Vijender Chaitankar

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.

Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

The derivation of three‐dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone‐rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp‐GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self‐organizing 3D retina‐like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S‐opsin and no rhodopsin or L/M‐opsin is present. The transcriptome profile, by RNA‐seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518

NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.

Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor.

Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research.

The advent of high throughput next generation sequencing (NGS) has accelerated the pace of discovery of disease-associated genetic variants and genomewide profiling of expressed sequences and epigenetic marks, thereby permitting systems-based analyses of ocular development and disease. Rapid evolution of NGS and associated methodologies presents significant challenges in acquisition, management, and analysis of large data sets and for extracting biologically or clinically relevant information. Here we illustrate the basic design of commonly used NGS-based methods, specifically whole exome sequencing, transcriptome, and epigenome profiling, and provide recommendations for data analyses. We briefly discuss systems biology approaches for integrating multiple data sets to elucidate gene regulatory or disease networks. While we provide examples from the retina, the NGS guidelines reviewed here are applicable to other tissues/cell types as well.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Regulation of Noncoding Transcriptome in Developing Photoreceptors by Rod Differentiation Factor NRL

Purpose Transcriptome analysis by next generation sequencing allows qualitative and quantitative profiling of expression patterns associated with development and disease. However, most transcribed sequences do not encode proteins, and little is known about the functional relevance of noncoding (nc) transcriptome in neuronal subtypes. The goal of this study was to perform a comprehensive analysis of long noncoding (lncRNAs) and antisense (asRNAs) RNAs expressed in mouse retinal photoreceptors. Methods Transcriptomic profiles were generated at six developmental time points from flow-sorted Nrlp-GFP (rods) and Nrlp-GFP;Nrl−/− (S-cone like) mouse photoreceptors. Bioinformatic analysis was performed to identify novel noncoding transcripts and assess their regulation by rod differentiation factor neural retina leucine zipper (NRL). In situ hybridization (ISH) was used for validation and cellular localization. Results NcRNA profiles demonstrated dynamic yet specific expression signature and coexpression clusters during rod development. In addition to currently annotated 586 lncRNAs and 454 asRNAs, we identified 1037 lncRNAs and 243 asRNAs by de novo assembly. Of these, 119 lncRNAs showed altered expression in the absence of NRL and included NRL binding sites in their promoter/enhancer regions. ISH studies validated the expression of 24 lncRNAs (including 12 previously unannotated) and 4 asRNAs in photoreceptors. Coexpression analysis demonstrated 63 functional modules and 209 significant antisense-gene correlations, allowing us to predict possible role of these lncRNAs in rods. Conclusions Our studies reveal coregulation of coding and noncoding transcripts in rod photoreceptors by NRL and establish the framework for deciphering the function of ncRNAs during retinal development.

Epigenetic control of gene regulation during development and disease: A view from the retina

Complex biological processes, such as organogenesis and homeostasis, are stringently regulated by genetic programs that are fine-tuned by epigenetic factors to establish cell fates and/or to respond to the microenvironment. Gene regulatory networks that guide cell differentiation and function are modulated and stabilized by modifications to DNA, RNA and proteins. In this review, we focus on two key epigenetic changes - DNA methylation and histone modifications - and discuss their contribution to retinal development, aging and disease, especially in the context of age-related macular degeneration (AMD) and diabetic retinopathy. We highlight less-studied roles of DNA methylation and provide the RNA expression profiles of epigenetic enzymes in human and mouse retina in comparison to other tissues. We also review computational tools and emergent technologies to profile, analyze and integrate epigenetic information. We suggest implementation of editing tools and single-cell technologies to trace and perturb the epigenome for delineating its role in transcriptional regulation. Finally, we present our thoughts on exciting avenues for exploring epigenome in retinal metabolism, disease modeling, and regeneration.

Molecular Diagnosis of Inherited Retinal Diseases in Indigenous African Populations by Whole-Exome Sequencing.

Purpose A majority of genes associated with inherited retinal diseases (IRDs) have been identified in patients of European origin. Indigenous African populations exhibit rich genomic diversity, and evaluation of reported genetic mutations has yielded low returns so far. Our goal was to perform whole-exome sequencing (WES) to examine variants in known IRD genes in underrepresented African cohorts. Methods Whole-exome sequencing was performed on 56 samples from 16 families with diverse IRD phenotypes that had remained undiagnosed after screening for known mutations using genotyping-based microarrays (Asper Ophthalmics). Variants in reported IRD genes were identified using WES and validated by Sanger sequencing. Custom TaqMan assays were used to screen for identified mutations in 193 unrelated indigenous Africans with IRDs. Results A total of 3494 variants were identified in 217 known IRD genes, leading to the identification of seven different mutations (including six novel) in six genes (RHO, PRPF3, PRPF31, ABCA4, CERKL, and PDE6B) in six distinct families. TaqMan screening in additional probands revealed identical homozygous CERKL and PDE6B variants in four more patients. Conclusions This is the first report of WES of patients with IRDs in indigenous African populations. Our study identified genetic defects in almost 40% of the families analyzed, significantly enhancing the molecular diagnosis of IRD in South Africa. Thus, WES of understudied cohorts seems to present an effective strategy for determining novel mutations in heterogeneous retinal diseases.

A three-dimensional organoid model recapitulates tumorigenic aspects and drug responses of advanced human retinoblastoma

Persistent or recurrent retinoblastoma (RB) is associated with the presence of vitreous or/and subretinal seeds in advanced RB and represents a major cause of therapeutic failure. This necessitates the development of novel therapies and thus requires a model of advanced RB for testing candidate therapeutics. To this aim, we established and characterized a three-dimensional, self-organizing organoid model derived from chemotherapy-naïve tumors. The responses of organoids to drugs were determined and compared to relate organoid model to advanced RB, in terms of drug sensitivities. We found that organoids had histological features resembling retinal tumors and seeds and retained DNA copy-number alterations as well as gene and protein expression of the parental tissue. Cone signal circuitry (M/L+ cells) and glial tumor microenvironment (GFAP+ cells) were primarily present in organoids. Topotecan alone or the combined drug regimen of topotecan and melphalan effectively targeted proliferative tumor cones (RXRγ+ Ki67+) in organoids after 24-h drug exposure, blocking mitotic entry. In contrast, methotrexate showed the least efficacy against tumor cells. The drug responses of organoids were consistent with those of tumor cells in advanced disease. Patient-derived organoids enable the creation of a faithful model to use in examining novel therapeutics for RB.