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50 Fearon, E. R., Pardoll, D. M., Itaya, T., Golumbeck, P., Levitsky, H. 1., Simons, J. W., Karasuyama, H., Vogelstein, B. and Frost, P. (1990) Cell 60, 397~03

51 Golumbeck, P. T., Lazenby, A. J., Levitsky, H. I., Jaffee, L. M., Karasuyama, H., Baker, M. and Pardoll, D. M. (1991) &ieme 254, 713~16

52 Esumi, N., Hunt, B., ltaya, T. and Frost, P. (1991) Cancer Res. 51,1185 1189

53 P,.osenberg, S. A. (1992)./. Am. Med. Assoc. 268, 241~2419

54 Culver, K. W., Ram, Z., Wallbridge, S., lshii, H., Oldfield, E. H. and Blaese, tZ. M. (1992) &ieme 256, 1550-1552

55 Rosenberg, S. A., Spiess, P. and Lafreniere, R. (1986) Science 233, 1318 1321

56 Friedmann, T. (1991) Cancer Cells 3, 271-274

57 Hdl~ne, C. (1991) Eur. J. Cancer 27, 1466 1471

58 Weerasinghe, M., Licm, S. E., Asad, S., Read, S. E. andJoshi, S. (1991)./. Virol. 65, 5531-5534

59 Gage, F. H., Wolff, j. A., Rosenberg, M. B. (1987) Neuwscience 23, 795 807

60 Wolff, J. A., Fisher, L. J., Xu, L., Jinnah, H. A., Langlais, P. J., luvone, p. M., O'Malley, K. L., Rosenberg, M. B., Shimohama, S., Friedmann, T. and Gage,

F. H. (1989) Proc. Natl Acad. &i. USA 86, 9011-9014

61 P, osenberg, M. B., Friedmann, T., Robertson, R. C., Tuszynski, M., Wolff, J. A., Breakefield, X. O. and Gage, F. H. (1988) Science 242, 1575 1578

Theodore Friedmann Cemer f~r Molecular Genetics 0634,

Departmeut of Pediatrics, UCSD School of Medicim,,

La ffolla, CA 92093, USA.

From genome mapping to gene therapy

The origins of genome mapping go back a long way, and involve our ability to follow the inheri tance of different variants o f a gene or gene product, or a disease, or any other genetic marker, through a family. This process is k n o w n as segregation analysis. Unt i l 15 years ago, human geneticists had to rely on the rela- tively few markers available for map- ping, such as blood groups, colour blindness, or protein variants. W h e n Kan and Dozy I found a variant in the D N A sequence sur rounding the ~-g lobin gene, which segregated with the sickle-cell muta t ion, Solomon and Bodmer , o f the U K Imperial Cancer Research Fund ( ICRF) , first poin ted out that the direct study of the variation of human D N A sequences represents a powerful new mapping tool 2. These brief prescient papers laid the basis for the studies that led to the inception of the h u m a n genome project (HGP), in which the entire h u ma n genome will be mapped, character- ized functionally, and eventually sequenced (Box 1).

W h e n we first cloned the human globin genes 4, we realized that c loning was not only a method of amplifying a h u m a n gene, and of allowing it and its surrounding con- trol regions to be sequenced, bu t also that every cloned gene is specific to each individual and contains the par- ticular sequences which encode most o f the inherited features that deter- mine our genetic individuality. Although the sequences that we each

inherit are unique - with the excep- t ion of identical twins, there are about five mil l ion differences be tween the D N A sequences of any two people - the general order and stmcture of genes hardly vary at all be tween individuals. Mapping is therefore possible by combin ing data from all families with, for example, a particular disease.

Reverse genetics The combina t ion of the person-

specificity of cloning and the general application of mapping led to reverse genetics, or (or to use a more accu- rate term) positional c loning s. Pos- i t ional c loning first defines the position of a muta t ion [such as that causing D u c h e n n e muscular dys- trophy (DMD) or cystic fibrosis (CF)] using genetic linkage: data from hundreds of affected families are used to position the gene accurately on the genetic map. T h e n thc sequence of D N A between the nearest genetic markers (which segregate most re- liably with the disease), usually several hundred thousand base pairs in length, is isolated and the genetic and physical maps are superimposed. D N A sequence data show the n u m b c r and range of alleles that are present, and the mutat ion causing the disease is tentatively identified by a combina t ion of in tu i t ion and the study of the variability o f the genome.

The most dramatic feature about the mutated genes that cause D M D and CF, however, is that they were

discovered by reverse genetics nei ther of the corresponding pro- teins, dystrophin in the case of D M D , and the cystic fibrosis t ransmembrane conductance regulator (CFT1L) in the case of CF, were k n o w n to exist before they were isolated by Kunkcl ~' and Tsui 7, respectively.

Most of the mutat ions that cause severe and c o m m o n diseases (called Mendelian, as they are inherited by the simple Mendel rules) have now been identified. In the case o f CF, the disease is rarc, bu t nevertheless severe (affected individuals have an average life span o f - 2 0 years), and over 90% of carriers in the U K know of no family history. Carrier testing and prenatal diagnosis in families with a k n o w n 1 in 4 risk of having an affected child is an obvious and essential strategy for decreasing the disease incidence (even if no t all families will agree to it). Screening the broader com m un i ty is more problematic, although the use of PCP- on D N A obtained from a mouthwash, or from single hairs, makes genetic testing cheaper and easier 8. It may be that a battery of tests for carriers o f a set o f severe inheri ted diseascs could soon be made available to any individual, but this would have to be coupled with education and counselling.

C o m m o n multifactorial diseases An exciting prospect must be the

possibility of dissecting the entire hum an genome to find the genes which predispose an individual to c o m m o n diseases such as cancer, coronary artery disease and

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Box 1. The Human Genome Project

The Human Genome Project is a co-ordinated major international research effort with the ultimate goal, by the year 2005, of having sequenced the entire human genome and identified the estimated 100000 human genes 3.

As soon as a complete human 'generic' genomic sequence, derived by drawing on data from many individuals, is available, together with compar- able data from experimental animal models, it will be possible to ascribe biological functions to many specific genes. Finding genes that can cause disease will be a high priority, due to the cost and difficulty of DNA sequenc- ing, particularly of repetitive regions of the genome. A major feature of the HGP is that it can be divided up among different research groups, with indi- vidual laboratories focusing on specific chromosomes or chromosomal regions.

At an international level, the HGP is co-ordinated loosely by the Human Genome Organization (HUGO), whose role is to promote co-operation among investigators who work on genome projects. HUGO itself does not control research funding, most of which comes from national bodies such as the Medical Research Council (MRC) in the UK, and the National Institutes of Health (NIH)in the USA, or medical charities such as Genethon in France.

Collaboration and free exchange of data should increase efficiency and help reduce redundancy of effort. Progress on diverse research fronts is helped by the development of strategies incorporating milestones over the next decade or so. However, immense technological advances in mapping, sequencing and data-handling ability have already led to a revision of the dates by which it was anticipated that each specific goal would be achieved.

Alzheimer's disease. These diseases are sometimes Mendelian, but more often are caused by a combination of genetic predisposition and environ- mental factors (i.e. are multifactorial), or even due solely to environmental factors. Consider heart disease. A very small proportion of cases (i.e. type III hypercholesterolaemia) are due to a deletion or mutation of the gene encoding the low density lipoprotein (LDL) receptor, which imports cholesterol into the cell. An even smaller number of cases are due to a defect in the gene for the carrier protein apolipoprotein B. These cases are usually so severe that they resist environmental manipulation and require pharmacological or surgical treatment. A few cases of heart diseases are due to gross en- vironmental abuse - the individual w h o drinks a lot of whisky, eats lots of butter and smokes, can overcome a healthy genetic inheritance! Most heart attacks, however, are due to a mixture of genetic and environmen- tal causes, i.e. there is a genetic pre- disposition to heart disease, which requires environmental triggers, or abuse, for it to become apparent.

For diseases such as atherosclerosis, hypertension and cancer, the role of the HGP and of the genetic testing which wilt ultimately result will be to point to specific risks, thus enabling the individual or the clinician to modify the environment, or to inter- vene to lower the risk pharmacologi-

cally or through somatic gene ther- apy. Genetic testing, in this context, is essentially a 'green' technology. It is inexpensive, easy to apply in any country, and it can help to make the environment safer and healthier in a personal way for each of us, taking into account our essential individu- ality and choice, without modifying our genetic heritage.

G e n e t h e r a p y - f e a s i b i l i t y a n d ethics

Ultimately, somatic gene therapy will be added to the choices appro- priate to those individuals at high risk of any serious disease. Every week there is another feature in the press or on television discussing gene ther- apy. Sometimes it is described in terms of its potential to treat genetic disorders in a new and more effective way. Rarely, its wider potential to deliver genes for multifactorial con- ditions, such as cancer or heart dis- ease, is discussed. Invariably, the 'ethical dilemmas', that phrase so beloved of subeditors, appears as a bold heading. Is this new-found optimism justified, or is it 'flavour of the month' , a mixture of hype by the practitioners (who stand to win large research grants), the biotechnology companies (which might otherwise languish in the corporate stakes), the large numbers ofethicists now com- peting for a public platform on which to air their personal views, and the journalists (whose favourite

words seem to be 'cure' and 'break- through')?

I declare a bias immediately; I am a practitioner, but I also think that thc optimism is justified as a result of several new developments 9. Pilot studies in which the mutated gene for adenosine deaminase (ADA) is replaced with a normal one, in the lymphocytes of several children affected by severe com- bined immunodef ic iency (SCID) have shown a clear clinical improvement . N e w studies show that targeting normal genes using viral and other systems to enter cells (from bone marrow, the lung and gut, liver and skin) may be easier than at first thought, and that D N A will function more or less normally once it is introduced into the cell. Further data show that the risk of causing harm by inactivating an essential gene in the host cell, or by recombining to form a pathogen, seems low (although we cannot yet say how low). Finally, there is a growing consensus (epitomized by the U K Clothier Commit tee Report) t° that it is ethical to use gene therapy in a variety of somatic ways, but not in gametes or embryos, and indeed that gene therapy may provide a new, general enabling technology for protein and drug delivery, as well as for gene therapy, through the intro- duction of normal genes.

It is necessary to know the gene (and the mutation) before even thinking of gene therapy. If a dis- ease is due to a muta t ion in o n e gene, it may be 'dominant ' (i.e. it may occur when only one of the two copies of the gene is mutated, despite the presence of a normal gene) or 'recessive' (i.e. it occurs only when both alleles are defec- tive). Clearly, it will be easier to treat a recessive disease than a dominant one with gene therapy, since it is only necessary to intro- duce a normal gene and not to remove the mutated copy. H o w - ever, this reasoning breaks down in some cases - for instance, in the form of hypercholesterolaemia that is due to a defect in the LDL recep- tor, the pathology is quantitative, and introducing a normal gene may improve prognosis, even in adults where the disease is dominant . What may prove almost impossible to treat are diseases involving gene mutations which cause a pathologi- cal change during early fetal devel- opment (as in Down ' s syndrome).

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Del iver ing the gene Thanks to the H G P and related

advances, cloning a normal human gene is no longer a problem; about 10000 o f the coding sequences (and the cor responding larger genomic sequences) are n o w avail- able. However , in t roducing these into human cells in a ffmctional form is more difficult. Several approaches have been proposed; the most advanced technology involves the use ofretroviruses and adenoviruses that have been engin- eered for safety, and to accept human genes.

Are these viruses, which in their unmodif ied form can cause any- thing from colds (adenovirus) to cancer (mammary t u m o u r virus), truly safe? Probably yes, but it is not possible to be absolutely certain o f safety in this context; the field is too new, and since t reatment for some diseases may have to be repeated many times, even an unlikely event may eventually happen. The viruses are inactivated by removing most o f their pa thogenic se- quences, but they could recombine with wi ld - type viral sequences ei ther dur ing preparat ion o f the vector in cell culture, or with endogenous viral scqucnces in the patient. It is not clear whether any- thing worse than the natural virus might result. Clearly, unti l these questions are answered, gene ther- apy will be reserved for l ife-threat- ening diseases.

Other approaches, such as the use of human min ichromosomes , and the combina t ion o f the best features o f the different viral and biochemical approaches, are under study and may offer the best long- term prospects. In addit ion, it must be r emembered that access to a specific tissue is often the most difficult part o f a proposal; there are many neurological condi t ions where gene therapy w o u l d be attractive i f parts o f the central nervous system were selectively accessible.

Targeted therapy Several different tissues may have

to be targeted specifically for some diseases - in the case o f CF, the lung is the natural first target, since it is severely affected and accessible, but the gut should also be treated. Target ing gut epi thel ium is also attractive for o ther diseases. For instance, it nfight be possible to introduce genes coding for specific

receptors to alter the s tomach or intestinal l ining and thus provide t reatment for ulcers. The possibility o f in t roducing tumour-suppressor genes into those individuals wi th a predisposi t ion to cancer o f the colon is undoubted ly one o f the most excit ing prospects.

Many single-gene inher i ted dis- eases are due to mutat ions which affect hepatic function. This is the case for the haemophil ias , phenylke tonur ia and many o f the disorders o f cholesterol metab- olism. Genes can be integrated into hepatocytes by targeting via trans- ferrin receptors, which are abun- dant in the liver. A single lobe o f the liver can be removed, disaggre- gated, the gene in t roduced in vitro, and then the cells ' re -seeded ' into the hepatic vessels f rom whence they will repopulate the regenerat- ing tissue. This would , o f course, only be just if ied for serious disease, but if more benign targeting tech- niques could be devised, it might be possible to use liver as a target tissue for any b iochemica l inter- vent ion to modify the level o f a b lood metabolite.

Skin cells from any individual can be grown in vitro; great advances have been made in this area because o f the interest in skin grafting, and the fact that fibroblasts do not readily transform to cancer cells in culture. A human gene can be inserted into fibroblasts or ker- atinocytes using a retroviral vector, and the skin then grafted back to the donor. This has been at tempted, with some success, using the gene for human Factor IX, although synthesis o f the p ro - tein continues for only a short time. The long- te rm significance o f using fibroblasts and skin grafting will come if it is possible to insert genes coding for polypept ide hor - mones, together wi th the associ- ated sequences which al low response to physiological controls: for instance, inserting the entire normal insulin gene into fibroblasts and hop ing that there will be a physiological insulin response.

Gene therapy is coming, and soon, for serious disease. It is here already for A D A deficiency, a form o f SCID, where the gene is cloned, the affected cell is easy to access, and the biochemistry is well under- stood. O the r single-gene disorders, such as CF and haemophi l ia B, will fol low relatively quickly. H o w - ever, the true exci tement will be in

the novel use o f the technology to enable t reatment o f the c o m m o n multifactorial diseases, even those not due pr imari ly to a genetic defect, by genetic means at the somatic level. The obvious ex- amples are hypercholesterolaemia, cancer o f the colon and auto- immune disease; the t ime to begin th inking o f these approaches is now. A n d while the ethical con- cerns are real enough, they are not novel in medic ine or in tech- nology, and are wor th confront ing for the benefits that will come to patients and the communi ty .

A c k n o w l e d g e m e n t s The research o f the Genet ic

Therapy group o f St Mary 's Hos- pital Medical School, Imperial College London is suppor ted by the Medical Research Counci l , the Shin-Etsu Institute W i t h o u t Walls, and especially the Mul ler Trustees. I thank m y colleagues Charles Coutel le , Natasha Caplen, Steve Hart and Clare Huxley for their help in formulating these ideas.

References 1 Kan, Y. W. and Dozy, A. M. (1978)

Proc. NatI Acad. Sci. USA 75, 5631-5635

2 Solomon, E. and Bodmer, W. F. (1979) Lancet 1, 923

3 Genome Mapping and Sequencing (1992) Trends Biotechnol. 10, t-73

4 Little, P. F., Curtis, P., Coutelle, C., Van den Berg, J., Dalgleish, R., Malcolm, S., Courtney, M., Westaway, D. and Williamson, R. (1978) Nature 273,640-643

5 Collins, F. S. (1992) Nature Genet. 1, 3 7

6 Monaco, A. P. (1989) Tren& Biochem. Sci. 14, 412-415

7 Rommens, J. M., lammzzi, M. C., Kerem, B. S., Drumm, M. L., Melmer, G., Dean, M., RozmaheI, R., Cole, J. L., Kennedy, D., Hidaka, N , Zsiga, M., Buchwald, M., Iviordan, J. R., Tsui, L. C. and Collins, F. S. (1989) Science 245, 1059 1065

8 Williamson, IV. (1993) Nature (2-enet. 3, 195-201

9 Anderson, W. F. (1992) Science 256, 808-813

10 Clothier, C. (1992) Report oJ'the Com- mittee ou the Ethics of Getw Therapy, HMSO

Robert Williamson Department of Molecular Genetics, St Mary's Hospital Medical .School, Imperial Colh'ge oj

Science, Technoloj!y and Medicine, l'qorfolk [)lace, London, UK I/V2 IPG.

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