|Surgical organ perfusion method for somatic gene transfer: An experimental study on gene transfer into the kidney, spleen, lung and mammary gland|
|Prev||Chapter 2. Review of the literature||Next|
Several diseases are candidates for gene therapy. These can be divided primarily into inherited genetic diseases, which may originate from a single gene defect (Aalto-Setälä & Vuorio 1997) or be multigenic diseases. Furthermore, acquired multifactorial diseases, such as inflammatory diseases, including rheumatoid arthritis, joint arthrosis (Jorgensen et al. 1997, Apparailly 1998) and cancer, are considered amenable to gene therapy. One aim of gene therapy in genetic diseases is to correct the phenotype by delivering a functionally normal gene copy into an affected somatic cell which contains a mutated genotype without physically replacing the defective gene itself. This is also called gene augmentation (Chang & Wu 1994). In the case of acquired diseases, gene therapy aims to modify the individual’s inflammatory response by delivering anti-inflammatory or antiproliferative genes or by delivering tumor suppressor genes, mutated oncogenes or genes encoding immunostimulatory agents to prevent the growth of malignant tumors (Curiel et al. 1996) and to reduce metastatic disease. Although inherited diseases resulting from a genetic defect would be the most logical disorders to treat with gene therapy, most of the human clinical trials so far made have focused on cancer gene therapy.
Kidney-targeted gene therapy to combat both genetic and acquired renal diseases that lead to kidney destruction is being extensively studied (Imai & Isaka 1998, Kelley & Sukhatme 1999). Our understanding of the pathogenesis of renal diseases, such as glomerulonephritis and glomerulosclerosis, has been gained from many interventional studies. For example, the first blockage of transforming growth factor, TGFβ , by antibodies illustrated the influence of this factor on extracellular matrix production and the development of glomerulosclerosis in experimental animal models of renal disease (Border et al. 1990). The results of Border et al. (1990) pointed out that increased production and TGFβ 1 activity are associated with the development of acute mesangial proliferative glomerulonephritis. Furthermore, advances in recombinant DNA technology have enabled studies of the function of TGFβ genes and their products in vivo in transgenic animals by oocyte microinjection of TGFβ cDNA. Alternatively, the function of genes in the kidney can now be studied by in vivo transfer of mutated genes into the glomeruli (Isaka et al. 1993, Kitamura & Fine 1997, Wagner et al. 1995). Isaka et al. (1996) reported markedly reduced proteinuria and extracellular matrix accumulation in a rat model of glomerulonephritis, which was achieved by transferring and expressing decorin-cDNA, an antagonist for TGFβ , in skeletal muscle. By renal gene transfer, the local production of natural antagonists for fibrotic cytokine (IL-1,PDGF,TGFβ ) can be increased, which helps to extinguish the inflammatory process in the kidney (Moullier et al. 1997). Thus, inflammatory and fibrotic diseases of the kidney are interesting targets for gene therapy research.
The kidney is the main organ affected in a number of inherited genetic diseases, such as polycystic renal disease, Alport syndrome, Fabry`s disease, Denys-Drash syndromes, nephronophtisis, nail-patella syndrome and nephrotic syndrome (Knebelmann et al. 1993). The most common of these are polycystic renal disease, with a gene frequency of 1:500–1000, and Alport syndrome, with a gene frequency of 1:5000 (Gregory & Atkin 1993). Alport syndrome is an example of a hereditary renal disease that could be a suitable candidate for gene therapy by gene augmentation. This disease is mainly X-hromosome-linked and caused by mutations in the type IV collagen α5 chain (COL4A5) (Hostikka et al. 1990, Tryggvason et al. 1993, Antignac et al. 1992). Some autosomal recessive forms of Alport syndrome exist (Mochizuki et al. 1994). These mutations cause structural and functional defects in the type IV collagen specific to glomerular basement membrane (GBM) and therefore also in GBM itself (Butkowski et al. 1989). The symptoms of Alport syndrome are mild to moderate hematuria and proteinuria, and the disease usually progresses to end-stage renal failure in males, leading to a need for continuous dialysis or renal transplantation. By delivering a normal copy of the type IV collagen α5 chain gene into the glomerular endothelial and epithelial cells of patients with X-linked Alport syndrome, the GBM defect could theoretically be corrected and the renal failure prevented.
Autosomal dominant polycystic renal disease is another candidate for gene therapy in the future. This disease leads to a loss of renal function due to multiple developing cysts, which gradually replace normal renal tissue. The manifestations of this disease are, however, variable and the possibly developing renal failure is difficult to predict.
In renal transplantation, gene transfer can be used either to prevent acute allograft rejection or to avoid chronic graft loss by the delivery of organ antigenicity decreasing genes into the graft or genes that induce the tolerance of the recipient (Qin et al. 1996, Zeigler et al. 1996a, Wissing et al. 1997).
The lung is a particularly attractive target for gene therapy interventions for a number of reasons. The genetic basis of several pulmonary diseases has been elucidated. For example, the genes of cystic fibrosis, CF, and α1-antitrypsin, α1-AT, deficiency have been identified (Kerem et al. 1989). These diseases are relatively common fatal inherited disorders among Caucasians. Although the current therapies have improved survival, they are inefficient at achieving cure (Curiel et al. 1996).
Cystic fibrosis is an autosomal recessive disease affecting about 1 of 3000 Caucasians born. The CF gene has been isolated in chromosome 7, and more than 350 mutations have been identified. The gene consists of approximately 250 000 bp that encode an mRNA of 6.5 kb. The major cause of morbidity and mortality in CF is pulmonary disease characterized by viscous mucus secretion, chronic bacterial infection, airway inflammation and premature death from respiratory failure at around 29 years of age (Rosenstein & Zeitlin 1998). Identification of the CF gene product, the cystic fibrosis transmembrane conductance regulator, CFTR, has led to lung-directed somatic gene therapy research through targeted replacement of the defective CFTR gene in pulmonary epithelial cells by a normal copy (Hyde et al. 1993, Crystal et al. 1994, Alton & Geddes 1995, McElvaney 1996). The first reports of in vitro correction of the CF chloride channel defect were published in 1990 (Drumm et al. 1990), and in vivo CF gene expression was established in the airways of mice in 1992 (Rosenfeld et al. 1992). Phase I human gene therapy trials have been initiated after that (Crystal et al. 1995, Curiel et al. 1996). So far, CF gene therapy has been extensively studied (Mc Elvaney 1996, Alton & Geddes 1997). Despite this, gene transduction into the target cells, namely airway and alveolar epithelial cells, has not been reported to be very effective. Instead, transduction has been patchy and variable into the various pulmonary cells expressing the transgene (Goula et al. 1998, Griesenbach et al. 1998).
Gene therapy is also being considered for the treatment of α1‐antitrypsin deficiency, an autosomal recessive disease (Crystal et al. 1989, Crystal 1992, Schwaiblmair & Vogelmeier 1998). The α1-AT gene is located at chromosome 14, and it is characterized by a high level of gene pleomorphism, with more than 100 alleles described (Crystal et al. 1989). This disease is the second most common lethal hereditary disorder of the lung in Caucasians, with an incidence of about 1 per 6000 births. The α1‐AT protein is a serum glycoprotein that is synthesized and secreted primarily by the liver. Its major site of action is the lower respiratory tract. The patients with this genetic defect usually develop a chronic condition defined as panacinar emphysema in their fifth decade. The treatment of α1‐AT protein deficiency has focused on intravenous weekly to monthly infusions of purified human α1‐AT protein to maintain adequate serum levels. The limitations of this treatment include high cost and inconvenience as long‐term treatment. α1-AT gene transfer directly into the lung or the liver could correct the lung manifestations (Rosenfeld et al. 1991, Crystal 1992, Lemarchand et al. 1992, Saylors et al. 1998).
Surfactant protein B is a phospholipid‐associated polypeptide expressed by respiratory epithelial cells. It is essential for lung function, enhancing the spreading and stability of surfactant phospholipids, which reduce surface tension at the alveolar air‐liquid interface. Surfactant protein B deficiency is an autosomal recessive disease of full‐term newborn infants, which leads to congenital pulmonary alveolar proteinosis and lethal respiratory failure within the first year of life. Human surfactant protein B deficiency represents a logical candidate for gene therapy, and the first reports of adenovirus-mediated surfactant apoprotein gene delivery into respiratory epithelial cells have been published (Yei et al. 1994, Korst et al. 1995). Messina et al. reported (1996) successful surfactant protein A gene delivery into pulmonary epithelial cells using 8- to 12-week-old human fetal lung explants in vitro. These findings suggest that congenital neonatal pulmonary disorders, neonatal respiratory distress syndrome of premature infants and bronchopulmonary dysplasia, are suitable candidates for gene therapy.
Pulmonary vessels represent a potential target for gene therapy, although the etiology of many forms of pulmonary hypertension remains unknown. Adenovirus-mediated transfer of the human endothelial nitric oxide synthase (NOS) gene into small and medium-sized pulmonary vessels has been reported to reduce acute hypoxic vasoconstriction in rats (Janssens et al. 1996). Pulmonary fibrosis is another possible candidate for gene therapy in the future. Transfer of the cytokine TGF-β 1 gene into rat lung has been demonstrated to result, through overexpression of the TGF-β 1 gene, in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of extracellular matrix proteins, collagen, fibronectin, and elastin, and by the emergence of cells with the myofibroblast phenotype (Sime et al. 1997). This animal model of pulmonary fibrosis illustrates the role of TGF-β 1 in the pulmonary fibrotic process and provides a model for studying antifibrinogenic therapeutic gene transfer strategies by, for example, TGF-β 1 inactivation.
Inflammatory lung diseases, such as asthma, chronic bronchitis and adult respiratory distress syndrome, ARDS, may be treated by modulating pulmonary inflammation via gene transfer. Genes that encode enzymes producing anti-inflammatory prostaglandins could be introduced into pulmonary epithelium. It has been shown that mucosal interferon-gamma (IFNγ ) and IL-12 gene transfer inhibits the pulmonary allergic response in a mouse model for allergic asthma (Li et al. 1996, Hogan et al. 1998). Delivery of the catalase gene into pulmonary vascular endothelium may be utilized to provide augmented vascular antioxidant defenses in the context of ARDS and other pulmonary vascular diseases (Erzurum et al. 1993). Kolls et al. developed an adenoviral vector encoding a tumor necrosis factor-α(TNFα)-soluble receptor (AdTNF-R) that neutralizes TNF activity. Mice treated with this vector have been shown to produce measurable levels of plasma TNF-inhibitory activity. The authors also showed that administration of AdTNF-R protected against endotoxic shock and impaired the normal host immune response suggesting gene therapy of autoimmune diseases as well (Kolls et al. 1994).
Ischemia-reperfusion injury and chronic allograft rejection, which often manifests as bronchiolitis obliterans characterized by progressive fibrous obliteration of the airways in the transplanted lung, limit the survival of lung transplant recipients. Consequently, the survival of recipients is currently 70% and 60% at 12 and 24 months, respectively (Cassivi et al. 1999). So far, no effective therapy is available to prevent these fatal complications. AdV-mediated catalase or superoxide dismutase gene transfer into pulmonary endothelial cells has been shown to protect these cells against oxidant injury, which indicates the utility of this method in limiting reperfusion injury (Erzurum et al. 1993). Fyfe & Hedrick (1995) demonstrated that adenoviral delivery of interleukin-10, an anti-inflammatory and immunosuppressive cytokine, into the allograft endothelium inhibits monocyte adhesion in the recipient. Boehler et al. (1998) showed, by using a rat model of bronchiolitis obliterans, that intramuscular adenovirus-mediated IL-10 administration inhibits post-transplant fibrous airway obliteration. Gene therapy targeted to the allograft (or xenograft in the future) offers a promising approach to the prevention of reperfusion injury and graft rejection in transplantations (Chapellier et al. 1996, Jeppson et al. 1998).
There is considerable evidence to suggest that cancer has a genetic origin based, at least partially, on the development of somatic mutations in families of genes responsible for the critical functions of cellular DNA repair, division and control of growth. One example of this is the presence of mutations in the tumor suppressor gene p53, which have been found to be related to the development of cancer (Horio et al. 1993). This is supported by the finding that transduction of cancer cells with the wild-type p53 gene had a therapeutic effect on cancer in an orthotopic lung cancer model (Fujiwara et al. 1994). On the other hand, cellular proliferation can be inhibited in cancer by using antisense constructs of oncogenes (Mukhopadhyay et al. 1991).
Table 1. Gene therapy methods for treatment of cancer.
|Compensation of mutation||Augmentation of deficient tumor-suppressor gene, inhibition of expression of dominant oncogene|
|Immunopotentiation||Passive imunotherapy: augmentation by genetic enhancement of cell targeting or cell-killing capacity of tumor-infiltraing lymphocytesActive immunotherapy: augmentation of immune recognition of cancer cells|
|Molecular chemotherapy||Selective delivery of toxin or toxin gene to cancer cells|
|Chemosensitization||Selective delivery of genes enhancing potency of chemotherapeutics into tumor cells|
The first clinical gene therapy protocols approved by regulatory agencies have been dealing with cancer gene therapy in widely spread diseases (Rosenberg et al. 1993, Tursz et al. 1996, Roth 1998). There are several strategies to exploit gene transfer as a tool to target specific molecular defects intrinsic to cancer cells, for example to enhance tumor chemosensitivity or to augment tumor immunogenicity (Dranoff 1998). The general principles of cancer gene therapy are presented in table 1. Animal models have shown the therapeutic potential of genetically modified tumor cell vaccines. This strategy entails surgical removal of tumor, ex vivo culture of cancer cells with genes that produce immunostimulatory agents (e.g. IL-2, IL-4, GM-CSF), followed by vaccination of the patient with the genetically modified autologous cancer cells (Golumbek et al. 1991). Melanoma, lung cancer, hepatocellular carcinoma (Tursz et al. 1996, Dubinett et al. 1998, Roth 1998), malignant mesothelioma, prostate cancer (Sanda et al. 1994, Harrison & Glode 1997), renal cancer (Golumbek et al. 1991), bladder cancer (Lee et al. 1994), leukemia, malignant brain tumors (Puumalainen et al. 1998a), metastatic breast carcinoma and colorectal cancer are examples of malignant diseases considered in gene therapy research (Rosenberg et al. 1993, Prince 1998).
Adenosine-deaminase (ADA) deficiency is a fatal recessively inherited disorder causing accumulation of adenosine and 2´-deoxyadenosine, which are toxic to lymphocytes. The patients suffer from severe combined immunodeficiency and develop life-threatening infections. ADA cDNA gene transfer into T-lymphocytes has been reported to increase the number of T cells and ADA in the blood of treated patients (Blaese & Anderson 1990).
The most prevalent hereditary metabolic storage disease, Gaucher disease, is due to genetically deficient activity of the glucocerebrosidase –enzyme, which normally cleaves the glucose residue from ceramide. This leads to an accumulation of glucocerebrosides in reticuloendothelial cells by the formation of the “Gaucher cells”, which are phagocytes filled with distended lysosomes. The disease is characterized by hepatosplenomegaly in the absence of central nervous system involvement. Gaucher cells are found in the liver, spleen, lymph nodes and bone marrow. The spleen may often enlarge massively, which makes it prone to rupture upon the slightest trauma. Hypersplenism and bone marrow replacement together contribute to anemia and leukopenia. The condition afflicts about one out of 40 000 people in the general population, but among a subset of Jewish people the odds are 1 in 400. The current treatment for the disease consists of recombinant alglucerase enzyme injections every two weeks. This is one of the most expensive drugs in the world. Gene therapy for Gaucher disease is currently under consideration (Eto & Ida 1996). The lysosomal storage lesions in the liver and spleen have been shown to be corrected by autologous implants of ex vivo human β -glucuronidase cDNA transduced fibroblasts in a transgenic animal model of mucopolysaccharidosis type VII (Sly syndrome) (Moullier et al. 1993, Naffakh et al. 1994) or by direct intravenous administration of vectors (Ohashi et al. 1997).
Fabry disease is an X‐linked recessive disorder, which is caused by insufficient activity of the enzymes important for the biodegradation of old red blood cells. One of these enzymes is called ceramidetrihexosidase (alpha‐galactosidase A) (Coppola et al. 1994). Fabry disease is primarily a disorder of men, but females may also exhibit some of the typical symptoms. The patients may develop problems with their kidneys, nerves, and blood vessels, most notably those that supply the heart and the brain. These changes may lead to kidney failure, premature myocardial infarction or stroke. The treatment is currently directed towards alleviating the signs and symptoms of the condition. To date, reports of corrective gene transfer trials in Fabry disease have appeared (Medin et al. 1996).
Phenylketonuria and ornithine transcarbamylase deficiency (Chang & Wu 1994) are rare inherited metabolic diseases considered for enzyme replacement gene therapy. Gene therapy trials for the treatment of one of the diseases in the Finnish heritage, aspartylglycosaminuria (AGU), are in their preclinical phase (Peltola et al. 1998).
Familial hypercholesterolemia caused by homozygous low-density lipoprotein (LDL) receptor deficiency leads to premature development of atherosclerotic cardiovascular disease. LDL receptors are expressed in most cells of the body, but it is the hepatic expression of these receptors that regulates cholesterol homeostasis. The transfer of LDL receptor genes to hepatocytes is a potential cure for hypercholesterolemia in these patients. Kozarsky et al. (1994) demonstrated correction of LDL receptor deficiency by adenoviral gene transfer in rabbits. Partial correction of serum lipid abnormalities has been attained in human by using autologous genetically modified hepatocytes (Grossman et al. 1994, Crystal 1995b). Treatment of obesity by gene transfer in a mouse model has been reported (Murphy et al. 1997).
In addition to familial hypercholesterolemia, there are several other cardiovascular diseases, such as hypertension, that are candidates for gene therapy. Chen et al. (1997) found that transfer of the human kallikrein-binding protein encoding gene, the kallistatin gene, into the liver of spontaneously hypertensive rats by intraportal infusion of AdV vectors resulted in 4 weeks’ sustained reduction of blood pressure.
Gene therapy is also considered for the treatment of disorders involving vascular thrombosis, thromboangiitis obliterans and other types of vasculitis, the prevention of arteriosclerosis and restenosis of the vascular grafts after bypass operations or angioplasty (Chen et al. 1994, Laitinen et al. 1997, Ylä-Herttuala 1997). Chiche et al. reported (1998) that adenoviral-mediated cGMP-dependent protein kinase (PKG) gene transfer into vascular endothelial cells in culture inhibits the proliferation and induces apoptosis of vascular smooth muscle cells via nitric oxide, NO. This predicts a possibility of PKG gene transfer to prevent restenosis after angioplasty. The other aims of gene therapy in vascular diseases include suppression of inflammation, prevention of vasoconstriction, induction of vascular dilatation and acceleration of neovascularization (Laitinen et al. 1998, Svensson & Schwartz 1998). Stimulation of neovascularization in ischemic diseases can be established by transfer of the vascular endothelial growth factor (VEGF) gene (Baumgartner et al. 1998). Acsadi et al. (1991) have shown the possibility of direct gene transfer into rat myocardial cells. Losordo et al. (1998) used direct myocardial injection of the phVEGF gene as the sole therapy for myocardial ischemia. This suggests the possibility of gene therapy for both acquired and genetic heart diseases.
Hemophilia B, Christmas disease, is an X-linked coagulation disorder due to defective synthesis of clotting factor IX in the liver. The current treatment of this disease consists of transfusion of factor IX concentrates into the patients periodically and upon evidence of bleeding. The results of Baru et al. (1995) demonstrate the potential use of liposome-encapsulated DNA to correct the factor IX deficiency. In this study, human factor IX cDNA was demonstrated in murine liver and spleen and, in very small amounts, in lungs, heart and kidneys two days after intravenous delivery of the vectors.
Bubeník et al. (1995) pretreated leukemic mice with intraperitoneal injections of cyclophosphamide, after which irradiated cells, genetically engineered to produce IL-2, were injected intravenously. These cells functioned as a source of cytokine (IR-IL-2 cells) in the mice. This treatment cured a significant proportion of the leukemic mice treated. Human clinical trials for gene therapy of leukemia are going on (Crystal 1995b).
Descamps et al. (1994) reported erythropoietin gene transfer and expression in mice via intravenous injections. There are reports concerning gene therapy for human immunodeficiency virus (HIV) infection, especially the use of ex vivo ribozyme gene transfer to protect autologous T cells from further infection with HIV-1 and thus to aim to slow down the progression of the disease to the AIDS phase, but the results obtained so far are not very encouraging (Sikorski & Peters 1998).
The intestinal epithelium is readily accessible for gene transfer, but the rapid turnover of the epithelium limits the attainment of long-term benefits from the delivered genes. Foreman et al. (1998) reported adenoviral in vivo transduction of intestinal epithelium, and Kawaguchi et al. (1998) demonstrated the possibility of administering ex vivo transduced intestinal stem cells into the small intestine in a rat model. These methods would be applicable to the treatment of metabolic disorders or intestinal diseases, such as enzyme defects. Both type I insulin-dependent and type II non-insulin-dependent diabetes mellitus are possible targets for gene therapy, and many reports on animal models for the gene therapy of diabetes have been published (Murphy et al. 1997, Efrat 1998). However, the regulation of insulin secretion from transduced cells is one of the major limiting factors in the gene therapy of diabetes mellitus. Hepatitis and various liver diseases are also candidates for gene therapy treatment (Kaido et al. 1996)
Human gene therapy trials for malignant brain tumors, of which the most prevalent is malignant glioma, have been going on for some years. In spite of the developments in both surgical methods and irradiation therapy, the prognosis of these patients has not improved. Gene therapy offers a promising addition to these therapies. In this strategy, the tumor is surgically resected as completely as possible and retrovirus vectors producing packaging cells are injected as multiple injections directly into the brain tissue around the margins of the remaining cavity. The retrovirus vectors contain the herpes simplex virus enzyme, thymidine kinase (HS-tk), gene to be transferred into the remaining proliferative tumor cells. After two weeks, the patient receives intravenous ganciclovir, a natural substrate for HS-tk, which causes death of both the thymidine kinase-expressing glioma cells and through the by-stander effect the non-expressing glioma cells as well. (Oldfield et al. 1993, Puumalainen et al. 1998a,b). Simons et al. (1999) found that transfer of the inhibitor of apoptosis (IAP) gene into cerebellar granule neurons caused a delay of apoptosis after potassium withdrawal. This suggests that neuronal gene transfer could be used to protect the brain in some metabolic disorders and e.g. Alzheimer´s disease in the future.
Such neurological conditions as Parkinson´s disease, cerebral ischaemia, Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase deficiency) (Lowenstein et al. 1998) and the known single gene disorders are also being considered as candidates for gene therapy (Wolff 1993, Kennedy 1997), as are also retinitis pigmentosa and other retinal diseases (Mioyshi et al. 1997).