CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Jin-Soo Kim,Jin Hyoung Kim,Taeyoung Koo,Jeong Hun Kim,Daesik Kim,Hee-Yeon Cho,Dong Hyun Jo,Jeungeun Kim,Sung Wook Park

doi:10.1038/s41467-018-04175-y

Abstract

LbCpf1, derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. Here we show that LbCpf1 can be used for therapeutic gene editing in a mouse model of age-related macular degeneration (AMD). The intravitreal delivery of LbCpf1, targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. Importantly, LbCpf1 targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. Unlike aflibercept, LbCpf1 targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases.

Highlights

LbCpf[1], derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications
We selected the TS3 and TS3 CRISPR RNA (crRNA), which target the exon 1 of vascular endothelial growth factor A (Vegfa) and exon 8 of hypoxia inducing factor 1a (Hif1a) genes, respectively (Fig. 1b), because they both resulted in high ratio of out-of-frame indels as a representative mutation pattern with high efficiencies (Fig. 1a; Supplementary Fig. 1)
We found that associated virus (AAV)-CRISPR from Prevoltella and Francisella 1 (Cpf1)-Vegfa or -Hif1a did not affect the size of the opsin-positive area in the retina, which is closely related to cone function (Fig. 4d, e), suggesting that AAV-Cpf1-mediated Vegfa or Hif1a gene disruption provides a safe therapeutic window

Summary

Introduction

LbCpf[1], derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. The intravitreal delivery of LbCpf[1], targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. LbCpf[1] targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. LbCpf[1] targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases. There are several reports demonstrating that LbCpf[1] exhibits a higher genome-wide specificity compared to SpCas[922,23]

Methods

Results

Conclusion