In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

Abstract

Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp) Cas9, delivered by lentiviral vectors (LVs), can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa) gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs) induce in vitro indel formation at high frequencies (44%–93%). A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP+ RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration.

Brain volumetric alterations accompanied with loss of striatal medium-sized spiny neurons and cortical parvalbumin expressing interneurons in Brd1+/− mice

“…Finally, the sections were mounted on glass slides and coverslipped with Prolong Diamond mounting media (Thermofisher Scientific, Waltham, USA). The quality of staining of PV+ and VVA+ interneurons was evaluated using Zeiss LSM710/780 laser-scanning confocal microscopes controlled by ZEN 2009/2011 software (Carl Zeiss, Jena, Germany), respectively, as previously described 58 . 3-dimensional confocal images were taken by collecting stack images (135 μm × 135 μm) with a z-plane step size of 1.5 μm throughout the section depth giving a total of 14–16 stacks per image.…”

Methods to label, image, and analyze the complex structural architectures of microvascular networks

“…Advances in new imaging techniques allow for in vivo imaging that provides the opportunity for higher throughput and fully temporal measurements in various tissues. The latest in vivo gene editing techniques, such as CRISPR/Cas9, have made targeted genetic alterations easier, yet there are still challenges remaining with regards to efficiency and off‐target binding of genetic payloads . At the same time, advances in 3D bioprinting, biomaterial research, and patient‐specific primary cell culture allow for more advanced in vitro models, although there is still difficulty with cell collection in these systems for subsequent analysis .…”

CRISPR/Cas9-Mediated Treatment Ameliorates the Phenotype of the Epidermolytic Palmoplantar Keratoderma-like Mouse

“… 30 , 31 So far, only a few studies have demonstrated in vivo gene editing following LV delivery of Cas9. 31 , 32 , 33 , 34 In this study, we chose LV as the delivery tool and suggested a better therapeutic efficacy over the renewal cycle of the epidermis. Then, the disrupted cytoskeletal integrity, abnormal differentiation, and irregular proliferation were ameliorated.…”