Abstract
Biologic therapies to promote fracture-healing such as use of bone
morphogenetic proteins (BMPs) are being increasingly employed in multiple
clinical scenarios. However, it has been challenging to design therapies that
deliver sufficient quantities of protein over a sustained time period. A
potential solution is the application of gene therapy that transfers genetic
information to host cells at the fracture site, resulting in the continuous
and localized production of the desired proteins. This approach has
demonstrated tremendous potential in preclinical animal models of
fracture-healing. This article will review the current state of gene therapy
approaches to fracture-healing with an emphasis on potential clinical
applications.
There are approximately 6.5 million fractures per year in the United
States, and between 5% and 10% of these result in nonunion or delayed
union1.
Collectively, this represents a substantial cause of morbidity, missed work,
and medical cost2.
Biologics that promote bone-healing are needed in the treatment of established
nonunions as well as in the acute treatment of certain fractures associated
with either extensive bone loss or soft-tissue injury. There are many clinical
options for stimulating bone formation, but each has substantial limitations.
To date, autologous bone remains the gold-standard graft material. However,
its harvest can cause substantial morbidity, including hematoma formation,
infection, numbness at the incision site, and persistent
pain3,4.
In addition, the limited quantity of autologous bone available for harvest may
not be sufficient for the treatment of large
defects5.
Recombinant human bone morphogenetic proteins (rhBMPs) have recently
emerged as a bone-graft substitute. RhBMP-2 has been approved by the U.S. Food
and Drug Administration for use in anterior lumbar interbody fusions and for
the treatment of open tibial
fractures6. RhBMP-7
(osteogenic protein-1) has been approved under a humanitarian device exemption
for the treatment of recalcitrant long-bone nonunions and for use in revision
posterolateral spinal
arthrodeses6. There
is evidence that BMPs are more effective than autograft for promoting
fracture-healing and spinal fusion; consequently, the introduction of BMPs has
been met with a great deal of enthusiasm by the orthopaedic
community7-10.
However, the use of BMP has not been optimized. High doses of growth factor
are needed to produce an adequate bone-formation response. Presently, rhBMP is
being administered at doses that are a million times greater than its normal
concentration in bone, and there are concerns about both the safety and the
cost of such supraphysiologic
doses11. Clinical
trials and preclinical studies have both shown a potential for ectopic bone
formation as well as
edema6. These
observations might partly be attributed to the collagen carriers used to
deliver BMP, which have been hypothesized to be inefficient protein delivery
systems11,12.
These concerns have led to investigations of alternative protein delivery
mechanisms to promote bone repair. Regional gene therapy offers a novel
approach to a difficult clinical problem. Genetic sequences encoding for
growth factors can be transferred to cells at the fracture site, resulting in
the production of osteogenic proteins in a localized, sustained, and
physiologic manner. Preclinical animal models have demonstrated the tremendous
potential of these
techniques13-19.
In the future, regional gene therapy may be one part of a comprehensive
tissue-engineering strategy that will include a spectrum of treatment options
such as autologous bone-grafting, use of recombinant growth factors,
autologous bone-marrow injection, and stem cell therapies. It is envisioned
that gene therapy options will initially be available for the most severe
clinical situations such as massive bone loss and recalcitrant nonunions.
Despite its tremendous promise, the clinical application of gene therapy must
be approached with caution. Thus far, clinical trials of gene therapy for
inflammatory arthritis and metabolic diseases have led to two
deaths20,21.
Any substantial morbidity will not be accepted in the treatment of nonfatal
musculoskeletal conditions.
The purpose of this review is to define the scientific basis of gene
therapy as it relates to fracture-healing, review the various gene-therapy
strategies, compare the results of preclinical models, and address the
obstacles that must be overcome in order to make gene therapy a clinical
reality.
Gene therapy is the transfer of genetic information to cells for
therapeutic purposes. This was originally envisioned as a means of replacing a
single defective gene in Mendelian disorders such as osteogenesis imperfecta.
However, in the setting of bone repair, gene therapy functions as a biologic
protein delivery system. A complementary deoxyribonucleic acid sequence (cDNA)
encoding for a therapeutic protein is transferred to target cells, resulting
in production of the desired protein at the fracture site. The duration of
protein production largely depends on the gene transfer technique being used.
Both short-term and long-term protein production is possible. Most
bone-healing applications require only short-term production; however, the
treatment of massive bone defects may necessitate a longer
duration22.
Vectors are used to enhance the transfer of cDNA sequences into target
cells. This process is termed "transduction" when a viral vector
is used and "transfection" when a nonviral vector is
used23,24.
Viruses have evolved to be efficient transducing agents because their survival
depends on it. When a viral vector is used, the cDNA either integrates into
the host genome, resulting in stable long-term expression, or remains outside
the genome as a circular plasmid called an episome. The latter results in a
relatively shorter period of expression but avoids the dangers of insertional
oncogenesis. Portions of the viral vector genome are deleted to render the
virus replication incompetent; nevertheless, there are safety concerns with
the use of viral vectors. The viruses could undergo recombination with other
viruses present in the host cell, resulting in replication competence and
virulence. Alternatively, certain viruses (e.g., adenovirus) are immunogenic,
and they induce a robust inflammatory response that limits the duration of
gene expression and poses danger to the
patient20,25,26.
Nonviral vectors are appealing because they are relatively safe; however, at
the present time, clinical applications are limited because of their poor
transfection efficiency and short period of gene expression. The selection of
an appropriate vector will vary for each application and will be based on the
desired duration of gene expression, the target cell type, and the safety
profile.
There are two fundamental gene-therapy strategies, termed "in
vivo" and "ex vivo" (Fig.
1). In vivo gene transfer occurs within the host, with direct
administration of the vector systemically or locally at a specific anatomic
site. Alternatively, host cells can be harvested from a specific location,
expanded in culture, transduced ex vivo in tissue culture, and then
reimplanted at the desired location.
Four critical elements are essential for bone repair. First, the process is
initiated by bioactive molecules including cytokines and growth factors that
recruit osteoprogenitors and stimulate their differentiation. Second, a
scaffold or matrix is needed to provide a surface for cellular attachment and
cell growth. In normal fracture-healing, this may be provided by fibrin clot
and the surrounding bone. Third, responding cells, including osteoprogenitors
and osteoblasts, are needed to synthesize and mineralize the osteoid matrix.
Finally, a good vascular supply is essential to provide the cells and
nutrients for
growth12.
Bone grafts can be classified according to whether they provide cells,
signals, or scaffolds. Cancellous chips, hydroxyapatite, and demineralized
bone matrix are all osteoconductive materials that provide a scaffold on which
cells can grow. Osteoinductive agents provide the signals that drive bone
formation. For instance, BMPs encourage bone-healing through the endochondral
ossification pathway by promoting mesenchymal stem cell chemotaxis and
proliferation, the differentiation of osteoprogenitors, and
angiogenesis16.
Osteogenic materials such as autologous corticocancellous graft and
bone-marrow aspirate contain all of the necessary elements of bone formation
and are able to generate new bone without any additional elements from the
host11.
Gene therapy is a tool that can be used to deliver osteoinductive proteins
at a desired location. It may be a more efficient growth-factor delivery
system than are the current methods of rhBMP delivery, which have substantial
limitations, such as a short duration of action. The commercially available
products deliver BMPs with a type-I collagen carrier. There is an initial
burst of BMP release with a half-life of less than ten minutes, followed by a
second phase of gradual release with a half-life of between three and five
days27,28.
A more prolonged expression of BMP might enhance the fracture-healing process.
Gene therapy may offer a solution. Cells present within the body can be
genetically manipulated to produce growth factors in the area of interest.
This would provide a more physiologic delivery system, with continuous in vivo
production of protein at a relatively constant level for a sustained
period.
Stem-cell-based therapies are another emerging option for promoting bone
regeneration. Stem cells are defined by their distinct ability to self-renew
and to differentiate into multiple cell types. The cell that has been most
extensively studied for orthopaedic applications is the mesenchymal stem
cell29,30.
This is an adult stem cell that is found in tissues of mesoderm origin such as
bone marrow, adipose tissue, muscle, and skin. When exposed to the appropriate
growth factors, these multipotent cells can differentiate into chondrocytes or
osteocytes and may contribute to bone formation. Mesenchymal stem cells seeded
onto scaffolds such as hydroxyapatite have induced healing of critical-sized
bone defects in several animal
models31-33.
Moreover, percutaneous injection of bone-marrow aspirates has been used to
treat tibial nonunions with moderate
success34-36.
Connolly summarized his cumulative experience with 100 patients between 1986
and 199537. Eighty
percent of the patients displayed increased bone formation in response to
injected marrow preparations. The efficacy of injections of bone-marrow
aspirate seems to be influenced by the concentration and total number of
mesenchymal stem cells as well as by their osteogenic
potential36.
Hernigou et al. reported healing of fifty-three of sixty tibial nonunions
treated with concentrated bone-marrow
cells36. A
threshold concentration of >1000 progenitors/cm3 was required
for healing.
Stem-cell-based gene therapy may be another way to increase the power of
these techniques. Mesenchymal stem cells can be genetically modified to
overexpress osteogenic proteins such as BMPs. These growth factors stimulate
the mesenchymal stem cells to differentiate into bone-forming cells through
autocrine signaling, and they recruit host osteoprogenitors through paracrine
signaling12,38.
In vivo gene therapy is the direct administration of a vector to a patient.
The vectors can be delivered either systemically or locally, but only local
delivery has been used in preclinical fracture-healing models, to our
knowledge. The most common method is percutaneous injection of viral vectors
carrying the BMP cDNA, although alternative delivery methods have been
described39-41.
The primary advantage of an in vivo approach is ease of use. A one-step
procedure that can be carried out in a minimally invasive manner would be less
expensive than ex vivo therapy. The disadvantages are lower transduction
efficiency, lack of control over the target cell population, the risks
associated with direct viral inoculation such as a robust immune response, and
the potential for genetic recombination resulting in virulence.
The percutaneous injection of adenoviruses containing either the cDNA for
BMP-2 or BMP-6 has been used in preclinical fracture-healing
models13,42-45.
When injected at the fracture site, these BMP-containing adenoviruses cause
gene expression in the bone and surrounding soft tissues for up to six
weeks46. Baltzer et
al. created critical-sized femoral defects in a rabbit model and locally
injected adenoviral vectors carrying human BMP-2 cDNA (Ad-BMP-2)
44. The treated
animals displayed healing radiographically and histologically by twelve weeks,
whereas fibrous nonunions developed in control animals. Similarly, Betz et al.
injected Ad-BMP-2 into criticalsized femoral defects in rats and assessed
healing with plain radiographs, microcomputed tomography scans, histological
analysis, and strength testing at eight weeks after
surgery13.
Seventy-five percent (eighteen) of twenty-four treated femora displayed
osseous union on plain radiographs, and trabecular bone was reestablished
across the fracture site in 50% (twelve). The ability to establish
radiographic evidence of union is promising; however, the mechanical strength
of the treated limbs was only 25% of the strength of the control femora and
there was histological evidence of incomplete healing in half of the animals.
These observations indicate that it is important to look beyond radiographic
parameters when assessing the outcomes of gene-therapy-mediated
bone-healing.
The timing of the administration of the in vivo gene therapy may affect its
efficacy. Betz et al. injected Ad-BMP-2 into critical-sized femoral defects in
rats at zero, one, five, and ten days after the injury and reported that
delayed administration (carried out at five or ten days) resulted in a greater
prevalence of union as seen radiographically and increased mechanical strength
at the fracture
site45. A possible
explanation for these observations is that delayed delivery coincides with the
arrival of osteoprogenitor cells at the fracture
site47. This
finding may affect gene-therapy treatment of acute open fractures. However, it
would not be relevant to other applications such as the treatment of
established nonunions.
Many of the original in vivo experiments were performed with use of
first-generation adenoviral vectors that are easy to produce and are efficient
transducing
agents13,42-45.
However, these vectors also evoke a strong immune response to the adenoviral
proteins, which limits the duration of gene expression and may be hazardous
for the
patient20,25,26,48.
One possible way to optimize the results of in vivo strategies is by modifying
the viral vector. Researchers are working to develop less immunogenic vectors
such as the adeno-associated virus, which is a replication-deficient virus
that has no association with immunogenicity or human
disease49. Other
researchers have described "gutless" adenoviruses that are
produced by deleting the viral encoding
genes50,51.
There are also nonviral forms of in vivo gene therapy. There has been
substantial interest in the development of plasmid-based delivery systems
because no viral vectors are necessary. Gene-activated matrices, which are
scaffolds impregnated with cDNA plasmids, have been evaluated in preclinical
models. The concept is that the gene-activated matrix serves as a local
bioreactor by releasing plasmids that then transfect the local cell
population. The scaffold localizes the cDNA in the area of need, provides a
sustained release of plasmids as it degrades, and offers a surface for
cellular attachment. A variety of scaffolds, including collagen, demineralized
bone matrix, hydroxyapatite, and polylactic glycolic acid, have been
used39,40,52,53.
A comprehensive discussion of the characteristics of these materials is beyond
the scope of this review; however, it is important to recognize that they
differ from one another with respect to immunogenicity, inflammatory response,
degradation rate, and biologic activity.
Gene-activated matrices improve bone regeneration in rat femur and tibia
models. To our knowledge, Fang et al. were the first to use gene-activated
matrices for bone
repair41. They
implanted collagen sponges loaded with plasmids encoding BMP-4, parathyroid
hormone, or a combination of both into a 5-mm rat femur osteotomy site. The
control animals, treated with empty collagen sponges, displayed fibrous tissue
and minimal healing, whereas the groups treated with the gene-activated
matrices had osseous ingrowth. Furthermore, the combination of BMP-4 and
parathyroid hormone produced greater healing than did either agent alone.
Bonadio et al. coated collagen sponges with a human parathyroid hormone
plasmid and implanted them into canine tibial
defects39. The
gene-activated matrices transduced 30% to 50% of the cells at the fracture
site, resulting in gene expression for approximately six weeks. They induced
healing of 1-cm and 1.6-cm gaps with new bone; however, they did not induce
successful healing of a 2-cm critical-sized defect.
Compared with viral delivery systems, gene-activated matrices (like all
plasmid delivery methods) have low transfection efficiency, and strategies are
being developed to address this problem. Huang et al. described a method of
condensing DNA plasmids to facilitate cell
uptake40. When
loaded on scaffolds and placed in critical-sized rat cranial defects, the
condensed DNA generated greater amounts of bone ingrowth, osteoid deposition,
and mineralization compared with the amounts generated by noncondensed DNA
gene-activated matrices. Other methods of improving transfection efficiency
have been described as
well53,54.
Ultimately, the clinical utility of gene-activated matrices may be limited by
their poor transfection efficiency. The reliance on local cells to serve as
protein production bioreactors may reduce their efficacy in biologically
compromised situations where these cell populations are limited.
The paradigm of combining a gene delivery system and a scaffold can also be
applied to viral vectors. For instance, collagen scaffolds saturated with
Ad-BMP-2 induce robust bone formation when implanted subcutaneously in
rats55. Ito et al.
immobilized recombinant adeno-associated virus (rAAV) encoding vascular
endothelial growth factor (VEGF) and receptor activator of nuclear
factor-kappa B ligand (RANKL) onto the cortical surface of mouse femoral
allografts with a freeze-drying
process56.
Remarkably, rAAV retained its infectivity through freeze-drying and
rehydration. When placed in femoral defects, the coated grafts underwent a
process of incorporation (bone resorption, vascularization, and remodeling),
whereas uncoated allografts generated a foreign-body reaction and remained as
a necrotic graft that was susceptible to fracture. From a manufacturing
standpoint, the rAAV may be freeze-dried onto a surface and stored at
—80°C for prolonged periods of time, so it potentially could be
packaged as an off-the-shelf product.
Ex vivo gene therapy involves several sequential steps. Host cells are
harvested, expanded in tissue culture for a period of days, transduced to
express the gene of interest, and then reimplanted at the fracture site, where
they produce the desired protein. An advantage of ex vivo therapy is that
specific target cells can be selectively transduced; this is in contrast to in
vivo strategies, in which all of the cells in the fracture region are exposed
to the vector. Also, in vitro transduction of cells is more efficient than in
vivo transduction, and the patient does not incur the risks of a direct viral
inoculation.
The downside of ex vivo therapy is that the current preclinical models
require a two-stage procedure; autologous cells are harvested on one day and
reimplanted on another. This greatly increases resource utilization and cost.
An alternative is the development of allogeneic lines that are held in cell
banks. However, this introduces immunologic issues and is unlikely to become a
clinical reality. One-stage therapies (cell harvest, transduction, and
reimplantation) are being
developed57.
There are four critical components to any ex vivo gene therapy strategy:
the cell type, cDNA sequence, vector, and delivery system. The ideal cell
source should be easily harvested without substantial patient morbidity.
Furthermore, the cells must be expandable in culture, efficiently transduced,
and able to express the protein at sufficient levels. Bone-marrow aspirates,
skin fibroblasts, muscle-derived cells, and adipose-derived cells have all
been used in preclinical
models16,18,58-64.
These cell sources are easily accessed. Bone-marrow aspiration is a
well-described technique, and adequate amounts of muscle and skin fibroblasts
can be easily procured with a surgical
biopsy15,36,65.
Lipoaspirates can be obtained with liposuction or from the infrapatellar fat
pad58,66.
In the setting of ex vivo gene therapy, it has been hypothesized that cell
sources with the potential to express an osteoblast phenotype contribute to
fracture-healing through both paracrine and autocrine
mechanisms38. The
cells not only overexpress BMPs that stimulate host cells (paracrine), but
they themselves also respond to the BMP (autocrine) by undergoing osteogenic
differentiation and directly contributing to bone repair. This point was
demonstrated in a mouse radial defect model. When BMP-producing bone-marrow
cells were implanted at the fracture site, some of the cells incorporated into
the callus12.
Furthermore, Gazit et al. highlighted the potential benefit of using an
osteogenic cell
source38. They
transfected both mesenchymal stem cells and nonosteogenic Chinese hamster
ovary cells with BMP-2 and compared their ability to induce healing of mouse
radial defects. The mesenchymal stem cells were more effective even though the
Chinese hamster ovary cells actually produced greater amounts of BMP-2.
Intuitively, it seems reasonable to use an osteogenic cell source. However,
further research is needed to determine if the autocrine contribution to bone
formation is clinically relevant. It may be that the cell source only needs to
function as a protein delivery vehicle. Presently, it is not known if there is
an optimal cellular delivery vehicle.
The next critical component is the selection of cDNA sequences.
Gene-therapy approaches to bone-healing have focused on the delivery of BMPs,
especially BMP-2. However, within the BMP family there are over thirty growth
factors with varied osteogenic
capabilities67-69.
Kang et al.70 and
Li et al.71
compared the abilities of multiple BMPs to induce ectopic bone formation in
murine models and reported that BMP-6 and BMP-9 were most effective.
Additional work is needed to determine which BMPs are best suited for
gene-therapy applications. Also, limited work has been done to explore other
growth factors that might be used in gene-therapy strategies, such as
platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and
VEGF, to name a few.
Several studies have shown that a combination of growth factors may be more
effective than a single
gene17,18,41,72,73.
For instance, BMP-2 and BMP-7 are more effective when delivered in
combination72,73.
Also, BMP-induced bone formation is potentiated by adding ancillary genes that
promote other aspects of
bone-healing17,18,41.
This is intuitive since physiologic bone formation involves multiple cellular
and biochemical pathways. Peng et al. showed that VEGF interacts
synergistically with BMP-2 and
BMP-417,18.
VEGF is a potent angiogenic stimulator that contributes to endochondral
ossification by promoting cartilage resorption and mineralization. In a mouse
calvarial bone-defect model, bone formation more than doubled when mesenchymal
stem cells expressed both VEGF and BMP-4 rather than BMP-4
alone18. In a
follow-up study, Peng et al. showed a similar relationship between BMP-2 and
VEGF17.
Interestingly, the ratio of VEGF to BMP is very important. At high levels,
VEGF is actually counterproductive with regard to bone-healing. This work
demonstrates that gene-therapy strategies can be maximized by identifying
other genes that interact synergistically with BMPs and optimizing the dosing
regimens.
The aforementioned studies focused on transferring cDNA sequences that
encode for growth factors; an alternative strategy is transcription
factor-based gene
therapy57,74-78.
These proteins can regulate the expression of multiple genes within a pathway
and therefore might represent a more efficient mechanism to "turn
on" the bone-healing process. LIM mineralization protein-1 is an
intracellular signaling molecule involved in osteoblast differentiation, and
it upregulates the expression of multiple
BMPs74. The LIM
mineralization proteins can promote bone formation in rabbit and murine
models57,79,80.
Sonic hedgehog, a secreted factor involved in bone formation, has been used
successfully in an ex vivo calvarial bone-defect
model81. The
concern regarding this strategy is that we do not understand all of the
downstream genes upregulated by this signaling pathway and this may lead to
unforeseen biologic responses.
Once the cDNA sequences have been selected, there are many vectors
available to transfect the ex vivo cells. The choice of vector affects the
duration of protein expression, the safety profile, and the efficiency of
transduction or transfection. Adenovirus has been used most frequently;
however, it evokes a strong immune response that may limit the duration of
gene expression and pose safety
hazards20,25,26,48.
For these reasons, there is interest in developing alternative vectors, such
as adeno-associated viruses that are modified to be less
immunogenic49. The
lentivirus has also emerged as an attractive alternative because it provides
long-term gene expression and has minimal
immunogenicity82-84.
Rat bone-marrow cells transduced with lentivirus express BMP for up to twelve
weeks in vitro and at least four weeks in
vivo82
(Fig. 2). Furthermore,
experiments suggest that the sustained production of BMP induced by lentivirus
transduction restores the biomechanical characteristics of bone more
effectively than does adenoviral
transduction82,85
(Fig. 3). For this reason,
lentivirus may prove useful for the treatment of larger defects.
Naked DNA and liposomes are two nonviral options for ex vivo strategies
that are receiving increased attention. Nonviral approaches are appealing
because they pose less of a safety risk; however, these techniques have poor
transfection efficiencies. Nucleofection, a new technique based on
electropermeabilization, has improved transfection
efficiency86,87.
Aslan et al. found that human mesenchymal stem cells expressed BMP-2 and BMP-9
for two to three weeks following nucleofection and were able to induce ectopic
bone formation when implanted in
mice87. Further
work needs to be done to determine if nucleofection is a viable alternative to
viral transduction strategies.
Once the cells have been transduced or transfected, they are combined with
a cellular delivery vehicle, typically a scaffold, and reimplanted at the
desired location. The scaffold serves multiple functions; it contains the
cells at the desired location, provides protection, and contributes to the
bone-healing process by providing a surface for cellular attachment and
mineralization. The scaffold also imparts physical characteristics that
facilitate molding, handling, and surgical application. When choosing a
cellular delivery system, one must consider biomaterial properties such as
porosity, degradation characteristics, and surface chemistry as well as
biologic properties such as the inflammatory response and the ability of the
scaffold to support cell attachment, bone ingrowth, matrix deposition, and
angiogenesis.
Type-I collagen-based scaffolds are attractive because the protein is
abundant in bone, promotes mineralization, binds noncollagenous matrix
proteins, and has a well-established safety
profile11,88,89.
There are multiple other options, including biologic scaffolds (hyaluronic
acid-based carriers or demineralized bone matrix), porous ceramics
(hydroxyapatite and tricalcium phosphate), or biodegradable polymers
(poly-l-lactide and
poly-l-lactide-co-glycolide)90.
Numerous studies have demonstrated the ability of ex vivo gene therapy to
promote bone formation and promote healing of critical-sized defects in animal
models15,17-19,38,58,60,81,82,87,91-95
(Table I). One of us (J.R.L.)
and colleagues used an ex vivo gene transfer strategy to induce healing of
8-mm femoral defects in a rat
model16.
Bone-marrow cells were harvested from rats, expanded in tissue culture,
transduced with an Ad-BMP-2 vector, and then implanted in critical-sized
femoral defects (Fig. 4).
Histomor-phometric analysis showed the pattern of bone formation in the
femoral defects treated with the BMP-2-producing bone-marrow cells to be more
robust than the pattern in defects treated with recombinant BMP-2 protein. It
was hypothesized that the more robust bone formation associated with the
BMP-producing bone-marrow cells could be the result of the more continuous and
physiologic release of the BMP as well as the combined autocrine/paracrine
mechanism. Similar results were obtained when transduced adipose-derived stem
cells were used in a cellular delivery
vehicle60.
Shen et al. used muscle-derived cells to promote healing of critical-sized
defects in a rat
model19. Muscle
cells were harvested, cultured, transduced with Ad-BMP-4, seeded onto collagen
sponges, and reimplanted into a 7-mm femoral defect. By twelve weeks
postoperatively, twelve of thirteen defects displayed radiographic signs of
union and histological evidence that the proximal and distal cortices had
remodeled and the medullary canal was reestablished. Biomechanical testing
showed that the strength of the treated femora had returned to 77% of normal.
The control animals received muscle cells transduced with a nonosteogenic
gene, and a fibrous nonunion developed in all of them.
Transferred cDNA segments include the gene of interest (i.e., BMP) and also
a promoter region that controls expression. Classically, gene therapy for the
promotion of bone-healing has featured viral promoters that are constitutively
active. Therefore, once a cell has been transduced, the transferred gene is
constantly expressed for either the life span of the cell or for as long as
the transferred gene persists. It would be advantageous to have more control
over gene expression, so that gene expression could be stopped after
bone-healing is complete.
Regulated gene therapy is an emerging strategy that aims to provide safer
and more precise gene therapy by controlling the duration, temporal sequence,
and magnitude of transgene expression. One such approach is to construct
vectors that have inducible promoter regions such as a tetracycline-sensitive
promoter96-98.
This allows gene expression to be either turned on (Tet-On) or off (Tet-Off)
by exogenously administering tetracycline or its analog doxycycline. In a
murine model, researchers have shown that BMP expression and consequent bone
formation can be controlled by using vectors with Tet-On and Tet-Off
promoters93,96,97.
Physiologic bone formation is partially regulated by the dynamic
interaction between agonist and antagonist molecules. For instance, BMP-4 and
its antagonist noggin demonstrate coordinated expression during embryogenesis
and
skeletogenesis59.
To date, most gene therapy applications have neglected the potential
importance of recreating this agonist-antagonist relationship. This may
explain observations of bone overgrowth or exuberant callus formation in
association with some gene-therapy applications. Peng et al. reported that the
combined delivery of both BMP-4 and noggin is more effective for inducing bone
formation than is delivery of BMP-4
alone59.
Critical-sized calvarial defects were created in a murine model and implanted
either with mesenchymal stem cells expressing both BMP-4 and noggin or with
BMP-4 alone. The combined approach more accurately replicated the architecture
and anatomy of normal bone, whereas delivery of BMP-4 alone resulted in
overabundant bone formation that exceeded the boundaries of the original
defect.
Preclinical studies have demonstrated that gene therapy has great potential
to promote fracture-healing. However, in order for this technology to become a
clinical reality, several obstacles must be addressed. First, cost-effective
and clinically feasible delivery systems need to be developed. Utilization of
gene therapy should not require the surgeon or hospital to develop a
specialized skill set. Either an off-the-shelf product or a single-stage ex
vivo strategy would be
ideal57. A
potential option is a two-stage technique that involves cell harvest, cell
expansion, and then reimplantation of the transduced cells. Although this
strategy is similar to the current approach used for autologous chondrocyte
implantation, it may not be cost-effective.
Second, further advances in the understanding of the biologic aspects of
gene therapy are needed. Efficacy will be increased by optimizing the four
critical components: cells, cDNA, vector, and delivery mechanism. Presently,
there are four viable cell sources: bone-marrow, muscle, adipose-derived stem
cells, and skin fibroblasts. Research is needed to determine if any of them
are superior for particular applications. With respect to cDNA, most
preclinical models have used BMPs. Authors of future studies will need to
investigate alternative growth factors, compare the individual BMPs, and
develop combination therapies. At present, viral vectors are the most viable
option. Adenovirus and retrovirus have been used in many preclinical models.
Alternative vectors, such as lentivirus and adeno-associated virus, that offer
long-term gene expression require further study.
In the future, gene therapy may be one option within a comprehensive
tissue-engineering strategy for fracture treatment. We envision that it will
be available for the most difficult clinical situations such as massive bone
loss and recalcitrant nonunions. As gene therapy approaches clinical
application, enthusiasm should be tempered by caution and due diligence
regarding patient safety. ?
Praemer A, Furner S, Rice DP, editors.
Musculoskeletal conditions in the United States. Rosemont, IL:
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