Kevin Gregory-Evans - Academia.edu (original) (raw)

Uploads

Papers by Kevin Gregory-Evans

Research paper thumbnail of Mutations in LRP5 or FZD4 underlie the common FEVR locus on chromosome 11q13

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of The Association Of MMP/TIMP Ratios In Neovascular AMD

Research paper thumbnail of Transcriptional Regulation and Expression of the Dominant Drusen Gene<i>FBLN3</i>(<i>EFEMP1</i>) in Mammalian Retina

Investigative Ophthalmology & Visual Science, Nov 1, 2003

PURPOSE. To determine the important transcriptional control elements and sites of expression of f... more PURPOSE. To determine the important transcriptional control elements and sites of expression of fibulin-3 in mammalian retina. METHODS. Sequencing and 5Ј rapid amplification of cDNA ends (RACE) were undertaken to characterize the genomic sequence upstream of the FBLN3 coding sequence. Reporter deletion-mutation constructs were used in luciferase transfection assays to determine the important regulatory motifs. Fibulin-3 expression in mouse and human retina was studied by in situ hybridization and RT-PCR. The effect of 17␤-estradiol on fibulin-3 production was studied in COS-7 and ARPE-19 cells. RESULTS. Two untranslated exons were fully sequenced completing the characterization of FBLN3 that comprises 12 exons. Reporter assays suggest that the FBLN3 proximal promoter is contained within 425 bp upstream of exon 1. Important regulatory elements include three Sp1-binding sites, a Tant motif (trans-activating) and an estrogen response element (ERE) binding site (trans-repressing). No TATA or CAAT regulatory boxes were identified. RT-PCR suggests that the fibulin-3 gene is expressed in murine and human RPE, and in situ studies confirm that Fbln3 is expressed in the outer and inner nuclear layers, but strikingly not in the ganglion cell layer. Fibulin-3 expression in ARPE-19 cells could be modified by varying the amount of estrogen in the cell culture medium. CONCLUSIONS. The 5Ј end of the FBLN3 gene has been characterized, and the important upstream motifs regulating its transcription have been identified. Fibulin-3 is expressed in adult retina and at early stages in human and mouse development. Estrogen may be an important regulator of fibulin-3 expression, and this highlights a novel mechanism by which circulating estrogen may control the composition of the retinal extracellular matrix.

Research paper thumbnail of Nonsense suppression rescues cell death and photoreceptor protein expression in CDH23 patient iPSC-derived retinal organoids

Investigative Ophthalmology & Visual Science, Jul 22, 2019

Research paper thumbnail of Retinal therapy with induced pluripotent stem cells; leading the way to human clinical trials

Expert Review of Ophthalmology, Jan 2, 2019

Introduction: Retinal cell transplantation is a promising approach to delay progression and rever... more Introduction: Retinal cell transplantation is a promising approach to delay progression and reverse blindness caused by infections, genetic inheritance, or associated with aging. Areas covered: The demand for transplantable retinal cells is broad and several cell sources exist that can supply such cells. The recent development of protocols that reprogram adult cells to their pluripotent potential (induced pluripotent stem cells; iPSCs) and the possibility of differentiating iPSCs to many retinal types make these cells very attractive for cell therapeutics in the degenerating retina.iPSCs overcome ethical dilemmas in using fetal cells and with autologous transplantation of iPSC derived retinal cells, immunosuppression will no longer be needed. Importantly, gene editing of iPSCs derived from patients with genetic diseases will allow autologous transplantation of healthy differentiated retinal cells. Expert commentary: Before marketing clinically useful retinal cells, differentiated from iPSCs, a multistep clinical trial is performed. Currently, 23 trials are described in the literature and they report no adverse reactions to cell transplantation. Early visual assessment tests demonstrate small visual benefits in a proportion of participants, with future results anticipated in the near future.

Research paper thumbnail of Proinflammatory Events in the P23H-1 Rat Model of Retinal Degeneration

Research paper thumbnail of Rip3 knockdown rescues photoreceptor cell death in pde6c zebrafish model of achromatopsia

Investigative Ophthalmology & Visual Science, Jun 16, 2013

Research paper thumbnail of Seconadry Ischemic Events Following Non-Arteritic Retinal Artery Ischaemia in Four Uk Centres

Investigative Ophthalmology & Visual Science, May 10, 2007

Research paper thumbnail of Macula Dysfunction in Subretinal Neovascular Membranes Results in Significant Pupillary Escape

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of Characterization of the Genomic and Transcriptional Structure of the CRX Gene: Substantial Differences Between the Human and Mouse Genes

Investigative Ophthalmology & Visual Science, Dec 1, 2002

We have previously shown that there is a temporal difference in human CRX gene expression compare... more We have previously shown that there is a temporal difference in human CRX gene expression compared with that of mouse Crx. We have now characterized these genes at the genomic and transcriptional levels and here we expand on this earlier report. Human CRX spans 25 kb and has six exons, and mouse Crx spans 15 kb and has four exons. We isolated seven human and two mouse mRNAs generated by alternative splicing of a variable 5Ј untranslated region. The human and mouse genes share an evolutionarily conserved promoter, which contains OTX/CRX type and SP1/AP2 binding sites and drives expression of two conserved transcripts in both species. Additionally, the human gene has a second human-specific promoter, which has OTX/CRX type binding sites and drives expression of five other transcripts. Band shift assays have shown that six of the seven candidate OTX/CRX elements bind CRX in vitro, possibly implying that the gene can regulate its own expression. These data may account for the differences in temporal expression in vivo we have previously reported between these two species.

Research paper thumbnail of Role of Receptor Interacting Protein Kinases in the Retinal Degeneration of S334ter and P23H Mutant Rat Models of Retinitis Pigmentosa

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Research paper thumbnail of Photoreceptor precursor cell integration into rodent retina after treatment with novel glycopeptide PKX-001

bioRxiv (Cold Spring Harbor Laboratory), Nov 22, 2020

Cell replacement therapy is emerging as an important approach in novel treatments for neurodegene... more Cell replacement therapy is emerging as an important approach in novel treatments for neurodegenerative diseases. Many problems remain, in particular improvements are needed in the survival of transplanted cells and increasing functional integration into host tissue. These problems arise because of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, toxic compounds released by dying tissues and nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) and showed 2.8-fold improvement in transplant cell survival after pre-treatment with a novel glycopeptide (PKC-100). In this study we extended these studies to look at cell survival, maturation and functional integration in an in vivo rat model of rhodopsinmutant retinitis pigmentosa causing blindness. We found that only when human photoreceptor precursor cells (PPCs) were pre-incubated with PKX-100 prior to transplantation, did the cells integrate and mature into cone photoreceptors expressing S-opsin or L/M opsin. In addition, ribbon synapses were observed in the transplanted cells suggesting they were making synaptic connections with the host tissue. Furthermore, optokinetic tracking and electroretinography responses in vivo were significantly improved compared to cell transplants without PKX-100 pre-treatment. These data demonstrate that PKX-100 promotes significant long-term stem cell survival in vivo, providing a platform for further investigation towards the clinical application to repair damaged or diseased retina.

Research paper thumbnail of Pharmacological Therapy Using Aminoglycosides Can Override the Genetic Defect Causing Choroideraemia in the Zebrafish Model

Investigative Ophthalmology & Visual Science, May 14, 2008

Research paper thumbnail of Fibulin–3 gene mutation leads to upregulation of chaperone proteins and apoptosis in retinal pigment epithelium

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of Oculo-oto-dental Syndrome: Linkage to 20q13.1

Investigative Ophthalmology & Visual Science, Dec 1, 2002

Research paper thumbnail of High–Density SNP Genome Scan Identifies a New Locus for Autosomal Dominant Oculo–Otodental Dysplasia

Investigative Ophthalmology & Visual Science, 2006

Research paper thumbnail of Prolonged Neuroprotection of Degenerating Photoreceptors in the S334ter4 rat Retina via Single Intravitreal Injection of Mouse Embryonic Stem Cells Expressing a GDNF Transgene

Investigative Ophthalmology & Visual Science, May 14, 2008

Research paper thumbnail of Rod And Cone Photoreceptors Die By Different Cell-death Mechanisms In The Pde6c Mutant Zebrafish Model Of Achromatopsia

Investigative Ophthalmology & Visual Science, Mar 26, 2012

Research paper thumbnail of Nonsense suppression drugs and their potential in the treatment of eye disease

Journal of Clinical & Experimental Ophthalmology, Jul 30, 2015

Research paper thumbnail of RNA-based therapies in animal models of Leber congenital amaurosis causing blindness

Precision Clinical Medicine, Mar 12, 2020

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in ... more Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in which ∼ 35% of gene mutations are in-frame nonsense mutations coding for loss-of-function premature termination codons (PTCs) in mRNA. Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein. A limitation of nonsense suppression is that nonsense-mediated decay (NMD) degrades PTC-containing RNA transcripts. The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo. Using a high-throughput approach in the recessive cep290 zebrafish model of LCA (cep290;Q1223X), we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren. We observed reduced retinal cell death and improved visual function. With these positive data, we next investigated whether this strategy was also applicable across species in two mammalian models: Rd12 (rpe65;R44X) and Rd3 (rd3;R107X) mouse models of LCA. In the Rd12 model, cell death was reduced, RPE65 protein was produced, and in vivo visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation. In the Rd3 model, however, no beneficial effect was observed with Ataluren alone or in combination with Amlexanox. This variation in response establishes that some forms of nonsense mutation LCA can be targeted by RNA therapies, but that this needs to be verified for each genotype. The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.

Research paper thumbnail of Mutations in LRP5 or FZD4 underlie the common FEVR locus on chromosome 11q13

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of The Association Of MMP/TIMP Ratios In Neovascular AMD

Research paper thumbnail of Transcriptional Regulation and Expression of the Dominant Drusen Gene<i>FBLN3</i>(<i>EFEMP1</i>) in Mammalian Retina

Investigative Ophthalmology & Visual Science, Nov 1, 2003

PURPOSE. To determine the important transcriptional control elements and sites of expression of f... more PURPOSE. To determine the important transcriptional control elements and sites of expression of fibulin-3 in mammalian retina. METHODS. Sequencing and 5Ј rapid amplification of cDNA ends (RACE) were undertaken to characterize the genomic sequence upstream of the FBLN3 coding sequence. Reporter deletion-mutation constructs were used in luciferase transfection assays to determine the important regulatory motifs. Fibulin-3 expression in mouse and human retina was studied by in situ hybridization and RT-PCR. The effect of 17␤-estradiol on fibulin-3 production was studied in COS-7 and ARPE-19 cells. RESULTS. Two untranslated exons were fully sequenced completing the characterization of FBLN3 that comprises 12 exons. Reporter assays suggest that the FBLN3 proximal promoter is contained within 425 bp upstream of exon 1. Important regulatory elements include three Sp1-binding sites, a Tant motif (trans-activating) and an estrogen response element (ERE) binding site (trans-repressing). No TATA or CAAT regulatory boxes were identified. RT-PCR suggests that the fibulin-3 gene is expressed in murine and human RPE, and in situ studies confirm that Fbln3 is expressed in the outer and inner nuclear layers, but strikingly not in the ganglion cell layer. Fibulin-3 expression in ARPE-19 cells could be modified by varying the amount of estrogen in the cell culture medium. CONCLUSIONS. The 5Ј end of the FBLN3 gene has been characterized, and the important upstream motifs regulating its transcription have been identified. Fibulin-3 is expressed in adult retina and at early stages in human and mouse development. Estrogen may be an important regulator of fibulin-3 expression, and this highlights a novel mechanism by which circulating estrogen may control the composition of the retinal extracellular matrix.

Research paper thumbnail of Nonsense suppression rescues cell death and photoreceptor protein expression in CDH23 patient iPSC-derived retinal organoids

Investigative Ophthalmology & Visual Science, Jul 22, 2019

Research paper thumbnail of Retinal therapy with induced pluripotent stem cells; leading the way to human clinical trials

Expert Review of Ophthalmology, Jan 2, 2019

Introduction: Retinal cell transplantation is a promising approach to delay progression and rever... more Introduction: Retinal cell transplantation is a promising approach to delay progression and reverse blindness caused by infections, genetic inheritance, or associated with aging. Areas covered: The demand for transplantable retinal cells is broad and several cell sources exist that can supply such cells. The recent development of protocols that reprogram adult cells to their pluripotent potential (induced pluripotent stem cells; iPSCs) and the possibility of differentiating iPSCs to many retinal types make these cells very attractive for cell therapeutics in the degenerating retina.iPSCs overcome ethical dilemmas in using fetal cells and with autologous transplantation of iPSC derived retinal cells, immunosuppression will no longer be needed. Importantly, gene editing of iPSCs derived from patients with genetic diseases will allow autologous transplantation of healthy differentiated retinal cells. Expert commentary: Before marketing clinically useful retinal cells, differentiated from iPSCs, a multistep clinical trial is performed. Currently, 23 trials are described in the literature and they report no adverse reactions to cell transplantation. Early visual assessment tests demonstrate small visual benefits in a proportion of participants, with future results anticipated in the near future.

Research paper thumbnail of Proinflammatory Events in the P23H-1 Rat Model of Retinal Degeneration

Research paper thumbnail of Rip3 knockdown rescues photoreceptor cell death in pde6c zebrafish model of achromatopsia

Investigative Ophthalmology & Visual Science, Jun 16, 2013

Research paper thumbnail of Seconadry Ischemic Events Following Non-Arteritic Retinal Artery Ischaemia in Four Uk Centres

Investigative Ophthalmology & Visual Science, May 10, 2007

Research paper thumbnail of Macula Dysfunction in Subretinal Neovascular Membranes Results in Significant Pupillary Escape

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of Characterization of the Genomic and Transcriptional Structure of the CRX Gene: Substantial Differences Between the Human and Mouse Genes

Investigative Ophthalmology & Visual Science, Dec 1, 2002

We have previously shown that there is a temporal difference in human CRX gene expression compare... more We have previously shown that there is a temporal difference in human CRX gene expression compared with that of mouse Crx. We have now characterized these genes at the genomic and transcriptional levels and here we expand on this earlier report. Human CRX spans 25 kb and has six exons, and mouse Crx spans 15 kb and has four exons. We isolated seven human and two mouse mRNAs generated by alternative splicing of a variable 5Ј untranslated region. The human and mouse genes share an evolutionarily conserved promoter, which contains OTX/CRX type and SP1/AP2 binding sites and drives expression of two conserved transcripts in both species. Additionally, the human gene has a second human-specific promoter, which has OTX/CRX type binding sites and drives expression of five other transcripts. Band shift assays have shown that six of the seven candidate OTX/CRX elements bind CRX in vitro, possibly implying that the gene can regulate its own expression. These data may account for the differences in temporal expression in vivo we have previously reported between these two species.

Research paper thumbnail of Role of Receptor Interacting Protein Kinases in the Retinal Degeneration of S334ter and P23H Mutant Rat Models of Retinitis Pigmentosa

Investigative Ophthalmology & Visual Science, Jun 11, 2015

Research paper thumbnail of Photoreceptor precursor cell integration into rodent retina after treatment with novel glycopeptide PKX-001

bioRxiv (Cold Spring Harbor Laboratory), Nov 22, 2020

Cell replacement therapy is emerging as an important approach in novel treatments for neurodegene... more Cell replacement therapy is emerging as an important approach in novel treatments for neurodegenerative diseases. Many problems remain, in particular improvements are needed in the survival of transplanted cells and increasing functional integration into host tissue. These problems arise because of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, toxic compounds released by dying tissues and nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) and showed 2.8-fold improvement in transplant cell survival after pre-treatment with a novel glycopeptide (PKC-100). In this study we extended these studies to look at cell survival, maturation and functional integration in an in vivo rat model of rhodopsinmutant retinitis pigmentosa causing blindness. We found that only when human photoreceptor precursor cells (PPCs) were pre-incubated with PKX-100 prior to transplantation, did the cells integrate and mature into cone photoreceptors expressing S-opsin or L/M opsin. In addition, ribbon synapses were observed in the transplanted cells suggesting they were making synaptic connections with the host tissue. Furthermore, optokinetic tracking and electroretinography responses in vivo were significantly improved compared to cell transplants without PKX-100 pre-treatment. These data demonstrate that PKX-100 promotes significant long-term stem cell survival in vivo, providing a platform for further investigation towards the clinical application to repair damaged or diseased retina.

Research paper thumbnail of Pharmacological Therapy Using Aminoglycosides Can Override the Genetic Defect Causing Choroideraemia in the Zebrafish Model

Investigative Ophthalmology & Visual Science, May 14, 2008

Research paper thumbnail of Fibulin–3 gene mutation leads to upregulation of chaperone proteins and apoptosis in retinal pigment epithelium

Investigative Ophthalmology & Visual Science, May 1, 2004

Research paper thumbnail of Oculo-oto-dental Syndrome: Linkage to 20q13.1

Investigative Ophthalmology & Visual Science, Dec 1, 2002

Research paper thumbnail of High–Density SNP Genome Scan Identifies a New Locus for Autosomal Dominant Oculo–Otodental Dysplasia

Investigative Ophthalmology & Visual Science, 2006

Research paper thumbnail of Prolonged Neuroprotection of Degenerating Photoreceptors in the S334ter4 rat Retina via Single Intravitreal Injection of Mouse Embryonic Stem Cells Expressing a GDNF Transgene

Investigative Ophthalmology & Visual Science, May 14, 2008

Research paper thumbnail of Rod And Cone Photoreceptors Die By Different Cell-death Mechanisms In The Pde6c Mutant Zebrafish Model Of Achromatopsia

Investigative Ophthalmology & Visual Science, Mar 26, 2012

Research paper thumbnail of Nonsense suppression drugs and their potential in the treatment of eye disease

Journal of Clinical & Experimental Ophthalmology, Jul 30, 2015

Research paper thumbnail of RNA-based therapies in animal models of Leber congenital amaurosis causing blindness

Precision Clinical Medicine, Mar 12, 2020

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in ... more Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in which ∼ 35% of gene mutations are in-frame nonsense mutations coding for loss-of-function premature termination codons (PTCs) in mRNA. Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein. A limitation of nonsense suppression is that nonsense-mediated decay (NMD) degrades PTC-containing RNA transcripts. The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo. Using a high-throughput approach in the recessive cep290 zebrafish model of LCA (cep290;Q1223X), we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren. We observed reduced retinal cell death and improved visual function. With these positive data, we next investigated whether this strategy was also applicable across species in two mammalian models: Rd12 (rpe65;R44X) and Rd3 (rd3;R107X) mouse models of LCA. In the Rd12 model, cell death was reduced, RPE65 protein was produced, and in vivo visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation. In the Rd3 model, however, no beneficial effect was observed with Ataluren alone or in combination with Amlexanox. This variation in response establishes that some forms of nonsense mutation LCA can be targeted by RNA therapies, but that this needs to be verified for each genotype. The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.