The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in BEST1, 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 Ca²⁺-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 in vitro and in vivo for protein expression, localisation following transduction, restoration of chloride conductance in vitro and safety following sub-retinal injection in vivo. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells in vitro. 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.

Bestrophin-1 is a homopentameric Ca2+-gated anion channel which localises to the basolateral plasma membrane of retinal pigment epithelium (RPE) cells. Homozygous and compound heterozygous mutations in the BEST1 gene are associated with autosomal recessive bestrophinopathy (ARB), a retinopathy characterised by altered electrooculogram (EOG), deposits in the retina, and is often associated with the risk of developing angle-closure glaucoma. The mechanism by which mutations in bestrophin-1 cause disease remains unknown. Expression of four ARB-causing bestrophin-1 proteins in polarised MDCKII cells, a cell model for RPE, results in mutant proteins mislocalisation and degradation. Furthermore, when the ability of the mutant proteins to conduct Cl- ions was investigated in HEK293 cells by whole-cell patch-clamp, a reduction in the Cl- current was observed in all mutants compared to the WT.The use of a combination of the small molecules bortezomib and 4-phenylbutyrate (4PBA) successfully restored the expression and trafficking of all four ARB-causing bestrophin-1 proteins. Importantly, 4PBA was also able to restore the ability of the mutant channel to conduct Cl- ions. Biotinylation of cell surface proteins shows that the number of active channels at the plasma membrane of HEK293 cells increases following 4PBA treatment. The functional rescue achieved with 4PBA supports the hypothesis that ARB-associated missense mutations reduce the number of functional channels that reach the cell membrane rather than altering other aspects of channel function. The results presented in this thesis suggest that 4PBA may be a promising therapy for the treatment of ARB and the other bestrophinopathies resulting from missense mutations in BEST1, particularly as 4PBA is already approved for long-term use in infants and adults. These finding also pave the way for the use of small molecule therapies to treat conformational diseases caused by mutation in other protein expressed in the RPE.

<p>Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The <i>VMD2</i> gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.</p><p>In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a <i>C. elegans</i> protein family, expressed in retina, was identified as the <i>VMD2</i> gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the <i>VMD2</i> gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. <i>VMD2</i> expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation.</p><p>Analysis of mouse <i>Vmd2</i> and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. <i>Vmd2</i> expression levels were highest around birth.</p>

Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The VMD2 gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD. In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a C. elegans protein family, expressed in retina, was identified as the VMD2 gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the VMD2 gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. VMD2 expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation. Analysis of mouse Vmd2 and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. Vmd2 expression levels were highest around birth.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

Mutations in BEST1, the gene encoding for Bestrophin-1 (Best1), cause five, clinically distinct inherited retinopathies: Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). Little is known regarding how BEST1 mutations cause disease and why mutations cause multiple disease phenotypes. Within the eye, Best1 is a homo-oligomeric, integral membrane protein that is exclusively localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Here, it regulates intracellular Ca2+ signaling and putatively mediates anion transport. Since defects in localization and oligomerization are known to underlie other channelopathies, we investigated how mutations causal for BVMD, AVMD, ARB, ADVIRC, and RP impact the localization and oligomerization of Best1. We generated replication-defective adenoviral vectors encoding for WT and 31 mutant forms of Best1 associated with these five diseases and expressed them in confluent, polarized Madin-Darby canine kidney and/or RPE cells. Localization was assessed via immunofluorescence and confocal microscopy. Oligomerization was examined using live-cell fluorescence resonance energy transfer (FRET) as well as reciprocal co-immunoprecipitation experiments. We report that all 31 BVMD, AVMD, ARB, ADVIRC, and RP mutants tested can reciprocally co-immunoprecipitate with and exhibit comparable FRET efficiencies to WT Best1, indicative of unimpaired oligomerization. While all RP and ADVIRC mutants were properly localized to the basolateral plasma membrane, many but not all AVMD, ARB, and BVMD mutants were mislocalized to intracellular compartments. When co-expressed with WT Best1, mislocalized mutants predominantly co-localized with WT Best1 in intracellular compartments. Studies involving four ARB truncation mutants reveal that the first 174 amino acids are sufficient to mediate oligomerization with WT Best1 and that amino acids 472-585 are not necessary for proper trafficking. We conclude that, although mislocalization is a common result of BEST1 mutation, it is not an absolute feature of any individual bestrophinopathy. Moreover, we show that some recessive mutants mislocalize WT Best1 when co-expressed, indicating that mislocalization cannot, on its own, generate a disease phenotype, and that the absence of Best1 at the plasma membrane is well tolerated.