KR-20260065891-A - lipid nanoparticles having nucleic acid cargo and ionizable lipids

Abstract

Preferably, lipid nanoparticles (LNPs) are disclosed that encapsulate a nucleic acid cargo comprising messenger ribonucleic acid (mRNA). The LNP comprises at least an ionizable lipid fraction and a stabilizer fraction. The stabilizer fraction preferably comprises at least one polyethylene glycol (PEG) lipid. Additionally, the ionizable lipid fraction comprises at least one ionizable glycerol dialkyl glycerol tetraether (GDGT) lipid. Additionally, pharmaceutical compositions comprising LNPs, for example, mRNA vaccines, are disclosed. In a further aspect, the present invention relates to ionizable GDGT lipids and a method for preparing the same.

Inventors

• 하이더, 막스
• 쿠에헨베르게르, 줄리안
• 우름, 데이비드

Assignees

• 노보아크 게엠베하

Dates

Publication Date

20260511

Application Date

20240830

Priority Date

20230901

Claims (10)

1. As a lipid nanoparticle (LNP) encapsulating nucleic acid cargo, The above LNP is at least, - ionizable lipid fraction and - Stabilizer fraction; Includes, The above ionizable lipid fraction comprises at least one ionizable glycerol dialkyl glycerol tetraether (GDGT) lipid; The above at least one ionizable GDGT lipid comprises at least one ionizable head group S; preferably, the S is as follows: and In the above chemical formula, In each case, R a is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine, and In each case, R b is independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and In each case, R c is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide. LNP selected from a group composed of
2. In Article 1, The above nucleic acid cargo is an LNP containing messenger ribonucleic acid (mRNA).
3. In Article 1 or Article 2, The above-mentioned stabilizer fraction is an LNP comprising at least one polyethylene glycol (PEG) lipid.
4. In any one of paragraphs 1 to 3, The above LNP is an LNP that further comprises a sterol lipid fraction, preferably containing cholesterol.
5. As a GDGT lipid suitable for use as an ionizable lipid in LNP, The above GDGT lipid comprises at least one ionizable head group S, wherein S is as follows: and In the above chemical formula, In each case, R a is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine, and In each case, R b is independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and In each case, R c is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide. GDGT lipids selected from the group consisting of
6. In Article 5, The above S is as follows: and In the above chemical formula, In each case, R a is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine, and in each case, R b is independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol. GDGT lipids selected from the group consisting of
7. In Article 5 or 6, The above head device S is: and In the above chemical formula, In each case, R is independently selected from H and alkyl; preferably, the alkyl is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl, heptyl, and iso-heptyl. GDGT lipids selected from.
8. As an ether lipid fraction, Comprising the GDGT lipid of any one of claims 5 to 7, An ether lipid fraction that can be obtained by substitution with an ionizable head group S after extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidocaldarius culture.
9. As a pharmaceutical composition, A pharmaceutical composition comprising an LNP of any one of claims 1 to 4 and preferably at least one excipient.
10. A method for producing GDGT lipids according to any one of claims 5 to 7, Steps below: - A step of obtaining a lipid fraction containing one or more precursor GDGT lipids from an archaeol culture, preferably a sulfolobus culture, more preferably a sulfolobus acidocaldarius culture; - A step of purifying one or more of the above-mentioned precursor GDGT lipids; and - A step of preparing a GDGT lipid having at least one ionizable head group S by contacting the above-mentioned one or more precursor GDGT lipids with one or more reagents; A method including

Description

lipid nanoparticles having nucleic acid cargo and ionizable lipids The field of the present invention relates particularly to lipid nanoparticles (LNPs) having a nucleic acid cargo used in messenger ribonucleic acid (mRNA) vaccines, as well as ionizable lipids suitable for use in LNPs. LNPs have recently been the focus of attention because LNP-based mRNA vaccines against SARS-CoV-2 (primarily elasomeran, marketed as Spikevax® by Moderna Inc., and tozinameran, marketed as Comirnaty® by Biontech SE/Pfizer Inc.) have been administered to millions of individuals. LNPs as delivery systems for RNA-based vaccines are generally reviewed in the literature [Aldosari et al., 2021]. LNPs for mRNA delivery are also discussed in detail in the literature [Hou et al., 2021]. LNPs are useful for the delivery of not only vaccines but also other therapeutic nucleic acids. For example, fetishiran is an LNP-based drug for RNA interference therapy in transthyretin-mediated amyloidosis (see reference [Zhang et al., 2019]). Generally, LNPs having nucleic acid cargo (or payload) comprise a lipid layer as well as microdomains of lipids and encapsulated nucleic acids. They have a central diameter of 10 nm to 1000 nm (e.g., determined by dynamic light scattering, DLS) and may adopt, for example, spherical or polyhedral shapes. They may be multilayer depending on their specific lipid composition. LNPs comprise ionizable lipids, particularly lipids that protonate at endosome pH (e.g., pH 4.5 to 6.5, preferably pH 4.5 to 6.0, particularly in the range of pH 4.5 to 5.5), i.e., lipids when in an endosome. Typically, LNPs further comprise stabilizers, such as polyethylene glycol (PEG) lipids, which reduce LNP aggregation, enzymatic degradation, opsonization, and immunogenicity. Additionally, LNPs typically contain other types of lipids (often referred to as "helper lipids"), such as phosphatidylcholine or phosphatidylethanolamine, to improve properties such as delivery efficacy, tolerability, or biodistribution. Finally, LNPs may contain cholesterol or other sterols to regulate membrane integrity and rigidity. LNP is disclosed, for example, in US patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127, as well as US 2013/0245107 A1. WO 2017/099823 A1 discloses an promoted-blood-removal-insensitive LNP comprising a cationic lipid, a polyethylene glycol (PEG)-lipid, a sterol, and a helper lipid, wherein the helper lipid does not include phosphatidylcholine. WO 2020/061284 A1 and WO 2019/089818 A1 also relate to LNPs having PEG lipids. The literature [Eygeris et al., 2021] is a review of the chemistry of LNPs for RNA delivery. It introduces PEG lipids in LNP formulations. WO 2014/143806 A1 discloses lipid particles having PEG lipids. WO 2020/219941 A1 discloses additional LNP and formulations containing LNP. WO 2021/123332 A1 relates to a cationic lipid and an LNP containing said cationic lipid that is useful for the delivery of nucleic acids into living cells. Despite recent advancements in the field, there is still a need for improved LNPs, particularly in relation to storage stability and/or transfection efficiency. For example, the LNP-based SARS-CoV-2 vaccine Comirnaty® must generally be stored at -90°C to -60°C (Summary of Product Characteristics, Version 2022.09.13, EMEA/H/C/005735 - II/0143, European Medicines Agency - EMA). Additionally, increased transfection efficiency would allow for lower doses, thereby reducing potential side effects. FIGS. 1 to 10 illustrate particularly desirable ionizable GDGT lipids. Parentheses indicate that the length of the segment within the parentheses may be 0 to 6 carbon atoms (n=0-6). In each case, " R1 " or " R2 " may be independently selected from the definition given immediately below the structure. Curly bonds indicate that both R and S enantiomers are desirable. Examples Example 1 - Purification of Precursor GDGT Lipids Biomass Extraction: 240 g of dried biomass of * Sulfolobus acidocaldarius * was mixed with 360 g of diatomite (Dionex ASE Prep DE), and the resulting 600 g of solid was shaken to homogeneously disperse it. Eighteen extraction cells (100 mL Dionex ASE 350 stainless steel) equipped with cellulose filters were loaded with approximately equal amounts of dispersed solid (30 to 35 g each) and placed in a Dionex ASE 350 accelerated solvent extractor. The cells were extracted with 3 L of a 2 :1 CHCl₃:MeOH mixture at 80°C and 1150 psi for 30 minutes each. The extracted liquid fractions were combined, and the mixture was concentrated to 200 mL (Heidolph Hei-Vap Core, 50°C, 100 mbar). 50 mL of aqueous 2N HCl was added, and the heterogeneous two-phase mixture was heated to 65°C for 24 hours. After cooling to room temperature, the mixture was neutralized with 50 mL of NaHCO₃ , the phases were separated, the aqueous phase was extracted with Et₂O (3 x 90 mL), the combined organic extract was dried with Na₂SO₄ , filtered, and the solvent was evaporated to obtain 4.4 g (1.8% w/w) of crud