Gene therapy and genetic disease

Well over 4000 genetic diseases have been characterized to date. Many of these are caused by lack of production of a single gene product or are due to the production of a mutated gene product incapable of carrying out its natural function. Gene therapy represents a seemingly straightforward therapeutic option that could correct such genetic-based diseases. This would be achieved simply by facilitating insertion of a 'healthy' copy of the gene in question into appropriate cells of the sufferer.

Although simple in concept, the application of gene therapy to treat/cure genetic diseases has, thus far, made little impact in practice. The slow progress in this regard is likely due to a number of factors. These include:

• The number of genetic diseases for which the actual gene responsible has been identified and studied are relatively modest, although completion of the human genome project should rapidly accelerate identification of such genes.

• As discussed previously, none of the first-generation gene-delivering vectors have proven fully satisfactory.

• Some genetic diseases are quite complex, with several organs/cell types being affected. In most instances, it has proven difficult in practice to introduce the required gene into all the affected cell types.

• Regulation of expression levels of the genes transferred has proven problematic.

• Drug companies often display greater interest in applying gene therapy to more prevalent diseases, such as cancer. The patient population suffering from many genetic diseases is relatively modest. In some instances, a limited patient population may not be sufficient to allow the developing company to recoup the cost of drug development.

Some of the genetic conditions for which the defective gene has been pinpointed are summarized in Table 14.4. Many of the initial attempts to utilize gene therapy in practice focused upon haemoglobinopathies (e.g. sickle cell anaemia and thalassaemias). These conditions were amongst the first genetic disorders to be characterized at a molecular level, with the defect centring around the haemoglobin a- or P-chain genes. Furthermore, the target cells in the bone marrow could be removed and subsequently replaced with relative ease. However, these conditions proved to be a difficult initial choice for the gene therapist. The production of the appropriate quantities of functional haemoglobin is dependent not only upon the presence of a- and P-globin genes of the correct

Table 14.4 Some examples of genetic diseases for which the defective gene responsible has been identified


Defective genes protein product

Haemophilia A

Factor VIII

Haemophilia B

Factor IX



Sickle cell anaemia

ß -globin

Familial hypercholesterolaemia

Low-density protein receptor

Severe combined immunodeficiency

Adenosine deaminase

Severe combined immunodeficiency

Purine nucleoside phosphorylase

Niemann-Pick disease


Gaucher's disease


Cystic fibrosis

Cystic fibrosis transmembrane regulator



Leukocyte adhesion deficiency



Ornithine transcarbamylase


Arginosuccinate synthetase


Phenylalanine hydroxylase

Maple syrup disease

Branched chain a-ketoacid dehydrogenase

Tyrosinaemia type 1

Fumarylacetoacetate hydrolase

Glycogen storage deficiency type 1A




Mucopolysaccharidosis type VII


Mucopolysaccharidosis type I



Galactose-1-phosphate uridyl transferase

sequence, but also upon detailed regulation of gene expression. Such tight regulation of expression of transferred genes is beyond the capability of gene therapy technology as it currently stands.

Another early genetic disease for correction by gene therapy was SCID. One form of this disease is caused by a lack of adenosine deaminase (ADA) activity. ADA is an enzyme that plays a central role in the degradation of purine nucleosides (it catalyses the removal of ammonia from adenosine, forming inosine, which, in turn, is usually eventually converted to uric acid). This leads to T- and B-lymphocyte dysfunction. Lack of an effective immune system means that SCID sufferers must be kept in an essentially sterile environment.

When compared with treating diseases such as thalassaemia, regulation of the level of expression of a corrected ADA gene was believed to be less important for a successful therapeutic outcome. (In most, though not all, metabolic diseases caused by an enzyme deficiency, it appears that expression of even a fraction of normal enzyme levels is sufficient to ameliorate the disease symptoms.)

Gene therapy trials aimed at counteracting ADA deficiency were initiated in 1990. The first recipient was a 4-year-old SCID sufferer. The protocol used entailed the isolation of the child's peripheral lymphocytes, followed by the in vitro introduction of the human ADA gene into these cells, using a retroviral vector. After a period of expansion (by culture in vitro), these treated cells were re-injected into the patient. As the lymphocytes (and, by extension, the corrective gene) had a finite life span, the therapy was repeated every 6-8 weeks. This approach appeared successful, in that it has resulted in a marked and sustained improvement in the recipient's immune function. Critically, however, interpretation of this outcome was made more difficult owing to the later revelation that the patient also initiated more conventional SCID therapy just prior to the gene therapy treatment. A second retroviral-based trial aiming to treat a different form of SCID has also been discussed earlier in this chapter.

Haematopoietic (and indeed other) stem cells are attractive potential gene therapy recipient cells because 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 haematopoietic stem cells has, thus far, proven 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 hypercholesterolemia and cystic fibrosis. Familial hypercholesterolemia 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. The 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 CFTR, a 170 kDa protein that serves as a chloride channel in epithelial cells. Inheritance of a mutant cftr gene from both parents results in the cystic fibrosis phenotype. While various organs are affected, the most severely affected are the respiratory epithelial cells. These cells have, unsurprisingly, become the focus of attempts at corrective gene therapy. Cystic fibrosis is the most common inherited mono-genetic disease in Europe and the USA, and sufferers have a typical life expectancy of less than 40 years. Over a third of the 100 or so gene therapy trials thus far undertaken to treat inherited disorders have specifically targeted cystic fibrosis.

Several vectors have been used in an attempt to deliver the cystic fibrosis 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 per cent of the target cell population receive and express the CFTR gene. This level of integration has not been achieved so far; and, furthermore, gene expression has often been transient.

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