Current Gene Therapy - Volume 9, Issue 6, 2009

Numerous ovarian gene therapy strategies are in clinical phases based on concepts of replacement/ knock out of deregulated gene, suicide gene strategies, strengthening of the immune response against a tumor, inhibition of tumor angiogenesis and growth factors. Non-viral delivery systems have potential advantages over currently widely used viral vectors and other classical vectors for delivering therapeutic gene of interest. The present review provides a comprehensive overview of potential of various delivery systems currently in use. Non-viral formulations used in ovarian gene therapy include injecting naked DNA, liposomes, polyplexes, lipopolyplexes, nanoparticles, gene gun and ultrasound/microbubble mediated gene delivery. In addition to improving vector delivery, the DNA constructs need to be optimised for both efficient and long-term transgene expression. Minicircles using minimal immunological defined gene expression (MIDGE) technology, are a promising future alternative to plasmid for use in non-viral ovarian gene therapy in terms of biosafety, improved gene transfer, potential bioavailability, minimal size and little immune reaction. The review explores the best route of administration for ovarian cancer gene therapy given its peritoneal dissemination which poses a major challenge in treating ovarian cancer patients. Enhancement of therapeutic index can be further achieved by overcoming barriers both at cellular and nuclear levels. Selective tumor targeting with minimal toxicity using folate modified, incorporating nuclear localization signal and PEGylated stealth liposome's represents a popular approach and needs to be exploited in ovarian gene therapy.

Lentiviral vectors (LV) are competent gene transfer vehicles, as used for both research and gene therapy applications, because of their stable integration in non-dividing and dividing cells and long-term transgene expression. Along with our understanding that LV offer solutions for gene therapy, biosafety concerns have uncovered risks due to insertional mutagenesis, the generation of replication competent lentiviruses (RCL) and vector mobilization. Researchers therefore continue to devote significant efforts in designing LV with improved efficacy and biosafety features. The choice of a particular LV system for experimental studies is often driven by functional considerations, including increased productivity and/or transduction efficiency. The design of safer vectors has also directly benefited researchers allowing them to conduct experimental studies with lower risk. Currently, vectors combine improved safety features (that decrease the risk of recombination and vector mobilization) with increased transduction efficiency. Hence, risks associated with the inadvertent transduction of cells of the investigator gain greater importance in assessing the overall risk of these vectors and become an important biosafety concern. This review outlines the different strategies used to improve LV biosafety by comparing state-of-the-art and emerging LV production systems and highlighting biosafety issues that can arise during their contained use. The few existing national and international biosafety recommendations that specifically address the use of LV in research are discussed and recommendations for most common research activities using LV are proposed.

Mitochondrial genetics has become an emerging area of research in the field of modern therapeutics. Mitochondrial genome is the source of 13 polypeptides which are components of subunits of complexes of electron transport chain and are used in the generation of ATP by oxidative phosphorylation. Any mutation and/or defects in these mitochondrial genes may cause diseases ranging from neurodegenerative diseases, diabetes mellitus to cancer. In an ideal condition mtDNA should be mutation free. There are various mechanisms for the repair of diseased cell or mitochondrial DNA. Nowadays, nucleic acid based therapeutics has become of interest and represent a new area of research. However, problem consistently encountered is safe and effective delivery of DNA to the mitochondria for therapeutic benefits. There are numerous barriers which are to be surpassed for successful delivery of nucleic acid to the cell interior and ultimately to the mitochondria. For efficient and effective DNA delivery to the mitochondrial matrix, a suitable carrier system is required to be designed and developed. In the present review we have discussed briefly about mitochondrial DNA and related diseases, various barriers encountered in the delivery of DNA, internalization processes, delivery strategies and methods for targeted delivery of DNA to the mitochondria.

Gene therapy provides the possibility of long term treatment for the severest of congenital disorders. In this review we will examine the recent advances in gene therapy for genodermatoses. Congenital diseases of the skin exhibit a wide range of severity and underlying causes and there are many possible therapeutic avenues. Gene therapy approaches can follow three paths-in vivo, ex vivo and fetal gene therapy, though the later is currently theoretical only it can provide potential results for even the most severe congenital diseases. All approaches utilize the many different vector systems available, including viral and the emerging use of non- viral integrating vectors. In addition, the use of RNAi based techniques to prevent dominant mutant protein expression has been explored as a therapy for specific dominant disorders such as keratin mutation disorders. Progress has been rapid in the past few years with some initial successful clinical trials reported. However, there are still some issues surrounding long term expression, transgene sustainability and safety issues that need to be addressed to further shift from experimental to clinically therapeutic applications. With the continuing development, merger and refinement of existing techniques there is an ever increasing likelihood of gene therapies becoming available for the more severe genodermatoses within the next decade or shortly thereafter.

Although cell transfection by viral vectors is highly efficient, undesirable side effects including immunogenicity, toxicity and carcinogenesis have to be taken into consideration before their clinical applications. In contrast, most nonviral vectors, such as chitosan, are advantageous due to their biocompatibility, biodegradability, low toxicity and immunogenicity. However, the tranfection efficiency of chitosan as gene vector is rather low because of its low stability and low buffering capacity. Recent technological progress in chemical modification of chitosan has led to improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability. These advances have led to a better understanding of the relationship between the physicochemical characteristics of a non-viral vector and its transfection efficiency. In this review, we summarize the obstacles encountered during the transfection process of chitosan and its derivatives, and then focus on strategies to overcome these obstacles. An accurate method for determining the rate-limiting step and intracellular unpacking kinetics of chitosan and its derivatives is also presented. Lastly, gene-silencing chitosan/ small interfering RNA (chitosan/siRNA) complexes and prospects of feasible methods for enhancing the transfection efficiency of chitosan and its derivatives are discussed.

Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders.