'Gene therapy' has been aggressively pursued for more than twelve years with little success. The death of a healthy teenager in a clinical trial in 1999 alerted the public to the hazards involved. Although regulations are tightened up, the technical and scientific problems remain unsolved. Diseases are not understood; animal models are misleading; vectors for delivering genes are ineffective and unsafe; and the effects of genes delivered cannot be predicted.
The most damning criticism of gene therapy, is that it is a simplistic, reductionist solution to complex diseases that must be understood in terms of the human being as a whole in his or her social, ecological environment. An in-depth analysis from Dr. Mae-Wan Ho and Prof. Joe Cummins
Gene therapy is based on the idea of introducing genes into human cells in order to cure diseases. Billions have been invested, and hundreds of clinical trials carried out since 1990, mostly in the United States, but there has not been a single documented case of the miracle cure that was promised [1].
It took the death of a healthy teenager Gelsinger in an early phase clinical trial in September 1999 to alert the public to the hazards of gene therapy. The US Food and Drug Administration (FDA) and the National Institutes of Health (NIH) responded to widespread concern. Clinical trials were suspended. A public enquiry turned up 652 cases of serious adverse events that went unreported, along with seven other deaths. David Baltimore, Nobel laureate and president of the biotech company Caltech with interests in gene therapy, declared, " I disagree we’ve had any benefit from gene therapy trials so far, many of us are now asking, what the hell are we doing putting these things into people?"
Administrative changes were put in place amid calls for more research, and clinical trials resumed with further promises. Although more stringent regulation can tighten up the protocols and ensure quality control, most of the inherent technical and scientific problems remain unsolved. Some of the necessary research that should have been done long ago is only now being carried out, revealing findings that confirm our worst fears.
These problems are not new. The NIH’s 1995 report documents a plethora of scientific and clinical risks associated with gene therapy research [2], many highlighted independently in an ISIS report [3].
The NIH expert panel found that all gene transfer vectors were ineffective and little is understood on how they interact with the host. Basic studies on disease pathology and physiology - which are critical for designing treatment – have not been done. It was not possible to extrapolate from animal experiments. In the cases of cystic fibrosis, cancer and AIDS, animal models do not have the major manifestation of the human disease. Gene transfer frequency is extremely low. There were no controls, and biochemical or disease endpoints were not defined.
The panel concludes, "only a minority of clinical studies... have been designed to yield useful basic information". It expressed "concern at the overselling of results of laboratory and clinical studies by investigators and their sponsors, either academic, federal, or industrial, leading to the widespread perception that gene therapy is further developed and more successful than it actually is".
Gene therapy is currently directed towards changing the genetic makeup of the cells in the body of an individual only (somatic gene therapy). Most countries outlaw gene therapy on germ cells (germline gene therapy) - which would change the genetic makeup of the next generation – on account of the its obvious eugenics implications. But there have nevertheless been calls for gene therapy on the unborn and on human embryos, as the result of the publicity generated by the human genome project.
The human genome project has dominated scientific research for the past 12 years, raising hopes and fears in equal measure. But it is unlikely to deliver, especially if it continues to be misguided by a discredited genetic determinist paradigm that attributes all human conditions simplistically to the genes [4] (see "Genetically modified organisms, 25 years on", this series).
Among the promises of the human genome project and genomics research are the possibilities of replacing ‘bad’ genes in gene therapy, including germline gene therapy, of ‘genetic enhancement’ and ‘designer babies’ to create superior human beings.
In reality, the only concrete offering from the human genome sequence is hundreds of patented gene tests. The high costs of the tests have prevented them from being used in cases where it might benefit patients in providing diagnosis. At the same time, healthy subjects who have tested positive are likely to suffer from genetic discrimination and risk losing employment and health insurance. The value of diagnosis for conditions for which there is no cure is highly questionable. The claim to identify putative ‘bad’ and ‘good’ genes is also fuelling the return of eugenics, which has blighted the history of much of the 20th century. This is exacerbated by the dominant genetic determinist mindset that makes even the most pernicious applications of gene technology seem compelling.
A prominent band of scientists and ‘bioethicists’ are advocating human genetic engineering, not just in ‘gene therapy’ for genetic disease, but in positively enhancing and improving the genetic makeup of children whose parents can pay for the privilege, and have no qualms about human reproductive cloning either (see "Why clone humans?", this series). In many ways, this is the subtlest form of hype for business to prosper. Novartis Foundation invited arch-eugenicist Arthur Jensen, to speak at a scientific meeting on intelligence in Britain in 1999. Jensen is best known for his insistence that black people are genetically inferior in intelligence to white people, and hence all efforts at enhancing the education of disadvantaged black children are bound to fail.
Recent developments have brought us closer to creating genetically engineered human beings, and more [5]. The United States Food and Drugs Administration suspended an experiment in gene therapy because of concerns that it might alter the germ line, a possibility that has been pointed out by many previously [3, 6]. The recombinant DNA Advisory Committee (RAC) of the National Institutes of Health met to consider the implications; regarded the risks to be ‘extremely low’ and germline modification acceptable as one of the ‘side-effects’.
Meanwhile, researchers isolated male germ-line stem cells from the testis of mice and genetically modified them in vitro. The modified stem cells were then injected into the testes of genetically infertile mice, which the cells successfully colonised, and matured into sperms.
This is so easily done that it may become the method of choice for all genetic engineered animals in future, including human beings. The testis of genetically infertile mice is so readily colonised by the male germ-line stem cells that it is an open door to corporate control of male reproduction. It has already been suggested that human males undergoing irradiation and chemotherapy treatments for cancer that destroy stem cells could have their male germ-line stem cells removed and frozen, to be re-transplanted after the cancer is eradicated [7]. This is a short step from genetic manipulation of the stem cells in vitro.
In vitro fertilisation, human nuclear transfer cloning, surrogate motherhood, have all passed with relatively little comment from the establishment, as these were all aimed at manipulating reproduction in women. Adding male reproduction certainly increases the possible routes for germ-line gene therapy (see Box 1). Germ-line gene therapy has enormous impacts on the social fabric of human societies, and should not be allowed in the name of ‘scientific progress’, particularly as it is based on a discredited, outmoded paradigm that has largely ignored both physical risks and ethical implications
Box 1
Routes for Germline Gene Therapy
Via female germ cells and embryos
Via male germ cells
In gene therapy, an artificial construct – consisting, in the minimum, of a promoter driving the expression of a gene, and the gene itself - is delivered, either by viral vectors, or as naked DNA into cells. There are two main ways to carry out gene therapy, ex vivo and in vivo. In the ex vivo procedure, the constructs are transfected (or transduced) into cells outside the body, and the resulting transgenic cells are reintroduced into the body. In the in vivo procedure, the constructs are introduced into the body by numerous routes depending on the locating of target cells [3], emphasizing the ease with which cells take up foreign DNA. These include rubbing on the skin, applying in drops to the eyes, inhalation, swallowing, injection or perfusion into the bloodstream or directly into the tissues such muscle or solid tumours.
The only limited success stories so far have been associated with the ex vivo procedure, which avoids most, if not all the risks of in vivo procedures. In April 2002, a team in London’s Great Ormond Street Hospital in Britain used gene therapy to cure a child with X-linked severe combined immunodeficiency disease (SCID) [7]. They followed the approach taken earlier by the team at the Hospital Necker-Enfants Malades in Paris, which involved ex vivo manipulation of bone marrow stem cells. Adrian Thrasher, who led the London team, is unaware of any UK protocols for treating single-gene disorders by injection of viral vectors in vivo. He believes the Gelsinger case did have some impact on clinical trials in the UK, which tend to be much more cautious.
The identification and successful isolation of stem cells (both adult and embryonic) may make ex vivo gene therapy the preferred procedure for some diseases.
Four main types of disease are targeted for gene therapy: rare single-gene inherited disorders such as cystic fibrosis and sickle-cell anaemia, multi-factorial disorders such as cardiovascular disease and diabetes, cancers and infectious diseases [8].
Single-gene inherited disorders are perhaps the most clear-cut candidates for gene therapy, as it involves introducing a normal gene to make up for the defective mutant gene.
Among the first candidates for gene therapy was cystic fibrosis, a mutation in the gene, cystic fibrosis transmembrane conductance regulator (CFTR). But 12 years on, "the goal of attaining clinically relevant and persistent correction of the cystic fibrosis defect in humans has not been achieved" [9]. Difficulties include delivery of the vector to the cell, lack of persistent gene expression in targeted cells, and immune responses to viral gene products, transgenes, or the cells targeted by the vectors. Furthermore, mice with deletion of the CFTR gene or the common human CFTR mutations do not develop lung diseases like people. These are some of the same problems identified in the 1995 NIH report.
Multi-factorial disorders, like coronary heart disease or diabetes, involve many genes and are strongly influenced by environmental factors. Studies from Finland, US to China have all documented the overwhelming influence of diet and exercise in reducing type 2 diabetes as well as heart disease [10].
The American Heart Association (AHA) expert panel on clinical trials of gene therapy in coronary angiogenesis found gene therapy unsatisfactory, especially in comparison to conventional treatments, and expressed concerns over safety [11].
Gene therapy for coronary angiogenesis involves delivering growth factor genes into the heart to stimulate blood vessels to grow. But the Heart Association experts pointed out that "no process-specific stimuli or growth factor has ever been identified", and "regrowth of blood vessels is a complex process that involves multiple levels of stimulators, inhibitors and modulators". They levelled strong criticism that the gene therapists aren’t even using the right genes.
The Heart Association experts were also concerned over the mode of delivery and the ‘optimal dose schedule’, which they said "is unknown". In one study, the adenovirus vectors persisted and caused dysregulation of a number of host genes. They stated that "preclinical and clinical studies should be preceded by tissue distribution studies to define the myocardial uptake and retention or expression of growth factors".
One of the major technical hurdles for delivering foreign genes is the form in which the constructs are delivered. Although naked DNA is widely used for modifying germ cells, this does not work as well for somatic cells therapy, for which viral vectors are routinely used.
"The ideal vector would be [one that could be] easily produced in pure form at high titers, would efficiently and stably transduce non-proliferating cells in vivo, and would enable long-term transgene expression without producing cytotoxic effects, inflammation, or immune responses. Such a vector also might be capable of tissue-specific targeting and transgene expression and allow for pharmacologically or physiologically regulated transgene expression." [12, p.546]
To this, we would only add the requirement that it could target genes to specific positions in the genome so as to minimise unintended genetic effects due to the random gene insertion (see Box 2).
Unfortunately, such an ideal vector has not yet been developed. Plasmid vectors are easy to produce and manipulate and capable of stably transducing cells. But they are inefficient in delivering transgenes to non-proliferating cells - which constitute most of the cells in the body - and can cause immune responses directed against CpG repeat sequences that are plentiful in plasmids of bacterial origin. All the problems of gene delivery are the same as those involved in creating other GMOs (see "GMOs 25 years on", this series).
Box 2
The Ideal Vector
There are several kinds of viral vectors, all of which carry risks of generating new viruses by recombination, or by activating endogenous viruses. As they insert into the genome at random, they can cause genetic disturbances (position effects) including cancer. In addition, some are immunogenic, and can trigger acute fatal reactions. The main vectors used are as follows.
Retroviral vectors such as murine leukaemia virus-derived vectors, were among the first used, but are no longer regarded as first choice because of several drawbacks. Low yields, inability of virus to infect non-dividing cells, lack of stable expression and recombination within cells are feared to cause activation of pre-existing, dormant retroviruses.
Adenoviral vectors were used for epithelial cells specifically, and was the first choice for cystic fibrosis. They can infect non-dividing cells, but not stem cells, so treatment has to be repeated at intervals. The vector is immunogenic and even the first application can cause inflammatory events. After repeated applications, the cells will no longer become infected. Antibodies against adenovirus are widespread within human populations even without gene therapy. The teenager Gelsinger died from a high dose of adenovirus, leading to liver failure followed by multi-organ failure. Post-mortem revealed that many organs were infected with high concentrations of adenovirus, contrary to the anticipated cell-specificity of adenovirus infection. As with retroviral vectors, gene delivered with adenoviral vectors are frequently shut down.
Adeno-associated viral vectors (AAV) are not pathogenic, and are thought to integrate at a defined position in chromosome 19. However, this site-specific integration is linked to the viral rep gene involved in viral replication. AAVs can carry only a small amount of foreign DNA and infect cells poorly. Immune responses occur also against AAVs. Antibodies to AAFs circulate in about 80% of the population. Moreover, a helper virus (usually herpes simplex or adenovirus) is required for AAV production, with danger of contamination as well as recombination to generate infectious viruses.
Recently, researchers in the Department of Medicine, University of Washington Seattle, reported that the AAV does not integrate at specific sites [13]. The AAV integrated into at least six different chromosomes. Although it was most frequently found in chromosome 19, the insertion was not at the specific intended site. Furthermore, insertions were "associated with chromosomal deletions and other rearrangements", or genome scrambling.
In another experiment, newborn transgenic mice with the mucopolysaccharide storage disease MPSVII were treated with recombinant AAVs carrying the enzyme that breaks down the mucopolysaccharide. A high proportion of the mice were found to develop hepatocellular carcinoma, 33% in one group of 12 treated animals at the age of 12 months, and 17% developed angiocarcinoma at the age of 12-18 months [14].
The cancers were found to be specific to rAAV, as they were absent in mice with bone marrow transplant and in transgenic mice carrying the same enzyme cassette but without the rAAV.
Lentiviral vectors, a subgroup of retroviruses, are capable of infecting non-dividing, but not truly quiescent cells. The AIDS associated virus HIV-1 is currently the candidate, after disarming the genes that cause disease. However, cell lines used for packaging may contain the disarmed genes, and give them back to the vector to generate pathogenic viruses. Like other retroviruses, these might activate endogenous retroviruses within recipient cells.
Apart from these main classes of viral vectors, others have been developed, including herpes simplex virus and baculovirus, an insect virus that’s being modified to control insect pests in agriculture, and has been found to infect all kinds of mammalian cells [15].
Even bacterial pathogens that can gain access into mammalian cells are being exploited as vectors, including Agrobacterium, widely used in genetic modification of plants, that was also found to transfer genes into mammalian cells by a process analogous to bacterial conjugation [16]. There is no limit to the dangerous agents that are being developed for gene therapy.
Random integration of viral vectors into genomes give rise to unintended effects known collectively as position effects. These affect both the inserted gene and the host genes in its vicinity. Not only can the introduced genes become inactive, but under adverse circumstance, may even cause cancer. The long-terminal repeats of the viral vectors typically contain strong promoters that can make genes such as proto-oncogenes (genes potentially capable of causing cancer) over-express. Alternatively, the insert may inactivate tumour suppressor genes. Sometimes, the transgenes themselves may also cause cancer.
Researchers in Heinrich-Pette-Institute, Hamburg, and Hannover Medical School, and their colleagues found that a retroviral vector carrying a marker gene, thought to be ‘biologically inactive’, actually induced leukemia in all the mice [17]. The risk of cancer from random insertion of viral vectors into the genome is about 10-7 per insertion. But when the marker gene dLNGFR was inserted into mouse bone marrow cells with a retroviral vector and transplanted serially into irradiated mice, all the mice developed leukemia. The 10 mice receiving secondary transplant developed hematopoietic disorders related to leukemia within 22 weeks, including six that succumbed to overt acute myeloid leukemia analogous to human AML M5 phenotype. The tertiary recipients of the bone marrow transplant developed lethal acute myeoloid keukemia within 4 months.
The disease appeared to have resulted from a combination of vector inserting in a position that activates an oncogene and the transgene product interfering with cancer suppression.
Although cancer itself is a risk of gene therapy, it is also the major target for gene therapy, for economic, if not good medical reasons [18]: "Apart from cystic fibrosis, most monogenetic diseases are very rare. Economically more relevant are "popular diseases" such as cancer or AIDS".
There are many attempts to target genes more precisely [3]. For example, to turn specific genes off, antisense RNA or RNA cutting enzymes (ribozymes) are used. But the effects are transient. ‘Chimeroplasty’ uses a 25 nucleotide inverted repeat hairpin sequence to introduce precise DNA changes in a target gene [19], though it did not succeed [20]. The best reported results of targeted gene insertion come from cell cultures transformed with a viral vector containing a human gene sequence into which the foreign genes are spliced [21]. This resulted in homologous recombination with the human gene, but it still gave random gene insertions that outnumber targeted insertions 10 to 1.
Cancer gene therapy has indeed taken over as the more active research area. A recent review states [22], "Although no cures can consistently be expected from today’s cancer gene therapy, the rapid progress may imply that such cures are a few short years away."
Cancer gene therapy targets cancer cells, cancer blood supply, the immune system and the bone marrow (see Box 3).
Box 3
Targets of Gene Therapy for Cancer
1. Cancer cells
Cancer suppressor genes
Suicide genes
Cytokine genes
2. Tumour blood supply
Inhibiting angiogenesis genes by antisense
Anti-angiogenesis genes
3. Enhancing anti-tumour immune reaction
Modifying dendritic cells, with tumour antigens or cytokines
ModifyingT lymphocytes, with receptor for tumour antigen
One of the main candidate genes is the tumour suppressor gene p53, which induces cell death if DNA damage is extensive. Viral-mediated p53 gene therapy is in clinical trials with non-small cell lung cancer and head and neck cancers.
Another candidate is the ‘suicide gene’. This gene induces cell death by encoding an enzyme capable of converting a precursor drug to a toxic compound. The suicide gene is delivered to the target cells in a viral vector by injection into the tumour before the precursor is given. The Herpes Simplex Virus (HSV) thymidine kinase is an example, it phosphorylates the drug ganciclovir 1000 times more efficiently than the mammalian enzyme. This blocks DNA synthesis, leading to cell death. Clinical trials are already taking place.
Anti-angiogenic gene therapy uses inhibitors of blood vessel formation in tumours. The vascular endothelial growth factor is a key mediator of tumour-induced blood-vessel formation. It can be inhibited by blocking its translation or transcription. Antisense cDNA was found to reduce tumour size on average by two-thirds. The combination of the inhibitors angiostatin and endostatin was found to be synergeristic in causing complete loss of tumorigenicity in 40% treated mice in a murine leukemia model.
Another approach is through genetic enhancement of anti-tumour immune responses. The major cell-based strategy uses dendritic cells, modified either to present specific tumour antigens to immune effector cells, or to be more efficient in activating an anti-tumour immune response. A second strategy involves genetic modification of T cells to alter their antigen specificity and increase their responsiveness to tumour antigens.
Dendritic cells are antigen-presenting cells that can initiate a potent immune response. After acquiring tumour proteins, dendritic cells process the proteins and present peptides to CD4+ T cells, activating the latter. Activated CD4+ cells express CD40, which in turn activates CD8+ cytotoxic T cells that act against the tumour. Genes encoding tumour antigens have been transduced into dendritic cells to enhance the immune response.
To boost T lymphocyte anti-tumour activity, cells have been isolated from patients and transduced with chimeric T cell receptor that targets tumour-specific antigen.
Cytokine-based therapy aims also to enhance the immune response to tumours. The genes used include those encoding the interleukins, IL –1b, IL-2, IL-4, IL-12, as well as GM-CSF and IFN-g. Clinical trials have been performed on patients with malignant mesotheliomas and melanomas by injecting vectors with transgenes into tumours, to infect both tumour cells and dendritic cells. At best, a partial clinical response has been recorded in some of the patients.
The profusion of cancer gene therapy reflects the desperate attempts of the simplistic gene-centred science to cope with the complex reality of the organism. Decades of cancer research focusing on molecular genetics have brought us no closer to understanding the causes of cancer [23] while many cancers have been increasing at alarming rates.
The stepwise development of human cancer is clinically well-recognised: initiation, promotion and progression [24], but trying to establish causal links between genetic alterations to different disease manifestations is something else.
One of the hallmarks of cancer cells is genetic instability, both at the level of single nucleotides and the chromosomes. Thousands of point mutations and small deletions are typically present in cancer cells, as well as large-scale chromosomal translocations, duplications, rearrangements and disturbances in chromosome numbers. This suggests that deficiencies in either DNA repair or in chromosomal partitioning during cell division, or both, may be involved in cancer progression. The emerging theory is that genetic instability affecting chromosomal organisation, rather than mutations in specific cancer promoting or suppressive genes, may be more important in cancer development [25, 26].
A cancerous cell does not stop dividing. Cell division is a complex process, involving not just precise copying of the genes but also their exact distribution to the two daughter cells so that each has two copies of every chromosome. Anything that disturbs this process can result in genomic imbalance. Damage to the genes that monitor the intricate copy and delivery process, the so-called guardians of the genome, can result in an altered chromosome balance in the daughter cells. Mutations in those key genes can initiate aneuploidy, so there may be a role for mutation in cancer production.
However, many other disturbances can start the process going wrong, such as chemicals from the environment, radiation or any form of stress, or indeed, stray foreign DNA jumping into the genome, as in gene therapy. It doesn’t have to be a gene mutation.
Once genomic imbalance (aneuploidy) starts, it will tend to get worse: the imbalance in chromosomes will result in further disturbances to cell division, a positive feedback effect. However, this is counteracted by the reduced survival of aneuploid cells and the body will tend to get rid of them, until some eventually escape the immune system and grow out of control. There does seem to be a positive correlation between the number of chromosomal alterations within a tumour and the malignant potential of the cancer.
As every cancer is genetically different, it will be very difficult to target cancer cells with specific drugs, let alone specific genes. So the key is prevention.
Recognition of the diverse factors that can disturb cell division means that the multitude of chemicals that pollute our environment must be screened for their capacity to induce aneuploidy. Most of these don’t cause mutation, but may well disturb chromosome separation.
Finally, the phenomenon of cancer remission needs to be much more thoroughly investigated. Remissions can occur after various types of stimulus to the whole body, such as change of diet or life-style, and many other non-specific influences. Cancer is primarily a systemic disease of the whole organism, and only secondarily a disease of particular cells or of genes in those cells.
The same kind of simplistic approach characterises other forms of gene therapy. The expression of the introduced gene is not the only, nor the main problem, its regulated expression within the body is the key to normal functioning. Unfortunately, most foreign genes are introduced with aggressive viral promoters that simply make them over-express in an unregulated way. The underlying assumption is that the single gene product is necessary and sufficient to provide a cure. But this does not even work for so-called single gene disorders.
Helge Grosshans of Heidelberg University, Germany, has stated this problem most succinctly [18]: "Gene therapy follows a simple principle: causal therapy instead of symptomatic treatment. Accordingly, expectations were high....By now, however, it has become evident that particularly in those cases where the idea of "causal therapy" appears most appropriate, i.e., monogenic diseases, success is minimal. This is due, among other factors, to the cell being a very complex and dynamic system. A change in the genetic make-up that causes a cellular defect also brings about a number of compensating mechanisms. Mere addition of the "health" gene does not automatically re-create the original situation, because the compensatory mechanisms will not necessarily be turned off again." p.145
Also, "A newly synthesised normal protein will appear abnormal to an immune system that has never been exposed to it" [27].
In other words, the cell, and ultimately the entire organism functions as a whole, so practically every part of it will have changed when even a single gene is mutated. Consequently, restoring that gene is unlikely to put things right, and may even result in the gene product being targeted by the body’s immune defence.
Most of all, the procedure of gene therapy is itself hazardous [18]: "The additional steps of gene therapy, such as integration and expression, would present additional problems and safety risks. A therapeutic chemical can be broken down and will be eliminated from the body within a certain period of time. Foreign DNA, on the other hand might stay in the body until death and even be transferred into additional cells or passed on to future generations." p. 144
The simplistic gene-centred approach has failed because it is fundamentally at odds with the complex reality of the organic whole. In contrast, indigenous cultures all over the world never lost touch with the organic reality that encompasses an entire way of life. Contemporary western science is beginning to rediscover this sense of the whole across the disciplines [28]. It is a challenge for western and indigenous scientists to work in equal partnership towards restoring sustainable, healthy ways of life to all.
Article first published 06/07/02
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