Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.

Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases

Neuronal Mitochondrial Dysfunction Activates the Integrated Stress Response to Induce Fibroblast Growth Factor 21.

Stress adaptation is essential for neuronal health. While the fundamental role of mitochondria in neuronal development has been demonstrated, it is still not clear how adult neurons respond to alterations in mitochondrial function and how neurons sense, signal, and respond to dysfunction of mitochondria and their interacting organelles. Here, we show that neuron-specific, inducible in vivo ablation of the mitochondrial fission protein Drp1 causes ER stress, resulting in activation of the integrated stress response to culminate in neuronal expression of the cytokine Fgf21. Neuron-derived Fgf21 induction occurs also in murine models of tauopathy and prion disease, highlighting the potential of this cytokine as an early biomarker for latent neurodegenerative conditions

Investigation of Retinal Morphology Alterations Using Spectral Domain Optical Coherence Tomography in a Mouse Model of Retinal Branch and Central Retinal Vein Occlusion

<div><p>Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize <i>in vivo</i> longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.</p></div

Correlation of angiographic findings with histology seven days after laser photocoagulation.

<p>Representative images after (<b>A</b>-<b>D</b>) single vein occlusion, (<b>E-G</b>) multiple vein occlusion, and (<b>H</b>-<b>J</b>) a control eye. (<b>A</b>, <b>E</b>, <b>H</b>) Whole eye hematoxylin & eosin sections [scale bars 500 μm], (<b>B</b>, <b>F</b>, <b>I</b>) magnified cutouts of the peripheral retina [scale bars 150 μm], and (<b>C</b>, <b>G</b>, <b>J</b>) fluorescein angiograms. At the laser site destruction of the outer retinal layers is evident (inset <b>D</b>) with a subretinal scar-like lesion (black arrows), representing collateral damage. (<b>E</b>) These changes are more pronounced in eyes where all veins have been occluded. (<b>B</b>, <b>F</b>) Interestingly, the inner retinal layers distal to the interventional site are thinner than in the control (<b>I</b>), whereas the outer nuclear layer and the photoreceptor outer segments are of normal dimensions. The outer plexiform layer is partially missing. The cell density in the ganglion cell layer is reduced in eyes after laser to (<b>F</b>) multiple veins and after (<b>B</b>) single vein laser in the affected area of the retina.</p

Experimental retinal vein occlusion in mice.

<p>(<b>A</b>) Location of experimental vein thrombosis. (<b>B</b>) Confirmation using ultra-widefield retinal imaging three days after occlusion. The vein appears dilated distally with reduced blood vessel caliber proximal to the intervention site. (<b>C&D</b>) Color retinal imaging using ultra-widefield technology illustrating the situation immediately after application of the laser burn. The veins appear white at the occlusion site and stasis of the blood column is observed. (<b>C</b>: overview, <b>D</b>: laser site at higher magnification).</p

Reperfusion patterns after single retinal vein occlusion.

<p>(<b>A</b>–<b>C</b>) Angiographic findings observed 7–14 days after experimental retinal vein occlusion are displayed. (<b>A</b>) Reperfusion had occurred in some eyes at week one. (<b>B</b>, <b>C</b>) Collateral blood vessels were evident in others. (<b>C</b>) These collaterals observed in an eye two weeks after experimental RVO were particularly prominent. (<b>D</b>, <b>E</b>) The optical coherence tomography scans taken horizontally from this eye highlight the depth location of the different vascular plexuses in the retina. While the normal major retinal veins (blue and dark green arrowhead) lie adjacent to the inner limiting membrane, the collaterals, representing dilated preexisting blood vessels, are found deeper in the retina (light green and green arrowhead in <b>E</b>). (<b>F</b>) Collateral blood vessels in a human with chronic BRVO for comparison. The vein is blocked at an arterio-venous crossing (red arrow) and appears dilated distal to the occlusion site. Tortuous collateral veins (yellow arrowhead) span from the dilated occluded vein to a proximal venous branch.</p

Angiographic findings with different laser patterns seven days after experimental retinal vein occlusion.

<p>(<b>A</b>) High-resolution fluorescein angiogram (Heidelberg Spectralis HRA) displays the structure of the blood vessels at the level of the capillaries in a control mouse. (<b>B-D</b>) Images represent the distinct angiographic patterns after different types of laser application. (<b>B</b>) Successful experimental retinal vein occlusion of a single vessel with blood flow still absent proximal to the interventional site. Some drop out of capillaries in the drainage area of the affected vein is observed, which is particularly marked adjacent to the laser site. (<b>C</b>) In some experimental eyes the arterial side of the circulation is affected too, and a complete breakdown of capillary perfusion is manifest in a sector of the retina. (<b>D</b>) In an experimental animal, which had all veins occluded, capillary drop out is more widespread and affects all quadrants to a similar extent. Some veins show reperfusion.</p

Optical coherence tomography and thickness measurements.

<p>(<b>A</b>) Mean total retinal thickness (means±SEM) over 4 weeks. The retinal thickening is marginally greater after occlusion of all retinal veins (CRVO) than after blockage of a single vein (BRVO) up to about day 7 (* p = 0.0426). At later time points the retinal thickness is decreased in the affected areas after both CRVO and BRVO, but there is no longer a statistical difference between the two laser patterns. The mean retinal thickness in affected quadrants is significantly different (p < 0.001) from control eyes (grey line) at all time points except day 7. (<b>B</b>-<b>H</b>) BRVO phenotype after occlusion of a single vein. (<b>C</b>) Fluorescein angiogram. (<b>D</b>-<b>H</b>) Serial scans taken at the location highlighted on photograph (<b>B</b>). The red arrowhead marks a retinal artery at the right border of the catchment area of the occluded vein. (<b>I</b>-<b>O</b>) CRVO phenotype after occlusion of multiple veins. (<b>J</b>) Fluorescein angiogram. (<b>K</b>-<b>O</b>) Horizontal scans taken through the optic nerve head. (<b>D, K, L</b>) Hyperreflectivity with washed-out retinal structures and thickening of the retina are observed within the first few days. (<b>F-H</b>, <b>M-O</b>) Thereafter progressive thinning of the inner retinal layers is evident. (<b>F</b>) A sliver of subretinal fluid (yellow arrows) is apparent [scale bars 200 μm].</p