The below mentioned article provides a close look on the gene therapy designed for inherited diseases.
Different genetic disorders are amenable to varying degrees to treatment by gene therapy. Common non-Mendelian genetic diseases may involve a complex interplay between different genetic loci and/or environmental factors, and so possible gene therapy approaches may not be .straightforward. Single gene disorders, where individuals are severely affected and where there is no effective treatment, are more obvious candidates for gene therapy.
Increasingly, genes underlying a variety of such diseases are being isolated and characterized as a result of positional cloning and candidate gene approaches. However, differing pathogeneses means that certain single gene disorders will be more amenable to gene therapy approaches than others (Table 23.4).
Recessively Inherited Disorders are Conceptually the Easiest Inherited Disorders to Treat by Gene Therapy:
Those disorders where the disease results from a simple deficiency of a specific gene product are generally the most amenable to treatment: high level expression of an introduced normal allele should be sufficient to overcome the genetic deficiency.
Recessively inherited disorders have been of particular interest as candidates for gene therapy because the mutations are almost always simple loss-of-function mutations. Affected individuals have deficient expression from both alleles and so the disease phenotype is due to complete or almost complete absence of normal gene expression. Heterozygotes, however, have about 50% of the normal gene product and are normally asymptomatic.
Additionally, there is, in at least some cases, wide variation in the normal levels of gene expression, so that a comparatively small percentage of the average normal amount of gene product may be sufficient to restore the normal phenotype. It is also often observed that the severity of the phenotype of recessive disorders is inversely related to the amount of product that is expressed.
As a result, even if the efficiency of gene transfer is low, modest expression levels for an introduced gene may make a substantial difference. This is quite unlike dominantly inherited disorders where heterozygotes with loss-of-function mutations have 50% of the normal gene product and may yet be severely affected.
Although recessively inherited disorders are, in principle, amenable to gene augmentation therapy, certain disorders are less amenable than others. In addition to the question of accessibility of the disease tissue, some disorders may be difficult to treat for other reasons. A good example is provided by β-thalassemia which results from mutations in the β- globin gene, HBB.
This is a severe disorder affecting hundreds of thousands of people worldwide, and superficially would appear to be an excellent candidate for gene therapy: the gene is very small and has been characterized extensively, the disorder is recessively inherited and affects blood cells.
An initial attempt at gene therapy for this disorder in 1980 failed, largely because of inefficient gene transfer and poor expression of the introduced β-globin genes. Even though we now know much about how this gene is expressed, there have been no subsequent gene therapy attempts.
This is due to the problem of the very tight control of gene expression required following insertion of a normal β-globin gene into the desired cells: the amount of β-globin product made must be equal to the amount of β-globin. If too much β-globin were to be made, the imbalance between β-globin and β- globin chains would result in an α-thalassemia phenotype.
The First Apparently Successful Gene Therapy was Initiated in 1990 for Adenosine Deaminase Deficiency:
For years we have been accustomed to the applications of molecular genetics in the diagnosis of disease, and, more recently, to the isolation and characterization of novel disease genes. Now we are living in a decade where molecular genetics is poised, at last, to deliver novel treatments for human disorders. Exciting though this prospect is, the limitations of the current technologies are apparent.
Even now, gene therapy has not cured any patient. Instead, current gene therapy trials are providing forms of treatment for some disorders: there may be amelioration of the disease, but the effects are temporary, and treatments have to be repeated at regular intervals.
The first apparently successful gene therapy was initiated on 14 September 1990. The patient, Ashanthi DeSilva, was just 4 years old and was suffering from a very rare recessively inherited disorder, adenosine deaminase (ADA) deficiency (see Fig. 23.12). ADA is involved in the purine salvage pathway of nucleic acid degradation, and is a housekeeping enzyme which is synthesized in many different types of cell.
An inherited deficiency of this enzyme has, however, particularly severe consequences in the case of T lymphocytes, one of the major classes of immune system cells. As a result, ADA patients suffer from severe combined immunodeficiency.
This severe disorder was particularly amenable to gene therapy for a variety of reasons: the ADA gene is small, and had previously been cloned and extensively studied; the target cells are T cells which are easily accessible and easy to culture, enabling ex vivo gene therapy; the disorder is recessively inherited and, importantly, gene expression is not tightly controlled (normal individuals show a huge range in enzyme levels, from 10% to 5,000% of the average levels).
The observation that allogeneic bone marrow transplantation can cure the disorder suggested that engraftment of T cells alone may be sufficient, and transfer of normal ADA genes into ADA-T cells was noted to result in restoration of the normal phenotype.
Alternative treatments for ADA deficiency do exist. Indeed, the treatment of choice is bone marrow transplantation from a perfectly HLA-matched sibling donor, which provides a cure in about 80% of cases. For children where this is not an option, an alternative is enzyme replacement therapy, consisting of weekly intramuscular injections of ADA conjugated to polyethylene glycol (PEG). PEG stabilizes the ADA enzyme, allowing it to survive and function in the body for days.
Inevitably, however, enzyme replacement therapy does not provide full immune reconstitution and so life expectancy is still likely to be shortened (T cells are required for mounting effective immune responses against invading microorganisms, and in preventing cancer).
The ADA gene therapy approach involved essentially four steps:
(i) Cloning a normal ADA gene into a retroviral vector;
(ii) Transfecting the ADA recombinant into cultured ADA-T lymphocytes from the patient;
(iii) Identifying the resulting ADA+T cells and expanding them in culture;
(iv) Re-implanting these cells in the patient (see Fig. 23.13).
This approach is necessarily a treatment, not a cure — which would instead require successful transfer into bone marrow stem cells. The trouble here is that human bone marrow stem cells are very difficult to isolate, although enrichment is possible using the monoclonal antibody CD34 (which selectively binds a population of cells that includes totipotent stem cells).
There is also the problem that the insertion of retroviral vectors into bone marrow stem cells is very inefficient. The compromise of targeting the differentiated T lymphocytes has meant that stable expression of the introduced ADA genes can be maintained over several weeks.
As a result, however, repeated injections were given, initially every 1-2 months, and subsequently once every 3-6 months. Evidence that the treatment was having the desired effect in kick-starting the patient’s immune system was obtained from various measures of antibody and T-cell function.
In parallel, there has been evidence of clinical improvement: the frequency of infections has dramatically decreased when compared with the incidence before treatment. The efficacy of ADA gene therapy is still difficult to assess because Ashanthi and other patients were simultaneously treated with PEG-ADA.
Since the pioneering work on ADA deficiency, gene therapy trials have been initiated for a few inherited disorders:
For the reasons given above, ADA deficiency presented a favourable target for gene therapy. Since this pioneering work, gene therapy has been initiated for a few additional inherited disorders (Table 23.5), while progress for others has been frustratingly slow. Different recessively inherited disorders have been targets for in vivo or ex vivo gene augmentation therapy and, in the one case where a dominantly inherited disorder has been treated, the patient was a homozygote. The following examples are simply illustrative of current progress and difficulties.
Familial Hypercholesterolemia (FH):
This disorder is caused by a dominantly inherited deficiency of low density lipoprotein (LDL) receptors, which are normally synthesized in the liver, and is characterized by premature coronary artery disease. About 50% of heterozygous affected males die by 60 years of age, unless treated. Because FH is such a common single gene disorder, homozygotes are occasionally seen.
They suffer precocious onset of disease and increased severity, with death from myocardial infarction commonly occurring in late childhood. Gene therapy for FH was first applied with some apparent success to a 28-year-old woman who is homozygous for a pathogenic missense mutation in the LDLR gene. She suffered a myocardial infarction at the age of 16 and required coronary artery bypass surgery at the age of 26.
The liver, being a solid internal organ, may not seem to be an ideal choice for targeting gene therapy, and its major cell-population, the differentiated hepatocyte, is refractory to infection with retroviruses, the most widely used vector system. However, hepatocytes can be cultured in vitro and, under such conditions, are susceptible to retroviral infection.
Ex vivo gene therapy became a possibility when animal experiments showed that cultured hepatocytes could be injected via the portal venous system — the veins which drain from the intestine directly into the liver — after which they appear to seed in the liver.
The gene therapy involved surgical removal of a sizeable portion of the left lobe of the patient’s liver, disaggregation of the liver cells and plating in cell culture prior to infection with retroviruses containing a normal human LDLR gene.
The genetically modified cells were infused back into the patient through a catheter implanted into a branch of the portal venous system. The patient’s LDL/high density lipoprotein (HDL) ratio subsequently declined from 10-13 before gene therapy to 5-8, and such improvement was maintained over a long period.
Cystic Fibrosis:
Cystic fibrosis is an autosomal recessive disorder that results in defective transport of chloride ions through epithelial cells, and results from mutations in a gene, CFTR, which encodes a cAMP-regulated chloride channel. The primary expression of the defect is in the lungs: a sticky mucus secretion accumulates which is prone to chronic infections. Because there are no methods to culture lung cells routinely in the laboratory, in vivo gene therapy approaches have been adopted.
As respiratory epithelial cells are differentiated, retroviral vectors cannot be used. Instead, gene therapy trials have used adenovirus vectors or liposomes to transfer a suitably sized CFTR minigene, either through a bronchoscope or through the nasal cavity. The first adenovirus-based protocol began in 1993 and, although preliminary data have confirmed gene transfer into respiratory epithelium in vivo, there have been major concerns regarding the safety of the procedure.
The first patient to be treated with a high dose of recombinant adenovirus experienced transient pulmonary infiltrates and alterations in vital signs, before recovering uneventfully. This experience prompted recognition of the need to confirm the maximum tolerated adenovirus dose. The liposome-based gene therapy trials are regarded as safer procedures, but the efficiency of gene transfer is expected to be much lower.
Duchenne Muscular Dystrophy:
DMD is a severe X-linked recessive disorder: affected males suffer progressive muscle deterioration, are confined to a wheelchair in their teens and die usually by the third decade. The target tissue is skeletal muscle, and initial interest in treatment for this disorder focused on cell therapy because of the unique cell biology of muscle.
As well as muscle fibers (or myofibers — very long, post-mitotic, multinucleate cells), skeletal muscle contains mononucleate myoblasts which are normally quiescent but can divide and subsequently fuse with myofibers to repair muscle damage.
Although implanting normal or genetically modified myoblasts into diseased muscles appeared attractive, difficulties have been evident with this approach in humans, despite promising pilot studies with myoblast transfer in mice.
Suitable gene therapy approaches have also been difficult to conceive, largely because of the lack of a suitable gene transfer system. Retroviral vectors cannot be used because adult skeletal muscle fibers are post-mitotic and, hence, not susceptible to retroviral infection.
Adenovirus vectors have been used to deliver genes to muscle fibers in vivo and, although the post-mitotic state of muscle nuclei allows the expression to persist, the need for expression to continue over the course of a lifetime (which would be required for successful therapy) remains doubtful.
For this, and many other disorders, one would like to see a vector system that combined the stable expression conferred by integrative vectors such as retroviruses with the wide target cell range of vectors such as adenoviruses. A final problem is the sheer size of the dystrophin coding sequence (~14 kb), although a very large central segment appears not to be crucially important (see England et al, 1990).