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We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (∼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.

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We report a potent cationic lipid, SST-02 ((3-hydroxylpropyl)dilinoleylamine), which possesses a simple chemical structure and is synthesized just in one step. Cationic lipids are key components of siRNA-lipid nanoparticles (LNP), which may serve as potential therapeutic agents for various diseases. For a decade, chemists have given enhanced potency and new functions to cationic lipids along with structural complexity. In this study, we conducted a medicinal chemistry campaign pursuing chemical simplicity and found that even dilinoleylmethylamine (SST-01) and methylpalmitoleylamine could be used for the in vitro and in vivo siRNA delivery. Further optimization revealed that a hydroxyl group boosted potency, and SST-02 showed an ID50 of 0.02 mg/kg in the factor VII (FVII) model. Rats administered with 3 mg/kg of SST-02 LNP did not show changes in body weight, blood chemistry, or hematological parameters, while the AST level decreased at a dose of 5 mg/kg. The use of SST-02 avoids a lengthy synthetic route and may thus decrease the future cost of nucleic acid therapeutics.

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In vivo biodistribution analyses, especially in tumors, of nucleic acids delivered with nanoparticles are important to develop drug delivery technologies for medical use. We previously developed wrapsome® (WS), an ~100 nm liposomal nanoparticle that can encapsulate siRNA, and reported that WS accumulates in tumors in vivo and inhibits their growth by an enhanced permeability and retention effect. In the present study, we evaluated the pharmacokinetics of nucleic acid-containing nanoparticles by combining dynamic positron emission tomography (PET) imaging and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. An 18-mer phosphorothioate oligodeoxynucleotide (ODN), trabedersen, was used as a model drug and was encapsulated in WS. Dynamic PET imaging and time-activity curve analysis of WS-encapsulated 64Cu-labeled ODNs administered to mice with MIA PaCa-2 subcutaneous xenograft tumors showed tumor accumulation (~3% injected dose per gram (%ID/g)) and liver accumulation (~30 %ID/g) at 24 h. Under these conditions, LC/MS/MS analysis showed that the level of intact ODNs was 1.62 %ID/g in the tumor and 1.70 %ID/g in the liver. From these pharmacokinetic data, the intact/accumulated ODN ratios were calculated using the following equation: intact/accumulated ODN ratio (%) = %ID/g LC/MS/MS, tissue, mean/%ID/g PET, tissue, mean × 100. Interestingly, the ratios for the tumor and kidney were maintained at 20-50% over 48 h after administration of the WS-encapsulated form. In contrast, the ratio for the liver rapidly decreased at 24 h, showing the same pattern as that for naked ODN. These different patterns indicate that WS effectively protected the ODN in the tumor and kidney, but protected it less efficiently in the liver. A combined approach of dynamic PET imaging and LC/MS/MS analysis will assist the development of nanoparticle-encapsulated nucleic acid drugs, such as those using WSs, to determine their detailed pharmacokinetics.

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The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2-7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.

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Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.

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