Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization.

Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome-editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knockdown using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

Comparison of chorioretinal layers in rhesus macaques using spectral-domain optical coherence tomography and high-resolution histological sections.

Nonhuman primates are important preclinical models of retinal diseases because they uniquely possess a macula similar to humans. Ocular imaging technologies such as spectral-domain optical coherence tomography (SD-OCT) allow noninvasive, in vivo measurements of chorioretinal layers with near-histological resolution. However, the boundaries are based on differences in reflectivity, and detailed correlations with histological tissue layers have not been explored in rhesus macaques, which are widely used for biomedical research. Here, we compare the macular anatomy and thickness measurements of chorioretinal layers in rhesus macaque eyes using SD-OCT and high-resolution histological sections. Images were obtained from methylmethacrylate-embedded histological sections of 6 healthy adult rhesus macaques, and compared with SD-OCT images from 6 age-matched animals. Thicknesses of chorioretinal layers were measured across the central 3 mm macular region using custom semi-automated or manual software segmentation, and compared between the two modalities. We found that histological sections provide better distinction between the ganglion cell layer (GCL) and inner plexiform layer (IPL) than SD-OCT imaging. The first hyperreflective band between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) appears wider on SD-OCT than the junction between photoreceptor inner and outer segments seen on histology. SD-OCT poorly distinguishes Henle nerve fibers from the outer nuclear layer (ONL), while histology correctly identifies these fibers as part of the outer plexiform layer (OPL). Overall, the GCL, inner nuclear layer (INL), and OPL are significantly thicker on histology, especially at the fovea; while the ONL, choriocapillaris (CC), and outer choroid (OC) are thicker on SD-OCT. Our results show that both SD-OCT and high-resolution histological sections allow reliable measurements of chorioretinal layers in rhesus macaques, with distinct advantages for different sublayers. These findings demonstrate the effects of tissue processing on chorioretinal anatomy, and provide normative values for chorioretinal thickness measurements on SD-OCT for future studies of disease models in these nonhuman primates.

In Vivo Multimodal Imaging of Drusenoid Lesions in Rhesus Macaques.

Nonhuman primates are the only mammals to possess a true macula similar to humans, and spontaneously develop drusenoid lesions which are hallmarks of age-related macular degeneration (AMD). Prior studies demonstrated similarities between human and nonhuman primate drusen based on clinical appearance and histopathology. Here, we employed fundus photography, spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF), and infrared reflectance (IR) to characterize drusenoid lesions in aged rhesus macaques. Of 65 animals evaluated, we identified lesions in 20 animals (30.7%). Using the Age-Related Eye Disease Study 2 (AREDS2) grading system and multimodal imaging, we identified two distinct drusen phenotypes - 1) soft drusen that are larger and appear as hyperreflective deposits between the retinal pigment epithelium (RPE) and Bruch's membrane on SD-OCT, and 2) hard, punctate lesions that are smaller and undetectable on SD-OCT. Both exhibit variable FAF intensities and are poorly visualized on IR. Eyes with drusen exhibited a slightly thicker RPE compared with control eyes (+3.4 µm, P=0.012). Genetic polymorphisms associated with drusenoid lesions in rhesus monkeys in ARMS2 and HTRA1 were similar in frequency between the two phenotypes. These results refine our understanding of drusen development, and provide insight into the absence of advanced AMD in nonhuman primates.

Effect of Syringe Design on the Accuracy and Precision of Intravitreal Injections of Anti-VEGF Agents.

PURPOSE: To evaluate the accuracy and precision of different syringe designs for intravitreal injection of anti-VEGF agents. METHODS: Volume output was measured from three syringe designs-1) 1.0 mL tuberculin syringe, 2) 1.0 mL syringe with low dead space plunger, and 3) 0.5 mL low-volume syringe-to deliver 50 µL of bevacizumab, ranibizumab, or aflibercept, each repeated four times by three different physicians for 108 total simulated injections. Volume output was calculated from difference in syringe weight before and after expelling the drug. Accuracy was determined by mean absolute percentage error (MAPE), and precision was measured by coefficient of variation (CV). RESULTS: Volume output from all three syringes was significantly different from 50 µL, with mean volumes of 58.0 ± 5.7 µL for the tuberculin syringe, 58.0 ± 4.0 µL for the low dead space syringe, and 55.5 ± 5.1 µL for the low-volume syringe (p < 0.00001 for all). The low-volume syringe was the most accurate (MAPE = 12.8 ± 7.8% vs. 17.3 ± 9.3% or 15.9 ± 8.1%), and the low dead space syringe was the most reproducible (CV = 0.068 vs. 0.091 or 0.097). There was no significant difference in volume output between different anti-VEGF agents. CONCLUSIONS: Intravitreal injections of anti-VEGF agents using a 1.0 mL tuberculin syringe demonstrate poor accuracy and precision. A lower capacity syringe may improve accuracy, while a low dead space plunger may improve precision.

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

PURPOSE: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. METHODS: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. RESULTS: Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. CONCLUSIONS: The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.