Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid ß, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid ß deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Lactose-Appended Hydroxypropyl-ß-Cyclodextrin Lowers Cholesterol Accumulation and Alleviates Motor Dysfunction in Niemann-Pick Type C Disease Model Mice.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of glycolipids such as free cholesterol, sphingomyelin, and gangliosides in late endosomes/lysosomes (endolysosomes) due to abnormalities in the membrane proteins NPC1 or NPC2. The main symptoms of NPC caused by free cholesterol accumulation in various tissues vary depending on the time of onset, but hepatosplenomegaly and neurological symptoms accompanied by decreased motor, cognitive, and mental functions are observed in all age groups. However, the efficacy of NPC treatment remains limited. Herein, we have fabricated lactose-appended hydroxypropyl-ß-cyclodextrin (Lac-HPßCD) and evaluated its lowering effects on cholesterol accumulation in NPC model mice. We reveal that Lac-HPßCD lowers cholesterol accumulation in the liver and spleen by reducing the amount of free cholesterol. Moreover, Lac-HPßCD reduces the amount of free cholesterol in the cerebrum and slightly alleviates motor dysfunction. These results suggest that Lac-HPßCD has potential for the treatment of NPC.

Fasting impairs type 2 helper T cell infiltration in the lung of an eosinophilic asthma mouse model.

Eosinophilic asthma is a form of bronchial asthma that is caused by the pulmonary infiltration of eosinophils and accounts for approximately half of the patients with severe asthma. Several cell types of the immune system in synergy with the epithelial cells of the lung provoke an inflammatory response in patients with asthma. Recently, the effect of fasting on immune cells and inflammation has attracted considerable attention. Therefore, we examined whether fasting may serve as novel preventive strategy in patients with asthma. In our study, we employed a previously established mouse model of eosinophilic asthma. C57BL/6 mice were inoculated intranasally with interleukin-33 and ovalbumin (OVA) in order to induce eosinophil infiltration in the lung and subjected to a 48-h long fasting period directly after or 7 days postinoculation. We used flow cytometry to characterise infiltrated immune cells in the lung and measured the quantity of inflammatory cytokines as well as antigen-specific immunoglobins (Ig) by ELISA. Our results indicated that fasting lowered the number of eosinophilic pulmonary infiltrates in the eosinophilic asthma model mice. Furthermore, fasting suppressed anti-OVA IgG1 production. Fasting suppressed Th2 cytokine production by impairing Th2 accumulation in the lung. The findings suggest that fasting may be a novel preventive strategy for eosinophilic asthma.

Lactose-appended ß-cyclodextrin as an effective nanocarrier for brain delivery.

The blood-brain barrier (BBB) prevents the permeability of drugs into the brain, and as such limits the management of various brain diseases. To overcome this barrier, drug-encapsulating nanoparticles or vesicles, drug conjugates, and other types of drug delivery systems (DDSs) have been developed. However, the brain-targeting ability of nanoparticles or vesicles is still insufficient. Recently, among the various brain-targeting ligands previously studied for facilitating transcellular BBB transport, several sugar-appended nanocarriers for brain delivery were identified. Meanwhile, cyclodextrins (CyDs) have been used as nanocarriers for drug delivery since they can encapsulate hydrophobic compounds with high biocompatibility. Therefore, in this study, we created various sugar-appended ß-cyclodextrins (ß-CyDs) to discover novel brain-targeting ligands. As a result, of the six sugar-appended CyDs, lactose-appended ß-CyD (Lac-ß-CyD) showed greater cellular uptake in hCMEC/D3 cells, human brain microvascular endothelial cells, than other sugar-appended ß-CyDs did. In addition, the permeability of Lac-ß-CyD within the in vitro human BBB model was greater than that of other sugar-appended ß-CyDs. Moreover, Lac-ß-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-ß-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-ß-CyD has the potential to be a novel carrier for drugs across the BBB.

Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.

A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-ß-cyclodextrin conjugate (GUG-ß-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-ß-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-ß-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-ß-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.