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Wu W, Tang L, D'Amore PA, Lei H. Application of CRISPR-Cas9 in eye disease. Exp Eye Res 2017;161:116-123.Abstract
The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease (Cas)9 is an effective instrument for revising the genome with great accuracy. This system has been widely employed to generate mutants in genomes from plants to human cells. Rapid improvements in Cas9 specificity in eukaryotic cells have opened great potential for the use of this technology as a therapeutic. Herein, we summarize the recent advancements of CRISPR-Cas9 use in research on human cells and animal models, and outline a basic and clinical pipeline for CRISPR-Cas9-based treatments of genetic eye diseases.
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Xiong W, Cepko C. Distinct Expression Patterns of AAV8 Vectors with Broadly Active Promoters from Subretinal Injections of Neonatal Mouse Eyes at Two Different Ages. Adv Exp Med Biol 2016;854:501-7.Abstract

The retinal expression patterns were analyzed following the injection of serotype 8 adeno-associated virus (AAV8) vectors that utilize two broadly active and commonly used sets of transcription regulatory sequences. These include the human cytomegalovirus (CMV) immediate early (IE) enhancer/promoter and the hybrid CAG element (also known as CAGGS or CBA) composed of a partial human CMV IE enhancer and the chicken β-actin promoter and intron. Subretinal delivery to postnatal day 0 (P0) or 6 (P6) mouse eyes resulted in efficient labeling of retinal cells, but with very distinct patterns. With P0 delivery, AAV8-CMV-GFP selectively labelled photoreceptors, while AAV8-CAG-GFP efficiently labeled both outer and inner retinal neurons, including photoreceptors, horizontal cells, amacrine cells and retinal ganglion cells. With P6 delivery, both vectors led to efficient labeling of photoreceptors and Müller glia cells, but not of inner retinal neurons. Our results suggest that the cell types that express the genes encoded by subretinally delivered AAV8 vectors are determined by both the timing of the injection and the regulatory sequences.

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Yamada T, Yang Y, Hemberg M, Yoshida T, Cho HY, Murphy PJ, Fioravante D, Regehr WG, Gygi SP, Georgopoulos K, Bonni A. Promoter decommissioning by the NuRD chromatin remodeling complex triggers synaptic connectivity in the mammalian brain. Neuron 2014;83(1):122-34.Abstract
Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.
Yang M, Delcroix V, Lennikov A, Wang N, Makarenkova HP, Dartt DA. Genomic DNA activates the AIM2 inflammasome and STING pathways to induce inflammation in lacrimal gland myoepithelial cells. Ocul Surf 2023;Abstract
PURPOSE: Primary Sjögren's syndrome (pSS) is an autoimmune disease that mainly attacks the lacrimal glands causing severe aqueous-deficient dry eye. Clinical evidence indicates the DNA sensing mechanism in the pathogenesis of pSS. The purpose of the present study is to determine the pro-inflammatory effect of self-genomic DNA (gDNA) on myoepithelial cells (MECs), which along with acinar and ductal cells is a major cell type of the lacrimal gland. METHOD: MECs primary culture was acquired from female C57BL6J mice. Genomic DNA was extracted from the spleen of the same animal. The MECs were challenged with self-gDNA. The cytokine secretion was detected using supernatant by enzyme-linked immunosorbent assay (ELISA). The activation of inflammasomes was determined using FAM-FLICA. Cryosections of NOD.B10.H2b mouse model of pSS were obtained for immunofluorescence microscopy (IF), with Balb/C as control. RESULT: Treatment with gDNA activated AIM2 inflammasome assembly and function, leading to secretion of interleukin (IL)-1β and IL-18 in MECs. The stimulation of IL-1β secretion by gDNA appeared to be solely at the post-translational level, whereas IL-18 secretion was a combination of increased protein synthesis and post-translational modification. Genomic DNA also induced the activation of STimulators of INterferon Genes (STING), which correlated to the activation of STING in the lacrimal gland from the NOD.B10.H2b mouse. STING activation led to the secretion of IFN-β via Nuclear Factor-κB (NF-κB). The IFN-β further enhances the secretion of IL-1β. The contractility of MECs was disabled by treatment with gDNA or poly AnT, independent of the level of intracellular [Ca2+]. CONCLUSION: Self-gDNA induces a proinflammatory response in lacrimal gland MECs by activating both the AIM2 inflammasome and STING and thus may contribute to the pathogenesis of pSS.
Yavuz Saricay L, Hoyek S, Ashit Parikh A, Baldwin G, Bodamer OA, Gonzalez E, Patel NA. A case of Aicardi syndrome associated with duplication event of Xp22 including SHOX. Ophthalmic Genet 2023;44(6):591-594.Abstract
BACKGROUND: Aicardi syndrome is a neurodevelopmental disorder characterized by a triad of partial or complete agenesis of the corpus callosum, infantile spasms, and pathognomonic chorioretinal lacunae. METHODS: Examination, multimodal imaging, and genetic testing were used to guide diagnosis. RESULTS: We report a case of a pediatric patient who was initially diagnosed with refractory infantile spasms. The patient was unresponsive to conventional antiepileptic therapy, and genetic testing with whole exome and mitochondrial genome sequencing could not identify the underlying cause, so vigabatrin was initiated. The ophthalmic examination under anesthesia for vigabatrin toxicity screening revealed chorioretinal atrophy in the retinal periphery of both eyes, with two 3-disc diameter chorioretinal lacunae superotemporal and inferonasal to the optic nerve in the left eye. Given the neuroimaging findings of corpus callosum hypoplasia with polymicrogyria and ocular findings, the patient was diagnosed with Aicardi syndrome. Genetic testing revealed a novel duplication event at the Xp22 locus. CONCLUSIONS: Aicardi syndrome, albeit a rare condition, should always be considered in the differential diagnosis when investigating a female child with refractory seizures in early childhood. Genetic testing may help further our understanding of AIS and the search for a genetic etiology.
Yin Z, Rosenzweig N, Kleemann KL, Zhang X, Brandão W, Margeta MA, Schroeder C, Sivanathan KN, Silveira S, Gauthier C, Mallah D, Pitts KM, Durao A, Herron S, Shorey H, Cheng Y, Barry J-L, Krishnan RK, Wakelin S, Rhee J, Yung A, Aronchik M, Wang C, Jain N, Bao X, Gerrits E, Brouwer N, Deik A, Tenen DG, Ikezu T, Santander NG, McKinsey GL, Baufeld C, Sheppard D, Krasemann S, Nowarski R, Eggen BJL, Clish C, Tanzi RE, Madore C, Arnold TD, Holtzman DM, Butovsky O. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFβ-mediated checkpoints. Nat Immunol 2023;24(11):1839-1853.Abstract
The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with β-amyloid (Aβ) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-β (TGFβ) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFβ pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFβ signaling provides a promising therapeutic intervention for AD.
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Zabaleta N, Bhatt U, Hérate C, Maisonnasse P, Sanmiguel J, Diop C, Castore S, Estelien R, Li D, Dereuddre-Bosquet N, Cavarelli M, Gallouët A-S, Pascal Q, Naninck T, Kahlaoui N, Lemaitre J, Relouzat F, Ronzitti G, Thibaut HJ, Montomoli E, Wilson JM, Le Grand R, Vandenberghe LH. Durable immunogenicity, adaptation to emerging variants, and low-dose efficacy of an AAV-based COVID-19 vaccine platform in macaques. Mol Ther 2022;30(9):2952-2967.Abstract
The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
Zeng Z, Zhang J, Jing S, Cheng Z, Bofill-Mas S, Maluquer de Motes C, Hundesa A, Girones R, Seto D, Zhang Q. Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom. Genome Announc 2016;4(6)Abstract

The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

Zhang C, Owen LA, Lillvis JH, Zhang SX, Kim IK, Deangelis MM. AMD Genomics: Non-Coding RNAs as Biomarkers and Therapeutic Targets. J Clin Med 2022;11(6)Abstract
Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the world's leading cause of blindness in the aging population. Although the clinical stages and forms of AMD have been elucidated, more specific prognostic tools are required to determine when patients with early and intermediate AMD will progress into the advanced stages of AMD. Another challenge in the field has been the appropriate development of therapies for intermediate AMD and advanced atrophic AMD. After numerous negative clinical trials, an anti-C5 agent and anti-C3 agent have recently shown promising results in phase 3 clinical trials, in terms of slowing the growth of geographic atrophy, an advanced form of AMD. Interestingly, both drugs appear to be associated with an increased incidence of wet AMD, another advanced form of the disease, and will require frequent intravitreal injections. Certainly, there remains a need for other therapeutic agents with the potential to prevent progression to advanced stages of the disease. Investigation of the role and clinical utility of non-coding RNAs (ncRNAs) is a major advancement in biology that has only been minimally applied to AMD. In the following review, we discuss the clinical relevance of ncRNAs in AMD as both biomarkers and therapeutic targets.
Zhang Q, Jing S, Cheng Z, Yu Z, Dehghan S, Shamsaddini A, Yan Y, Li M, Seto D. Comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in China reveals similar yet divergent genomes. Emerg Microbes Infect 2017;6(11):e92.Abstract
Human adenovirus type 14 (HAdV-B14p) was originally identified as an acute respiratory disease (ARD) pathogen in The Netherlands in 1955. For approximately fifty years, few sporadic infections were observed. In 2005, HAdV-B14p1, a genomic variant, re-emerged and was associated with several large ARD outbreaks across the U.S. and, subsequently, in Canada, the U.K., Ireland, and China. This strain was associated with an unusually higher fatality rate than previously reported for both this prototype and other HAdV types in general. In China, HAdV-B14 was first observed in 2010, when two unrelated HAdV-B14-associated ARD cases were reported in Southern China (GZ01) and Northern China (BJ430), followed by three subsequent outbreaks. While comparative genomic analysis, including indel analysis, shows that the three China isolates, with whole genome data available, are similar to the de Wit prototype, all are divergent from the U.S. strain (303600; 2007). Although the genomes of strains GZ01 and BJ430 are nearly identical, as per their genome type characterization and percent identities, they are subtly divergent in their genome mutation patterns. These genomes indicate possibly two lineages of HAdV-B14 and independent introductions into China from abroad, or subsequent divergence from one; CHN2012 likely represents a separate sub-lineage. Observations of these simultaneously reported emergent strains in China add to the understanding of the circulation, epidemiology, and evolution of these HAdV pathogens, as well as provide a foundation for developing effective vaccines and public health strategies, including nationwide surveillance in anticipation of larger outbreaks with potentially higher fatality rates associated with HAdV-B14p1.
Zhang H, Daheron L, Cerna-Chavez R, Place EM, Huckfeldt RM, Pierce EA, Garita-Hernandez M. Generation of a human induced pluripotent stem cell line (OGIi001) from peripheral blood mononuclear cells of a healthy male donor. Stem Cell Res 2024;74:103280.Abstract
We have successfully derived a novel human induced pluripotent stem cell (hiPSC) line using non-integrative Sendai virus. This hiPSC line was generated from a healthy male adult donor, aged 55, and subjected to thorough characterization and extensive quality control. The analysis confirmed the expression of undifferentiated stem cell markers, demonstrated the ability to differentiate into the three germ layers, and revealed the absence of any chromosomal abnormalities.
Zhang C, Zhang Q, Wang F, Liu Q. Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish. Biochem Biophys Res Commun 2015;465(4):651-7.Abstract

Proteomic analysis of the mouse photoreceptor sensory cilium identified a set of cilia proteins, including Poc1 centriolar protein b (Poc1b). Previous functional studies in human cells and zebrafish embryos implicated that Poc1b plays important roles in centriole duplication and length control, as well as ciliogenesis. To study the function of Poc1b in photoreceptor sensory cilia and other primary cilia, we expressed a tagged recombinant Poc1b protein in cultured renal epithelial cells and rat retina. Poc1b was localized to the centrioles and spindle bundles during cell cycle progression, and to the basal body of photoreceptor sensory cilia. A morpholino knockdown and complementation assay of poc1b in zebrafish showed that loss of poc1b led to a range of morphological anomalies of cilia commonly associated with human ciliopathies. In the retina, the development of retinal laminae was significantly delayed and the length of photoreceptor outer segments was shortened. Visual behavior studies revealed impaired visual function in the poc1b morphants. In addition, ciliopathy-associated developmental defects, such as small eyes, curved body axis, heart defects, and shortened cilia in Kupffer's vesicle, were observed as well. These data suggest that poc1b is required for normal development and ciliogenesis of retinal photoreceptor sensory cilia and other cilia. Furthermore, this conclusion is supported by recent findings that mutations in POC1B gene have been identified in patients with inherited retinal dystrophy and syndromic retinal ciliopathy.

Zinn E, Vandenberghe LH. Adeno-associated virus: fit to serve. Curr Opin Virol 2014;8C:90-97.Abstract

Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.

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