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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-associated X-linked Retinitis Pigmentosa.

Sat, 2019-05-25 20:49
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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-associated X-linked Retinitis Pigmentosa.

Hum Gene Ther. 2019 May 20;:

Authors: Giacalone JC, Andorf JL, Zhang Q, Burnight E, Ochoa D, Reutzel AJ, Collins MM, Sheffield V, Mullins RF, Han IC, Stone E, Tucker BA

Abstract
In a screen of 1000 consecutively ascertained families we recently found that mutations in the gene RPGR are the 3rd most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant AAV vectors, RPGR is an obvious early target for AAV based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e. potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5 and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull down assays respectively. By making synonymous substitutions within the repetitive region of RPGR we were able to stabilize the plasmid and subsequently generate a clinical grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull down experiments we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e. analysis revealed normal binding to PDE6D, INPP5E and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an IND application.

PMID: 31106594 [PubMed - as supplied by publisher]

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Sat, 2019-05-25 20:49
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Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Microvasc Res. 2019 05;123:50-57

Authors: Giacalone JC, Miller MJ, Workalemahu G, Reutzel AJ, Ochoa D, Whitmore SS, Stone EM, Tucker BA, Mullins RF

Abstract
Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

PMID: 30571950 [PubMed - indexed for MEDLINE]

Evaluation of serum and ocular levels of membrane attack complex and C-reactive protein in CFH-genotyped human donors.

Sat, 2019-05-18 16:56
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Evaluation of serum and ocular levels of membrane attack complex and C-reactive protein in CFH-genotyped human donors.

Eye (Lond). 2018 11;32(11):1740-1742

Authors: Chirco KR, Flamme-Wiese MJ, Wiley JS, Potempa LA, Stone EM, Tucker BA, Mullins RF

Abstract
BACKGROUND: There is a considerable body of evidence demonstrating a link between the membrane attack complex (MAC) and age-related macular degeneration (AMD), and between C-reactive protein (CRP) and AMD. Both the MAC and the monomeric form of CRP (mCRP) accumulate within the choriocapillaris in AMD. However, the precise contribution of these species to AMD pathophysiology has not been fully elucidated.
METHODS: We sought to directly assess CRP and MAC levels between human serum and ocular tissues from the same CFH Y402H genotyped donors using ELISA of serum and RPE/choroid proteins.
RESULTS: The Y402H polymorphism was associated with significantly increased MAC in RPE/choroid samples, but not in the serum, in a previously unstudied cohort. While MAC levels in the choroid were independent of circulating levels, choroidal CRP was correlated to serum levels.
CONCLUSIONS: These data provide further evidence for local activation of complement within the choriocapillaris in AMD.

PMID: 30013157 [PubMed - indexed for MEDLINE]

Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Sat, 2019-05-11 13:34
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Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Exp Eye Res. 2019 May 07;:

Authors: Voigt AP, Whitmore SS, Flamme-Wiese MJ, Riker M, Wiley LA, Tucker BA, Stone EM, Mullins RF, Scheetz TE

Abstract
The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

PMID: 31075224 [PubMed - as supplied by publisher]

Two-photon Polymerized Poly(caprolactone) Retinal Cell Delivery Scaffolds and their Systemic and Retinal Biocompatibility.

Sat, 2019-05-11 13:34
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Two-photon Polymerized Poly(caprolactone) Retinal Cell Delivery Scaffolds and their Systemic and Retinal Biocompatibility.

Acta Biomater. 2019 May 02;:

Authors: Thompson JR, Worthington KS, Green BJ, Mullin NK, Jiao C, Kaalberg EE, Wiley LA, Han IC, Russell SR, Sohn EH, Guymon CA, Mullins RF, Stone EM, Tucker BA

Abstract
Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.

PMID: 31055121 [PubMed - as supplied by publisher]

Choriocapillaris Degeneration in Geographic Atrophy.

Sat, 2019-05-11 13:34
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Choriocapillaris Degeneration in Geographic Atrophy.

Am J Pathol. 2019 Apr 30;:

Authors: Sohn EH, Flamme-Wiese MJ, Whitmore SS, Workalemahu G, Marneros AG, Boese EA, Kwon YH, Wang K, Abramoff MD, Tucker BA, Stone EM, Mullins RF

Abstract
Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculas from controls (n=99), early AMD (n=35), or clinically diagnosed with geographic atrophy (GA; n=9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross sectional area of capillary lumens divided by length) and of vascular lumen-to-stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni corrected P (pcorr)=0.024) with greater loss in GA (pcorr <10-9) even in areas of intact retinal pigment epithelium. In contrast, changes in lumen-to-stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P >0.05), suggesting that changes in the choriocapillaris are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration contributes to dry AMD, and that these changes are predominant in the microvasculature. Addressing the capillary loss in AMD remains an important translational target.

PMID: 31051169 [PubMed - as supplied by publisher]

Subretinal pseudocyst: A novel optical coherence tomography finding in age-related macular degeneration.

Sat, 2019-04-27 10:39
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Subretinal pseudocyst: A novel optical coherence tomography finding in age-related macular degeneration.

Eur J Ophthalmol. 2019 Apr 25;:1120672119846437

Authors: Sacconi R, Mullins RF, Lutty GA, Borrelli E, Bandello F, Querques G

Abstract
PURPOSE: To report the presence of a new structural optical coherence tomography finding, namely, subretinal pseudocysts, in a patient affected by age-related macular degeneration.
METHODS: Case report including multimodal imaging discussion.
CASE REPORT: We report a case of a 77-year-old woman affected by age-related macular degeneration from 7 years. Best corrected visual acuity was counting fingers and 20/40 in the right and left eye, respectively. The left eye was affected by type 1 macular neovascularization treated by 34 intravitreal injections of anti-vascular endothelial growth factor (22 ranibizumab and 12 aflibercept injections). Interestingly, structural optical coherence tomography showed the persistence of a subretinal cystoid space (i.e. 'subretinal pseudocyst') after the last anti-vascular endothelial growth factor treatment, even in absence of other signs of exudation.
CONCLUSIONS: Subretinal pseudocysts are a new structural optical coherence tomography entity. We reported for the first time the evidence that pseudocysts may develop in the subretinal space in a case of age-related macular degeneration.

PMID: 31018677 [PubMed - as supplied by publisher]

Wide-Field Swept-Source OCT and Angiography in X-Linked Retinoschisis.

Sat, 2019-04-27 10:39
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Wide-Field Swept-Source OCT and Angiography in X-Linked Retinoschisis.

Ophthalmol Retina. 2019 Feb;3(2):178-185

Authors: Han IC, Whitmore SS, Critser DB, Lee SY, DeLuca AP, Daggett HT, Affatigato LM, Mullins RF, Tucker BA, Drack AV, Stone EM

Abstract
PURPOSE: Retinal vascular and structural changes, particularly outside of the central macula, are not well characterized in X-linked retinoschisis (XLRS). We aim to describe wide-field swept-source OCT (SS-OCT) and swept-source OCT angiography (SS-OCTA) findings in XLRS.
DESIGN: Retrospective, cross-sectional study at a tertiary referral center.
PARTICIPANTS: Nine consecutive male patients with molecularly confirmed XLRS.
METHODS: All patients underwent complete ophthalmic examination with multimodal imaging, including SS-OCT with SS-OCTA (PLEX Elite 9000; Carl-Zeiss Meditec Inc., Dublin, CA). Images were then reviewed by 2 retinal specialists as independent graders to determine the frequency and distribution of retinal structural and vascular abnormalities.
MAIN OUTCOME MEASURES: Structural and vascular abnormalities seen on SS-OCT and SS-OCTA in patients with XLRS, with attention to the retinal layers involved, the regional distribution of schitic spaces in the posterior pole, and vascular abnormalities within the superficial and deep capillary plexuses.
RESULTS: Eighteen eyes from 9 male patients (mean age, 20 years; range 9-40) with molecularly confirmed XLRS were included. Median best-corrected visual acuity measured 20/63 (range, 20/25-10/300). A total of 17 of 18 eyes (94.4%) were noted to have schitic spaces on SS-OCT, and these were observed to be predominantly within the inner nuclear layer in all 17 eyes. A regional variation in the distribution of cysts was noted, with schitic spaces within the ganglion cell layer (13/17 eyes; 76.5%) observed to be perifoveal and those within the outer nuclear layer (8/17 eyes, 47.1%) observed to be mostly extramacular. All eyes had vascular abnormalities on SS-OCTA, including an irregular foveal avascular zone and flow loss within the deep capillary plexus corresponding to the distribution of the schisis.
CONCLUSIONS: Wide-field SS-OCT and SS-OCTA provide detailed visualization of structural and vascular changes in XLRS and may be helpful for monitoring disease progression or treatment response in clinical trials for the disease.

PMID: 31014769 [PubMed - in process]

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Sat, 2019-04-27 10:39
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Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Stem Cells Transl Med. 2019 Apr 19;:

Authors: Scruggs BA, Jiao C, Cranston CM, Kaalberg E, Wang K, Russell SR, Wiley LA, Mullins RF, Stone EM, Tucker BA, Sohn EH

Abstract
Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019.

PMID: 31004408 [PubMed - as supplied by publisher]

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Sat, 2019-04-13 06:04
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Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Genes (Basel). 2019 Apr 05;10(4):

Authors: Bohrer LR, Wiley LA, Burnight ER, Cooke JA, Giacalone JC, Anfinson KR, Andorf JL, Mullins RF, Stone EM, Tucker BA

Abstract
Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

PMID: 30959774 [PubMed]

Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants.

Sat, 2019-04-13 06:04
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Assessment of Adeno-Associated Virus Serotype Tropism in Human Retinal Explants.

Hum Gene Ther. 2018 04;29(4):424-436

Authors: Wiley LA, Burnight ER, Kaalberg EE, Jiao C, Riker MJ, Halder JA, Luse MA, Han IC, Russell SR, Sohn EH, Stone EM, Tucker BA, Mullins RF

Abstract
Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

PMID: 29160116 [PubMed - indexed for MEDLINE]

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

Sat, 2019-04-06 02:41
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CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Curr Protoc Stem Cell Biol. 2018 02 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 - indexed for MEDLINE]

Histochemical Analysis of Glaucoma Caused by a Myocilin Mutation in a Human Donor Eye.

Sat, 2019-03-30 02:06
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Histochemical Analysis of Glaucoma Caused by a Myocilin Mutation in a Human Donor Eye.

Ophthalmol Glaucoma. 2018 Sep-Oct;1(2):132-138

Authors: van der Heide CJ, Alward WLM, Flamme-Wiese M, Riker M, Syed NA, Anderson MG, Carter K, Kuehn MH, Stone EM, Mullins RF, Fingert JH

Abstract
Objective: Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation.
Design: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes.
Subjects: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined.
Methods: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork.
Main Outcome Measure: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects.
Results: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes.
Conclusions: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.

PMID: 30906929 [PubMed]

POSTERIORLY INSERTED VITREOUS BASE: Preoperative Characteristics, Intraoperative Findings, and Outcomes After Vitrectomy.

Fri, 2019-03-22 23:47
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POSTERIORLY INSERTED VITREOUS BASE: Preoperative Characteristics, Intraoperative Findings, and Outcomes After Vitrectomy.

Retina. 2019 Feb 15;:

Authors: Sohn EH, Strohbehn A, Stryjewski T, Brodowska K, Flamme-Wiese MJ, Mullins RF, Eliott D

Abstract
PURPOSE: To determine the preoperative characteristics, intraoperative and postoperative complications, and outcomes of eyes with posteriorly inserted vitreous base.
METHODS: In this retrospective, observational, consecutive case series at 2 academic centers, 37 patients were studied who had posteriorly inserted vitreous base noted during vitrectomy. Posteriorly inserted vitreous base was defined as the insertion of the posterior hyaloid membrane being located posterior to the vortex veins. Fifteen eyes were analyzed in a histopathologic study of donor eyes to determine the average distance of the ora serrata from the vortex veins as this distance is uncertain.
RESULTS: Posteriorly inserted vitreous base was identified during vitrectomy in 31 eyes with rhegmatogenous retinal detachment (84%), 4 with macular hole (11%), 1 with vitreous hemorrhage, and 1 with epiretinal membrane. Adjunctive buckle was used in 24%; 54% had 360° laser. Average number of tears seen preoperatively in those with rhegmatogenous retinal detachment was 3.1. Thirty percent had new breaks identified intraoperatively. Forty-one percent had lattice degeneration; new breaks were found in 40% of eyes with lattice. Thirteen percent of rhegmatogenous retinal detachments developed proliferative vitreoretinopathy. Average distance from the ora serrata to the vortex veins was 7.6 mm.
CONCLUSION: Any eye undergoing vitrectomy may have posteriorly inserted vitreous base, but those with a high number of retinal breaks and lattice near the equator may be at highest risk. Redetachment and proliferative vitreoretinopathy still occur despite knowledge of the disorder and adjuvant treatments.

PMID: 30883531 [PubMed - as supplied by publisher]

How Much Green Does It Take to Be Orange? Determining the Cost Associated with Trauma Center Readiness.

Fri, 2019-02-22 12:36
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How Much Green Does It Take to Be Orange? Determining the Cost Associated with Trauma Center Readiness.

J Trauma Acute Care Surg. 2019 Feb 13;:

Authors: Ashley DW, Mullins RF, Dente CJ, Johns TJ, Garlow LE, Medeiros RS, Atkins EV, Solomon G, Abston D, Ferdinand CH

Abstract
BACKGROUND: Readiness Costs are real expenses incurred by trauma centers to maintain essential infrastructure to provide emergent services on a 24/7 basis. Although the components for readiness are well described in the American College of Surgeon's Resources for Optimal Care of the Injured Patient, the cost associated with each component is not well defined. We hypothesized that meeting the requirements of the 2014 Resources for Optimal Care of the Injured Patient would result in significant costs for trauma centers.
METHODS: The state trauma commission in conjunction with trauma medical directors, program managers, and financial officers of each trauma center standardized definitions for each component of trauma center readiness cost and developed a survey tool for reporting. Readiness costs were grouped into four categories: Administrative/Program Support Staff, Clinical Medical Staff, In-House Operating Room, and Education/Outreach. To verify consistent cost reporting, a financial auditor analyzed all data. Trauma center outliers were further evaluated to validate variances. All Level I/Level II trauma centers (n=16) completed the survey on 2016 data.
RESULTS: Average annual readiness cost is $10,078,506 for a Level I trauma center and $4,925,103 for Level II's. Clinical medical staff was the costliest component representing 55% of costs for Level I's and 64% for Level II's. Although education/outreach is mandated, Level I and II trauma centers only spend approximately $100,000 annually on this category (1-2%) demonstrating a lack of resources.
CONCLUSIONS: This study defines the cost associated with each component of readiness as defined in the Resources for Optimal Care of the Injured Patient manual. Average readiness cost for a Level I trauma center is $10,078,506 and $4,925,103 for a Level II. The significant cost of trauma center readiness highlights the need for additional trauma center funding to meet the requirements set forth by the American College of Surgeons.
LEVEL OF EVIDENCE: Economic & Value-based Evaluations, level III.

PMID: 30768564 [PubMed - as supplied by publisher]

PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq.

Fri, 2019-02-15 11:26
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PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet scRNA-Seq.

Cell Rep. 2019 Feb 12;26(7):1951-1964.e8

Authors: Tyler SR, Rotti PG, Sun X, Yi Y, Xie W, Winter MC, Flamme-Wiese MJ, Tucker BA, Mullins RF, Norris AW, Engelhardt JF

Abstract
Toolsets available for in-depth analysis of scRNA-seq datasets by biologists with little informatics experience is limited. Here, we describe an informatics tool (PyMINEr) that fully automates cell type identification, cell type-specific pathway analyses, graph theory-based analysis of gene regulation, and detection of autocrine-paracrine signaling networks in silico. We applied PyMINEr to interrogate human pancreatic islet scRNA-seq datasets and discovered several features of co-expression graphs, including concordance of scRNA-seq-graph structure with both protein-protein interactions and 3D genomic architecture, association of high-connectivity and low-expression genes with cell type enrichment, and potential for the graph structure to clarify potential etiologies of enigmatic disease-associated variants. We further created a consensus co-expression network and autocrine-paracrine signaling networks within and across islet cell types from seven datasets. PyMINEr correctly identified changes in BMP-WNT signaling associated with cystic fibrosis pancreatic acinar cell loss. This proof-of-principle study demonstrates that the PyMINEr framework will be a valuable resource for scRNA-seq analyses.

PMID: 30759402 [PubMed - in process]

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Fri, 2019-02-15 11:26
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CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Prog Retin Eye Res. 2018 07;65:28-49

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 - indexed for MEDLINE]