Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review
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 newspaper › Journal article › Research › peer-review
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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 -