Current Gene Therapy - Volume 4, Issue 3, 2004

Radiation has been a well-established modality in cancer treatment for several decades. Significant improvements have been achieved in radiotherapy over the years due to technological advances and development of facilities for delivery of charged particles such as protons. Nonetheless, the potential for tumor control with radiotherapy must always be carefully balanced with the risk for normal tissue damage. In addition, tumor cells outside the immediate field of radiation exposure or that have metastasized to distant sites are not destroyed. Gene therapy offers many exciting possibilities by which the overall efficacy of radiotherapy may be improved, while minimizing unwanted side effects. This review highlights several of the most promising gene transfer approaches that are currently being evaluated in combination with radiation in the treatment of cancer. Results from studies utilizing genes encoding molecules that function in apoptosis, radiosensitization, immune up-regulation, angiogenesis, DNA repair, normal tissue protection from radiation damage, and tumor targeting are discussed. The evidence indicates that many of these innovative gene-based strategies have great potential to augment radiotherapy, as well as other established forms of cancer treatment, in the near future.

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines / chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virusfree vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines / chemokines are presented.

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are neurodegenerative diseases, which cause progressive paralysis and premature death in affected adults and children. The treatment rational for these diseases is to halt or delay the degeneration of motor neurons but to date there are no effective drugs. This may however change with recent advances in gene therapy using lentiviral vectors. These vectors can transfer genes to motor neurons with high efficiency and give long term expression. One of these vector systems, based on the equine infectious anaemia virus (EIAV), can insert genes into the cells of the central nervous system after remote delivery including delivery into the muscle by exploiting retrograde transport pathways. This opens up the exciting possibity of rescuing the denervation of key muscle groups in patients by simple injections of these neurotropic lentiviral vectors into the muscle. This review will describe the general features of lentiviral vectors derived from the EIAV. It will then describe some key examples of gene transfer and genetic correction in animal models of motor neuron disease. The prospects for the clinical evaluation of lentiviral vectors for the treatment of human motor neuron disease will be outlined.

Various disorders of bone and mineral metabolism are diagnosed to be defective in genes related to cellular growth and differentiation. Gene therapy to introduce normal copy of defective genes into cells and tissues to compensate for silent, minimally expressed or mutated genes can be accomplished by multiple approaches. Although each bone disease / disorder would require a case-wise evaluation of potential strategies for best possible outcome, considerations for the gene therapy approaches are: 1) introduction of a therapeutic gene into cells without changing any of its native biological properties, 2) minimal or total absence of immunogenic and toxic effects from introduced vectors, geneticallymodified cells or conditionally-expressed proteins, while achieving a therapeutic effect, 3) cell-type or tissue-specific, regulated expression of a therapeutic protein, and 4) restricting or abolishing the expression of disease triggering genes at the RNA or DNA levels. Although most of the currently available therapies for osteoinduction are pharmacological in nature, molecular understanding of biologically-driven factors provides greater opportunity to test their potential as therapeutic proteins. Strategies of gene therapy complement this approach through efficient delivery of genes encoding therapeutic proteins to target sites. The present review will attempt to give a comprehensive account of existing therapies for osteoinduction and discuss the potential and limitation of vector-mediated gene therapy for bone diseases.

The recent discovery of several molecules that negatively modulate the migration and growth of endothelial cells, collectively referred to as inhibitors of angiogenesis, has made it possible to test the hypothesis that control of angiogenesis might be an effective strategy in controlling tumor growth, as well as ameliorating the course of other lifethreatening diseases. Angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes products of the proteolysis of larger molecules with a different function, such as angiostatin and endostatin, natural modulators of vascular endothelial growth factor activity, such as sFLT-1, and some cytokines with a marked antiendothelial activity, such as IL-12 and interferon-a. Pre-clinical studies have clearly indicated that most of these factors exert cytostatic rather than cytotoxic effects, thus implying the need for long-term administration in order to obtain a prolonged therapeutic effect. This feature of angiostatic therapy and the difficulty in synthesizing large amounts of recombinant functional proteins have prompted several studies, which have investigated their delivery by a gene therapy approach. This review addresses the several experimental approaches attempted to date, points out the constraints that have delayed clinical application, and envisions possible areas of integration between antiangiogenic gene therapy and other established therapeutic options against cancer.

Electroporation-based gene therapy has become a “hot field” for non-viral gene delivery. This review summarizes the progress made in intramuscular and intratumoral electrogenetransfer, which include new applications and modifications. The progress in dendritic cell (DC) and stem cell transfection by use of electroporation has also been discussed. Rapid progress during the past two years clearly demonstrates the great potential of this technology, but there are challenges faced by both in vitro and in vivo applications, which include how to enhance the transfection efficiency for intratumoral delivery, how to extend the duration of gene expression for intramuscular injection, and how to increase the survival rate for in vitro cell transfection. Resolving these issues will shed new light on this field.

Since their discovery during the end of the 80's the number of diseases found to be associated with defects in the mitochondrial genome has grown significantly. Organs affected by mutations in mitochondrial DNA (mtDNA) include in decreasing order of vulnerability the brain, skeletal muscle, heart, kidney and liver. Hence neuromuscular and neurodegenerative diseases represent the two largest groups of mtDNA diseases. Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is however no satisfactory treatment available to the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by administering alternative metabolic carriers of energy. Conventional biochemical treatment having reached an impasse, the exploration of gene therapeutic approaches for patients with mtDNA defects is warranted. For now mitochondrial gene therapy appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the development of an efficient mitochondrial transfection vector. In this review we describe the current state of the development of mitochondria-specific DNA delivery systems. We summarize our own efforts in exploring the properties of dequalinium and other similar cationic bolaamphiphiles with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells. Further, we outline some unique hurdles that need to be overcome if the development of such delivery systems is to progress.

Replacing the function of diseased organs by transplantation has proved to be highly successful in the past four decades. The immune system poses the most significant barrier to the long term survival of the transplanted organs. Without lifelong treatment with powerful immunosuppressive agents to keep the immune response at bay, organ grafts will invariably be rejected. However, current immunosuppressive agents are non-specific and leave transplant recipients more susceptible to opportunistic infections and tumour development. Achieving donor specific tolerance would eliminate the need for lifelong treatment with these agents and thereby, avoid the associated side effects. There have been many exciting new developments in immunopharmacology and in the understanding of the mechanisms of rejection and tolerance. These developments on their own, or in combination with the use of gene therapy techniques may allow the induction of transplantation tolerance in human recipients of allografts in the future. Pretransplant exposure to donor MHC antigens has been highly successful strategy for tolerance induction in experimental models. Pretransplant blood transfusion, and more recently administration of donor bone marrow, has been used in an attempt to prolong graft survival in human. However, using fully allogeneic donor bone marrow carries the risk of graft versus host disease. Gene therapy may allow this approach to be used without this risk. Here, we review efforts to induce transplantation tolerance by gene transfer of donor MHC genes to recipient derived cells and show that this may be a potential strategy to achieve the holy grail of organ transplantation: donor specific tolerance.

Adenoviral gene therapy has shown promise in both preclinical and clinical settings, but several hurdles need to be overcome before it can reach its full therapeutic potential. One such hurdle is the need for targeting the right cell type, while avoiding liver uptake and hence side effects. This review will focus on transductional targeting strategies, in which the adenoviral particle is physically targeted to specific surface receptors expressed on the target cell. This can be achieved by using either bifunctional adaper molecules, which bind to the adenoviral particle on one side and to the targeted receptor on the other, or genetic targeting strategies. Adapter molecules comprise both chemically conjugated targeting moieties and recombinant fusion proteins, the latter having the advantage of being a homogeneous population. Genetic retargeting strategies include fiber or fiber knob chimerism, genetic incorporation of targeting ligands in the fiber or other capsid locales, or a combination of both (‘complex mosaics’). Since sequestration of virions in the liver presents a major problem for the therapeutic utility of adenoviral gene therapy after systemic administration, blockade of liver uptake has become an increased area of investigation. Strategies encompass blockade of the adenovirus interaction with its cognate receptor CAR, by either using the soluble ectodomain of CAR, or ablation of CAR-interacting amino acid residues in the fiber knob. In addition, inhibition of interaction with additional adenovirus receptors, such as integrins or heparan sulphate proteoglycans, hold promise for decreasing liver uptake and hence adenoviral toxicity.

The clinical application potential of monoclonal antibodies concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Intravenous injection is a simple and obvious mode of administration of purified therapeutic antibodies to patients but may not always be appropriate for longterm treatments for a variety of reasons. One limitation concerns the elevated costs of recombinant proteins certified for human use. Moreover, hour-long infusions require a hospital environment and are often associated with mild to very severe side effects. This makes large-scale clinical applications of a number of monoclonal antibodies with demonstrated therapeutic activity impossible or, at least, severely compromised. In vivo production of therapeutic antibodies in patients, through either genetic modification of their tissues or implantation of antibody-producing cells, might represent an attractive alternative to overcome these drawbacks. Moreover, this method should also provide other benefits. Continuous and sustained delivery of antibodies at a low, but therapeutic level should prevent, or at least delay, induction of neutralizing anti-idiotypic immune responses, which sometimes develop when massive doses of purified immunoglobulins are repeatedly injected into patients. Additionally, it should also limit variations in the bioavailability of therapeutic antibodies that are often detrimental to the efficacy of treatments. The present review reports on the recent developments of gene / cell therapies aiming at the in vivo production and systemic delivery of monoclonal antibodies with the final goal of treating severe chronic diseases.