AS Biology CourseworkRetinal Gene Therapy in Patients with ChoroideremiaBy David WrightThe ProblemChoroideremia is a recessive genetic disorder caused by a genetic defect that leads to blindness due to mutations in the CHM gene (1). The disease causes a gradual loss of vision over a period of time; sufferers report that their central vision is affected by a ring of darkness that gradually extends towards the periphery of the eye (1). The sufferers are almost exclusively male, because the males need only one mutated copy of the gene from the X chromosome (one of two sex chromosomes) whereas the female requires both mutated X chromosomes (18). Because the disease is a result of an X chromosome, fathers cannot pass the trait onto their offspring (as they provide the Y chromosome during fertilisation) meaning that choroideremia is passed on by the mother, who provides the X chromosome (18). As the disease occurs over a long period of time, sufferers of the genetic disease can often only become aware of the problem later on in life(2) usually within the 5th decade(1). It is estimated that there are around 1000 sufferers of choroideremia in the UK (9), with a prevalence of around 1 in 50,000 (1).What are scientists doing to come up with a solution?In an attempt to combat the disease, a team of scientists lead by a Dr Robert MacLaren asked Can gene therapy be applied to neurons in the retina to help sufferers with choroideremia? Gene therapy is the process of altering the alleles present within a cell to cure a genetic disorder (19). Gene therapy has never been used on patients suffering choroideremia before, and this is the first attempt to target a gene within the photoreceptors of the eye specifically (3). Dr Robert MacLaren and his team decided to run a clinical trial in which 6 patients had an intravitreal ciliary neurotrophic factor implant (a gene they believed could help patients regain some or all of their vision was injected into a specific part of the retina) (1). The method the doctors used required the patients to undergo very invasive surgery of the eye.

Figure 1 Retinal Gene Therapy: Diagram showing the insertion of a surgical needle into the eye and touching the retinas photoreceptor cells. Once in place, the needle injects the replacement gene into the necessary area (20).They required a way insert the CHM gene into the affected cells (the official name of this gene is choroideremia Rab escort Protein 1). For this they required a viral vector. In molecular biology a viral vector is when genetic material is placed into a virus (or vehicle) that is then placed into the area of the body the scientist is targeting (4). The virus transfers the gene over to the affected cells when the virus hijacks the cell. The chromosomes within the affected cell will incorporate the healthy genes and this will affect the synthesis of proteins as the genetic code of the DNA molecule has changed. During transcription, the free-floating RNA molecules will align a different way to what they once did when the cell contained the mutated CHM gene. This means the newly assembled RNA molecule will be translated in a different way too, and the protein will fold in a different way causing its function to be different. In most cases of gene therapy to date, the virus itself is usually incapable of damaging the cell as the genome required for viral replication is removed. As it cannot reproduce, the virus transduces the medical solution to the cell, but then dies shortly after (transduction is the process by which foreign DNA is introduced into another cell via a viral vector) (21).Scientists have used a similar method to treat Cystic Fibrosis; in one clinical trial scientists used a viral vehicle to transfer a protein straight to affected cells in the brain in an effort to improve neuron function (5).

Figure 2 A viral vehicle and the process of transduction: A diagram showing a viral vector packed within a vesicle transporting and transducing a gene into the nucleus of a cell where the gene will be replicated (7).Dr Robert MacLaren and his team decided to use a Woodchuck hepatitis virus as their vehicle (6). They decided to use the Woodchuck hepatitis virus as the virus has been known to enhance AAV-mediated transgene expression (6).As mentioned before, once they had acquired a means to transduce the gene into the healthy cells, they next had to get the vehicle near the affected cells. This required a particular invasive surgery of the eye; the retina of the sufferers eye was detached from the underlying epithelium layer and the genome was injected via a sterile syringe into the affected area. The scientists then waited to see what the results would be.How effective is the solution?Choroideremia is a genetic disease that occurs exclusively in males. To date, there is no other treatment available to sufferers of this disease (8).Gene therapy is an appropriate form of treatment as the disease itself is caused by a genetic defect. Somatic therapy, the process of changing the alleles of body cells, will provide the sufferer with short term relief from the disease. The method of treatment Dr Robert MacLaren adopted relieved symptoms due to the mutated gene being replaced with healthy alleles. As long as the cell is alive, it will no longer express the symptoms of choroideremia. It is important to note that the therapy is not permanent.One problem that may arise during gene therapy treatments is that the patients immune system may detect the viral vehicle and attack the pathogen before it can carry out the transduction. The body would prevent the mutated cells from being replaced.

How reliable is the data?Figure 3 Changes in visual function from baseline to 6 months after gene therapy(6)Figure 3 shows that there has been some improvement in the patients that underwent treatment. Improvement was measured by the process of standardised refraction. Considering that there have been no previous medical advancements for choroideremia, the results collected by Dr Robert MacLaren are promising as improvement has been found, whereas before, choroideremia was a disease medical organizations had no solution for, and sufferers had no choice but to rely on blindness aids such as guide dogs and canes. The clinical trial has treated little more than a handful of sufferers (6 patients) and thus provides insufficient evidence that this method would be a final solution to the disease. A larger sample group numbering within the thousands is needed, to show that the medical solution is plausible once all the factors have been included such as sex, age, weight, genetic history etc. The therapy must also be repeated to prove that their results are reliable and to ensure the validity of the treatment.The implications of the methodEthical Implication- Animal TestingLike most other medical methods, gene therapy is first tested on animals before being tested on humans. In the case of gene therapy there are usually two different clinical tests performed on an animal. The first test is to see whether the viral vehicle will reproduce and damage the animal (10). They need to do this in order to make sure that the virus will not reproduce when in humans, and cause damage to the patient. Many people disagree with the idea of animal testing& believe that the animal should not be made to suffer unnecessarily in ways that do not directly benefit the animal itself. This is in contrast to defenders of animal testing who state that modern laws now require them to limit the suffering of animals and to care for them properly. They argue that without animal testing, drugs such as antibiotics and painkillers would not exist and that at this moment in time, animals play a vital role in the future development of medicine.

Social/Economical Implication- Who can afford the treatment?Gene therapy is a new method of treatment, and not all genetic therapy methods have been thoroughly clinically tested. This particular treatment was performed in a clinical trial, and requires many more years of trial and error before the UK will approvethe treatment to be commercially viable. Once approved, there is the issue of cost. Retinal gene therapy is hugely expensive(11), estimated to be around 1,000,000 per patient (22)and it is very unlikely that the NHS would be able to support gene therapy as an option to most of its patients. This means that only the more privileged citizens would be able to afford treatment. This could likely start a debate as to whether this is socially ethical or not. Why should only the rich be able to have this treatment? That being said, as choroideremia has no known cure and gene therapy is amongst the only medical options available at this time, this implication could be unavoidable. Benefits of the SolutionChoroideremia is a genetic disease. The only way to cure genetic diseases is to use gene therapy (16). Whilst drug treatments have not been devised for choroideremia, should drug treatments be made available at some point in the future they will often give the patient debilitating side-effects ranging from simple nausea to extreme pain (17). Gene therapy would not provide the patient with this experience. The relief of blindness would give the patient a degree of independence once again. Choroideremia affects a man throughout his life, but as mentioned previously, symptoms are usually at their worst within the 5th decade of life. They would have been familiar with their independence in younger years and being robbed of their eyesight could mean they would require a carer or a guide dog with them at all times. If the mans eyesight was restored as a result of gene therapy, the man could regain some of his independence. This would have a positive effect on the mental and emotional wellbeing of the man.Relieving as many people as possible of choroideremia could help the NHS financially as they would no longer have to supply as many people with support such as guide dogs. The finances could then be focused to whatever field needs the money.Gene therapy is a hotly debated subject right now and many people, scientists and ethicists alike, believe the cons of gene therapy outweigh the pros. The success of any and all trials of gene therapy could give the pro-gene therapy argument extra clout.Whilst currently illegal at the moment, gene therapy could wipe out all the hereditary disease known to man if gene therapy was applied to the gametes of people with genetic disorders.

Risks of the SolutionPatients are required to undergo retinal detachment (1). This is a delicate method of work which requires concentration and a steady hand. It isnt unreasonable to think that something could go wrong during such a procedure. A fully qualified surgeon could still cause irreparable damage to the cells beneath the retina or even the retina itself.Surgery is conducted in a surgical theatre at a hospital. Every care is taken to maintain a clean and bacterial free environment, but mistakes do happen. The surgeons themselves could be carrying a pathogen and may not know it (as symptoms may not have shown yet). The surgeons would have opened up the eye and detached the retina. The eyes internal structure is open and vulnerable to infection during this time. The surgeon has to breathe, of course, and whilst breathing, some of the pathogens could be inadvertently passed onto the patient in this vulnerable area. Theres always a risk of this ocurring during surgery.During gene therapy, vectors are needed to transfer the desired alleles into the mutated cells. In the clinical trial performed by Dr MacLaren, a viral vector was used. The virus would have had its reproductive genome extracted from it, preventing it from replicating itself and causing damage to the patients cells. The virus itself can still be detected by the patients immune system and an immune response may be triggered should the patients immune system detect the pathogen. Alternative forms of treatmentChoroideremia has no known cure. Gene therapy is, to the knowledge of the scientists involved in the clinical trial, the only known study to have produced positive results. The treatments mentioned below are therefore speculative and would require a significant amount of financial investment and time. The treatments are not available as of yet and may only be available within the distant future.Retinal MicrochipsIn February 2013, Germans scientists at Retina Implant used artificial retinas in a clinical trial (9). They placed one implant behind one of the eyes of each patient (9 in total) and recorded the results. Eight of the 9 patients recorded a significant improvement in light perception with 5 of them being able to detect moving patterns and 3 of the patients able to read letters (12).The implant itself is a three millimeter square chip, with 1,500 pixels. A photodiode receives the light through the eye and the microchip dicerns what the eye is looking at. The aim of the implant is to replace the photoreceptors that have degenerated and then allow the bodys retinal circuitry to do the rest (12). The implant itself was not tested on patients with choroideremia, but in theory, should be able to work on choroideremia sufferers too. The implant requires an external power supply, placed behind the ear. The power supply would need to be replaced or recharged which means that for a duration of time the patients would be robbed of their eyesight again. In order to have the implant placed behind the eye, surgery is needed which leaves the patient open to infections (just like the retinal gene therapy would). The procedure, like the retinal gene therapy, is likely to be expensive and could raise the previously mentioned socio-economical argument. However, it may be easier to reduce the costs of artifical implants if the NHS managed to purchase a contract with a manufacturer, than it would be to reduce the costs incurred by gene therapy. One potential benefit of using artifical technology, is that the patients immune system is much less likely to attack it. With retinal gene therapy, there is a risk that the immune system will attack and destroy the virus, thereby preventing transduction. As plastics and metals are not coated in antigens, the risk is much more reduced as the immune system cannot detect whether the implant is foreign or not.

Figure 4: A detailed diagram of how the implant is placed within the eye.Retinal Stem Cell TherapyA stem cell is an unspecialised cell that can be turned into a specialised cell (a cell with a specific function). The two main types of stem cell are embryonic stem cells and adult stem cells (13); embryonic stem cells can specialise into any cell in the human body where as adult stem cells are usually limited to specialising into cells where they may be found, for example when an adult stem cell is found in the bone marrow, it may only be able to specialise into red blood cells.The aim of stem cell therapy is to specialise stem cells into cells that are needed by the body, in this case retinal cells such as photoreceptors. Retinal cells do not undergo mitosis to reproduce upon cell death (14) and so once theyre gone theyre usually gone for good. If stem cells were specialised as retinal cells they could be injected into the area of the eye Theoretically this could prevent age-related blindness. In the case of choroideremia, the stem cells could be specialised as retinal cells and then scientists could perform surgery on the retina to discard the cells affected by the choroideremia mutation and replace them with the new, unaffected retinal cells made from the stem cells. There are advantages and disadvantages to using stem cell therapy. It has been reported that stem cells can be found within the retina. If retinal stem cells can be provided by the patient themselves, this could prevent the issue of an immune response as the stem cells will have the recognisable antigen coating. Stem cells could be taken from the patient via retinal surgery, grown in a culture in a laboratory, specialised within said laboratory, and then injected into the required area during surgery. The whole process would require the patient to undergo surgery twice; this whole process is called autologous transplantation (14). Whilst it is true that stem cells can be found within a healthy retina, it is not known wheter or not stem cells can be found in a patient whos retinal cells are experiencing degeneration. The number of stem cells found in the healthy retina is likely to be low, and so it will be difficult for scientists to extract them efficiently. Also, whilst stem cells have been found in the retina, it is not known whether or not the stem cell can be used to replicate the photoreceptors needed (14).If a patient had a hereditary disease, as is the case with choroideremia sufferers, any stem cells extracted from them would also contain mutated genes. This would render the advantage of being able to treat a patient with only their own cells, and any stem cell based treatment would require a donor. Having a donor provide the stem cells means the doctor runs the risk of an immune response within the patient that would attack the viral vector and prevent the stem cell treatment from working. If doctors opted to use stem cell therapy, it is likely they would have to use gene therapy to treat the patients mutated stem cells first(14) if they wanted to prevent the need for a donor. This would then beg the question why use gene therapy to treat stem cells to then treat the patient, when you can cut straight to the chase and use gene therapy directly on the patient?. This is another reason that make the use of gene therapy for choroideremia an appropriate form of treatment.All stem cells used during the therapeutic treatment must be 100% specialised, as unspecialised stem cells have been found to have a propensity to form cancerous tumours within the body (15).Evaluation of the SourcesRetinal Gene Therapy in Patients with Choroideremia: Initial Findings from a Phase Clinical Trial-January 2014- Author: Dr R.E. MacLarenThis is a primary source as the report has come directly from the scientists involved in the treatment I have based my report on. The report published by Dr MacLaren and his team was peer-reviewed and commented on by Drs Hendrik P.N Scholl & Jos A Sahel, scientists not involved in the trial or publication of the report and both are experts within the same scientific field as Dr MacLaren and his team.The funding for Dr MacLarens research was procured from the Health Innovation Challenge Fund. Being financed by an external organization can lead to the manipulation of data so as to appease to the benefactors expectations, causing damage to the reliability of the study. However, to allay concerns on funding bias, the publisher of this report stated:The funding source had no role in data gathering or interpretation, or in writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. The report was concise and clearly stated the variations between the patients, such as age and the stage of retinal cell degeneration each patient was currently at (which could affect results). Because of this, I believe that the report given to me by Dr MacLaren is reliable and valid.---------------------------------------------------------------------------------------------------------------------------http://www.ffb.ca/eye_conditions/choroideremia.html?PHPSESSID=6d92cdc3aa6245ec744d26349d4f5d08 The Foundation Fighting Blindness (Canada) February 15th 2012 Author of the article unattainable Peer Reviewed by Dr Ian MacDonaldThis is a secondary source taken from a charity website called the Foundation Fighting Blindness. As the authors of this article are not directly involved in research, they do not fall under the influence of funding bias. The charity is an advisory body that aims to make information on sight-related diseases more available to the public and provides detailed descriptions and explanations on each disease listed on their webpage.The articles published on the website are reviewed on a regular basis by scientists within the field of work the article is based on. For this reason I think this source is reliable as it provides an unbiased overview of the disease I have written my report on.