Blood brain barrier function8/11/2023 Screening for effective ASO sequences in vitro Here we report a proof-of-concept study that intravenously injected Toc-HDO can efficiently knock down a target molecule in mouse BMECs, and that this novel platform technology for administering a chemically synthesized oligonucleotide without any additional delivery vectors allows modulation of the BBB function in vivo. Given that ASOs are being most actively developed among the oligonucleotide agents 13, our Toc-HDO has a great potential for gene silencing in various organs and tissues in vivo. When targeted to the liver, the effect of Toc-HDO was as much as 20 times that of the parent ASO 10. In the previous study 10, we proved that intravenously administered α-tocopherol-conjugated HDO (Toc-HDO), in which α-tocopherol (a delivery ligand) is covalently conjugated to the 5′-end of the cRNA, binds to serum lipoproteins in blood circulation, and is distributed along the physiological transport pathway of α-tocopherol 12. HDO is composed of an ASO as the parent strand, having a gapmer structure (DNA nucleotides flanked by a few locked nucleic acid (LNA) nucleotides 11), duplexed with the complementary RNA (cRNA). We recently developed a new “heteroduplex oligonucleotide” (HDO) approach that achieves highly efficient gene silencing in vivo 10. Therefore, an alternative simple and safe strategy is needed the best one would be conventional systemic administration of a chemically synthesized oligonucleotide without any additional delivery vectors. However, hydrodynamic injection is highly invasive because of the volume overload and high hydrostatic pressure involved, and delivery along with extracted endogenous lipoprotein may have adverse effects caused by blood derivatives. To downregulate gene expression in BMECs, ASO has not been employed so far but siRNA was used siRNA was hydrodynamically injected 6, 7, 8 or delivered along with extracted endogenous lipoprotein 9. Among various types of oligonucleotides, antisense oligonucleotide (ASO) and short-interfering RNA (siRNA) have been widely studied as conventional methods 4, 5. Oligonucleotide-based gene silencing is a useful strategy, which is being actively developed as both an experimental tool and a therapeutic platform, to modulate biological function at the molecular level in vivo 4, 5. Moreover, the ability to manipulate pathological molecules in BMECs can lead to the development of a new class of molecular targeted therapy for a variety of intractable neurological disorders such as multiple sclerosis, Alzheimer’s disease, and stroke 1, 2. To expand our understanding of how the BBB functions and interacts with its environment, it is important to establish a platform technology to be able to control gene expression in brain microvascular endothelial cells (BMECs), which are core components of the BBB, in vivo. The blood-brain barrier (BBB) is no longer regarded only as a substantial barrier for drug delivery to the brain, but also as a dynamic interface that adapts to the needs of the brain and responds to physiological changes the BBB is affected by and can even promote diseases 1, 2, 3. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 ( OAT3) gene in brain microvascular endothelial cells in mice. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases.
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