VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy

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VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy. / Alsing, Sidsel; Doktor, Thomas Koed; Askou, Anne Louise et al.

In: Molecular Therapy - Nucleic Acids, Vol. 28, 06.2022, p. 58-76.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Alsing, Sidsel ; Doktor, Thomas Koed ; Askou, Anne Louise et al. / VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy. In: Molecular Therapy - Nucleic Acids. 2022 ; Vol. 28. pp. 58-76.

Bibtex

@article{e49ef3210e87410489baf6adbeeb2973,
title = "VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy",
abstract = "Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.",
keywords = "agoshRNA, agshRNA, Dicer-independent shRNAs, in vivo efficacy, Non-coding RNAs, off-target effects, Pol II-driven miRNA scaffold, retinal gene therapy, RNA interference, VEGF",
author = "Sidsel Alsing and Doktor, {Thomas Koed} and Askou, {Anne Louise} and Jensen, {Emilie Grarup} and Ulvi Ahmadov and Kristensen, {Lasse Sommer} and Andresen, {Brage Storstein} and Lars Aagaard and Corydon, {Thomas J.}",
year = "2022",
month = jun,
doi = "10.1016/j.omtn.2022.02.019",
language = "English",
volume = "28",
pages = "58--76",
journal = "Molecular Therapy - Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",

}

RIS

TY - JOUR

T1 - VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy

AU - Alsing, Sidsel

AU - Doktor, Thomas Koed

AU - Askou, Anne Louise

AU - Jensen, Emilie Grarup

AU - Ahmadov, Ulvi

AU - Kristensen, Lasse Sommer

AU - Andresen, Brage Storstein

AU - Aagaard, Lars

AU - Corydon, Thomas J.

PY - 2022/6

Y1 - 2022/6

N2 - Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

AB - Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

KW - agoshRNA

KW - agshRNA

KW - Dicer-independent shRNAs

KW - in vivo efficacy

KW - Non-coding RNAs

KW - off-target effects

KW - Pol II-driven miRNA scaffold

KW - retinal gene therapy

KW - RNA interference

KW - VEGF

UR - http://www.scopus.com/inward/record.url?scp=85126453187&partnerID=8YFLogxK

U2 - 10.1016/j.omtn.2022.02.019

DO - 10.1016/j.omtn.2022.02.019

M3 - Journal article

C2 - 35356684

AN - SCOPUS:85126453187

VL - 28

SP - 58

EP - 76

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

SN - 2162-2531

ER -