REVIEWS PROGRESS AND PROBLEMS WITH THE USE OF VIRAL VECTORS FOR GENE THERAPY Clare E Thomas Anja Ehrhardt and Mark A Kay Gene therapy has a history of controversy Encouraging results are starting to emerge from the clinic but questions are still being asked about the safety of this new molecular medicine With the development of a leukaemia like syndrome in two of the small number of patients that have been cured of a disease by gene therapy it is timely to contemplate how far this technology has come and how far it still has to go TERMINAL REPEAT A short non coding DNA sequence found at each end of the viral genome which contains elements required for the replication and packaging of the viral DNA CAPSID A protein shell that encapsulates the viral genetic material Departments of Pediatrics and Genetics Stanford University School of Medicine Stanford California 94305 USA Correspondence to M A K e mail markay stanford edu doi 10 1038 nrg1066 346 The science of gene therapy has a turbulent history Initially perceived as a revolutionary new technology with the promise to cure almost any disease provided that we understood its genetic or molecular basis enthusiasm rapidly waned as clinical trial after clinical trial failed to show efficacy1 The stumbling block seemed to be the vehicles that were used to deliver the therapeutic genes to the target tissue early recombinant viral vectors were inefficient failed to persist in host cells and transgene expression was typically short lived Then in 1999 an adverse patient reaction to an adenovirus vector during a clinical safety trial led to the realization that the failure to understand the biology of vector interactions with the human immune system could have fatal consequences BOX 1 The year 2000 brought the first gene therapy success in which three children were cured of a fatal immunodeficiency disorder but this therapy has subsequently caused a leukaemia like disease in 2 of the 11 patients who have been treated BOX 1 Such severe blows have overshadowed the substantial progress that has been made in the development of gene transfer technologies over recent years The message we have extracted from a history of anticipation and disappointment is that the future success of gene therapy will be founded on a thorough understanding of vector biology and pharmacology Over the past few years intense efforts have been concentrated on understanding the molecular basis of how viruses and viral vectors interact with the host MAY 2003 VOLUME 4 Our findings have allowed us to develop vectors with improved efficiency specificity and safety and some clinical successes have recently been achieved This article highlights some of the advances in the development of viral vectors as well as discussing the substantial challenges that remain before gene therapy can truly fulfil all of its promises From pathogen to medicine Viruses are highly evolved biological machines that efficiently gain access to host cells and exploit the cellular machinery to facilitate their replication Ideal virus based vectors for most gene therapy applications harness the viral infection pathway but avoid the subsequent expression of viral genes that leads to replication and toxicity This is achieved by deleting all or some of the coding regions from the viral genome but leaving intact those sequences usually the TERMINAL REPEAT sequences that are required in cis for functions such as packaging the vector genome into the virus CAPSID or the integration of vector DNA into the host chromatin The expression cassette of choice is then cloned into the viral backbone in place of those sequences that were deleted The deleted genes encoding proteins that are involved in replication or capsid envelope proteins are included in a separate packaging construct to provide helper functions in trans The packaging cells into which the vector genome and packaging construct are co transfected then produce the recombinant vector particles FIG 1 www nature com reviews genetics 2003 Nature Publishing Group REVIEWS DISSEMINATED INTRAVASCULAR COAGULATION Inappropriate blood clotting TRANSDUCTION The introduction of genetic material into a cell using a viral vector TITRE A measure of vector concentration that is usually expressed as the number of transducing units or the number of particles per millilitre After production in a packaging cell line the recombinant vector particles are purified and quantified TITRED Purification strategies have traditionally relied on the separation of vector particles from cellular components by density gradient centrifugation usually a caesium chloride gradient however this process is laborious difficult to scale up for industrial purposes and can sometimes damage the vector particles and reduce the infectious titre of the vector stock Advances in column chromatographic methods for the purification of several classes of vector have alleviated these concerns2 3 and most of the main classes of vector that are described here are now able to be grown and purified to the high titres required for administration to humans The main groups of viral vectors Gene therapy was first conceived as a treatment for hereditary single gene defects4 Today acquired diseases such as cancer5 cardiovascular disease6 neurodegenerative disorders7 and infectious disease8 are the subject of most gene therapy research FIG 2 Given the diversity of disease targets that are potentially amenable to gene transfer it has become clear that there can be no single vector that is suitable for all applications Perhaps the only characteristics that are required by all vectors are the abilities to be reproducibly and stably propagated and purified to high titres to mediate targeted delivery that is to deliver the transgene specifically to the tissue or organ of interest without widespread vector dissemination Box 1 Adverse events in gene therapy 1999 adenovirus vector causes patient death In September 1999 18 year old Jesse Gelsinger took part in a gene therapy clinical trial at the University of Pennsylvania in Philadelphia Gelsinger suffered from a partial deficiency of ornithine transcarbamylase OTC a liver enzyme that is required for the safe removal of excessive nitrogen from amino acids and proteins OTC deficiency leads to an accumulation of ammonia in the bloodstream which in turn causes an elevation of ammonium ions in the brain leading to encephalopathy brain damage and coma The University of Pennsylvania
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