Current Gene Therapy - Volume 6, Issue 2, 2006

Cystic fibrosis (CF) was one of the first inherited disorders for which gene therapy was seriously considered as a realistic option for treatment, and as such, it has long provided a paradigm for gene therapy of inherited diseases. However, despite the cloning of the cystic fibrosis transmembrane conductance regulator gene in 1989, over 15 years later a practical gene therapy for CF has not eventuated. There are a number of reasons for this, and analysis of the specific issues that have delayed the successful development of gene therapy for CF also provides general insights into the practical complexities involved in the development of gene therapy for inherited disorders. The issues which have prevented the application of gene therapy for CF to date include the lack of suitable gene delivery technologies, the complexities of the interactions between the host and vector, the biology of the lung airways, and the nature of the pathology found in individuals with CF. We will discuss the history of CF gene therapy with specific reference to these and other issues that preoccupy the field at present: namely, the question of what vectors appear to be suitable for airway gene delivery in CF, what cells must be targeted, how airway epithelium defences can be overcome or eluded to allow efficient gene delivery, how to ensure safe and long-term transgene expression and the need to identify relevant surrogate success measures that can be used to assess the outcome of gene therapy in CF patients.

The implantation of genetically-modified non-autologous cells in immuno-protected microcapsules is an alternative to ex vivo gene therapy. Such cells delivering a recombinant therapeutic product are isolated from the host's immune system by being encapsulated within permselective microcapsules. This approach has been successful in pre-clinical animal studies involving delivery of hormone or enzymes to treat dwarfism, lysosomal storage disease, or hemophilia B. Recently, this platform technology has shown promise in the treatment for more complex diseases such as cancer. One of the earliest strategy was to augment the chemotherapeutic effect of a prodrug by implanting encapsulated cells that can metabolise prodrugs into cytotoxic products in close proximity to the cancer cells. More recent approaches include enhancing tumor cell death through immunotherapy, or suppressing tumor cell proliferation through anti-angiogenesis. These can be achieved by delivering single molecules of cytokines or angiostatin, respectively, by implanting microencapsulated cells engineered to secrete these recombinant products. Recent refinements of these approaches include genetic fusion of cytokines or angiostatin to additional functional groups with tumor targeting or tumor cell killing properties, thus enhancing the potency of the recombinant products. Furthermore, a COMBO strategy of implanting microencapsulated cells to deliver multiple products targeted to diverse pathways in tumor suppression also showed much promise. This review will summarise the application of microencapsulation of genetically-modified cells to cancer treatment in animal models, the efficacy of such approaches, and how these studies have led to better understanding of the biology of cancer treatment. The flexibility of this modular system involving molecular engineering, cellular genetic modification, and polymer chemistry provides potentially a huge range of application modalities, and a tremendous multi-disciplinary challenge for the future.

Diabetes mellitus invariably induces retinopathy which causes a loss of vision that is the major cause of blindness in people of working age across most ethnic groups. Although there have been major advances in gene therapy technologies, there is still no effective cure-all gene therapy for diabetes mellitus. This may be due to (i) involvement of multiple genes that may have different influences on diabetes across different ethnic groups, (ii) immune response to viral vectors, (iii) local, specific transfection only and not into systemic circulation, (iv) lack of stable long-term expression, and (v) lack of control of gene expression. Hence, a separate approach to gene therapy of diabetic retinopathy is necessary due to the difficulties in treating the underlying diabetes. Diabetic retinopathy is the inevitable microvascular complication in the retina from diabetes mellitus. There are possible genetic bases in several pathophysiological pathways for diabetic retinopathy, including oxidation of retinal cells, polyol accumulation pathways, increased non-enzymatic glycation in retinal cells and the release of growth factors by endothelial cells. We review the candidate genes in these putative pathways for diabetic retinopathy and discuss the challenges for gene therapy. The eye is an isolated system with a strong blood-retinal barrier and therefore provides a challenge for delivery of drugs and vectors from the systemic circulation using traditional approaches. Newer delivery approaches include the use of nanoparticles, liposomes, and iontophoresis. We also consider the social and health economic dimension of diabetic retinopathy gene therapy. Diabetic retinopathy is the most common cause of blindness for people of working age. The loss of visual acuity caused by diabetic retinopathy creates a detrimental impact on the patient's quality of life. This results in quality-of-life costs to the individual, the health care system and to society. Significant progress has been made in gene therapy approaches for diabetic retinopathy, and it appears that this is an important area for continued research in order to improve visual outcomes and reduce the healthcare costs of diabetic retinopathy in our communities.

Adenoviral (Ad) vectors can efficiently transduce a broad range of cell types and have been used extensively in preclinical and clinical studies for gene delivery applications. The presence of preexisting Ad immunity in the majority of human population and a rapid development of immune response against the Ad vector backbone following the first inoculation with the vector have impeded clinical use of these vectors. In addition, a number of animal inoculation studies have demonstrated that high systemic doses of Ad vectors invariably lead to initiation of acute inflammatory responses. This is mainly due to activation of innate immunity by vector particles. In general, vector and innate immune responses drastically limit the vector transduction efficiency and the duration of transgene expression. In order to have a predictable response with Ad vectors for gene therapy applications, the above limitations must be overcome. Strategies that are being examined to circumvent these drawbacks of Ad vectors include immunosuppression, immunomodulation, serotype switching, use of targeted Ad vectors, microencapsulation of Ad vectors, use of helper-dependent (HD) Ad vectors, and development of nonhuman Ad vectors. Here we review the current understanding of immune responses to Ad vectors, and recent advances in the strategies for immune evasion to improve the vector transduction efficiency and the duration of transgene expression. Development of novel strategies for targeting specific cell types would further boost the utility of Ad vectors by enhancing the safety, efficacy and duration of transgene expression.

Lysosomal storage disorders (LSD) are a group of approximately 40 genetic diseases that are caused by the deficiency of one or more lysosomal enzymes. The incidence of LSD is estimated to be approximately 1 in 7500 live births, which makes this one of the more prevalent groups of genetic diseases in humans. The loss in enzymatic activity leads to the accumulation of undegraded substrates within lysosomes, resulting in distension of the organelle and subsequent cellular malfunction. Although palliative treatments such as enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) have been shown to be effective for some of the LSD such as Gaucher, Fabry and MPS I, they are not available as yet, or ineffective, for a large number of other LSD patients. To fulfill this unmet medical need, gene therapy is being considered as an alternate or adjunctive therapy for this group of disorders. A goal of gene therapy for LSD is to introduce a normal copy of the DNA for the lysosomal enzyme into a depot organ such as the liver or muscle with the intent that this will lead to the sustained production and reconstituion of therapeutic levels of the enzyme in the affected tissues. Here, we review the utility of various gene therapy strategies under consideration for the treatment of the LSD, including viral and non-viral gene transfer approaches, as well as stem cell transplantation.

Nonviral gene transfer is markedly enhanced by the application of in vivo electroporation (also denoted electrogene transfer or electrokinetic enhancement). This approach is safe and can be used to deliver nucleic acid fragments, oligonucleotides, siRNA, and plasmids to a wide variety of tissues, such as skeletal muscle, skin and liver. In this review, we address the principles of electroporation and demonstrate its effectiveness in disease models. Electroporation has been shown to be equally applicable to small and large animals (rodents, dogs, pigs, other farm animals and primates), and this addresses one of the major problems in gene therapy, that of scalability to humans. Gene transfer can be optimized and tissue injury minimized by the selection of appropriate electrical parameters. We and others have applied this approach in preclinical autoimmune and/or inflammatory diseases to deliver either cytokines, anti-inflammatory agents or immunoregulatory molecules. Electroporation is also effective for the intratumoral delivery of therapeutic vectors. It strongly boost DNA vaccination against infectious agents (e.g., hepatitis B virus, human immunodeficiency virus-1) or tumor antigens (e.g., HER-2/neu, carcinoembryonic antigen). In addition, we found that electroporation-enhanced DNA vaccination against islet-cell antigens ameliorated autoimmune diabetes. One of the most likely future applications, however, may be in intramuscular gene transfer for systemic delivery of either endocrine hormones (e.g., growth hormone releasing hormone and leptin), hematopoietic factors (e.g., erythropoietin, GM-CSF), antibodies, enzymes, or numerous other protein drugs. In vivo electroporation has been performed in humans, and it seems likely it could be applied clinically for nonviral gene therapy.