Publications by authors named "Susie E Barker"

11 Publications

  • Page 1 of 1

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

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

Long-term survival of photoreceptors transplanted into the adult murine neural retina requires immune modulation.

Stem Cells 2010 Nov;28(11):1997-2007

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

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.
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http://dx.doi.org/10.1002/stem.520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272388PMC
November 2010

The drusenlike phenotype in aging Ccl2-knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages.

Invest Ophthalmol Vis Sci 2009 Dec 2;50(12):5934-43. Epub 2009 Jul 2.

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

Purpose: Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD.

Methods: The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry.

Results: The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice.

Conclusions: These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.
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http://dx.doi.org/10.1167/iovs.09-3462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801148PMC
December 2009

Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice.

J Gene Med 2009 Jun;11(6):486-97

Division of Molecular Therapy, UCL Institute of Ophthalmology, London, UK.

Background: Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product.

Methods: We evaluated cellular and humoral responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results: Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65(-/-) mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions: These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration.
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http://dx.doi.org/10.1002/jgm.1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841821PMC
June 2009

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector.

Biochemistry 2007 Nov 13;46(45):12930-44. Epub 2007 Oct 13.

Department of Chemistry, Christopher Ingold Laboratories, University College London, United Kingdom.

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.
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http://dx.doi.org/10.1021/bi701014yDOI Listing
November 2007

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

Effective gene therapy with nonintegrating lentiviral vectors.

Nat Med 2006 Mar 19;12(3):348-53. Epub 2006 Feb 19.

Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.
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http://dx.doi.org/10.1038/nm1365DOI Listing
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

Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

J Drug Target 2004 May;12(4):185-93

Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.
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http://dx.doi.org/10.1080/10611860410001724459DOI Listing
May 2004