© 2003 Nature PublishingGroup346 | MAY 2003 | VOLUME 4 www.nature.com/reviews/geneticsREVIEWSThe science of gene therapy has a turbulent history.Initially perceived as a revolutionary new technologywith the promise to cure almost any disease — pro-vided that we understood its genetic or molecular basis— enthusiasm rapidly waned as clinical trial after clini-cal trial failed to show efficacy1. The stumbling blockseemed to be the vehicles that were used to deliver thetherapeutic genes to the target tissue; early recombinantviral vectors were inefficient, failed to persist in hostcells and transgene expression was typically short-lived.Then, in 1999, an adverse patient reaction to an aden-ovirus vector during a clinical safety trial led to the real-ization that the failure to understand the biology ofvector interactions with the human immune systemcould have fatal consequences (BOX 1). The year 2000brought the first gene-therapy success in which threechildren were cured of a fatal immunodeficiency disor-der, but this therapy has subsequently caused aleukaemia-like disease in 2 of the 11 patients who havebeen treated (BOX 1). Such severe blows have overshad-owed the substantial progress that has been made in thedevelopment of gene-transfer technologies over recentyears. The message we have extracted from a history ofanticipation and disappointment is that the future suc-cess of gene therapy will be founded on a thoroughunderstanding of vector biology and pharmacology.Over the past few years, intense efforts have been con-centrated on understanding the molecular basis ofhow viruses and viral vectors interact with the host.Our findings have allowed us to develop vectors withimproved efficiency, specificity and safety, and someclinical successes have recently been achieved. This arti-cle highlights some of the advances in the developmentof viral vectors, as well as discussing the substantialchallenges that remain before gene therapy can trulyfulfil all of its promises.From pathogen to medicineViruses are highly evolved biological machines that effi-ciently gain access to host cells and exploit the cellularmachinery to facilitate their replication. Ideal virus-basedvectors for most gene-therapy applications harness theviral infection pathway but avoid the subsequent expres-sion of viral genes that leads to replication and toxicity.This is achieved by deleting all, or some, of the codingregions from the viral genome, but leaving intact thosesequences (usually the TERMINAL REPEAT sequences) that arerequired in cis for functions such as packaging the vectorgenome into the virus CAPSID or the integration of vectorDNA into the host chromatin. The expression cassette ofchoice is then cloned into the viral backbone in place ofthose sequences that were deleted. The deleted genesencoding proteins that are involved in replication orcapsid/envelope proteins are included in a separatepackaging construct to provide helper functions in trans.The packaging cells into which the vector genome andpackaging construct are co-transfected then produce therecombinant vector particles (FIG. 1).PROGRESS AND PROBLEMS WITHTHE USE OF VIRAL VECTORS FORGENE THERAPYClare E. Thomas, Anja Ehrhardt and Mark A. KayGene therapy has a history of controversy. Encouraging results are starting to emerge from theclinic, but questions are still being asked about the safety of this new molecular medicine. Withthe development of a leukaemia-like syndrome in two of the small number of patients that havebeen cured of a disease by gene therapy, it is timely to contemplate how far this technology hascome, and how far it still has to go.TERMINAL REPEATA short non-coding DNAsequence found at each end ofthe viral genome, which containselements required for thereplication and packaging of theviral DNA.CAPSIDA protein shell that encapsulatesthe viral genetic material.Departments of Pediatricsand Genetics, StanfordUniversity School ofMedicine, Stanford,California 94305, USA.Correspondence to M.A.K.e-mail:[email protected]:10.1038/nrg1066© 2003 Nature PublishingGroupNATURE REVIEWS | GENETICS VOLUME 4 | MAY 2003 | 347REVIEWSDISSEMINATED INTRAVASCULARCOAGULATIONInappropriate blood clotting.TRANSDUCTIONThe introduction of geneticmaterial into a cell using a viralvector.TITREA measure of vectorconcentration that is usuallyexpressed as the number oftransducing units, or thenumber of particles permillilitre.The main groups of viral vectorsGene therapy was first conceived as a treatment forhereditary single-gene defects4. Today, acquired diseasessuch as cancer5, cardiovascular disease6,neurodegenera-tive disorders7and infectious disease8are the subject ofmost gene-therapy research (FIG. 2). Given the diversityof disease targets that are potentially amenable to genetransfer, it has become clear that there can be no singlevector that is suitable for all applications. Perhaps the onlycharacteristics that are required by all vectors are the abili-ties to be reproducibly and stably propagated and puri-fied to high titres, to mediate targeted delivery (that is,to deliver the transgene specifically to the tissue or organof interest without widespread vector disseminationAfter production in a packaging cell line, the recom-binant vector particles are purified and quantified(TITRED). Purification strategies have traditionally reliedon the separation of vector particles from cellular com-ponents by density gradient centrifugation (usually acaesium chloride gradient); however, this process islaborious, difficult to scale up for industrial purposesand can sometimes damage the vector particles andreduce the infectious titre of the vector stock.Advancesin column-chromatographic methods for the purifica-tion of several classes of vector have alleviated these con-cerns2,3and most of the main classes of vector that aredescribed here are now able to be grown and purified tothe high titres required for administration to humans.Box 1 | Adverse events in gene therapy1999: adenovirus vector causes patient deathIn September 1999, 18-year-old Jesse Gelsinger took part in a gene-therapy clinical trial at the University of Pennsylvaniain Philadelphia. Gelsinger suffered from a partial deficiency of ornithine transcarbamylase (OTC), a liver enzyme that isrequired for the safe removal of excessive nitrogen from amino acids and proteins. OTC deficiency leads to anaccumulation of ammonia in the bloodstream, which, in turn, causes an elevation of ammonium ions
View Full Document
Unlocking...