International Journal of Molecular Sciences

Research

14 pages, 4248 KiB

Open AccessArticle

Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice

by Kabhilan Mohan, Sushil Kumar Dubey, Kyungsik Jung, Rashmi Dubey, Qing Jun Wang, Subhash Prajapati, Jacob Roney, Jennifer Abney and Mark Ellsworth Kleinman

Int. J. Mol. Sci. 2023, 24(6), 5186; https://doi.org/10.3390/ijms24065186 - 8 Mar 2023

Viewed by 1753

Abstract

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with [...] Read more.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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18 pages, 2305 KiB

Open AccessArticle

Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection

by Xinyan Zhang, Jinxian Xu, Brendan Marshall, Zheng Dong, Yutao Liu, Diego G. Espinosa-Heidmann and Ming Zhang

Int. J. Mol. Sci. 2023, 24(5), 4322; https://doi.org/10.3390/ijms24054322 - 21 Feb 2023

Viewed by 1373

Abstract

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways [...] Read more.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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19 pages, 5457 KiB

Open AccessArticle

Analysis of Electric Field Stimulation in Blue Light Stressed 661W Cells

by Sharanya Bola, Pallavi Subramanian, Daniela Calzia, Andreas Dahl, Isabella Panfoli, Richard H. W. Funk and Cora Roehlecke

Int. J. Mol. Sci. 2023, 24(4), 3433; https://doi.org/10.3390/ijms24043433 - 8 Feb 2023

Viewed by 1465

Abstract

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected [...] Read more.

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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16 pages, 3074 KiB

Open AccessArticle

Congenital Stationary Night Blindness: Clinical and Genetic Features

by Angela H. Kim, Pei-Kang Liu, Yin-Hsi Chang, Eugene Yu-Chuan Kang, Hung-Hsuan Wang, Nelson Chen, Yun-Ju Tseng, Go Hun Seo, Hane Lee, Laura Liu, An-Ning Chao, Kuan-Jen Chen, Yih-Shiou Hwang, Wei-Chi Wu, Chi-Chun Lai, Stephen H. Tsang, Meng-Chang Hsiao and Nan-Kai Wang

Int. J. Mol. Sci. 2022, 23(23), 14965; https://doi.org/10.3390/ijms232314965 - 29 Nov 2022

Cited by 7 | Viewed by 4213

Abstract

Congenital stationary night blindness (CSNB) is an inherited retinal disease (IRD) that causes night blindness in childhood with heterogeneous genetic, electrophysical, and clinical characteristics. The development of sequencing technologies and gene therapy have increased the ease and urgency of diagnosing IRDs. This study [...] Read more.

Congenital stationary night blindness (CSNB) is an inherited retinal disease (IRD) that causes night blindness in childhood with heterogeneous genetic, electrophysical, and clinical characteristics. The development of sequencing technologies and gene therapy have increased the ease and urgency of diagnosing IRDs. This study describes seven Taiwanese patients from six unrelated families examined at a tertiary referral center, diagnosed with CSNB, and confirmed by genetic testing. Complete ophthalmic exams included best corrected visual acuity, retinal imaging, and an electroretinogram. The effects of identified novel variants were predicted using clinical details, protein prediction tools, and conservation scores. One patient had an autosomal dominant CSNB with a RHO variant; five patients had complete CSNB with variants in GRM6, TRPM1, and NYX; and one patient had incomplete CSNB with variants in CACNA1F. The patients had Riggs and Schubert–Bornschein types of CSNB with autosomal dominant, autosomal recessive, and X-linked inheritance patterns. This is the first report of CSNB patients in Taiwan with confirmed genetic testing, providing novel perspectives on molecular etiology and genotype–phenotype correlation of CSNB. Particularly, variants in TRPM1, NYX, and CACNA1F in our patient cohort have not previously been described, although their clinical significance needs further study. Additional study is needed for the genotype–phenotype correlation of different mutations causing CSNB. In addition to genetic etiology, the future of gene therapy for CSNB patients is reviewed and discussed. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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24 pages, 5039 KiB

Open AccessArticle

Depleted Calcium Stores and Increased Calcium Entry in Rod Photoreceptors of the Cacna2d4 Mouse Model of Cone-Rod Dystrophy RCD4

by Vittorio Vellani, Giovanna Mauro and Gian Carlo Demontis

Int. J. Mol. Sci. 2022, 23(21), 13080; https://doi.org/10.3390/ijms232113080 - 28 Oct 2022

Cited by 2 | Viewed by 1442

Abstract

Unidentified pathogenetic mechanisms and genetic and clinical heterogeneity represent critical factors hindering the development of treatments for inherited retinal dystrophies. Frameshift mutations in Cacna2d4, which codes for an accessory subunit of voltage-gated calcium channels (VGCC), cause cone-rod dystrophy RCD4 in patients, but [...] Read more.

Unidentified pathogenetic mechanisms and genetic and clinical heterogeneity represent critical factors hindering the development of treatments for inherited retinal dystrophies. Frameshift mutations in Cacna2d4, which codes for an accessory subunit of voltage-gated calcium channels (VGCC), cause cone-rod dystrophy RCD4 in patients, but the underlying mechanisms remain unknown. To define its pathogenetic mechanisms, we investigated the impact of a Cacna2d4 frameshift mutation on the electrophysiological profile and calcium handling of mouse rod photoreceptors by patch-clamp recordings and calcium imaging, respectively. In mutant (MUT) rods, the dysregulation of calcium handling extends beyond the reduction in calcium entry through VGCC and surprisingly involves internal calcium stores’ depletion and upregulation of calcium entry via non-selective cationic channels (CSC). The similar dependence of CSC on basal calcium levels in WT and MUT rods suggests that the primary defect in MUT rods lies in defective calcium stores. Calcium stores’ depletion, leading to upregulated calcium and sodium influx via CSC, represents a novel and, so far, unsuspected consequence of the Cacna2d4 mutation. Blocking CSC may provide a novel strategy to counteract the well-known pathogenetic mechanisms involved in rod demise, such as the reticulum stress response and calcium and sodium overload due to store depletion. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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10 pages, 4110 KiB

Open AccessCommunication

Tropism of the Novel AAVBR1 Capsid Following Subretinal Delivery

by Lara Carroll, Hironori Uehara, Xiaohui Zhang and Balamurali Ambati

Int. J. Mol. Sci. 2022, 23(14), 7738; https://doi.org/10.3390/ijms23147738 - 13 Jul 2022

Viewed by 1594

Abstract

A serious limitation of current adeno-associated viral (AAV) capsids employed for subretinal delivery is achieving adequate lateral spread beyond the injection site, required for the efficient delivery of gene therapy to the outer retina and/or RPE. AAVBR1 is a unique AAV with exceptional [...] Read more.

A serious limitation of current adeno-associated viral (AAV) capsids employed for subretinal delivery is achieving adequate lateral spread beyond the injection site, required for the efficient delivery of gene therapy to the outer retina and/or RPE. AAVBR1 is a unique AAV with exceptional tropism for CNS microvasculature following systemic delivery. Here, we used in vivo and ex vivo analysis to show that subretinal delivery of AAVBR1.GFP in mice achieves superior tropism to RPE and outer retina than either AAV2.GFP or AAV8.GFP, two of the most common capsids used for subretinal delivery. At a low (5 × 10⁸ vg) subretinal dose, the AAVBR1.GFP signal was visible by 48 h and significantly surpassed peak fluorescence of other AAVs in retina and RPE. The co-injection of AAVBR1.GFP with the AAVBR1-specific heptapeptide, NRGTEWD, significantly blocked the AAVBR1.GFP signal, but had no effect on AAV2.GFP fluorescence, confirming that AAVBR1’s enhanced tropism for RPE and outer retina derives from this 7AA modification within the capsid-binding motif. Enhanced dispersal and consequent transduction suggest that AAVBR1 can be employed at a lower dosage than the standard AAV2 capsid to achieve equivalent expression for gene therapy, warranting further evaluation of its utility as a therapeutic vehicle for subretinal delivery. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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Review

Jump to: Research, Other

16 pages, 8080 KiB

Open AccessReview

Primary versus Secondary Elevations in Fundus Autofluorescence

by Rait Parmann, Stephen H. Tsang and Janet R. Sparrow

Int. J. Mol. Sci. 2023, 24(15), 12327; https://doi.org/10.3390/ijms241512327 - 2 Aug 2023

Cited by 1 | Viewed by 1119

Abstract

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven [...] Read more.

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven retinal diseases to highlight the possible pathways to increased fundus autofluorescence. ABCA4- and RDH12-associated diseases benefit from known mechanisms whereby gene malfunctioning leads to elevated bisretinoid levels in RPE cells. On the other hand, peripherin2/RDS-associated disease (PRPH2/RDS), retinitis pigmentosa (RP), central serous chorioretinopathy (CSC), acute zonal occult outer retinopathy (AZOOR), and ceramide kinase like (CERKL)-associated retinal degeneration all express abnormally high fundus autofluorescence levels without a demonstrated pathophysiological pathway for bisretinoid elevation. We suggest that, while a known link from gene mutation to increased production of bisretinoids (as in ABCA4- and RDH12-associated diseases) causes primary elevation in fundus autofluorescence, a secondary autofluorescence elevation also exists, where an impairment and degeneration of photoreceptor cells by various causes leads to an increase in bisretinoid levels in RPE cells. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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12 pages, 1367 KiB

Open AccessReview

Base and Prime Editing in the Retina—From Preclinical Research toward Human Clinical Trials

by Tiffany Yee and Katherine J. Wert

Int. J. Mol. Sci. 2022, 23(20), 12375; https://doi.org/10.3390/ijms232012375 - 16 Oct 2022

Cited by 5 | Viewed by 3146

Abstract

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases that are one of the leading causes of vision loss in young and aged individuals. IRDs are mainly caused by a loss of the post-mitotic photoreceptor neurons of the retina, [...] Read more.

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of diseases that are one of the leading causes of vision loss in young and aged individuals. IRDs are mainly caused by a loss of the post-mitotic photoreceptor neurons of the retina, or by the degeneration of the retinal pigment epithelium. Unfortunately, once these cells are damaged, it is irreversible and leads to permanent vision impairment. Thought to be previously incurable, gene therapy has been rapidly evolving to be a potential treatment to prevent further degeneration of the retina and preserve visual function. The development of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) base and prime editors have increased the capabilities of the genome editing toolbox in recent years. Both base and prime editors evade the creation of double-stranded breaks in deoxyribonucleic acid (DNA) and the requirement of donor template of DNA for repair, which make them advantageous methods in developing clinical therapies. In addition, establishing a permanent edit within the genome could be better suited for patients with progressive degeneration. In this review, we will summarize published uses of successful base and prime editing in treating IRDs. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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Other

Jump to: Research, Review

9 pages, 1306 KiB

Open AccessCase Report

Large Benefit from Simple Things: High-Dose Vitamin A Improves RBP4-Related Retinal Dystrophy

by Vasily M. Smirnov, Baptiste Wilmet, Marco Nassisi, Christel Condroyer, Aline Antonio, Camille Andrieu, Céline Devisme, Serge Sancho, José-Alain Sahel, Christina Zeitz and Isabelle Audo

Int. J. Mol. Sci. 2022, 23(12), 6590; https://doi.org/10.3390/ijms23126590 - 13 Jun 2022

Cited by 4 | Viewed by 1784

Abstract

Inherited retinal diseases (IRD) are a group of heterogeneous disorders, most of which lead to blindness with limited therapeutic options. Pathogenic variants in RBP4, coding for a major blood carrier of retinol, retinol-binding protein 4, are responsible for a peculiar form of [...] Read more.

Inherited retinal diseases (IRD) are a group of heterogeneous disorders, most of which lead to blindness with limited therapeutic options. Pathogenic variants in RBP4, coding for a major blood carrier of retinol, retinol-binding protein 4, are responsible for a peculiar form of IRD. The aim of this study was to investigate if retinal function of an RBP4-related IRD patient can be improved by retinol administration. Our patient presented a peculiar white-dot retinopathy, reminiscent of vitamin A deficient retinopathy. Using a customized next generation sequencing (NGS) IRD panel we discovered a novel loss-of-function homozygous pathogenic variant in RBP4: c.255G >A, p.(Trp85*). Western blotting revealed the absence of RBP4 protein in the patient’s serum. Blood retinol levels were undetectable. The patient was put on a high-dose oral retinol regimen (50,000 UI twice a week). Subjective symptoms and retinal function markedly and sustainably improved at 5-months and 1-year follow-up. Here we show that this novel IRD case can be treated by oral retinol administration. Full article

(This article belongs to the Special Issue Retinal Degeneration—From Genetics to Therapy)

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