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Gene therapy: lessons learnt from the past decade It is more than a decade since the first approved clinical trial to put genes into the cells of human beings was initiated. In that first trial, investigators at the US National Institutes of Health used a modified mouse leukaemia virus to insert a DNA marker into lymphocytes being used to treat cancer.1 A year later, a similar viral technique was used as treatment for two girls who had severe combined immunodeficiency with mutant adenosine deaminase.2 Ten years and more than 3000 patients later, it is appropriate to ask, "Where are we now?"
Where are we now? Initial speculation that gene therapy would quickly revolutionise medicine has clearly been wrong. As is often the case when entirely new areas of work develop, there was an overoptimistic view of the pace of progress and an underestimation of the problems remaining to be overcome. In something of a knee jerk reaction, many scientific pundits declared gene therapy dead or, at best, a potentially useful research tool that was being aggressively oversold by a few biotechnology companies. In reality, the science of gene transfer was progressing quickly in a classic reiterative process, where lessons learned from the early clinical studies were redirecting the course of research. Gene therapy is now a robust scientific discipline encompassing specialties and subspecialties, and an array of new reagents are nearing availability for specific clinical applications. Delivery One of the challenges that was not fully appreciated was the difficulty encountered in delivering genes to the cells that need correction. Since the techniques for modifying mutant genes directly (gene correction) were far too inefficient to be useful clinically, it was necessary to treat genetic disorders by adding a normal copy of the mutant gene to the cells (addition gene therapy). Both viral and non-viral techniques for gene delivery have been tested clinically.3 Viral methods have proved to be the most efficient to date, particularly in applications that require stable integration of the delivered gene. The molecular weight of the DNA encoded by clinically relevant genes is very large and this mass becomes even larger (greater than one million) when it is packaged into a replication inactivated virus. The viral vectors used in gene therapy are inactivated to remove any chance that they might themselves cause disease. However, by crippling replication, the mechanism that viruses normally use to spread genes in the body is also inactivated. The laws of physics concerned with diffusion of large molecules then govern the spread of the vector. This diffusion is often further limited by the small intercellular spaces through which the viral particles must move and by the presence of viral binding ligands on the surface of the cells they are trying to move beyond. Although ex vivo gene treatment should avoid some of these delivery problems, the limited tropism and the dependence on the cell cycle of early gene transfer vectors gave disappointing results in initial efforts to target cells such as haematopoietic stem cells.4 Physiological regulation Physiological regulation of the added genes is another major challenge. To achieve a long term effect, integration of the added gene into the chromosomal DNA of the host may be essential. Unfortunately, the most efficient integrating gene transfer systems use small viral vectors such as retroviruses that are unable to accommodate full length human genes containing all of their original regulatory sequences. In most viral vectors, genomic gene sequences are replaced by smaller cDNAs, thereby losing the regulatory information (enhancers, etc) encoded in the deleted introns. Furthermore, short viral or heterologous cellular promoters are often substituted, resulting in a gene expression pattern that may be fundamentally different from the expression of the normal endogenous gene. Random integration of gene transfer vectors at different chromosomal locations can also adversely influence gene expression. In addition, some of the early attempts to use these gene transfer systems have encountered problems related to the development of immune responses directed towards the viral vectors and even against some of the transgenes expressed in the treated cells.5,6 These technical limitations forced investigators to look for candidate disorders outside the traditional genetic diseases. Current trials Clinical trials are currently addressing a very broad range of potential delivery systems and disease targets (figure, tables 1 and 2, and box). Of the 313 trials listed in the public database maintained by the US National Institute of Health, 70% are involved in the treatment of cancer. The preponderance of cancer related trials (figure, table 2) may surprise readers who think of gene therapy as a treatment option for genetic diseases (table 1). However, in the broader context, gene therapy is another form of drug delivery, and this accounts for the wide variety of applications of the "technique." These applications include treatments aimed at a diverse list of disorders including arthritis, HIV infection, dozens of different types of cancers, and extremely rare genetic diseases. With a few exceptions, the number of patients enrolled in any given trial is small (generally fewer than 20). This is mainly because of the requirement for ex vivo manipulation of the individual patient's cells.
Direct in vivo gene transfer Direct in vivo gene transfer (often called the holy grail of gene therapy) is being tested with increasing frequency and with some encouraging results. The types of gene transfer reagents being used in the clinic range from injections of simple DNA molecules and DNA-lipid complexes to genetically modified hybrid animal viruses. Naked DNA alone, either in the form of an oligonucleotide or as a plasmid, is the simplest form of gene transfer reagent that can be used to transfect some cell types directly. For example, direct DNA injections are being used as a new generation of vaccines where the DNA directs the expression of an antigen.7 One of the more encouraging results in recent reports comes from the use of injections of DNA encoding vascular endothelial growth factors to promote angiogenesis in tissues affected by vascular insufficiency.8
 Proportion of protocols for human gene therapy trials relating to various types of disease Viral vectors The list of modified viruses that can be used in gene therapy experiments continues to increase, as does the understanding of the biology and immunology of these systems (box). Early experiments using adenovirus showed a rapid and important immune response against viral proteins and transgenes.5 Subsequently, investigators have worked to produce a new generation of adenoviral vectors without viral genesthe so called "gutted" adenovirus.9 These highly modified adenoviral vectors have shown appreciable long term persistence in a number of animal model studies. There are also exciting reports on the use of the human adeno-associated virus as a gene transfer vector.10 This ubiquitous virus is not associated with any serious human disease and has the useful property of being able to infect mature differentiated cells such as muscle or neurons. The main drawbacks to using the adeno-associated virus vector system are the limited coding capacity of the vector (about 4.0 kb) and the laborious production systems.
Encouraging results have been reported from long term animal studies of gene transfer and expression by direct injection of vector into the brain, muscle, and liver.11,12 These data have led to an increased interest in adeno-associated virus and expanded its use in human gene therapy trials. One of the most exciting applications is adeno-associated vector injection into muscle. A trial was recently initiated to inject factor IX expressing adeno-associated vectors into the muscles of patients with haemophilia B, and similar approaches have been proposed for retinitis pigmentosa, familial hypercholesterolaemia, and muscular dystrophy. Nucleotide exchange Viral mediated gene transfer methods result in new genetic material being added to the existing cells' genome - they do not correct the underlying genetic defect that causes the disease. Revolutionary changes are under way in non-viral mediated gene transfer and gene correction, which may lead to a paradigm shift in our thinking about the types of diseases that can be treated by gene therapy. A new experimental strategy to correct single nucleotide mutations in genomic DNA has recently been developed.13,14 A chimeric oligonucleotide comprising RNA and DNA residues in a duplex conformation was used to target the desired nucleotide exchange. The approach was based on the observation that RNA-DNA hybrids were highly active in homologous pairing reactions in vitro. The duplex conformation was designed with 2'-O methylated RNA-DNA stems and poly-T hairpin loops for chemical and thermal stability as well as resistance to helicases and RNA and DNA nucleases. The RNA-DNA sequence is complementary to that of the target gene, except that it contains one mismatched nucleotide when aligned with the genomic DNA sequence. It seems this unpaired nucleotide is recognised by endogenous repair systems, resulting in an alteration of the DNA sequence of the targeted gene. If these methods can be developed for clinical application, they could mediate the correction of mutations that cause disease. This would be a major advance for gene therapy.
 Three dimensional crystal structure of a hammerhead ribozyme Gene therapy-related links on world wide web European Society of Gene Therapy at www.cbt.ki.se/ewgt/ Hopeful future Gene therapy today is at a stage similar to that experienced in the 1980s by recombinant proteins and monoclonal antibodies in medicine - that is, much hope and speculation but very few products. Recombinant proteins such as insulin, erythropoietin, and various cytokines are now common in medical practice. The future of gene therapy is equally exciting, and the next few years should prove interesting. Footnotes Competing interests: RMB is employed by Kimeragen, Inc and has consulted for ARIAD Pharmaceuticals, Genetic Therapy/Systemix/Novartis, and Baxter Healthcare. RMB has shares or stock options in Kimeragen, ARIAD, and Cell Genesys Inc. Richard A Morgan interim chiefa R Michael Blaese chief scientific officerb. a Clinical Gene Therapy Branch, National Institutes of Health, Bethesda, MD 20892, USA, b Kimeragen Inc, Newtown, PA 18940, USA Correspondence to: R A Morgan rmorgan@nhgri.nih.gov References
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1 Antitrypsin deficiency