Kazutaka Nishina

High-Density Lipoprotein Facilitates In Vivo Delivery of α-Tocopherol–Conjugated Short-Interfering RNA to the Brain

We originally reported the use of vitamin E (α-tocopherol) as an in vivo vector of short-interfering RNA (siRNA) to the liver. Here, we apply our strategy to the brain. By combining high-density lipoprotein (HDL) as a second carrier with α-tocopherol-conjugated siRNA (Toc-siRNA) in the brain, we achieved dramatic improvement of siRNA delivery to neurons. After direct intracerebroventricular (ICV) infusion of Toc-siRNA/HDL for 7 days, extensive and specific knock-down of a target gene, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), was observed in both mRNA and protein levels, especially in the cerebral cortex and hippocampus. This new delivery method achieved a much more prominent down-regulation effect than conventional silencing methods of the brain gene, i.e., ICV infusion of nonconjugated siRNA or oligonucleotides. With only 3 nmol Toc-siRNA with HDL, BACE1 mRNA in the parietal cortex could be reduced by ∼ 70%. We suppose that this dramatic improvement of siRNA delivery to the brain is due to the use of lipoprotein receptor-mediated endocytosis because the silencing efficiency was significantly increased by binding of Toc-siRNA to the lipoprotein, and in contrast, was clearly decreased in lipoprotein-receptor knockout mice. These results suggest exogenous siRNA could be used clinically for otherwise incurable neurological diseases.

Efficient in vivo delivery of siRNA into brain capillary endothelial cells along with endogenous lipoprotein.

The brain capillary endothelial cell (BCEC) is a major functional component of the blood-brain barrier and is an underlying factor in the pathophysiology of various diseases, including brain ischemia, multiple sclerosis, and neurodegenerative disorders. We examined gene silencing in BCECs by using endogenous lipoprotein to introduce short-interfering RNA (siRNA) in vivo. A cholesterol-conjugated 21/23-mer siRNA targeting organic anion transporter 3 (OAT3) mRNA (Chol-siOAT3) was intravenously injected into mice after its incorporation into extracted endogenous lipoproteins. Chol-siOAT3 was not delivered to neurons or glia, but was successfully delivered into BCECs and resulted in a significant reduction of OAT3 mRNA levels when injected after its incorporation into high-density lipoprotein (HDL). Efficient delivery was not achieved, however, when Chol-siOAT3 was injected without any lipoproteins, or after its incorporation into low-density lipoprotein (LDL). Investigations in apolipoprotein E (ApoE)-deficient and LDL receptor (LDLR)-deficient mice revealed that the uptake of HDL-containing Chol-siOAT3 was mainly mediated by ApoE and LDLR in mice. These findings indicate that siRNA can be delivered into BCECs in vivo by using endogenous lipoprotein, which could make this strategy useful as a new gene silencing therapy for diseases involving BCECs.

DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field.

Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences.

Enteral siRNA delivery technique for therapeutic gene silencing in the liver via the lymphatic route

An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver.

Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain

Recently, nanocarriers that transport bioactive substances to a target site in the body have attracted considerable attention and undergone rapid progression in terms of the state of the art. However, few nanocarriers can enter the brain via a systemic route through the blood-brain barrier (BBB) to efficiently reach neurons. Here we prepare a self-assembled supramolecular nanocarrier with a surface featuring properly configured glucose. The BBB crossing and brain accumulation of this nanocarrier are boosted by the rapid glycaemic increase after fasting and by the putative phenomenon of the highly expressed glucose transporter-1 (GLUT1) in brain capillary endothelial cells migrating from the luminal to the abluminal plasma membrane. The precisely controlled glucose density on the surface of the nanocarrier enables the regulation of its distribution within the brain, and thus is successfully optimized to increase the number of nanocarriers accumulating in neurons.There are only a few examples of nanocarriers that can transport bioactive substances across the blood-brain barrier. Here the authors show that by rapid glycaemic increase the accumulation of a glucosylated nanocarrier in the brain can be controlled.

Short interfering RNA and the central nervous system: development of nonviral delivery systems

The development of gene silencing therapies for neurological diseases has placed great importance on the delivery of short interfering RNA (siRNA) to the central nervous system (CNS). However, delivery of siRNA to neurons, glia and brain capillary endothelial cells (BCECs) has not been well established. This editorial describes different approaches that are being used to efficiently deliver siRNA to the CNS via intravenous, intracerebroventricular, or intranasal administration.

Synthesis and properties of double-stranded RNA-bindable oligodiaminogalactose derivatives conjugated with vitamin E

RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs (siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-2,6-dideoxy-(1-4)-β-D-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with α-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal was suggested to bind to RNA duplexes without inhibiting RNAi activity.

Synthesis and properties of vitamin E analog-conjugated neomycin for delivery of RNAi drugs to liver cells

RNA interference (RNAi) is a promising tool to regulate gene expression by external double stranded RNAs (dsRNAs) such as siRNAs. As an efficient method to deliver siRNAs to liver cells, we propose a novel strategy using vitamin E (VE)-conjugated neomycin derivatives. With the aim of delivering RNAi-based drugs to liver cells, several tripod-type and prodrug-type neomycin derivatives were synthesized, all of which were thermodynamically stabilized RNA duplexes. The prodrug-type derivative 7 and the tripod-type derivative 10 were delivered to liver cancer cells and successfully induced RNAi activity. These results indicated the potential use of natural aminoglycosides as carriers of RNAi drugs.

Can anti-AQP4 antibody damage the blood-brain barrier?

Background: Aquaporin 4 (AQP4) is a water-channel protein predominantly expressed in astrocyte end feet that make up the blood-brain barrier (BBB). Recently, anti-AQP4 antibody has been identified as a specific biomarker of neuromyelitis optica (NMO). However, whether anti-AQP4 antibodies damage the BBB is unclear. Methods: We evaluated BBB damage in patients with NMO and multiple sclerosis by measuring albumin leakage (AL) and studied its correlation with anti-AQP4 antibody. Results: No obvious difference in AL was observed between patients with and without anti-AQP4 antibodies. In the multivariate analysis, anti-AQP4 antibody was not associated with BBB damage. Of the anti-AQP4-positive patients, 58.0% had normal AL values, and the degree of BBB damage was unrelated to the anti-AQP4 antibody titer. In addition, 41.9% of anti-AQP4-positive patients showed no gadolinium enhancement of the MRI. Conclusion: These results indicate that the presence of anti-AQP4 antibody alone in plasma is insufficient to disrupt the BBB.