Gene therapy: into the future of medicine

Jonas Araújo de Souza discusses different approaches and the relevance to modern medicine

The sequencing of the human genome has been compared to putting a man on the moon, and it will certainly change health care, but the most important work lies ahead, in determining how to put the information to medical use. In this context, applications such as gene therapy are being explored. What was once seen as a science fiction dream is now becoming a real possibility.

An introduction to gene therapy

Gene therapy is a new form of drug delivery that leads the patient's own cells to produce a therapeutic agent.^1,2 It could potentially eliminate the need for repeated administration of proteins or drugs. Applications of gene therapy not only include rare inherited diseases but extend to common acquired disorders, including tumours (predominantly malignant melanoma) and haematological disorders, cardiovascular disease, and the acquired immunodeficiency syndrome (fig 1). Gene therapy therefore could be a key element of medical practice in the future.

Gene therapy was tried in 1970 and again in 1980 without success, but the knowledge and techniques were primitive by today's standards. The first attempt in what might be called the modern era of gene therapy began in September 1990 at the United States National Institutes of Health, when doctors treated two children with severe combined immunodeficiency due to adenosine deaminase deficiency. The doctors took the children's own white blood cells, altered them by adding the gene for the missing enzyme, and transplanted the altered cells back into the children. Gene treatment ended after two years, but integrated vector and ADA gene expression in T cells persisted.³

There are several approaches to gene therapy. The most commonly used in clinical trials is the "gene addition" approach, which seeks to compensate for a defective gene by providing cells with a corrective gene.⁴ A gene expressing a cytokine may be inserted to enhance or stimulate an immune response to cancer cells; a "prodrug" convertase gene (also known as a suicide gene) may be inserted to convert a non-active prodrug into an active metabolite, so confering the target cells' susceptibility to drug treatment.

But there are some barriers. Firstly, the gene is often quite large, making the construction of a delivery device (vector) problematic and often impossible. Secondly, the delivery vector is usually a virus and consequently can infect with high efficiency, but because most higher eukaryotic organisms respond immunologically to viruses, antibody production often impairs the therapy, particularly on secondary administrations. Thirdly, the expression of many genes is rigorously regulated and context dependent, which makes the achievement of the correct balance and function of expression challenging.

Another approach has focused on gene targeting or gene replacement (fig 2), whereby the defective gene is removed and a normal gene is inserted at the same position in the chromosome.⁴ The molecular process controlling the replacement of a defective gene with a normal one is called homologous recombination. The principle is simple. The gene is artificially created using a short string of nucleotides, called an oligomer. With one exception, the new gene is exactly complementary to the section of the gene in which the error is located, with the exception being at the site of the error. The nucleotide complementary to the one that is supposed to be in the DNA sequence of the normal gene is then inserted. The oligomer binds to its complementary sequence on the DNA, and by design creates a bulge at the site of the mismatch. This bulge is eventually detected by the cell's internal DNA repair mechanisms. Repair enzymes remove the erroneous nucleotide and replace it with a nucleotide complementary to the one in that position in the oligomer, which happens to be the correct nucleotide. The main problem here is that the frequency with which the normal gene copy goes where it is designed to go is extremely low.

Delivering the genes

The molecular manipulation or alteration of a designated "target" population of cells can be effected in one of two ways: by exvivo modification, with the material being reintroduced into the body after modification, or by in-vivo modification, done in situ. Both ways need tools for delivering the genes. These tools are the vectors, the vehicles of gene delivery and the Achilles heel of gene therapy. Thus far, the problem has been an inability to deliver genes efficiently and to obtain sustained expression (see table).

Most gene therapy has involved the use of viruses as carriers of the gene.⁵ This has proved to be the most efficient method to date, performing many of the tasks necessary to achieve successful gene transfer, such as binding to a target cell and delivering the viral genome to the nucleus in a stable way. Retroviruses are the most used viral vectors. They are a group of viruses whose RNA genome is converted to DNA in the infected cell, which is an actively dividing cell, whereas most tissues in adults consist primarily of non-dividing (somatic) cells. Retroviruses can induce insertional mutagenesis as well as potentially transforming these cells. Adenoviruses, which are a family of DNA viruses that can infect both dividing and non-dividing cells, causing benign respiratory tract infections in humans, are the second most used vectors for gene delivery. The current generation of adenoviral vectors is immunogenic (elicits an immune response), which reduces the length of time available for the expression of the gene and makes repeated administration of the vector impossible. Promising viral vectors are adenoassociated virus and lentivirus. Adenoassociated virus is a simple, non-pathogenic, single stranded DNA virus. This virus infects a wide range of cells, including lung and muscle cells, and it integrates its genes within the host's. Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells. The bestknown lentivirus is the human immunodeficiency virus (HIV), which has been disabled and developed as a vector for in-vivo delivery.

These viruses may lack virtually all viral genes except those required for infecting mammalian cells. If properly prepared, these viruses are so defective that after they infect the appropriate target cell they cannot replicate or infect other cells. However, the human immune system can fight off the virus, and attemps to deliver genes in viral vectors have been confronted by these host responses, such as the ill fated gene therapy that led to the death of Jesse Gelsinger. He was an 18 year old patient who suffered from ornithine transcarbamylase (OTC) deficiency, an inherited disorder, that, in its most common form, causes death in affected newborn males because of their inability to properly process nitrogen in food proteins. Gelsinger's death was the result of an immune reaction to the engineered adenovirus that researchers had infused into his liver.

Some experiments using this vector showed a rapid immune response against its proteins.⁶ The immune reaction is potent, eliciting both the "cellular" and the "humoral" response. In the cellular response, virally infected cells are killed by cytotoxic T lymphocytes. The humoral response results in the generation of antibodies to viral proteins, and it will prevent any subsequent infection if the person is given a second injection of the recombinant virus. In fact, these events resemble the immune responses to any viral infection. To overcome these immunological problems, vectors have been engineered to minimise the expression of viral antigens. Another approach is to prevent activation of T helper cells by administering an immunosuppressive drug along with the vector. When using viral vectors one should consider the pre-existing host immunity as well as the remote possibility of target cells having factors that may trigger the synthesis of viral proteins.

Non-viral methods of delivery

There are also non-viral methods of gene delivery. They cause a relatively small immune response and can deliver larger amounts of DNA than viral vectors can. But they are limited by a low rate of gene transfer and poor long term expression, especially when delivered in vivo. Liposomes are small vesicles created from lipids that resemble those making up membranes of mammalian cells, allowing these vectors to fuse with cell membranes.⁷ Direct injection of naked DNA plasmids is possible, but relatively few cells take up the DNA (1-3%), leading to a small production of the encoded protein. The most important use of naked DNA plasmids is in vaccine development, since the small amount of protein produced can elicit a protective immune response.⁸

Gene therapy researchers are trying to find ways to improve each vector, as well as to match vector characteristics with diseases that they will target most successfully. It is likely that there will be a lot of specialised vectors rather than one universal vector. There will be some for situations when doctors want short term expression - for example, expression of a toxic gene product in cancer cells - some for situations when doctors want long term expression, such as for most genetic diseases, some that deliver large chunks of DNA, and some that carry smaller pieces.

Inserting a gene is not the only problem. The vector must also contain a mechanism for activating the therapeutic gene, as this is not automatic. Hence the vector must include a timing and regulatory "device." These mechanisms, which allow genes to be turned on and off and change levels of the therapeutic protein over time, are the gene's promoters. They are complex and sometimes quite large, so placing them into a therapeutic vector is difficult. Recent vectors include portions of the gene's own promoter. This allows the therapeutic gene to be expressed as naturally as possible. Other constructions attach promoters that can be externally controlled. For example, it is possible to regulate the expression of a gene of interest, such as one encoding a hormone, by means of hybrid microbial and mammalian proteins and microbial DNA regulatory elements that respond to drugs. The goal of regulating genes through the use of endogenous biological signals, such as glucose levels in diabetes, is also being researched.⁹

Prospects for the future

The bottom line in any kind of biomedical research is its relevance. Despite the excitement gene therapy can cause, the field is still in its infancy. Several clinical trials of gene therapy have been completed or are under way. They can provide information that cannot be concluded from tests in animals. Although this therapy is theoretically good in principle, it has proved difficult to demonstrate its efficacy in practice. Thus, the success rate in clinical trials has been relatively low. Such results can reduce the enthusiasm for genetic approaches, but the field is still new and the pace and surprises of new discoveries are amazing.

I wish to thank Carlos Eduardo Motta, University of Rio de Janeiro State, Brazil, for his invaluable help in preparing this article.

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