Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy
<p>The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in <em>BEST1</em>, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The correspondi...
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| Format: | Thesis |
| Language: | English |
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2017
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| _version_ | 1826301017885507584 |
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| author | Wood, S |
| author2 | Maclaren, R |
| author_facet | Maclaren, R Wood, S |
| author_sort | Wood, S |
| collection | OXFORD |
| description | <p>The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in <em>BEST1</em>, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The corresponding protein, bestrophin-1, is localised to the basolateral membrane of the retinal pigment epithelium (RPE), where it is thought to function as a Ca<sup>2+</sup>-activated Cl- channel. Currently, there are no treatments for these conditions. In recent years, gene therapy has emerged as an exciting treatment option for inherited retinal disorders (IRDs). Gene delivery to retinal cells using a recombinant adeno-associated virus (rAAV) has produced positive results in several IRDs. Given the recessive nature of ARB, this thesis proposes that the rAAV-mediated delivery of bestrophin-1 to the RPE could represent a potential therapy. The aims of this thesis were to produce and compare rAAV vectors <em>in vitro</em> and <em>in vivo</em> for protein expression, localisation following transduction, restoration of chloride conductance <em>in vitro</em> and safety following sub-retinal injection <em>in vivo</em>. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells <em>in vitro</em>. Immunocytochemistry (ICC) revealed bestrophin-1 expression that was localised to the cytosol. Whole-cell patch-clamping revealed a significant increase in chloride conductance in HEK293 cells transduced with AAV-BEST1 vectors which was then ablated upon the removal of chloride from the buffers. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) indicated that the bestrophin-1 protein was successfully transcribed and translated from the BEST1 coding sequence (CDS). Sub-retinal injections of AAV-BEST1 produced bestrophin-1 expression in the RPE of wild-type C57BL/6 mice however significant retinal thinning was seen at higher doses of vector. In conclusion, rAAV-mediated transfer of bestrophin-1 to the RPE has potential to be a future therapy for ARB, however safety issues need to be addressed and an RPE-specific promoter could be more suitable.</p> |
| first_indexed | 2024-03-07T05:25:59Z |
| format | Thesis |
| id | oxford-uuid:e0925bc0-8f36-4a76-9366-bc7dc316c5af |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:25:59Z |
| publishDate | 2017 |
| record_format | dspace |
| spelling | oxford-uuid:e0925bc0-8f36-4a76-9366-bc7dc316c5af2022-03-27T09:48:14ZDevelopment of AAV-mediated gene therapy for autosomal recessive bestrophinopathyThesishttp://purl.org/coar/resource_type/c_db06uuid:e0925bc0-8f36-4a76-9366-bc7dc316c5afGene therapyOphthalmologyEnglishORA Deposit2017Wood, SMaclaren, R<p>The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in <em>BEST1</em>, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The corresponding protein, bestrophin-1, is localised to the basolateral membrane of the retinal pigment epithelium (RPE), where it is thought to function as a Ca<sup>2+</sup>-activated Cl- channel. Currently, there are no treatments for these conditions. In recent years, gene therapy has emerged as an exciting treatment option for inherited retinal disorders (IRDs). Gene delivery to retinal cells using a recombinant adeno-associated virus (rAAV) has produced positive results in several IRDs. Given the recessive nature of ARB, this thesis proposes that the rAAV-mediated delivery of bestrophin-1 to the RPE could represent a potential therapy. The aims of this thesis were to produce and compare rAAV vectors <em>in vitro</em> and <em>in vivo</em> for protein expression, localisation following transduction, restoration of chloride conductance <em>in vitro</em> and safety following sub-retinal injection <em>in vivo</em>. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells <em>in vitro</em>. Immunocytochemistry (ICC) revealed bestrophin-1 expression that was localised to the cytosol. Whole-cell patch-clamping revealed a significant increase in chloride conductance in HEK293 cells transduced with AAV-BEST1 vectors which was then ablated upon the removal of chloride from the buffers. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) indicated that the bestrophin-1 protein was successfully transcribed and translated from the BEST1 coding sequence (CDS). Sub-retinal injections of AAV-BEST1 produced bestrophin-1 expression in the RPE of wild-type C57BL/6 mice however significant retinal thinning was seen at higher doses of vector. In conclusion, rAAV-mediated transfer of bestrophin-1 to the RPE has potential to be a future therapy for ARB, however safety issues need to be addressed and an RPE-specific promoter could be more suitable.</p> |
| spellingShingle | Gene therapy Ophthalmology Wood, S Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title | Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title_full | Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title_fullStr | Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title_full_unstemmed | Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title_short | Development of AAV-mediated gene therapy for autosomal recessive bestrophinopathy |
| title_sort | development of aav mediated gene therapy for autosomal recessive bestrophinopathy |
| topic | Gene therapy Ophthalmology |
| work_keys_str_mv | AT woods developmentofaavmediatedgenetherapyforautosomalrecessivebestrophinopathy |