TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models

Abstract

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Targeting TLR4 with ApTOLL Improves Heart Function in Response to Coronary Ischemia Reperfusion in Pigs Undergoing Acute Myocardial Infarction

“…In cases when ventricular fibrillation/ventricular tachycardia occurred, we administered a biphasic DC shock (10–20 joules), combined with direct manual chest compressions. After 45 min of LAD occlusion, the artery was unblocked, and after 10 min of reperfusion, APTOLL was administered intravenously in slow bolus at a dose of 0.078 mg/kg (equivalent dosage previously tested in mice [ 13 ]) and diluted in distilled water.…”

“…ApTOLL is a new therapeutic tool designed to antagonize TLR4. ApTOLL aptamer is a single-stranded oligonucleotide that recognizes and specifically binds to TLR4 by structural interaction, as described [ 13 ]. Once folded under physiological conditions, aptamers acquire unique three-dimensional structures based on their nucleotide sequence, conferring target affinity and specificity [ 14 , 15 , 16 ].…”

“…Once folded under physiological conditions, aptamers acquire unique three-dimensional structures based on their nucleotide sequence, conferring target affinity and specificity [ 14 , 15 , 16 ]. The protective effect of ApTOLL was recently reported against experimental strokes, a setting in which TLR4 played a crucial role in the initiation of ischemic injury [ 13 ].…”

Aptamer Chimeras for Therapeutic Delivery: The Challenging Perspectives

“…The approach adopted in which aptamers were used as both the transport mediator and payload was effective to promote the delivery and of the functional dopamine aptamer ligands from the peripheral injection site into the brain. Fernandez et al have recently reported the generation of DNA aptamers capable to specifically bind and antagonize the Toll-like receptor 4 (TLR4) [ 103 ]. Developing drugs targeting TLR4 has become promising for stroke and multiple pathologies in which it is implicated [ 104 ].…”

“…They show that the truncated ApTLR#4FT specifically bind to and antagonize the TLR4 in vitro and in vivo. In a rat model, the ApTLR#4FT revealed to be protective against brain injury, in a TLR4-mediated manner, and showed to be able to cross the BBB and reach the ischemic mice brain region [ 103 ].…”