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Stem cells are attractive potential recipients cells, as they are immortal. Successful introduction of the target gene into these cells should facilitate ongoing production of the gene product in mature blood cells, which are continually derived from the stem cell population. This would likely remove the requirement for repeat gene transfers to the affected individual.
The routine transduction of stem cells has, thus far, proved technically difficult. They are found only in low quantities in the bone marrow, and there is a lack of a suitable assay for stem cells. However, recent progress has been made in this regard and routine transduction of such cells will likely be achievable within the next few years.
Additional genetic diseases for which a gene therapy approach is currently being evaluated include familial hypercholesterolaemia and cystic fibrosis. Familial hypercholesterolaemia is caused by the absence (or presence of a defective form of) low-density lipoprotein receptors on the surface of liver cells. This results in highly elevated serum cholesterol levels, normally accompanied by early onset of serious vascular disease. Gene therapy approaches that have been attempted thus far to counteract this condition have entailed the initial removal of a relatively large portion of the liver. Hepatocytes derived from the liver are then cultured in vitro, with gene transfer being undertaken using retroviral vectors. The corrected hepatocytes are then usually infused back into the liver via a catheter. Although studies in animals have been partially successful, transduction of only a small proportion of the hepatocytes is normally observed. Subsequent expression of the corrective gene can also be variable. In vivo approaches to hepatic gene correction, using both viral and non-viral approaches, are also currently being assessed.
The cystic fibrosis (cf) gene was first identified in 1989. It codes for a 170 kDa protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which serves as a chloride channel in epithelial cells. Inheritance of a mutant cftr gene from both parents results in the CF phenotype. While various organs are affected, the most severely affected are the respiratory epithelial cells, which have, unsurprisingly, become the focus of attempts at corrective gene therapy.
Several vectors have been used in an attempt to deliver the cf gene to the airway epithelial cells of sufferers. The most notable systems include adenoviruses and cationic liposomes. Vector delivery to the target cells can be achieved directly by aerosol technology. Delivery of cftr cDNA to airway epithelial cells (and subsequent gene expression) has been demonstrated with the use of both vector types. However, in order to be of therapeutic benefit, it is essential that 5-10% of the target cell population receive and express the cftr gene. This level of integration has not been
NUCLEIC ACID THERAPEUTICS 485
Table 11.5. Some therapeutic strategies being pursued in an attempt to treat cancer using a gene therapy approach. Refer to text for details
Modifying lymphocytes in order to enhance their anti-tumour activity Modifying tumour cells to enhance their immunogenicity Inserting tumour suppressor genes into tumour cells
Inserting toxin genes in tumour cells in order to promote tumour cell destruction Inserting suicide genes into tumour cells
Inserting genes, such as a multiple drug resistance (mrd) gene, into stem cells to protect them from chemotherapy-induced damaged Counteracting the expression of oncogenes in tumour cells by inserting an appropriate antisense gene
achieved so far and, furthermore, gene expression has often been transient. However, it is considered likely that ongoing developments in this field will render gene therapy a useful treatment for CF within the earlier part of the twenty-first century.
Gene therapy and cancer
To date, the majority of gene therapy trials undertaken aim to cure not inherited genetic defects, but cancer. The average annual incidence of cancer reported in the USA alone stands at ca. 1.4 million cases. Survival rates attained by pursuit of conventional therapeutic strategies (surgery, chemo-therapy, radio-therapy) stands at about 50%. Gene therapy will likely provide the medical community with an additional therapeutic tool with which to combat cancer within the next 10-15 years.
Initial gene therapy trials aimed at treating/curing cancer began in 1991. Various strategic approaches have since been developed in this regard (Table 11.5). Numerous trials aimed at assessing the application of gene therapy for the treatment of a wide variety of cancer types are now under way (Table 11.6).
While many of the results generated to date provide hope for the future, thus far gene therapy has failed to provide a definitive cure for any cancer type. The lack of success is likely due to a number of factors, including: