Gene Therapy For Spine Fusion

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GENE THERAPY FOR SPINE FUSION

Safdar N. Khan, MD, Chisa Hidaka, MD, Harvinder S. Sandhu, MD, Federico P. Girardi, MD,

Frank P. Cammisa Jr, MD, and Ashish D. Diwan, MD, PhD

Spine fusions stabilize adjacent vertebral segments by achieving bone union, eliminat- ing motion. The preferred method of spine fusion involves decortication of the host bed and transplantation of autologous bone graft from the iliac crest. Of the 289,000 spinal procedures performed in the United States each year, nearly all require augmentation with a bone graft material.39 Although autogenous bone grafting is the gold standard, this procedure has significant morbidity, including increased blood loss and concomitant risk of blood trans- fusion as well as persistent pain or infection at the harvest site. These problems can affect 20% of patients undergoing this procedure.50 Because of these significant problems, the enhancement of spine fusion is an active area of research.

One novel therapeutic modality that may improve fusion rates is gene therapy. Gene therapy is a technique in which nucleic acid, usually DNA, is transferred to target cells for a therapeutic effect. Although originally con- ceived as a modality for treatment of genetic diseases, gene therapy has begun to show promise for repair and regeneration of musculoskeletal tissues, including the spine.

Various methods continue to be developed for therapeutic gene transfer, and many of these are reviewed in this article, with particular emphasis on methods that have shown promise in musculoskeletal tissues, including the spine. Several studies in which gene transfer has been used specifically to enhance spine fusion in animal models are reviewed.

GENE EXPRESSION AND REGULATION

Humans have 23 pairs of chromosomes that are located within the nucleus of every somatic cell of the body. Each chromosome comprises DNA and proteins and contains at least 150,000 genes. Gene expression is regu- lated in a manner so that only a fraction of the genes are expressed for any particular cell, de- termining the unique biologic properties, or phenotype, for that cell. In simplest terms, gene expression can be described as the transcription of particular sequences of DNA (of which genes are made) into messenger ribonucleic acid (mRNA), which, in turn, is translated into proteins. The resulting proteins, which may or

From the SpineCare Institute (SNK, HSS, FPG, FPC), Spine Service (ADD, FPC), and Laboratory for Soft Tissue Research (CH), Hospital for Special Surgery; Belfer Gene Therapy Core Facility (CH) and Department of Orthopaedic Surgery (HSS, FPG, FPC), Weill Medical College of Cornell University, New York, New York; and the University of New South Wales, Sydney, Australia (ADD)

ORTHOPEDIC CLINICS OF NORTH AMERICA

VOLUME 31 * NLiMBER 3 * JULY 2000 473

may not be modified further after translation, govern many of the cell’s functions.

The underlying principle of gene therapy is to transfer a therapeutic gene to a target cell. Expression of this trunsgene leads to its transcription into mRNA followed by translation of the mRNA into protein by the target cell. Depending on the function of the gene product (protein), the target cell may then be stimulated to proliferate, form matrix, or otherwise alter its biologic characteristics. With most gene therapy vectors, activation of DNA transcription is designed to be constitutive, that is, continuous, at high levels. This continuous activation usually is accomplished by including a constitutively active promoter sequence of DNA in front of the therapeutic DNA sequence within the gene therapy construct. In- vestigations have also shown the feasibility of constructing vectors with regulatable or tissue-specific promoters that can direct genes to be expressed only under specific conditions (e.g., the presence of tetracycline) or in specific tissues (e.g.,

TARGETED GENE TRANSFER

Targeted gene transfer, which is known as trunsfection, can be accomplished using one of two major techniques: in vivo or ex vivo (Fig. 1). For ex vivo gene therapy, cells from target tissue are isolated and cultured and are genetically modified in cell culture media. These genetically modified cells are transplanted back into the patient. The advantages of ex vivo therapy include closer control over the manip- ulations of the target cells and the ability to combine the altered cells with biomaterials before reimplantation onto the host. Also in ex vivo gene therapy, the patient is not neces- sarily exposed to the gene transfer vector but only to the modified cells. Ex vivo therapy is limited to tissues for which culture settings and transplantation techniques are well estab- l i ~ h e d . ~ For in vivo therapy, the vector is sim- ply introduced to the target tissue, by local injection during surgery, for example, and gene transfer occurs in the cells within the tissues. Although simple in concept, this method is not always straightforward when treating such tis-

sues as cortical bone and cartilage, which are not penetrated easily.

Two major classes of vectors are available for gene transfer: nonviral and viral. Although viral vectors are generally much more effective for gene transfer than nonviral vectors, viral vectors have other disadvantages, such as host immunity. The various vectors currently being studied in musculoskeletal tissues-in particular, for spine fusion-are reviewed subse- quently.

Nonviral Vectors

Nonviral methods to introduce genes into target cells include plasmid-mediated transfer, liposomal transfection, and particle-mediated gene transfer.

Plasmids

Plasmids are circular constructs of naked DNA that were identified because of their ability to transmit antibiotic resistance to bacteria. DNA encoding therapeutic gene products can be incorporated (cloned) into these plasmids, then transferred to cells. The advantages of plasmids are the virtually unrestricted size of genes that can be introduced and the relative lack of toxicity. Similar to other nonviral strat- egies, however, this method results in relative inefficient gene transfer.

Plasmid-mediated transfer of the vascular endothelial growth factor (VEGF) to induce new blood vessel growth in ischemic limbs was studied in a clinical trial by Baumgartner et a1.2 Gene transfer was performed in 10 limbs of nine patients with nonhealing ischemic ul- cers resulting from peripheral arterial disease. Naked plasmid DNA encoding the isoform of human VEGF was injected directly into the muscles of the ischemic limbs. Gene expression was documented by an increase in serum levels of VEGF monitored by enzyme-linked immunosorbent assay. The ankle-brachial in- dex improved significantly, newly visible collateral blood vessels were shown by contrast angiography in seven limbs, and magnetic res- onance angiography showed evidence of improved distal flow in eight limbs. Ischemic ul- cers healed or improved markedly in four of seven limbs, including successful limb salvage

GENE THERAPY FOR SPINE FUSION 475

A

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B

Figure 1. A, Various methods for introducing the gene of interest into the target cell (transfection). I, Naked DNA or plasmid (Naked DNA with the therapeutic gene can be introduced into the target cell); II, viral transfection (Viral mediated gene transfer can be accomplished with retroviruses, adenoviruses or other viruses); 111, microinjection (physical methods like a “gene gun” can be used to introduce genes into cells); IV, liposomal transfer involves the fusion of cationic liposomes across the negatively charged cell membrane introducing genes into the cell. C = cytoplasm; G = target cell genome; N = nucleus. 6, Incorporation of the gene of interest into the target cell genomic DNA. g = gene of interest; tg = transgene.

Illustration continued on following page

U 0 0 protein

C

Figure 1 (Continued). C, 1, Transcription of the transgene to messenger RNA; 2, Translation of the mRNA to protein; 3, Post-translational modification of the protein. The protein (which is the end product of expression of the therapeutic gene) can act i) intracellularly, ii) in an autocrine manner by stimulating receptors on the host cell surface, or iii) in a paracrine manner by acting on cells at a distance from the secreting cell. Transgene expression. ECM = extracellular matrix; C = cytoplasm; tg = transgene; tRNA = transfer RNA; mRNA = messenger RNA.

in three patients recommended for below-knee amputation. The authors concluded that intramuscular injection of naked plasmid DNA could achieve constitutive overexpression of VEGF sufficient to induce therapeutic angiogenesis in selected patients.

Plasmids have been incorporated onto three-dimensional structural matrices called gene-activated polymer matrices (GAMs). The GAMs act as scaffolds that allow cells to mi- grate onto them, come into contact with the plasmid DNA, take up the DNA, then produce the plasmid-encoded protein. Fang et all7 and others” used GAMs to enhance fracture healing in rat and canine models. The GAMs con- tained plasmids encoding bone morphogenetic protein (BMP)-4 plasmid or a fragment of parathyroid hormone (PTH1-34). Plasmid- mediated gene transfer stimulated bridging of the 5-mm gap in the rat femora 9 weeks after implantation with the BMP-4 or the PTH1-34

single-plasmid GAM preparation. Implanta- tion with the two-plasmid GAM preparation (BMP-4 plus PTH1-34) resulted in bridging of the gap in approximately 4 weeks. These results showed that plasmid DNA could be transferred into repair cells in vivo, which could then stimulate bone growth after local overexpression of osteogenic factors. A similar experiment was conducted in a canine model. Surgical defects 8 mm in diameter and 8 mm deep were created in distal femora and prox- imal tibiae of the experimental animals. GAMs encoded with BMP-4 alone, PTH1-34 alone, or BMP-4 plus PTH1-34 were placed within the defects. The results of this canine study were similar to that of the previous rat study, with substantial bone formation observed 10 weeks postoperatively. At 23 weeks, there was significant evidence of remodeling and recorti- calization in the bone defects of the experimental animals.


Liposomes

Liposomes are lipid vesicles that have been used extensively to enhance plasmid-mediated transfection of cells. This technique has been used to enhance spine fusion (see later). Liposomal transfection involves the coupling of cationic liposomes with anionic DNA. Li- posomes are able to fuse to the negatively charged lipid cell membrane, allowing the plasmid DNA that has been coupled to it to be delivered to the inside of a cell. As with naked DNA plasmid, the main advantage of lipofection is that the size of the gene to be delivered is unlimited. Although inefficient gene transfer has been the main limitation of this method, innovations, such as the ligation of certain viral components to liposomes, has enhanced liposome-mediated gene transfer significantly?

Tomita et a P showed direct gene transfer in vivo in the articular cartilage of rats by liposomes, which were modified by ligands from the hemagglutinating virus of Japan (HVJ or Sendai virus). This study showed expression of a nontherapeutic marker transgene in chondrocytes in the superficial and middle zones of the articular cartilage indicating ex- cellent penetration in vivo by this method. Nakamura et a13 used a similar HVJ-liposome suspension to transfer the cDNA encoding platelet-derived growth factor (PDGFI-B into a patellar ligament injury model in rats. Rats treated with this method showed enhanced expression of PDGF-B and induction of angiogenesis and accelerated collagen matrix dep- osition in a ligament healing model 4 weeks posttransfection. Enhancement of spine fusion using liposomes to transfer the LIM-related mineralization protein-1 is described subse- quently.

Particle-Mediated Gene Transfer

Particle-mediated gene transfer involves gene delivery by bombardment of target cells with microprojectiles coated with DNA. Under the influence of an accelerating force (e.g., high- voltage electric discharge, helium pressure), DNA-coated microparticles, such as tungsten or gold, penetrate target cells to deliver the DNA.

The advantage of this technique is the ability to circumvent the cell membrane that acts as a barrier to physicochemical methods of gene transfer. As a result, a wide variety of cell and tissue types can be transfected in this manner. Multiple genes with a large amount of DNA can be delivered in this manner. Dis- advantages include transient gene expression and lack of stable gene expression. This technique has been used with varying success to transfer genes into muscle fibers and kin.^,'^,^^

Viral Vectors

Currently, the most efficient vectors are based on viruses. Viral vectors are usually designed so that genes required for viral replication are replaced by a specific therapeutic gene of interest. The resulting recombinant vector is unable to replicate and unable to cause infections, as wild-type viruses are able to do. They are still able to deliver genetic material directly to a cell's nucleus, however, resulting in efficient gene transfer.

Retrovirus

Recombinant retroviral vectors have been used in human trials since 1991 and have shown safety over this significant period. Gene transfer to the synovium using a retroviral vector encoding an anti-inflammatory molecule, interleukin-1 receptor antagonist, is currently the only human clinical trial being conducted in orthopedics-related gene therapy.16

Retroviruses are single-stranded RNA viruses with approximately an 8-kilobase genome size. Because the retrovirus genome is made up of RNA, it must be converted into DNA by the target cell before expression can occur. Retroviral vectors are integrating vectors. In other words, the viral genome must be integrated into the target cell genome before expression is p0ssible.4~ Integration can occur only during cell division, and retroviruses can transfer genes only to dividing cells. Because targeting dividing cells is accomplished most efficiently in vitro, retroviral gene transfer usually requires an ex vivo technique.

The advantages of recombinant retroviral gene transfer are that these viruses are able to transfer genes to a wide array of different cells, and the genes of interest are integrated reliably and efficiently into the target cell genome. Only genes of limited sizes (<6 kilobases) can be transferred into the virion core, however.

There have been concerns about the potential ability of the recombinant retroviruses to convert into replication competent viruses. Advances in the development of screening methods to detect these aberrant viruses have made their production virtually impossible, however.'* There is a further theoretic concern of insertional mutagenesis in which the retrovirus may become integrated adjacent to a cancer-causing gene (proto-oncogene), which may lead to neoplastic transformation. The proba- bility of such an insertion is low, however, and it takes several mutations for a cell to become truly neoplastic.

Engstrand et all5 evaluated the transplantation of allogeneic osteoprogenitor cells transfected with a retroviral vector encoding BMP-2 to induce bone. Abundant ectopic bone formation was shown in 85% of the treated animals, suggesting that retrovirus-mediated transfer of the BMP-2 gene may have therapeutic potential in applications, such as spine fusion, requiring enhanced bone formation.

Adenoviruses

Adenoviruses are double-stranded DNA viruses with a genome size of approximately 35 kilobases. The wild-type is a minor human pathogen, able to cause mild upper respiratory and other infections. Recombinant adenovirus vectors usually carry deletions in several genes called early genes, which are necessary for adenovirus replication. First-generution vectors typ- ically carry deletions of early genes E l and E3, whereas newer vectors also carry deletions of E4. The so-called gutless vectors have virtually no adenovirus genes.1s,21,a These various deletions are designed not only to make room in the viral genome for one or more therapeutic genes of choice, but also to minimize the inflammatory and other effects of the adenovirus particles.1°

Adenoviral vectors have shown consis- tently the highest efficiency gene tran~fer.~,**,~'

Because adenoviral vectors are able to transfer genes to nondividing cells, they can be used for direct in vivo gene transfer. This ability is considered to be the greatest advantage of adenoviral gene transfer over retroviral gene transfer. Adenoviral vectors are able to transfer genes to nondividing cells because, in contrast to retroviral vectors, adenoviral vectors do not require integration into the host genome for expression. Instead the adenovirus genome remains episornal, separate from the host genome; this means that expression of the therapeutic gene is limited to the life span of the transfected cell itself, that is, that expression is transient. Although this transience may be a disadvantage for the treatment of genetic diseases, transient gene transfer may be ap- propriate for applications such as spine fusion. The major disadvantages of adenoviral transfection include host immune response directed against the viral surface proteins.

Lieberman et alE used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells for the delivery of BMP-2 to heal a critical-sized femora1 segmental defect in a rat model. The genetically modified cells were implanted with inactivated demineralized bone matrix as a substrate. At 2 months, 92% of the defects healed radiographically, and there was no significant difference in biome- chanical testing between the groups that had been treated with genetically modified cells and those that had been treated with rhBMP- 2 alone. Their results showed that BMP-2-producing bone marrow cells created by means of adenoviral gene transfer produced sufficient protein to heal a segmental femoral defect in a rat model.

Other Viral Vectors

Other viral vectors that are currently being developed include adeno-associated virus (AAV), herpes simplex virus (HSV), and len- tivirus. AAV is a single-stranded DNA virus in the parvovirus family whose wild-type is not pathogenic to humans. Although the wild- type genome appears to integrate specifically to the end of chromosome 19 in the human genome, the integration of the recombinant AAV is less well characterized. AAV appears to be able to transfer genes permanently to


some cell lines, such as hematopoietic stem and has been shown to transfer genes

efficiently to mouse joints (synovium and chondrocytes on the surface of cartilage), es- pecially if the joint is inflamed.% Recombinant HSV, similar to the related herpes pathogen, has shown tropism for neural In addition, recombinant HSV has been shown to transfer genes efficiently to tendons.

STUDIES IN GENE THERAPY FOR SPINE FUSION

Pathologic conditions of the spine are of- ten treated by intersegmental spinal arthrodesis using internal fixation devices and iliac crest autogenous bone grafts. Although auto- grafts are the gold standard for initiating fusion, there are several disadvantages with its use, including insufficient amount of graft material available for use (particularly in chil- dren, during revision surgery, and for treating large osseous defects), significant postoperative morbidity at the donor site in 8% of pa-

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tients (e.g., infection, pain, hemorrhage, nerve injury), increased operative time, operative blood loss, and additional cost.% It is estimated that nonunion occurs in 35% of patients with a single-level fusion and in more patients when multiple level fusions are attem~ted.~ Technologic advances in the capability of fixation devices to stabilize the spinal column rig- idly have been optimized; however, successful spinal fusion still relies on the biologic ability to form bone union between adjacent segments. Although the mechanical facilitation of spinal fusion with internal fixation has de- creased the incidence of nonunions, pseudarthrosis still occurs in 10% to 15% of patient^.^ Consequently, active research efforts are being focused on this area.

One novel area of research that has shown significant promise is gene therapy (Fig. 2). To date, much of the work in gene therapy for the enhancement of spine fusion has centered around the transfer of genes encoding BMPs and related proteins. The BMPs are a large family of osteoinductive proteins whose expression is important for skeletal patterning

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Figure 2. Gene therapy to augment spinal fusion. 1, Transfected cells (Cells transfected ex vivo with the gene of interest. These cells could be bone marrow cells, myoblasts, or fibroblasts). 2, Carrier (i.e., collagen, biphasic calcium phosphate, hydroxyapatite, demineralized bone matrix. 3, CelVcarrier composite. 4, Posterolateral lumbar interbody fusion, where the cell-carrier composite can be used alone or with autogenous bone graft. 5, Cellkarrier composite placed in the hollow of an intervertebral body fusion cage. 6, Anterior interbody fusion with celucarrier composite.

480 KHAN et a1

and development. Numerous preclinical studies in a variety of animal spine fusion models have shown the ability of purified recombinant BMP-2 and BMP-7 to enhance bone dep- osition at the fusion site.*

Although the use of recombinant BMPs has shown promise in smaller animals, there is evidence to suggest that the high doses required for successful treatment of larger animals, including humans, may not be achieva- ble through currently available means. The development of an ideal delivery system to deliver BMP remains a clinical problem. An ideal bioconductive system should allow controlled release of the protein, have adequate exposure to inducible cells, be immunologically inert, be biodegradable, and enable cell proliferation and angiogenesis to Numerous approaches, including demineralized bone matrix, fibrin, ceramics, collagen, polylactic acid, and titanium, have been used to enhance the ability to deliver the growth factor to the fusion bed. No carrier has been shown consis- tently to deliver BMPs efficiently in a sustained fashion, however, as well as maintain the growth factor concentration within a therapeutic window. Potentially, genes encoding osteoinductive growth factors may be transferred to target cells, inducing the production of large amounts of the growth factor in a controlled and sustained fashion for a predictable period of time. Four studies are reviewed in which BMP-2 gene, or a BMP-related gene, the LIM mineralization protein-1 (LMP-1) gene, were transferred to cells at the site of spinal arthrodeses in animal models to improve bone fusion (see Table 1 for summary).

Riew et aF7 attempted to prolong the bone-inducing effect of BMP-2 using an adenoviral vector carrying the human BMP-2

*References 8,9, 11, 19, 22, 23,26, 27,30,38,40,41,52.

gene to transduce marrow-derived mesenchy- ma1 stem cells in New Zealand White rabbits. In their model, they isolated and expanded bone marrow mesenchymal cells from the re- sected ribs. Immunocytochemistry was used to show that approximately 80% of mesenchy- ma1 cells could be modified genetically to overexpress BMP-2 protein by treatment with an adenoviral vector encoding human BMP-2 (Adv-BMP-2). A similar efficiency of gene transfer was shown with the control vector encoding a marker gene Escherichiu coli P-galactosidase (Adv-@-gal).

Four weeks after rib harvest, the rabbits underwent spinal fusion at L5-6. Genetically modified mesenchymal stem cells loaded onto collagen sponges were placed between the transverse processes of L-5 and L-6 with Adv- BMP-2 transduced mesenchymal stem cells placed on the left side and the Adv-P-gal cells on the right.

After the second postoperative week, all rabbits were examined weekly using a radio- graph. Of the five study rabbits, one showed radiographic evidence of new bone formation on the side implanted with Adv-BMP-2 5 weeks after surgery. No new bone was noted on the control Adv-@-gal side. No new bone formation was observed on either side in the other four study rabbits. Rabbits were sacrificed 7 weeks after the operation, and histologic examination of the rabbit with new bone revealed mature bone with a trabecular struc- ture. Histology was unremarkable in the control side. The authors concluded that it was possible to transduce mesenchymal stem cells with hBMP-2 gene so that the transformed cells could produce BMP-2 in vivo that would exert an osteoinductive effect. These initial results, however, warranted further work on the longevity and efficiency of the gene transfer.

In a related study, Alden et all investigated the effect of percutaneous spinal fusion

Table 1. ANIMAL STUDIES ON GENE THERAPY FOR SPINE FUSION

Author Animal Model Gene Vector Outcome ~ ~~ ~

Riew et aP7 Rabbit h BMP-2 Recombinant adenovirus 20% fusion Boden et a16 Athymic rat LMP-1 Plasmid DNA with pCMV2 promoter 100% fusion Alden et all Athymic rat hBMP-2 Recombinant adenovirus (percutaneous 100% fusion

Wang et ale Syngeneic Lewis rat hBMP-2 Recombinant adenovirus 100% fusion injection along paraspinal muscles)


using BMP-2 using adenovirus-mediated gene transfer techniques in an athymic (immunocompromised) rat model. The 12 rats were divided into three groups of four rats each. All groups underwent paraspinal percutaneous injection with 7.5 pL of virus by a Hamilton microsyringe inserted through a 19-gauge guide needle placed at the junction of the spinous process and the lamina on each side. The groups were as follows: (1) Adv-BMP-2 bilaterally; (2) Adv-BMP-2 on the right, Adv-P-gal on the left; and (3) Adv-P-gal bilaterally. Com- puted tomography (CT) scans of the lumbo- sacral junction were obtained at weeks 3,5,8, and 12. Twelve weeks postinjection, the rats were sacrificed and studied histologically.

Examination of CT scans at each time point revealed new bone formation adjacent to the spinous processes and laminae at each of the Adv-BMP-2-injected sites, but no changes were observed at the Adv-P-gd control sites. Histology of the Adv-BMP-2 sites 12 weeks postinjection revealed extensive endochondral bone formation within the paraspinal muscu- lature with well-developed vascular channels and areas of cartilage as well as trabecular bone and mature marrow elements. New bone formed was in direct continuity with the adjacent laminae, inferior facets, and spinous process. No new bone formation was detected in the Adv-P-gal control animals. The authors concluded that in vivo endochondral bone formation was possible using direct adenoviral construct injection into the paraspinal muscu- lature and suggested that this approach potentially could be used for spine fusion in the fu- ture.

Wang et a146 transfected marrow cells with an adenoviral vector to produce BMP-2 in a rat intertransverse spinal fusion model. Bone marrow was harvested from syngeneic Lewis rats and expanded in tissue culture. These cells were genetically modified using Adv-BMP-2. The transfected cells were then soaked onto a guanidine-extracted, deactivated, demineralized bone matrix carrier and implanted into the rat spine between the transverse processes of L-L5. Fifty-six rats were divided into seven groups: (1 ) adenovirus-modified BMP-2-producing marrow cells, (2) recombinant BMP-2 protein, (3) demineralized bone matrix alone, (4) cells transfected with a control gene, (5) de-

cortication of the transverse processes alone, (6) autogenous iliac crest bone graft, and (7) normal (not genetically modified) bone marrow cells with demineralized bone matrix. Outcome assessment to determine fusion was performed by inspection, plain radiographs, manual palpation, and histologic evaluation.

At 4 weeks, all rats that had been implanted with adenovirus-modified BMP-2- producing marrow cells showed 100% fusion. All rats (100%) that had been implanted with recombinant BMP-2 alone also had complete arthrodesis by 4 weeks. All other groups did not show fusion at 8 weeks' time. The data of this study confirmed that regional gene therapy using BMP-2-producing bone marrow cells produced by adenoviral gene transfer could be used successfully in a rat model of intertransverse spine fusion, opening further avenues for its potential use in humans.

Boden et a16 investigated the feasibility of achieving lumbar spine fusion by liposomal- mediated transfer of a different osteoinductive protein gene, the LIM mineralization protein- 1 (LMP-1) gene. LMP-1 is an intracellular protein that plays a key role in the BMP-6 stimulation of osteoblasts. Because LMP-1 is an intracellular signaling molecule, the technique of gene therapy is best suited to deliver the LMP-1 cDNA (copy of the gene sequence that is necessary to synthesize the protein) within the cell.

Marrow fibroblasts were isolated from the hind limbs of rats and transfected with a plasmid containing LMP-1 in the forward orientation (study group) or the reverse orientation (control group) using lipofection. Once the cells were transfected, they were soaked onto a devitalized bone matrix carrier.

Fourteen athymic (immunocompromised) rats were used. Implants were composed of devitalized bone matrix carrier loaded with bone marrow cells transfected with either the LMP-1 gene (active) or the reverse LMP-1 (control). In the pilot phase, two rats received subcutaneous implants on the right (active) and left (control) sides of the chest. The same rats received implants to the lumbar (active) and the posterior thoracic (control) spine. In the experimental phase, 12 rats received active and control implants in the thoracic (Tll-12) and lumbar (L5-6) spine. Rats that received mar-

row cells transfected with the active gene in the thoracic region received cells with the control gene in the lumbar region and vice versa. Four weeks postoperation, all rats were killed. The samples of the rats that received subcutaneous implants underwent high-definition radiographs and undecalcified histology. All thoracic and lumbar spines in the experimental group underwent assessment of fusion by manual palpation, radiography, and undecalcified histology.

Examination of the subcutaneous implants from the two pilot rats revealed complete bone formation with marrow and osteoblast-lined trabeculae on the active side (carrier plus marrow cells with active LMP-1 cDNA) with no bone formation on the control side (carrier plus marrow cells with reverse LMP-1 cDNA). The two lumbar spines of the pilot rats that were implanted with the active LMP-I cDNA were completely fused, whereas the two thoracic control fusion sites that had been implanted with reverse LMP-1 cDNA failed to show new bone formation.

In the experimental group, three of the twelve rats died of perioperative anesthetic complications. In the remaining nine rats, complete arthrodesis was shown manually, radiographically, and histologically in nine of nine (100%) sites receiving marrow cells transfected with the active LMP-1 cDNA, whereas zero of nine (0%) sites receiving marrow cells transfected with inactive LMP-1 cDNA were fused. The authors concluded that the local delivery of LMP-1 cDNA to bone marrow cells was feasible and efficacious for enhancing spine arthrodesis.

PROSPECTS FOR GENE THERAPY IN SPINE FUSION

Pseudarthrosis in spine fusion continues to be a significant clinical problem, enough to warrant the search for alternate biologic methods to ensure satisfactory and depend- able fusions. The spectrum of gene therapy has opened new horizons toward reaching that goal.

The studies of Riew et aP7 and Wang et a1& represent a strategy in which gene transfer is used to achieve sustained-release BMP-2 delivery. Instead of using a slow-release carrier imbued with recombinant BMP-2, cells are ge-

netically modified to overexpress the protein, turning them into a biologic BMP-2 factory, which can be implanted into the site of spine fusion. Riew et aP7 only achieved 20% fusion, however, whereas Wang et a146 did not show any advantage in terms of fusion rates over recombinant protein. Although these studies show feasibility, they do not show a clear advantage of using gene transfer in place of recombinant BMP-2. In a related fracture-healing study of Lieberman et a1,% in which the same BMP-2 gene transfer model as Wang's was used, a histologic difference was detected in the bone formed by genetically modified cells when compared with bone formed by recombinant protein. The genetically modified cells appeared to form bone with a finer trabecular architecture than that formed by recombinant BMP-2. This observation suggests that gene transfer and local expression of BMP-2 may lead to biologic effects that are different from the addition of exogenous protein. Studies in larger animals, in which the amount of recombinant protein required has been a limiting factor, may be necessary to show a true advantage of the gene transfer over the addition of recombinant protein.

The study of Alden et al' points to the in- triguing possibility for developing a percutaneous method of spine fusion based on gene transfer techniques. Their study was in an immunocompromised animal, however. For a clinical application of this strategy, development of a vector with the efficiency of gene transfer of adenovirus, but without its immunogenic properties, is required.

Perhaps the most promising of the studies reviewed is that of Boden et a1,6 not only because of the rate of fusion that they were able to achieve but also because of their choice of gene for transfer. LMP-1, being an intracellular molecule, cannot be delivered efficiently ex- cept by gene transfer. The effects of this protein are so powerful that the low-level transfection achieved by lipofection was sufficient for inducing a potent biologic effect. The use of LMP-1 in a gene transfer strategy has been developed out of Boden's in-depth studies on the effects of BMP-6 at the molecular level. Boden's work is exemplary and points to some important considerations regarding the gene therapy strategy as a whole.

As the human genome project progresses, and new technologies such as the gene chip


and DNA array become available, the gene expression profiles of many cell types (such as osteoblasts) as well as many biologic processes (such as bone formation and healing) will be elucidated in great detail. Such studies may il- luminate, for example, the differences in gene expression in fusion masses of patients who do not achieve fusion (e.g., smokers) versus those who do, providing target genes for overexpression or suppression to enhance fusion. Such studies may lead to the discovery of novel genes, such as LMP-1, which may be more potent than secreted gene products, such as the BMPs in enhancing spine fusion.

Vector development is likely to lead to the production of improved vectors that are less immunogenic and that allow for controllable gene expression. Vectors with the in vivo gene transfer efficiency of adenovirus but without its immunogenic effects may lead to the development of percutaneous methods of spine fusion. Such vectors may be viral, nonviral, or a hybrid, incorporating the most advanta- geous aspects of each. Controlled gene expression, through the use of inducible or tissue- specific promoters, for example, is also likely to lead to increased safety.

CONCLUSION

Gene therapy is a novel therapeutic tech- nology that holds much promise for the im- provement of spinal arthrodesis. Studies have shown some efficacy in using plasmid-mediated and adenovirus-mediated gene transfer of BMP and related genes in animal models. Current areas of research, including the eluci- dation of gene expression profiles during bone formation and the development of new gene transfer vectors, promise the development of clinically applicable techniques in the near fu- ture.

References

1. Alden TD, Pittman DD, Beres EJ, et a1 Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy. J Neurosurg 90:109-114,1999

2. Baumgartner I, Pieczek A, Manor 0, et a1 Constitutive expression of ph VEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 971114- 1123,1998

3. Benn SI, Whitsitt JS, Broadley KN, et al: Particle-mediated gene transfer with transforming growth factor

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Spine Service Hospital for Special Surgery

535 East 70th Street, New York, NY 10021

e-mail: [email protected]

Gene Therapy For Spine Fusion

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