Current Gene Therapy - Volume 4, Issue 1, 2004

Adenovirus (Ad) targeting is a novel approach for the design and administration of therapeutic agents wherein the agent is rationally designed to localize and restrict transgene expression to the site of disease in a self-directed manner, usually via exploitation of unique biophysical and genetic properties specific to the diseased tissue. The ablation of promiscuous native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and adenovirus receptor (CAR), to achieve CAR-independent vector tropism. Herein, we describe advances in Ad targeting that are predicated not only on fundamental understanding of vector / cell interplay, but also on the specific transcriptional profiles of target tissues. Further, targeting is discussed in the context of improving the safety and efficacy of clinical approaches utilizing adenoviral vectors and replication competent oncolytic agents. In summary, existing results suggest a critical linkage between targeted agents and increases in therapeutic utility.

Efficient and regulated co-expression of multiple genes is an important consideration in the design of gene therapy vectors. While the augmentation of a single therapeutic gene is often sufficient for gene therapy of simple mendelian disorders, strategies for the treatment of complex disorders and infectious diseases necessitate the introduction of multiple genes into the cell. Complex disorders such as cancer often involve mutations in multiple genes and a combination strategy targeting different defective genes simultaneously are often more effective than any single strategy. Likewise, approaches for treating infectious diseases such as HIV-1 (human immunodeficiency virus) often involve the blocking of multiple steps of the viral replication pathway simultaneously to prevent the emergence of resistant strains of the virus. Even for the treatment of single gene defects, the additional incorporation of a selectable marker gene is often necessary to achieve sustained expression of the therapeutic gene in the cells. Among the several different strategies to coexpress multiple genes, the incorporation of an IRES (internal ribosome entry site) into gene therapy vector design represents one of the more promising strategies. IRES functions as a ribosome-landing pad for the efficient internal initiation of translation ensuring coordinate expression of several genes and are located at the 5'UTR (5' untranslated regions) of these genes. Currently, the most popular IRES utilized for gene therapy is the IRES from the EMCV (encephalomyocarditis virus). However, the major caveat with present vector systems utilizing this IRES is that the expression of the downstream gene is significantly less efficient than the upstream gene. This review will examine the growing list of naturally occurring and synthetic IRESes and how they can be exploited for human gene therapy.

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and “immune-privileged” tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, “in-stent” intimal hyperplasia / restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL / Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.

Viruses that kill the host cell during their replication cycle have attracted much interest for the specific killing of tumor cells and this oncolytic virotherapy is being evaluated in clinical trials. The rationale for using replicative oncolytic viruses is that viral replication in infected tumor cells will permit in situ viral multiplication and spread of viral infection throughout the tumor mass thus overcoming the delivery problems of gene therapy. Improved understanding of the life cycle of viruses has evidenced multiple interactions between viral and cellular gene products, which have evolved to maximize the ability of viruses to infect and multiply within cells. Differences in viral-cell interactions between normal and tumor cells have emerged that have led to the design of a number of genetically engineered viral vectors that selectively kill tumor cells while sparing normal cells. These viruses have undergone further modifications to carry adjunct therapy genes to increase their anti-cancer abilities. Since these viruses kill cells by oncolytic mechanisms differing from standard anticancer therapies, there is an opportunity that synergistic interactions with other therapies might be found with the use of combination therapy. In this review, we focus on the oncolytic Herpes Simplex Virus-1 (HSV-1) vectors that have been examined in preclinical and clinical cancer models and their use in combination with chemo-, radio-, and gene therapies.

A link between common chromosome fragile sites and frequent chromosomal deletions in cancer was observed two decades ago and led to the hypothesis that genes at fragile sites may play a role in tumor development. In 1996, the human fragile histidine triad gene, FHIT, was identified by positional cloning of the chromosome region spanning the carcinogen-sensitive, common fragile site, FRA3B at 3p14.2. Loss or inactivation of the FHIT gene in a large fraction of human tumors results in absence or reduction of Fhit protein. In vitro analyses and in vivo tumorigenicity studies show that restoration of Fhit protein induces tumor suppression in 50% of tumor cell lines tested. Viral vector-mediated FHIT gene transfer to Fhit-deficient mice not only prevents but reverses the carcinogen-induced tumor development in vivo, in accordance with the oncosuppressive properties of Fhit protein. The strong proapoptotic activity following Fhit infection of cancer cells strengthens the case for further exploration of FHIT gene therapy in cancer prevention and treatment.

Vectors derived from human immunodeficiency virus type 1 (HIV-1) are an attractive option for many gene therapy applications as they can transduce non-cycling cell populations, and can integrate their genome into the host cell chromosome. The rationale underlying the design of most retroviral vector systems is to segregate the viral cis sequences, which are required for transfer of the viral genome, from the trans sequences that encode viral proteins. This allows the efficient production of replication incompetent virus and has been successfully applied to the generation of HIV-1 vectors. Nonetheless, the possibility that recombination events in the vector production system can generate replication-competent virus, combined with the pathogenic nature of HIV-1, raises major bio-safety issues. Numerous HIV-1 vectors have now been reported, with each generation significantly improved in ways designed to reduce the risk of replication-competent virus being produced. However, progress in vector design needs to be complemented by the development of methods for the quantitation of the probability of replication competent virus being produced. Assaying individual events in the multi-step pathway that can lead to the production of replication-competent virus, rather than relying on the detection of replication-competent virus per se, will be important for quality control purposes. This review will specifically examine the approaches to HIV-1 vector design that have been postulated as increasing bio-safety, possible methods for evaluating bio-safety and whether these approaches are likely to be sufficient to overcome resistance to the use of HIV-1 for clinical application. In addition, we discuss the possible justifications for developing vectors from lentiviruses other than HIV-1.

Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy-the transfer of genetic material for therapeutic purposes-has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined.

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailormade' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

The success of gene therapy strongly depends on an efficient delivery system to allow local transfer and expression of the therapeutic gene in the target organ or tissue. Vector systems have been improved and many show promise. There are two different categories of delivery vehicles: non-viral and viral vectors, both with advantages and disadvantages that must be taken into consideration in view of the final aim. Compared to other solid organs, the kidney offers the main advantage of access by different routes that dictate different sites of transfection. Thus, the choice of the delivery vehicle and administration route has to take account which cells are to be specifically targeted by the gene transfer approach. This concept will be discussed in the first part of the review. Using a gene therapy approach, improvements of renal function and interstitial inflammation have been achieved in experimental models of glomerulonephritis and tubulo-interstitial damage. Gene therapy applied to renal transplantation has shown promising results in rodents, almost controlling acute rejection. Finally, the development of animal models resembling the clinical features of human genetic renal disorders offers a first step towards new treatments among which gene therapy could become reality in the near future. The main findings concerning the suitability of gene therapy for slowing the progression of kidney diseases, and preventing acute renal graft rejection, or treating genetic disorders, are discussed.

Hypertrophic scar and keloid are common and difficult to treat diseases in plastic surgery. Results of wound healing research over the past decades have demonstrated that transforming growth factorβ (TGFβ) plays an essential role in cutaneous scar formation. In contrast, fetal wounds, which heal without scarring, contain a lower level of TGFβ than adult wounds. How to translate the discovery of basic scientific research into the clinical treatment of wound scarring has become an important issue to both clinicians and basic researchers. The development of gene therapy techniques offers the potential to genetically modify adult wound healing to a healing process similar to fetal wounds, and thus reduces wound scarring. This article intends to review the roles of TGFβ in the formation of wound scarring, the possible strategies of antagonizing wound TGFβ, and our preliminary results of scar gene therapy, which show that wound scarring can be significantly reduced by targeting wound TGFβ.