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BDSRA 2015 CLN6 Mole

2015 CLN6 Mole

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BDSRA 2015 CLN6 Mole

  1. 1. Access to UCL e-resources UCL Library Services, Gower St., London WC1E 6BT 020 7679 7700 E-mail: lease note om 1 August 2009, UCL will no longer be using the Athens login for access to electronic sources. You may still see links labelled "Athens login" on various resources, but these will longer work for UCL members. The new access route for UCL users is outlined in this guide. ccessing electronic resources via the UCL Library Services website CL users are advised to access resources through the lists of ejournals ttp:// and databases ( ailable on MetaLib. This will guarantee that you get free access to all the resources to which u are entitled as a UCL member. If you are on-site you will not need to log in to resources. If u are off-site, you will be prompted for your UCL userid and password automatically. This echanism uses a so-called “proxy server” which simulates on-site access once you have tered your UCL login. ow do I log in to UCL e-resources? any electronic resources do not require a login from computers on the UCL network. However, you are using a computer which is not on the UCL network, then when you follow links to Mole & Ali laboratory: Variant Late Infantile Batten Disease Sophia kleine Holthaus1,2, Sara Mole1, Sander Smith2, Robin Ali2 1MRC Laboratory of Molecular Cell Biology Gower Street, London WC1E 6BT United Kingdom Dr. Sara Mole 2Institute of Ophthalmology 11-43 Bath Street, London EC1V 9EL United Kingdom Prof. Robin Ali •  Loss of vision is one of the first symptoms in (juvenile) Batten disease and is characterised by loss of photoreceptors in the retina. It precedes severe neurological symptoms and reduces quality of life of patients and affected families signifcantly. •  Most of the therapies that are currently under investigation for Batten disease target the brain. It is unlikely that these therapies have a beneficial effect on the eye. •  A treatment for the eye may delay the onset of the visual decline or slow down the progression of the vision loss. In the future, a treatment for the eye and the brain could be combined. •  In comparison to the brain the eye is less complex, smaller and easy to access, which facilitates the delivery of therapeutic components. •  For more information on gene therapy for the eye and the structure of the eye and retina please see concept poster “Gene therapy for the eye”. What is the purpose of a gene therapy for the eye? What mouse model is good to test a therapy for the eye? •  Loss of vision is a key symptom in juvenile Batten disease. However, all mouse models that represent juvenile Batten disease show only a mild visual decline and a slow degeneration of the retina making it difficult to test therapies in the eye. •  The Cln6 mouse, a model that represents a variant form of late infantile Batten disease, is a much better model because it shows an early onset and faster progression of the retinal degeneration. In addition, the Cln6 mouse is deficient in a transmembrane protein which poses very similar challenges for the development of a gene therapy as the mouse models of juvenile Batten disease. Aim of project The aim of this project is to investigate how feasible a gene therapy is to improve vision in Batten disease. The ultimate goal of this study is to improve quality of life of patients. Step 1: Characterisation of vision loss in untreated Cln6 mice Test for photoreceptor function 3w 1M 2M 3M 4M 5M 6M Healthy Mutant Age Healthy retina Mutant retina Photo- receptors Photo- receptors Step 2: Delivery of viral vector carrying healthy Cln6 CLN6 virus AAV8.CLN6 virus, high dose Step 3: AAV8.CLN6 viral treatment of photoreceptors in Cln6 mice Healthy Mutant Mutant, very high dose Mutant, high dose Mutant, moderate dose Mutant, low dose Test for photoreceptor function in treated mutant mice 1 month 2 months 4 months 6 months CLN6 virus Mutant, high dose Mutant, moderate dose Mutant, low doseHealthy retina Photo- receptors Photo- receptors Step 4: Looking for a new target It is important to understand when Cln6 mice start to loss vision and how it progresses over time. We found that: •  Photoreceptor function is progressively reduced from 3 weeks of age in Cln6 mice (mutant) compared to healthy animals (A). •  Predominately photoreceptors are dying in the retina of mutant animals, whereas the other two layers of the retina appear preserved (B). A B The characterisation of the mutant Cln6 mice shows that the function of photoreceptors is reduced and that photoreceptors are dying. Subretinal injections are ideal to deliver a virus to photoreceptors throughout the retina. The cartoon depicts the procedure in a human eye (A). We performed subretinal injections of a virus carrying Cln6 (AAV8.Cln6) and show that the majority of photoreceptors are targeted in the retina of a healthy mouse eye (B). A B Step 5: New virus targeting inner retina Next, we injected mutant CLN6 mice subretinally with the AAV8.CLN6 virus and tested photoreceptor function. We found that: •  A very high dose of the virus causes severe loss of photoreceptor function indicating too much virus is detrimental. •  Lower doses do not seem to improve photoreceptor function as the treated mutant eyes show a p r o g r e s s i v e r e d u c t i o n o f photoreceptor function similar to untreated mutant eyes. We investigated the structure of the retina to find out whether the viral treatment slowed down the photoreceptor loss. We found: •  The injection of the virus introduced the expression of Cln6 in photoreceptors across all doses. •  However, none of the viral doses p r e v e n t e d t h e l o s s o f photoreceptors in the mutant mice. CLN6 bipolar Photo- receptors Inner retina, e.g. bipolar cells Healthy retina As the treatment of photoreceptors is not sufficient to treat the retinal degeneration in Cln6 mice, we investigated where Cln6 is expressed in a healthy retina. For this purpose we performed Cln6 antibody staining on human retina. We found that: •  Cln6 is present in photoreceptors but the levels of Cln6 are much higher in the inner layer of the retina (green) (A). This is surprising because the mutant Cln6 mice do not present with a severe loss of cells in the inner retina. •  Cln6 is present in bipolar cells (red), a cell type of the inner retina, that receives signals from photoreceptors and is involved in transmitting the signals to the brain (B). Healthy retina, magnified B A New virus Green virus Photo- receptors New virus Inner retina (bipolar cells) Old AAV8 virus Until recently, there was not a virus available to target the inner retina as most virus cannot physically reach the middle layer of the retina. However, very recently we imported a new virus that can reach the inner retina and allows us to treat cells in the inner retina in Cln6 mice. In addition, we can inject both the new and old vector to target the inner retina and photoreceptors in mutant animals. Summary •  In Cln6 mice photoreceptor function and photoreceptor cells are predominantly lost. •  However, gene therapy targeting photoreceptors is not sufficient to prevent the retinal degeneration in Cln6 mice. •  Cln6 is present in photoreceptors but it is present to a much higher level in the inner retina. •  We now have a viral vector that targets the inner retina and we are currently treating mutant Cln6 mice with this new vector. In addition, we can treat now simultaneously the inner and outer retina when injecting the old and new virus in mutant mice. Old AAV8 virus This project was generously funded by: