Phylogenetic and Recombination Analysis of Clinical Vitreous Humor-Derived Adenovirus Isolates Reveals Discordance Between Serotype and Phylogeny.

Purpose: To sequence, identify, and perform phylogenetic and recombination analysis on three clinical adenovirus samples taken from the vitreous humor at the Bascom Palmer Eye Institute. Methods: The PacBio Sequel II was used to sequence the genomes of the three clinical adenovirus isolates. To identify the isolates, a full genome-based multiple sequence alignment (MSA) of 722 mastadenoviruses was generated using multiple alignment using fast Fourier transform (MAFFT). MAFFT was also used to generate genome-based human adenovirus B (HAdV-B) MSAs, as well as HAdV-B fiber, hexon, and penton protein-based MSAs. To examine recombination within HAdV-B, RF-Net 2 and Bootscan software programs were used. Results: In the course of classifying three new atypical ocular adenovirus samples, taken from the vitreous humor, we found that all three isolates were HAdV-B species. The three Bascom Palmer HAdV-B genomes were then combined with over 300 HAdV-B genome sequences, including nine ocular HAdV-B genome sequences. Attempts to categorize the penton, hexon, and fiber serotypes using phylogeny of the three Bascom Palmer samples were inconclusive due to incongruence between serotype and phylogeny in the dataset. Recombination analysis using a subset of HAdV-B strains to generate a hybridization network detected recombination between nonhuman primate and human-derived strains, recombination between one HAdV-B strain and the HAdV-E outgroup, and limited recombination between the B1 and B2 clades. Conclusions: The discordance between serotype and phylogeny detected in this study suggests that the current classification system does not accurately describe the natural history and phylogenetic relationships among adenoviruses.

OUTCOMES OF INFECTIOUS PANUVEITIS ASSOCIATED WITH SIMULTANEOUS MULTI-POSITIVE OCULAR FLUID POLYMERASE CHAIN REACTION.

PURPOSE: To evaluate features of infectious panuveitis associated with multiple pathogens detected by ocular fluid sampling. METHODS: Single-center, retrospective, consecutive case series of patients with aqueous/vitreous polymerase chain reaction testing with >1 positive result in a single sample from 2001 to 2021. RESULTS: Of 1,588 polymerase chain reaction samples, 28 (1.76%) were positive for two pathogens. Most common pathogens were cytomegalovirus (n = 16, 57.1%) and Epstein-Barr virus (n = 13, 46.4%), followed by varicella zoster virus (n = 8, 28.6%), Toxoplasma gondii (n = 6, 21.4%), herpes simplex virus 2 (n = 6, 21.4%), herpes simplex virus 1 (n = 6, 21.4%), and Toxocara (n = 1, 3.6%). Mean initial and final visual acuity (logarithm of the minimum angle of resolution) were 1.3 ± 0.9 (Snellen ∼20/400) and 1.3 ± 1.1 (Snellen ∼20/400), respectively. Cytomegalovirus-positive eyes (n = 16, 61.5%) had a mean final visual acuity of 0.94 ± 1.1 (Snellen ∼20/175), whereas cytomegalovirus-negative eyes (n = 10, 38%) had a final visual acuity of 1.82 ± 1.0 (Snellen ∼20/1,320) ( P < 0.05). Main clinical features included intraocular inflammation (100%), retinal whitening (84.6%), immunosuppression (65.4%), retinal hemorrhage (38.5%), and retinal detachment (34.6%). CONCLUSION: Cytomegalovirus or Epstein-Barr virus were common unique pathogens identified in multi-PCR-positive samples. Most patients with co-infection were immunosuppressed with a high rate of retinal detachment and poor final visual acuity. Cytomegalovirus-positive eyes had better visual outcomes compared with cytomegalovirus-negative eyes.

Phylogenetic and Genomic Characterization of Whole Genome Sequences of Ocular Herpes Simplex Virus Type 1 Isolates Identifies Possible Virulence Determinants in Humans.

Purpose: There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses. Methods: The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease. Results: A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG. Conclusions: There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Morning glory disc anomaly associated with moyamoya disease and pituitary stalk duplication.

Purpose: We report a case of a 10-year-old with Moring glory disc anomaly (MGDA) associated with Moyamoya disease and pituitary stalk duplication. Observations: A 10-year-old Asian child presented with decreased vision in the right eye and bilateral nystagmus. Both dilated fundus exam and magnetic resonance imaging (MRI) of the orbit confirmed MGDA of the right eye. MRI of the brain demonstrated duplication of the pituitary stalk. Magnetic resonance angiography (MRA) of the brain revealed bilateral severe narrowing (greater on the right side) of the distal supraclinoid internal carotid arteries with bilateral reconstitution at the carotid terminus and prominent collaterals, suggestive of Moyamoya disease. Conclusions: Patients with MGDA should undergo neuroimaging due to the associated central nervous system (CNS) anomalies.

Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity.

Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3-/-) knockout mice. The corneal stroma in F3-/- mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3-/- mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3-/- mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease. KEY MESSAGES: • Fibulins are expressed throughout the body at varying levels. • Fibulin-3 has a tissue-specific pattern of expression within the eye. • Lack of fibulin-3 leads to structural deformities in the cornea. • The retina and RPE remain structurally and functionally healthy in the absence of fibulin-3 in both mice and humans.

Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones.

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.

Delivery of Antisense Oligonucleotides to the Cornea.

Antisense oligonucleotides (ASOs) are synthetic nucleic acids that recognize complementary RNA sequences inside cells and modulate gene expression. In this study, we explore the feasibility of ASO delivery to the cornea. We used quantitative polymerase chain reaction to test the efficacy of a benchmark ASO targeting a noncoding nuclear RNA, Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), in a human corneal endothelial cell line, ex vivo human corneas, and in vivo in mice. In vivo delivery was via intravitreal or intracameral injections as well as topical administration. The anti-MALAT1 ASO significantly reduced expression of MALAT1 in a corneal endothelial cell line. We achieved a dose-dependent reduction of target gene expression in endothelial tissue from ex vivo human donor corneas. In vivo mouse experiments confirmed MALAT1 reduction in whole corneal tissue via intravitreal and intracameral routes, 82% and 71% knockdown, respectively (P < 0.001). Effects persisted up to at least 21 days, 32% (P < 0.05) and 43% (P < 0.05) knockdown, respectively. We developed protocols for the isolation and analysis of mouse corneal endothelium and observed reduction in MALAT1 expression upon both intravitreal and intracameral administrations, 64% (P < 0.05) and 63% (P < 0.05) knockdown, respectively. These data open the possibility of using ASOs to treat corneal disease.

Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo.

The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.

A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye - Implications for Ocular Gene Therapy.

Purpose: Temporal and reversible control of protein expression in vivo is a central goal for many gene therapies, especially for strategies involving proteins that are detrimental to physiology if constitutively expressed. Accordingly, we explored whether protein abundance in the mouse retina could be effectively controlled using a destabilizing Escherichia coli dihydrofolate reductase (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP). Methods: We intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: DHFR fused to yellow fluorescent protein (DHFR.YFP) and mCherry. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. Ocular TMP levels post treatment were quantified by LC-MS/MS. Protein abundance was measured by fundus fluorescence imaging and western blotting. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment. Results: Without TMP, DHFR.YFP was efficiently degraded in the retina. TMP achieved ocular concentrations of ∼13.6 µM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Oral gavage and TMP eye drops stabilized DHFR.YFP as quickly as 6 hours, whereas continuous TMP drinking water could stabilize DHFR.YFP for ≥3 months. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. Long-term TMP treatment had no impact on retina function/structure and had no effect on >99.9% of tested genes. Conclusions: This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.

Defective retinal depolarizing bipolar cells in regulators of G protein signaling (RGS) 7 and 11 double null mice.

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gß5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gß5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gß5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gß5S is undetectable, whereas levels of the photoreceptor-specific Gß5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gß5S expression in retina, the DBC-related defects in Gß5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gß5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.