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Genes can be described as the basic units of heredity. All living organisms depend on genes because they hold information for building and maintaining cells and encode traits that can be passed down to offspring. Often times, genes can be altered during replication and this can change protein encoding, resulting in the formation of non-functional or ineffective proteins. Such cases are termed as 'genetic disorders' and they can have profound effects on the health of an organism.
Gene therapy is one of the prime methods of correcting defective genes as a means to treat such individuals. In its simplest description, this therapy involves altering, inserting or removing genes within an organism's cells and tissues to treat diseases. Changing the gene, and correcting the defective nucleotides, allows for the production of effective and functional proteins which can meet the body's needs. The most common method of executing gene therapy involves inserting the normal (therapeutic) gene into a non specific region in the genome without acting on the defective gene. However, as scientists begin to better understand the mechanisms of this therapy, new approaches are being tried. For example, a more recent approach currently being studied involves altering the regulation (the turning on or off) of the defective gene as opposed to the nucleotide sequence in the gene itself.
Gene therapy is administered through many different vectors, which are carrier molecules that are used to deliver the therapeutic gene to the patient's target cell. Broadly, these vectors can be divided into virus vectors and non viral vectors. Viruses typically release genetic material into the host cell; however by replacing their pathogenic viral genes with the therapeutic gene scientists can use viruses to deliver genes to target cells in patients with genetic disorders. Hence, a normal (wild type) copy of the defective gene can be inserted to allow the body to synthesize its required proteins. Viral vectors include the use of retroviruses, adenoviruses, or the herpes simplex viruses. Non viral vectors include the use of liposomes to deliver the genetic information, and another example involves chemically linking the gene to a carrier molecule which would bind to surface receptors on the target cell and be endocytosed.
Currently, there are no government approved gene therapy products or procedures; however there is active ongoing research in using gene therapy as a viable treatment option. Many scientific trials have been performed in animals over the past few years but only recently are trials being actively executed in humans to treat genetic disorders. One such trial involves evaluating gene therapy to treat immunodeficiency in patients with adenosine deaminase deficiency (ADA deficiency).
Adenosine deaminase (ADA) is an enzyme that metabolizes purines and it is essential for life. The enzyme is primarily required for the breakdown of adenosine that is obtained from foods to make nucleic acids through the salvage pathway (a pathway of the synthesis of essential nucleotides using nucleotide intermediates). Deficiency of this enzyme occurs through an autosomal (on chromosome 20) recessive (need two copies of the defective gene (from mother and father) to inherit the disease) disorder and this leads to a toxic accumulation of purines in the body which is detrimental to the development of T cells and B cells. Thus, patients with ADA deficiency often have an impaired immune response and suffer from recurrent infections. Conventional treatment for ADA deficiency relies on injecting patients with polyethylene modified bovine ADA, (PEG-ADA) which is a formulation of the adenosine deaminase enzyme. However this treatment is expensive as it requires life-long injections and it fails to provide a sustained improvement to patients' immune systems.
In a recent study conducted at the San Raffaele Foundation, doctors used gene therapy to treat 10 children (median age of 1.7 years) with ADA deficiency and they followed them for 1 year to monitor any improvements in health or any adverse effects that may have occurred (Aiuti et. al, 2009). The study involved obtaining CD34+ cells (stem cells found in the body with surface glycoproteins for adhesion) from each child's own bone marrow, transducing the cells with a retroviral vector which carried the gene for the production of the ADA enzyme, and infusing the cells back into the children. Overall, the study found that after gene therapy, the presence of the ADA enzyme in the patients could be clearly documented (through monitoring its enzymatic activity in blood cells). After 1 year, the median ADA activity in the blood was found to be significantly higher (497nmol/hr/mg vs. 65nmol/hr/mg) in the patients than what was observed before therapy. This increased activity also "...resulted in a significant reduction of toxic levels of purine metabolites in red cells at 1 year as compared with levels at diagnosis in the same patients" (Aiuti et. al, 2009). However, within the study, one of the patients had episodes of autoimmune thrombocytopenia (low blood platelet counts) and required the reintroduction of PEG-ADA injections 5 months after the gene therapy treatment.
Hence, within the study, of the 10 patients that were treated, 9 showed signs of restoration of their immune system and a certain level of protection against severe infections. Doctors also found that the cells infused with the ADA gene passed down a copy of the gene to all their daughter cells and this allowed for sustained expression of the ADA enzyme which resulted in detoxification of the purine metabolites. Additionally, the authors found that the intracellular expression of the ADA gene thorough gene therapy was far more effective than using PEG-ADA, which is the conventional method of treatment for the disease. Overall, authors found gene therapy as a safe and effective treatment for immunodeficiency associated with ADA deficiency and they noted that gene therapy can be extended to treat other such congenital diseases.
Indeed, gene therapy is also being studied as a form of treatment for other congenital diseases. One such example is a study conducted at University College London to treat Leber congenital amaurosis (LCA) with is a rare, genetic eye disease that is often detected at birth. The disease causes poor vision at first which, over three decades of life, develops into complete blindness. LCA is an autosomal recessive disorder which makes rod cells in the retina unable to respond to light. The disease occurs as a result of a mutation of one of several genes, including RPE65, which is expressed in the retinal pigment epithelium cells. This study included 3 young adult patients (17 to 23 year olds) with early onset of abnormal retinal development caused by a mutation in the RPE65 gene. The study involved subretinally (beneath the retina) delivering a recombinant adeno-associated virus vector which was infused with the RPE65 gene in addition to a 1400 base pair fragment of the human RPE65 gene promoter region, and terminated by a poly(A) tail (polyadenylation site) (Bainbridge et. al, 2008).
Before the study began, each patient had little or no low light vision but some limited vision in good lighting conditions. After the gene therapy treatment, doctors found no clinically significant improvement in the visual acuity of the three patients. There were no changes observed in how the retina responded to light or flash patterns and for two patients there was no overall change in retinal function. However, the third patient showed some improvement in detecting light as he was able to see "â€¦small spots of light that were 1/25th as bright as those that could be seen before treatment" (Bainbridge et. al, 2008). The study also found no adverse events that could be associated with the gene therapy. In the one patient where improvement in the retinal function was found, the doctors could not be sure whether the improvement was entirely due to the injected RPE65 gene in the retina. The authors of the study stated that such information can only be obtained through a biopsy of the retina and that would be unsafe and unethical. Thus the conclusion of the study was that further research is needed because there is a chance that RPE65 gene therapy can lead to modest visual improvements in patients with a highly degenerated retina.
Research in gene therapy however, is not just being conducted on autosomes but use of the therapy in congenital diseases associated with sex-linked chromosomes is also being studied. A study by Cartier N. et al, 2009, examined the effects of gene therapy on patients with Adrenoleukodystophy (ALD) which is a rare genetic disorder that leads to progressive brain damage and eventually death. ALD is caused by a defect in the ABCD1 gene which leads to a deficiency in adenosine-5'-triphosphate (ATP) binding cassette transporter, a transmembrane protein that uses ATP to transport various substances across cell membranes. In this study, two ALD patients had CD34+ cells removed from their own bone marrows, and these cells were then infused with a lentiviral vector (lentiviruses are slow incubating retroviruses) containing the wild type ABCD1 gene. These cells were then re-infused back into the two patients and the effects were observed. The study found that 14 to 16 months after the infusion the slow destruction of the myelin sheaths in the cerebral region had stopped in the two patients. Hence the authors concluded that lentiviral gene therapy can provide significant clinical benefits in ALD patients.
As new findings occur in the applications of gene therapy, there are also new avenues being explored in enhancing gene therapy and finding new approaches. One example is exploring the use of small interfering ribonucleic acid (siRNA) molecules which are 20-25 nucleotides long, double stranded RNA molecules that play a role in the expression of specific genes. Hence, it is believed that siRNA molecules can be extremely useful in imparting gene therapy to patients in the future. However, the challenge lies in the need to deliver the molecules intracellularly to the specific tissues, organs and cells that express the target gene. A recent study by Davis M. et. al, 2010, utilized nanotechnology to deliver the siRNA molecules through tiny polymers covered with transferrin (a blood plasma protein that delivers iron). The nanoparticles contained a 'chemical sensor' that recognized when the molecule was inside the cell and when it was safe to give off the siRNA to regulate a target gene expression. The study involved a phase 1 clinical trial whereby doses of the nanoparticles were intravenously infused in patients with tumors. It was observed that the nanoparticles were able to go inside the tumor cells and release the siRNA molecule as was evident by the measured disablement of ribonucleotide reductase (an enzyme that catalyzes the formation of deoxyribonucleotides).
Gene therapy is not just being studied in rare congenital diseases but it is also being explored in relatively more common diseases such as heart failure and Alzheimer's disease. For example, recently scientists at the Columbia University Medical Centre were able to restore the heart's ability to pump blood in over 39 heart failure patients by introducing the SERCA2a gene in patients where this gene was depressed. The SERCA2a gene raised the levels of the enzyme it coded for allowing better regulation of the cycling of calcium in the body, which directly affected how well the heart contracted (Stiles, 2010).
The future of gene therapy appears to be promising in treating many diseases. There are however certain problems that would need to be overcome and these include ways to target specific cells and tissues, delivering genes to the target cells effectively and ensuring the safe use of viral vectors. Additionally, gene therapy also has its limitations in that the therapeutic gene must integrate with the human DNA and for this to occur the target cells must remain functional and stable. Problems with integrating the gene into the DNA or the rapid division of cells may prevent gene therapy from providing long-term benefits and patients may need to undergo multiple rounds of gene therapy. Furthermore, the introduction of viral vectors may induce strong immune responses in certain patients and there is also a fear that the once the virus is inside the patient it may recover its ability to cause disease. Gene therapy also has only been studied in disorders of a single gene, however some very common disorders (eg. diabetes, high blood pressure) are multigene disorders and thus they would be difficult to treat effectively by gene therapy. Lastly, if the therapeutic gene is integrated in the wrong place in human DNA (for example in a tumor suppressing gene) it could potentially cause a tumor to develop in such patients. Hence gene therapy still needs to be fully studied and well understood and therefore, although it shows great potential, it appears to be still many years away from being freely utilized as an accepted health care procedure.
Aiuti, A, Cattaneo, F, Galimberti, S, Benninghoff, Cassani, B. et al (2009). Gene Therapy for Immunodeficiency due to Adenosine Deaminase Deficiency. The New England Journal of Medicine, 360(5), Retrieved from www.nejm.org/doi/full/10.1056/NEJMoa0805817
Bainbridge, J, Cattaneo, A, Smith, S, Barker, Robbie, S. et al (2008). Effect of Gene Therapy on Visual Function in Leber Congenital Amaurosis. The New England Journal of Medicine, 358(21), Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18441371
Cartier, N, Abina, S, Bartholomae, C, Veres, G, Schmidt, M. et al (2009). Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy. Science, 326(5954), Retrieved from http://www.sciencemag.org/content/326/5954/818.abstract
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