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CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Tue, 2018-04-03 03:00
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CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Prog Retin Eye Res. 2018 Mar 22;:

Authors: Burnight ER, Giacalone JC, Cooke JA, Thompson JR, Bohrer LR, Chirco KR, Drack AV, Fingert JH, Worthington KS, Wiley LA, Mullins RF, Stone EM, Tucker BA

Abstract
Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

PMID: 29578069 [PubMed - as supplied by publisher]

Evaluation of sFLT1 protein levels in human eyes with the FLT1 rs9943922 polymorphism.

Tue, 2018-04-03 03:00
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Evaluation of sFLT1 protein levels in human eyes with the FLT1 rs9943922 polymorphism.

Ophthalmic Genet. 2018 Jan-Feb;39(1):68-72

Authors: Chirco KR, Lewis CJ, Scheetz TE, Johnston RM, Tucker BA, Stone EM, Fingert JH, Mullins RF

Abstract
PURPOSE: Age-related macular degeneration (AMD) is a devastating disease characterized by central vision impairment in individuals with advanced age. Neovascular AMD is a form of end-stage disease in which choroidal vessel outgrowth occurs beneath the retina. While many hypotheses have been raised as to what triggers the formation of pathological choroidal neovascular membranes, the exact mechanism for their initiation remains unresolved. Polymorphisms in the FLT1 gene have previously been associated with neovascular AMD risk, including the rs9943922 single nucleotide polymorphism (SNP). Here, we aimed to determine the association between the high-risk FLT1 genotype and FLT1 protein levels in human retina or retinal pigment epithelium (RPE)/choroid tissue.
METHODS: Retina and RPE/choroid tissue from 10 human donor eyes was selected from a collection of eyes genotyped for the rs9943922 SNP. Differences in soluble and membrane bound FLT1 protein levels were assessed for retina versus RPE/choroid donor tissue using ELISA and Western blotting analyses. Genotype-associated changes in FLT1 protein levels were also evaluated.
RESULTS: We found soluble FLT1 levels in the RPE/choroid tissue to be approximately three times higher than that of the retina (p < 0.001), while both samples have similar levels of the membrane bound form. When tissue with the rs9943922 SNP was compared with controls, no significant genotypic differences in FLT1 protein levels were observed.
CONCLUSIONS: Based on these data, we conclude that the rs9943922 SNP in the FLT1 gene does not result in a large difference in FLT1 protein levels, regardless of whether it is the soluble or the membrane bound form.

PMID: 28949775 [PubMed - indexed for MEDLINE]

CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Mon, 2018-03-12 21:21
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CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Curr Protoc Stem Cell Biol. 2018 Feb 28;44:5B.7.1-5B.7.22

Authors: Giacalone JC, Sharma TP, Burnight ER, Fingert JF, Mullins RF, Stone EM, Tucker BA

Abstract
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.

PMID: 29512106 [PubMed - in process]

Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa.

Mon, 2018-03-12 21:21
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Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa.

Stem Cell Res. 2017 May;21:58-70

Authors: Sharma TP, Wiley LA, Whitmore SS, Anfinson KR, Cranston CM, Oppedal DJ, Daggett HT, Mullins RF, Tucker BA, Stone EM

Abstract
Retinitis pigmentosa (RP) is a heterogeneous group of monogenic disorders characterized by progressive death of the light-sensing photoreceptor cells of the outer neural retina. We recently identified novel hypomorphic mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1) gene that cause early-onset RP. To model this disease in vitro, we generated patient-specific iPSCs and iPSC-derived retinal organoids from dermal fibroblasts of patients with molecularly confirmed TRNT1-associated RP. Pluripotency was confirmed using rt-PCR, immunocytochemistry, and a TaqMan Scorecard Assay. Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived retinal organoids. Patient-specific iPSCs and iPSC-derived retinal organoids exhibited a deficit in autophagy, as evidenced by aberrant accumulation of LC3-II and elevated levels of oxidative stress. Autologous stem cell-based disease modeling will provide a platform for testing multiple avenues of treatment in patients suffering from TRNT1-associated RP.

PMID: 28390992 [PubMed - indexed for MEDLINE]

Two-photon polymerization for production of human iPSC-derived retinal cell grafts.

Mon, 2018-03-05 17:09
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Two-photon polymerization for production of human iPSC-derived retinal cell grafts.

Acta Biomater. 2017 Jun;55:385-395

Authors: Worthington KS, Wiley LA, Kaalberg EE, Collins MM, Mullins RF, Stone EM, Tucker BA

Abstract
Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials' inability to guide cell alignment. In this work, we used two-photon polymerization to create 180μm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3µm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25μm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases.
STATEMENT OF SIGNIFICANCE: Cell replacement therapy is an important goal for investigators aiming to restore neural function to those suffering from neurodegenerative disease. Cell delivery scaffolds are frequently necessary for the success of such treatments, but traditional biomaterials often fail to facilitate the neuronal orientation and close packing needed to recapitulate the in vivo environment. Here, we use two-photon polymerization to create prototype cell scaffolds with densely packed vertical pores for photoreceptor cell loading and small, interconnected horizontal pores for nutrient diffusion. This study offers a thorough characterization of how two-photon polymerization parameters affect final structural outcomes and printing time. Our findings demonstrate the feasibility of using two-photon polymerization to create scaffolds that can align neuronal cells in 3D and are large enough to be used for transplantation. In future work, these scaffolds could comprise biodegradable materials with tunable microstructure, elastic modulus and degradation time; a significant step towards a promising treatment option for those suffering from late-stage neurodegeneration, including retinal degenerative blindness.

PMID: 28351682 [PubMed - indexed for MEDLINE]

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies.

Mon, 2018-02-26 14:55
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Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies.

Acta Biomater. 2017 Jul 15;57:293-303

Authors: Chirco KR, Worthington KS, Flamme-Wiese MJ, Riker MJ, Andrade JD, Ueberheide BM, Stone EM, Tucker BA, Mullins RF

Abstract
Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies.
STATEMENT OF SIGNIFICANCE: Age-related macular degeneration (AMD) is a devastating disease affecting more than 600 million people worldwide. Endothelial cells of the choriocapillaris (CECs) are among the first cell types lost in early AMD, and cell replacement therapy is currently the most promising option for restoring vision in patients with advanced AMD. In order to study CEC replacement strategies we have generated a 3D choroid scaffold using a novel decellularization method in human RPE/choroid tissue. To our knowledge, this is the first report describing decellularization of human RPE/choroid, as well as recellularization of a choroid scaffold with CECs. This work will aid in our development and optimization of cell replacement strategies using a tissue scaffold that is similar to the in vivo environment.

PMID: 28483697 [PubMed - indexed for MEDLINE]

Bestrophinopathy: An RPE-photoreceptor interface disease.

Mon, 2018-02-05 09:46
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Bestrophinopathy: An RPE-photoreceptor interface disease.

Prog Retin Eye Res. 2017 May;58:70-88

Authors: Guziewicz KE, Sinha D, Gómez NM, Zorych K, Dutrow EV, Dhingra A, Mullins RF, Stone EM, Gamm DM, Boesze-Battaglia K, Aguirre GD

Abstract
Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

PMID: 28111324 [PubMed - indexed for MEDLINE]

MMP19 expression in the human optic nerve.

Mon, 2018-01-22 04:35
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MMP19 expression in the human optic nerve.

Mol Vis. 2016;22:1429-1436

Authors: Chirco KR, Hazlewood RJ, Miller K, Workalemahu G, Jampol LM, Lesser GR, Mullins RF, Kuehn MH, Fingert JH

Abstract
PURPOSE: The defining feature of glaucoma is excavation of the optic nerve head; however, the mechanism of this loss of tissue is not well understood. We recently discovered a copy number variation upstream of matrix metalloproteinase 19 (MMP19) in a large, autosomal dominant pedigree with a congenital malformation of the optic disc called cavitary optic disc anomaly (CODA). Patients with CODA have abnormal optic discs that exhibit an excavated shape similar to cupping seen in glaucoma. The goal of this study is to characterize the localization of MMP19 within the human optic nerve.
METHODS: The MMP19 protein in the optic nerve was evaluated with western blot analysis and with immunohistochemistry in sagittal and en face/cross sections of optic nerves obtained from healthy human donor eyes.
RESULTS: The MMP19 protein was detected in the human optic nerve, retina, and RPE/choroid with western blot analysis, with highest expression in the retina and the optic nerve. Using immunohistochemistry, MMP19 was localized within the optic nerve to the extracellular space within the septa that separate bundles of optic nerve axons into fascicles. The presence of MMP19 within the optic nerve septa was further confirmed by the colocalization of MMP19 to this structure with type IV collagen. Strong labeling of MMP19 was also detected in the arachnoid layer of the optic nerve sheath. Finally, immunohistochemistry of the optic nerve cross sections demonstrated that MMP19 shows a peripheral to central gradient, with more abundant labeling along the edges of the optic nerve and in the arachnoid layer than in the center of the nerve.
CONCLUSIONS: Abundant MMP19 was detected in the optic nerve head, the primary site of pathology in patients with CODA. The localization of MMP19 to the optic nerve septa is consistent with its predicted secretion and accumulation within the extracellular spaces of this tissue. Moreover, the lateral localization of MMP19 observed in the optic nerve cross sections suggests that it might have a role in regulating adhesion to the optic nerve to the scleral canal and remodeling the extracellular matrix that provides the structural integrity of the optic disc. Dysregulation of MMP19 production might, therefore, undermine the connections between the optic nerve and the scleral canal and cause a collapse of the optic disc and the development of CODA. Similar processes might also be at work in the formation of optic disc cupping in glaucoma.

PMID: 28003733 [PubMed - indexed for MEDLINE]

Using Patient-Specific Induced Pluripotent Stem Cells and Wild-Type Mice to Develop a Gene Augmentation-Based Strategy to Treat CLN3-Associated Retinal Degeneration.

Mon, 2018-01-22 04:35
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Using Patient-Specific Induced Pluripotent Stem Cells and Wild-Type Mice to Develop a Gene Augmentation-Based Strategy to Treat CLN3-Associated Retinal Degeneration.

Hum Gene Ther. 2016 Oct;27(10):835-846

Authors: Wiley LA, Burnight ER, Drack AV, Banach BB, Ochoa D, Cranston CM, Madumba RA, East JS, Mullins RF, Stone EM, Tucker BA

Abstract
Juvenile neuronal ceroid lipofuscinosis (JNCL) is a childhood neurodegenerative disease with early-onset, severe central vision loss. Affected children develop seizures and CNS degeneration accompanied by severe motor and cognitive deficits. There is no cure for JNCL, and patients usually die during the second or third decade of life. In this study, independent lines of induced pluripotent stem cells (iPSCs) were generated from two patients with molecularly confirmed mutations in CLN3, the gene mutated in JNCL. Clinical-grade adeno-associated adenovirus serotype 2 (AAV2) carrying the full-length coding sequence of human CLN3 was generated in a U.S. Food and Drug Administration-registered cGMP facility. AAV2-CLN3 was efficacious in restoring full-length CLN3 transcript and protein in patient-specific fibroblasts and iPSC-derived retinal neurons. When injected into the subretinal space of wild-type mice, purified AAV2-CLN3 did not show any evidence of retinal toxicity. This study provides proof-of-principle for initiation of a clinical trial using AAV-mediated gene augmentation for the treatment of children with CLN3-associated retinal degeneration.

PMID: 27400765 [PubMed - indexed for MEDLINE]

Drusen on Demand? Authors Describe a Novel Culture System for Generating subRPE Deposits.

Mon, 2018-01-08 00:57
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Drusen on Demand? Authors Describe a Novel Culture System for Generating subRPE Deposits.

Invest Ophthalmol Vis Sci. 2017 02 01;58(2):720

Authors: Mullins RF

PMID: 28152142 [PubMed - indexed for MEDLINE]

CLINICOPATHOLOGICAL CORRELATION IN A PATIENT WITH PREVIOUSLY TREATED BIRDSHOT CHORIORETINOPATHY.

Mon, 2018-01-08 00:57
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CLINICOPATHOLOGICAL CORRELATION IN A PATIENT WITH PREVIOUSLY TREATED BIRDSHOT CHORIORETINOPATHY.

Retin Cases Brief Rep. 2017 Fall;11(4):344-347

Authors: Sohn EH, Chirco KR, Folk JC, Mullins RF

Abstract
PURPOSE: Birdshot chorioretinopathy (BCR) is a bilateral, chronic uveitis primarily involving the posterior segment that often results in progressive vision loss. Histopathology on eyes with BCR has been limited, but we had the rare opportunity to study the eyes of a donor with BCR. We sought to compare immunolabeling in the eyes of this donor who was treated with immunosuppression for over 30 years to age-matched controls.
METHODS: From each eye, a macular punch and superotemporal regions were used for cryostat sectioning, and immunohistochemistry was performed on the sections using antibodies directed against CD45, intercellular adhesion molecule-1, IBA1, and GFAP. The vasculature-binding lectin, Ulex europaeus agglutinin-I (UEA-I), was also used to perform lectin histochemistry.
RESULTS: At death, her visual acuity was 20/25 right eye, 20/250 left eye with extensive chorioretinal atrophy, vascular attenuation, and disk pallor. Compared with controls, the BCR donor had extensive degeneration of the outer nuclear layer and retinal pigment epithelium as well as choroidal thinning with inner retinal preservation. Loss of UEA-I+ choroidal endothelial cells was extensive, and atypical intercellular adhesion molecule-1 labeling and IBA+ microglia/macrophages were present along with widespread GFAP labeling throughout the retina.
CONCLUSION: The BCR may cause progressive chorioretinal and optic atrophy with long-standing increased leukocyte abundance throughout the retina and microglial activation especially at the retina-choroid interface.

PMID: 27465484 [PubMed - indexed for MEDLINE]