Current Gene Therapy - Volume 3, Issue 4, 2003

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of “pseudotyped” AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Damage of articular cartilage is a frequent clinical problem and is commonly considered to be irreversible. Fullthickness defects may lead to the formation of fibrous repair tissue of minor mechanical quality, while partial-thickness lesions hardly show any repair response. Surgical approaches often fail to restore the articular surface, facing the problem of incomplete chondrogenesis or rapid degradation of the repair tissue. However, advances in molecular biology have revealed the potential of growth factors, differentiation factors, and cytokines in directing cellular differentiation and metabolic activity. Anabolic factors including members of the TGF-ß superfamily, IGF-1, FGF, or HGF have proven their potential to stimulate chondrogenesis and synthesis of cartilage-specific matrix components, allowing the formation of a hyaline cartilage-like repair tissue in experimental studies. In addition, anti-catabolic or anti-inflammatory molecules, such as IL-4, IL-10, IL-1Ra, and TNFsR may also exert beneficial effects by inhibiting excessive cartilage degradation. Although it is questionable whether regeneration of hyaline cartilage implying a complete restoration of the articular surface by a tissue that is identical with the original can ever be achieved, all these molecules have been considered as suitable tools for cartilage repair. The transfer of the respective genes into the joint, possibly in combination with the supply of chondroprogenitor cells, might be an elegant method to achieve a sustained delivery of such therapeutic factors at the required location in vivo. This review focuses on the therapeutic molecules, the suitability of different viral and nonviral vectors for intraarticular gene transfer and the lessons learned from gene therapy studies on various animal models.

Radiotherapy is, after surgery, the most widely used form of cancer treatment but the main limitation of radiation treatment is damage to normal tissues. This may be overcome by targeted radiotherapy - the selective delivery to malignant deposits of cytotoxic radionuclides bound to tumour-seeking agents. Several gene transfer techniques are being evaluated which combine gene therapy and targeted radiotherapy, rendering malignant cells more sensitive to radiation or causing them to take up radioactive drugs - providing tumour cell kill with reduced damage to normal tissues. This review examines several aspects of targeted radiotherapy / gene therapy strategies. As well as identification of suitable gene / radiopharmaceutical combinations, expression of the transgenes must be confined to tumour cells via tumour specific transcriptional regulation or via delivery vehicles which specifically target tumour cells. The inability of current delivery vehicles to target every cell within a tumour mass must be addressed by maximising collateral cell damage in targeted radiotherapy strategies via radiation mediated bystander effects and suitable in vitro models are described, which allow assessment of promising gene transfer strategies in scenarios where tumour heterogeneity of transgene expression and cell proliferative state are considered.

The hepatitis B virus (HBV) infection is a public health problem worldwide, particularly in East Asia. The current therapy of HBV infection is mostly based on chemical agents and cytokines that have been shown to provide limited efficacy and are also toxic to the human body. Gene therapy is a new therapeutic strategy against HBV infection, involving the transmission of gene drugs into liver cells by specific delivery systems and methods. Although this new anti-HBV infection technique is under active investigation, various promising anti-HBV viral gene drugs have been developed for gene therapy, including antisense RNA and DNA, hammerhead ribozymes, dominant negative HBV core mutants, single chain antibody, co-nuclease fusion protein, and antigen. In order to optimize their antiviral effects and / or enhance anti-HBV immunity, various novel gene delivery systems have also been developed to (specifically) deliver such DNA constructs into liver cells; some of them are viral vectors, such as adenoviral vectors, retroviral vectors and poxviral vectors, and even hepatitis B viral for its hepatocellular specificity. Others are non-viral vectors, in which naked DNA and liposomes are frequently used for DNA vaccine or nucleotide analogs for inhibiting HBV DNA polymerase. This review addresses various aspects of gene therapy for HBV infection, including gene drugs, delivery methods, animal model, and liver transplantation with combination therapy. It also discusses the problems that remain to be solved.

Virus-mediated oncolysis is a rapidly growing field with the potential to dramatically alter the future of cancer therapy. Replication-selective viruses are superior to non-replicating vectors in several aspects, such as the amplification of the initial low-dose viral inoculum up to 103-104-fold, lateralization into neighboring cells, introduction of novel cell killing mechanisms, and a potential for a safety profile. However, due to their capacity to replicate, the importance of tumor selectivity is further underscored. Of the replication-selective viruses, adenoviruses (Ad) possess several attributes that appear essential for targeting and eliminating tumor cells. These include susceptibility to genomic modifications, convergence with cellular pathways implicated in carcinogenesis, and a high oncolytic capacity. A primary tumor targeting strategy of oncolytic Ad is based on re-engineering the viral genome viruses to construct conditionally replicative adenoviruses (CRAds). In this regard, modification of CRAd genome is traditionally designated as type I or type II. Type I CRAds are based on mutation or deletion of early Ad genes. Type II CRAds are based on the placement of essential early Ad genes under tissue / tumor-specific regulatory elements in a heterologous context. Thus, both strategies confer varying degrees of tumor-specific replication. Recent data, however, indicate that type III CRAds, embodying the paradigms of both type I and II, offer better replication selectivity for tumor cells while maintaining efficient oncolysis. These characteristics of CRAds yield therapeutic indices unprecedented heretofore in cancer therapy. However, other biological aspects of CRAds should also be addressed before these agents prove as first-line antitumor agents. When these issues are resolved, novel tumor cell killing potential of CRAds may truly be realized and dramatically alter future cancer therapy.