Elliot H Choi, Anahita Daruwalla, Susie Suh, Henri Leinonen, Krzysztof Palczewski

CRISPR-Cas-based genome editing technologies could, in principle, be used to treat a wide variety of inherited diseases, including genetic disorders of vision. Programmable CRISPR-Cas nucleases are effective tools for gene disruption, but they are poorly suited for precisely correcting pathogenic mutations in most therapeutic settings. Recently developed precision genome editing agents, including base editors and prime editors, have enabled precise gene correction and disease rescue in multiple preclinical models of genetic disorders. Additionally, new delivery technologies that transiently deliver precision genome editing agents in vivo offer minimized off-target editing and improved safety profiles. These improvements to precision genome editing and delivery technologies are expected to revolutionize the treatment of genetic disorders of vision and other diseases. In this Perspective, we describe current preclinical and clinical genome editing approaches for treating inherited retinal degenerative diseases, and we discuss important considerations that should be addressed as these approaches are translated into clinical practice.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1 . We discovered an accumulation of ceramides in the AdipoR1 ^–/– retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1 ^–/– mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1- related retinopathy.

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre- LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreER ^T2 coding sequence is fused with the native RPE-specific 65 kDa protein ( Rpe65 ) gene for cotranslational expression of CreER ^T2 . Cre ^+/– mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre ^+/– mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre ^+/– mice. These data indicate that Rpe65 ^CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.

External ventricular drainage is a common and invaluable neurosurgical procedure and is one of the first procedures learned and performed independently by neurosurgical residents. As accuracy and precision are paramount to EVD placement, attention to technique is paid early in a resident's training. With the advancement of virtual technology, it has become increasingly possible to move away from traditional training situations and human error, and towards automated assistance and superior cyber learning environments. Although there is significant room for improvement, there are promising results with computerized placement guides and virtually augmented practice. Here, we provide a review of the updates on EVD placement techniques, technology and training, all of which serve to improve the precision, accuracy and efficiency of EVD placement.