In the last century, there have been a number of discoveries regarding the treatment of human disease and genetic conditions. The current on-going research is in the field of gene therapy, an experimental technique that uses genes to treat and replace the defective genes of an affected person. Instead of treating disease symptoms, this has the potential to correct the underlying cause (1). Besides its high costs and ethical concerns (therapy involving germ line treatment), this technique also poses a considerable amount of risk.
Thus, gene therapy is currently only being tested on the diseases for which there is no cure. This article shall look primarily into cystic fibrosis, as well as X-linked severe combined immunodeficiency (SCID), as examples to describe the potential of gene therapy in medicine. Cystic fibrosis is a single gene disorder, with an autosomal recessive pattern of inheritance. The gene is located on the long ‘q’ arm of chromosome 7 and is called the cystic fibrosis transmembrane conductance regulator (CFTR) gene (2).
The gene product is an apical membrane chloride channel regulated by nucleotide binding domains and phosphorylation sites (3). This channel maintains the salt-water balance on epithelial surfaces such as in the lungs or pancreas. Mutations in the CFTR gene result in structural changes in the CFTR protein. These changes disturb a process called mucocillary clearance. Thus in the lung, thickened mucus results in repeated bacterial colonisation by strains such as Staphylococcus aureus and Haemophilus influenzae. These significantly increase the risk of inflammation which ultimately leads to respiratory failure. 4). The most common mutation in the CFTR gene is a 3 base pair deletion in exon 10 (which comprises a codon for phenylalanine), called the AF508 mutation (Refer to Figure 1). This accounts for approximately 70% of CFTR gene mutations.
Currently, over 400 CF-associated mutations have been identified (5). Despite this, CF has been a candidate for treatment by gene therapy. This is due to the following reasons (6): 1) The genetics of CF are simple as it is a single gene disorder. 2) The lung, the most affected organ, is easily accessible. ) There is no long-term treatment for the disease. The airway has been the principal target for CF gene therapy, because it is the respiratory aspects of the disease which have been associated with the highest rates of morbidity and mortality. The primary basis of treatment is to deliver the cloned CFTR gene to the airway epithelial cells via an in vivo approach. This is performed using two methods: 1)Adenoviralmediated gene transfer: Adenovirus is a suitable candidate vector for gene transfer to the airway as it naturally infects the respiratory epithelial cells.
They therefore make useful vectors in the transfer of the healthy CFTR gene into the epithelial cells of the lungs (7). These adenoviruses are then grown in epithelial cells in the laboratory along with plasmids that have had the healthy CFTR gene incorporated into their DNA. This DNA, called cDNA is then incorporated into the double-stranded DNA of the adenoviruses. The adenoviruses that have taken up the healthy CFTR gene are introduced into the nostrils of the patient and are drawn into the lungs during inhalation.
They inject their DNA, which includes the healthy CFTR gene, into the epithelial cells of the lungs (7). Refer to figure 2) 2) Cationic liposome-mediated DNA transfer: This method utilises a charge interaction between plasmid DNA (negatively charged) and a mixture of cationic and neutral lipids to form a complex of lipid-coated DNA. These reach the targeted epithelium cells via inhaled aerosols (8). (Refer to figure 3) Now while these two methods are the most studied and tested, they are not always effective. Patients may develop immunity to the adenoviruses, which then cannot inject its cDNA into the epithelial cells. The changes induced by the adenoviruses also do not last very long, only about 10-14 days after DNA injection (9).
In the liposome-mediated transfer, the aerosol might not be fine enough to pass through the bronchiole membrane. However, recently, this has returned positive results. A phase 2b trial conducted by the UK Cystic Fibrosis Consortium observed that the CF symptoms were much more stabilised in patients who received the gene treatment than those who took a placebo, over the course of 12 months (10). Thus gene therapy using liposome-mediated transfers is gaining more recognition as a possible treatment for cystic fibrosis. X-linked SCID (SCID-X1) affects the male population primarily nd accounts for 50-60% of all cases of SCID.
It is caused by defects in the interleukin-2 receptor y-chain gene (11) and is characterised by the lack of T-cell and B-cell development. Like cystic fibrosis, this disorder arises due to mutations in a single gene and thus is suitable for gene therapy treatments. Treatment is performed by removing bone marrow from the affected patient, and isolating and enriching haematopoietic stem cells. The normal gene is then inserted ex vivo using a Moloney retrovirus-mediated transfer of y-chain receptor cDNA into CD34+ cells (refer to figure 4).
Between March 1999 and April 2002, nine patients with SCID-X1 underwent a gene therapy trial at the Necker-Enfants Malades Hospital in Paris (12). Gene therapy initially corrected immune dysfunction in eight out of the nine patients. However, four patients developed leukaemia, and one of them died. The other three were treated with chemotherapy and were among seven children who exhibited long-term immune reconstitution. Gene-corrected Tcells have persisted in them for more than 10 years (13).
Overall, this treatment confirmed the expectation that ex vivo gene therapy for SCID-X1 can result in long-term correction of the SCID phenotype. However, the risk of developing acute leukaemia related to treatment, which involved the use of retroviral vectors, must be taken into consideration. Research should be conducted into the development of safer viral vectors (12). Gene therapy, like any other form of treatment, has its advantages and disadvantages. On one hand, it seeks to root out the cause of diseases which have no cure, rather than only treating its symptoms. However, treatments vary from disease to disease.
In the case of cystic fibrosis, the effects of treatment do not last very long, and in SCID-X1, the treatment has led to risks of leukaemia. There is an ethical concern that it could modify human capabilities, thus altering the standards of normal human life. Gene therapy is also a very expensive form of treatment and hence should be regulated effectively. Gene therapy has a remarkable therapeutic potential (14) and this should be exploited. Through effective research and regulation, gene therapy has the potential to cure genetic diseases, eliminate any possible side effects and usher in a new standard of healthcare.
