TY - JOUR

T1 - Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs

AU - Holmgaard, Andreas Braae

AU - Askou, Anne Louise

AU - Jensen, Emilie Grarup

AU - Alsing, Sidsel

AU - Bak, Rasmus O

AU - Mikkelsen, Jacob Giehm

AU - Corydon, Thomas J

N1 - Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

PY - 2021/1

Y1 - 2021/1

N2 - The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.

AB - The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.

KW - Cas9

KW - age-related macular degeneration

KW - chemically modified single guide RNAs

KW - gene knockout

KW - nonviral gene therapy

KW - retina pigment epithelium

KW - ribonucleoproteins

KW - short-lived nuclease activity

KW - vascular endothelial growth factor A

U2 - 10.1016/j.ymthe.2020.09.032

DO - 10.1016/j.ymthe.2020.09.032

M3 - Journal article

C2 - 33022212

VL - 29

SP - 191

EP - 207

JO - Molecular Therapy

JF - Molecular Therapy

SN - 1525-0016

IS - 1

ER -