Gene therapy approaches for modulating bone regeneration

Advanced Drug Delivery Reviews 42 (2000) 121–138

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Gene therapy approaches for modulating bone regeneration Shelley R. Winn a , *, Yunhua Hu a , Charles Sfeir b , Jeffrey O. Hollinger a a

Department of Surgery, School of Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA b Department of Periodontology and Oral Molecular Biology, School of Dentistry, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA

Abstract Following injury, bone has the ability to regenerate itself to a form and function nearly indistinguishable from the pre-injury state. However, if the injury is beyond a critical limit, recovery will not occur without therapeutic interventions. Autografts and implants with banked bone continue as the treatments of choice, although each exhibits limitations and liabilities. Alternatives have included the utilization of bone-graft substitutes that may incorporate bone derivatives and soluble signaling molecules such as mitogens and morphogens. In addition, an evolving treatment modality, gene therapy, offers an exciting avenue for bone regeneration. This review presents some of the current concepts for developing a rational gene therapy approach in bone regeneration.  2000 Elsevier Science B.V. All rights reserved. Keywords: Gene therapy; Fracture repair; Poly(alpha-hydroxy) acids; Collagen; Tissue engineering; Gene-activated matrix

Contents 1. Introduction ............................................................................................................................................................................ 2. Gene therapy to regenerate bone............................................................................................................................................... 2.1. Overview ........................................................................................................................................................................ 2.2. Vector systems ................................................................................................................................................................. 2.3. Fracture repair: current issues ........................................................................................................................................... 2.4. Bone graft substitutes ....................................................................................................................................................... 2.5. Morphogen / cytokine delivery ........................................................................................................................................... 2.6. Tissue engineering ........................................................................................................................................................... 3. Targeted gene delivery ............................................................................................................................................................ 3.1. GAM technology for fracture repair .................................................................................................................................. 3.2. Gene therapy for oral bone ............................................................................................................................................... 3.3. Future applications ........................................................................................................................................................... 4. Conclusions ............................................................................................................................................................................ Acknowledgements ...................................................................................................................................................................... References .................................................................................................................................................................................. *Corresponding author. Oregon Health Sciences University, L352A, Division of Plastic and Reconstructive Surgery, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USA. Tel. / fax: 1 1-503-4944-692. E-mail address: [email protected] (S.R. Winn).

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1. Introduction Damaged bone will spontaneously heal provided the defect is not over a critical limit. When the

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damage or trauma elicits critical-sized defects, interventive therapies are necessary, if one expects the defect to heal. Conventional therapies of autografting, followed by grafts of allogeneic banked bone, can promote reasonable clinical outcome (reviewed in Refs. [1,2]). However, there are recognized limitations to these conventional therapies that may be addressed with alternative treatments [3,4]. Alternatives have included the utilization of bone-graft substitutes, such as metallics, calcium-phosphates, calcium sulfate, and polymers such as collagens and poly(a-hydroxy) acids (reviewed in Refs. [5,6]). In addition, biologics have been included as additives to some of these bone-graft substitutes, including bone derivatives and soluble signaling molecules such as mitogens and morphogens (reviewed in Refs. [7–9]). Furthermore, recent advances in cell and molecular biology have enabled researchers in the bone tissue engineering field to incorporate cell and gene therapies [10–16]. Inadequate clinical outcome from traditional therapies has provided the incentive for researchers to develop clinical alternatives. The evolving field of tissue engineering in the musculoskeletal system attempts to mimic many of the components found in the intact, healthy subject. Those components consist of a biological scaffold, cells, extracellular matrix and signaling molecules. The bone biomimetic provides structural architecture for the regeneration and ingrowth of osseous tissue at the site of injury [6,8,17,18]. Progress to date has primarily emphasized suitable biomaterials incorporating an osteoinductive class of signaling molecules known as the bone morphogenetic proteins, or BMPs [7,8,18,19]. Furthermore, these types of biomaterials, as well as cells, genes, or genetically-modified cells within a tissue engineered construct, offer a platform for gene therapy, the subject for this review [10–16].

2. Gene therapy to regenerate bone

2.1. Overview Gene therapy was initially envisioned as the insertion of a functioning gene into cells of a host to compensate for an inborn error of metabolism, such as a hereditary genetic abnormality, or to provide a

new function in a cell, such as producing a growth factor or even killing cancer cells [20]. The target cells can either be the nonreproductive cells of the body, termed the somatic cells, or the reproductive cells, i.e., the germ-line cells [20]. Somatic cell gene therapy, which targets such tissues as muscle, lung, brain, skin, breast, colon, cells of the blood, etc., is technically feasible and is ethically acceptable for applications in humans. However, somatic cell gene therapy can correct hereditary abnormalities for only one generation. In contrast, germ-line gene therapy can eliminate hereditary defects for all subsequent generations. Although germ-line gene therapy has been applied successful in animal model systems, at this time, the technology has not been shown to be technically feasible, and moreover, is not ethically acceptable for human use. In addition to somatic versus germ-line therapy, the delivery of genetic material, i.e., the cDNA, can be either indirectly incorporated into target tissues by an ex vivo approach, or directly into a target cell via in situ innoculation. The ex vivo method can include autologous, as well as allogeneic cell transplantation. For the autologous method, target cells are removed from the body, generally transduced with viral vectors containing recombinant genes, and re-inserted into the chosen tissue [16,21–27]; reviewed in Refs. [9,20]. In preclinical settings, allogeneic strategies include timing and repeated dosing of stem cell administration, ex vivo modification of the transplant, microchimerism induction for postnatal transplantation, and direct gene targeting [28]. The in situ approach of somatic gene therapy introduces the cDNA directly into the targeted tissue. A variety of techniques have been developed and many of the gene transfer methods are outlined in Table 1. Some of these include the delivery of ‘‘naked DNA’’ in a liquid buffer [29], or formulations in liposome carriers [30]. Particle bombardment technology has been utilized as a pre-clinical tool for gene transfection of several nerve cell systems [31] and clinically to deliver plasmid gene constructs for immunization [32]. In addition, DNA has been formulated in a number of polymer systems to attain sustained release, such as hydrogel suspensions [33], and polymer encapsulation for oral [34] and intraarterial [35] delivery. DNA has also been incorporated into the structural matrix, the so-called gene acti-

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Table 1 Vector systems for gene therapy Gene transfer methods

Transduction efficiency

Integration efficiency

Chemical Transfection a Lipofection / cytofection

Low Medium

Low Low

Physical Electroporation a Microinjection Particle bombardment*

Low High High

Low Low Low

Fusion Liposomes a

Low

Low

Receptor-mediated endocytosis DNA–protein complexes Viral envelope / capsid–DNA complexes

High High

Low Low

Recombinant viruses Adenovirus a Adeno-associated virus (AAV)a Herpes simplex Lentivirus (e.g., HIV-based) Moloney murine leukemia virus (MMLV)a

High High Low High High

Low High Low High High

a

Approved for clinical trial use. Adapted from Ref. [20].

vated matrix formulation [11], an important consideration for tissues like bone that prefer a scaffold for regenerating. Recombinant viruses as vectors have been the preferred method for the Recombinant DNA Advisory Committee (RAC)-approved clinical gene therapy protocols for genetic diseases, cancer, HIV infection, autoimmune and cardiovascular diseases [20].

2.2. Vector systems Vectors for gene therapy applications have been described as either a disabled virus or a DNA structure used as a vehicle to transfer genes into cells [20]. Vectors enable the delivery of cDNA into the appropriate cells, either ex vivo or in situ, and render a cell capable of expressing the transgene product. Several vector systems are available, including replication-deficient recombinant viruses, as well as DNA molecules / complexes (Table 1). Retroviruses, being the most thoroughly characterized vector systems, are RNA viruses that replicate via a DNA intermediate [36]. Retroviruses are a

popular gene therapy vector since they stably integrate into the host chromosome during the host cell’s replication cycle. The retroviral vector most frequently used for human clinical trials has been the Moloney murine leukemia virus (MMLV) (Table 1) [36]. Isolates of endogenous MMLV have been classified into three broad categories based on their infectivity or host range patterns for tissue culture cells, their cross-interference patterns, and serological relatedness [37,38]. Ecotropic MMLVs replicate in mouse cells and, in general, do not infect cells of other species. Xenotropic MMLVs grow on cells from other species and will not grow on cell of the mouse [39]. Lastly, amphotropic MMLVs possess the ability to replicate in mouse cells, as well as other species [37]. For use as a vector in gene therapy applications, the MMLV is rendered replication-deficient by deleting the gag, pol and env DNA sequences [36]. The replication-deficient vector can carry up to 9 kilobases (Kb) of genetic information within an expression cassette [36]. Generally, the vector containing an expression cassette enters a dividing target cell

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through a specific receptor. Once in the cytoplasm, the vector RNA is converted by reverse transcriptase into proviral DNA, is randomly integrated into the target cell genome, and thereafter, the expression cassette generates its product [36]. Thus, the target cell’s genotype should be permanently altered. In cases of treating hereditary or chronic disorders, integration into the host genome is tolerable, although risky. For example, during integration of the proviral DNA, insertional mutagenesis can occur if the inserted DNA disrupts housekeeping genes or activates other genes, e.g., an oncogene [36]. Furthermore, local toxicity can result from chronic overexpression of the expression cassette product. Other limitations include low titers, sensitivities of retroviral vectors to inactivation, and they infect, integrate and express only in dividing target cells [36]. The inability of murine retroviral vectors to infect nondividing cells has been overcome by deriving vectors from lentiviruses [40,41]. Lentiviruses (Table 1), such as the human immunodeficiency virus type 1 (HIV-1), have been shown to infect non-proliferating cells [42]. Recombinant lentiviral vectors based on HIV can efficiently transduce human macrophages [40,41], as well as primary tissues such as brain [43], liver [44], muscle [44], retinal tissue [45] and islet cells [46]. These HIV vectors have been pseudotyped with the vesicular stomatitis virus G glycoprotein, which enables the ability to transduce a broad range of tissues, and can be concentrated to high titers [40,41]. However, there are safety concerns since HIV-1 is the etiologic agent for acquired immune deficiency syndrome (AIDS), and as described for MMLV-based vectors, random insertion into the host genome may activate cellular oncogenesis. Adenovirus vectors have also been thoroughly evaluated in pre-clinical and clinical gene therapy trials [20,36]. Approximately 7.5 Kb of genetic information in an expression cassette can be accommodated within these vectors [36], although newer ‘‘gutless’’ adenoviral vectors allow the insertion of much larger sized DNA [47–49]. The virus is rendered replication-deficient by deleting the E1 and generally the E3 early gene sequences [36]. Entry of adenovirus vectors is dependent on the primary adenovirus receptor CAR, i.e., coxsackievirus-adenovirus receptor [50,51], and the secondary a v b 3 / a v b 5

integrin surface receptors for the adenovirus penton [36,52,53]. Once in a cytoplasmic endosome, the linear, double stranded DNA with the expression cassette is delivered into the nucleus, and product expression is generated in an epichromosomal manner [36]. Adenovirus vectors are efficient, can be produced in high titers, achieve high levels of expression following transduction, and can transfer genes to both replicating and nonreplicating cells [9,36]. Since the genetic material remains epichromosomal, problems associated with insertional mutagenesis are avoided. Adenoviral-based vectors are limited by their nonspecific immunologic reactions they elicit, as well as the potential for autoimmune reactions to the transgene-encoded proteins [54,55]. In fact, a recent death of a patient receiving adenovirus-based gene therapy for treating an X-linked defect of the urea cycle has resulted in the RAC citing numerous changes to increase the safety of adenovirus vectors [56]. Clearly, patient safety is a paramount issue, and the RAC will continue it’s discussions of guideline revisions. Although transfer efficiency and levels for the adenoviral vectors are generally high, the stability of the expression cassette production is low, hence the duration of expression is generally weeks to months [36]. Thus, for sustained transgene expression, adenoviral vectors require readministration. The adeno-associated viral (AAV) vectors are an attractive option since AAV is naturally defective, is readily integrated into the target cell’s genome, does not cause disease in humans and does not contain genes necessary to elicit an immune response [57]. Other viral vectors are available (Table 1) and more information can be obtained within several review articles [36,58,59]. Two additional nonviral-based techniques will be described: liposome–DNA complexes and the direct administration of DNA or DNA complexes. In contrast to the viral-based vectors, liposome– DNA complexes and direct administration of pure DNA complexes can transfer expression cassettes nearly without size limitations. Additionally, these systems offer advantages such as proven stability under a variety of conditions, utilization in a number of delivery systems, and are less immunogenic than the viral vectors [60,61]. The major drawback of these systems is that gene transfer is very inefficient,

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requiring large quantities of materials, sustained release or repetitive administration to achieve clinical success. The delivery of pure DNA complexes will be discussed in detail in Section 3.1.

2.3. Fracture repair: current issues Fracture repair in healthy adult animal models has been characterized to identify the interactions of cells and soluble factors, as well as the temporal and spatial relationships among cells, the extracellular matrix (ECM), and soluble signaling factors [62– 68]. Following bone injury, activation of complement ensues, and lacerated blood vessels elicit extravasation that initiates the cascade for cell signaling. Proteolytic degradation of the ECM, as well as other molecules, liberates chemotactic signals attracting monocytes and macrophages to the wound environment. Activated macrophages release fibroblast growth factors (FGFs) and vascular endothelial growth factors (VEGFs), stimulating endothelial cells to express plasminogen activator and procollagenase [69]. Degranulating platelets elaborate growth factors from the a granules to attract polymorphonuclear leukocytes (PMNs), lymphocytes, monocytes and macrophages. At this point, the injury environment experiences a decrease in oxygen tension and pH, conditions needed for activities of PMNs and macrophages. PMNs remove microbial infestations and micro debris, whereas the macrophages clear larger-sized debris. Macrophages may develop into polykaryon, multinucleated giant cells, to maintain protection against sustained invaders. Macrophages synthesize and secrete a variety of factors at the wound site. These include growth factors to stimulate cell activity, recruit cells, and initiate mitogenesis and chemotaxis throughout the injury repair cascade. Following fracture, a repair blastema develops at 3 to 5 days, consisting of collagen isotypes, cells, such as fibroblasts and macrophages, and the formation of new blood vessels. Selective binding of modulating factors, such as FGF-I, FGF-II, PDGF, TGF-b and the BMPs, to the collagenous component of the wound repair environment makes these factors available to responsive cells. For example, osteoprogenitor cells localized to periosteum and endosteum are chemotactically attracted to the fracture

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site (e.g., TGF-b, BMPs), interact with the granulation tissue collagen, and differentiate into chondroblasts and osteoblasts with the influence of signaling molecules such as BMPs. The activities associated with cell anchorage, cell transductive mechanisms, and cell–factor interactions induces cellular differentiation to specific phenotypes for wound repair. With the gradual differentiation of cells, accumulation of cell expression products, and maturation of the extracellular matrix over the course of several weeks, callus formation results. The callus is comprised of vascular elements, stromal products, various cell types, and cartilage-like structure. Initially, woven bone is formed, characterized by cellular, randomly oriented spicules of immature bone. Thereafter, woven bone matures to lamellar bone, which is less cellular than woven bone, consisting of parallelfibered bone oriented to support and stabilize fracture fragments. Haversian remodeling occurs a few weeks henceforth, indicative of a regenerated structure. A variety of cells, growth factors and morphogens contribute to the fracture healing process that restores structure by 2 to 3 months nearly equivalent to the pre-injured tissue. A partial list of these cells and factors include: mesenchymal [70], fibroblasts [71] and endothelial [71] cells, parathyroid hormone [72], IGFs [73–75], TGF-b [76,77], FGFs [78–81], VEGF (for angiogenic pulse [82,83]), the BMPs [84–86] (PDGF) and cytokines (the interleukins IL-1, IL-6, and IL-11 [71,87,88]). The clinical relevance of cells and their by-products in the wound repair process is that the combination of growth factors, cell attachment molecules, and matrix substratum must interact to drive the cellular machinery responsible for synthesizing new bone. When cellular machinery is either limited in quantity, due to material defects, or defective in responsiveness (e.g., indicative of the aged, osteoporotic condition), one can predict a reduction in bone regeneration dynamics. One of the major challenges in orthopedics arise when the injury results in osseous defects that are difficult to heal. Complex fractures, e.g., crushed or splintered, segmental defects, and large open fractures, resulting in large voids or at sites with reduced vascularity are at risk for nonunion [89,90]. The large open complex fractures with extensive damage and contamination present the greatest risk for nonunion [91]. Nonunions represent a significant

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clinical challenge since they cause disabling pain, a reduction in the quality of life and are associated with expensive chronic care. Delayed unions are bone fractures that fail to heal in the usual 2 to 6 month period [89]. Nonunions, typically defined as a fracture that fails to unite after a 6-month treatment regime [89], continue to occur at an unacceptably high rate of incidence with a variety of etiologies. A number of treatment options are available, including physical stabilization, fixation and bone grafting [89]. Bone grafts and bone graft substitutes function as osteoconductive materials to provide a scaffold for the adjacent host capillaries, cells and tissues to take residence and grow. These treatments, along with the delivery of growth factors, morphogens and gene products, are presented as treatment options to heal complex fractures.

2.4. Bone graft substitutes Thorough reviews have been presented that provide an inclusive overview of candidate materials to restore form and function to deficient osseous sites [6,92–107]. The common theme is that contemporary bone repair candidates are not ideal. In general, it has been a difficult task to design, define and manufacture a synthetic material with optimal and desired properties as a bone-graft substitute. Some of the resorbable synthetic materials, e.g., poly(a-hydroxy) acid polymers, can be fabricated as a bonegraft substitute that mimics the porous characeristics of cancellous bone and can exhibit a degradation profile that approximates a species-specific regenerative process. These types of biomimetics have incorporated cytokines, morphogens, plasmid DNA and cells in an effort to develop a tissue engineered equivalent as a bone biomimetic. Tissue engineering technology is a powerful method to develop candidate bone regenerative materials.

2.5. Morphogen /cytokine delivery As a therapy to regenerate bone, the recombinant human BMPs have attracted the greatest enthusiasm from clinicians. However, while recombinant human BMPs were reported 10 years ago [108], outcome from only three clinical studies is available [109–

111]. In these studies, superphysiological doses of rhBMP-2 and OP-1 (i.e., rhBMP-7) ranging from 1.7 to 3.4 mg were utilized. Although indications for an efficacious outcome were observed, flooding a wound environment with massive doses of potent morphogens, such as BMPs, generates cautious optimism. Could potent osteoinductive factors elicit the bone induction process at sites removed from the implant and thereby compromise clinical success? This issue would be limited if an optimized delivery system was developed to localize the morphogens to the implant materials, or release them in a sustained, predictable manner. The therapeutic delivery or presentation of BMPs, or other potent bioactive factors, requires a well characterized delivery system to effectively and safely present molecules at the implant site [8,18,112]. Recent efforts have utilized the percent rhBMP-2 retention from several biomaterials implanted and retrieved at various intervals from the rat pectoralis implant site [18,112]. Typical rhBMP-2 pharmacokinetic release profiles consisted of an initial burst effect with a half-life less than 10 min, followed by a secondary release phase characterized by a half-life of 1 to 10 days [18,112]. The secondary release phase was strongly biomaterial-dependent. Sustained release of rhBMP-2 was observed from an absorbable collagen sponge and a composite of polylactide infiltrated with a collagen type I sol– gel containing the rhBMP-2 (Fig. 1). In contrast, rhBMP-2 suspended within bovine bone mineral did not exhibit a sustained secondary release profile of rhBMP-2 (Fig. 1). By the day 1 assay interval, nearly 95% of the rhBMP-2 available for release was depleted from the bone mineral (Fig. 1). Fig. 2 depicts the structural architecture of the resorbable biomaterials we have utilized in many studies. The collagen is an atellopeptide bovine dermal type I while the polylactide is a molecular mass (Mr ) 100 000 monomer. Each has been processed to exhibit interconnecting pores in the 50–200 mm range. In addition to the influence of biomaterials, the rhBMP-2 structural features have also been shown to influence rhBMP-2 pharmacokinetics [8,113]. The initial burst effect was dependent on the protein isoelectric point (pI). These studies demonstrated that a succinylated rhBMP-2 with a pI of approxi-

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Fig. 1. rhBMP-2 pharmacokinetics. The rhBMP-2 in solution was infiltrated / interacted with various biomaterials to assess the percent retention at various explant intervals. Sustained release of rhBMP-2 was observed from a collagen sponge and a composite of poly(lactide) infiltrated with a collagen type I sol–gel containing the rhBMP-2. In contrast, rhBMP-2 suspended within bovine bone mineral did not exhibit a sustained secondary release profile of rhBMP-2.

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mately 3 resulted in as much as a 99% burst effect of released rhBMP-2 by day 1 (Fig. 3). This is in striking contrast to the native, unmodified rhBMP-2 protein with a pI of approximately 9, which exhibited a burst effect near 70% by day 1, retaining approximately 30% of the rhBMP-2 protein in the collagen sponges (Fig. 3). In addition, chemical modification of rhBMP-2 by plasmin cleavage impacts protein retention within a collagen implant [18]. By day 1, approximately 8% of the plasmincleaved rhBMP-2 was retained within the implant (Fig. 3). An additional 5% of the plasmin-cleaved rhBMP-2 loaded into the collagen sponge was released from the implant between days 1 and 13. Clearly, the quantity of unmodified rhBMP-2 retained and released from the collagen implants was superior to either the plasmin-cleaved or succinylated rhBMP-2 modified molecules. Other growth factors have stimulated clinical interest (for example, insulin-like growth factor, platelet derived growth factor [114], and fibroblast growth factors-1 [81] and -2 [14]). At this time, these agents have not generated as much enthusiasm as the BMPs among clinicians for bone regenerative

Fig. 2. Scanning electron micrographs of porous scaffolds utilized for site-specific delivery of proteins and plasmid DNA. (Left) collagen macrostructure that represents a candidate porous delivery scaffold for BMPs, as the protein or plasmid DNA in a GAM, which exhibits interconnecting pores. (Right) poly(lactide) monomer with a 100 000 molecular mass also utilized to deliver factors and plasmid DNA to an osseous wound that is characterized by an interconnecting open-pore meshwork. Scale bars 5 200 mm.

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Fig. 3. Effect of plasmin cleavage and succinylation on rhBMP-2 pharmacokinetics. Both the initial retention and the subsequent release of rhBMP-2 was faster for the chemically modified than the unmodified rhBMP-2 proteins. The dose retained at the implant site was dependent on the chemical structure of the rhBMP-2.

therapies. Difficulties promoting a clinical effect with growth factors may be due to insufficient dosing and temporal inconsistencies [115]. Other factors may include, asynchrony between kinetics of signaling receptor expression and growth factor availability [110]; transient active half-life of growth factors (i.e., minutes [116,117]); inadequate responding cell populations [70]; and diffusivity, sequestration by plasma membranes, and lysis in the wound healing environment [14].

2.6. Tissue engineering Tissue engineering is a relatively new field that combines technologies from such diverse areas as cell and molecular biology, engineering, biochemistry, materials science, medical implant and transplantation science, and immunology [118]. Tissue engineers design and fabricate three-dimensional substitutes to mimic and restore the structure–function properties of the target (replaced) structure [9,118]. Advances in tissue engineering have gener-

ated polymers for orofacial and orthopedic implants, and have developed composites as bone graft substitutes. Bone regeneration with a tissue engineered formulation should be convenient for the surgeon, and should exhibit an ability to localize, position, and sustain combinations of cueing molecules (e.g., BMPs), DNA plasmids or cells at the wound site [119]. First and foremost, the tissue engineered formulation must provide the role of a substratum [8,70,120]. The substrate provides a site for attachment and differentiation of host pluripotent cells. In addition to offering the role as a vehicle for the sustained release of cueing molecules, such as the BMPs, the polymer scaffolds have recently been infiltrated with various cell types to develop a true tissue engineered bone biomimetic [15]. Autologous ex vivo stem cell therapy [121] is an important development in bone engineering to consider as a supplemental therapy for bone grafting procedures, as well as, the potential for augmenting the bone cell population in osteoporotic patients. Mesenchymal stem cells (bone marrow-derived) have been harvested, expanded in tissue culture, and re-implanted into recipient pre-clinical animal models to regenerate bone [12,122–124]. These studies have included autologous, allogeneic and xenogeneic implant strategies. Rather than harvesting, expanding, and re-implanting autologous cells, a significant advantage would be to have an allogeneic, osteoprogenitor cell type (OPC) available for immediate application [125]. Recent efforts in our laboratory have been geared toward establishing BMP-responsive OPC lines that could be utilized to supplement the osteogenic cell population in a tissue engineered scaffold [125]. Human osteoprogenitor cells were transfected with a conditional immortalizing plasmid construct in which the transgene was stably incorporated into the host cell genome (Fig. 4, OPC). These OPCs have been delivered within a porous polylactide fabric to restore critical-sized defects (CSDs) in a athymic rat calvaria model [15]. Recent efforts have suggested that modifications to the seeding densities and holding conditions prior to implantation will further improve the treatment modality. Moreover, the scope for our current bone biomimetic tissue engineered construct will incorporate BMP plasmids expressed

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Fig. 4. Gene therapy strategy for developing a tissue engineered bone biomimetic. Human osteoprogenitor cells were transfected with a conditional immortalizing plasmid construct in which the transgene was stably incorporated into the host cell genome (OPC1). These OPCs have been further modified with liposomes or adenoviral vectors containing protein expression cassettes for epichromosomal transgene action. Various types of these genetically-modified OPCs have been delivered within a porous poly(a-hydroxy) acid fabric to restore osseous critical-sized defects.

in an episomal fashion, to provide a mini bioreactor to constituitively express BMP activity (Fig. 4). This is similar to the ex vivo approach recently described by Lieberman et al. [16] and similar to our previous efforts to engineer OPCs to express human ciliary neurotrophic factor (CNTF) [126]. These types of tissue engineered constructs containing BMP-producing cells should minimize the therapeutic dose of BMPs required in a clinical carrier (currently mg quantities) and still effectively recruit locally responsive cells to differentiate into osteoblasts. This later point is especially important in the geriatric patient population with limited numbers of functioning osteoblasts [70,127,128].

3. Targeted gene delivery Alternatives to delivery of high doses of potent morphogens / growth factors (e.g., rhBMP-2 [109,110]) at the osseous wound site could include regional gene therapies.

An example of regional, targeted gene therapy is ex vivo adenoviral gene transfer to generate BMP-2producing bone-marrow cells [13,16]. The Lieberman group determined critical-sized defects in rats could regenerate with adenoviral gene transfer of autogenous marrow cells delivered and positioned by allogeneic, inactivated demineralized bone matrix [16]. Although this accomplishment is highly significant, the authors noted concerns about safety of the adenoviral vector, such as the immunological sequelae (both from the vector and the allogeneic delivery system), and the fate of BMP-transfected cells. Another ex vivo method of regional, targeted gene therapy involved cultured periosteal cells transduced retrovirally with the BMP-7 and delivered with poly(glycolic) acid to restore critical-sized calvarial defects in rabbits [129]. Despite the advantages of a retroviral vector (i.e., gene incorporation into the cell genome, thus prolonging expression), data were not persuasive to validate efficacy. Delivery of genetic material directly into the host wound site via in situ / in vivo delivery has been described

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with a number of viral vectors, and has similarly been achieved through a localized, direct delivery of plasmid genetic material [9,11,14].

3.1. GAM technology for fracture repair One of the recent developments to deliver plasmid DNA directly to the repair cells involved in fracture repair has been described as a gene activated matrix (i.e., GAM) [9,11,14]. The GAM porous architecture provides scaffolding to promote cell ingrowth and delivers the plasmid DNA that has been incorporated into the degradable matrix. The local granulation tissue fibroblasts, along with capillaries, migrate into the GAM, uptake and transiently express the local plasmid DNA. The transfected reactive cells secrete the plasmid-encoded proteins to stimulate and augment the bone regenerative cascade. As previously described, some of the advantages of delivering pure DNA complexes are the virtually unlimited size of recombinant plasmid constructs, ready availability of low-cost straightforward methods for DNA production, ability to combine DNA with pharmaceutical delivery systems and carriers [130], and the proven safety of pure DNA [14]. Targeted administration of plasmid DNA from GAMs to regenerate bone in ostectomy gaps in rats was reported by Fang et al. [11]. They used lyophilized bovine tracheal collagen incorporating DNA plasmids encoding human parathyroid hormone peptide fragment 1-34 (hPTH1-34) and / or mouse BMP4. While each plasmid elicited a favorable response of new bone filling the gaps, the GAM implants containing both plasmids, in contrast to the individual plasmids, resulted in an increase in the rate of new bone formation. Similar results have been demonstrated in a canine preclinical tibial critical defect model [14]. In this model, implantation of GAMs containing hPTH1-34 plasmid DNA demonstrated retention and expression of plasmid DNA for at least 6 weeks [14]. Bone induction was observed in a stable, reproducible, dose- and time-dependent manner [14]. Until recently, low transfection efficiency for DNA plasmids delivered in situ has been a limitation. However, recent data has demonstrated a 30– 50% expression range of b-galactosidase following targeted administration of plasmid DNA encoding this reporter gene [14]. In addition to improved

transfection efficiency, plasmid DNA has a stable, flexible chemistry compatible with polymer-based drug delivery systems [9]. In terms of safety-related issues, systemic toxicity from the DNA turnover should not be a concern [131], and quiescent, nonhealing tissue should be impacted minimally by plasmid gene transfer [29]. Lastly, plasmid DNA is economical and relatively simple to manufacture [132,133]. However, care must be taken to avoid unmethylated CpG dinucleotides. Oligodeoxynucleosides containing the unmethylated CpG motif (CpG ODN) are immunostimulatory, can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells and NK cells, and switch on T helper 1 (Th1) immunity [134,135]. Select pure DNA, which can be administered locally as a recombinant plasmid construct, is takenup by the local reactive cells, and the transgene product is expressed by an epichromosomal action in the host cell (Fig. 4). Having local cells secrete factors such as hPTH1-34 and BMP-2, -4 or -7, provides physiologic levels of these potent factors, which should circumvent safety issues related to both the relative short half-lives of these molecules, as well as the issues surrounding massive dosing presently required in the clinical setting [109–111].

3.2. Gene therapy for oral bone Recent advances in molecular biology have paved the way for gene therapy interventions in many oral health diseases. Many research groups are carrying on gene therapy experiments for oral-specific applications, i.e., oral cancer [136–139] and salivary gland treatments [140,141]. Preclinical studies for malignancies of the oral cavity have shown that suicide gene therapy can reduce tumor volumes in an oral squamous cell carcinoma animal model [137] and in human head / neck cancers engrafted in nude mice and treated with the HSVtk / gancicilovir strategy [138]. Additionally, since tissue access is relatively straightforward in the oral cavity, gene transfer to salivary glands is emerging as a potential target for use in systemic gene therapies. Kagami et al. [140], assessed the feasibility of using gene transfer to salivary glands to direct the in situ systemic delivery of therapeutic proteins. A replication-deficient recombinant adenovirus vector

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that encodes human a 1 -antitrypsin (h a 1 -AT), was used as a marker protein and administered by retrograde ductal instillation to the submandibular glands of male rats. Low serum levels of h a 1 -AT were detected using a sensitive enzyme-linked immunosorbent assay (ELISA). This data demonstrated that salivary glands is a reasonable target site for the systemic delivery of low levels of therapeutic proteins using gene transfer technology. Prolonged expression of the reporter gene b-galactosidase was reported in the acinar cells of the submandibular and sublingual glands of the rat [141]. This study demonstrated that the uptake of retrograde ductal injected retroviral vectors could be enhanced by up regulating acinar cell division. Another line of investigations regarding salivary glands therapies are carried out by the same research group and are reviewed by Baum et al. [142]. Other oral-specific applications of interest to our laboratory are the regeneration of mineralized structures in the oral cavity. In particular, degeneration of cementum leading to periodontal disease, and dentin defects resulting from dentin caries or mechanical trauma. Periodontal regeneration. Periodontitis affects the composition and integrity of periodontal structures at the dento-gingival junction, alveolar bone, cementum and periodontal ligament. Periodontitis causes the destruction of connective tissue matrix and cells, loss of fibrous attachment, resorption of alveolar bone and often leads to tooth loss. The major goal for periodontal regeneration is to reverse the destructive effects of this disease. Successful periodontal regeneration will result in the formation of new cementum, new connective tissue attachment with functionally oriented periodontal ligament fibers, and coronal apposition of new supporting bone. A thorough presentation describing periodontal applications in tissue engineering can be reviewed in an article by Miller et al. [143]. Recent data indicate that molecules isolated from cementum matrix (cementum-derived attachment protein, CAP) [144] are capable of influencing the differentiation of mesenchymal cells and exhibit inductive properties important for regeneration. These molecules promote the migration, attachment and proliferation of periodontal fibroblasts, can activate their synthetic activities and induce their differentiation [145,146]. Other studies have suggested

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that products released during disruption of Hertwig’s epithelial root sheath, which coincide with root development, may act by recruiting follicle cells to the area of cementogenesis. These products may include membrane associated glycoproteins, e.g., laminin [147], and amelogenin [148]. Studies in our laboratory are underway to evaluate the feasibility of delivering recombinant amelogenin plasmid DNA in a GAM configuration to regenerate a periodontal defect. Details are presented in Section 3.3. Dentin regeneration. During mechanical cutting of tooth structure to repair dental caries, or as a result of a traumatic injury, exposures of the pulp chamber can occur. The traditional means of treatment is to perform a direct pulp capping procedure. This involves applying a layer of calcium hydroxide to the exposed area, followed by a covering layer of glass ionomer cement [149]. When calcium hydroxide is applied directly to pulp tissue, necrosis is observed in the adjacent pulp tissue, and inflammation of the contiguous tissue occurs. Dentin bridge formation occurs at the junction of the necrotic and inflamed tissues. Another possible method to treat pulp exposure is to stimulate pulp cells to form reparative dentin (reviewed in [150]). Reparative dentinogenesis has been observed with recombinant human (rh) BMP-2, BMP-4, OP-1 and TGF-b (reviewed in Ref. [150]). Nakashima demonstrated that rhBMP-2 and -4, but not TGF-b, elicited reparative dentinogenesis in a canine model of partially amputated dental pulps [151]. OP-1 has also been shown to induce reparative dentin formation in experimental models of large direct pulp exposures in permanent teeth [152,153]. Other methods for regenerating dentin can be reviewed in an article by Charette and Rutherford [150]. Studies in our laboratory are underway to evaluate the feasibility of delivering recombinant rhBMP-2, -4 or OP-1 plasmid DNA in a GAM configuration to regenerate a dentin defect targeting an odontoblast-specific gene. Details are presented in Section 3.3.

3.3. Future applications Many obstacles, with the majority being consistent with the drug development process, continue to exist for the successful application of human gene transfer [36]. Issues related to the production of clinical-

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grade vectors, and the ability to generate consistent and reliable results with appropriate, safe and consistent vectors, will continue to challenge the field. However, with the plethora of human genes available from the human genome project, the potential applications of somatic gene therapy for understanding and treating human diseases is staggering. We are optimistic that the appropriate course of actions will be taken and that new technologies, such as the GAM approach, will provide exciting new avenues in the areas of wound repair, augmentation and regeneration. One such area for future applications lies in the ability to stimulate an up-regulation of osteogenic activity in the aged, osteoporotic individual. We propose to stimulate the local environment by presenting hPTH1-34 and / or rhBMP-2 plasmid DNA to induce the bone regenerative process that would be at a pace consistent with the non-geriatric counterpart. Our logic for selecting hPTH1-34 and / or hBMP-2 as regulators for bone regeneration is that during the process of bone regeneration in the osteoporotic, transfection of wound healing fibroblasts by the osteo-anabolic peptide hPTH1-34 [154– 158] and hBMP-2 (a recruiting and differentiating factor) will provide a significant boost to the healing process. This logic is derived from known functional properties of the selected molecules [157–161]. Moreover, of the BMPs available, BMP-2 may be superior to the previously described use of BMP-4 by Fang et al. [11]. Selection is based on our experience testing osteoinduction [162–164], data indicating this molecule is a proliferation factor for pre-osteoblast-like cells, a differentiating factor for osteoblasts, it’s presence in the osseous wound healing continuum, and recent evidence BMP-2 is chemotactic for osteoblast-like phenotypes [84,159,165–170]. For applications in regenerating oral structures, experiments are underway to deliver recombinant amelogenin to the tooth root surface using GAM technology. The rationale to use recombinant amelogenin to regenerate the periodontium is based on a series of tissue culture experiments. Recombinant amelogenin and / or recombinant human BMP-2 were added to a base medium with or without an osteogenic supplement of 10 mM b-glycerophosphate, dexamethasone and ascorbic acid phosphate

[125]. Various types of medium were placed in contact with OPC1 [125], odontoblast [171] and cementoblast cells [172]. Alkaline phosphatase enzyme activity and extracellular calcium production were measured at 9 and 16 days. The cementoblast cells exhibited significant increases in the expression of alkaline phosphatase and calcium deposition as compared to the odontoblast and osteoprecursor cells (unpublished data). To regenerate dentin structures, our approach has been to specifically target the odontoblast and preodontoblast cells. To achieve this goal, a recently cloned odontoblast specific promoter of the Dentin Matrix Protein 3 (DMP3) gene [173] is being used as a tool to specifically target expression to the odontoblast (or pre-odontoblast). The DMP3 promoter will be cloned 59 to growth factors such as rhBMP-2, -4 or OP-1 to trigger dentin regeneration. In this case, gene therapy is being used as pharmacological agent and targeted to a specific cell type.

4. Conclusions Restoring damaged bone to a form and function that is equivalent to its pre-injury state is the gold standard for bone regeneration. Even though bone tissue demonstrates the ability to regenerate itself under a majority of conditions, if the injury exceeds a critical limit, regeneration will not occur. Without interventions, these damaged states may result in bone that exhibits a nonunion fracture. A variety of physical techniques, as well as bone grafts and bone graft substitutes, have been developed to overcome complex, potential nonunion fractures. Many of these treatments continue to exhibit limitations. An evolving treatment modality, gene therapy, offers an exciting avenue for bone regeneration. This review has presented some of the current concepts for developing a rational gene therapy approach in bone regeneration.

Acknowledgements The authors gratefully acknowledge our colleagues David Buck, Charlie DuBois, Amy Pugh, Xi Gong and Rich Sipe at the Oregon Health Sciences Uni-

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versity. In addition, we extend our appreciation to our collaborators, Jeffrey Bonadio, Martha Somerman, Arthur Veis and John Wozney.

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