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Post-natal somatic cell gene therapy. KW CulverHuman Gene Therapy Research Institute, Des Moines, Iowa 50309-9976, USA., USA Correspondence Address: PMID: 0008568710
Keywords: Animal, Clinical Trials, Ethics, Medical, Gene Therapy, Gene Transfer Techniques, Genetic Diseases, Inborn, therapy,HIV Infections, therapy,Human, Neoplasms, Experimental, therapy,
For decades, people have dreamed of the ultimate cure for disease, the correction of the gene defect itself. Significant advances in human genetics have occurred since the structure of DNA was identified by James Watson and Francis Crick 40 years ago [Table - 1]. By 1993, more than 3000 of the more than 100,000 genes in the human genome have been mapped. This growth in our understanding of the fundamentals of human genetics has led to the initiation of the first human gene therapy experiment[1]. On September 14, 1990 at the National Institutes of Health, a 4-year-old girl with adenosine deaminase (ADA) deficiency received the first gene therapy treatment. This child, who suffered from severe combined immunodeficiency (SCID) due to the ADA deficiency, has manifested significant improvement in her immune system as a result of the gene therapy. With more than 4000 genetic diseases, many of whom have no satisfactory therapy, the prospect of a genetic cure using "gene therapy" stands as a great monument of hope for patients, families and health care providers.
Gene therapy is the insertion of a functioning gene into the cells of a patient to correct an inborn error of metabolism (i.e. genetic abnormality or birth defect) or provide a new function to a cell (e.g. insertion of an immunostimulatory gene into cancer cells to vaccinate a patient against their own cancer). This is a very broad definition that includes the potential treatment of all genetic disorders as well as cancer, infectious diseases and other acquired disorders through the genetic modification of cells of the human body to prevent or eliminate disease. Somatic cells are the non-reproductive cells of the body (e.g. skin, muscle, bone, brain, cells of the blood, etc.). Gene therapy that would alter the reproductive cells (sperm or ova) is termed germline gene therapy. At this time in the evolution of human gene therapy, somatic cell gene therapy is the only form that is technically feasible and ethically acceptable for human use. Historically, gene therapy had been thought of as a treatment for classical inherited genetic diseases such as thalassemia. However, this concept has broadened in several ways. First, gene therapy may become a useful treatment for a variety of acquired diseases such as HIV infection, not just congenital disorders. Second, we are no longer limited to the concept that genetic correction can only be achieved by the correction of the defective cell. For instance, the insertion of the insulin gene into skin or endothelial cells may be much more practical and efficient than attempting to recover and genetically correct pancreatic islet cells. There is also no reason why one must necessarily insert the hemophilia Factor VIII or IX genes into liver cells, where the clotting factors are normally made. Clotting factor genes maybe more easily inserted into tissues that can be repeatedly removed from the body and grown in the laboratory than hepatocytes, which require a surgical resection. Third, we now know cancer is a genetic disease, a series of genetic aberrations within one cell that results in autonomous, uncontrolled growth. The ability to manipulate the genetic make-up of tumour cells, restoring normal cellular patterns or to use alteration in the genetic constitution of tumour cells as means for their selective destruction offers tremendous therapeutic potential.
The ability to transfer genes into living cells has been available for more than 20 years. In general the early techniques were inefficient and not practical for clinical use. However, during the last 10 years, several novel and more efficient techniques for the transfer of genes into living cells have been developed. Unfortunately, techniques that replace the defective gene with the normal gene, a process termed homologous recombination, remains too inefficient for consideration for clinical trials[2]. In the last 3 years, the development of improved methods for both ex vivo and in vivo gene transfer has processed into clinical trials[3]. The available gene transfer techniques include chemical, physical, receptor-mediated endocytosis an recombinant virus vectors [Table - 2]. A vector is a disabled virus that can be used a vehicle for gene transfer. Each technique has its own theoretical advantages and disadvantages. Many of these methods will find a niche in the clinical application of gene therapy. One discriminating feature among gene transfer methods is related to whether or not the transferred gene is integrated into the DNA of the largest cell. For example, it a corrective gene is to be inserted into hematopoietic stem cells (HSC), a process, which will result in highly efficient, stable integration of the inserted gene, is needed. Otherwise, as the stem cell proliferates and differentiates, the inserted gene will be progressively diluted out and eventually lost. By contrast, gene insertion into terminally differentiated, non-proliferative tissue such as skeletal muscle or liver might not need integration as a feature of the gene transfer system. At this point in time, the application of gene transfer to humans has predominantly utilized murine retroviral vectors, which stably integrate genes into the target cell genome.
In vitro gene transfer methods: All of the methods listed in [Table - 2] can be used to transfer genes in vitro. Gene transfer in vitro with murine retroviral vectors (MRV) has been used most widely in clinical trials. This ex vivo method have the advantage of eliminating the possibility of gene transfer into germline tissues. However, in vitro methods are only suitable for those tissues that can be removed from the body, genetically altered and returned to the patient (e.g. cells of the hematopoietic system). Unfortunately, many tissues of the body cannot be removed and reimplanted (e.g. brain). Due to these limitations, in vitro gene transfer will be primarily applied to hematopoietic cells, skin, endothelial and tumour cells. For gene therapy to become widely applicable to patients, a variety - of new in vivo gene transfer techniques will need to be developed. In vivo gene transfer methods: This can be accomplished in 3 general ways: 1) By the direct injection of "naked" DNA (not coated or bound to antibody, protein or lipid) directly with a syringe and needle into a specific tissue (e.g. muscle, thyroid), 2) DNA can be injected in artificially generated lipid vesicles (liposomes) which allow the DNA to survive in vivo and bind to cells, or 3) DNA can be conjugated to a carrier (e.g. antibodies to a specific cell surface moiety or attachment to galactose or transferrin) to target DNA to specific tissues. An advantage of these systems is their simplicity, low risk and decreased expense compared to recombinant viral vectors. The disadvantages include poor gene transfer efficiency and a low level of stable integration of the injected DNA. Therefore, long-term expression without repeated administration of the vector will be a limitation with these methods, especially in the as proliferating cells. However, the new DNA may continue to express its genes for months in cell that do not regularly proliferate (e.g. muscle). Several circumstances exist in which the direct DNA gene transfer method may have a place in clinical gene therapy application. First, this form of gene transfer may be effective in cancer therapy where transient expression may be sufficient to induce tumour cell destruction. Second, DNA transfer might be useful for repeated injection into non-proliferative target tissues if permanent expression is not absolutely required (e.g. hormone production). Third, the direct injection of DNA into the body might be a useful method for immunization. Animal studies have shown that the insertion of influenza DNA into skin or muscle cells results in transient expression of the gene by the cell inducing a systemic immune response[4]. Recombinant virus vectors, in particular, the Moloney murine leukemia virus (MoMLV) vectors are the most commonly used method in human clinical gene therapy protocols[5]. These vectors are produced by replacing the vital genes required for the production of replication-competent viruses with the genes desired for transfer. This genetic engineering technique produces replication defective or replication incompetent viruses that are unable to produce a productive viral infection while maintaining the ability to bind to the cell surface[6]. Once the virus binds to the cell, it is internalised and the vector genes are inserted into the target cell chromosomes. Once integrated, the vector genes become a stable part of the cell's inheritance, being passed along to all cell progeny during normal cell division. This feature is critical for the development of curative gene therapy methods for genetic diseases. Another feature of the MoMLV vector system, is that the target cell population must be proliferating to allow gene transfer. Totipotent hematopoietic stem cells (HSC) are not generally actively proliferating cells. Therefore, the use of retroviral vectors for effective stem cell gene transfer requires manipulations to induce stem cell proliferation. While this feature has delayed the clinical application of gene transfer to HSC, this feature allows some selective targeting of gene transfer into tumour cells since they generally have a much higher index of proliferation than the surrounding normal tissues. One potential disadvantage of retroviral vectors is the fact that they randomly integrate into the host cell genome. Random integration means that vector gene expression may vary between cells presumably due to differences in the local environment of the chromosome where neighbouring genes may influence gene expression. For many genes, a very wide range of gene expression from cell to cell will not be a problem. However, certain systems may require stringent regulation to maintain normal cellular metabolism (e.g. thalassemia). Random integration also provides for the possibility that the integration event could occur with a gene that is absolutely required for normal cellular function or survival. In such a case, the transduced cell might be killed. With millions of cells being treated for human therapy, the loss of an occasional cell would be of little consequence. However, a more serious theroretical problem with random integration may result if vector insertion activates an oncogene or inactivates a tumour suppressor gene. Either of these events could potentially lead to the eventual transformation of that cell to a malignant phenotype, a process termed insertional mutagenesis. This is a theoretical possibility since the use of replication incompetent retrovial vectors has never been reported to result in malignant transformation in any in vivo system. The only other virus-mediated gene transfer system approved for human use is the adenovirus[7]. These vectors are derived from a common virus that produces infections of the upper respiratory tract. These vectors have a natural tropism for respiratory epithelium. In order to improve their safety, they have been rendered replication-deficient by deletion of the E1 structural genes. However, one of the risks of this gene transfer method, is that wild type Adenoviruses may infect the same cell with their own E1 gene allowing the production of infectious recombinant replication-competent vector particles. This vector system has been primarily developed for transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into airway cells of patients with cystic fibrosis. Cystic fibrosis is an autosomal recessive disorder that results in defective transport of chloride ions through epithelial cells. Insertion of a normal copy of the MR gene corrects the chloride transport defect in vitro[8]. The injection of MR adenoviral vectors into the airway of animals results in highly efficient gene transfer into cells lining the airway. Adenovirus vectors have also been shown to have the ability to transfer genes into most tissues of the body (e.g. lung, brain, muscle). The primary advantages of this vector are high efficiency gene transfer regardless for the proliferate state of the tissue, production at very high titer (1011/ml) vs retroviral vectors (106/ml) and delivery by an aerosol. As in true of all vector systems, there are several disadvantages to the use of adenoviral vectors. First, these vector appear to be able to infect nearly all cells, expressing their genes in each infected cell. This lack of discrimination could result in toxicity in normal surrounding tissues. Tissue-specific promoters may be used to overcome this problem. One example of a tissue-specific promoters is ? fetoprotein (?FP). Transfer of the ?FP promoter coupled to the gene of interest into a wide array of cell types will result in expression of the vector genes only within the cells that normally synthesize ?FP. Since ?FP is made almost exclusively by hypatocellular carcinoma cells, this system may limit cell destruction to hepatoma cells alone. Second, this vector does not integrate. Therefore, long-term expression without repeated administration of the vector is a problem, especially in the proliferating cell types. However, in cell types that do not regularly proliferate (e.g. muscle), the adenovirus may continue to express its genes for months. Third, the repeated aerosolization of the adenovirus into the airway may induce specific anti-viral antibody that will limit the repeated administration of the therapeutic adenovirus vectors. Genetic engineering of adenovirus vectors to allow specific integration and expression precisely in the desired target cell will markedly increase their utility for clinical use. Other virus vectors in pre-clinical development include the adeno-associated virus (AAV), which is based upon a replication-defective human "dependent" parvovirus[9]. This virus is considered "dependent" because it requires co-infection with another virus such as adenovirus or herpes virus to produce a productive infection. Without co-infection, the virus appears to integrate at a specific site (non-random) on chromosome 19, remaining latent until a co-infectious agent arrives. This virus is ubiquitous and infects many cell types. It is thought to be a non-pathogenic integrating virus and appears to have great potential as a vector for clinical use. Herpes simplex virus vectors have the unique advantage of being tropic for the central nervous system, establishing life long latent infections in neurons. Genetic attenuation of these viruses to prevent lytic destruction of the infected cell can theoretically be made by deleting virulence genes[10]. These vectors could potentially be used to deliver genes into neurons for the treatment of neurologic diseases such as Parkinson's disease or for the treatment of CNS tumours. Vaccinia virus, which was first employed as the vaccination that resulted in the eradication of small pox, is being used as a vector to immunize against a variety of infectious agents. The first human use of recombinant vaccines for human use has centred on HIV infection. A phase I trial involving 37 healthy individuals has shown no severe side effects and an immune response to the HIV-1 envelope proteins expressed by the vector. However, none of the individuals developed neutralizing antibodies to the HIV antigen.
There have been 4 gene therapy experiments approved for the treatment of genetic diseases in the USA [Table - 3] The first was an attempt to treat a rare autosomal recessive disorder called ADA deficiency. The observation that ADA gene corrected T-lymphocytes have an increased survival advantage in vitro and in vivo allowed the initiation of gene therapy in 1990[11]. Two children, now 7 and 12 years are currently being treated at the National Institute of Health (NIH). They have received IV infusions of autologous ADA gene-corrected T-cells periodically for the past 3 years. Persistence of ADA gene-corrected T-lymphocytes in vivo has been documented in both the children. Both demonstrated persistent improvement of in vivo humoral and cellular immune functions. The genetic correction of HSC is a potential single treatment cure for ADA deficiency. Nine children worldwide have received ADA gene-corrected stem cells. Problems with inefficient gene transfer persist hampering the development of this curative approach. These technological problems should be solved in the next 5 years allowing ADA deficiency to become curable with gene therapy. Familial Hypercholesterolemia (FH) is a rare dominant disorder that is caused by a lack of receptor expression required for uptake of low density lipoprotein (LDL). These individuals have serum cholesterol levels ranging to 1000 mg/dl with a mean of 600-700 mg/ dl resulting in the development of coronary heart disease in childhood. An initial attempt at the genetic correction of LDLr (LDL receptor) deficiency was initiated in 1992 at the University of Michigan[11]. In this study, a piece of liver, about 200-300 gms, was resected, hepatocytes were grown in the laboratory, genetically altered with a murine retroviral vector and then the cells were reinfused through the hepatic artery into the remaining liver. Early results have shown a persistent survival of the genetically-altered hepatocytes, a 20-40% decrease in the serum cholesterol levels and a heightened response to cholesterol lowering medications. No toxicity related to the transfer of the gene has been identified in any of the 3 patients treated. Cystic fibrosis (CF) is an autosomal recessive genetic disorder resulting in severe lung disease and poor growth due to chronic lung infections and an inability to absorb nutrients from the digestive tract[12]. Transfer of the CFTR gene into CFTR deficient cells in vivo using adenoviral vectors or liposomes has corrected the biochemical abnormality without significant toxicity. Based upon these pre-clinical data, there are 5 approved human clinical trials in the US using adenoviral vectors for the treatment of CF. The first protocol began enrolling patients in April, 1993. Early results have confirmed gene transfer and CFTR expression in airway epithelial cells, but have had increasing adverse side effects with increasing doses of viruses. The clinical utility of this approach, which is expected to require repeated treatments to be fully effective, remains uncertain. A trial using liposomes is underway in the United Kingdom. The most common glycogen storage diseases are Hunters and Hurler's syndrome, with the most common lipid storage disease being Gaucher's disease. Alone, these disorders do not represent large numbers of patients, but taken together, storage diseases are among the 10 most common genetic abnormalities. In vitro and in vivo animal experimentation has suggested that many of these disorders may be amenable to treatment with gene insertion into HSC. Once the ability to stably express the desired genes in the appropriate hematopoietic lineage has been documented, the opportunity for cure of this group of disease becomes a distinct possibility. The first human trials will focus on the insertion of the glucocerebrosidase gene into the HSC in patients with Gaucher's disease. Three studies have been approved and are expected to begin soon.
Cancer: Major advances in our understanding of how and why cancer occurs has proven that cancer is a complex genetic disease that results in the abnormal proliferation of a clone of cells. This understanding of the genetic basis for cancer allows entirely new approaches to the treatment of cancer [Table - 4]. For instance, the deletion of tumour suppressor genes can be theoretically corrected at the genetic level by the insertion of a normal copy of the gene, perhaps before the development of cancer. Likewise, the over expression of an oncogene may be blocked at the genetic level by the insertion of an anti-sense gene that will bind to the oncogene disabling its ability to express. Cancer cells by virtue of their high proliferative index are ideal targets for murine retroviral vectors, who require that the target cells be proliferating for gene integration and expression. An early group of clinical studies have focussed upon the insertion of “marker” genes into the bone marrow of patients in remission from leukemia and neuroblastoma who are undergoing autologous, purged bone marrow transplantation. The first of these studies has identified leukemia relapses that occurred from the gene "marked" marrow even though there was no evidence of leukemic cells in the infused marrow at the lime of transplatation[13]. Apparently, the systemic chemotherapy was effective in eliminating the systemic disease, but the marrow purging techniques were suboptimal. This fundamental biological question could only be adequately answered with gene marking using retroviral vectors, since the vector will only insert into dividing cells and the gene is passed onto all daughter cells unlike other forms of gene transfer. A second series of clinical studies are in preparation to evaluate different methods of marrow purging in human autologous bone marrow transplantation. The genetic manipulation of stem cells may also be theoretically used to protect them from the toxic effects of chemotherapy. This approach to the gene therapy of cancer maybe accomplished by the insertion of the multiple drug resistance type-1 (MDR-1) gene into stem cells prior to administration of high dose, myelosuppressive chemotherapy[14]. The MDR-1 gene was isolated from tumour cells and functions to pump chemo-therapy drugs (i.e. daunorubicin, doxorubicin, vincristine, vinblastine, VP-16, VM-26, taxol, actinomycin-D) from the tumour cells. This tumour resistance mechanism represents one of the ways in which tumours develop resistance to chemotherapy drugs. The use of ex vivo retrovira) vector- mediated insertion of the MDR-1 gene into murine marrow cells has demonstrated significant protective effects in vivo, when the animals were treated with high doses of taxol. Human clinical trials are in development to study these properties in the high dose chemotherapy treatment of disseminated breast cancer and ovarian cancer. Another approach to the genetic therapy of cancer is gene transfer into T-lymphocytes. Since T-lymphocytes are critical for the prevention and elimination of tumours, investigators are growing T-lymphocytes from tumour biopsies. Researchers are inserting the TNF alpha cytokine gene into these tumour-infiltrating lymphocytes (TIL) in an effort to increase their anti-tumour efficacy[15]. These experiments are currently in an early toxicity trial stage. These pace of these human experiments have been slowed due to a poor efficiency of gene transfer into human TIL and a down regulation of cytokine expression by the TIL[16]. The most common type of clinical trials involves the injection of gene-modified human autologous or allogeneic tumour cells. These initial experiments are an attempt to immunize tumour-bearing patients against their own tumour by injecting genetically altered cells that are expected to increase the host immune reactivity to the tumour. Human trials have been approved involving the in vitro insertion TNF?, IL-2, IL-4 or GM-CSF using retroviral vectors into melanoma, neuroblastoma, breast cancer, colorectal and renal cell carcinoma cells[17],[18],[19],[20]. These "tumour vaccines" are produced by surgically removing tumour from the body and genetically- altering the tumour in the laboratory. Once the tumor has been shown to produce the inserted gene product, the tumour cells are reinjected subcutaneously (SQ) into the patient. Melanoma and renal cell carcinoma have been the primary focus of the tumour vaccine studies since they may be more immunogenic than other tumours and therefore more likely to respond. The first attempt at the genetic modification of tumors in situ were initiated in 1992. The first involved the direct injection of liposomes and the second, the in vivo gene transfer with murine retroviral vectors. Liposomesare being used to increase the immunogenicity of malignant melanoma by transferring DNA encoding the HLA-137 gene[21]. The liposomes are taken up by tumor cells via phagocytosis. The tumour cells express the foreign HLA-137 antigen transiently on their surface. Animal studies have shown that tumour cells expressing foreign antigens on their cell surface induce a significant anti-tumour immune response. The direct application of murine retroviral -mediated gene therapy was applied to the treatment of brain beginning in December 1992[22]. In this protocol, murine fibroblast cells that are producing retroviral vectors (retroviral vector producer cells or VPC) are directly implanted into growing brain tumours. The gene being transferred into the surrounding brain tumour cells is the Herpes simplex-thymidine kinase (HS-tK) gene which confers a sensitivity to the anti-Herpes drug, ganciclovir (eytovene or GCV)[2]. In a series of animal experiments, this technique resulted in gene transfer into an average of 60% of tumour cells and was capable of mediating complete tumour destruction in mice with experimental tumors[4]. No associated toxicity or evidence of systematic spread of the retroviral vectors with this form of in vivo gene transfer has been observed. Eight patients with recurrent glioblastoma multiforme or metastatic tumours have been treated with the stereotaxic implantation of HS-tK VPC without any evidence of toxicity related to the implantation of the murine cells or treatment with ganciclovir. Five have demonstrated evidence of anti-tumour efficacy with a decrease in size and cystic changes within the tumour. Three additional clinical trials have been approved using this technique. The first is a trial in adult patients that will focus on the direct injection of HS-tK into the walls of the tumour bed at the time of tumour resection. Repeated injections of VPC will be administered through an Ommaya reservoir into the tumour bed. Two additional trials will apply this technique to recurrent astrocytomas in children. Other in vivo gene transfer protocols for cancer have been recently approved using the direct injection of retroviral vector supernate into tumour deposits. One group has approval to directly inject retroviral vector supernate into endobronchial lung cancers. in this experiment the retroviral vectors will carry genes that target the genetic mechanisms responsible for the malignancy[25]. If lung tumours are deficient in the p53 tumour suppressor gene, a p53 vector will be used to transfer a normal copy of the p53 gene. In lung cancers that over-express the k-RAS oncogene, a vector containing an anti-sense k-RAS gene will be used. The anti- sense k-RAS vector will produce mirror image RNA molecules that will bind the ones being produced by the onogene. RNA:RNA hybrids are then degraded by the cell. Experiments in animals have demonstrated that either the insertion of the tumour suppressor gene or the anti-sense oncogene can result in in vivo destruction of the injected tumour. Another group of researchers plans to directly inject retroviral vector supernate containing an interferon vector into melanoma tumour deposits. At this time, neither of these research groups has begun human experimentation. Another approach to the treatment of brain tumours targets one to the methods that tumours appear to use to hide from the immune system. This method uses an anti-sense copy of insulin-like growth factor 1 (IGF-1) to block tumour cell production of IGF-1[26]. Animal experiments have demonstrated that insertion of an anti-sense W-1 gene results in immunologic rejection of the genetically altered tumour after re-implantation as well as non-genetically altered tumour cells at other sites in the body. This trial is expected to begin in early 1994. HIV: Since the HIV-induced immunodeficiency results when the human immunodeficiency virus (HIV) makes T-lympocyte "sick", genetically altering HIV infected T lymphocytes maybe a useful therapy. Many scientists around the world are working on genetic methods for the modification of the body tissues that might prevent HIV infection, prevent the spread of HIV in the body of genetically turning off the ability of HIV to grow or by preventing the development of the HIV induced immunodeficiency [Table - 5]. The first gene transfer trial of the experimental treatment of HIV infection was approved in 1992 in the US[27]. This protocol does not actually use the transferred gene for therapy, but rather involves insertion of the Herpes simplex-thymidine kinase "suicide" gene for use in the event that one of the genetically-altered T - lymphocytes would become malignant. T-lymphocyte clones that will specifically attack HIV infected cells are grown from the blood of HIV infected patients with lymphoma, genetically-altered and reinfused IV into patients following cytoablation and bone marrow transplantation in an attempt to eradicate the lymphoma and the cellular pool of HIV infected cells. The protocol began in early 1993 and no results are available till this time. Three other trials have been approved and have not yet been initiated [Table - 5]. The first is the direct injection of DNA into muscle. The genetic material injected encodes for the HIV IIIB envelope gene. Investigators hope to induce an immunizing cytotoxic T-lymphocyte immune response in the recepient. Two other protocols focus on the genetic alteration of T-lymphocytes. In one, T-lymphocytes are altered by the insertion of a "dominant negative" gene called rev M10. In this experiment, a mutant rev gene produces an RNA that binds the HIV rev RNA inactivating it. Rev is an HIV regulatory protein that is involved in the transport of HIV RNA from the nucleus to the cytoplasm where it is packaged into an infectious virus particle. In a third trial, an HIV ribozyme gene is being inserted into T-lymphocytes. A ribozyme is a RNA molecule that cleaves after RNA's. In this trial, the inserted gene will produce a ribozyme that cleaves other HIV RNA preventing translation and production of infectious HIV virions.
Theonly form of human gene therapy being conducted today in the United States is somatic cell gene therapy. The Recombinant DNA Advisory Committee (RAC) of the NIH, who must approve all human gene therapy trials in the US will not consider germline gene therapy protocols. At this point in history, the technology for human germ line manipulation is too inefficient and early in development for consideration. The human gene therapy experiments in progress around the world involve patients with known fatal diseases and use a gene transfer method that is specific to that individual. Since these experiments are limited to single individuals, the many concerns expressed about germ line manipulation have not been pertinent to the somatic cell treatments.
There have been tremendous advances in our understanding of the human genome over the past 40 years that have provided the foundation for human gene therapy application. Currently the identification of genes is occurring at a much faster than the development of in vivo gene delivery methods and our understanding of gene expression. Advances in these areas are required for the widespread application of gene therapy. As these advances occur, we will witness a dramatic expansion in the number of somatic cell gene therapy trials on a worldwide basis. The first groups to benefit will most likely be patients with cancer. Over the next 5-10 years, gene therapy will become a standard form of therapy for certain forms of cancer. With the development of efficient gene transfer methods, the dream of congenital and acquired diseases will become a reality changing science and medicine forever.
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5]
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