ionizable cationic lipid; RNA delivery

To create dosage nanoparticles for immunization research, ALC-0315 is employed. ALC-0315 is an ionisable aminolipid that is responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. The ionisable lipid in the Moderna COVID-19 vaccine is not disclosed, but it is most likely heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. [1].

---

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

---

ALC-0315 achieves more potent siRNA-mediated knockdown in hepatocytes compared to MC3.[2]
Mice treated with 1 mg/kg siFVII encapsulated in LNPs containing either ALC-0315 or MC3 (siFVII-ALC-0315 or siFVII-MC3, respectively) exhibited significant knockdown of FVII mRNA, compared to control mice treated with siLuc-ALC-0315 or siLuc-MC3 (Figure 1A). Mice treated with siFVII-ALC-0315 at the same dose had greater FVII mRNA knockdown (1.6 ± 0.3 % residual mRNA, P = 0.0004), compared to mice treated with siFVII-MC3 (15.3 ± 3% residual mRNA, P = 0.002) (Fig. 1). Plasma proteins levels between siFVII-ALC-0315 (18 ± 8 %, P = 0.003) and siFVII-MC3 (6 ± 2% plasma protein, P = 0.02) treated mice did not significantly differ (Fig. 1B). There were no differences detected between male and female mice. Encapsulation of siRNA within LNPs was quantified, and there was no substantial difference in RNA loading between siFVII-MC3 (88%), siFVII-ALC-0315 (78%), siLuc-MC3 (90%) and siLuc-ALC-0315 (66%).

---

ALC-0315 achieves siRNA-mediated knockdown in HSCs, while knockdown by MC3 is minimal.[2]
Mice treated with siADAMTS13-ALC-0315 at the same dose had greater FVII mRNA and protein knockdown (31 ± 13% residual mRNA, P = 0.038, and 40 ± 20% plasma protein, P = 0.060), compared to mice treated with siADAMTS13-MC3 (86 ± 18% residual mRNA, P = 0.221, and 75 ± 9.5% plasma protein, P = 0.274) (Fig. 2A–B). Thus, mice treated with siADAMTS13-ALC-0315 resulted in a 69% knock down in ADAMTS13 mRNA expression while mice treated with siADAMTS13-MC3 did not have a statistically significant decrease.31 Between siADAMTS13-ALC-0315 and siADAMTS13-MC3, the difference in Adamts13 mRNA knock down was statistically significant (P = 0.0243).

The enzymatic activity of ADAMTS13 in plasma was determined by measuring the rate of cleavage of a fluorogenic substrate (Figure 2C). Samples from mice treated with siLuc-MC3 and siLuc-ALC-0315 exhibited high ADAMTS13 activity (5.2 ± 0.04 and 3.7 ± 0.04 RFU/sec, respectively) that were quenched in the presence of EDTA, an inhibitor of ADAMTS13 activity. Plasma from siADAMTS13-ALC-0315 and siADAMTS13-MC3-treated mice both showed a diminished ADAMTS13 activity (0.42 ± 0.02 RFU/sec and 2.4 ± 0.05 RFU/sec, respectively, both P < 0.05), indicating a significant decrease in activity compared to their respective siLuc-treated groups. Groups were not powered to detect statistical significance of sex-differences, however, the knockdown between males and females appeared to be the same. There was also no substantial difference in RNA loading between siADAMTS13-MC3 (94%), siADAMTS13-ALC-0315 (82%), siLuc-MC3 (94%) and siLuc-ALC-0315 (80%).[2]

To validate that ADAMTS13 was knocked down in HSCs, ADAMTS13 mRNA was measured in HSCs isolated from livers of mice treated with siLuc or siADAMTS13 encapsulated in ALC-0315 LNPs. mRNA encoding ADAMTS13, and housekeeping gene Ppia, were measured via qPCR. Extracted RNA yields were low, corresponding to the small population of cells isolated, but detection (cycle threshold) of Ppia was similar in samples from mice treated with siLuc and siADAMTS13 (32.5 ± 1.19 and 32.1 ± 0.76, respectively); ADAMTS13 mRNA was detected in samples from mice treated with siLuc (44.4 ± 4.6) but was not detected in RNA extracted from HSCs of mice treated with siADAMTS13, up to a maximum of 55 amplification cycles.[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.

---

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).

[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 leaves LNPs and other excipients as possible sources of allergens. The list of disclosed excipients in the Pfizer-BioNTech COVID-19 vaccine includes sucrose, sodium chloride, potassium chloride, disodium phosphate dihydrate, potassium dihydrogen phosphate, and water for injection. In the Moderna COVID-19 vaccine, the excipients are tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, and sucrose.11 Collectively these excipients are not classified as allergens. The LNPs in the Pfizer-BioNTech vaccine comprise four components: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ALC-0315 [(4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate)], and ALC-0159 (2-[(polyethylene glycol)2000]-N,N-ditetradecylacetamide). The first two components have been widely used in regulatory approved liposomal medicines (e.g., Doxil) and are also features in the Moderna COVID-19 vaccine. ALC-0315 is an ionisable aminolipid that is responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. The ionisable lipid in the Moderna COVID-19 vaccine is not disclosed, but it is most likely heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. The LNPs in the Pfizer-BioNTech COVID-19 vaccine contain low levels (<2 mol %) of ALC-0159, which contributes to nanoparticle stabilization by a steric mechanism through its poly(ethylene glycol) (PEG) moiety. In the Moderna COVID-19 vaccine, ALC-0159 is replaced with another PEGylated lipid (1,2-dimyristoyl-rac-glycero-3-methoxyPEG2000). There are speculations on a possible role for ALC-0159 (the PEGylated lipid) in triggering anaphylaxis, based on earlier reported anaphylactic reactions in some recipients of intravenously infused PEGylated nanomedicines.12 For example, with PEGylated nanomedicines such as Doxil, complement activation was initially thought to account for anaphylactoid reactions (the so-called complement activation-related pseudoallergy [CARPA] hypothesis); however, the validity of CARPA has been recently questioned and, instead, a direct role for macrophages and other immune cells have been proposed.13,14 Anaphylatoxins might play minor roles in potentiating anaphylactoid reactions; for instance, intradermal injection of low doses of anaphylatoxins (C3a, C4a, or C5a) in healthy volunteers was shown to induce immediate wheal-and-flare reactions.6 If LNP-based vaccines can trigger immediate local complement activation, then complement activation is expected to proceed in almost all vaccine recipients, but anaphylaxis with the Pfizer-BioNTech and Moderna vaccines is very rare, and complement activation alone cannot account for anaphylaxis episodes. With PEGylated nanomedicines such as pegnivacogin, anaphylactic reactions have been most notable in individuals with high titers of anti-PEG immunoglobulin G (IgG), but, again, not all individuals with high levels of such antibodies experienced allergic reactions.15 Thus, there are either inter-individual differences in susceptibility to antibody-triggered reactions or differences in the properties of anti-PEG antibodies. Nonetheless, the molecular basis of these reactions in humans remains unknown, but, in the murine model, antigen-induced anaphylaxis appears to proceed through the IgG, low-affinity FcγRIII, effector cells, and platelet-activating factor pathway.[1]

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

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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 2.5 mg/mL (3.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)