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Gene therapy: trials and tribulations
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"REVIEWS The basic concept of gene therapy is disarmingly simple ? introduce the gene, and its product should cure or slow down the progression of a disease. Encompassed within this idea are a number of goals, including the treatment of both inherited and acquired disease. This approach requires a technology capable of gene transfer in a wide variety of cells, tissues and whole organs, but the delivery vehicles needed to ferry genetic material into a cell still represent the ?Achilles heel? of gene therapy. An ideal vector should have the attributes outlined in BOX 1. At present, not all of these attributes can be found in any one vector, although distinct classes of vector have differ- ent combinations of attributes. The vectors available now fall into two broad cate- gories ? the non-viral and viral vectors. Non-viral vec- tors include naked DNA and LIPOSOMES 1 . Although non- viral vectors can be produced in relatively large amounts, and are likely to present fewer toxic or immunological problems, they suffer from inefficient gene transfer at present. Furthermore, expression of the foreign gene is transient. Given the need, in many diseases, for sustained and often high-level expression of the transgene, viral vectors are the most suitable vehicles for efficient gene delivery. Therefore the purpose of this article is to review the current status of the most commonly used viral vectors (for comprehensive reviews on gene therapy vec- tors, see REFS 2,3). We discuss viral vectors within the framework of the ?ideal vector?, and consider the implica- tions of recent experimental data and gene therapy trials on the uses and future development of these vectors. A survey of viral vectors All viruses have a genetic component that is essential for further propagation. Viral vectors are derived from viruses by replacing these genetic components with the therapeutic gene. In a cell (typically called a packaging cell), the essential components can be provided in trans, which enables the viral vector to be packaged and to deliver genes to the target cell. But this is a dead-end infection, because the vector lacks the essential compo- nents for viral propagation. Recombination between the vector and the viral genes encoding the essential com- ponents in the packaging cells can lead to the generation of infectious parental virus. Therefore the removal or the separation of genes encoding viral components essential for viral propagation reduces the risk of gener- ating infectious virus ? a principle frequently used in gene therapy vector design. The viral vectors can be divided into two general cat- egories ? integrating and non-integrating ? the for- mer holding the promise of life-long expression of the deficient gene product. At present, there are three main vectors (retroviral, lentiviral and adeno-associated viral) that can integrate in recipient cells, and one vector type (adenoviral) that is maintained as an EPISOME. Retroviral vectors. Vectors based on retroviruses were among the first to be designed, and they have been important in the technical and conceptual development of viral vectors as a whole 4?7 . Retroviruses have three essential genes, which can be provided separately in GENE THERAPY: TRIALS AND TRIBULATIONS Nikunj Somia and Inder M. Verma The art and science of gene therapy has received much attention of late. The tragic death of 18-year-old Jesse Gelsinger, a volunteer in a Phase I clinical trial, has overshadowed the successful treatment of three children suffering from a rare but fatal immunological disease. In the light of the success and tragedy, it is timely to consider the challenges faced by gene therapy ? a novel form of molecular medicine that may be poised to have an important impact on human health in the new millennium. LIPOSOMES Artificial lipid vesicles. Liposomes fuse with the cell membrane to deliver their contents, such as DNA for gene therapy. EPISOMES DNA molecules that are maintained in the nucleus without integrating into the chromosomal DNA. NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 91 The Laboratory of Genetics, The Salk Institute, 10,010 North Torrey Pines Road, La Jolla, California 92037, USA. e-mail: verma@salk.edu � 2000 Macmillan Magazines Ltd 92 | NOVEMBER 2000 | VOLUME 1 www.nature.com/reviews/genetics REVIEWS inactivating) vectors 11 , in which the viral regulatory ele- ments have been deleted. On integration, all viral pro- moter/enhancer activity is lost and the transcription of the transgene is under the control of a heterologous promoter. The use of retroviral vectors has also lead the way in the technological production, storage and distribution of commercial vectors on the scale required for human clinical trials. A wide variety of packaging cell lines and vectors with improved TRANSDUCTION efficiencies are now used that include features such as tissue-specific pro- moters, inducible promoters, INTERNAL RIBOSOMAL ENTRY SITES (IRESs) and env proteins with modified target specificity 3,12 . The main limitation of retroviral vectors has been their inability to infect non-dividing cells, meaning that tissues such as brain, eye, lungs and pancreas are not amenable to direct in vivo gene delivery. Furthermore, on transplantation of transduced cells in the host, tran- scription of the transgene is often extinguished 13,14 . These two serious limitations have lead many scientists to search for vectors that can infect non-dividing cells, as well as integrate into the host chromosome. Retroviral vectors continue to be extensively used for introducing genes into dividing cells, such as tumour cells and dividing HAEMATOPOIETIC cells. Lentiviral vectors. Lentiviruses, such as human immun- odeficiency virus (HIV), are part of the retrovirus fami- ly, but have acquired the unusual property of transduc- ing non-dividing cells 15,16 . The mechanism of this KARYOPHILIC attribute is still in contention, but it clearly offers an opportunity missing in its distant cousins, the prototypic retroviruses such as Moloney murine leukaemia virus (M-MLV). The HIV genome, in addi- tion to coding for the usual Gag, Pol and Env proteins, also codes for the accessory proteins 17 Tat, Rev, Nef, Vif, Vpu and Vpr. Although the precise involvement of each of these accessory proteins in the aetiology of AIDS is still a matter of debate, none of them is obligatory (except for Rev and Tat) for virus propagation in vitro. The first-generation lentiviral vectors relied largely on substitution of viral Env protein with vesicular stom- atitis virus G protein (VSVG), which relieved them of their dependence on CD4, the T-cell receptor protein required for lentivirus infection 18 . Instead, the vectors showed a wider TROPISM by infecting cells known not to express CD4 protein, including neurons, hepatocytes, muscle fibres and retinal cells. Although the first-gener- ation vectors fulfilled many of the criteria of an ideal vector, they were viewed with some suspicion because of the possibility of recombination and generation of infectious HIV. To minimize some of these concerns, several research groups have taken the initiative of sys- tematically deleting as many viral accessory genes as possible while maintaining the key feature of infection of non-dividing cells 19,20 . The latest lentiviral vector sys- tem retains less than 25% of the viral genome in the packaging constructs and less than 5% of the viral genome in the vector construct 20 . Extra features that have improved vectors include the central polypurine packaging cells: gag encodes viral structural proteins, pol encodes reverse transcriptase/integrase and env encodes viral envelope glycoprotein. The idea of separating gag and pol from env offered a packaging cell line in which the chances of generating replication-competent retro- viruses (RCR) were significantly reduced (FIG. 1) ? a concept in vector design first applied to retroviruses. Retroviral vectors were also important for the develop- ment of strategies, such as changing the envelope pro- tein, to modify the range of target cells from ecotropic (infecting only rodent cells) to xenotropic (infecting most mammalian cells except rodent cells), amphotrop- ic (infecting all mammalian cells) and pantropic (infect- ing various species) 8?10 . Another conceptual break- through in generating safe and transcriptionally regulable vectors was the development of SIN (self- Box 1 | Properties of the ideal gene therapy vector Easy production The vector should be easy to produce at high titre on a commercial scale. This consideration stems from the wide range of cell numbers that must be transduced ? from a handful of stem cells capable of reconstituting the entire haematopoietic repertoire to 10 11 or more cells to infect 5?10% of the liver. For widespread use, the vector should be amenable to commercial production and processing (such as concentration technology for delivery in small volumes), and should have a reasonable shelf-life for transport and distribution. Sustained production The vector, once delivered, should be able to express its genetic cargo over a sustained period or expression should be regulable in a precise way. Different disease states have different requirements (for example, regulated expression in diabetes and lifetime expression in haemophilia). Immunologically inert The vector components should not elicit an immune response after delivery. A humoral antibody response will make a second injection of the vector ineffective, whereas a cellular response will eliminate the transduced cells. Tissue targeting Delivery to only certain cell types is highly desirable, especially where the target cells are dispersed throughout the body (such as in the haematopoietic system), or if the cells are part of a heterogeneous population (such as in the brain). It is also important to avoid certain cells, such as dendritic cells, the ?professional? antigen-presenting cells of the body, because of their role in mediating the immune response. Cell or tissue-targeted vectors present a great challenge, but also offer rich dividends for gene therapy approaches. Size capacity The vector should have no size limit to the genetic material it can deliver. The coding sequence of a therapeutic gene can vary from 350 base pairs for insulin, to over 12,000 base pairs for dystrophin. Furthermore, addition of appropriate regulatory sequences may be required for efficient transduction and expression of the foreign genetic material. Replication, segregation or integration The vector should allow for site-specific integration of the gene into the chromosome of the target cell, or should reside in the nucleus as an episome that will faithfully divide and segregate on cell division. Site-specific integration is a very desirable attribute because it eliminates the uncertainty of random integration into the host chromosome, and endogenous regulatory regions will control its expression under physiological conditions. The ability of the vector to be maintained as an episome could make the genetic elements independent of local chromatin environments, but faithful replication and segregation is needed if the vector is to be effective in systems such as stem cells. Infection of dividing and non-dividing cells As large numbers of cells (such as neurons, hepatocytes and myocytes) are postmitotic, vectors capable of efficiently transducing non-dividing cells are very desirable. TRANSDUCTION The introduction of a gene into a target cell by a viral vector. INTERNAL RIBOSOME ENTRY SITE A sequence that is inserted between the coding regions for two proteins and allows efficient assembly of the ribosome complex in the middle of a transcript, leading to translation of the second protein. HAEMATOPOIESIS The programme of cellular differentiation leading to the formation of blood cells. � 2000 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 93 REVIEWS discussing their use with regulatory authorities. Like other integrating vectors, the lentiviral vectors will have the disadvantage of non-specific integration in the chromosome. The duration of expression of the trans- gene in lentiviral vectors also needs further testing. Adeno-associated viral vectors. Adeno-associated virus (AAV) is a small, non-pathogenic, single-stranded DNA virus that has turned out to be an efficient and useful delivery vehicle 30 . The virus is a member of the depen- doviruses and, as the name suggests, requires extra genes to replicate 31 . These genes are provided by adenovirus (hence the name, as AAV was found associated with this virus) or herpes virus. AAV itself has two genes: rep, which codes for replication and integration functions of the virus; and cap, which codes for the structural compo- nents of the virus. On either side of rep and cap are two inverted terminal repeats (ITRs), which define the begin- ning and the end of the virus, and contain DNA sequences needed to pack the viral genome into CAPSIDS. tract (cPPT), which allows internal initiation of second- strand DNA synthesis and probably aids in the trans- port of the pre-integration complex to the nucleus 21,22 ; and the woodchuck post-transcriptional regulatory ele- ment (WPRE), which further improves the transduc- tion and translational efficiency of lentiviral vectors 23 . Use of SIN lentiviral vectors further reduces the chance that recombination will generate replication- competent HIV 24,25 . Recently, packaging-cell lines in which most of the accessory gene products have been deleted have also been developed (D. Trono and T. Kafri, personal communication). The improved minimal lentiviral vectors can efficiently infect most cell types tested (FIG. 2). Some investigators have used non-human lentivirus- es, such as simian immunodeficiency virus, feline immunodeficiency virus and equine infectious anaemia virus, to generate efficient vectors capable of transduc- ing non-dividing cells 26?29 . There are no clinical trials with lentiviral vectors at present, but several groups are Figure 1 | Retrovirus-based vectors. a | The retroviral genome contains the gag, pol and env genes. The ? sequence is the packaging sequence (in red) that differentiates the viral RNA from all other RNA in the cell, and is recognized by the viral proteins for packaging. b | The vector genome. The gag, pol and env genes are replaced by the therapeutic gene. c | The packaging cell. The gag and pol genes are separated from the env gene, making regeneration of a replication competent virus unlikely. The retrovirus is a membrane-bound virus, and the Env protein is expressed on the cell surface. The vector genomes, by virtue of the ? sequence, are encapsulated along with the Pol and Gag proteins and are assembled under the membrane. The virus buds off from the packaging cell, resulting in the retroviral vector, which is used to infect the target cell. Animated online Figure 2 | In vivo gene delivery into mice and rats using lentiviral vectors. The gene coding for green fluorescent protein (GFP) was placed into lentiviral vectors, and these were injected into various tissues. a,b | Efficient gene transduction (green cells) to a | liver, and b | muscle. c,d | Different promoters driving transgene expression. c | The cytomegalovirus (CMV) promoter or d | the rhodopsin promoter driving GFP expression. Note the high-level expression of GFP in the retinal pigment epithelium (RPE) with the CMV promoter, and in photoreceptor cells with the rhodopsin promoter. e,f | A third-generation SIN lentivirus vector expresses GFP under the control of the CMV promoter e | in the retina and f | in the brain. ONL, outer nuclear layer; INL, inner nuclear layer. (See REFS 19,77.) KARYOPHILIC Literally, attracted to the nucleus ? a nuclear localization signal in a protein is karyophilic. TROPISM The range of cells that can be productively infected by a virus. CAPSID The proteinaceous coat surrounding a virus. � 2000 Macmillan Magazines Ltd 94 | NOVEMBER 2000 | VOLUME 1 www.nature.com/reviews/genetics REVIEWS liver and brain 30 . This long-term expression results from randomly integrated vectors and some vector DNA that persists as extra-chromosomal DNA. At present, it is not clear what proportion of expression originates from integrated or extra-chromosomal DNA 36 . The main problem with AAV vectors is that the rep gene and some of the adenoviral helper genes are cyto- static and cytotoxic to the packaging cells, and so it has been difficult to scale up production of these viral vec- tors. No cell lines have been reported that stably pro- duce high-titre AAV vectors carrying a therapeutic gene. Current clinical trials with AAV vectors rely on transient production systems, which may be suitable for proof of principle, but a more efficient production sys- tem is urgently required 37 . Another problem is the cod- ing capacity of the vector, which is restricted to around 4.5 kilobases. However, two groups have extended the packaging capacity of these vectors by using the obser- vation that AAV genomes concatomerize after transduc- tion. When two vectors, one encoding the first half and the other encoding the second half of a protein, were transduced into cells, head-to-tail stitching of the viral genomes resulted in the reconstitution of a functional gene, effectively increasing the size of the gene that can be delivered. It remains to be seen if concatomerized vectors will be stable and have sustained expression 38,39 . Adenoviral vectors. The adenoviruses are a family of DNA tumour viruses that cause benign respiratory tract infections in humans 40 . The genome contains over a dozen genes, and on infection the virus remains episo- mal in the nucleus, and can transduce genetic material into both dividing and non-dividing cells. Replication- defective recombinant adenoviral vectors can be gener- ated at high titres by deleting several viral genes, includ- ing E1A, E1B, E3, E4 and E2A (REF. 41). Most recently, ?gutless? adenoviral vectors (in which all the viral genes are removed and provided in trans) have also been gen- erated 42,43 . All adenoviral vectors retain the ability to transduce dividing and non-dividing cells efficiently, and it is relatively easy to generate high-titre commer- cial-grade recombinant vectors. The challenge for adenoviral vectors concerns the persistence of expression of the transgene. All adenoviral vectors so far, with the exception of gutless vectors (for which data are still preliminary) 44 , express the transgene in adult animals for only a short time (between 5 and 20 days post-infection) 45 . In immuno-compromised ani- mals, expression in long-lived cells, such as muscle cells and neurons, is observed for long periods. It is now gen- erally recognized that the short-term expression from recombinant adenoviral vectors is because of the immune response (see below). These vectors will, how- ever, continue to be used in situations in which high- level but transient expression of the foreign gene is required, for example in RESTENOSIS and cancer. Immune response: the bane of gene therapy The biggest challenge facing all viral vectors is the immune response of the host. The host defence mecha- nism functions at both the cellular level, by generating The viral vector is produced by replacing the rep and cap genes with the therapeutic gene. The Rep and Cap proteins are produced in trans in the packaging cell, as are the adenoviral proteins needed for replicating the virus (FIG. 3). AAV can integrate in a site-specific location on chromosome 19 through the action of the Rep pro- tein. However, as the vector does not code for Rep, it does not have this highly desirable attribute after entry into the target cell 32 . Another interesting feature of AAV vec- tors with respect to chromosomal integration is their propensity for homologous recombination. The vector has been shown to correct point mutations and deletions in selectable reporter genes integrated into the chromo- some, although this occurs at a very low frequency 33 . The cell tropism of the virus, and hence the vector, is relatively broad, and most cell types are receptive to gene transfer to some extent. Efforts are underway to define the receptors and residues in the capsid proteins responsible for interaction with the cell surface, and this could lead to the design of new capsids with restricted or targeted tropism 34,35 . When tested in mice, dogs and monkeys, gene expression from AAV vectors is sus- tained in tissues with long-lived cells, such as muscle, Figure 3 | Adeno-associated viral vectors. a | The adeno-associated virus (AAV) genome contains the sequences essential for transduction ? the inverted terminal repeats (ITRs) and the genes rep and cap. b | In the vector genome, rep and cap are replaced by the therapeutic gene. c | The Rep and Cap gene proteins provided by the packaging constructs are required to produce single-stranded DNA genomes that are encapsulated by the coat proteins. AAV ? a non-enveloped virus ? is assembled in the nucleus. The helper proteins from adenovirus (Ad) needed for replication (E1A, E1B, E2A, E4orf6 and VA RNA) are not shown here. Ad replication is lytic, and this is the exit route for AAV. It has been difficult to separate Rep and Cap coding regions and still get production of high titre virus. As a result generation of replication competent virus is still possible. However, the target cell still needs helper functions from adenovirus for replication of the replication competent virus. RESTENOSIS Stenosis is the blocking of a blood vessel that can be cleared by mechanical disruption. Restenosis is the recurrence of the blockage caused, for example, by unchecked proliferation and migration of vascular smooth muscle cells. � 2000 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 95 REVIEWS pathology, in the context of a genetic background that influences their immune response. The Gelsinger tragedy Jesse Gelsinger was barely 18 years old when he became the first patient to die in a Phase I gene therapy clinical trial. Although many patients have experienced severe adverse effects and even death during Phase I safety and toxicity studies, Jesse was the first patient in whom death could be directly attributed to the vector ? an adenoviral vector. Jesse suffered from deficiency of ornithine transcar- bamylase (OTC), a metabolic enzyme required to break down ammonia. The total lack of this enzyme leads to death shortly after birth, owing to a build-up of ammo- nia. The partial presence of enzyme activity also leads to an accumulation of ammonia, but can be controlled by drugs and dietary intake. The aetiology of the disease, its associated morbidity, and the need for rapid production of the enzyme suggested that transient production of OTC by adenoviral vectors could extend the lifespan of OTC-deficient newborns, to allow implementation of a drug and dietary regime 54 . The Phase I trial consisted of a study in which a cohort of patients with partial OTC activity were given escalating doses of second-generation (deleted for the E1 and E4 genes) adenoviral vectors. Jesse was in the last cohort, receiving up to 6 x 10 13 recombinant adenovirus- es particles containing the OTC gene. Within hours of intra-hepatic administration, he began to experience severe complications and died two days later. What went wrong? Was too much virus infused? Perhaps not, as another patient getting the same dose of the same vector did not suffer the same consequences. Were Jesse?s aden- oviral antibody titres higher? Again, there is no clear answer, as other patients with higher antibody titres did not have the same reaction. Perhaps there were other mitigating causes, like other viral infections or higher lev- els of ammonia before vector transduction. Are the ani- mal models really reliable? Should we be screening patients for genetic variations, as immune responses are so heterogeneous? These are just some of the questions that have been raised, and several expert committees are now in the process of defining and refining new mea- sures for gene therapy trials (LINKS). The first successes The field of gene therapy also has cause to celebrate. Alain Fischer and colleagues in Paris have suc- cessfully treated three young babies (1?11 months old), who suffer from a fatal form of severe combined immunodeficiency (SCID) syndrome 55 . SCID-XI is an X-linked disorder characterized by an early block in T- and natural killer (NK) cell differentiation, due to muta- tions of the gene encoding the ?C cytokine receptor subunit common to several interleukin receptors. A mutation in the ?C subunit leads to disruption of sig- nals required for growth, survival and differentiation of lymphoid progenitor cells. Haematopoietic stem cells from the patients were transduced ex vivo with a recombinant mouse cytotoxic T cells, and at the humoral level, by generating antibodies to viral proteins. Cellular immunity elimi- nates the transduced cells, whereas humoral immunity precludes the repeat administration of the vector because the subsequent antibody response will be boosted by MEMORY CELLS 45,46 . The host immune system may also recognize the transgene product as foreign, and induce both cellular and humoral immunity 47 . To minimize the cellular response, most vectors have been designed to prevent the synthesis of viral proteins following transduction. However, adenoviral vectors present a unique problem, because even the inactivated recombinant adenoviral vectors can elicit potent cyto- toxic T-cell responses against viral proteins 46 . Therefore it is difficult to see how gutless vectors, which still require the full complement of viral structural proteins for efficient transduction, can bypass this host immune response. The humoral response is also most pertinent to adenoviral vectors because they do not integrate, and so suffer loss by cell division and by DNA degradation, necessitating a repeat infection with the vector. The host raises neutralizing antibodies against viral proteins, thereby precluding any further infection. As there are scores of adenoviral SEROTYPES, one strategy to overcome this problem might be to use different serotypes 40 . Retroviral, lentiviral and AAV vectors do not seem to suffer from cytotoxic-T cell responses. It could be that the vectors are completely replication-defective, and that the incoming viral proteins do not elicit a cytotoxic T-cell response. Alternatively, the titres of at least the recombi- nant retro- or lentiviral vectors tested so far might not be sufficiently high to elicit an immune response. Antibody responses are also less of a concern, as retro- and lentivi- ral vectors integrate into the host genome and may not require subsequent transduction. Furthermore, for vec- tors engineered with the VSVG protein, there are several strains of VSV that have different serotypes 48 . Antibodies to AAV-based vectors have been detected, which has pre- vented transduction by a second injection of the vector 49 , but this may also be overcome by using another serotype of AAV 50 . Why some vectors are more immunogenic than oth- ers is a matter of considerable interest, and early hints indicate that antigen-presenting DENDRITIC CELLS may be important. In contrast to AAV-based vectors, it seems that adenoviral vectors efficiently transduce dendritic cells 51 . The route of administration also influences the immunological outcome 52 , and there is the question of pre-immunity in the host. Over 70% of the population may be carrying antibodies to adenovirus and AAV. What function might these pre-existing antibodies have in the efficiency of transduction or the toxicity of viral vectors? Are there sites in the body where the humoral response can be bypassed by the introduced vectors? Finally, the transgene itself may be highly immunogenic, particularly in hosts in which the trans- gene product was never made, due to either complete gene deletion or aberrant expression 53 . So, the gene ther- apy strategy in such hosts will also require the induction of TOLERANCE. Ultimately, individual patients may well require a therapeutic regimen tailored to their specific MEMORY CELLS Immune cells that are primed, after an initial exposure to an antigen, to make a rapid response to subsequent exposure to the same antigen. SEROTYPES Antigenically distinct forms that elicit different antibody responses by the immune system. DENDRITIC CELLS These cells present antigen to T cells, and stimulate cell proliferation and the immune response. TOLERANCE The lack of an immune response to a specific foreign protein. � 2000 Macmillan Magazines Ltd 96 | NOVEMBER 2000 | VOLUME 1 www.nature.com/reviews/genetics REVIEWS preparation with enzymatic activity) was administered to the patients in addition to the vector expressing the ADA gene. This may have prevented the selective advan- tage observed in the successful French trial 56?58 . The use of modified MLV vectors and the extensive manipula- tion of the stem cells (use of cytokines to stimulate cell proliferation) before transduction are a testimony to the continuous and incremental progress made in the field. We believe that with the availability of lentiviral vectors capable of transducing resting stem cells, the efficiency of transduction will improve even further 59,60 . Haemophilia (A and B) is another excellent model system for gene therapy because the deficient protein does not have to be provided from its normal cellular source. Therefore, several vectors have been designed that transduce a range of cells to produce and secrete factor IX protein. Both the factor IX knockout mice and haemophilic dogs have turned out to be extremely ben- eficial pre-clinical model systems 53,61,62 . Further model systems will continue to be useful for pre-clinical stud- ies, and promising results have recently been obtained in a mouse model of ?-thalassaemia 63 . In another exciting human study, Kathryn High and colleagues at the University of Pennsylvania have treated several haemophilia B (factor IX-deficient) patients in a Phase I clinical trial with AAV vectors that contain the human factor IX gene 37 . The recombinant vector was injected intramuscularly, and preliminary results indicate that factor IX protein can be found in the serum of a patient. Although the levels of the factor IX protein expected to be produced by the low doses of injected AAV are not curative 44 , the treated patients did show some clinical benefits. No factor IX inhibitors were found, but neither could they be expected, because very low amounts of factor IX were being secreted. This is still a preliminary study, but nevertheless it bodes well for success in treating haemophilia. The next phase It was not long ago that the ?battlecry? of the gene thera- py community was ?titres, titres, titres?. Then it switched to ?delivery, delivery, delivery?, and now it is ?expression, expression, expression?. We have the appropriate titres of desirable vectors for delivering genes to patients. The emphasis now is on efficiency, safety and duration of expression. The issue of safety will always remain pre- dominant, and the trend is to generate ?minimal vectors? carrying the least amount of viral information needed for successful transduction. Significant progress in vector development is occur- ring in the area of tissue- or cell-specific expression. For example, there have been encouraging advances in the targeting of vectors to specific tissues. The ideal here is that the original tropism of the virus is ablated and a new specificity generated. To this end, two strategies have generally been pursued. The first strategy involves engineering viral proteins responsible for binding the cellular receptors that subsequently mediate viral entry. For retroviruses, these are the envelope proteins. They have been altered to add new binding domains to the envelope, and although this does target leukaemia viral vector containing the ?C receptor gene and infused back into the young patients. After ten months, ?C transgene expression in T- and NK cells was detected in the patients but, more importantly, T-, B- and NK-cell counts and function were comparable to those of age-matched controls. To all appearances, the recipients are clinically cured, and the fantastic promise of gene therapy is realized. Some concerns remain: only ten months of data are available, and expression of the transgene may be ?shut-off?. Also, very few patients have been treated so far. It remains to be seen if this approach will work for other diseases, because the success with SCID-XI is probably owing to the strong selective advantage provided to the transduced lymphoid prog- enitors. Only those haematopoietic cells that express the ?C receptor subunit can survive and differentiate. In earlier trials of SCID patients suffering from adenosine deaminase deficiency (ADA), PEG?ADA (a protein Figure 4 | Regulation of gene expression. a | The Tet system. Two expression cassettes need to be delivered to the target cell. One expresses the tTA protein, which either binds to tetracycline (red) or the tet operator (tetO, yellow), and the other expresses the therapeutic gene under the control of tetO. Gene expression is conditional on the binding of tTA to tetO, and hence on the absence of tetracycline. b | A dimerization-regulated system. Three expression constructs need to be delivered to the target cells that code for a DNA-binding domain, a transactivation domain and the therapeutic gene under the control of operator sequences of the DNA-binding domain. The DNA-binding domain binds to the operator sequence, but cannot trigger gene expression without the transactivation domain. These two domains can be dimerized by a ligand, and the dimer activates gene expression. � 2000 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 97 REVIEWS activates transcription. This mutant has been engi- neered with a DNA-binding domain from yeast (GAL4) and a transcriptional transactivation domain from a viral protein. Activation of a transgene downstream of the GAL4-binding sequence is conditional on the pres- ence of RU486, which can be administered orally. However, RU486 is not inert ? it is an antagonist of the progesterone receptor and is used as a method of birth control. Variants of this system use a steroid hor- mone receptor from a different organism ? for exam- ple, the ecdysone receptors of insects such as Drosophila 73 and Bombyx 74 . In the presence of the insect hormone ecdysone, expression of the receptors in target cells activates transcription of genes placed downstream of ecdysone receptor DNA-binding sites. Agonists of these receptors, such as muristerone A, are commercial- ly available and seem to be non-toxic 75 . Finally, a synthetic dimerizer strategy 75 has been successfully used in vivo to produce regulated amounts of erythropoietin 76 in an AAV vector system. The system involves ligand-binding sites on two proteins that can be brought together by a small molecule, resulting in dimerization (FIG. 4). The pro- tein FKBP (FK506 binding protein) can be linked to a transactivation domain (FKBP?TA) and a DNA-bind- ing domain (FKBP?DB). The target gene is linked to DNA-binding sites specific for the DNA-binding domain. The FKBP?DB will bind to the DNA-binding sites, and in the presence of the linker (a synthetic dimer of FK506, FK1012), FKBP?TA will form dimers with FKBP?DB and turn on transcription of the reporter gene. The immunogenicity of these proteins requires further investigation. Perspectives The last two decades have witnessed the birth of the field of gene therapy, which has generated great hopes and great hypes. The promise of influencing the out- come of a vast array of diseases, ranging from birth defects to neurological disorders and from cancer to infectious diseases, although far-reaching, is not beyond reach. With the completion of the sequence of the human genome, over 50,000 genes will be available to the practitioners of gene therapy. The potential benefits the binding of the retroviral viral vectors to the desired cell type, it has been difficult to engineer a high-titre tar- geted vector. There is a coordination of binding and subsequent fusion between the virus and cell mem- branes, which has been difficult to achieve for the modi- fied envelopes, resulting in low titres 64 . This type of engineering has also been reported for adenoviral 65 and for AAV vectors 35 , and although there can be a loss of titre for these vectors, the approach is promising. The second strategy relies on adaptor molecules that bridge the virus and the cell, and has been applied to retrovi- ral 66 ,adenoviral 65 and AAV vectors 67 . The adaptors are modular and comprise a component that binds the virus (either a fragment of the cellular receptor or part of a monoclonal antibody) and a component that rec- ognizes the target molecule (a ligand or antibody). However, generating these vectors at high titres and testing their utility in vivo are problems that need to be overcome. Although tissue- and cell-specific expression will continue to command interest, we suspect that regulat- ed expression of the transgene will become an impor- tant focus for practitioners of gene therapy. Fortunately, several promising systems are available and are being explored. All are based on the idea that the expression of the therapeutic gene can be regulated by an inducible, co-expressed transcription factor. The induction should be reversible, the inducer must be non-toxic (and preferably active on oral administration), and the inducer should not activate other genes. Furthermore, the components of the regulatory system should not be immunogenic in the host. The most widely used regulation system is based on bacterial tetracycline resistance regulation (the Tet sys- tem). The bacterial protein, TetR, binds to its target DNA, the tet operator (tetO), only in the absence of tetracycline or its non-antibiotic analogue, doxycycline (Doc). Bujard and colleagues 68 have engineered TetR and appended it to a eukaryotic transcriptional activa- tor (tTA). The transgene, downstream of tetO, has to be delivered to the target cell along with the coding sequence for tTA. In the presence of tetracycline, the tar- get gene is ?off?, and in the absence of tetracycline, the target gene is ?on? (FIG. 4, FIG. 5). This system has also been extended, by ingenious manipulation, to generate rtTA, which binds DNA and activates transcription in the presence of tetracycline 69 . Modulation of the amount of tetracycline also modulates the amount of protein pro- duced, and if tTA is produced from a tissue-specific pro- moter, this can enable spatial control of gene expression. As tTA is based on a bacterial gene, it may be recognized as foreign by the human immune system, but so far it has been tolerated in animal models. It has proved to be applicable to a wide variety of systems, including those using AAV and lentiviral vectors 70,71 . A second system is based on steroid hormones and relies on the observation that binding of the hormone to its receptor activates gene transcription. An early sys- tem was based on a truncated version of the proges- terone receptor 72 . This mutant receptor binds proges- terone antagonists, such as RU486, and, paradoxically, a b Figure 5 | Regulated expression of a gene therapy vector. Regulated expression is illustrated with a lentiviral vector in which the gene coding for green fluorescent protein (GFP) is under the control of the Tet system. GFP expression in the brain is a | predominately ?off? in the presence of tetracycline and b | ?on? in its absence 71 . � 2000 Macmillan Magazines Ltd 98 | NOVEMBER 2000 | VOLUME 1 www.nature.com/reviews/genetics REVIEWS to accepted guidelines is incumbent on all investigators participating in clinical trials, and those wilfully violat- ing the recommended practices will have to pay the con- sequences. The field of gene therapy has also been rocked by charges of conflict of interest, an area relative- ly new in biomedical science. Harmonized guidelines need to be put in place to allay the public?s concern of real or perceived conflicts of interest. The science of gene therapy has many hurdles ahead, but they are sur- mountable. for human health are vast, so how can the biomedical community move forward to realize this potential? Geneticists will continue to identify the genetic con- tribution to disease. Virologists will generate safe and efficient viral vectors, and molecular biologists will help to design vectors capable of cell- and tissue-specific expression of the foreign genes carried by the transduc- ing vectors. Immunologists will work out ways to pre- vent unwanted immunological consequences of the delivery vehicles and their cargo. Cell biologists will devise ways to facilitate gene transfer to various tissues and will take the lead in identifying stem cells. Clinicians will carry out clinical trials on humans with the best vectors that the scientists can supply. To achieve success- ful gene therapy, all branches of biology will have to contribute to this endeavour. Society has an enormous stake in science, and scien- tists have an obligation not to promise more than they can deliver. Gene therapy is a young science that has undergone extreme scrutiny in the recent past. 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A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl Acad. Sci. USA 93, 13565?13570 (1996). References 42 and 43 describe the generation of new adenoviral, ?gutless?, vectors in which all the viral genes required for viral propagation are provided in trans. The gutless vectors show long-term expression of the transgene. Links DATABASE LINKS CD4 | OTC | OTC gene | SCID-XI | ?C cytokine receptor subunit | ADA | ADA gene | Haemophilia A | Haemophilia B | factor IX | factor IX knockout mice | haemophilic dogs | ?-thalassaemia | ecdysone receptor FURTHER INFORMATION NIH recombinant DNA advisory committee meeting 8?10 March, 2000 | The Institute for Human Gene Therapy | Verma laboratory homepage � 2000 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 99 REVIEWS 44. Morral, N. et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc. Natl Acad. Sci. USA 96, 12816?12821 (1999). 45. Dai, Y. et al. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc. Natl Acad. Sci. USA 92, 1401?1405 (1995). 46. Kafri, T. et al. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc. Natl Acad. Sci. USA 95, 11377?11382 (1998). Even physically inactivated adenoviral particles can generate a cytotoxic T-cell response, raising concerns about adenoviral vectors as suitable tools for long-term gene therapy. 47. Tripathy, S. K., Black, H. B., Goldwasser, E. & Leiden, J. M. 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