Current Gene Therapy - Volume 5, Issue 2, 2005

The natural function of viruses is to deliver their genetic material to cells. Among the most effective of viruses in doing that is Simian Virus-40 (SV40). The properties that make SV40 a successful virus make it an attractive candidate for use as a gene delivery vehicle: high titer replication, infectivity for almost all nucleated cell types whether the cells are dividing or resting, potential for integration into cellular DNA, a peculiar pathway for entering cells that bypasses the cells' antigen processing apparatus, very high stability, and the apparent ability to activate expression of its own capsid genes in trans. Exploiting these and other characteristics of wild type (wt) SV40, increasing numbers of laboratories are studying recombinant (r) SV40-derived vectors. Among the uses to which these vectors have been applied are: delivering therapy to inhibit HIV, hepatitis C virus (HCV) and other viruses; correction of inherited hepatic and other protein deficiencies; immunizing against lentiviral and other antigens; treatment of inherited and acquired diseases of the central nervous system; protecting the lung and other organs from free radical-induced injury; and many others. The effectiveness of these vectors is a reflection of the adaptive evolution that produced their parent virus, wt SV40. This article explores how and why these vectors work, their strengths and their limitations, and provides a functional model for their exploitation for experimental and clinical applications.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone has the unique ability to regenerate and continuously remodel itself throughout life. However, clinical situations arise when bone is unable to heal itself, as with segmental bone loss, fracture non-union, and failed spinal fusion. This leads to significant morbidity and mortality. Current attempts at improved bone healing have been met with limited success, fueling the development of improved techniques. Gene therapy in many ways represents an ideal approach for augmenting bone regeneration. Gene therapy allows specific gene products to be delivered to a precise anatomic location. In addition, the level of transgene expression as well as the duration of expression can be regulated with current techniques. For bone regeneration, the gene of interest should be delivered to the fracture site, expressed at appropriate levels, and then deactivated once the fracture has healed. Delivery of biological factors, mostly bone morphogenetic proteins (BMPs), has yielded promising results both in animal and clinical studies. There has also been tremendous work on discovering new growth factors and exploring previously defined ones. Finally, significant advances are being made in the delivery systems of the genes, ranging from viral and non-viral vectors to tissue engineering scaffolds. Despite some public hesitation to gene therapy, its use has great potential to expand our ability to treat a variety of human bone and musculoskeletal disorders. It is conceivable that in the near future gene therapy can be utilized to induce bone formation in virtually any region of the body in a minimally invasive manner. As bone biology and gene therapy research progresses, the goal of successful human gene transfer for augmentation of bone regeneration draws nearer.

The lung represents an important target for gene therapy: for correction of genetic abnormalities such as cystic fibrosis, for lung cancer therapy, and for vaccination. Genes in the form of expression plasmids can be delivered both by the intravenous route and via the airways. So-called “naked” DNA can be delivered by both of these methods, but gene expression is low. Successful delivery is usually accomplished by complexing the DNA with cationic lipids or with polycations. This review will discuss the efficacy of delivery for particular purposes by various methods and complexing agents, as well as issues of biodistribution, inflammatory reactions, and improvements in formulations. Non-viral gene delivery to the lung has a long history of development, and it is now poised to represent a significant addition to the medical arsenal.

Locoregional administration of a genetic construct by means of in vivo, in situ isolated perfusion (IP) of a target organ or extremity is a method that may increase in vivo efficacy. Vascular isolation and perfusion minimizes systemic exposure and thereby reduces unwanted side effects. Isolated hepatic perfusion (IHP) is the most extensively studied IP model, especially in gene therapy protocols for inborn errors of metabolism. To achieve stable transduction most frequently retroviruses are used in IHP. IHP is combined with hepatectomy or vascular ligation of liver lobes to induce liver regeneration increasing transduction efficacy. When adenoviruses are used in IHP high transduction percentages of hepatocytes can be achieved without significant toxicity. In tumor models adenoviral IHP has been performed, but has not been very successful up till now. Isolated limb perfusion (ILP) is a promising treatment modality in pre-clinical cancer gene therapy studies. After ILP a homogeneous distribution of transduced cells was demonstrated especially at the viable rim of the tumor and around tumor associated vessels. Moreover complete tumor responses have been observed. Isolated pulmonary perfusion (IPP) results in selective expression in the perfused lung and the duration of expression is longer than after systemic administration. In rats a significant decrease of tumor nodules upon IPP can be achieved. Furthermore other less studied perfusion models are discussed: isolated kidney perfusion (IKP), isolated spleen perfusion (ISP) and isolated cardiac perfusion (ICP). IP is a methodology that delivers vectors highly selectively, with a long exposure time and high concentrations at the target side. This results in higher transduction rates and thereby may improve therapeutic effects.

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly crossreactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

Cancer cells are known to express cell surface molecules such as specific antigens or cytokine receptors, e.g., EGFR, Fas / CD95, gp100, HER-2 / neu, IL-13Rα2, and MAGE. Among them, interleukin-13 receptor (IL-13R) a2 chain is expressed on certain types of cancer cells including glioblastoma, AIDS Kaposi's sarcoma, and head and neck cancer. This protein is one of the receptor components for IL-13, a Th2 cell-derived pleiotropic immune regulatory cytokine. IL- 13Rα2 chain on these cancer cells can be targeted with a receptor-directed cytotoxin termed IL13-PE to induce specific cancer cell killing, however, this molecule does not mediate cytotoxicity to cells that do not express or express low levels of IL-13Rα2. In order to achieve a broad therapeutic window for IL13-PE, plasmid-mediated gene transfer of IL-13Rα2 in cancer cells was employed in vitro and in vivo. Cancer cells transfected with IL-13Rα2 demonstrated increased binding to IL-13 and sensitivity to IL13-PE in vitro. In vivo intratumoral gene transfer of IL-13Rα2 profoundly enhanced the antitumor activity of IL13-PE, providing complete elimination of established tumor in some xenografts. In this review article, current findings from IL-13Rα2 gene transfer in a variety of human cancer models in nude mice are summarized. In addition, safety issues and possible future directions utilizing this therapeutic approach are discussed.

Endogenous and environmental oxidation is increasingly becoming an important factor associated with numerous disorders in both children and adults. The lung is particularly prone to oxidation, as the gas exchange organ is continuously exposed to a great deal of airborne oxidants. Lung oxidation-induced toxicity is a critical clinical problem that is currently lacking cure. For example, treatment for acute respiratory distress syndrome (ARDS), a common type of acute diffuse lung injury, is strictly supportive. Alkylating chemotherapeutics and many methyl chemicals can cause acute or chronic lung injury, which is also difficult to treat. Many new approaches are being tried to improve the treatment of lung oxidation and alkylation; one of these is the use of DNA repair proteins, such as base excision repair proteins that are largely involved in repairing DNA damage caused by oxidation and alkylation. Recent advances have revealed their promising potential for treating oxidation toxicity. Here we discuss discoveries that have led to this possibility, including pioneering research into the cellular signaling transduction and molecular mechanisms of DNA repair proteins. In conclusion, when combined with other therapeutic measures such as anti-oxidant chemicals and enzymes, DNA repair proteins may have great potential for treating acute and chronic lung toxicity induced by oxidation and alkylation.

Dendritic cells (DCs) are the most effective antigen presenting cells (APCs) to elicit both primary and secondary T-cell response that is critical for antitumor immunity and elimination of intracellular pathogens. Therefore, DCs pulsed ex vivo with antigens have the potential used as cell-based vaccines against tumors. Viral vectors derived from adenoviruses have been extensively used to pulse DCs ex vivo by delivering genes encoding immunomodulatory molecules and tumor antigens to DCs since these vectors are relatively safe, effective in inducing the maturation of DCs, and can accommodate large expression cassettes encoding antigens. One of the hurdles for gene delivery to DCs by adenovirus (Ad) vectors, however, is low transfection efficiency of DCs due to the paucity of Ad receptor on DCs. To overcome this obstacle, targeted Ad vectors have been made by modifying viral capsid proteins. These targeted Ad vectors not only enhance the gene delivery to DCs, but also allow in vivo gene delivery to DCs, thus avoiding ex vivo manipulation of DCs.

Type 1 diabetes results from insulin deficiency caused by autoimmune destruction of insulin-producing pancreatic β cells. Islet transplantation, β cell regeneration, and insulin gene therapy have been explored in an attempt to cure type 1 diabetes. Major progress on islet transplantation includes substantial improvements in islet isolation technology to obtain viable and functionally intact islets and less toxic immunosuppressive drug regimes to prevent islet graft failure. However, the availability of human islets from cadaveric pancreata is limited. Regeneration of pancreatic β cells from embryonic or adult stem cells may overcome the limited source of islets and transplant rejection if β cells are regenerated from endogenous stem cells. However, it is difficult to overcome the persisting hostile β cell-specific autoimmune response that may destroy the regenerated β cells. Insulin gene therapy might overcome the weakness of islet transplantation and β cell regeneration with respect to their vulnerability to autoimmune attack. This method replaces the function of β cells by introducing various components of the insulin synthetic and secretory machinery into non- β cells, which are not targets of β cell-specific autoimmune responses. However, there is no regulatory system that results in the expression and release of insulin in response to glucose with satisfactory kinetics. Although there is no perfect solution for the cure of type 1 diabetes at the present time, research on a variety of potential approaches will offer the best choices for the cure of human type 1 diabetes.