Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3

Mark E. Kleinman; Hiroki Kaneko; Won Gil Cho; Sami Dridi; Benjamin J. Fowler; Alexander D. Blandford; Romulo J.C. Albuquerque; Yoshio Hirano; Hiroko Terasaki; Mineo Kondo; Takashi Fujita; Balamurali K. Ambati; Valeria Tarallo; Bradley D. Gelfand; Sasha Bogdanovich; Judit Z. Baffi; Jayakrishna Ambati

doi:10.1038/mt.2011.212

Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3

Mark E. Kleinman, Hiroki Kaneko, Won Gil Cho, Sami Dridi, Benjamin J. Fowler, Alexander D. Blandford, Romulo J.C. Albuquerque, Yoshio Hirano, Hiroko Terasaki, Mineo Kondo, Takashi Fujita, Balamurali K. Ambati, Valeria Tarallo, Bradley D. Gelfand, Sasha Bogdanovich, Judit Z. Baffi, Jayakrishna Ambati^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

84 Scopus citations

Abstract

The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these naked siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.

Original language	English (US)
Pages (from-to)	101-108
Number of pages	8
Journal	Molecular Therapy
Volume	20
Issue number	1
DOIs	https://doi.org/10.1038/mt.2011.212
State	Published - Jan 2012
Externally published	Yes

Funding

We thank R. King, L. Xu, M. McConnell, C. Payne, G.R. Pattison, D. Robertson, and G. Botzet for technical assistance, and R. Mohan, P.A. Pearson, A.M. Rao, G.S. Rao, K. Ambati, and B. Savage for discussions. J.A. was supported by National Eye Institute/National Institutes of Health (NIH) grants R01EY018350, R01EY018836, R01EY020672, R21EY019778, RC1EY020442, the Doris Duke Distinguished Clinical Scientist Award, the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, the Dr E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, the Senior Scientist Investigator Award [Research to Prevent Blindness (RPB)], and a departmental unrestricted grant from the RPB; M.E.K. by NIH K08EY021757 and Foundation Fighting Blindness; J.Z.B. by NIH K08EY021521 and American Health Assistance Foundation; M.E.K. and J.Z.B. by University of Kentucky Physician Scientist Awards and International Retinal Research Foundation; S.B. and B.J.F. by NIH T32HL091812; B.K.A. by NIH R01EY017182, R01EY017950, VA Merit Award and Department of Defense; J.A. is named as an inventor on a patent application filed by the University of Kentucky on ultrashort siRNAs as TLR3 antagonists.

ASJC Scopus subject areas

Drug Discovery
Genetics
Molecular Medicine
Molecular Biology
Pharmacology

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10.1038/mt.2011.212

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Kleinman, M. E., Kaneko, H., Cho, W. G., Dridi, S., Fowler, B. J., Blandford, A. D., Albuquerque, R. J. C., Hirano, Y., Terasaki, H., Kondo, M., Fujita, T., Ambati, B. K., Tarallo, V., Gelfand, B. D., Bogdanovich, S., Baffi, J. Z., & Ambati, J. (2012). Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3. Molecular Therapy, 20(1), 101-108. https://doi.org/10.1038/mt.2011.212

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title = "Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3",

abstract = "The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these naked siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.",

author = "Kleinman, {Mark E.} and Hiroki Kaneko and Cho, {Won Gil} and Sami Dridi and Fowler, {Benjamin J.} and Blandford, {Alexander D.} and Albuquerque, {Romulo J.C.} and Yoshio Hirano and Hiroko Terasaki and Mineo Kondo and Takashi Fujita and Ambati, {Balamurali K.} and Valeria Tarallo and Gelfand, {Bradley D.} and Sasha Bogdanovich and Baffi, {Judit Z.} and Jayakrishna Ambati",

note = "Funding Information: We thank R. King, L. Xu, M. McConnell, C. Payne, G.R. Pattison, D. Robertson, and G. Botzet for technical assistance, and R. Mohan, P.A. Pearson, A.M. Rao, G.S. Rao, K. Ambati, and B. Savage for discussions. J.A. was supported by National Eye Institute/National Institutes of Health (NIH) grants R01EY018350, R01EY018836, R01EY020672, R21EY019778, RC1EY020442, the Doris Duke Distinguished Clinical Scientist Award, the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, the Dr E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, the Senior Scientist Investigator Award [Research to Prevent Blindness (RPB)], and a departmental unrestricted grant from the RPB; M.E.K. by NIH K08EY021757 and Foundation Fighting Blindness; J.Z.B. by NIH K08EY021521 and American Health Assistance Foundation; M.E.K. and J.Z.B. by University of Kentucky Physician Scientist Awards and International Retinal Research Foundation; S.B. and B.J.F. by NIH T32HL091812; B.K.A. by NIH R01EY017182, R01EY017950, VA Merit Award and Department of Defense; J.A. is named as an inventor on a patent application filed by the University of Kentucky on ultrashort siRNAs as TLR3 antagonists.",

year = "2012",

month = jan,

doi = "10.1038/mt.2011.212",

language = "English (US)",

volume = "20",

pages = "101--108",

journal = "Molecular Therapy",

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Kleinman, ME, Kaneko, H, Cho, WG, Dridi, S, Fowler, BJ, Blandford, AD, Albuquerque, RJC, Hirano, Y, Terasaki, H, Kondo, M, Fujita, T, Ambati, BK, Tarallo, V, Gelfand, BD, Bogdanovich, S, Baffi, JZ & Ambati, J 2012, 'Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3', Molecular Therapy, vol. 20, no. 1, pp. 101-108. https://doi.org/10.1038/mt.2011.212

TY - JOUR

T1 - Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3

AU - Kleinman, Mark E.

AU - Kaneko, Hiroki

AU - Cho, Won Gil

AU - Dridi, Sami

AU - Fowler, Benjamin J.

AU - Blandford, Alexander D.

AU - Albuquerque, Romulo J.C.

AU - Hirano, Yoshio

AU - Terasaki, Hiroko

AU - Kondo, Mineo

AU - Fujita, Takashi

AU - Ambati, Balamurali K.

AU - Tarallo, Valeria

AU - Gelfand, Bradley D.

AU - Bogdanovich, Sasha

AU - Baffi, Judit Z.

AU - Ambati, Jayakrishna

N1 - Funding Information: We thank R. King, L. Xu, M. McConnell, C. Payne, G.R. Pattison, D. Robertson, and G. Botzet for technical assistance, and R. Mohan, P.A. Pearson, A.M. Rao, G.S. Rao, K. Ambati, and B. Savage for discussions. J.A. was supported by National Eye Institute/National Institutes of Health (NIH) grants R01EY018350, R01EY018836, R01EY020672, R21EY019778, RC1EY020442, the Doris Duke Distinguished Clinical Scientist Award, the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, the Dr E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, the Senior Scientist Investigator Award [Research to Prevent Blindness (RPB)], and a departmental unrestricted grant from the RPB; M.E.K. by NIH K08EY021757 and Foundation Fighting Blindness; J.Z.B. by NIH K08EY021521 and American Health Assistance Foundation; M.E.K. and J.Z.B. by University of Kentucky Physician Scientist Awards and International Retinal Research Foundation; S.B. and B.J.F. by NIH T32HL091812; B.K.A. by NIH R01EY017182, R01EY017950, VA Merit Award and Department of Defense; J.A. is named as an inventor on a patent application filed by the University of Kentucky on ultrashort siRNAs as TLR3 antagonists.

PY - 2012/1

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N2 - The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these naked siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.

AB - The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these naked siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.

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Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3

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