A new era of orthopedic surgery has been marked by gene therapy, an approach that provides options for treating patients with orthopedic conditions. Orthopedic surgeons must understand the terminology, fundamental concepts, and current research and development of gene therapy. Researchers used different gene therapy approaches, multiple gene vectors, and a multitude of cytokines to produce a list of orthopedic diseases that may be studied through these perspectives in the future. As gene therapy applications for bone healing, articular disorders, skeletal muscle injuries, bone tumors, and infections are explored, many new treatment strategies for musculoskeletal conditions will broaden the spectrum of practice for orthopedic surgeons.
The Scope of Gene Therapy
On September 14, 1990, a 4-year-old girl with adenosine deaminase deficiency, which is a severe combined immunodeficiency disease that is the hereditary equivalent of acquired immune deficiency disease (AIDS), was treated with ex vivo viral vector auto-lymphocytes that had been genetically engineered. The patient was treated by W. French Anderson and colleagues at the National Institutes of Health in Bethesda, Maryland, USA.1
In 1998, 320 clinical trials were performed throughout the world. However, the era of clinical gene therapy is considered to have begun in 1992 when 20 clinical trials were performed in the United States. In 2004, the number of clinical trials performed worldwide may have reached 4 digits.
In the early 1990s, the language of gene therapy research and development seemed unfamiliar to surgeons, especially because it included many new expressions and abbreviations. This tutorial discusses basic principles of gene therapy and examines the relationship between gene therapy and orthopedic surgeons. Although this tutorial will not cover basic cell and gene biology, related cell and gene technology expressions will be covered.
Gene therapy is the insertion of manufactured or controlled genes into a target tissue or organ. The goal of gene therapy is to cure or prevent diseases through the production of proteins.
A gene needs a carrier, or vector, to transfer it to the target organ, in which it is inserted. The gene is introduced into the vector by a laboratory cut-and-paste process using a variety of endonucleases, which are enzymes that split DNA sequences at specific sites.
The expression of a gene as an effective protein in the target organ must be controlled in duration and strength to achieve the desired therapeutic goals for each disease.2
The goal of gene therapy is to treat patients with inherited diseases and acquired diseases and provide vaccinations for infectious and non-infectious conditions.2
Although the genetic defects in inherited diseases are usually well-characterized, they require long-term gene therapy. Furthermore, the amount of expressed protein required by the gene may be variable. This is the case in the factor VIII protein in hemophilia and in the Hb A and Hb B proteins in sickle cell anemia.
The genetic defects in acquired diseases are less well-characterized than the genetic defects in heritable diseases. However, a shorter term of gene expression may be effective in acquired diseases. The availability of large clinical pools including information about diseases of global concern such as AIDS and cancer makes acquired diseases easier to study than heritable diseases in terms of gene expression. Vaccination for non-infectious diseases such as cancer is based on engineering an immune response to tumor cells. New vaccinations may be available for patients with infectious diseases such as AIDS. Better vaccines may be available for patients with other viral conditions such as measles, and for bacterial conditions such as tuberculosis (TB).
Modes of Gene Therapy
Depending on when the healthy protein is produced, 2 modes of gene therapy are possible, ex vivo, in which the healthy protein is produced outside the body, and in vivo, in which the healthy protein is produced inside the body (Table 1).
Table 1. Comparison between ex vivo and in vivo
|Needs sophisticated lab
||Does not need sophisticated lab
|Selection of cells simple
||Selection of cells difficult
|Controlled target cells
||Less target cell control
|Allows safety testing
||Does not allow safety testing
|No direct viral or other particle introduction
||Direct viral or other particle introduction
The vector may be delivered through a systemic mode to all cells in the body or by a local mode to only the targeted tissue.
Systemic gene therapy has the potential to target all cells and may be an effective treatment for patients with metastatic diseases, in which all cells may be affected.3 However, systemic delivery can also be associated with unwanted side effects and is therefore more dangerous than other modes of gene therapy. Systemic delivery is unable to penetrate tissues with a poor blood supply, such as cartilage and menisci.
Local gene therapy is applied by injecting the vector into the specific tissue. Injections are made directly or indirectly.4 In direct gene therapy, the vector is injected directly into the specified target tissue. Direct gene therapy is easier to implement than indirect gene therapy. Direct gene therapy has been used effectively to target knee and ankle joints, skeletal muscles, the spine, anterior cruciate ligaments, medial collateral ligaments, and menisci as well as other tendons and ligaments. However, the cells targeted through direct gene therapy may not continue to be targeted after the virus has been injected. Furthermore, the target cells cannot be tested after being exposed to the vector.
In the indirect approach, the target cell is removed from the body, exposed to the vector in vitro, and then reinserted into the body. Thus, indirect gene therapy is termed "ex vivo gene therapy." Indirect gene therapy is technically more demanding than direct gene therapy because the target cells must be harvested from the body and reinserted. However, indirect gene therapy has the advantage of allowing a physician to accurately select a particular cell. In addition, the cell can be tested in vitro to ensure protein expression and to evaluate the cell for possible unpredicted effects of the gene expression. Indirect gene therapy has been used effectively to target the spine, articular cartilage, and skeletal muscles of animals. Recently, indirect gene therapy has been used to target human metacarpophalangeal joints using bone marrow and mesenchymal stem cells, osteoprogenitor muscle cells, fibroblasts, peripheral buffy-coat blood cells, chondrocytes, and synovial cells.4
Gene Transfer Technology
The ideal vector for gene transfer into the target organ must fulfill many criteria, which cannot all be met by current technology. The ideal delivery system accommodates a broad size and range of DNA, targets to a specific cell type, provides long-term gene expression, is available in a concentrated form, and prevents DNA replication. The ideal delivery system must also be non-toxic and non-immunogenic.
Vectors are vehicles that carry the engineered gene to the target cell. Common vectors are viral (Table 2) because they manipulate genes easily, are available, and have an excellent ability to infect living cells. A viral vector must be rendered replication incompetent to prevent uncontrolled trans-gene production and must have part of its genome removed to allow trans-gene insertion. Some nonviral vectors are also available, and recently hybrid vectors (viral and nonviral) have been developed, as well as viral replication-competent vectors.
Retrovirus (murine-leukemia virus) is an excellent vector with long-term expression, no immunogenic protein in its genome, and no pre-existing human immunity. However, its use is limited to dividing cells, its preparations are stable for only a short time after engineering, and it may have replication potential.4
Adenovirus (AdV), on the other hand, transduces a broad spectrum of tissues spanning the respiratory epithelium, vascular endothelium, cardiac and skeletal muscles, central nervous system, and peripheral nerves. Consequently, AdV has a broad range for administration through intravenous, intrabiliary, intravesical, intracranial, intramuscular, and intraperitoneal administration, as well as through aerosolization. Nevertheless, AdV remains episomal in the target cell, which means that their concentration becomes diluted with cell division. Adenovirus is a common virus and may initiate a host immune response. Finally, the human host cell may provide an enzymatic catalyst (E1) for adenovirus replication.4
The adeno-associated virus (AAV) is a single-strand DNA non-enveloped virus that integrates well into the genome of non-dividing cells in chromosome 19. The adeno-associated virus is not associated with a known human disease, is stable in chemical preparations, and is easy to purify, concentrate, and store. Disadvantages of AAV are limited carriage capacity and difficulty in producing large quantities.5
Herpes simplex virus
The herpes simplex virus (HSV) and other less common viruses such as vaccinia, papilloma, Simian virus 40, polyoma, picornavirus, lentivirus, and others are also in the pipeline for gene transfer research.6,7
Table 2. Viral gene delivery vectors.
||Most developed,easy production
||Random insertion, infects only dividing cells, variable long-term expression.
||High titer, high infectivity, easy production, infects non-dividing cells, broad range of host cells
||Episomal DNA, toxicity, persistent viral protein expression.
||Non-pathogenic, high titer, integrates in a selective site, stable expression, infects non-dividing cells, broad range of host cells
||Small (~4k base), difficult production, poorly developed
||Infects non-dividing cells, large packaging (~35k base), high titer
||Poorly developed, toxicity, transient expression
Nonviral vectors are good vectors to use because they can be manufactured and have no replication potential. Nonviral vectors include liposomes, DNA-coated gold particles, simple DNA-ligand complexes, and purified uncomplexed plasmid DNA.8
Liposomes form a delivery system that permits drug targeting to an intracellular location. This delivery system allows in vivo gene therapy to deliver large hydrophilic transgenes across the plasma membrane and into the nucleus for direct access to the cell transcription machinery.9,10
The gene gun (colloidal gold) system is a simple delivery system with a high efficiency potential. The gene gun technology paints the surface of gold colloid particles with ligand protein or DNA and directly bombards them under high pressure through the cell membrane (Table 3).11,12
Table 3. Nonviral gene delivery vectors.
||Inexpensive, non-toxic, non-immunogenic, large carriage capacity
||Episomal DNA expression, not all cell types lipofect well
||Episomal DNA expression, often poor expression
|Gene gun (colloidal gold)
||Access to tissues in vivo difficult, episomal DNA expression, long-term after-effect of gold particles unknown
Gene Expression Control
Efficient gene transfer to the proper target cell does not ensure automatic effective treatment of the patient. Gene expression must be regulated to the appropriate level and appropriate time-duration for the patient to be effectively treated.13
The regulation of gene expression is achieved in nature through gene promoters, which are regions of the DNA that are located near a gene that is essential for efficient transcription. Genetic researchers have developed 3 kinds of induced gene promoters: viral, eukaryotic, and hybrid. These induced gene promoters, which work only in the presence of exogenous stimuli such as heavy metals, dexamethazone, or mifepristone (RU486) or an endogenous stimulus, facilitate disease activity inducible promoters. Disease activity inducible promoters form an automatic promoter system that works only during disease flare-ups, preventing induced gene promotors from raising levels of human interleukin (IL)-1 in rheumatoid arthritis flares.
For most gene therapy protocols, a prolonged duration of gene expression is more critical for successful treatment than the level of gene expression (especially in patients with hemophilia or Gaucher's disease). Nevertheless, in some chronic diseases, including rheumatoid arthritis and osteoarthritis, the control of the duration and level of gene expression is critical because prolonged effect of high concentrations of anti-inflammatory and chondroprotective molecules may be dangerous.14
Genes and Tumors
Malignant tumors are linked to 3 types of genes that are always present in the genome, namely, oncogenes, tumor suppressor genes, and fusion genes.15
Oncogenes are activated from normal "proto-oncogenes" to make a dominant oncogene that keeps cells permanently "turned on." Oncogenes release proteins that encode for aberrant growth factors, growth factor receptors, and direct signal transducers from the cell membrane to the nucleus.
Tumor suppressor genes encode for proteins that restrict cell proliferation and act as "growth-brakes." Defects in tumor suppressor genes are present in many malignant conditions like the retinoblastoma-1 gene defect, and the p53 gene defect. The p53 gene defect occurs in approximately 50% of patients with cancer.16
Fusion genes produce abnormal chromosome shapes, such as the Philadelphia chromosome, which is present in chronic myeloid leukemia and Ewing's sarcoma.17
Obstacles to Gene Therapy
Gene therapy is one of the most difficult medical fields, and gene therapy researchers face many obstacles. The DNA delivery systems and their pharmacokinetics, especially in vivo research, have possible hazards, and the duration of expression of transferred genes required for certain treatments may be unknown. Only retrovirus and AAV can be integrated into the host genome, whereas other vectors are episomal and are diluted by host cell division. The adverse effects of heterologous gene expression related to immune response and potentially dangerous viral replication also present a challenge.
Furthermore, many ethical issues connected to gene therapy are of global concern, such as gene transfer into the germ-line tissue for "frivolous" purposes, prophylactic gene therapy, alteration of normal characters, and intentional production of mutant viral, bacterial, or tumor DNA molecules.13
Orthopedic Gene Therapy
Recent advances in gene therapy have had little impact on the day-to-day practice of orthopedics. This situation is likely to change as the volume of new information in the field of genetics reaches all aspects of medical practice. The general strategy for orthopedic gene therapy may be oriented around transfer techniques such as in vivo, ex vivo, or target cell-oriented techniques based on the abundance of specialized cell lines (synoviocyte, chondrocyte, fibroblast, osteoblast, osteoclast, marrow stem cells) present in the musculoskeletal system.18
Patients with osteogenesis imperfecta, familial osteoarthritis, osteopetrosis, some crystal arthropathies, certain forms of osteoporosis, muscular dystrophy, mucopolysaccharidosis, hemophilia, Marfan's syndrome, Ehlers-Danlos syndrome, and other connective tissue hereditary disorders may benefit from ongoing gene therapy trials.19-21
Gene therapy trials are likely to be used to treat patients with acquired diseases such as arthritis, collagen diseases (systemic lupus erythematosus, scleroderma, and others), cartilage damage, ligament and tendon repair, fractures (especially delayed union, nonunion, and spinal fusion), infections such as TB and coccidiodomycosis, some forms of osteoporosis, and osteosarcomas and other tumors. Gene therapy trials are less likely to be used in patients with heritable diseases.22
Patients with autoimmune rheumatic conditions will benefit from gene therapy when genes that code for immunosuppressive proteins are developed. Perhaps the only clinical gene therapy trial licensed to treat patients with nonfatal acquired conditions is the trial for rheumatoid arthritis, which was conducted at Harvard Medical School, Boston, Mass. In this trial, 9 patients participated in a double-blinded, prospective-controlled trial. Researchers studied the intraarticular expression of the IL-1 receptor antagonist gene in the patients' small hand joints after 1 week of gene therapy. The preliminary data were encouraging.23
Enhancing osteogenesis for defective fracture healing has potential as a gene therapy approach because several growth-promoting substances have been identified at the site of fractures. These growth-promoting substances belong to 2 groups of proteins; growth factors that are peptide-signaling molecules, and growth factors that are cytokines, or immunomodulatory factors.24 Although these proteinaceous molecules could be produced by recombinant DNA technology, recombinant proteins have a short half-life, and some classes of intracellular factors are ineffective when administered as exogenous proteins. Thus, the use of regional or localized gene therapy holds greater promise than recombinant DNA technology in controlling the progress of bone repair.25
Gene therapy research on healing other tissues is also ongoing. Recombinant human bone morphogenic protein (rhBMP)-7 has been used to produce a new articular surface on allograft bone, and rhBMP-2 was used in full thickness articular cartilage defects. Recombinant human bone morphogenic protein-2 also accelerates the healing of the tendon-bone junction when it is incorporated in a collagen sponge. Growth differentiation factor (GDF) 5, GDF- 6, and GDF-7 and bone morphologic protein (BMP)-12 carry more hope for direct ligament repair. Insulin-like growth factor was found to be beneficial in controlling post-traumatic muscle wasting. Regeneration of degenerative intervertebral disks may be possible through direct gene delivery of transforming growth factor beta.26
Musculoskeletal infections may benefit from gene technology in many aspects; the TB standard Bacille Calmette Guerin (BCG) substrains were found to lack almost 100 genes in their genome.27 Consequently, new vaccines are being developed. New genetically engineered mutant strains of the Mycobacterium are being generated to render the TB and BCG substrains averulant.28 Skeletal mycosis could respond well to certain antibiotics that are known to be systemically toxic. Liposomal amphoterecin B avoids nephrotoxicity and other toxic reactions by being encapsulated inside the Liposome vector, delivering the drug only intracellularly, and enabling administration of high doses that were previously impossible to use.29
Cancer Gene Therapy
The logical strategies for cancer gene therapy are to interrupt "oncogene" function and/or to restore "tumor suppressor gene" function. Oncogene function and tumor suppressor gene functions are the known molecular regulators of tumor cell growth. Although these strategies are needed to carry out gene therapy, they are difficult to achieve by current gene technology because each cell, including metastatic cells, must be targeted during the suitable cell cycle. Thus, alternative strategies have been developed. Among them are cell-targeted suicide and tumor vaccination, either by ectopic cytokine expression or by immune enhancement. More recent approaches explored the strategies of tumor infarction and drug resistance gene transfer.
Cell-targeted suicide is the expression of analog tumor genes that exhibit a negatively selectable phenotype to cancer cells. An example of such an analog tumor gene is the HSV thymine kinase gene, which expresses a drug-metabolizing enzyme that converts the prodrug gancyclovir to a toxic metabolite through phosphorylation. Cell suicide occurs after the administration of gancyclovir. Neighbor tumor cells are also killed as a result of the "bystander effect."30
Tumor vaccination entails engineering an immune response to tumor cells by inducing a tumor to secrete cytokines such as IL2 or IL3, inducing tumor cells to express a strong rejection to the major histocompatibilty complex antigen, or inducing tumor cells to express lymphocyte costimulatory molecules (B7-1), which increase the numbers and the activity of the lymphocytic cellular phase of immunity.31,32
Tumor infarction is an antiangiogenic gene therapy approach in which induced markers on tumor endothelial cells render the cells susceptible to the intravenous injection of truncated tissue factor, which acts as a selective thrombogen and can cause complete tumor resgression.33,34
Drug resistance gene transfer is based on the fact that malignant cells survive the cytotoxic effect of chemotherapy by expressing genes that inactivate toxic drugs. These genes can be engineered to have an opposite effect by protecting normal host tissues from the toxic effects of chemotherapy. The multidrug resistance gene 1 encodes the multidrug transport protein, which is capable of pumping a variety of known toxic chemotherapeutics such as Adriamycin (Pharmacia & Upjohn SpA, Milan, Italy), vinca alkaloids, taxol, and others extracellularly. The multidrug resistance gene 1 has been proposed to render bone marrow resistant to toxic chemotherapy.35,36
A new biologic era of orthopedic surgery has been initiated by scientific advances that have resulted in the development of gene therapy and tissue engineering approaches for treating musculoskeletal disorders. Orthopedic surgeons must understand the terminology, fundamental concepts, and current research in this developing field. Different gene therapy approaches, multiple gene vectors, a multitude of cytokines, a growing list of potential scaffolds, and putative stem cells are being studied. As gene therapy applications for bone healing, articular disorders, skeletal tumors and infections, and skeletal muscle injuries are explored, innovative methodologies ensuring the safety of patients can lead to many new treatments for patients with musculoskeletal conditions, broadening the spectrum of practice for orthopedic surgeons.
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