Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.
Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.
Kleinman
Mark E
ME
Department of Ophthalmology, University of Kentucky, Lexington, Kentucky 40506, USA.
Yamada
Kiyoshi
K
Takeda
Atsunobu
A
Chandrasekaran
Vasu
V
Nozaki
Miho
M
Baffi
Judit Z
JZ
Albuquerque
Romulo J C
RJ
Yamasaki
Satoshi
S
Itaya
Masahiro
M
Pan
Yuzhen
Y
Appukuttan
Binoy
B
Gibbs
Daniel
D
Yang
Zhenglin
Z
Karikó
Katalin
K
Ambati
Balamurali K
BK
Wilgus
Traci A
TA
DiPietro
Luisa A
LA
Sakurai
Eiji
E
Zhang
Kang
K
Smith
Justine R
JR
Taylor
Ethan W
EW
Ambati
Jayakrishna
J
D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology; D018664:Interleukin-12 / Q000276:immunology; D008268:Macular Degeneration / Q000150:complications / Q000235:genetics / Q000628:therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000276:immunology* / Q000517:prevention & control* / Q000628:therapy; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051196:Toll-Like Receptor 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D034741:RNA, Small Interfering; D051196:Toll-Like Receptor 3; D042461:Vascular Endothelial Growth Factor A; D018664:Interleukin-12; D007371:Interferon-gamma