Loo FA, van Beunin-
gen HM, et al: Overexpression of active
TGF-beta-1 in the murine knee joint:
Evidence for synovial-layer-dependent
chondro-osteophyte formation. Osteo-
arthritis Cartilage 2001;9:128-136.
57. Smith P, Shuler FD, Georgescu HI, et al:
Genetic enhancement of matrix synthe-
sis by articular chondrocytes: Com-
parison of different growth factor genes
in the presence and absence of

interleukin-1. Arthritis Rheum 2000;43:
1156-1164.
58. Hidaka C, Goodrich LR, Chen CT, War-
ren RF, Crystal RG, Nixon AJ: Acceler-
ation of cartilage repair by genetically
modified chondrocytes over expressing
bone morphogenetic protein-7. J Orthop
Res 2003;21:573-583.
59. Grande DA, Mason J, Light E, Dines D:
Stem cells as platforms for delivery of
Christopher H. Evans, PhD, et al
Vol 13, No 4, July/August 2005 241
genes to enhance cartilage repair. J Bone
Joint Surg Am 2003;85(suppl 2):111-116.
60. Madry H, Padera R, Seidel J, et al: Gene
transfer of a human insulin-like growth
factor I cDNA enhances tissue engi-
neering of cartilage. Hum Gene Ther
2002;13:1621-1630.
61. Hidaka C, Ibarra C, Hannafin JA, et al:
Formation of vascularized meniscal tis-
sue by combining gene therapy with
tissue engineering. Tissue Eng 2002;8:
93-105.
62. Nishida K, Kang JD, Gilbertson LG, et
al: Modulation of the biologic activity
of the rabbit intervertebral disc by
gene therapy: An in vivo study of
adenovirus-mediated transfer of the
human transforming growth factor

beta 1 encoding gene. Spine 1999;24:
2419-2425.
63. Gerich TG, Kang R, Fu FH, Robbins PD,
Evans CH: Gene transfer to the rabbit
patellar tendon: Potential for genetic
enhancement of tendon and ligament
healing. Gene Ther 1996;3:1089-1093.
64. Pascher A, Steinert AF, Palmer GD, et
al: Enhanced repair of the anterior cru-
ciate ligament by in situ gene transfer:
Evaluation in an in vitro model. Mol
Ther 2004;10:327-336.
65. Lou J, Tu Y, Burns M, Silva MJ, Manske
P: BMP-12 gene transfer augmentation
of lacerated tendon repair. J Orthop Res
2001;19:1199-1202.
66. Nakamura N, Hart DA, Boorman RS, et
al: Decorin antisense gene therapy im-
proves functional healing of early rab-
bit ligament scar with enhanced col-
lagen fibrillogenesis in vivo. J Orthop
Res 2000;18:517-523.
67. Evans CH, Ghivizzani SC, Robbins PD:
Orthopaedic gene therapy. Clin Orthop
2004;429:316-329.
68. Evans CH: Editorial: Orthopaedics:
Gene therapy’s dark horse. Gene Ther
2004;11:343.
69. Evans CH, Scully SP: Orthopaedic gene
therapy. Clin Orthop 2000;379(suppl):S2.

70. Evans CH, Palmer G, Pascher A, Stein-
ert A, Betz O, Ghivizzani SC: Genetical-
ly enhanced tissue engineering without
cell culture or manufactured scaffolds,
in Sandell LJ, Grodzinsky AJ (eds): Tis-
sue Engineering in Musculoskeletal Clin-
ical Practice. Rosemont, IL: American
Academy of Orthopaedic Surgeons,
2004, pp 389-394.
Gene Therapy for the Treatment of Musculoskeletal Diseases
242 Journal of the American Academy of Orthopaedic Surgeons