Q J Med 2001; 94: 337-339
© 2001 Association of Physicians
Editorial |
Human gene therapy: are we still expecting too much, too soon?
Department of Medicine, Cambridge University, Addenbrooke's Hospital, Cambridge
With the publication of the first complete draft of the human genome,1 we have for the first time a wild-type sequence for very nearly every human gene. Although we are still in the early stages of mastering the technology required to extract the maximum scientific benefit from this vast databank, it is already clear that it will make a contribution to our understanding of biology of unrivalled breadth and magnitude. We can expect yet a further increase in the pace of discovery in the new ‘post-genomic era’.
Even before the whole genomic sequence became available, human molecular genetics had uncovered the fundamental inborn genetic variation which accounts for disease susceptibility in a wide range of diseases, particularly those for which errors in a single gene are largely responsible. With these advances has come the possibility of identifying individuals who will suffer a particular disease in the earliest stages of pregnancy. For a number of severe inherited disorders, such as cystic fibrosis, this has already become clinical reality. While the ethical issues surrounding genetic testing remain difficult, the technology is straightforward.
The obvious progression from gene diagnostics is gene therapy. If errors in a particular gene are responsible for a deleterious phenotype, then the most direct route of treatment would be to correct that genetic defect. Two things would seem to be required for successful gene therapy: knowledge of which genes were responsible for which diseases and a reliable and safe technique for introducing the correct DNA sequence, at least into those cells which need it. The availability of the human genome sequence database will help the first of these requirements, while recent reports suggest that after a decade of attempts, progress is finally being made on the technology to introduce functional genetic material in a clinical setting (for example, see references 2 and 3). As a result, the expectations for gene therapy have never been higher. Will it live up to these expectations and revolutionize twenty-first century medical practice?
The underlying assumption of most gene therapy procedures, irrespective of the disease being targeted, is that restoration of a wild-type DNA sequence will alone be sufficient to normalize the phenotype of the individual. This assumption has been supported by the concept that the DNA encodes every component of the cell as well as when it should be produced and in what quantity. Surely, then, a correct set of DNA plans will ensure a correctly functioning cell?
Unfortunately, this assumption is patently invalid: a liver cell and a neuron from the same individual share an identical DNA sequence, but perform very different functions. The phenotype of a cell depends not only on its particular DNA sequence, but also the current concentrations and locations of all the different protein (and possibly even non-protein) components. A liver cell may differ from a neuron in part because it contains a different array of transcription factor proteins. Large as it is, the human genome databank is likely only to contain a tiny fraction of all the information needed to make a human cell. In principle, we could use this information to make the DNA itself as well as all the protein components to put in a ‘synthetic cell’, but we would not know how much of each component to add nor where in our cell to put them. A cell is such a complex system that the overwhelming majority of combinations of the starting materials would fail to function as a stable cell. Our ‘synthetic cell’ would simply degenerate, despite containing a wild-type DNA sequence and all the correct components.
Once we consider the whole array of post-genomic information that is present in even the simplest cell, it becomes immediately clear why cloning is possible4 but de novo creation of a living cell is not. Should some of the proteins in a cell be phosphorylated, glycosylated or proteolytically processed? We would need to consider acetylation of histones, farnesylation of G-proteins, subcellular localization of transcription factors, polymerization of actin and tubulin and so on. No amount of interrogation of the human genome sequence will answer these questions. Cloning Dolly the sheep involved transfer of DNA into an oocyte: a specialized cell set-up to convert the information in the DNA into a whole organism. To have created Dolly from scratch would have required much more information. The DNA tells you how to make each protein component of the cell, and how to change the levels of the protein: it does not tell you the absolute amount of the protein that should be present under any given circumstances. Thus, it is not unreasonable to assume that the entire human genome contains only a minute proportion of the information density necessary to create a human cell from isolated molecules.
What are the implications of these observations for the future of gene therapy? It suggests that restoring the wild-type DNA sequence after the disease phenotype has manifested itself will, in most cases, not reverse the phenotype. Worse still, it may not even prevent further progression of the disease. Consider a typical monogenic disorder which might be considered for gene therapy in the future such as Duchenne Muscular Dystrophy. Mutations in the dystrophin gene mildly affect the function of skeletal muscle,5 so that shortly after birth the muscle is able to function almost as well as wild-type muscle. Over a period of years, however, the response to this defect is the accumulation of extracellular matrix around the muscle fibres. After some time, the loss of muscle function is more a result of the presence of this fibrosis than the original mutation in the dystrophin gene. This leads to a vicious cycle in which further loss of muscle function leads to more matrix deposition, and eventually to the complete failure of the muscle. To intervene with gene therapy once the fibrotic cycle has begun will be largely ineffective: the disease has progressed to the point where the mutation in dystrophin (although the initial trigger) is no longer the driving force of the disease.
Duchenne Muscular Dystrophy may be the rule rather than the exception: cystic fibrosis, hypertrophic cardiomyopathy and Marfan syndrome may all be similar. Once the protein distribution pattern of a cell or organ has been disrupted beyond a certain point, even the wild-type genome may not have the wherewithal to correct it. This hypothesis is graphically illustrated by the process of ageing. Much of the degeneration in tissue architecture and function may not result from accumulated genetic mutation, but from accumulated damage to the protein distribution. Wrinkled skin results from excess cross-linking of the dermal collagen through formation of advanced glycation end-products (AGEs). Cataracts result from disorganization of the crystallin proteins in the lens. Reduced kidney function results from damage to the glomerular basement membrane. All this degeneration could plausibly occur in the presence of an entirely normal genomic DNA sequence.
Is this an argument for halting research into gene therapy? Certainly not. Instead it may provide some guidelines for devising successful gene therapy strategies. Some diseases may be amenable to gene therapy even when the phenotype has become established: for example, repair of the gene encoding cerebrosidase may ameliorate the symptoms in Gaucher's disease and restoration of a functional adenosine deaminase gene may indeed prevent the symptoms of ADA deficiency. The important thing is to ascertain whether the absence of the trigger protein remains the driving force behind the pathology at the time when gene therapy is contemplated. The second issue is therefore one of timing. The earlier the gene therapy intervention can be made, the more likely it is to be successful. Unfortunately, this is at odds with the demands of safety monitoring for a new clinical technique. As with most new therapies, the benefit-to-risk ratio is greatest for those most severely affected by the disease being targeted. While treating these individuals may successfully demonstrate the safety of the technique, it may dramatically underestimate the efficacy. We must guard against taking an overly negative overview of the efficacy of gene therapy if the first few clinical studies produce surprisingly little clinical benefit.
The one application of gene therapy which seems certain to have a golden future, albeit many years from now, is germ-line modification. The recent success of cloning suggests that the genome sequence does indeed code for the majority of the differences between individuals (rather than this information depending to any significant degree on, for example, the levels or locations of certain proteins in the oocyte). Of course, even this may be an over-simplification: injection of various proteins into a frog oocyte can cause the development of a tadpole with two heads without a single base change to the DNA sequence.6 Nevertheless, it seems likely that restoration of the wild-type DNA sequence provided to the fertilized embryo will, in the large majority of cases, avoid the development of inherited disease. Unfortunately, despite offering the greatest hope of success, germ-line gene modification raises both the biggest safety hurdles and the most demanding ethical issues. Both these obstacles will have to be overcome, in addition to the technical difficulties of gene transfer, before germ-line gene therapy becomes a reality.
We are reaching a critical phase in gene therapy research. Finally, some of the technical difficulties which have hampered progress are being solved, and we can expect the first trials to begin reporting efficacy data in the next few years. It is important that we remain realistic in our expectations of what gene therapy can achieve. There is some danger that in the euphoria surrounding the completion of the human genome sequence that we expect too much from gene therapy approaches, such that the early results are disappointing. Ensuring that gene therapy makes the difficult transition from a bright idea to a clinical reality will require some wise decisions on which diseases to target, as well as attention to the details of the protocol: correct timing of the intervention may be as important as choosing the right gene.
References
1. Lander ES, et al. Initial sequencing and analysis of the human genome. Nature2001; 409:860–921.[Medline]
2.
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science2000; 288:669–72.
3. Dick JE. Gene therapy turns the corner. Nature Med2000; 6:624–6.[Web of Science][Medline]
4. Campbell KH, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature1996; 380:64–6.[Medline]
5. Hoffman EP, Brown RH, Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell1987; 51:919–28.[Web of Science][Medline]
6. Sokol S, Christian JL, Moon RT, Melton DA. Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell1991; 67:741–52.[Web of Science][Medline]
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