Publications by authors named "Anastasios Georgiadis"

17 Publications

  • Page 1 of 1

Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts.

Invest Ophthalmol Vis Sci 2019 07;60(8):3170-3177

Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom.

Purpose: Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer.

Methods: HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment.

Results: LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD.

Conclusions: ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.
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http://dx.doi.org/10.1167/iovs.18-26260DOI Listing
July 2019

Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.

Stem Cell Res Ther 2018 06 13;9(1):156. Epub 2018 Jun 13.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK.

Background: The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required.

Methods: We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions.

Results: Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures.

Conclusions: Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.
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http://dx.doi.org/10.1186/s13287-018-0907-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998504PMC
June 2018

Prevention of Photoreceptor Cell Loss in a Cln6 Mouse Model of Batten Disease Requires CLN6 Gene Transfer to Bipolar Cells.

Mol Ther 2018 05 2;26(5):1343-1353. Epub 2018 Mar 2.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London EC1V 2PD, UK. Electronic address:

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6 mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.
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http://dx.doi.org/10.1016/j.ymthe.2018.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993939PMC
May 2018

Harnessing the Potential of Human Pluripotent Stem Cells and Gene Editing for the Treatment of Retinal Degeneration.

Curr Stem Cell Rep 2017 18;3(2):112-123. Epub 2017 Apr 18.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL UK.

Purpose Of Review: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors.

Recent Findings: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect.

Summary: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina.
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http://dx.doi.org/10.1007/s40778-017-0078-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445184PMC
April 2017

Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration.

Stem Cell Reports 2017 06 25;8(6):1659-1674. Epub 2017 May 25.

Department of Genetics, UCL Institute of Ophthalmology, London EC1V 9EL, UK; NIHR Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust, City Road, London EC1V 2PD, UK. Electronic address:

The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1 mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470175PMC
June 2017

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Hum Mol Genet 2017 01;26(2):305-319

UCL Institute of Ophthalmology, 11-43 Bath Street, London, UK.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.
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http://dx.doi.org/10.1093/hmg/ddw387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351934PMC
January 2017

Enhanced Ccl2-Ccr2 signaling drives more severe choroidal neovascularization with aging.

Neurobiol Aging 2016 Apr 4;40:110-119. Epub 2016 Jan 4.

Department of Genetics, UCL Institute of Ophthalmology, London, UK; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK. Electronic address:

The impact of many inflammatory diseases is influenced by age-related changes in the activation of resident and circulating myeloid cells. In the eye, a major sight-threatening consequence of age-related macular degeneration is the development of severe choroidal neovascularization (CNV). To identify the molecular pathways and myeloid cell populations involved in this increased neovascular response, we characterized the immune status of murine choroid and retina during aging and in the context of experimental CNV. In the choroid, but not in the retina, advancing age is associated with proinflammatory upregulation of CCL2-CCR2 signaling. Genetic excision of CCL2 diminishes age-related inflammatory changes in the choroid, with reduced recruitment of proinflammatory myeloid cells and attenuation of CNV. These findings indicate that CCL2-driven recruitment of myeloid cells contributes to increased severity of CNV with age. Similar mechanisms may be involved in other age-related inflammatory diseases.
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http://dx.doi.org/10.1016/j.neurobiolaging.2015.12.019DOI Listing
April 2016

IL-4 regulates specific Arg-1(+) macrophage sFlt-1-mediated inhibition of angiogenesis.

Am J Pathol 2015 Aug 13;185(8):2324-35. Epub 2015 Jun 13.

School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom; Institute of Ophthalmology, University College London, London, United Kingdom; School of Clinical Sciences, University of Bristol, Bristol, United Kingdom; National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital and University College London Institute of Ophthalmology, London, United Kingdom. Electronic address:

One of the main drivers for neovascularization in age-related macular degeneration is activation of innate immunity in the presence of macrophages. Here, we demonstrate that T helper cell type 2 cytokines and, in particular, IL-4 condition human and murine monocyte phenotype toward Arg-1(+), and their subsequent behavior limits angiogenesis by increasing soluble fms-like tyrosine kinase 1 (sFlt-1) gene expression. We document that T helper cell type 2 cytokine-conditioned murine macrophages neutralize vascular endothelial growth factor-mediated endothelial cell proliferation (human umbilical vein endothelial cell and choroidal vasculature) in a sFlt-1-dependent manner. We demonstrate that in vivo intravitreal administration of IL-4 attenuates laser-induced choroidal neovascularization (L-CNV) due to specific IL-4 conditioning of macrophages. IL-4 induces the expression of sFlt-1 by resident CD11b(+) retinal microglia and infiltrating myeloid cells but not from retinal pigment epithelium. IL-4-induced suppression of L-CNV is not prevented when sFlt-1 expression is attenuated in retinal pigment epithelium. IL-4-mediated suppression of L-CNV was abrogated in IL-4R-deficient mice and in bone marrow chimeras reconstituted with myeloid cells that had undergone lentiviral-mediated shRNA silencing of sFlt-1, demonstrating the critical role of this cell population. Together, these data establish how lL-4 directly drives macrophage sFlt-1 production expressing an Arg-1(+) phenotype and support the therapeutic potential of targeted IL-4 conditioning within the tissue to regulate disease conditions such as neovascular age-related macular degeneration.
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http://dx.doi.org/10.1016/j.ajpath.2015.04.013DOI Listing
August 2015

Long-term effect of gene therapy on Leber's congenital amaurosis.

N Engl J Med 2015 May 4;372(20):1887-97. Epub 2015 May 4.

From the UCL (University College London) Institute of Ophthalmology (J.W.B.B., M.S.M., V.S., S.J.R., S.E.B., C.R., A.G., F.M.M., S.G.B., P.J.G., V.A.L., K.B., A.V., G.E.H., F.W.F., M.N., A.T.M., M.M., A.S., A.J.S., G.R., R.R.A.) and the Department of Civil, Environmental, and Geomatic Engineering (N.T.), UCL, and Moorfields Eye Hospital (J.W.B.B., M.S.M., V.S., S.J.R., A.G., K.B., G.H., A.M., M.M.), London, and the Department of Psychology, Durham University, Durham (M.N.) - all in the United Kingdom; the College of Veterinary Medicine, Michigan State University, East Lansing (F.M.M., S.M.P.-J.), and the Kellogg Eye Center, University of Michigan Medical School, Ann Arbor (K.L.F., D.A.T., R.R.A.); the Center for Human Genetics, KU Leuven (T.J.L.R.), and the Department of Ophthalmology, UZ Leuven, Campus Sint-Rafaël (I.C.) - both in Leuven, Belgium; Rotterdam Eye Hospital, Rotterdam, the Netherlands (S.Y., L.I.B.); and the Oregon Retinal Degeneration Center, Ophthalmic Genetics Service, Casey Eye Institute, Oregon Health and Science University, Portland (R.G.W.).

Background: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited.

Methods: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings.

Results: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG.

Conclusions: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).
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http://dx.doi.org/10.1056/NEJMoa1414221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497809PMC
May 2015

Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

Nat Biotechnol 2013 Aug 21;31(8):741-7. Epub 2013 Jul 21.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL UK.

Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
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http://dx.doi.org/10.1038/nbt.2643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826328PMC
August 2013

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

Development 2012 Jul 23;139(13):2340-50. Epub 2012 May 23.

Department of Genetics, Institute of Ophthalmology, NIHR Biomedical Research Centre for Ophthalmology, University College London, London EC1V 9EL, UK.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.
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http://dx.doi.org/10.1242/dev.070813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367444PMC
July 2012

The tight junction associated signalling proteins ZO-1 and ZONAB regulate retinal pigment epithelium homeostasis in mice.

PLoS One 2010 Dec 30;5(12):e15730. Epub 2010 Dec 30.

Department of Genetics, UCL Institute of Ophthalmology, University College London, London, United Kingdom.

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015730PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012699PMC
December 2010

Corrigendum to "Local Administration of an Adeno-Associated Viral Vector Expressing IL-10 Reduces Monocyte Infiltration and Subsequent Photoreceptor Damage During Experimental Autoimmune Uveitis".

Mol Ther 2006 Apr 3;13(4):829. Epub 2005 Nov 3.

Division of Molecular Therapy, Institute of Ophthalmology, University College, London, 11-43 Bath Street, London EC1V 9EL, UK; Molecular Immunology Unit, Institute of Child Health, London, UK. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2005.09.013DOI Listing
April 2006

Regulation of PCNA and cyclin D1 expression and epithelial morphogenesis by the ZO-1-regulated transcription factor ZONAB/DbpA.

Mol Cell Biol 2006 Mar;26(6):2387-98

Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, United Kingdom.

The tight junction protein ZO-1 inhibits G1/S-phase transition by cytoplasmic sequestration of a complex formed by CDK4 and the transcription factor ZONAB. Canine ZONAB is the homologue of human DbpA, an E2F target gene that is overexpressed in different carcinomas. Since the ZONAB target genes that are involved in G1/S-phase transition are unknown, we employed the mammary epithelial cell line MCF-10A and cDNA arrays to screen for such genes. We identified genes encoding cell cycle and replication proteins whose expression was altered due to increased ZONAB expression. For proliferative cell nuclear antigen and cyclin D1 genes, we show that increased mRNA levels resulted in increased protein levels and we identified ZONAB-responsive elements in their promoters by using different approaches, including chromatin immunoprecipitation assays. RNA interference and overexpression of ZONAB affected the proliferation of both MCF-10A and MDCK cells as well as the differentiation of MDCK cells into polarized cysts in three-dimensional cultures. These results indicate that ZONAB regulates the transcription of genes that are important for G1/S-phase progression and links tight junctions to the transcriptional control of key cell cycle regulators and epithelial cell differentiation.
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http://dx.doi.org/10.1128/MCB.26.6.2387-2398.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1430269PMC
March 2006

Local administration of an adeno-associated viral vector expressing IL-10 reduces monocyte infiltration and subsequent photoreceptor damage during experimental autoimmune uveitis.

Mol Ther 2005 Aug;12(2):369-73

Division of Molecular Therapy, Institute of Ophthalmology, University College, London, 11-43 Bath Street, London EC1V 9EL, UK.

Autoimmune posterior uveitis is a chronic, potentially blinding inflammatory disease of the eye. It is commonly treated with immunosuppressive drugs that have adverse long-term effects. Advances in gene transfer techniques have enabled long-term, stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. Here we report for the first time that subretinal injection of rAAV-2 encoding murine IL-10 into the retina of C57BL/6 mice significantly decreases the median experimental autoimmune uveitis (EAU) disease severity. This protection is shown to be due to a decrease in the number and activation status of infiltrating monocytes during EAU, as determined by costimulatory molecule expression and nitrotyrosine detection. No differences within splenocyte proliferative responses or serum antibody levels were detected, emphasizing the potential of gene therapy strategies in ameliorating autoimmune responses in local microenvironments without unwanted systemic effects.
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http://dx.doi.org/10.1016/j.ymthe.2005.03.018DOI Listing
August 2005

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy.

Blood 2004 Nov 1;104(9):2714-21. Epub 2004 Jul 1.

Imperial College London, Gene Therapy Research Group, Section of Cell and Molecular Biology, Sir Alexander Fleming Bldg, Imperial College Road, London, SW7 2AZ, United Kingdom.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.
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http://dx.doi.org/10.1182/blood-2004-02-0627DOI Listing
November 2004

Long-term evaluation of retinal function in Prph2Rd2/Rd2 mice following AAV-mediated gene replacement therapy.

J Gene Med 2003 Sep;5(9):757-64

Department of Molecular Genetics, Institute of Ophthalmology, University College London, UK.

Background: Prph2(Rd2/Rd2) mice have a retinal degeneration due to a null mutation for the Prph2 gene which encodes a photoreceptor-specific glycoprotein, peripherin2, essential for outer-segment formation. We have previously shown improvement of photoreceptor function at a single time point following AAV-mediated gene replacement therapy. Here we quantify the functional rescue over a 15-week time course and present a detailed analysis of the improvement in retinal function.

Methods: An AAV2 vector, AAV.rho.rds, carrying a peripherin2 c-DNA, was in injected subretinally into 10-day-old Prph2(Rd2/Rd2) mice. One group was injected at a single time point while in a second group the injections were repeated after 5 days. The effect of treatment was analysed histologically using electron microscopy and electroretinography (ERG) was used to assess functional changes. Treated mice were recorded at regular intervals over 15 weeks. Untreated contralateral eyes served as internal control.

Results: A significant increase in b-wave amplitude was first noted 3 weeks after treatment of 10-day-old Prph2(Rd2/Rd2) mice and persisted for up to 14 weeks. An increase in the area of retina exposed to vector resulted in a significant increase in both b-wave amplitude and persistence.

Conclusions: In this study AAV-mediated gene replacement in Prph2(Rd2/Rd2) mice resulted in a significant functional improvement over a period of 14 weeks. These results support the utility of gene therapy approaches as treatment for photoreceptor dystrophies.
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http://dx.doi.org/10.1002/jgm.401DOI Listing
September 2003