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From: Bone Regeneration and Repair: Biology and Clinical Applications Edited by: J. R. Lieberman and G. E. Friedlaender © Humana Press Inc., Totowa, NJ
9
Gene Transfer Approaches to Enhancing Bone Healing
Oliver Betz, PhD, Mark Vrahas, MD, Axel Baltzer, MD, Jay R. Lieberman, MD, Paul D. Robbins, PhD, and Christopher H. Evans, PhD
THE CLINICAL NEED FOR NEW METHODS TO ENHANCE BONE HEALING
Although bone is one of the few organs in the body that can heal spontaneously and restore func- tion without scarring, it has been recognized since the time of Hippocrates that repair is not always satisfactory. Bone healing is inadequate when the loss of bone through, for example, tumor resection or traumatic injury, is extensive enough to produce a critical-sized defect. Healing may also be impaired in much smaller defects, and nonunion following fracture occurs in 5–10% of cases (1–3).
Beginning with the pioneering experimental studies of John Hunter in 18th-century London, non- invasive approaches to the problem, such as splinting, were superceded by surgical methods to enhance bone healing. Recent decades have seen significant advances in the way orthopedic surgeons treat prob- lems in bone healing. In particular, improved handling of soft tissues and the development of advanced methods of fixation using closed techniques have led to greater rates of success (4). Moreover, heal- ing has been greatly improved by the introduction of autografting, which has become the gold standard of repair for osseous defects. However, this exposes patients to additional surgical procedures with their associated morbidity, and the amounts of bone available for autografting are limited. Allograft- ing avoids this, but raises concerns about the transmission of disease, harvesting and storage of donor tissue, and possible immune reactions (5,6). Moreover, bone allografting has a failure rate of 30% or higher (7).
BIOLOGICAL APPROACHES TO BONE HEALING
The need to improve the clinical response has led to greater interest in the biology of bone healing with the notion that, if we understood natural osteoregenerative processes, it should prove possible to harness them for clinical use. Best understood are the rodent fracture repair models pioneered by Einhorn and colleagues (8). They have helped identify five stages of endochondral healing. Initially there is a hematoma and inflammation, which is superceded the formation of a cartilaginous callus, later invaded by blood vessels as it calcifies, resorbs, and becomes replaced by bone. Different genes are expressed at different stages of this process. In the mouse, type II collagen and aggrecan, which signal the formation of a cartilaginous callus, appear approx 9 d after fracture. One of the first indi- cations of the osteogenic process within callus is the expression of type I collagen, followed by the early osteogenic markers alkaline phosphatase, osteopontin, and osteonectin. Subsequent matrix min- eralization is associated with expression of type X collagen, bone sialoprotein, and osteocalcin (9).
Additional research into the biology of bone formation has identified several potent osteogenic proteins (10,11). The best studied of these are the bone morphogenetic proteins (BMPs), which, at
nanomolar concentrations, powerfully induce new bone formation both within osseous lesions and at ectopic sites, such as skeletal muscle (12–15). The US Food and Drug Administration has recently approved recombinant, human bone morphogenic proteins BMP-2 and BMP-7 for restricted clinical use. Although these are potent osteogenic agents, their clinical application is complicated by delivery problems (16). The main limitation is the need for delivery systems that provide a sustained, biologi- cally appropriate concentration of the osteogenic factor at the site of the defect. Delivery needs to be sustained, because these factors have exceedingly short biological half-lives, usually of the order of minutes or hours, rather than the days or weeks needed to stimulate a complete osteogenic response.
Delivery also needs to be local to avoid ectopic ossification and other unwanted side effects.
Because systemic delivery by intravenous, intramuscular, or subcutaneous routes fails to satisfy these demands, there has been much interest in developing implantable slow-release devices from which the BMP can progressively leach. Typically, such devices comprise a biocompatible matrix impreg- nated with very large amounts of recombinant BMP; in the clinic they are most frequently used with autologous bone grafts. The device is surgically implanted at the site of the defect and thus satisfies the need for local delivery. However, release is not uniform over time. In most cases, there is an initial rapid efflux (“dumping”) of the protein, which spikes the surrounding tissue with wildly supraphysiological concentrations of growth factor. Subsequent release, although slower, provides much lower, subopti- mal concentrations of protein. Another drawback is the denaturation of the growth factor at body tem- perature before it is released from the matrix. Moreover, the carrier, usually bovine collagen, can pro- voke inflammation. Clearly, such systems, although capable of increasing osteogenesis, are clumsy and inefficient (16,17). Research into the genetic manipulation of bone healing is based on the hypothesis that gene transfer can do better.
GENE THERAPY APPROACHES TO ENHANCING BONE HEALING
Advances in gene transfer technology provide the opportunity to overcome the technical limita- tions described above (18–20). The concept, shown in Fig. 1, is to transfer genes encoding osteo- genic factors to osseous lesions. When the transgene is expressed, the lesion becomes an endogenous, local source of the factors needed for bone healing. Thus the gene transfer approach offers great poten- tial as a delivery system that meets the requirement of sustained and local delivery of the growth fac- tor at the appropriate concentrations. Moreover, unlike the recombinant protein, the growth factor synthesized in situ as a result of gene transfer undergoes authentic posttranslational processing and is presented to the surrounding tissues in a natural, cell-based manner. This may explain why gene delivery is often more biologically potent than protein delivery. A good example of this from another area of gene therapy research is provided by the work of Makarov et al. (21), who have shown that the treatment of arthritic rats with cDNA encoding the interleukin-1 receptor antagonist is 104 times more potent than treatment with the corresponding recombinant protein. Similar gains in potency may be achieved by local delivery of osteogenic genes to sites of osseous defect. The use of gene transfer to enhance bone repair has been previously reviewed in refs. 18, 19, and 20).
A GENE TRANSFER PRIMER
Because cells do not spontaneously take up and express exogenous genes, successful gene transfer requires vectors. These can be divided into those that are derived from viruses and those that are not.
The properties of the most advanced viral vectors are listed in Table 1. With the exception of lenti- virus, all of these have been used in human clinical trials.
Retroviral vectors have the ability to integrate their genetic material into the chromosomal DNA of the cells they infect. This is a major for advantage for settings where long-term transgene expres- sion is required. However, because the insertion site is random, there is a possibility of insertional mutagenesis. Although this possibility is extremely low, the first instances of insertional mutagenesis
are now emerging from human clinical trials (23), and this has resurrected huge concerns about the safety of these vectors.
Because genetically enhanced bone healing should not require long-term transgene expression, use can be made of nonintegrating vectors such as adenovirus and adeno-associated virus (AAV). Both of these are DNA viruses that deliver genes episomally to the nuclei of the cells they infect. The most com- monly used adenovirus vectors (so-called first-generation adenovirus vectors) have the advantage of being straightforward to construct and produce at high titers. They readily infect a wide range of divid- ing and nondividing cells, and usually achieve high levels of transgene expression. The big drawback of adenovirus vectors is the high antigenicity of both the virions themselves and cells infected with first-generation adenovirus. The latter problem can be eliminated by using a third-generation, so-called gutted adenovirus vector that contains no viral coding sequences, but these are difficult to manufacture.
Moreover, the antigenicity of the virions is not reduced by removing viral DNA. It remains to be seen whether immune reactions limit the clinical use of adenovirus in human bone healing.
AAV is far less antigenic than adenovirus and causes no known disease in humans. Recombinant AAV vectors are of great current interest because of the perception that they are very safe. However, they are difficult to make and they do not infect all cell types well. Their carrying capacity is limited to about 4 kb, but this is probably adequate for the types of cDNAs needed to promote bone healing.
As far as it is possible to tell, AAV seems to infect both dividing and nondividing cells.
Vectors derived from herpes simplex virus are difficult to manufacture, often cytotoxic, and of little immediate and obvious utility to bone healing at the present time.
Nonviral vectors (Table 2) can be as simple as naked, plasmid DNA. To enhance gene transfer effi- ciency, the DNA can be associated with carrier molecules such as various types of liposomes and syn- thetic or natural polymers. There is also interest in using physical techniques, such as electroporation, Fig. 1. Schematic representation of ex vivo and in vivo gene therapy strategies for enhancing bone healing.
(From ref. 18.)
Table 1
Common Viral Vectors and Their Salient Properties
Vector Key properties Comment
Oncoretrovirusa Inserts DNA into host chromosome Requirement for cell division usually (retrovirus) Insertional mutagenesis a safety issue limits use to ex vivo protocols
Packaging capacity ~8 kb Commonly derived from Moloney Only transduces dividing cells murine leukemia virus
Straightforward to manufacture Human use has been associated with
Medium titers leukemia
Lentivirusa Inserts DNA into host chromosome Commonly derived from HIV (retrovirus) Insertional mutagenesis a safety issue Not yet used in human clinical trials
Packaging capacity ~8 kb
Transduction not limited by cell division Moderately difficult to manufacture Medium titers
Adeno-associated W.t. inserts DNA into host chromosome Generally considered to be the safest virus (AAV) —a rare event with recombinant AAV of the viral vectors
vectors In clinical trials
Packaging capacity ~4 kb
Not all cell types are readily transduced Manufacture very difficult
Adenovirus Noninsertional Ease of production, high infectivity,
First- and second-generation vectors, and wide tropism ensure common packaging capacity ~8 kb experimental use, especially for Both virus and cells transduced by early- in vivo gene delivery
generation vectors are highly antigenic Human use has been associated with
High infectivity one death
In vivo use associated with inflammation Transduction not limited by cell division Straightforward to manufacture at high titer
Herpes simplex Noninsertional Major clinical application may be in
virus Very large packaging potential the CNS, where it has a natural
Often cytotoxic tropism and latency
High infectivity
Transduction not limited by cell division Very difficult to manufacture
High titers possible
aBoth oncoretrovirus and lentivirus are members of the Retroviridae family.
to improve gene transfer efficiency. Nonviral vectors are usually cheaper and safer than viral vectors, but far less efficient. Gene transfer with nonviral vectors is known as transfection. Gene transfer with viral vectors is known as transduction.
Regardless of the vector, genes may be transferred to sites in the body by ex vivo or in vivo strate- gies (Fig. 1). Other things being equal, in vivo methods are simpler, cheaper, and more expeditious, because they involve no extracorporal manipulation of the target cells. However, they raise greater safety concerns. Ex vivo methods do not involve the direct introduction of vectors into the body, and allow the target cells to be isolated, manipulated, tested, and optimized before reimplantation. Under conditions where soft tissue support for osteogenesis is compromised, ex vivo protocols allow the introduction of genetically modified osteoprogenitor cells to enhance repair.
More detailed reviews of gene therapy in an orthopedic context are to be found in refs. 24–28.
EX VIVO GENE TRANSFER
Nearly all investigators in this area have used the ex vivo approach pioneered by Lieberman and colleagues (29,30). Using a rat critical-sized-defect model, Lieberman’s group employed a recom- binant, first-generation adenovirus to transfer a human BMP-2 cDNA to osteogenic stromal cells recovered from bone marrow. This population of cells probably includes mesenchymal stem cells (MSCs). Under the transcriptional regulation of the human cytomegalovirus early promoter, the trans- duced cells expressed high levels of human BMP-2. These cells were seeded onto a collagenous matrix and surgically implanted into critical-sized defects. Under conditions where control defects failed to heal, defects receiving the genetically modified cells reproducibly achieved osseous union (29,30) (Fig. 2).
BMP-2 gene therapy produced a better response than recombinant BMP-2 protein in healing osse- ous defects in rats. Although both approaches led to osseous union, the recombinant protein gener- ated atypical new bone filled with lacey, delicate trabeculae, which formed a shell around the defect.
The gene transfer method, in contrast, led to new bone with an authentic three-dimensional trabecu- lar structure, remodeling to form a neocortex (30).
Table 2
Common Types of Nonviral Vectors Naked DNA
DNA combined with cationic and anionic liposomes (many different formulations) DNA–protein complexes (many different formulations)
DNA–polymer complexes (many different synthetic and natural polymers) Electroporation
Ballistic projection (“gene gun”)
Fig. 2. Healing of rat segmental bone critical-sized defect by ex vivo BMP-2 gene transfer. Animals were sacrificed 2 mo postoperatively and were treated in one of the following ways: (A) BMP-2 producing bone marrow cells created via adenoviral gene transfer; (B) 20 μg of rhBMP-2; (C) -galactosidase-producing bone marrow cells (cells infected with an adenovirus containing lacZ gene); (D) noninfected rat bone marrow cells;
or (E) guanidine-extracted demineralized bone matrix alone. Dense trabecular bone formed within the defects that had been treated with the BMP-2-producing cells, and the bone remodeled to form a new cortex. The defects that had been treated with rhBMP-2 healed but were filled with lacelike trabecular bone. Minimal bone repair was noted in the other three groups. (From ref. 30 with permission.)
Subsequent investigators have confirmed the success of the ex vivo approach using cells derived from skin, muscle, fat, and peripheral blood using, in addition to BMP-2, other osteogenic proteins such as BMP-4 and BMP-7 (31–36). In common with marrow-derived osteoprogenitors, cells derived from muscle, fat, and, according to Krebsbach et al. (37), even skin fibroblasts, have the ability to differen- tiate into bone under the influence of appropriate biological cues. Thus, when genetically modified, they aid osteogenesis not only as a local source of osteogenic factors, but also as an additional source of osteoprogenitor cells that enhance repair through both paracrine and autocine processes. The notion that mature fibroblasts can transdifferentiate into osteoblasts is unfamiliar, but the utility of fibro- blasts is supported by the recent work of Gugala et al. (38). These investigators compared the osteo- genic properties of human MSCs, human skin fibroblasts, and the human fetal lung cell line MRC-5.
Cells were transduced with adenovirus carrying BMP-2 cDNA and injected intramuscularly into immu- nodeficient mice. There was no statistically significant difference in the amount of bone formed by the three different types of human cells.
Among tissues other than skin that may contain osteoprogenitor cells, fat could be the most conve- nient for eventual human application. Most individuals are more than happy to donate adipose tissue, which is readily biopsied; adipose-derived stem cells are straightforward to culture, can be easily expanded, and transduced. Moreover, their abundance and proliferative properties do not appear to decline with the age of the donor. According to a recent paper by Dragoo et al. (34), fat provides a richer source of osteoprogenitor cells than bone marrow, and, when genetically modified to express BMP-2, they are more efficient osteoprogenitors. These cells are also able to heal large segmental defects in rats (Lieberman et al., unpublished).
Despite the above successes, the use of first-generation adenovirus vectors remains a concern because the cells it transduces express viral proteins and thus become antigenic. Several strategies are being employed to obviate this concern. One is to make adenoviral transduction of MSCs more efficient. The major cell-surface receptor for the most commonly used recombinant adenovirus vector, serotype 5, is the Coxsackie and adenovirus receptor (CAR). It is poorly expressed on MSCs, thus requiring very high multiplicities of infection; even then, only about 20% of the cells are transduced (39). Tsuda et al.
(39) have used modified adenovirus whose coat carries the tripeptide sequence RGD, which enhances interaction with cell-surface integrins and thus engenders greater uptake. Cells transduced with the modified virus produce greater amounts of BMP-2 and are more osteogenic in vivo. It should thus be possible to reduce the antigenic load by administering fewer modified MSCs. A similar, alternative approach uses serotype 35 adenovirus, that enters cells in a CAR-independent fashion (40).
Although the above strategies may reduce the antigenic burden, they will not eliminate it. For this reason there is interest in using vectors that express no foreign, antigenic proteins. Abe et al. (41) have successfully used a “gutted” adenovirus for this purpose. Recombinant retrovirus is also successful in animal models (42), although, as discussed above, there are renewed concerns about the safety of such vectors. AAV is another candidate vector that has shown success when delivered in vivo (43,44) (see next section). Avoiding viral vectors altogether, Park et al. (45) used liposomes to transfect MSCs and heal mandibular defects in rats, by an ex vivo strategy. Healing with liposome gene delivery was slower than healing with adenoviral vectors, but was otherwise indistinguishable. Given the resistance of MSCs to transfection, this result is quite remarkable.
A major drawback of ex vivo gene delivery is the need to culture autologous cells from each patient. There is thus interest in using allogeneic cells so that a universal donor could be established.
This endeavor is encouraged by the possibility that MSCs can be successfully allografted. However, in a rat segmental-defect model, allogeneic MSCs transduced with BMP-2 healed the defect only if the immunosuppressant FK 506 was administered (46). Although the need for FK 506 was only transient, its clinical use in bone healing may raise difficult safety concerns. Transient immunosup- pression has also been used experimentally for the in vivo delivery of osteogenic genes (47,48) (see next section).
IN VIVO GENE DELIVERY
Two in vivo strategies have emerged. One involves the implantation of plasmid DNA incorporated into a collagen sponge (gene activated matrix, GAM). The other involves the direct injection of vector.
GAM technologies were developed by Bonadio and Goldstein (49,50), and have the advantage of using plasmid DNA. The GAM is stable upon storage, and is surgically inserted directly into the osseous lesion. Cells from the area of the lesion migrate into the matrix, where they encounter, take up, and express the DNA. GAMs containing plasmids encoding PTH 1-34 and BMP-4 healed 5-mm femoral defects in rats that would not otherwise heal (49). When used in a critical sized tibial defect in dogs, a GAM containing PTH 1-34 cDNA resulted in 6 wk of transgene expression. Although impres- sive amounts of new bone were deposited in response, they were insufficient to heal the defect (50).
Human clinical trials are pending.
One of the advantages of adenoviral vectors is their ability to infect cells in situ, a property com- patible with in vivo gene delivery. Most investigators have avoided in vivo gene delivery for bone healing, because the intramuscular injection of adenovirus vectors containing osteogenic genes leads to very little bone formation. The problem appears to lie with the immune response to the adenovirus, because considerable bone formation occurs when immunodeficient animals are used (51), or when an immunosuppressant, such as cyclophosphamide, is administered (47,48).
Nevertheless, Baltzer et al. were able to heal critical-sized defects in the femurs of immunocom- petent rabbits by the direct, intralesional injection of adenovirus carrying a BMP-2 cDNA (Fig. 3).
Studies were conducted with a rabbit femoral critical-sized (1.3-cm)-defect model (52). Injection of a first-generation adenovirus vector carrying the human BMP-2 cDNA into such defects produced osseous union, judged radiologically and histologically, under conditions where control defects receiv- ing an irrelevant gene failed to heal (53). Injection of similar vectors carrying marker genes showed that the greatest expression of the transgene occurred in the musculature surrounding the defect, with significant expression also occurring in the gap scar and the cut ends of the bone. Marker gene expres- sion was observed in marrow cells and lining osteoblasts. Lung, liver, and spleen were also sampled.
There was transient transgene expression in the liver, but not elsewhere (52).
The direct injection of Ad.BMP-2 also heals critical-sized femoral defects in rats (Betz et al., unpub- lished), further supporting the notion that the intraosseous environment, unlike the intramuscular one, supports osteogenesis in response to adenoviral delivery of an osteogenic transgene to immunocompe- tent animals. The critical difference may involve the degree to which the immune system and inflam- matory responses are activated. The key question of whether redosing of the same osteogenic adeno- virus will continue to promote bone formation has not yet been addressed.
The immune response to adenovirus may be further blunted by delivering the virus in conjunction with a collagenous matrix in a modified GAM strategy. Both Franceschi et al. (35) and Sonobe et al.
(54) have used this tactic successfully to form bone intramuscularly and subdermally in immunocom- petent rodents. The adenoviral burden may be also be reduced by using more effective serotypes of adenovirus (40), or administering the virus at times when its receptor is maximally expressed. The CAR used by the type 5 adenovirus is induced upon fracture and, in mice, its expression peaks at d 5 (55). The tactic of transient immunosuppression also works experimentally (47,48), but its clinical applicability is questionable.
As an alternative to adenovirus, recombinant AAV vectors carrying BMP-2 (43) or BMP-4 (44) elicit bone formation after direct injection. Transcutaneous electroporation of plasmid carrying BMP-2 cDNA also stimulates bone formation in muscle (56).
WHICH GENES?
Most experiments have focused on the use of transgenes expressing BMPs. Of this group, BMP-2 and BMP-7 cDNAs have advantages for overcoming the regulatory barriers to clinical application,
because the corresponding recombinant proteins have been widely tested in humans and shown to be safe and somewhat effective. Recent research by Helms’s group, however, suggests that BMP-6 and BMP-9 cDNA are more effective osteogenic agents when delivered by adenovirus vectors (57).
Growth factors are not the only class of gene product capable of eliciting bone formation. Boden’s group has identified a transcription factor, LMP-1, that promotes osteogenesis at tiny concentrations (36,58). Because LMP-1 acts intracellularly, gene transfer is a particularly pertinent delivery system for this protein, although advances in peptide delivery are also providing new avenues. The remark- able potency of LMP-1 is at least partially explained by its ability to induce expression of multiple, different BMPs and other osteogenic factors, thus providing a rich osteogenic environment within the osseous lesion (59).
The value of combining factors has been demonstrated in a rat calvarial defect model, where heal- ing was greater when BMP-4 and VEGF transgenes were coexpressed than when either was expressed alone (60).
The types of gene products of potential use in the gene treatment of osseous lesions are listed in Table 3.
Fig. 3. Healing of rabbit segmental bone critical-sized defect by in vivo BMP-2 gene transfer. Defects were treated with Ad.BMP-2 (panels A–D) or Ad.luciferase (panels E–H) and radiographed at the time of surgery (panels A and E) and after 5 wk (panels B and F), 7 wk (panels C and G) and 12 wk (panels D and H). Defects treated with Ad.BMP-2 undergo osseous union, as judged radiologically, whereas those treated with Ad.luci- ferase do not. (From ref. 53 with permission.)
APPLICATIONS
Gene transfer has numerous applications under circumstances where it is necessary to form bone.
Long bone fractures, non- and delayed unions, as well as segmental defects, are obvious examples that have attracted the most experimental attention.
Spine fusion is another area of considerable interest, and progress has been made in the use of an abbreviated ex vivo procedure in which adenovirus carrying LMP-1 cDNA is used to transduce buffy coat cells from peripheral blood intraoperatively (36). The cells are applied to a collagenous matrix and implanted. This procedure is effective in rabbits, and is now being evaluated in nonhuman primates.
Successful spine fusion in a rabbit model has also been achieved with the used of MSCs expressing a BMP-2 transgene (61). Percutaneous injection of adenovirus carrying cDNA for BMP-2 or BMP-9 induces spine fusion in athymic, but not immunocompetent, rodents (33,62,63).
There are also many applications in the cranial and maxillofacial areas. There are numerous expe- rimental examples of healing cranial lesions in rodents using gene transfer. Chang et al. (64) have recently described the repair of large maxillary defects in pigs using BMP-2 gene transfer.
The need to form bone sometimes arises under circumstances where it is necessary not only to form new bone via osteoblasts, but also to prevent bone loss via osteoclasts. Aseptic loosening pro- vides one such example. An appropriate strategy in these conditions is to express genes whose prod- ucts inhibit the activities of the cytokines that promote bone loss (65–68). Discussion of this aspect is beyond the scope of this chapter, but overlaps with gene treatment of inflammatory diseases such as rheumatoid arthritis, reviewed in refs. 69–72.
CONCLUSION
Collectively, the preclinical data provide strong experimental support for the proposition that gene transfer provides a powerful method for healing osseous defects that will not otherwise heal.
However, although the application of gene therapy to clinical problems associated with bone healing has a persuasive logic and accumulating experimental support, there is a pressing need for transla- tional studies that convert preclinical concepts and findings into clinically useful modalities. Many fundamental questions still need to be answered, including which gene or gene combinations to use, whether to use in vivo or ex vivo delivery, and which vectors to employ. There has been little work in large animal models, and safety issues remain to be addressed. The latter is of particular importance as, for the majority of prospective patients, the procedure will be elective and the condition not life- threatening.
Table 3
Classes of Gene Products of Potential Use for Bone Healing
Class Examples Comment
Growth factors BMP-2,-4,-7,-9 Perform well in animal models.
IGF-1 TGF-1–3 PDGF
Transcription factors LMP-1, Cbfa-1 Intracellular site of action compatible with gene transfer.
LMP-1, very potent.
Angiogenic factors VEGF; FGF May act synergistically with other factors.
Antiinflammatories sTNFR Of potential use under conditions of excessive bone sIL-1R resorption, e.g., aseptic loosening.
IL-1Ra
Osteoclast blockers Osteoprotegerin Good results in models of aseptic loosening.
Given the numerous different clinical circumstances under which it is necessary to promote bone formation, there will probably be no single preferred method. Not all patients will require gene therapy, and not all gene therapies will be the same. Depending on circumstances, different vectors, genes, and strategies will be indicated.
One advantage of bone healing as a target for gene therapists is the existence of a robust, natural repair process, and the observation that, at least in animal models, healing is very responsive to mod- erate levels of gene expression for a limited period of time. Thus clinical success may be achieved with existing gene therapy technologies. This is not the case for most other areas of gene therapy.
ACKNOWLEDGMENTS
The authors’ work in this area has been supported by the Orthopaedic Trauma Association (CHE), National Institutes of Health grant number AR 050243 (CHE).
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