KR20240017196A - Delivery system for preventing or treating of cancer comprising modified rt-let7 as an active ingredient - Google Patents
Delivery system for preventing or treating of cancer comprising modified rt-let7 as an active ingredient Download PDFInfo
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- KR20240017196A KR20240017196A KR1020220093739A KR20220093739A KR20240017196A KR 20240017196 A KR20240017196 A KR 20240017196A KR 1020220093739 A KR1020220093739 A KR 1020220093739A KR 20220093739 A KR20220093739 A KR 20220093739A KR 20240017196 A KR20240017196 A KR 20240017196A
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- let7
- modified
- nucleic acid
- galnac
- liver cancer
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Abstract
본 발명은 딜리버리 시스템을 탑재한 변형된 RT-LET7을 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물에 관한 것으로, 본 발명에 따른 딜리버리 시스템을 탑재한 변형된 RT-LET7-8은 기존의 RT-LET7-8 대비 Let-7i-5p의 발현 억제를 증가시키고, 간암 세포 성장 억제를 증가시키며, TSP1 발현을 증가시키고, 및 대식세포 식작용 활성을 증가시킴을 확인하였다. 따라서 이를 간암의 예방 또는 치료용 약학 조성물로 이용할 수 있다.The present invention relates to a pharmaceutical composition for the prevention or treatment of liver cancer comprising a modified RT-LET7 equipped with a delivery system as an active ingredient. The modified RT-LET7-8 equipped with a delivery system according to the present invention is a pharmaceutical composition for preventing or treating liver cancer. It was confirmed that compared to RT-LET7-8, it increased the inhibition of Let-7i-5p expression, increased the inhibition of liver cancer cell growth, increased TSP1 expression, and increased macrophage phagocytosis activity. Therefore, it can be used as a pharmaceutical composition for preventing or treating liver cancer.
Description
본 발명은 변형된 RT-LET7을 유효성분으로 포함하는 암의 예방 또는 치료를 위한 딜리버리 시스템에 관한 것으로, 구체적으로, 딜리버리 시스템을 탑재한 변형된 RT-LET7을 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a delivery system for the prevention or treatment of cancer comprising modified RT-LET7 as an active ingredient. Specifically, the present invention relates to a delivery system for the prevention or treatment of cancer comprising a modified RT-LET7 equipped with a delivery system as an active ingredient. It relates to a pharmaceutical composition for therapeutic use.
간암은 전 세계적으로 다섯 번째로 빈발하는 암이지만 그로 인한 사망률은 3위에 해당하는 공격적인 암이다(Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004;50:556-573) 치료목적의 수술은 단지 15%에서 25% 정도의 환자에게만 가능하고 대부분의 간암 환자들은 국부적으로 진행하거나 전이되는 질병들에 의해 비교적 단기간 내에 사망한다(Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25:212-225) 간암의 주요 원인으로 B형 간염 바이러스(hepatitis B virus), C형 간염 바이러스(hepatitis C virus), 및 아플라톡신 B1(aflatoxin B1) 등이 잘 알려져 있다. 하지만, 지난 20년간 간암 환자의 전체적인 생존율은 크게 증가하지 않았고, 간암의 발달(development) 및 진전(progression) 기작은 여전히 잘 알려져 있지 않은 상태이다(Bruix J, et al Focus on hepatocellular carcinoma Cancer Cell 2004;5:215-219) 지금까지, 분자표적치료(molecular targeted therapy)가 성숙한 간암의 치료에 효과적인 것으로 나타났지만(Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives J Gastroenterol;45:794-807), 어떻게 이러한 유전적 변화가 간암 환자들 개개인에게 관찰되는 임상적 특징들을 야기하는지는 불명확하다.Liver cancer is the fifth most common cancer worldwide, but it is an aggressive cancer with the third highest mortality rate (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004;50:556-573). Surgery for curative purposes occurs in only 15% of cases. It is possible for only about 25% of patients, and most liver cancer patients die within a relatively short period of time due to locally progressing or metastatic disease (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25: 212-225) Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well known as major causes of liver cancer. However, the overall survival rate of liver cancer patients has not significantly increased over the past 20 years, and the mechanisms of development and progression of liver cancer are still not well known (Bruix J, et al Focus on hepatocellular carcinoma Cancer Cell 2004; 5:215-219) So far, molecular targeted therapy has been shown to be effective in treating mature liver cancer (Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives J Gastroenterol;45:794-807), it is unclear how these genetic changes give rise to the clinical characteristics observed in individual liver cancer patients.
HDACs(Histone deacetylases)는 종종 보조억제자(corepressors)나 다중-단백질 전사복합체(multi-protein transcriptional complexes)들에 의해 유전자 프로모터에 붙을 수 있으며, 그곳에서 DNA에 직접 결합하지 않고 크로마틴(chromatin) 변형을 통해 전사를 조절한다(Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF Histone deacetylases: unique players in shaping the epigenetic histone code Ann N Y Acad Sci 2003;983:84-100) 암호화된 사람 HDACs는 18개가 있으며, 이들은 클래스 I(HDAC 1, 2, 3 및 8), 클래스 II(HDAC 4, 5, 6, 7, 9 및 10), 클래스 III(SIRT 1-7), 및 클래스 IV(HDAC11) 효소들로 분류된다(Yang XJ, Seto E The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men Nat Rev Mol Cell Biol 2008;9:206-218) 히스톤 아세틸화 효소(acetyltransferases) 및 HDACs 모두 세포 증식, 분화 및 세포주기 조절에 관여한다는 사실이 알려져 있다(Witt O, Deubzer HE, Milde T, Oehme I HDAC family: What are the cancer relevant targets Cancer Lett 2009;277:8-21) 또한, HDACs의 병리학적 활성 및 조절감소(deregulation)가 암, 면역질환, 및 근이영양증(muscular dystrophy)과 같은 여러 질병들을 야기할 수 있다는 사실이 보고되었다(Yang XJ, Seto E HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention Oncogene 2007;26:5310-5318). HDAC6는 HDACs의 클래스 IIb 패밀리 멤버이고, 미세소관(MTs)[0004] 과 관련 있는 세포질내 탈아세틸화효소(cytoplasmic deacetylase)로 작용하며, 알파-튜뷸린(α-tubulin)을 탈아세틸화시킨다(Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18α. Nature 2002;417:455-458). Histone deacetylases (HDACs) can attach to gene promoters, often by corepressors or multi-protein transcriptional complexes, where they modify chromatin without binding directly to DNA. (Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF Histone deacetylases: unique players in shaping the epigenetic histone code Ann N Y Acad Sci 2003;983:84-100) There are 18 HDACs, which are class I (HDAC 1, 2, 3, and 8), class II (HDAC 4, 5, 6, 7, 9, and 10), class III (SIRT 1-7), and class IV ( HDAC11) enzymes (Yang It is known that both and HDACs are involved in cell proliferation, differentiation, and cell cycle regulation (Witt O, Deubzer HE, Milde T, Oehme I HDAC family: What are the cancer relevant targets Cancer Lett 2009;277:8-21). , it has been reported that pathological activity and deregulation of HDACs can cause various diseases such as cancer, immune diseases, and muscular dystrophy (Yang XJ, Seto E HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention Oncogene 2007;26:5310-5318). HDAC6 is a member of the class IIb family of HDACs, acts as a cytoplasmic deacetylase associated with microtubules (MTs), and deacetylates alpha-tubulin (α-tubulin). Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18α. Nature 2002;417:455-458).
한편, miRNA는 세포 내에 존재하는 20-25 핵산(nucleotide) 길이의 작은 RNA(endogenous small RNA)의 일종으로 단백질을 합성하지 않는 DNA에서 유래되어 헤어핀-구조 전사체(hairpin-shaped transcript)로부터 생성이 된다. miRNA는 표적 mRNA의 3'-UTR의 상보적인 서열에 결합하여 그 mRNA의 번역 억제 또는 불안정화를 유도하여, 궁극적으로 그 표적 mRNA의 단백질 합성을 억제하는 리프레서(repressor) 역할을 하게 된다. 하나의 miRNA는 여러 개의 mRNA를 타겟팅하며, mRNA 역시 여러 개의 miRNA에 의해 조절될 수 있다고 알려져 있다.Meanwhile, miRNA is a type of endogenous small RNA (20-25 nucleotides) long that exists within cells. It is derived from DNA that does not synthesize proteins and is generated from a hairpin-shaped transcript. do. MiRNAs bind to the complementary sequence of the 3'-UTR of a target mRNA and induce translational inhibition or destabilization of the mRNA, ultimately acting as a repressor that inhibits protein synthesis of the target mRNA. It is known that one miRNA targets multiple mRNAs, and mRNAs can also be regulated by multiple miRNAs.
한편, 대식세포들은 식균작용에 의해 질병세포(암세포)들을 탐식하는데, 그것은 항체들의 Fc 절편이 대식세포들 막표면의 Fc 수용체와의 결합을 매개함으로써 일어난다. 그러나 종양들은 정상 면역 조절 기구 전복을 통해 대식세포들을 포함한 면역세포들의 공격에서 벗어날 수 있다. 그러한 기전의 하나가 정상 세포들에서 발현되는 하나의 단백질인 CD47이다. CD47은 SIRPα(signal-regulatory protein α)라 부르는 대식세포의 수용체와 상호작용하여 이것이 대식세포들에게 “나를 잡아먹지 말라(탐식 차단)”는 신호 전달을 이끌음으로써 정상 세포들을 떠나게 한다. 동일하게 암세포들에 의한 CD47 발현도 그들로 하여금 암세포들이 항체와 결합했을 경우에서 조차 대식세포들에 대한 저항성을 갖게 하는 것이다. 따라서 종양들에 많은 수의 대식세포들이 접근하지만 “탐식 차단” 신호가 꺼지지 않으면 암세포들에게 작용할 수없는 것이다. 이에대한 하나의 치료 전략이 CD47에 대한 단일클론 항체를 이용한 “탐식 차단” 신호를 차단하는 것이다.Meanwhile, macrophages phagocytose disease cells (cancer cells) through phagocytosis, which occurs by mediating the binding of Fc fragments of antibodies to Fc receptors on the membrane surface of macrophages. However, tumors can escape attack by immune cells, including macrophages, by overturning normal immune regulatory mechanisms. One such mechanism is CD47, a protein expressed in normal cells. CD47 interacts with a receptor on macrophages called SIRPα (signal-regulatory protein α), which leads to the transmission of a “don’t eat me” signal to macrophages, causing normal cells to leave. Likewise, CD47 expression by cancer cells makes them resistant to macrophages even when the cancer cells bind to antibodies. Therefore, although large numbers of macrophages approach tumors, they cannot act on cancer cells unless the “phagocytosis blocking” signal is turned off. One treatment strategy for this is to block the “phagocytosis” signal using a monoclonal antibody against CD47.
따라서, 본 발명은 Let-7i-5p의 발현 억제제인 RT-LET7을 변형시켜 기존의 RT-LET7 보다 효과가 상승됨을 통해 이를 간암의 예방 또는 치료용 약학 조성물 로 사용될 수 있음을 확인하였고, 또한 변형된 RT-LET7에 의한 대식세포 식작용을 조절할 수 있음을 통해 CD47-양성 간암의 치료용 면역항암제 약학 조성물로 사용될 수 있음을 확인하여 본 발명을 완성하였다.Therefore, the present invention modified RT-LET7, an expression inhibitor of Let-7i-5p, and confirmed that it can be used as a pharmaceutical composition for the prevention or treatment of liver cancer by increasing the effect over the existing RT-LET7. The present invention was completed by confirming that it can be used as an immunotherapy pharmaceutical composition for the treatment of CD47-positive liver cancer by controlling macrophage phagocytosis by RT-LET7.
본 발명의 목적은 Let-7i-5p를 표적화하는 핵산분자를 제공하는 것이다.The object of the present invention is to provide a nucleic acid molecule targeting Let-7i-5p.
또한, 본 발명의 다른 목적은 상기 핵산분자를 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer containing the nucleic acid molecule as an active ingredient.
또한, 본 발명의 다른 목적은 상기 핵산분자를 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide an immunotherapy pharmaceutical composition for the treatment of CD47-positive liver cancer, comprising the nucleic acid molecule as an active ingredient.
또한, 본 발명의 다른 목적은 서열번호 1로 표시되는 핵산분자에서, 뉴클레오티드 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형되고, 일부 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된, N-Acetylgalactosamine(GalNAc)이 결합된 것을 특징으로 하는 Let-7i-5p를 표적화하는 핵산분자를 제공하는 것이다.In addition, another object of the present invention is to modify the nucleic acid molecule represented by SEQ ID NO: 1 by modifying the nucleotide sequence to 2'-O-Methoxyethyl (methoxyethyl), and to modify some of the backbone to phosphorothioate. To provide a nucleic acid molecule targeting Let-7i-5p, which is modified with N-Acetylgalactosamine (GalNAc) bound.
상기 과제를 해결하기 위하여, 본 발명은 Let-7i-5p를 표적화하는 핵산분자로서, 상기 핵산분자는 서열번호 1의 염기서열로 표시되고, 뉴클레오티드 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형된 핵산인 것을 특징으로 하며, 일부 또는 전체 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것을 특징으로 하고, N-Acetylgalactosamine(GalNAc)이 결합된 것을 특징으로 하는 핵산분자를 제공한다.In order to solve the above problem, the present invention is a nucleic acid molecule targeting Let-7i-5p, the nucleic acid molecule is represented by the base sequence of SEQ ID NO: 1, and the nucleotide sequence is 2'-O-Methoxyethyl (methoxyethyl) A nucleic acid molecule characterized in that it is a modified nucleic acid, in which part or the entire backbone is modified with phosphorothioate, and in which N-Acetylgalactosamine (GalNAc) is bound. to provide.
또한, 본 발명은 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7을 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer, comprising modified RT-LET7 bound to a hepatocyte targeting moiety as an active ingredient.
또한, 본 발명은 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7을 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물을 제공한다.In addition, the present invention provides an immunotherapy pharmaceutical composition for the treatment of CD47-positive liver cancer, comprising modified RT-LET7 bound to a hepatocyte targeting moiety as an active ingredient.
본 발명에 따른 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7은 기존 간암세포의 Let-7i-5p를 억제하는 안티센스 microRNA(AS-miRNA)인 RT-LET7 및 변형된 RT-LET7 대비 간암에서의 종양 세포 성장을 현저하게 억제하는 것을 확인할 수 있었고, 딜리버리 시스템을 탑재한 변형된 RT-LET7은 간암세포에서 Let-7i-5p를 억제해 TSP1을 증가시키고, 증가한 TSP1은 CD47과 결합해 대식세포의 SIRPα와 상호작용을 막게되며, 따라서 간암세포의 CD47과 대식세포의 SIRPα 결합을 막아 암세포에 대한 대식세포의 면역활성을 높여 항암효과를 보이는 기전을 통해 대식세포 식작용 활성을 현저히 증가시킴을 확인하여, 이를 간암의 예방 및 치료용 약학 조성물 또는 CD47-양성 간암의 예방 또는 치료용 면역항암제 약학 조성물로 이용할 수 있음을 확인하였다.The modified RT-LET7 combined with the hepatocyte targeting moiety according to the present invention is compared to RT-LET7 and modified RT-LET7, which are antisense microRNAs (AS-miRNA) that inhibit Let-7i-5p of existing liver cancer cells. It was confirmed that tumor cell growth in liver cancer was significantly suppressed, and the modified RT-LET7 equipped with a delivery system increased TSP1 by inhibiting Let-7i-5p in liver cancer cells, and the increased TSP1 bound to CD47. It blocks the interaction with SIRPα of macrophages, and thus significantly increases macrophage phagocytosis activity through a mechanism that increases the immune activity of macrophages against cancer cells by blocking the binding of CD47 of liver cancer cells to SIRPα of macrophages, showing an anticancer effect. It was confirmed that this could be used as a pharmaceutical composition for the prevention and treatment of liver cancer or an immunotherapy pharmaceutical composition for the prevention or treatment of CD47-positive liver cancer.
도 1은 본 발명의 일 실시예에 있어서, Let-7i-5p의 발현 억제제인 RT-LET7의 다양한 변형 구조(도 1a) 및 억제 효율(도 1b)을 나타낸 도이다(기본형: RT-LET7, 변형: RT-LET7-2, RT-LET7-4, RT-LET7-6 및 RT-LET7-8).
도 2는 본 발명의 일 실시예에 있어서, 본 발명의 변형된 RT-LET7인 RT-LET7-8이 HCC 세포주에서 유의미하게 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다(도 2a; SNU-387, 도 2b; SNU-368, 도 2c; SNU-423).
도 3은 본 발명의 일 실시예에 있어서, 본 발명의 변형된 RT-LET7인 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 3a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 3b; 기본형 RT-LET7 및 변형 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 3c; 기본형 RT-LET7 및 변형 RT-LET7-8가 처리된 HCC 세포의 대식세포 식작용 활성).
도 4는 GalNAc이 결합된 RT-LET7-8의 HCC 세포에서 Let-7i-5p 발현 억제 효율 및 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다.
도 5는 본 발명의 일 실시예에 있어서, Gal-LNP가 결합된 RT-LET7-8의 HCC 세포에서 Let-7i-5p 발현 억제 효율 및 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다.
도 6은 본 발명의 일 실시예에 있어서, GalNAc이 결합된 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 6a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 6b; GalNAc이 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 6c; GalNAc이 결합된 RT-LET7-8이 처리된 HCC 세포의 대식세포 식작용 활성).
도 7은 본 발명의 일 실시예에 있어서, Gal-LNP가 결합된 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 6a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 6b; Gal-LNP가 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 6c; Gal-LNP가 결합된 RT-LET7-8이 처리된 HCC 세포의 대식세포 식작용 활성).
도 8은 본 발명의 일 실시예에 있어서, 간세포 표적 딜리버리 시스템(GalNAc 및 Gal-LNP) 결합에 대한 모식도를 간단히 나타낸 도이다(도 8a; Gal-LNP 결합, 도 8b; Old GalNAc 결합, 도 8c; D-GalNAc 결합).
도 9는 본 발명의 일 실시예에 있어서, 구조가 상이한 GalNAc(D-GalNAc)이 결합된 RT-LET7-8의 HCC 세포에서 Let-7i-5p 발현 억제 효율 및 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다.
도 10은 본 발명의 일 실시예에 있어서, 구조가 상이한 GalNAc(D-GalNAc)이 결합된 RT-LET7-8에 의한 HCC 세포주의 생장 억제를 확인한 도이다.
도 11은 본 발명의 일 실시예에 있어서, 구조가 상이한 GalNAc(D-GalNAc)이 결합된 RT-LET7-8에 의한 HCC 세포주의 TSP1 발현변화 및 대식세포의 활성 증가를 확인한 도이다.Figure 1 is a diagram showing various modified structures (Figure 1a) and inhibition efficiency (Figure 1b) of RT-LET7, an expression inhibitor of Let-7i-5p, in one embodiment of the present invention (basic type: RT-LET7, Variants: RT-LET7-2, RT-LET7-4, RT-LET7-6 and RT-LET7-8).
Figure 2 is a diagram confirming the effect of RT-LET7-8, a modified RT-LET7 of the present invention, in significantly and continuously inhibiting Let-7i-5p in HCC cell lines in one embodiment of the present invention (Figure 2a ; SNU-387, Figure 2b; SNU-368, Figure 2c; SNU-423).
Figure 3 is a diagram confirming the inhibition of growth of HCC cell lines and increased activity of macrophages by RT-LET7-8, a modified RT-LET7 of the present invention, in an embodiment of the present invention (Figure 3a; MTT and cells Tumor formation characteristics of Let-7i-5p through survival analysis, Figure 3b; Changes in TSP1 expression through Western blot analysis after treatment with basic RT-LET7 and modified RT-LET7-8, Figure 3c; Basic type RT-LET7 and Macrophage phagocytosis activity of HCC cells treated with modified RT-LET7-8).
Figure 4 is a diagram confirming the efficiency of suppressing Let-7i-5p expression in HCC cells of RT-LET7-8 conjugated with GalNAc and the effect of continuously suppressing Let-7i-5p.
Figure 5 is a diagram confirming the efficiency of suppressing Let-7i-5p expression and the effect of continuously suppressing Let-7i-5p in HCC cells of RT-LET7-8 bound to Gal-LNP, in an embodiment of the present invention. am.
Figure 6 is a diagram confirming the inhibition of growth of HCC cell lines and increased activity of macrophages by GalNAc-conjugated RT-LET7-8 in an embodiment of the present invention (Figure 6a; Let through MTT and cell survival analysis Tumor formation characteristics of -7i-5p, Figure 6b; TSP1 expression change through Western blot analysis after treatment with GalNAc-conjugated RT-LET7-8, Figure 6c; Treatment with GalNAc-conjugated RT-LET7-8 Macrophage phagocytosis activity of HCC cells).
Figure 7 is a diagram confirming the inhibition of growth of HCC cell lines and increased activity of macrophages by RT-LET7-8 coupled with Gal-LNP in an embodiment of the present invention (Figure 6a; MTT and cell survival analysis Tumor formation characteristics of Let-7i-5p, Figure 6b; TSP1 expression change through Western blot analysis after treatment with Gal-LNP-conjugated RT-LET7-8, Figure 6c; Gal-LNP-conjugated RT- Macrophage phagocytosis activity of HCC cells treated with LET7-8).
Figure 8 is a schematic diagram briefly showing the binding of the hepatocyte target delivery system (GalNAc and Gal-LNP) in one embodiment of the present invention (Figure 8a; Gal-LNP binding, Figure 8b; Old GalNAc binding, Figure 8c ; D-GalNAc binding).
Figure 9 shows the efficiency of suppressing Let-7i-5p expression in HCC cells of RT-LET7-8 bound with GalNAc (D-GalNAc) with a different structure, and the continuous expression of Let-7i-5p in an embodiment of the present invention. This is a province that confirmed the suppressing effect.
Figure 10 is a diagram confirming the inhibition of growth of HCC cell lines by RT-LET7-8 bound to GalNAc (D-GalNAc) with a different structure, in one embodiment of the present invention.
Figure 11 is a diagram confirming the change in TSP1 expression and increase in macrophage activity in HCC cell lines by RT-LET7-8 conjugated with GalNAc (D-GalNAc) of different structures, according to an embodiment of the present invention.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
일 측면에서, 본 발명은 서열번호 1로 표시되는 핵산분자로서, Let-7i-5p를 표적화하는 핵산분자에 관한 것이다.In one aspect, the present invention relates to a nucleic acid molecule represented by SEQ ID NO: 1, which targets Let-7i-5p.
일 구현예에서, 본 발명의 "핵산분자"는 뉴클레오티드 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형된 것일 수 있다.In one embodiment, the "nucleic acid molecule" of the present invention may have a nucleotide sequence modified by 2'-O-Methoxyethyl (methoxyethyl).
일 구현예에서, 본 발명의 "핵산분자"는 일부 또는 전체 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것일 수 있으며, 바람직하게는 서열번호 1의 뉴클레오티드 중 5‘ 말단으로부터 1 내지 4번째 뉴클레오티드 및 3‘ 말단으로부터 1 내지 4번째 뉴클레오티드의 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment, the "nucleic acid molecule" of the present invention may be one in which part or the entire backbone has been modified with phosphorothioate, preferably from 1 to 5' from the 5' end of the nucleotides of SEQ ID NO: 1. The backbone of the 4th nucleotide and the 1st to 4th nucleotides from the 3' end may be modified with phosphorothioate, but is not limited thereto.
일 구현예에서, 본 발명의 "핵산분자"는 간세포 표적 모이어티(Moiety)가 결합된 것일 수 있다.In one embodiment, the “nucleic acid molecule” of the present invention may be one to which a hepatocyte targeting moiety is bound.
또한, 상기 간세포 표적 모이어티(Moiety)는 간 조직 타겟 약물 전달 시스템으로 알려진 것이라면 제한 없이 사용될 수 있으며, 예를 들면 N-Acetylgalactosamine(GalNAc), Gal-LNP(Galactosyl lipidoid nanoparticle), Lipid-siRNA, Antibody-siRNA, Peptide-ASO, Stable nucleic acid lipid particle, Exosome, Spherical nucleic acid, DNA cage 등이 있다(Nat Rev Drug Discov. 2020 Oct;19(10):673-694.).In addition, the hepatocyte targeting moiety can be used without limitation as long as it is a known liver tissue targeting drug delivery system, for example, N-Acetylgalactosamine (GalNAc), Gal-LNP (Galactosyl lipidoid nanoparticle), Lipid-siRNA, Antibody -siRNA, Peptide-ASO, Stable nucleic acid lipid particle, Exosome, Spherical nucleic acid, DNA cage, etc. (Nat Rev Drug Discov. 2020 Oct;19(10):673-694.).
또한, 상기 Gal-LNP(Galactosyl lipidoid nanoparticle)는 콜레스테롤, DSPC, C16-PET2000-ceramide 및 α-galactosyl ceramide로 구성된 것일 수 있다.Additionally, the Gal-LNP (Galactosyl lipidoid nanoparticle) may be composed of cholesterol, DSPC, C16-PET2000-ceramide, and α-galactosyl ceramide.
또한, 상기 N-Acetylgalactosamine(GalNAc)은 화학식 1 또는 2로 표시되는 것일 수 있고, 바람직하게는 화학식 2로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In addition, the N-Acetylgalactosamine (GalNAc) may be represented by Formula 1 or 2, and preferably may be represented by Formula 2, but is not limited thereto.
일 측면에서, 본 발명은 상기 핵산분자를 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising the nucleic acid molecule as an active ingredient.
일 구현예에서, 본 발명의 "조성물"은 Let-7i-5p의 발현을 억제하는 것일 수 있다.In one embodiment, the “composition” of the present invention may inhibit the expression of Let-7i-5p.
일 구현예에서, 본 발명의 "조성물"은 간암 세포 성장을 억제하는 것일 수 있다.In one embodiment, the “composition” of the present invention may inhibit liver cancer cell growth.
일 구현예에서, 본 발명의 "조성물"은 TSP1(thrombospondin-1) 발현을 증가시키는 것일 수 있다.In one embodiment, the “composition” of the present invention may increase TSP1 (thrombospondin-1) expression.
일 구현예에서, 본 발명의 "조성물"은 대식세포 식작용 활성을 증가시키는 것일 수 있다.In one embodiment, the “composition” of the present invention may increase macrophage phagocytosis activity.
일 측면에서, 본 발명은 상기 핵산분자를 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물에 관한 것이다.In one aspect, the present invention relates to an immunotherapy pharmaceutical composition for the treatment of CD47-positive liver cancer comprising the nucleic acid molecule as an active ingredient.
일 구현예에서, 본 발명의 "TSP1"은 CD47 수용체를 점유하여 CD47-SIRPα의 상호작용을 방해할 수 있다.In one embodiment, “TSP1” of the present invention can occupy the CD47 receptor and interfere with the interaction of CD47-SIRPα.
일 구현예에서, 본 발명의 "핵산분자"는 let-7i-p-TSP1 신호전달 축을 조절할 수 있으며, 대식세포와 HCC 사이의 CD47-SIRPα 상호작용을 CD47-TSP1 상호작용으로 전환함으로써 대식세포의 HCC 세포에 대한 식작용을 재-활성화하는 것일 수 있다.In one embodiment, the “nucleic acid molecule” of the present invention is capable of regulating the let-7i-p-TSP1 signaling axis, and converts the CD47-SIRPα interaction between macrophages and HCC to CD47-TSP1 interaction, thereby promoting the activation of macrophages. It may be to re-activate phagocytosis on HCC cells.
일 구현예에서, 본 발명의 "간암"은 Let-7i-5p 고발현 간암일 수 있으며, 간세포암종(hepatocellular carcinoma)일 수 있고, stage Ⅰ, Ⅱ, Ⅲ, ⅣA 또는 ⅣB 병기(phase)의 간세포암종일 수 있으며, 초기 병기보다 치료가 어려운 stageⅢ 내지 Ⅳ 병기의 간세포암종인 것이 더욱 바람직하다.In one embodiment, the “liver cancer” of the present invention may be liver cancer with high Let-7i-5p expression, may be hepatocellular carcinoma, and may be stage Ⅰ, Ⅱ, Ⅲ, ⅣA or ⅣB liver cells. It may be carcinoma, and it is more preferable that it is stage III to IV hepatocellular carcinoma, which is more difficult to treat than the early stage.
일 구현예에서, 본 발명의 "간암"은 TSP1 저발현 간암일 수 있으며, 간세포암종일 수 있고, stage Ⅰ, Ⅱ, Ⅲ, ⅣA 또는 ⅣB 병기(phase)의 간세포암종일 수 있으며, 초기 병기보다 치료가 어려운 stageⅢ 내지 Ⅳ 병기의 간세포암종인 것이 더욱 바람직하다.In one embodiment, the “liver cancer” of the present invention may be TSP1 low-expressing liver cancer, may be hepatocellular carcinoma, may be stage Ⅰ, Ⅱ, Ⅲ, ⅣA or ⅣB hepatocellular carcinoma, and may be stage Ⅰ, Ⅱ, Ⅲ, ⅣA or ⅣB hepatocellular carcinoma. It is more preferable to have stage III to IV hepatocellular carcinoma, which is difficult to treat.
본 발명에서 사용되는 용어, "발현 억제"란 표적 유전자의(mRNA로의) 발현 또는(단백질로의) 번역 저하를 야기하는 것을 의미하며, 바람직하게는 이에 의해 표적 유전자 발현이 탐지 불가능해지거나 무의미한 수준으로 존재하게 되는 것을 의미한다.As used herein, the term "expression inhibition" means causing a decrease in the expression (to mRNA) or translation (to protein) of a target gene, preferably thereby rendering the target gene expression at an undetectable or meaningless level. It means coming into existence.
본 발명에서, 사용된 용어 "예방"이란 본 발명에 따른 조성물의 투여에 의해 간암의 발생, 발달 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term “prevention” used refers to all actions that inhibit or delay the occurrence, development, and recurrence of liver cancer by administering the composition according to the present invention.
본 발명에서 사용된 용어 "치료"란 본 발명에 따른 조성물의 투여로 간암 및 이로 인한 합병증의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of liver cancer and its complications by administering the composition according to the present invention. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.
본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 개체의 상태 등에 따라 달라질 수 있다. The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, and condition of the subject.
본 발명의 약학적조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효 용량 수준은 개체의 건강상태, 감염증의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term “pharmaceutically effective amount” refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is Factors including health status, type and severity of infection, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
일 구현예에서, 상기 약학 조성물은 경구형 제형, 외용제, 좌제, 멸균 주사용액 및 분무제를 포함하는 군으로부터 선택되는 하나 이상의 제형일 수 있다.In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 본 발명의 조성물은, 조성물 총 중량에 대하여 상기 단백질을 0.0001 내지 10 중량 %로, 바람직하게는 0.001 내지 1 중량 %를 포함한다.The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
본 발명의 약학 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid. Calcium, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 경구 투여 하는 것이 가장 바람직하다. 투여량은 개체의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다.The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the desired method, and oral administration is most preferred. The dosage range varies depending on the individual's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.
<실시예 1> 기존 RT-LET7의 변형된 RT-LET7 제작<Example 1> Production of RT-LET7, a modified version of the existing RT-LET7
Let-7i-5p의 억제제인 RT-LET7(서열번호1: AACAGCACAAACUACUACCUCA에 4단계 과정으로 한 cycle씩 진행하여, RNA oligo 1mer 씩 3‘ 말단에서 5’말단까지 Deblocking -> Coupling -> Oxidation -> Capping의 과정으로 순차적으로 진행한다. 이때, 변형된 염기를 사용하는데, Nat Rev Drug Discov. 2020 Oct;19(10):673-694.에 따르면, 2’-Ribose의 변형(2’-O-methyl, 2‘-MOE)은 변형시키지 않은 RNA보다 핵산분해효소로부터 저항성을 증가시키고, 세포질내에서 안정성을 증가시키는데 기여한다. 또한, 생체 조직내에서 반감기를 증가시키는 역할도 하여, 결과적으로 약물의 효과를 증대시키는 역할을 한다. 더욱이, 상보적인 RNA에 강하게 부착하여, RNase에 의해 표적 유전자가 더 효과적으로 분해되는데 도움을 준다. 추가로, RNA 염기사이에 phosphorothioate로 치환하였을 때, RNase의 활성에는 영향을 주지 않으며, 표적 RNA에 효율적으로 결합한다. 그리고, 세포와 체내에서 알부민과 같은 단백질과 결합하여, 핵산분해효소로부터 보호되고, 이에 따라, 약물이 소변으로 배출되는 것을 막아주는 역할을 하여, 체내에서 약물의 지속시간을 늘려, 약물의 효과를 증대시키는 역할을 하는 이유 때문에 Ribose와 phosphorothioate 변형을 진행하였다. 변형된 RNA 염기 2‘-OMe rA Phosphoramidite(Glen Research, Sterling, VA, cat. 10-3100), 2‘-OMe rC Phosphoramidite(Glen Research, cat. 10-3115), 2‘-OMe rG Phosphoramidite(Glen Research, cat. 10-3120), 2‘-OMe rU Phosphoramidite(Glen Research, cat. 10-3130), 2'-MOE rA Phosphoramidite(Glen Research, cat. 10-3200), 2'-MOE rC Phosphoramidite(Glen Research, cat. 10-3211), 2'-MOE rG Phosphoramidite(Glen Research, cat. 10-3220), 2'-MOE rU Phosphoramidite(Glen Research, cat. 10-3231)를 합성한다. 합성과정에서 phosphorothioate로 치환이 필요한 부분은 Sulfurizing Reagent II(Glen Research, cat. 40-4037-10)를 활용하여 합성한다(바이오니아 합성). 이때, 아무변형을 하지 않은 RNA oligo를 RT-LET7, 모든 염기를 2’-O-methyl로 변형하고, phosphorothioate로 변형한 RNA oligo를 RT-LET7-2, 5’말단과 3’말단 4곳을 변형시킨 염기를 RT-LET7-6으로 명명하였다. 그리고, 모든 염기를 2’-MOE로 변형하고, phosphorothioate로 변형한 RNA oligo를 RT-LET7-4, 5’말단과 3’말단 4곳을 변형시킨 염기를 RT-LET7-8로 명명하였다(도 1a).RT-LET7, an inhibitor of Let-7i-5p (SEQ ID NO: 1: AACAGCACAAACUACUACCUCA) proceeds one cycle in 4 steps, Deblocking -> Coupling -> Oxidation -> Capping of RNA oligo 1 mer from 3' end to 5' end The process proceeds sequentially. At this time, a modified base is used, and according to Nat Rev Drug Discov. 2020 Oct;19(10):673-694., the modification of 2'-Ribose (2'-O-methyl , 2'-MOE) increases resistance from nucleases compared to unmodified RNA and contributes to increasing stability in the cytoplasm. It also plays a role in increasing the half-life in biological tissues, resulting in the effect of the drug. It plays a role in increasing . Moreover, by strongly attaching to complementary RNA, it helps target genes to be degraded more effectively by RNase. Additionally, when phosphorothioate is substituted between RNA bases, it affects the activity of RNase. It efficiently binds to the target RNA and binds to proteins such as albumin in cells and the body, protecting it from nucleases and thus preventing the drug from being excreted in the urine. Ribose and phosphorothioate modifications were performed for the reason that they play a role in increasing the effect of the drug by increasing the duration of the drug.Modified RNA base 2'-OMe rA Phosphoramidite (Glen Research, Sterling, VA, cat. 10-3100) , 2'-OMe rC Phosphoramidite (Glen Research, cat. 10-3115), 2'-OMe rG Phosphoramidite (Glen Research, cat. 10-3120), 2'-OMe rU Phosphoramidite (Glen Research, cat. 10-3130) ), 2'-MOE rA Phosphoramidite (Glen Research, cat. 10-3200), 2'-MOE rC Phosphoramidite (Glen Research, cat. 10-3211), 2'-MOE rG Phosphoramidite (Glen Research, cat. 10-3220), 2'-MOE rU Phosphoramidite (Glen Research, cat. . 10-3231) is synthesized. During the synthesis process, parts that require substitution with phosphorothioate are synthesized using Sulfurizing Reagent II (Glen Research, cat. 40-4037-10) (Bioneer synthesis). At this time, the RNA oligo without any modification was used as RT-LET7, all bases were modified with 2'-O-methyl, and the RNA oligo modified with phosphorothioate was used as RT-LET7-2, with four positions at the 5' and 3' ends. The modified base was named RT-LET7-6. In addition, all bases were modified with 2'-MOE, and the RNA oligo modified with phosphorothioate was named RT-LET7-4, and the base with four modifications at the 5' and 3' ends was named RT-LET7-8 (Figure 1a).
<실시예 2> 변형된 RT-LET7의 처리에 의한 Let-7i-5p의 발현 억제 확인<Example 2> Confirmation of inhibition of Let-7i-5p expression by treatment with modified RT-LET7
도 1a와 같이 만들어진 변형된 RT-LET7를 트랜스펙션한 HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에서 총 RNA를 TRIzol 시약(Invitrogen, Carlsbad, CA)을 이용하여 분리한 후, 상기 두 miRNA에 특이적인 cDNA를 micscipt II RT 키트(Qiagen, Manchester, UK)를 이용하여 합성하였다. qRT-PCR은 SensiFASTTM SYBR NoROX Kit(Bioline, London, UK)로 수행되었다. HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에서 Let-7i-5p의 qRT-PCR 분석을 수행한 결과, 2’-MOE로 모든 염기를 치환 하였을 때(RT-LET7-4 및 RT-LET7-8), 2‘-O-methyl로 치환하였을 때(RT-LET7-2 및 RT-LET7-6) 보다 Let-7i-5p의 발현을 억제하는데 효과가 좋은 것을 확인하였다(도 1b).Total RNA was isolated from HCC cell lines (SNU-387, SNU-368, and SNU-423) transfected with the modified RT-LET7 as shown in Figure 1a using TRIzol reagent (Invitrogen, Carlsbad, CA). , cDNA specific for the above two miRNAs was synthesized using the micscipt II RT kit (Qiagen, Manchester, UK). qRT-PCR was performed with the SensiFAST™ SYBR NoROX Kit (Bioline, London, UK). qRT-PCR analysis of Let-7i-5p in HCC cell lines (SNU-387, SNU-368, and SNU-423) showed that when all bases were substituted with 2'-MOE (RT-LET7-4 and RT-LET7-8) was confirmed to be more effective in suppressing the expression of Let-7i-5p than when substituted with 2'-O-methyl (RT-LET7-2 and RT-LET7-6) (Figure 1b) ).
또한, 2’-MOE로 RT-LET7을 변형시켰을 때, HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에 트랜스펙션 후 최대 7일까지 배양 후 RNA 추출 후 Let-7i-5p의 발현 억제정도를 비교하였을 때, 2’-MOE로 변형시킨 후 phosphorothioate로 치환하였을 때, 아무 변형도 안한 RT-LET7에 비해서 Let-7i-5p의 발현 억제가 유의미하게 효율이 증대되는 것을 확인하였다(도 2).Additionally, when RT-LET7 was modified with 2'-MOE, it was cultured for up to 7 days after transfection into HCC cell lines (SNU-387, SNU-368, and SNU-423), and after RNA extraction, Let-7i-5p When comparing the degree of inhibition of expression, it was confirmed that when modified with 2'-MOE and then replaced with phosphorothioate, the efficiency of inhibiting the expression of Let-7i-5p was significantly increased compared to RT-LET7 without any modification. (Figure 2).
<실시예 3> MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성 확인<Example 3> Confirmation of tumor formation characteristics of Let-7i-5p through MTT and cell survival analysis
간암에서의 RT-LET7 처리시 인비트로 종양형성(in vitro tumorigenesis) 억제능을 확인하기 위해, RT-LET7를 이용하여 MTT 분석을 수행하였다. 구체적으로, MTT 분석을 위해, SNU-387 세포를 12-웰 플레이트에 분주하고 RT-LET7, RT-LET7-4, RT-LET7-8을 트랜스펙션한 뒤, MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 용액(Sigma) 0.5 mg/ml과 1시간 동안 인큐베이션한 뒤, SYNERGY H1 Multilabel 플레이트 리더기(Bio-Tek, Winooski, VT)를 이용하여 흡광도를 측정하였다. 그 결과, RT-LET7의 변형된 RT-LET7-8을 처리한 실험군에서 종양 세포 성장을 가장 우수하게 억제하는 것을 확인할 수 있었다(도 3a). To confirm the ability to inhibit in vitro tumorigenesis upon RT-LET7 treatment in liver cancer, MTT analysis was performed using RT-LET7. Specifically, for MTT analysis, SNU-387 cells were seeded in a 12-well plate and transfected with RT-LET7, RT-LET7-4, and RT-LET7-8, followed by MTT[3-(4,5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (Sigma) at 0.5 mg/ml for 1 hour, then absorbance was measured using a SYNERGY H1 Multilabel plate reader (Bio-Tek, Winooski, VT). Measured. As a result, it was confirmed that the experimental group treated with RT-LET7-8, a modified version of RT-LET7, suppressed tumor cell growth the best (Figure 3a).
<실시예 4> 변형된 RT-LET7에 의한 대식세포 식작용 조절 확인<Example 4> Confirmation of regulation of macrophage phagocytosis by modified RT-LET7
Let-7i-5p가 대식세포 식작용(macrophage phagocytosis)의 조절에 관여하는 TSP1의 발현을 조절하는지 확인하기 위해, RT-LET7과 변형된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 발현변화를 확인하였다. 그 결과, RT-LET7과 RT-LET7-8을 각각 처리하였을 때, TSP1의 발현이 증가되는 것을 확인할 수 있었다(도 3b).To determine whether Let-7i-5p regulates the expression of TSP1, which is involved in the regulation of macrophage phagocytosis, expression changes were measured through Western blot analysis after treatment with RT-LET7 and modified RT-LET7-8. Confirmed. As a result, it was confirmed that the expression of TSP1 increased when RT-LET7 and RT-LET7-8 were treated respectively (Figure 3b).
이러한 결과를 토대로, Let-7i-5p-TSP1 네트워크의 CD47과 SIRPα(signal regulatory protein α)의 상호작용에서 TSP1이 CD47 수용체를 점유하여 CD47-SIRPα의 상호작용을 방해하여 대식세포가 HCC를 식작용할 수 있게 하는지를 확인하기 위하여, 인비트로 식작용 분석을 수행하였다. 구체적으로, HCC 세포주인 SNU-387 각각 단일 세포 현탁액으로 만든 후, CFSE(abcam, Cambridge, UK)로 표지하였다. 그 후, C57BL/6 마우스로부터 복막 대식세포를 수득하고 SNU-387과 2시간 동안 공배양한 뒤, RT-LET7 및 변형된 RT-LET7-8로 각각 처리하였고, 양성 대조군으로 TSP1 재조합 단백질을 직접 처리한 HCC 세포와 비교하였다. 식작용 지수는 종양 세포를 포획하는 대식세포의 수를 대식세포의 총 수로 나눠 계산하였다. 그 결과, RT-LET7 및 변형된 RT-LET7-8가 처리된 HCC 세포 모두 대식세포 식작용 활성이 현저히 증가함을 확인하였고, RT-LET7 대비 변형된 RT-LET7-8에서 대식세포 식작용 활성이 더 증가함을 확인하였다(도 3c).Based on these results, in the interaction between CD47 and SIRPα (signal regulatory protein α) in the Let-7i-5p-TSP1 network, TSP1 occupies the CD47 receptor and interferes with the interaction of CD47-SIRPα, allowing macrophages to phagocytose HCC. To confirm whether this was possible, an in vitro phagocytosis analysis was performed. Specifically, each HCC cell line, SNU-387, was made into a single cell suspension and then labeled with CFSE (abcam, Cambridge, UK). Afterwards, peritoneal macrophages were obtained from C57BL/6 mice, co-cultured with SNU-387 for 2 hours, and then treated with RT-LET7 and modified RT-LET7-8, respectively. As a positive control, TSP1 recombinant protein was directly incubated. Comparison was made with treated HCC cells. The phagocytosis index was calculated by dividing the number of macrophages capturing tumor cells by the total number of macrophages. As a result, it was confirmed that the macrophage phagocytosis activity was significantly increased in both HCC cells treated with RT-LET7 and modified RT-LET7-8, and the macrophage phagocytosis activity was higher in modified RT-LET7-8 compared to RT-LET7. An increase was confirmed (Figure 3c).
이를 통해, RT-LET7을 변형시킨 RT-LET7-8가 let-7i-p-TSP1 신호전달 축을 조절할 수 있으며, 대식세포와 HCC 사이의 CD47-SIRPα 상호작용을 CD47-TSP1 상호작용으로 전환함으로써 대식세포의 HCC 세포에 대한 식작용을 재-활성화하는 것을 알 수 있다.Through this, RT-LET7-8, a modified version of RT-LET7, can regulate the let-7i-p-TSP1 signaling axis and convert the CD47-SIRPα interaction between macrophages and HCC into CD47-TSP1 interaction. It can be seen that the phagocytes re-activate the phagocytosis of HCC cells.
<실시예 5> 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8의 효율 검증<Example 5> Verification of the efficiency of RT-LET7-8 combined with hepatocyte targeting moiety
5-1. 딜리버리 시스템을 이용한 RT-LET7-8의 Let-7i-5p의 발현 억제 확인5-1. Confirmation of inhibition of Let-7i-5p expression by RT-LET7-8 using delivery system
딜리버리 시스템을 이용한 변형된 RT-LET7-8의 Let-7i-5p의 발현 억제 효과를 확인하기 위해, 약물 전달 시스템(drug delivery system)으로 알려진 N-Acetylgalactosamine(GalNAc) 또는 Gal-LNP(Galactosyl lipidoid nanoparticle)에 RT-LET7-8을 결합시킨 핵산분자를 ASGPR(asialoglycoprotein receptor)의 발현이 positive한 cell(Hep3B)과 negative인 cell(SNU-449)에 전달하였다. To confirm the inhibitory effect of modified RT-LET7-8 on the expression of Let-7i-5p using a delivery system, N-Acetylgalactosamine (GalNAc) or Galactosyl lipidoid nanoparticle (Gal-LNP), known as drug delivery system, was used. ), the nucleic acid molecule bound to RT-LET7-8 was delivered to positive cells (Hep3B) and negative cells (SNU-449) expressing ASGPR (asialoglycoprotein receptor).
구체적으로, Gal-LNP-RT-LET7-8는 알킬에폭사이드(Sigma-Aldrich)와 폴리아민(Sigma-Aldrich)을 결합하여 합성한 C12-SPM, DSPC(Distearoylphosphatidylcholine)(Sigma-Aldrich), 콜레스테롤(Sigma-Aldrich), C16-PEG2000-ceramide(Avanti Polar Lipids)와 α-galactosyl ceramide(Avanti Polar Lipids)를 각각 50:10:38.5:0.75:0.75의 비율로 혼합하여 Gal-LNP를 제조하였다. 이후 RT-LET7-8 용액(10 mg /mL) 을 10 mM citrate buffer(pH 3)에 섞어주고, Gal-LNP 와 RT-LET7-8을 7:1로 혼합 후 37 °C에서 30분간 배양하여 Gal-LNP에 RT-LET7-8이 탑재되도록 하였다. 이후 PBS(Sigma-Aldrich, cat. P5368)에 75분간 투석하여, 에탄올과 Gal-LNP가 탑재되지 않은 RT-LET7-8을 제거하였다(도 8a 참조). 이어서, GalNAc-RT-LET7-8는 modified RT-LET7-8의 5‘ 말단에 Trebler phosphoramidite(GLEN Research)를 접합하여 RT-LET7-8을 변형한 이후에 N-acetylgalactosamine(GLEN Research)을 접합하여 GalNAc-RT-LET7-8을 합성하였다(도 8b 참조). 이후 실시예 2와 동일한 방법으로 각 세포에서 Let-7i-5p 발현량을 확인하였다.Specifically, Gal-LNP-RT-LET7-8 is C12-SPM synthesized by combining alkyl epoxide (Sigma-Aldrich) and polyamine (Sigma-Aldrich), DSPC (Distearoylphosphatidylcholine) (Sigma-Aldrich), cholesterol ( Sigma-Aldrich), C16-PEG2000-ceramide (Avanti Polar Lipids) and α-galactosyl ceramide (Avanti Polar Lipids) were mixed at a ratio of 50:10:38.5:0.75:0.75, respectively, to prepare Gal-LNPs. Afterwards, RT-LET7-8 solution (10 mg/mL) was mixed with 10 mM citrate buffer (pH 3), Gal-LNP and RT-LET7-8 were mixed in a ratio of 7:1, and incubated at 37 °C for 30 minutes. RT-LET7-8 was loaded onto Gal-LNP. Afterwards, it was dialyzed against PBS (Sigma-Aldrich, cat. P5368) for 75 minutes to remove ethanol and RT-LET7-8 not loaded with Gal-LNP (see Figure 8a). Subsequently, GalNAc-RT-LET7-8 was modified by conjugating Trebler phosphoramidite (GLEN Research) to the 5' end of modified RT-LET7-8 and then conjugating N-acetylgalactosamine (GLEN Research). GalNAc-RT-LET7-8 was synthesized (see Figure 8b). Thereafter, the expression level of Let-7i-5p in each cell was confirmed in the same manner as in Example 2.
그 결과, GalNAc이 결합된 RT-LET7-8의 경우, ASGPR positive cell인 Hep3B 세포에서는 기존 RT-LET7-8 대비 Let-7i-5p의 발현 억제 효과가 유의하게 증가하였으나, ASGPR negative cell인 SNU-449 세포에서는 Let-7i-5p의 발현 억제 효과가 증가하지 않은 것으로 나타났다(도 4a). 반면에, Gal-LNP가 결합된 RT-LET7-8의 경우, ASGPR의 여부와 상관없이 Let-7i-5p의 발현 억제 효과가 유의하게 증가하는 것으로 나타났다(도 5a). 이러한 결과는 GalNAc은 간 세포 표면의 ASGPR 수용체와 결합하는 성질을 갖는 딜리버리 시스템이어서 ASGPR postive cell에만 효과가 있으나, Gal-LNP는 세포막과 유사한 구조를 가지기 때문에 ASGPR과 관계없이 효과를 나타낸 것으로 예상된다.As a result, in the case of GalNAc-conjugated RT-LET7-8, the inhibitory effect on expression of Let-7i-5p was significantly increased in Hep3B cells, ASGPR positive cells, compared to the existing RT-LET7-8, but in SNU-5p cells, ASGPR negative cells. In 449 cells, the inhibitory effect of Let-7i-5p expression did not appear to increase (Figure 4a). On the other hand, in the case of RT-LET7-8 combined with Gal-LNP, the effect of suppressing the expression of Let-7i-5p was significantly increased regardless of the presence of ASGPR (Figure 5a). These results show that GalNAc is a delivery system that binds to the ASGPR receptor on the surface of liver cells, so it is effective only on ASGPR postive cells, but Gal-LNP is expected to have an effect regardless of ASGPR because it has a structure similar to the cell membrane.
또한, Gal-LNP가 결합된 RT-LET7-8(Gal-LNP-RT-LET7-8)은 lipofectamine가 결합된 RT-LET7-8(Lipo-RT-LET7-8) 대비 Let-7i-5p의 발현이 현저히 감소되는 것으로 나타났다. 이는 lipofectamine의 경우 단순한 인지질로 구성되어 있어, 콜레스테롤, DSPC, C16-PET2000-ceramide, α-galactosyl ceramide로 구성된 Gal-LNP와 비교하였을 때, 간으로의 전달효율이나, 생체 내에서의 안정성이 현저하게 떨어진다는 것을 의미한다.In addition, RT-LET7-8 (Gal-LNP-RT-LET7-8) bound to Gal-LNP has higher levels of Let-7i-5p than RT-LET7-8 (Lipo-RT-LET7-8) bound to lipofectamine. Expression was found to be significantly reduced. In the case of lipofectamine, it is composed of simple phospholipids, so when compared to Gal-LNP composed of cholesterol, DSPC, C16-PET2000-ceramide, and α-galactosyl ceramide, the delivery efficiency to the liver and stability in vivo are significantly lower. It means falling.
이어서, Hep3B 세포에서 간세포 표적 모이어티(GalNAc 또는 Gal-LNP)가 결합된 RT-LET7-8의 7일 간의 효율 유지 검증을 수행하였다. 그 결과, 7일까지 Let-7i-5p의 발현 억제 효과가 유지되었으며, Lipo-RT-LET7-8과 비교해서도 Let-7i-5p의 발현을 안정적으로 억제하는 것으로 나타났다(도 4b 및 도 5b).Subsequently, the efficiency maintenance of RT-LET7-8 conjugated with hepatocyte targeting moiety (GalNAc or Gal-LNP) for 7 days was verified in Hep3B cells. As a result, the effect of suppressing the expression of Let-7i-5p was maintained until 7 days, and it was shown to stably suppress the expression of Let-7i-5p compared to Lipo-RT-LET7-8 (Figures 4b and 5b ).
5-2. 딜리버리 시스템을 이용한 RT-LET7-8의 간암에서 종양형성 억제 확인5-2. Confirmation of inhibition of tumor formation in liver cancer by RT-LET7-8 using delivery system
간암에서의 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8 처리 시 인비트로 종양형성(in vitro tumorigenesis) 억제능을 확인하기 위해, 실시예 3의 방법으로 MTT 분석을 수행하였다.To confirm the ability to inhibit in vitro tumorigenesis when treated with RT-LET7-8 combined with a hepatocyte targeting moiety in liver cancer, MTT analysis was performed using the method of Example 3.
그 결과, 간세포 표적 모이어티(Moiety)가 결합된 GalNAc-RT-LET7-8 및 Gal-LNP-RT-LET7-8의 경우 대조군 대비 간암세포의 종양형성을 효과적으로 억제하였으며, Lipo-RT-LET7-8와 비교해서도 72시간 경과하였을 때, 높은 종양형성 억제능을 보이는 것으로 나타났다(도 6a 및 도 7a).As a result, GalNAc-RT-LET7-8 and Gal-LNP-RT-LET7-8 combined with hepatocyte targeting moiety effectively inhibited tumorigenesis of liver cancer cells compared to the control group, and Lipo-RT-LET7- Compared to 8, it was found to have a high tumorigenesis inhibition ability after 72 hours (FIGS. 6a and 7a).
5-3. 딜리버리 시스템을 이용한 RT-LET7-8의 대식세포 식작용 조절 확인5-3. Confirmation of macrophage phagocytosis regulation of RT-LET7-8 using delivery system
RT-LET7의 표적 microRNA인 let-7i-5p의 발현 저하에 의해 let-7i-5p의 표적 단백질인 TSP1의 발현 변화를 확인하기 위해, 변형된 RT-LET7-8과 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 발현 변화를 확인하였다.To confirm changes in the expression of TSP1, the target protein of let-7i-5p, by lowering the expression of let-7i-5p, the target microRNA of RT-LET7, modified RT-LET7-8 and hepatocyte targeting moiety were used. After treatment with conjugated RT-LET7-8, expression changes were confirmed through Western blot analysis.
그 결과, 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8의 경우 간세포 표적 모이어티(Moiety)가 결합되지 않은 변형된 RT-LET7-8 대비 TSP1의 발현이 증가되는 것을 확인하였다(도 6b 및 도 7b).As a result, it was confirmed that in the case of RT-LET7-8 to which the hepatocyte targeting moiety was bound, the expression of TSP1 was increased compared to the modified RT-LET7-8 to which the hepatocyte targeting moiety was not bound (Figure 6b and Figure 7b).
이어서, 대식세포가 HCC를 식작용할 수 있게 하는지를 확인하기 위하여, 실시예 4의 방법으로 인비트로 식작용 분석을 수행하였다.Next, in order to confirm whether macrophages can phagocytose HCC, an in vitro phagocytosis assay was performed using the method of Example 4.
그 결과, 변형된 RT-LET7-8 대비 GalNAc-RT-LET7-8 및 Gal-LNP-RT-LET7-8에서 대식세포 식작용 활성이 더 증가함을 확인하였다(도 6c 및 도 7c).As a result, it was confirmed that macrophage phagocytosis activity was further increased in GalNAc-RT-LET7-8 and Gal-LNP-RT-LET7-8 compared to modified RT-LET7-8 (Figures 6c and 7c).
<실시예 6> D-GalNAc이 결합된 RT-LET7-8의 효율 검증<Example 6> Verification of efficiency of RT-LET7-8 combined with D-GalNAc
실시예 5를 통해 GalNAc의 경우 변형된 RT-LET7-8과 결합 시 Gal-LNP에 비해 간으로의 전달효율 및 생체 내에서의 안정성이 높다는 것을 확인하였다.Through Example 5, it was confirmed that GalNAc, when combined with modified RT-LET7-8, had higher liver delivery efficiency and in vivo stability compared to Gal-LNP.
따라서, 변형된 RT-LET7-8과 결합하여 RT-LET7-8의 효능을 현저히 향상시킬 수 있는 최적의 GalNAc을 탐색하기 위하여, 구조가 서로 상이한 GalNAc(D-GalNAc)을 5‘ 말단에 결합시킨 RT-LET7-8을 제작하고, 하기 실험을 통해 GalNAc이 결합된 변형된 RT-LET7-8의 효율을 비교하였다.Therefore, in order to search for the optimal GalNAc that can significantly improve the efficacy of RT-LET7-8 by combining with modified RT-LET7-8, GalNAc (D-GalNAc) with different structures was bound to the 5' end. RT-LET7-8 was produced, and the efficiency of the modified RT-LET7-8 coupled with GalNAc was compared through the following experiment.
6-1. D-GalNAc-RT-LET7-8의 Let-7i-5p의 발현 억제 확인6-1. Confirmation of inhibition of Let-7i-5p expression by D-GalNAc-RT-LET7-8
실시예 5에서 사용된 GalNAc(Old-GalNAc)과 새로운 GalNAc인 D-GalNAc이 결합된 RT-LET7-8을 제조하였으며, 상기 2종의 Old-GalNAc-RT-LET7-8(도 8b 참조) 및 D-GalNAc-RT-LET7-8(도 8c 참조)을 Hep3B 세포에 각각 전달하여 실시예 2와 동일한 방법으로 각 세포에서 Let-7i-5p 발현량을 확인하였다.RT-LET7-8 was prepared by combining GalNAc (Old-GalNAc) used in Example 5 and D-GalNAc, a new GalNAc, and the two types of Old-GalNAc-RT-LET7-8 (see Figure 8b) and D-GalNAc-RT-LET7-8 (see Figure 8c) was delivered to each Hep3B cell, and the expression level of Let-7i-5p in each cell was confirmed in the same manner as in Example 2.
그 결과, D-GalNac이 결합된 RT-LET7-8의 경우, ASGPR positive cell인 Hep3B 세포에서는 기존 Old-GalNAc-RT-LET7-8 대비 Let-7i-5p의 발현 억제 효과가 유의하게 증가하는 것을 확인하였다(도 9a).As a result, in the case of RT-LET7-8 combined with D-GalNac, the effect of suppressing the expression of Let-7i-5p was significantly increased in Hep3B cells, which are ASGPR positive cells, compared to the existing Old-GalNAc-RT-LET7-8. Confirmed (Figure 9a).
이어서, Hep3B 세포에서 Old-GalNAc-RT-LET7-8 및 D-GalNAc-RT-LET7-8의 14일 간의 효율 유지 검증을 수행하였으며, 효율 유지 검증 테스트는 온도 조건(4°C/실온)을 달리하여 수행하였다.Subsequently, efficiency maintenance verification of Old-GalNAc-RT-LET7-8 and D-GalNAc-RT-LET7-8 for 14 days was performed in Hep3B cells, and the efficiency maintenance verification test was performed under temperature conditions (4°C/room temperature). It was performed differently.
그 결과, Old-GalNAc-RT-LET7-8의 경우 7일까지 Let-7i-5p의 발현 억제 효과가 유지되었으나, 7일이 경과하는 경우 Let-7i-5p의 발현이 대조군과 비슷한 수준으로 회복되었다. 그러나, GalNAc-RT-LET7-8의 경우 14일 까지 Let-7i-5p의 발현을 안정적으로 억제하는 것으로 나타났다(도 9b, c).As a result, in the case of Old-GalNAc-RT-LET7-8, the effect of suppressing the expression of Let-7i-5p was maintained until 7 days, but after 7 days, the expression of Let-7i-5p recovered to a level similar to that of the control group. It has been done. However, in the case of GalNAc-RT-LET7-8, it was shown to stably suppress the expression of Let-7i-5p for up to 14 days (Figure 9b, c).
6-2. GalNAc-RT-LET7-8의 간암에서 종양형성 억제 확인6-2. Confirmation of inhibition of tumorigenesis in liver cancer by GalNAc-RT-LET7-8
간암에서의 GalNAc(Old-GalNAc)과 새로운 GalNAc인 D-GalNAc이 결합된 RT-LET7-8 처리 시 인비트로 종양형성(in vitro tumorigenesis) 억제능을 확인하기 위해, Old-GalNAc-RT-LET7-8 및 D-GalNAc-RT-LET7-8을 Hep3B 세포에 각각 전달한 후, MTT assay 및 BrdU assay를 수행하였다.To confirm the ability of Old-GalNAc-RT-LET7-8 to inhibit in vitro tumorigenesis when treated with RT-LET7-8, which is a combination of GalNAc (Old-GalNAc) and D-GalNAc, a new GalNAc, in liver cancer. and D-GalNAc-RT-LET7-8 were respectively delivered to Hep3B cells, and then MTT assay and BrdU assay were performed.
MTT assay는 실시예 3에 기재된 방법으로 수행하였으며, BrdU assay는 제조업체의 프로토콜에 따라 BrdU cell proliferation assay kit(Millipore)를 사용하여 수행하였다.The MTT assay was performed by the method described in Example 3, and the BrdU assay was performed using a BrdU cell proliferation assay kit (Millipore) according to the manufacturer's protocol.
MTT assay 결과, D-GalNAc-RT-LET7-8의 경우 Old-GalNAc-RT-LET7-8 대비 간암세포의 종양형성을 효과적으로 억제하는 것으로 나타났다(도 10a). 이어서, BrdU assay 결과도 MTT assay 결과와 마찬가지로 Old-GalNAc-RT-LET7-8 대비 D-GalNAc-RT-LET7-8이 처리된 Hep3B 세포의 Proliferation 저해능이 현저히 높아지는 것을 확인하였다(도 10b).As a result of the MTT assay, D-GalNAc-RT-LET7-8 was found to effectively inhibit tumor formation in liver cancer cells compared to Old-GalNAc-RT-LET7-8 (FIG. 10a). Subsequently, the BrdU assay results, similar to the MTT assay results, confirmed that the proliferation inhibition ability of Hep3B cells treated with D-GalNAc-RT-LET7-8 was significantly increased compared to Old-GalNAc-RT-LET7-8 (FIG. 10b).
6-3. GalNAc-RT-LET7-8의 대식세포 식작용 조절 확인6-3. Confirmation of regulation of macrophage phagocytosis by GalNAc-RT-LET7-8
RT-LET7의 표적 microRNA인 let-7i-5p의 발현 저하에 의해 let-7i-5p의 표적 단백질인 TSP1의 발현 변화를 확인하기 위해, Hep3B 세포에 Old-GalNAc-RT-LET7-8 및 D-GalNAc-RT-LET7-8을 각각 처리 후 웨스턴 블롯 분석을 통해 발현 변화를 확인하였다.To confirm changes in the expression of TSP1, the target protein of let-7i-5p, by decreasing the expression of let-7i-5p, the target microRNA of RT-LET7, Hep3B cells were incubated with Old-GalNAc-RT-LET7-8 and D- After treatment with GalNAc-RT-LET7-8, expression changes were confirmed through Western blot analysis.
그 결과, D-GalNAc-RT-LET7-8의 경우 Old-GalNAc-RT-LET7-8 대비 TSP1의 발현이 증가되는 것을 확인하였다(도 11a).As a result, it was confirmed that the expression of TSP1 was increased in the case of D-GalNAc-RT-LET7-8 compared to Old-GalNAc-RT-LET7-8 (FIG. 11a).
이어서, 대식세포가 HCC를 식작용할 수 있게 하는지를 확인하기 위하여, 실시예 4의 방법으로 인비트로 식작용 분석을 수행하였다.Next, in order to confirm whether macrophages can phagocytose HCC, an in vitro phagocytosis assay was performed using the method of Example 4.
그 결과, D-GalNAc-RT-LET7-8의 경우 Old-GalNAc-RT-LET7-8 대비 대식세포 식작용 활성이 더 증가함을 확인하였다(도 11b).As a result, it was confirmed that the macrophage phagocytosis activity of D-GalNAc-RT-LET7-8 was further increased compared to Old-GalNAc-RT-LET7-8 (FIG. 11b).
이러한 결과를 통해, 변형된 RT-LET7-8에 간세포 표적 딜리버리 시스템을 탑재하는 경우 타겟 기관인 간으로 전달성으로 높여 간세포에서 Let-7i-5p 발현 억제 효능, 간암에서 종양 형성 억제 효능 및 대식세포 식작용 작용 활성 효능이 현저히 증가시키는 것을 확인하였다. 특히, 간세포 표적 딜리버리 시스템 중 화학식 2로 표시되는 D-GalNAc이 결합된 변형된 RT-LET7-8의 경우 RT-LET7-8의 상기 언급된 간암 예방 또는 치료효과를 현저히 향상시키는 것을 알 수 있다.Through these results, when the modified RT-LET7-8 is equipped with a hepatocyte-targeting delivery system, it increases delivery to the target organ, the liver, thereby inhibiting Let-7i-5p expression in hepatocytes, inhibiting tumor formation in liver cancer, and phagocytosis of macrophages. It was confirmed that the functional activity efficacy was significantly increased. In particular, among the hepatocyte targeting delivery systems, the modified RT-LET7-8 combined with D-GalNAc represented by Formula 2 was found to significantly improve the above-mentioned liver cancer prevention or treatment effect of RT-LET7-8.
이상에서 살펴본 바와 같이, 본 발명의 구체적인 실시예를 상세하게 설명되었으나, 본 발명의 사상을 이해하는 당업자는 동일한 사상의 범위 내에서 다른 구성요소를 추가, 변경, 삭제 등을 통하여, 퇴보적인 다른 발명이나 본 발명 사상의 범위 내에 포함되는 다른 실시예를 용이하게 제안할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상술한 상세한 설명보다는 후술하는 특허청구의 범위에 의하여 나타내어지며, 특허청구의 범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.As discussed above, specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, thereby creating other regressive inventions. However, other embodiments that are included within the scope of the present invention can be easily proposed. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as
Claims (8)
상기 핵산분자는 서열번호 1의 염기서열로 표시되고,
상기 핵산분자는 뉴클레오티드 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형된 핵산인 것을 특징으로 하며,
상기 핵산분자는 일부 또는 전체 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것을 특징으로 하고,
상기 핵산분자는 간세포 표적 모이어티(Moiety)가 결합된 것을 특징으로 하며,
상기 간세포 표적 모이어티(Moiety)는 N-Acetylgalactosamine(GalNAc) 또는 Gal-LNP(Galactosyl lipidoid nanoparticle)인 것이고, 이때 N-Acetylgalactosamine(GalNAc)는 화학식 1 또는 화학식 2로 표시되는 것을 특징으로 하는, 핵산분자:
[화학식 1]
[화학식 2]
.As a nucleic acid molecule targeting Let-7i-5p,
The nucleic acid molecule is represented by the base sequence of SEQ ID NO: 1,
The nucleic acid molecule is characterized in that it is a nucleic acid whose nucleotide sequence has been modified by 2'-O-Methoxyethyl (methoxyethyl),
The nucleic acid molecule is characterized in that part or the entire backbone is modified with phosphorothioate,
The nucleic acid molecule is characterized by binding a hepatocyte targeting moiety,
The hepatocyte targeting moiety is N-Acetylgalactosamine (GalNAc) or Gal-LNP (Galactosyl lipidoid nanoparticle), where N-Acetylgalactosamine (GalNAc) is a nucleic acid molecule characterized in that it is represented by Formula 1 or Formula 2. :
[Formula 1]
[Formula 2]
.
상기 핵산분자는 서열번호 1의 뉴클레오티드 중 5‘말단으로부터 1 내지 4번째 뉴클레오티드의 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것인, 핵산분자.According to paragraph 1,
The nucleic acid molecule is one in which the backbone of the 1st to 4th nucleotides from the 5' end of the nucleotide of SEQ ID NO: 1 is modified with phosphorothioate.
상기 핵산분자는 서열번호 1의 뉴클레오티드 중 3‘말단으로부터 1 내지 4번째 뉴클레오티드의 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것인, 핵산분자.According to paragraph 1,
The nucleic acid molecule is one in which the backbone of the 1st to 4th nucleotides from the 3' end of the nucleotides of SEQ ID NO: 1 is modified with phosphorothioate.
상기 조성물은 Let-7i-5p의 발현을 억제하는 것인, 간암의 예방 또는 치료용 약학 조성물.According to clause 4,
The composition is a pharmaceutical composition for preventing or treating liver cancer, which inhibits the expression of Let-7i-5p.
상기 조성물은 TSP1(thrombospondin-1) 발현을 증가시키는 것인, 간암의 예방 또는 치료용 약학 조성물.According to clause 4,
The composition is a pharmaceutical composition for preventing or treating liver cancer, which increases TSP1 (thrombospondin-1) expression.
상기 조성물은 대식세포 식작용 활성을 증가시키는 것인, 간암의 예방 또는 치료용 약학 조성물.According to clause 4,
The composition is a pharmaceutical composition for preventing or treating liver cancer, which increases macrophage phagocytosis activity.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220093739A KR20240017196A (en) | 2022-07-28 | 2022-07-28 | Delivery system for preventing or treating of cancer comprising modified rt-let7 as an active ingredient |
| EP22853368.3A EP4372087A1 (en) | 2021-08-06 | 2022-07-29 | Composition for prevention or treatment of liver cancer, comprising modified rt-let7 as active ingredient |
| PCT/KR2022/011190 WO2023013990A1 (en) | 2021-08-06 | 2022-07-29 | Composition for prevention or treatment of liver cancer, comprising modified rt-let7 as active ingredient |
| AU2022324176A AU2022324176A1 (en) | 2021-08-06 | 2022-07-29 | Composition for prevention or treatment of liver cancer, comprising modified rt-let7 as active ingredient |
| CN202280054494.4A CN117795077A (en) | 2021-08-06 | 2022-07-29 | Composition for preventing or treating liver cancer comprising modified RT-LET7 as active ingredient |
| CA3228077A CA3228077A1 (en) | 2021-08-06 | 2022-07-29 | Composition for prevention or treatment of liver cancer, comprising modified rt-let7 as active ingredient |
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| KR1020220093739A KR20240017196A (en) | 2022-07-28 | 2022-07-28 | Delivery system for preventing or treating of cancer comprising modified rt-let7 as an active ingredient |
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| KR20120014893A (en) | 2009-04-08 | 2012-02-20 | 바스프 코포레이션 | Zoned catalysts for diesel applications |
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| KR20120014893A (en) | 2009-04-08 | 2012-02-20 | 바스프 코포레이션 | Zoned catalysts for diesel applications |
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