Current Gene Therapy - Volume 3, Issue 1, 2003

The efficacy of various currently available therapeutic strategies for bladder cancer is not always sufficient, especially for the advanced disease, recurrent superficial cancer, and treatment-resistant carcinoma in situ. Advances in genetic and molecular biology have led to novel approaches for cancer treatment. Gene therapy is currently one of the most promising strategies against various malignancies, and several clinical trials have been approved worldwide. Various strategies for modulating the genetic state have been applied in bladder cancer treatment, and encouraging results have been demonstrated both in vitro and in vivo. Although the therapeutic genes work dramatically when the transgenes are effectively expressed in the targeted cells, however, a sufficient rate of transduction cannot always be achieved. The most significant obstacle for clinical application of cancer gene therapy might be the method for sufficient delivery and expression of the therapeutic genes. Bladder is an easily accessible organ because of its anatomy, however, a glycosaminoglycan (GAG) layer on the bladder mucosa may protect integration of exo-delivered genetic vectors. Various strategies are applied for improving the transduction efficacy of the therapeutic genes into the bladder cancer cells. These strategies include the modification of adenoviral fibers, cotransduction of the materials for enhancing the viral infectivity, and disruption of the GAG layer. Recent advances in the field of gene therapy for bladder cancer are briefly summarized in this review.

Gene-directed enzyme prodrug therapy (GDEPT) is a two step therapeutic approach for cancer gene therapy. In the first step, the transgene is delivered into the tumor and expressed. In the second step a prodrug is administered and is selectively activated by the expressed enzyme. The first GDEPT system described was the thymidine kinase gene of the Herpes Simplex virus (HSVtk) in combination with the prodrug Ganciclovir (GCV). A large number of experiments have been performed with this system, in different types of tumors and initial studies in animal models were very promising. This encouraged investigators to move into clinical trials although poor results have been obtained so far. A large effort has been made with numerous different strategies to enhance HSVtk / GCV efficacy in cellular and in vivo models and very strong cytotoxic effects have been obtained. The present review describes the current state of preclinical research and summarizes the results of the clinical trials undertaken.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into selfinactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.

Cerebral ischemia induces many degenerative cellular reactions, including the release of excitatory amino acids, the formation of oxygen free radicals, Ca2+ overload, the activation of several cellular enzyme systems such as Ca2+dependent proteases, and the initiation of genomic responses that can affect the tissue outside the area of reduced blood flow. Furthermore, increasing evidence indicates that apoptosis contributes to the death of brain cells following cerebral ischemia. Several studies have shown that cerebral ischemia alters the expression of genes, some of which may play protective or harmful roles. Although many genes have the potential to treat cerebral ischemia, target genes or their translated products are often difficult to express, if at all, in brain cells. However, adenovirus-mediated gene transfer can overcome this disadvantage. To date, many treatment strategies have been developed for cerebral ischemia using target genes such as neuronal apoptosis inhibitory protein (NAIP), glial cell line-derived neurotrophic factor (GDNF), sensitive to apoptosis gene (SAG), 150-kDa oxygen-regulated protein (ORP150), etc. Moreover, new vectors and gene delivery systems are constantly being invented although there is no perfect vector to date. Gene therapy could constitute a powerful strategy to treat cerebral ischemia in the near future.

We discuss possible gene therapies for the treatment of ischemic diseases in the central nervous system (CNS). These therapies aim at the prevention of carotid artery restenosis, stimulation of angiogenesis for ischemic brain, protection of neurons against ischemia, and prevention of vasospasm due to subarachnoid hemorrhage (SAH).Carotid artery restenosis can perhaps be approached by preventing vascular smooth muscle cell proliferation via gene therapy in addition to surgical treatment. Cerebral angiogenesis therapy might be applicable to moyamoya disease. Gene therapies with VEGF and HGF to stimulate angiogenesis have been successful in muscle, however, efficacy in the CNS is unknown.Gene transfection efficiency of viral vectors has been poor in the CNS, and the safety of such vectors is questionable. Therefore, development of gene therapy is for neural protection and prevention of vasospasm due to SAH has been limited. Infusion of HVJ-AVE liposomes into monkey cerebrospinal fluid (CSF) space yielded wide-spread gene transfection. HVJ-AVE liposomes may be a promising vector for use in the human CNS.Few currently available gene therapies appear to be options for clinical treatment of cerebral ischemia despite many experimental designs. In addition to the inherent difficulties of treating the CNS, vectors and methods for introducing vectors into the CNS must be improved.

Much intensive research has gone into the development of safe and efficient methods for the delivery of therapeutic genes. In vivo electroporation is a non-viral delivery protocol in which plasmid DNA solutions are injected into targeted tissues, followed by electric pulses (typically 100 V, 50 ms). In general, in vivo electroporation enhances gene expression in targeted tissues by 2-3 orders of magnitude, as compared to the injection of plasmid DNA solutions without electric pulses, and the tissue damage appears to be minimal. Among the other advantages of this technique are that it can safely be administered repeatedly, and it is simpler and more economical to use than viral vectors, especially in clinical cases. Using this approach, highly efficient gene transfer has already been achieved in muscle and liver as well as in tumors. In fact, gene therapies for cancer utilizing in vivo electroporation have been proved effective in a number of experimental murine tumor models. The therapeutic genes delivered in those cases were diverse including, for example, cytokine genes (IL-12) and cytotoxic genes (TRAIL), making possible a wide range of therapeutic strategies. Moreover, systemic antitumor effects were also observed, suggesting that this approach may be effective for the treatment of metastatic as well as primary tumors.

Gene therapy, developing rapidly as a result of advances in molecular biology and the Human Genome Project, is now highlighted as a most hopeful technology of the 21st century. The major goal of gene therapy in diabetes mellitus (DM) is to maintain euglycemia in face of wide variations in dietary intake. Although some obstacles remain to be overcome, the risk-benefit ratio of gene therapy in DM is better than that of lifelong injections of insulin, and islet transplantation, which faces the problems of donor shortage and rejection. This review focuses on the recent advances in gene therapy of insulin-requiring diabetes, with particular emphasis on 1. the gene delivery systems by viral vectors, since most gene therapy approaches for DM involve the use of viral vectors, paying special attention to current efforts to overcome the disadvantages of adenovirus, adenovirus-associated virus and retrovirus vectors and targeting gene delivery for optimal efficiency of gene expression, 2. coupling the synthesis and release of the transgene insulin to serum glucose concentrations, especially with reference to the current promoters controlling at transcriptional level the ectopic insulin expression in autologous hepatocytes, 3. β-cell replacement strategies: engineering of β-cells, especially those derived from pluripotent stem cells, non β-cells, and on a new comer, the K cells. Recent advances in the use of stem cells for potential application in diabetes gene therapy are also discussed.