This dissertation provides a comprehensive analysis of structure-activity relationship of cationic-hydrophilic block copolymers as a building block of polymeric micelle for siRNA delivery. Our work should make a significant contribution toward the development of siRNA-based therapy, because the delivery system is believed to govern the feasibility of therapeutic siRNA as reviewed in chapter 1. Since the discovery of siRNA in 2001, methods based on RNAi machinery have been utilized to silence specific target genes, as a research tool to elucidate the gene function and also for the prevention of undesired disease gene expression. Some studies have already progressed to clinical trials, but almost all cases utilize local administration, an application limited to readily accessible tissues such as those found in the ocular and respiratory systems. Indeed, it is the unfavorable pharmaceutical property of siRNA that prevents the extensive practical application of RNAi based therapy. For example, siRNAs are known to rapidly degrade into non-effective fragments in the RNase-rich physiological environment. Additionally, systemically administered naked siRNAs are rapidly eliminated from circulation, with plasma-half lives reported to be as short as 0.03 hour in mice and 0.1 hour in rats. It is clear that an innovative carrier system for the systemic administration is required for further development of siRNA as a universal drug. Fundamental design and evaluation of a siRNA carrier, which could be developed into a system for therapeutic use, was a specific aim of this work.

An incorporation of siRNA into block copolymer micelle is of great interest for the improvement of pharmaceutical property of siRNA. However, little is known about the molecular design of a block copolymer that is suitable for siRNA delivery, because the short and rigid siRNA molecule was almost a new entity as a cargo molecule of block copolymer micelle. In order to explore the requirement of molecular structure for the successful siRNA transfection, we began with the screening of siRNA transfection efficacy of block copolymers with various compositions. In this regard, PEG-b-PLL and its derivatives (e.g.PEG-b-P[Asp(DET)/Lys]), which possess ethylene diamine structure with lowered basicity, were evaluated as a carrier for siRNA delivery in chapter 2. Cationic'segment of block copolymer is vital for the interaction with anionic nucleic acids: the protonated state of primary amine in Lys residue is quite stable with the pKa of nearly 10 in aqueous solution, allowing the stable complexation with the anionic phosphodiester in siRNA. On the contrary, ethylene diamine structure of Asp(DET) residue is less cationic than primary amine, hence the substitution of Lys for Asp(DET) may decrease the stability of the complex with siRNA. The looser complexation appears to be disadvantageous for siRNA delivery,however, Asp(DET) has been successfully enhanced plasmid DNA transfection by facilitating the endosomal escape of the plasmid DNA complex. Endosomal escape is believed to be based on the ethylene diamine structure which allows the molecule to act as a 'proton sponge' that buffers a proton influx during endosome acidification, leading to endosome swelling and disruption. Thus, block copolymers have been successfully optimized those structures for plasmid DNA transfection by utilizing a hybrid cationic segment of Lys and Asp(DET) to balance the plasmid DNA binding force and proton sponge effect. Given the similar requirement of stable complexation and endosomal escape for siRNA transfection, same strategy was considered to be adoptable for the development of siRNA carrier. As a result, siRNA was successfully complexed with all of PEG-b-PLL and its derivatives examined, and obtained remarkable resistance against enzymatic degradation upon the complexation with PEG-b-PLL, however, none of these block copolymers showed significant improvement in transfection efficacy. Even though the cationic moieties of block copolymers were substituted with PAsp(DET), these copolymers exhibited rather weak transfection efficacy than PEG-b-PLL and PEI. Furthermore, any attempts to compensate the instability of PAsp(DET)/siRNA complex by increasing Lys/Asp(DET) ratio or adding a hydrophobic segment (PLL(Z)) hardly improved transfection efficacy. It was suspected that the common PEG shell of block copolymer/siRNA complexes hindered the cellular uptake of them, however, homo PAsp(DET) still showed poor efficacy in siRNA transfection. One explanation for the paradoxical results between plasmid DNA and siRNA might be the difference in the intracellular destinations of each nucleic acid: siRNA has been supposed to be recruited into RNAi machinery in cytoplasm, while plasmid DNA should be transported into nucleus, where siRNA can not mediate RNAi. In this regard, PAsp(DET) derivatives, which may achieve highly-efficient plasmid DNA transfection by enhancing the nuclear transport of those cargo molecule, could have unfavorably distributed siRNAs to nucleus. Furthermore, siRNA may require to be in its intact form to be active in cytosol, because the enzymatic molecular recognitions of siRNA and target mRNA occur at each stage in RNAi process. Hence, the recruitment of siRNA into RNAi machinery might be susceptible to the hindrance associated with the surrounding cationic copolymers, which does not interfere the successful transfection in the case of plasmid DNA. Based on these considerations about the difference between plasmid DNA and siRNA, ideal siRNA carrier may well require a distinct development in the structure of block copolymer.

In chapter 3, we shifted our focus to nano-scaled structuring of block copolymers and siRNA as a fundamental step of the development of siRNA carrier. Although the successful complexation of siRNA with block copolymers were indicated by gel retardation analysis and EtBr assay in chapter 2, it provided no information on the resulting structure of the block copolymer:siRNA complexes. Knowing the detailed structure of the block copolymer complex was considered to be important, because block copolymers have been demonstrated to form not a simple but a variety of higher-ordered structure (e.g. micelle and PICsome) depending on the copolymer composition and mixing condition of different types of copolymers. Thus, siRNA may well be structured into various types of higher ordered assembly, whose property may affect the transfection efficacy, upon the complexation with block copolymers. As a mean to observe the nano-scaled structuring, we utilized light scattering techniques: static light scattering (SLS) for confirming the existence of large assembly and dynamic light scattering (DLS) for the size distribution analysis. Indeed, SLS analysis of PEG-b-PLL/siRNA complexes as a function of N/P revealed a distinct assembling behavior which was not known before. That is, PEG-b-PLL and siRNA formed higher-ordered structuring at precisely N/P=1.2, while no large assemblies were observed at any other N/P. Furthermore, size distribution analysis of the PEG-b-PLL/siRNA complex suggested the formation of gigantic higher-ordered structuring but not the typical PIC micelle,' and the complex was so sensitive to ionic strength that it was almost diminished even at 150 mM HC1. Although the relationship between this unprecedented assembling behavior and poor siRNA transfection efficacy of PEG-b-PLL has not been clarified yet,' we could conclude that PEG-b-PLL does not form PIC micelle with siRNA at physiological condition.

It was assumed that the incorporation of siRNA into PIC micelle is more difficult than that of plasmid DNA or oligo DNA, because the length of siRNA is less than one hundredth the length of plasmid DNA and its degree of conformational freedom is likely more restricted than single-stranded oligo DNA. Therefore, refinement of the block copolymer structure was considered to be necessary to support the micellar assembly with short and rigid siRNAs. While increased stability is needed to allow micelle formation, micelle was also required to promptly release siRNA in the cytoplasm of the target cell for recruitment of siRNAs into RNAi. Thus, ideal PIC micelle for siRNA delivery must meet the following rather conflicting requirements: stability of PIC micelles in extracellular media and efficient release of free siRNAs from the carrier in the cytoplasm of target cells following internalization. In order to meet the requirements, PEG-b-PLL was reacted with 2-iminothiolane to obtain PEG-b-(PLL-IM), with a portion of lysine residues bearing both mercaptopropyl and amidine groups. Thiol groups were utilized to form disulfide cross-links in a core of the micelle to confer environment-responsive stability. The disulfide cross-links are stable under non-reductive physiological conditions helping to maintain micellar structure, while they degraded in reductive conditions following core destabilization. Indeed, the core-shell type PIC micelle with a disulfide cross-linked core was prepared through the assembly of PEG-b-(PLL-IM). and siRNAs at a characteristic optimum mixing ratio. The PIC micelles showed spherical shape of approximately 70 nm in diameter with narrow distribution. Micellar structure was successfully maintained at physiological ionic strength, but was feasibly disrupted under reductive conditions due to the cleavage of disulfide cross-links, which is desirable for release of siRNAs in the intracellular reductive environment. In fact, the environment-responsive PIC micelles achieved 100-fold higher siRNA transfection efficiency compared to non-crosslinked PICs prepared using PEG-b-PLL, which were not stable at physiological ionic strength. Furthermore, PIC assemblies formed with PEG-b-(PLL-IM) at non-optimum ratios failed to show micellar structure and also failed in siRNA transfection. Most importantly, environment-responsive PIC micelles successfully prolonged the retention time of siRNA in blood circulation. These findings show that PIC micelle can be used as carriers for therapeutic siRNA, and that stable micellar structure is a crucial factor for efficient siRNA delivery.

In conclusion, we successfully developed the disulfide cross-like micelle as a new siRNA delivery system, which was derived from a comprehensive analysis of structure-activity relationships explored so far. The discovery of unprecedented assembling behavior of block copolymers and siRNA would provide a universal basis for all polymer formulation that is relevant to siRNA delivery. With all the incremental improvements that are being made to the development therapeutic siRNA around the world, RNAi-based drug would be available in the very near future.

特定の2本鎖RNAと相補的な塩基配列を持つ標的mRNAが選択的に分解され、その遺伝子の発現が抑制されるRNA干渉(RNAi)は1990年代に発見され、現在、疾患を惹起する遺伝子を抑制する次世代核酸医薬としての応用研究が世界各国で盛んに行われている中で、特定の遺伝子疾患に対応するRNAiを誘起するshort interfering RNA(siRNA)が数多く発見されている。しかし、実際の医用応用に至るにはsiRNAを体内で安定に存在させ、患部まで送達する技術の開発が不可欠となる。本論文ではこの点に着目し、siRNAの全身投与型送達システムの開発を目指し、siRNAの分解の抑制や機能発現に適したブロック共重合体を設計すると共に、siRNAとブロック共重合体との複合体形成の物理化学的特性解析を行い、その生物学的評価ではsiRNAの機能発現を評価する新規株化細胞を樹立し、物理化学的評価と生物学的評価の結果をブロック共重合体の設計にフィードバックさせながら、siRNA送達に最適な送達システムを構築している。さらに、マウスを用いたin vivo実験では、培養細胞実験において高い遺伝子抑制効果を示した送達システムを用いることにより、siRNAの血中滞留性が大幅に向上し、siRNAに特化した新規送達システムとしての有用性を示している。以下、各章毎に、本論文の審査結果の概要を述べる。

第一章の序論では、siRNAの発見に至る歴史的背景から核酸医薬としての可能性および問題点を概観するとともに、昨今のsiRNA医薬開発情勢を踏まえ、高分子ミセルを用いたsiRNAのための全身性送達システム開発の重要性を説いている。siRNAやRNAiの基礎研究については欧米の諸研究機関が先導的な役割を果たしているが、siRNAの医薬としての実用化には全身性送達システムの開発が不可欠であるため、本章において、ブロック共重合体を用いたsiRNA送達システムの開発を本論文の主題として位置付けている。

第二章では、siRNA送達に適切な機能を有する高分子材料設計法の探索として、構造の異なる様々なブロック共重合体の、siRNAとの複合体形成能および培養細胞へのsiRNA送達能評価を行っている。siRNA送達能の評価にあたり、検出の容易なレポーター遺伝子を恒常的に発現する株化細胞を独自に樹立し、ブロック共重合体の構造機能相関を正確かつ高速に解析する工夫を行っている。siRNAと高分子ミセルを形成するためのブロック共重合体としては、親水性鎖とカチオン性鎖からなるブロック共重合体であるpoly(ethylene)-block-poly(L-lysine) (PEG-b-PLL)を基本構造として評価を進めている。PEG-b-PLLは、siRNAの核酸類縁体に相当するplasmid DNA(pDNA)やアンチセンスDNAと高分子ミセルを形成し、in vitroやin vivoにおいて高い核酸送達能を有することが報告されてきた。本論文でもPEG-b-PLLとsiRNAによる複合体形成はゲル電気泳動やEtBr Assayより確認され、siRNAの酵素分解耐性は著しく向上したものの、培養細胞へのsiRNA送達能には向上はみられなかった。そこでPEG-b-PLLのsiRNA送達能を改善する方策として、カチオン性側鎖にエチレンジアミン構造を有する弱塩基性官能基(Asp(DET))を導入する方法を検討した。Asp(DET)構造は、pDNAの遺伝子導入において細胞内への核酸-ブロック共重合体複合体の取り込みを促進することが実証されている。しかし、PEG-b-PLLへのAsp(DET)構造の導入によりsiRNA送達能は減少し、ゲル電気泳動による解析からsiRNAとの複合体が不安定化したことが示唆された。上記の通り本章では、ブロック共重合体の構造機能相関評価を行うRNAi実験系を独自に構築したが、pDNA等の核酸類縁体のために設計されたブロック共重合体がsiRNA送達には有効でないことを明らかにし、これらの検討からsiRNA送達に特化したブロック共重合体設計指針を確立する必要性を指摘している。

第三章では、siRNA送達に優れたブロック共重合体設計指針として、内核にジスルフィド架橋を有するコア-シェル形の高分子ミセルを検討している。ジスルフィド架橋ミセルは、PEG-b-PLLの一級アミンにアミジン結合を介してチオール基を導入したPEG-b-(PLL-IM)とsiRNAから形成され、細胞外ではsiRNAを安定に保持し、細胞内の還元的環境ではジスルフィド架橋の開裂によりsiRNAを放出する環境応答性を備えている。この設計では、培養細胞においてsiRNAによる高い発現抑制効果を確認するとともに、マウスへの尾静脈投与において血中滞留性の大幅な向上をも確認している。一般に、血流中に投与された未修飾の siRNAは極めて速やかに腎排泄されることから、本システムにおける血中滞留性の向上はsiRNA用の送達材料に求められる機能として極めて重要である。本章では、生物学的評価に先立ち、PEG-b-(PLL-IM)とsiRNAとの複合体形成において、新たに微量サンプルでの光散乱測定法を導入し、従来は困難であった複合体形成過程の観測にも成功している。これにより、従来知られていなかった特殊な複合体形成挙動、即ちイミノチオレイン(IM)修飾を施されたPEG-b-PLLがsiRNAと特定の比率で混合された場合にのみ、siRNA内包高分子ミセルが形成されることを見出している。このミセル形成挙動の発見は、ブロック共重合体を用いたsiRNA製剤技術の重要な要素技術である。

第四章は総括として、一連の研究のまとめと今後の高分子ミセルを用いたsiRNA送達システムの展望についてまとめている。

以上、本論文ではPEG-b-(PLL-IM)を利用してsiRNAを内包した環境応答性高分子ミセルの創製に成功している。この内容は、siRNAの機能評価のために独自の生物評価系を確立し、この結果を高分子材料設計にフィードバックさせ、真に有効なsiRNA送達用の高分子材料設計指針を確立したものと評価できる。その内容は医工融合研究分野において極めて秀逸であり、世界的にも実用化の機運が高まるsiRNA医薬の開発に多大な福音を与えるものと判断される。

よって本論文は博士(工学)の学位請求論文として合格と認められる。