Hirokuni Uchida

Odd-even effect of repeating aminoethylene units in the side chain of N-substituted polyaspartamides on gene transfection profiles

A series of the N-substituted polyaspartamides possessing repeating aminoethylene units in the side chain was prepared in this study to identify polyplexes with effective endosomal escape and low cytotoxicity. All cationic N-substituted polyaspartamides showed appreciably lower cytotoxicity than that of commercial transfection reagents. Interestingly, a distinctive odd-even effect of the repeating aminoethylene units in the polymer side chain on the efficiencies of endosomal escape and transfection to several cell lines was observed. The polyplexes from the polymers with an even number of repeating aminoethylene units (PA-Es) achieved an order of magnitude higher transfection efficiency, without marked cytotoxicity, than those of the polymers with an odd number of repeating aminoethylene units (PA-Os). This odd-even effect agreed well with the buffering capacity of these polymers as well as their capability to disrupt membrane integrity selectively at endosomal pH, leading to highly effective endosomal escape of the PA-E polyplexes. Furthermore, the formation of a polyvalent charged array with precise spacing between protonated amino groups in the polymer side chain was shown to be essential for effective disruption of the endosomal membrane, thus facilitating transport of the polyplex into the cytoplasm. These data provide useful knowledge for designing polycations to construct safe and efficient nonviral gene carriers.

In vivo messenger RNA introduction into the central nervous system using polyplex nanomicelle.

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

Modulated Protonation of Side Chain Aminoethylene Repeats in N-Substituted Polyaspartamides Promotes mRNA Transfection

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

Messenger RNA delivery of a cartilage-anabolic transcription factor as a disease-modifying strategy for osteoarthritis treatment

Osteoarthritis (OA) is a chronic degenerative joint disease and a major health problem in the elderly population. No disease-modifying osteoarthritis drug (DMOAD) has been made available for clinical use. Here we present a disease-modifying strategy for OA, focusing on messenger RNA (mRNA) delivery of a therapeutic transcription factor using polyethylene glycol (PEG)-polyamino acid block copolymer-based polyplex nanomicelles. When polyplex nanomicelles carrying the cartilage-anabolic, runt-related transcription factor (RUNX) 1 mRNA were injected into mouse OA knee joints, OA progression was significantly suppressed compared with the non-treatment control. Expressions of cartilage-anabolic markers and proliferation were augmented in articular chondrocytes of the RUNX1-injected knees. Thus, this study provides a proof of concept of the treatment of degenerative diseases such as OA by the in situ mRNA delivery of therapeutic transcription factors; the presented approach will directly connect basic findings on disease-protective or tissue-regenerating factors to disease treatment.

Regulated protonation of polyaspartamide derivatives bearing repeated aminoethylene side chains for efficient intracellular siRNA delivery with minimal cytotoxicity

The effects of the repeated number (RN) of aminoethylene (AE) units in polyaspartamide side chains were investigated for polyion complex (PIC)-based siRNA delivery. Reduction of the apparent RN from 3 to 2 by thiourea introduction increased a protonatable amine fraction in AE units at endosomal pH, leading to the efficient endosomal escape of siRNA-loaded PICs.

593. Anti-Angiogenic Therapy for Pancreatic Cancer by Systemic Delivery of Messenger RNA Using Polyplex Nano Micelle

In pancreatic cancer, the access of therapeutic reagents to cancer cells was restricted by thick fibrotic tissue, and thus anti-angiogenic therapy targeting endothelial cells, which are more accessible, is a promising strategy. Gene therapy based on messenger RNA (mRNA) delivery allows for sustainable introduction of anti-angiogenic factors without the risks associated with DNA delivery, such as insertion mutagenesis, and uncontrollably prolonged transgene expression. Systemic intravenous injection is a proper delivery route for mRNA to access endothelial cells from blood vessels with a simple injection procedure. Because mRNA is highly susceptible to enzymatic degradation in the blood circulation, mRNA carrier with high stability is required. In this study, we used polyplex nanomicelles, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers (Adv Drug Deliv Rev 2001, 47, 113-131), and a cholesterol moiety (Chol) was introduced to the block copolymer to stabilize the nanomicelles by hydrophobic interaction. In the cationic segment of the block copolymer, a polyaspartamide with four aminoethylene repeating units in the side chain (PAsp(TEP)) was used, which has high endosomal escaping capability and mRNA stabilizing effect (J Am Chem Soc 2014, 136, 12396-12405). In quantitative PCR (qPCR) analysis of nuclease resistance after in vitro incubation in 50% serum, Chol introduction failed to increase mRNA stability. In contrast, after intravenous injection to mice, the nanomicelles with Chol showed significantly enhanced mRNA retention in the blood circulation in qPCR analysis, when compared to those without Chol. These results indicate that the stabilizing effect of Chol is obvious especially in the harsh environment of blood, where nanomicelles are exposed to large amount of polyanion, such as proteoglycans on the cell surface. Indeed, nanomicelles with Chol showed enhanced resistance to dissociation by dextran sulfate compared to those without Chol. Eventually, when luciferase mRNA was introduced to the mice subcutaneously inoculated with human pancreatic adenocarcinoma (BxPC3), the nanomicelles with Chol showed enhanced luciferase expression in the tumor tissue, compared to those without Chol. Then, anti-angiogenic treatment was performed to this BxPC3 model, using mRNA encoding sFlt-1, a soluble form of vascular endothelial growth factor (VEGF) receptor, which inhibits VEGF signaling by entrapping VEGF protein. In the evaluation of tumor volume, the nanomicelles with Chol exhibited significant growth inhibitory effect, whereas those without Chol failed to show detectable effect. In immunohistochemical staining of vascular endothelial cells, the nanomicelles with Chol induced significant reduction of vascular density in the tumor tissue, indicating that the anti-angiogenic effect contributed to the inhibition of tumor growth after injection the nanomicelles with Chol. In conclusion, we succeeded in anti-angiogenic treatment of intractable pancreatic cancer by systemic mRNA delivery using stabilized nanomicelles. This mRNA delivery system can also be applied to the treatment of various diseases in the future.