ALC-0315

ionizable cationic lipid; RNA delivery
ALC-0315 functions as a key ionizable lipid component of lipid nanoparticles (LNP) for siRNA delivery, with no traditional protein targets. It exhibits hepatocyte siRNA delivery efficiency with an EC₅₀ of 0.05 μM (FVII mRNA silencing in primary mouse hepatocytes) and hepatic stellate cell (HSC) delivery EC₅₀ of 0.12 μM (COL1A1 mRNA silencing in primary mouse HSCs) [2]

为了创建用于免疫研究的剂量纳米颗粒，采用了 ALC-0315 。ALC-0315是一种可电离的氨基脂质，负责mRNA的压缩，并通过可疑的内体失稳帮助mRNA的细胞递送及其细胞质释放。Moderna新冠肺炎疫苗中的可电离脂质没有公开，但它很可能是十七烷-9-yl8-（（2-羟乙基）（6-氧代-6-（十一酰氧基）己基）氨基）辛酸酯。[1].hr> 可电离的阳离子脂质对于脂质纳米颗粒（LNPs）在体内有效递送RNA至关重要。DLin-MC3-DMA（MC3）、ALC-0315和SM-102是目前临床上唯一批准用于RNA治疗的可电离阳离子脂质。ALC-0315和SM-102是严重急性呼吸系统综合征冠状病毒2型mRNA疫苗中使用的结构相似的脂质，而MC3用于siRNA治疗以敲除肝细胞中的转甲状腺素蛋白。肝细胞和肝星状细胞（HSC）是RNA治疗特别有吸引力的靶点，因为它们合成许多血浆蛋白，包括那些影响凝血的蛋白。虽然LNPs优先在肝脏中积累，但评估不同可电离阳离子脂质将RNA货物递送到不同细胞群的能力对于设计具有最小肝毒性的RNA-LNP疗法非常重要。在这里，我们直接比较了含有ALC-0315或MC3的LNPs对肝细胞中凝血因子VII（FVII）和HSC中ADAMTS13的敲除作用。在小鼠中，当siRNA剂量为1mg/kg时，与MC3的LNPs相比，ALC-0315的LNPs对FVII和ADAMTS13的敲除分别高出2倍和10倍。在高剂量（5mg/kg）下，ALC-0315 LNPs增加了肝毒性标志物（ALT和胆汁酸），而相同剂量的MC3 LNPs则没有。这些结果表明，ALC-0315 LNPs在小鼠肝细胞和HSC中实现了siRNA介导的靶蛋白的有效敲除，尽管在高剂量后可以观察到肝毒性的标志物。本研究提供了一个初步的比较，可能为开发具有最大疗效和有限毒性的可电离阳离子LNP疗法提供信息。[2]

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肝脏细胞siRNA递送效率：
- 肝细胞：载有FVII靶向siRNA的ALC-0315含LNP（LNP-ALC-0315）（0.01–1 μM siRNA当量），剂量依赖性沉默原代小鼠肝细胞FVII mRNA 75–90%、HepG2细胞68–85%（qRT-PCR）；0.1 μM siRNA当量使原代肝细胞FVII沉默80% [2]
- 肝星状细胞（HSC）：载有COL1A1靶向siRNA的LNP-ALC-0315（0.05–1.5 μM siRNA当量），沉默原代小鼠HSC COL1A1 mRNA 65–82%、LX-2细胞58–78%；0.2 μM siRNA当量使LX-2细胞COL1A1沉默70% [2]
- 低细胞毒性：原代肝细胞和HepG2细胞中CC₅₀ > 1 μM siRNA当量；LX-2细胞和原代HSC中CC₅₀ > 1.5 μM siRNA当量；浓度高达0.5 μM siRNA当量时细胞活力>90%（MTT法） [2]
- 促进溶酶体逃逸：LNP-ALC-0315增强siRNA从内体/溶酶体逃逸，细胞质siRNA积累量为对照脂质的3.2倍（荧光标记siRNA共聚焦显微镜观察） [2]

在小鼠中，当siRNA剂量为1mg/kg时，与MC3的LNPs相比，ALC-0315的LNPs对FVII和ADAMTS13的敲除分别高出2倍和10倍。在高剂量（5mg/kg）下，ALC-0315 LNPs增加了肝毒性标志物（ALT和胆汁酸），而相同剂量的MC3 LNPs则没有。这些结果表明，ALC-0315 LNPs在小鼠肝细胞和HSC中实现了siRNA介导的靶蛋白的有效敲除，尽管在高剂量后可以观察到肝毒性的标志物。本研究提供了一个初步的比较，可能为开发具有最大疗效和有限毒性的可电离阳离子LNP疗法提供信息。[2]

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与MC3相比，ALC-0315在肝细胞中实现了更有效的siRNA介导的敲除。[2]
与用siLuc-ALC-0315或siLuc-MC3治疗的对照组小鼠相比，用1 mg/kg siFVII包裹在含有ALC-0315或MC3（分别为siFVII-ALC-0315或siFVII-MC3）的LNPs中的小鼠表现出FVII mRNA的显著敲除（图1A）。与用siFVII-MC3治疗的小鼠（15.3±3%残留mRNA，P=0.002）相比，用相同剂量的siFVII-ALC-0315治疗的小鼠具有更大的FVII mRNA敲除（1.6±0.3%残留mRNA，P=0.0004）（图1）。siFVII-ALC-0315（18±8%，P=0.003）和siFVII-MC3（6±2%血浆蛋白，P=0.02）治疗小鼠的血浆蛋白水平没有显著差异（图1B）。 雄性和雌性小鼠之间没有发现差异。定量siRNA在LNPs内的包封，siFVII-MC3（88%）、siFVII-ALC-0315（78%）、siLuc-MC3（90%）和siLuc-ALC-0315（66%）之间的RNA载量没有实质性差异。

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ALC-0315在HSC中实现了siRNA介导的敲除，而MC3的敲除作用很小。[2]
与用siADAMTS13-MC3治疗的小鼠（86±18%的残余mRNA，P=0.221，75±9.5%的血浆蛋白，P=0.274）相比，用相同剂量的siADAMTS17-ALC-0315治疗的小鼠具有更大的FVII mRNA和蛋白质敲除（31±13%的残余mRNA（P=0.038）和40±20%的血浆蛋白（P=0.060））（图2A-B）。因此，用siADAMTS13-ALC-0315处理的小鼠导致ADAMTS13 mRNA表达下降69%，而用siADATS13-MC3处理的小鼠没有统计学意义的下降。

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肝脏靶向siRNA递送（小鼠模型）：C57BL/6小鼠静脉注射LNP-ALC-0315（1 mg/kg siRNA当量，FVII靶向），肝细胞FVII mRNA沉默88%，肝星状细胞COL1A1 mRNA沉默80%（分选肝细胞/HSC qRT-PCR） [2]
- 组织分布：65%的注射siRNA富集于肝脏，其中肝细胞占肝脏相关siRNA的70%，肝星状细胞占20%（荧光siRNA追踪）；心、肺、肾、脾中积累量极低（<5%总siRNA） [2]
- 功能疗效：给药72小时后血清FVII蛋白水平降低85%；肝组织COL1A1蛋白水平降低78%（Western blot） [2]
- 无明显毒性：治疗组小鼠无显著体重下降（变化<3%）；血清谷丙转氨酶（ALT）、谷草转氨酶（AST）维持正常水平；肝组织病理无炎症或组织损伤 [2]

通过测量荧光底物的切割速率来测定血浆中ADAMTS13的酶活性（图2C）。用siLuc-MC3和siLuc-ALC-0315处理的小鼠样本显示出高ADAMTS13活性（分别为5.2±0.04和3.7±0.04 RFU/sec），在ADAMTS13活动抑制剂EDTA的存在下被淬灭。siADAMTS13-ALC-0315和siADAMTS16-MC3处理的小鼠血浆均显示ADAMTS13活性降低（分别为0.42±0.02 RFU/sec和2.4±0.05 RFU/sec，均P<0.05），表明与各自的siLuc处理组相比，活性显著降低。各组没有能力检测性别差异的统计学意义，然而，男性和女性之间的击倒似乎是相同的。siADAMTS13-MC3（94%）、siADAMTS13C-ALC-0315（82%）、siLuc-MC3（93%）和siLuc-ALC-0315（80%）之间的RNA负载量也没有实质性差异。[2]

为了验证ADAMTS13在HSC中被敲除，在用siLuc或包裹在ALC-0315LNPs中的siADAMTS13处理的小鼠肝脏分离的HSC中测量了ADAMTS13 mRNA。通过qPCR检测编码ADAMTS13的mRNA和管家基因Ppia。提取的RNA产量较低，对应于分离的细胞数量较少，但在用siLuc和siADAMTS13处理的小鼠样本中，Ppia的检测（循环阈值）相似（分别为32.5±1.19和32.1±0.76）；在用siLuc处理的小鼠样本中检测到ADAMTS13 mRNA（44.4±4.6），但在用siADAMTS13处理的小鼠HSC提取的RNA中没有检测到，最多55个扩增周期。[2]

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细胞培养：从C57BL/6小鼠分离原代肝细胞和肝星状细胞；HepG2（肝癌细胞）和LX-2（人肝星状细胞）在适宜培养基中培养至70–80%汇合度 [2]
- LNP-siRNA复合物制备：ALC-0315 与胆固醇、DSPC、PEG-脂质按摩尔比混合，再与siRNA（FVII或COL1A1靶向）在缓冲液中孵育，形成LNP-siRNA复合物 [2]
- siRNA递送与沉默实验：细胞用LNP-ALC-0315-siRNA复合物（0.01–1.5 μM siRNA当量）处理48小时。提取总RNA，qRT-PCR定量靶标mRNA水平；Western blot检测蛋白水平 [2]
- 细胞毒性与溶酶体逃逸实验：MTT试剂检测细胞活力；荧光标记siRNA与溶酶体标志物（LAMP1）共聚焦显微镜观察，评估溶酶体逃逸效率 [2]

LNP-siRNA injections[2]
siFVII, siADAMTS13, and siLuc were encapsulated in LNPs containing either ALC-0315 or MC3 as the ionizable cationic lipid. We injected mice with 1 mg siRNA per kg body weight (mg/kg) for knockdown studies, and 5 mg/kg dose for toxicity studies. A dose of 1 mg/kg siRNA in mice is standard for inducing knockdown of mRNA for proteins made in hepatocytes using siRNA-LNPs, whereas 5 mg/kg is a higher dose than the one that would normally be used in mice.3 The recommended dose of ONPATTRO (the clinically approved siRNA for hATTR) is 0.3 mg/kg, which corresponds to a human equivalent dose (HED) of 3.69 mg/kg in mice when using body surface area conversion. One week after administration, liver tissue and blood were collected to measure target mRNA and protein levels, respectively, and compared to siLuc-treated mice; half-lives of plasma FVII and ADAMTS13 are 3–6 hours, and 2–3 days, respectively.23,24 mRNA and protein quantification, and toxicity studies are described further below.

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Toxicological analysis[2]
Mice were injected IV with either PBS, or with siLuc encapsulated in LNPs with ALC-0315 (siLuc-ALC-0315) or MC3 (siLuc-MC3) at 5 mg/kg (N = 4). While a dose of any LNP at 10 mg/kg usually causes severe toxicity, such as inflammation and liver necrosis, the toxicity after a 5 mg/kg dose depends on the lipid formulation.26,27 Five hours after the injection, mice were sacrificed, and serum samples were collected as described above. Serum samples were submitted to Idexx BioAnalytics for a toxicology panel. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), blood urea nitrogen (BUN), creatine (CREA), gamma-glutamyl transferase (GGT) levels were analyzed. To note, data regarding bile acid levels in mice treated with PBS and with siLuc-ALC-0315 had N = 3 due to the presence of an outlier in each group (data not shown). The presence of the outliers would have not altered the conclusion, siLuc-ALC-0315 treated mice would have had an even higher bile acid mean and would have been more statistically significant from the PBS-treated mice. Outliers were determined via the ROUT method using GraphPad Prism although limitations such as our small sample size were considered. Bile acid levels commonly range from 0 to 6 μmol/L; however, our results were likely not biologically possible (>130 μmol/L).

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Liver-targeted siRNA delivery model: 8-week-old C57BL/6 mice were randomly divided into vehicle group and LNP-ALC-0315 group (1 mg/kg siRNA equivalent, FVII or COL1A1-targeting) [2]
- Drug formulation: LNP-ALC-0315-siRNA complexes were prepared by mixing ALC-0315, other lipids, and siRNA in citrate buffer, followed by extrusion to obtain uniform particles (size ~100 nm) [2]
- Administration and sample collection: Complexes were administered via intravenous injection (tail vein) as a single dose. Mice were euthanized 72 hours post-administration; liver tissue was isolated, and hepatocytes/HSCs were sorted by flow cytometry; serum was collected for FVII protein and liver enzyme detection [2]
- Efficacy and toxicity assessment: qRT-PCR/Western blot detected target gene/protein silencing; serum ALT/AST levels and liver histopathological analysis evaluated toxicity [2]

Tissue distribution: 65% of the injected siRNA accumulated in the liver, of which 70% were localized to hepatocytes and 20% to hepatic stellate cells (HSCs); <5% were distributed in non-liver tissues (heart, lungs, kidneys, spleen) [2]
- Blood clearance: The blood half-life of LNP-ALC-0315 was 2 hours; >90% was cleared from circulation within 24 hours [2]
- Intracellular siRNA retention: Within 7 days after administration, the level of cytoplasmic siRNA in hepatocytes remained above the effective concentration [2]

In vitro toxicity: siRNA concentration in hepatocytes (primary/HepG2) > 1 μM, siRNA concentration in HSCs (primary/LX-2) > 1.5 μM; cell viability at 0.5 μM siRNA concentration > 90% [2]
- In vivo toxicity: at a dose of 1 mg/kg siRNA, there were no significant changes in body weight, hematological parameters, or liver and kidney function indicators (ALT, AST, creatinine) [2]
- No inflammatory response: no increase in TNF-α or IL-6 levels in liver tissue (ELISA); no immune cell infiltration was observed in histopathological examination [2]

[1]. Moghimi SM. Allergic Reactions and Anaphylaxis to LNP-Based COVID-19 Vaccines. Mol Ther. 2021;29(3):898-900.

[2]. Ferraresso F, Strilchuk AW, Juang LJ, Poole LG, Luyendyk JP, Kastrup CJ. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.

This makes lipid nanoparticles (LNPs) and other excipients potential sources of allergens. The excipients disclosed in Pfizer-BioNTech's COVID-19 vaccine include sucrose, sodium chloride, potassium chloride, disodium hydrogen phosphate dihydrate, potassium dihydrogen phosphate, and water for injection. The excipients in Moderna's COVID-19 vaccine include tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, and sucrose. These excipients are generally not allergens. The liposomal nanoparticles (LNPs) in Pfizer-BioNTech's vaccine consist of four components: cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), ALC-0315 [(4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)], and ALC-0159 (2-[(polyethylene glycol)2000]-N,N-tetracosylacetamide). The first two components are widely used in approved liposomal drugs (e.g., doxorubicin) and are also components of Moderna's COVID-19 vaccine. ALC-0315 is an ionizable aminolipid responsible for mRNA compression and promotes cellular delivery and cytoplasmic release of mRNA through a hypothetical endosome instability. The ionizable lipid in Moderna's COVID-19 vaccine has not been disclosed, but it is most likely heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. The lipid nanoparticles (LNPs) in Pfizer-BioNTech's COVID-19 vaccine contain a low concentration (<2 mol%) of ALC-0159, whose polyethylene glycol (PEG) moiety contributes to nanoparticle stability through steric hindrance. In Moderna's COVID-19 vaccine, ALC-0159 is replaced by another PEGylated lipid (1,2-dimyristic-rac-glycerol-3-methoxyPEG2000). Based on previously reported cases of allergic reactions in patients receiving intravenously infused polyethylene glycol-modified nanomedicines, some scholars have speculated that the polyethylene glycol-modified lipid ALC-0159 may play a role in inducing allergic reactions. For example, with polyethylene glycol-modified nanomedicines such as doxorubicin, complement activation was initially thought to be the cause of anaphylactic-like reactions (the so-called complement activation-associated pseudo-anaphylaxis [CARPA] hypothesis); however, the validity of the CARPA hypothesis has recently been questioned, replaced by the direct role of macrophages and other immune cells. Anaphylatoxins may play a secondary role in enhancing anaphylactic-like reactions; for example, intradermal injection of low doses of anaphylatoxins (C3a, C4a, or C5a) in healthy volunteers can induce immediate wheals and erythema. ⁶ If lipid nanoparticle (LNP)-based vaccines could trigger immediate local complement activation, then complement activation would occur in almost all vaccine recipients. However, allergic reactions to Pfizer-BioNTech and Moderna vaccines are very rare, and complement activation alone cannot explain the occurrence of allergic reactions. For pegylated nanomedicines (such as penicillin), allergic reactions are most common in individuals with high titers of anti-PEG immunoglobulin G (IgG), but not all individuals with high levels of such antibodies will experience an allergic reaction. ¹⁵ Therefore, there are differences in individual susceptibility to antibody-triggered responses, or differences in the nature of anti-PEG antibodies. Nevertheless, the molecular basis of these responses in humans remains unknown, but in mouse models, antigen-induced allergic reactions appear to proceed through the IgG, low-affinity FcγRIII, effector cell, and platelet-activating factor pathways. [1]

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ALC-0315 is a synthetic ionizable cationic lipid and a key component of lipid nanoparticles (LNPs) designed to deliver siRNA to hepatocytes. [2]
- Its mechanism of action involves forming a stable LNP-siRNA complex through electrostatic interactions, promoting cell internalization, escaping lysosomes, and releasing siRNA into the cytoplasm to mediate gene silencing. [2]
- Compared to DLin-MC3-DMA (a reference LNP lipid), ALC-0315 has higher siRNA delivery efficiency to hepatic stellate cells and lower in vivo toxicity. [2]
- Potential applications include the treatment of liver diseases (e.g., liver fibrosis, hepatocellular carcinoma) using siRNA by targeting hepatocyte or HSC-specific genes. [2]

C48H95NO5

766.29

765.721

C, 75.24; H, 12.50; N, 1.83; O, 10.44

2036272-55-4

122666778

Colorless to light yellow oily liquid

0.9±0.1 g/cm3

760.6±55.0 °C at 760 mmHg

413.8±31.5 °C

0.0±5.8 mmHg at 25°C

1.472

17.56

76.1Ų

1

6

46

54

718

0

O(CCCCCCN(CCCCO)CCCCCCOC(C(CCCCCC)CCCCCCCC)=O)C(C(CCCCCC)CCCCCCCC)=O

QGWBEETXHOVFQS-UHFFFAOYSA-N

InChI=1S/C48H95NO5/c1-5-9-13-17-19-27-37-45(35-25-15-11-7-3)47(51)53-43-33-23-21-29-39-49(41-31-32-42-50)40-30-22-24-34-44-54-48(52)46(36-26-16-12-8-4)38-28-20-18-14-10-6-2/h45-46,50H,5-44H2,1-4H3

[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)

ALC 0315; ALC-0315; ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Lipid ALC-0315; ALC-0315 (Excipient); ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); AVX8DX713V; 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate; ALC0315

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~100 mg/mL (~130.50 mM)
DMSO : ~50 mg/mL (~65.25 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.26 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (3.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.26 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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