WO2026013203A1 - Nanoparticules lipidiques améliorées - Google Patents

Nanoparticules lipidiques améliorées

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Publication number
WO2026013203A1
WO2026013203A1 PCT/EP2025/069780 EP2025069780W WO2026013203A1 WO 2026013203 A1 WO2026013203 A1 WO 2026013203A1 EP 2025069780 W EP2025069780 W EP 2025069780W WO 2026013203 A1 WO2026013203 A1 WO 2026013203A1
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Prior art keywords
vitamin
lnp
lipid
composition according
lnps
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English (en)
Inventor
Yuan DING
Igor Bendik
Nicolas Schmitt
Karin Kuratli
Zdravka MISIC
Jonathan Alan Medlock
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DSM IP Assets BV
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DSM IP Assets BV
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Publication of WO2026013203A1 publication Critical patent/WO2026013203A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

  • the present invention relates to lipid nanoparticles (LNPs) comprising vitamin A or a vitamin A derivative.
  • LNPs lipid nanoparticles
  • the LNPs according to the present invention have an improved stability and nucleic acid delivery efficacy.
  • Nucleic acid drugs such as siRNA, mRNA, and DNA offer therapeutic potential for gene therapy.
  • naked nucleic acid drugs are unstable in circulation and prone to degradation.
  • the negative charge of RNA and DNA limits their entry across the cell membrane which is also negatively charged. Therefore, materials have been developed in recent years for nucleic acid delivery, such as lipids (Jia, Y. et al., Lipid nanoparticles optimized for targeting and release of nucleic acid. Advanced Materials, 2024, 36, 2305300).
  • Lipid nanoparticles are currently the most promising clinical delivery vehicles for nucleic acid drugs.
  • Lipid nanoparticles are nanosized spherical vesicles made of cationic or ionizable lipids (Akinc, A., et al., A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nature biotechnology, 2008. 26(5): p. 561 -569) which are positively charged at low pH (enabling RNA complexation) and neutral at physiological pH (reducing potential toxic effects, as compared with positively charged lipids, such as liposomes).
  • Lipid nanoparticles usually comprise a helper lipid; cholesterol; and a polyethylene glycol (PEG) lipid, wherein each component impacts stability, transfection efficiency, and safety of the lipid nanoparticle.
  • PEG polyethylene glycol
  • the relative amounts of ionizable lipid, helper lipid, cholesterol and PEG need to be optimized for a given application and administration route.
  • Lipid type, size, and surface charge also impact the behavior of lipid nanoparticles in vivo (Let’s talk about lipid nanoparticles. Nature Reviews Materials, 2021. 6(2): p.99-99).
  • mRNA Messenger RNA
  • mRNA COVID-19 vaccines at unprecedented speed, demonstrating the clinical potential of lipid nanoparticle-mRNA formulations (Hou, X., et al., Lipid nanoparticles for mRNA delivery. Nature Reviews Materials, 2021. 6(12): p.1078-1094).
  • mRNA-based delivery technologies have further shown therapeutic potential in biomedical applications such as protein replacement therapies, cellular reprogramming, and cancer immunotherapies.
  • nucleic acid molecules such as mRNA
  • the nucleic acid molecules must be protected from degradation and delivered to specific target cells to produce the desired proteins. Stability of the nucleic acid molecules and their efficient delivery to the intracellular environment are crucial for the nucleic acid molecules to exert their intended function.
  • mRNA-carrying LNPs including vaccines
  • nucleic acid delivery systems which are safe, stable, and effective, protecting the nucleic acid from degradation and allowing efficient cellular uptake and nucleic acid release, even after longer storage and/or storage at non-freezing temperatures.
  • the present invention relates to the following items:
  • a lipid nanoparticle (LNP) composition comprising:
  • lipid phase comprising i. at least one ionizable cationic lipid, ii. at least one helper lipid, iii. at least one cholesterol or derivative thereof, iv. at least one PEG-lipid, and v. at least one vitamin A or derivative thereof;
  • the at least one helper lipid is selected from the group consisting of the following lipids: DSPC, DOPE, DOPS, DOPG, DOTAP, DOTMA, POPE, and POPC.
  • R is a C1-C25 hydrocarbon chain which may be linear or branched, and which may contain one or more unsaturations and/or ring systems.
  • a formulation comprising the LNP composition according to any one of the preceding items.
  • a vitamin A or vitamin A derivative to improve the nucleic acid delivery efficacy of a lipid nanoparticle (LNP), wherein the vitamin A or vitamin A derivative is added to the lipid components of the LNP and forms part of the LNP.
  • LNP lipid nanoparticle
  • Figures 1 A and B show the particle size of mRNA-loaded LNPs containing different vitamin A derivatives. Stability of particle size over time is a measure for the stability of the LNPs.
  • Figure 1A shows the particle size of LNPs containing vitamin A palmitate.
  • Figure 1B shows the particle size of LNPs containing vitamin A propionate.
  • Figures 2 A and B show the delivery efficiency of mRNA-loaded LNPs with and without vitamin A-palmitate after storage at 4°C.
  • the LNPs were loaded with nucleoside-modified luciferase encoding mRNA, stored at 4°C for different time intervals, and then transfected into human cells.
  • the luciferase activity after transfection is shown after storage for 4 weeks ( Figure 2A) and 6 weeks ( Figure 2B).
  • Figures 3 A and B show the delivery efficiency of mRNA-loaded LNPs with and without vitamin A-propionate after storage at 4°C.
  • the LNPs were loaded with nucleoside-modified luciferase encoding mRNA, stored at 4°C for different time intervals and then transfected into human cells.
  • the luciferase activity after transfection is shown after storage for 3 weeks ( Figure 3A) and 4 weeks ( Figure 3B).
  • Figures 4 A and B show the delivery efficiency of mRNA-loaded LNPs with and without vitamin A after storage at 4°C.
  • the LNPs were loaded with nucleoside-modified luciferase encoding mRNA, stored at 4°C for different time intervals and then transfected into human cells.
  • the luciferase activity after transfection is shown after storage for 4 weeks ( Figure 4A) and 6 weeks ( Figure 4B).
  • Figure 5 to 8 show the polydispersity indices (PDIs) for mRNA-loaded LNPs with and without vitamin A derivatives after storage at 4°C.
  • Figure 5 and Figure 7 show the PDI of mRNA-loaded LNPs having different lipid compositions but both containing vitamin A palmitate.
  • Figure 6 and Figure 8 show the PDI of mRNA-loaded LNPs having different lipid compositions but both containing vitamin A propionate.
  • Figure 5 shows the PDI of LNPs comprising vitamin A palmitate at time point 0 (TO) (Figure 5A), and after being stored at 4°C for 4 weeks (Figure 5B) and 6 weeks (Figure 5C).
  • Figure 6 shows the PDI of LNPs comprising vitamin A propionate at time 0 ( Figure 6A), and after being stored at 4°C for 3 weeks (Figure 6B).
  • Figure 7 shows the PDI of LNPs comprising vitamin A palmitate at time point 0 ( Figure 7A), and after being stored at 4°C for 4 weeks ( Figure 7B) and 6 weeks (Figure 7C).
  • Figure 8 shows the PDI of LNPs comprising vitamin A propionate at time 0 ( Figure 8A), and after being stored at 4°C for 4 weeks ( Figure 8B) and 6 weeks (Figure 8C)
  • the present invention is based on the finding that the addition of vitamin A or a vitamin A derivative can improve the stability and/nucleic acid delivery efficacy of a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the LNP compositions according to the present invention show improved stability and nucleic acid delivery efficacy after long-term storage for 4 weeks and more, and storage at non-freezing temperatures, such as at 4°C.
  • the present invention relates to a lipid nanoparticle (LNP) composition, comprising: (a) a lipid phase comprising i. at least one ionizable cationic lipid, ii. at least one helper lipid, iii. at least one cholesterol or derivative thereof, iv. at least one PEG-lipid, and v. at least one vitamin A or derivative thereof; and
  • said LNP is combined with a nucleic acid as the load.
  • the resulting LNP composition is a loaded LNP, more precisely, an LNP comprising a nucleic acid as the load.
  • the lipid nanoparticle (LNP) composition of the present invention is consisting of:
  • an LNP/ lipid phase comprising i. at least one ionizable cationic lipid, ii. at least one helper lipid, iii. at least one cholesterol or derivative thereof, iv. at least one PEG-lipid, and v. at least one vitamin A or derivative thereof;
  • nucleic acid delivery efficacy refers to the ability of the LNP to deliver its load, i.e. , the nucleic acid (e.g., an mRNA) to a target cell.
  • the delivery efficacy can be influenced by many factors, including the storage time and storage temperature.
  • the nucleic acid delivery efficacy is improved if the expression of the delivered nucleic acid in the target cell is higher than that of a nucleic acid delivered by a conventional/ comparable LNP that does not comprise the claimed lipid phase components (i)-(v), e.g., does not comprise a vitamin A or vitamin A derivative.
  • the term “load” means the cargo, i.e. , the material transported by the LNP.
  • LNPs are typically nanospheres that have a homogeneous matrix throughout the particles that hold (encapsulate) an active compound/ active ingredient.
  • the active ingredient comprised in the load can be any nucleic acid.
  • the load comprises a ribonucleic acid (RNA); more preferably, it comprises a messenger RNA (mRNA).
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid may be contained in an aqueous phase. Accordingly, the load may comprise further ingredients, such as a buffer.
  • LNP composition refers to a composition comprising, or consisting of, a) a lipid phase comprising different components making up the LNP, and b) a load comprising the nucleic acid as defined herein.
  • the nucleic acid may be provided in a solution to be mixed or added to a lipid nanoparticle or lipid nanoparticle solution, such that the nucleic acid may be encapsulated in the lipid nanoparticle.
  • the LNP/ lipid phase according to the present invention contains lipids from at least four categories of lipids: ionizable cationic lipids, helper lipids, cholesterols, and PEG-lipids. In addition, it comprises vitamin A or a derivative thereof. To obtain the LNP compositions according to the present invention, the said LNPs are further loaded with at least one kind of nucleic acid.
  • the ionizable cationic lipid may be any ionizable cationic lipid.
  • the ionizable cationic lipid is selected from the following:
  • the lipid phase of the LNP composition according to the present invention comprises 35-75 wt.-%, more preferably 40-70 wt.-%, even more preferably 45-65 wt.-%, and most preferably 50-60 wt.-% of the ionizable cationic lipid, based on the total weight of the LNP (i.e. , the total weight of components (i)-(v) of the lipid phase as defined herein), provided that the total wt.-% does not exceed 100%.
  • the helper lipid may be any helper lipid.
  • the helper lipid is selected from any of the following:
  • DSPC (1 ,2-Distearoylglycero-3-phosphorylcholine; also known as Distearoylphosphatidylcholine, 1 ,2-Distearoyl-sn-glycero-3-phosphocholine, or DL- 2,3-Bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate),
  • DOPE (1 ,2-Dioleoyl-glycero-3-phosphoethanolamine; also known as dioleoylphosphatidylethanolamine, or 1 ,2-Dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOPS Dioleoylphosphatidylserine
  • the helper lipid is selected from DSPC, DOPE, DOPS, and DOPG, or a mixture thereof. Most preferably, the helper lipid is DSPC or DOPE, or a mixture thereof.
  • the lipid phase of the LNP composition according to the present invention comprises 1-20 wt.-%, more preferably 3-15 wt.-%, even more preferably 5-12 wt.-%, and most preferably 6-11 wt.-% of the helper lipid, based on the total weight of the LNP, provided that the total wt.-% does not exceed 100%.
  • the cholesterol may be any cholesterol or cholesterol derivative.
  • the cholesterol is a hydroxy-cholesterol, which can be selected, for example, from the following: 25-hydroxycholesterol, 7a- hydroxycholesterol, and 20a- hydroxycholesterol.
  • the cholesterol is Cholest- 5-en-3[3-ol.
  • the lipid phase of the LNP composition according to the present invention comprises 10-40 wt.-%, more preferably 15-35 wt.-%, even more preferably 20-30 wt.-%, and most preferably 22-28 wt.-% of the cholesterol or cholesterol derivative, based on the total weight of the LNP, provided that the total wt.-% does not exceed 100%.
  • the PEG-lipid may be any PEG- lipid.
  • the PEG-lipid is selected from the following:
  • PEG-DMG (1 ,2-Dimyristoyl-glycero-3-methoxypolyethylene glycol 2000).
  • the PEG-lipid is ALC-0159.
  • the lipid phase of the LNP composition according to the present invention comprises 1 -15 wt.-%, more preferably 2-12 wt.-%, even more preferably 4-9 wt.-%, and most preferably 5.5-7.5 wt.-% of PEG-lipid, based on the total weight of the LNP, provided that the total wt.-% does not exceed 100%.
  • the vitamin A may be vitamin A or any derivative thereof.
  • the vitamin A or derivative thereof has the following chemical structure: wherein
  • R is a C1 -C25 hydrocarbon chain which may be linear or branched, and which may contain one or more unsaturation and/or ring systems.
  • unsaturation refers to double and/ or triple bonds which might be at any position of the hydrocarbon chain.
  • unsaturation is a double bond, most preferably, only one double bond is present in the hydrocarbon chain.
  • the hydrocarbon chain is an unsaturated or saturated linear hydrocarbon chain. Even more preferably, the hydrocarbon chain is saturated or a mono-unsaturated linear hydrocarbon chain.
  • the lipid phase of the LNP composition according to the present invention comprises at least 0.5 wt.-% of a vitamin A or a vitamin A derivative, more preferably at least 1 wt.-%, even more preferably at least 1 .5 or 2.0 wt.-%, most preferably at least 2.5 or 3.0 wt.-%, based on the total weight of the LNP.
  • the lipid phase of the LNP composition according to the present invention preferably comprises 0.5-10 wt.-%, more preferably 1 -8 wt.-%, even more preferably 2-6 wt.-%, and most preferably 3-5 wt.-% of the vitamin A or vitamin A derivative. Further suitable ranges for vitamin A or vitamin A derivative are 1 -5 wt.-%, 2-4 wt.-%, and 2.5-3.5 wt.-%, e.g., about 3 wt.-%. All based on the total weight of the LNP, provided that the total wt.-% does not exceed 100%.
  • the LNP compositions according to the present invention further comprise a load comprising a nucleic acid.
  • the LNPs can be used to deliver deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA).
  • the load comprises an RNA.
  • the nucleic acid carried by the LNP is a messenger RNA (mRNA).
  • Alternative RNAs that can be comprised in the LNP compositions according to the invention are, for example: antisense RNA (asRNA), small interfering RNA (siRNA), micro RNA (miRNA), small activating RNA (saRNA), and RNA aptamers.
  • CRISPR-associated protein (Cas) systems may also be included in the LNPs according to the present invention.
  • the nucleic acid may be contained in an aqueous phase. Accordingly, the load may comprise further ingredients, such as, for example, one or more buffers.
  • the LNP compositions according to the present invention may further comprise one or more solvents.
  • the solvent is an organic solvent. More preferably, the solvent is an alcohol. Ethanol is particularly preferred.
  • an aqueous solvent is preferred.
  • LNP compositions comprising, more preferably consisting of:
  • an LNP/ a lipid phase comprising: i. an ionizable cationic lipid, ii. a helper lipid, iii. Cholest-5-en-3[3-ol, iv. a PEG-lipid, and v. a vitamin A derivative; and
  • a particularly preferred LNP according to the present invention is an LNP comprising: (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) ALC -0159, and (v) at least 1 wt.-%, preferably at least 3 wt.-%, or about 3-5 wt.% of retinyl palmitate (vitamin A palmitate).
  • LNP is an LNP comprising: (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) ALC -0159, and (v) at least 1 wt.-%, preferably at least 3 wt.-%, or about 3-5 wt.% of retinyl propionate (vitamin A propionate).
  • a particularly preferred LNP composition according to the present invention is an LNP composition comprising, or consisting of: (a) an LNP/ lipid phase comprising: (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol,
  • LNP composition comprising, or consisting of:
  • an LNP/ lipid phase comprising: (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) ALC -0159, and (v) at least 1 wt.-%, preferably at least 3 wt.-%, or about 3-5 wt.% of retinyl propionate (vitamin A propionate); and
  • a key challenge in the development and manufacturing of mRNA-loaded LNPs is achieving a uniform particle size distribution, since high polydispersity can lead to reduced reproducibility, lower stability, and variable biological performance.
  • the present inventors found that by introducing vitamin A derivatives into the mRNA-loaded LNPs, the polydispersity index (PDI) of the mRNA-LNPs can be significantly reduced in comparison to mRNA-loaded LNPs that do not contain a vitamin A derivative.
  • the improved uniformity enhances product quality and supports scalable, robust manufacturing processes.
  • the present invention relates to a lipid nanoparticle (LNP) composition, in particular an LNP composition as described and embodied above, wherein the LNPs in the LNP composition have a lower polydispersity index (PDI) compared to LNPs that do not comprise a vitamin A or vitamin A derivative in their lipid phase.
  • LNP lipid nanoparticle
  • the polydispersity index of the LNPs comprised in the LNP composition is reduced by at least 10% (10% or more), more preferably by at least 15% (15% or more), even more preferably by at least 20% (20% or more), most preferably by at least 25% (25% or more).
  • the polydispersity index is reduced by 5-10%, more preferably by 10-15%, when compared to the polydispersity index of LNPs which do not comprise at least one vitamin A or derivative thereof in the lipid phase.
  • the LNPs of said LNP compositions are selected from the LNPs listed in Table 1 above.
  • LNPs comprising:
  • ALC-0315 (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) ALC -0159, and (v) retinyl palmitate (vitamin A palmitate); (i) ALC-0315, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) ALC -0159, and (v) retinyl propionate (vitamin A propionate);
  • SM-102 (i) SM-102, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) PEG-DMG, and (v) retinyl palmitate (vitamin A palmitate); or
  • SM-102 (i) SM-102, (ii) DSPC, (iii) Cholest-5-en-3[3-ol, (iv) PEG-DMG, and (v) retinyl propionate (vitamin A propionate).
  • the present invention relates to the use of a vitamin A derivative (preferably a vitamin A ester, more preferably vitamin A propionate or vitamin A palmitate) for improving (i.e., lowering) the polydispersity index of LNPs.
  • a vitamin A derivative preferably a vitamin A ester, more preferably vitamin A propionate or vitamin A palmitate
  • the LNP composition according to the present invention can be in a liquid or a solid form.
  • the LNP composition according to the present invention can be in a buffer solution or can be lyophilized.
  • the LNP composition according to the present invention may comprise further components, such as buffers and excipients.
  • the present invention relates to a formulation comprising the LNP composition according to the present invention.
  • the formulation is a pharmaceutical composition comprising the LNP composition according to the present invention.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration.
  • compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and/or transderrnal administration (e. g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.
  • liquid dosage forms e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs
  • injectable forms e.g., solid dosage forms (e.g., capsules, tablets, pills, powders, and granules)
  • dosage forms for topical and/or transderrnal administration e. g., ointments, paste
  • the LNP compositions according to the present invention or the formulations comprising the LNP compositions according to the present invention may be used in a method of treatment or prevention of a disease or condition.
  • the LNP compositions according to the present invention or the formulations comprising the LNP compositions according to the present invention may be used as a vaccine.
  • the present invention relates to the LNP composition according to the present invention or the formulation comprising the LNP composition according to the present invention for use as a medicament or for use in a method of treatment or prevention of a disease or condition.
  • the LNP composition according to the present invention or the formulation comprising the LNP composition according to the present invention is for use as a vaccine; preferably, carrying mRNA as their load. Uses in RNA-based therapy and genome editing are also envisioned.
  • the LNP compositions according to the present invention can be produced by any method for producing LNPs and LNP compositions known in the art.
  • individual lipid stock solutions can be prepared by dissolving the lipids and vitamins in absolute ethanol.
  • the required volumes of lipid and vitamin stocks can be mixed, based on the Cayman exploration kit protocol to prepare the desired composition.
  • LNPs may then be prepared using the NanoAssemblr® IgniteTM from Precision NanoSystems Inc. (Vancouver, BC, Canada).
  • the LNP compositions according to the present invention, its components and solutions may further be processed, e.g., by purification, pH adjustment, buffer exchange and/or a concentration step.
  • the processing may include filtering for removal of an organic solvent such as alcohol.
  • the LNP composition or formulation according to the present invention may also be sterilized before storage or use, e.g., by filtration through a 0.1 -0,5 pm filter.
  • the LNP compositions or formulations according to the present invention may also be processed by preparing or packing them for storage. This may include any of the following steps: adding a cryoprotectant; lyophilization; adding a buffering solution.
  • the present invention relates to the use of a vitamin A or vitamin A derivative to improve the stability and/or nucleic acid delivery efficacy of an LNP, wherein the vitamin A or vitamin A derivative is added to the LNP, for example to the lipid components/ lipid phase of the LNP.
  • a method for improving the stability and/or nucleic acid delivery efficacy of an LNP comprising the step of adding the vitamin A or vitamin A derivative to the LNP, for example to the lipid components/ lipid phase of the LNP.
  • the vitamin A or vitamin A derivative may be added during the process of producing the (loaded) LNP.
  • a vitamin solution may be prepared by dissolving the vitamins in absolute ethanol (or another solvent); the required volumes of lipid solutions and vitamin solution are then mixed, forming an LNP solution comprising the LNP; a nucleic acid solution comprising the nucleic acid is finally added, thereby forming the an LNP composition or LNP formulation comprising vitamin A-containing LNPs encapsulating the nucleic acid.
  • the ionizable lipid (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315, Lot. No. 0648122-13), PEG-lipid (2[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide) (ALC-0159, Lot. No. 0634881-30), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC, Lot. No. 0646647-25), Cholest-5-en-3[3-ol (Lot. No.
  • Firefly luciferase mRNA (CleanCap FLuc mRNA) was purchased from Tebubio GmbH, Offenbach, Germany.
  • Vitamin A palmitate also known as retinyl palmitate
  • Vitamin A propionate (also known as retinyl propionate) was obtained from Merck/Sigma-Aldrich (Buchs, Switzerland). Vitamin A was obtained from Merck KGaA (Germany).
  • Quant-itTM RiboGreen RNA Assay Kit used for encapsulation efficacy determination, was purchased from ThermoFisher (USA).
  • Ethanol (EtOH), Triton® X-100 and Amicon Ultra-15 filters were purchased from Merck KGaA (Germany, as END Millipore Corporation, USA). Acrodisc Syringe filters with Supor® membrane 0.2 pm were acquired by Pall Laboratory (USA). Sucrose, sodium chloride (NaCI), Phosphate-Buffered Saline (PBS; 1X, pH 7.4) and TRIS (tromethamine) buffer solution (0.1 M, pH 7.4) were supplied by VWR Chemicals (USA). All solvents and chemicals were of analytical grade, and the deionized water was obtained via Millipore filter system (USA).
  • Lipid nanoparticle compositions according to the present invention were prepared, comprising the ingredients shown in Table 2 (LNPs comprising vitamin A palmitate I retinyl palmitate), Table 3 (LNPs comprising vitamin A propionate I retinyl propionate), and Table 4 (LNPs comprising vitamin A).
  • mRNA 1 mg/mL mRNA was thawed in ice bath at room temperature and dissolved in sodium acetate buffer (0.05 M, pH 5.2) to reach an mRNA concentration of 83.3 ⁇ 2 pg/mL, measured by Nanodrop One (Thermo Fisher Scientific, USA).
  • Individual lipid stock solutions were prepared by dissolving the lipids or vitamins in absolute ethanol. The required volumes of lipid and vitamin stocks were mixed, based on the Cayman exploration kit protocol to prepare the desired formulation, then kept at room temperature. 10 mM Tris + 300 mM sucrose buffer pH 7.4 was used as diluent.
  • LNPs were prepared using the NanoAssemblr® IgniteTM from Precision NanoSystems Inc. (Vancouver, BC, Canada).
  • microfluidic chip IgniteTM NxGen dilution cartridge used (Precision NanoSystems, Canada) was of Y-channel design. Microfluidic mixing was undertaken at a standard flow rate ratio (FRR). After production, the loaded LNPs were diluted 25 times for solvent removal; the solution was then purified into Amicon centrifuge filter tubes and concentrated for 30 minutes at 4 °C and 2000g by using the CT 4 22 Centrifuge (Jouan, USA).
  • the resulting LNP compositions contained the ingredients as shown in Tables 2, 3 and 4 below.
  • Table 2 mRNA-loaded LNPs comprising vitamin A palmitate
  • Particle size was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Panalytical Ltd, Worcestershire, UK) equipped with a 633 nm laser and a detection angle of 173°. Three measurements of 10 runs each were performed at room temperature in a 1/10 dilution using PBS buffer pH 7.4. DLS standard disposable 1 mL cuvettes were used, and attenuation value was 8. The particle sizes of the prepared mRNA-loaded LNPs are shown in Figure 1 A and B.
  • Lipid nanoparticles comprising the ingredients shown in Tables 2 and 3 were successfully produced.
  • the particle size of the LNPs comprising 1 , 3 and 5% of vitamin A palmitate (Figure 1A) and vitamin A propionate (Figure 1 B) was around 95 and 100 nm, respectively, on day 1 (TO).
  • the LNPs comprising the different vitamin A derivatives were stable over up to 6 weeks of storage at 4°C, illustrating an excellent long-term stability, even at non-freezing temperatures.
  • the lipid nanoparticles according to the present invention containing vitamin A/ vitamin A derivative were properly formed and physically stable during storage at 4°C.
  • Lipid nanoparticle compositions were prepared as described in Example 1.1 above.
  • Vitamin A palmitate was incorporated into the LNP at different proportions: 3 % and 5 % of the total lipid composition, as shown in Table 2 above, and compared to its benchmark LNP comprising 0 % vitamin A or vitamin A derivative.
  • the LNP compositions were prepared via a Precision NanoSystems microfluidic device, each containing 267 ng of encapsulated luciferase mRNA.
  • the resulting mRNA-loaded LNP compositions were stored at 4°C. Luciferase expression was determined after 4 weeks and 6 weeks of storage at 4°C.
  • HEK293 cells were grown in Earle's balanced salt solution without L-glutamine and supplemented with 10% fetal bovine serum (Sigma-Aldrich Corp., St. Gallen, Switzerland), 2 mM glutamax (Life Technologies AG, Basel, Switzerland), 0.1 mM non-essential amino acids (Life Technologies), and 1 mM sodium pyruvate (Life Technologies) at 37°C in 5% CO2.
  • 10% fetal bovine serum Sigma-Aldrich Corp., St. Gallen, Switzerland
  • 2 mM glutamax Life Technologies AG, Basel, Switzerland
  • 0.1 mM non-essential amino acids Life Technologies
  • 1 mM sodium pyruvate Life Technologies
  • 7.5 x 1 o 4 cells were plated per well (90 pl) in white 96-well cell culture plates with clear bottom (Corning, Basel, Switzerland) in minimum essential medium (Eagle) with Earle's balanced salt solution without L-glutamine and without phenol red supplemented with 10% charcoal-treated fetal bovine serum (HyClone Laboratories, Inc., Logan, UT, USA), 2 mM glutamax, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate and grown overnight. The next day, the cells were treated with the mRNA loaded LNPs at >80% confluence, and the treatment took place for 24 hours. The luciferase expression was measured using the Bright-Glo luciferase assay Promega E2620, according to established protocols (Promega AG, Dubendorf, Switzerland).
  • Lipid nanoparticle compositions were prepared as described in Example 1 .1/ Table 3 above.
  • the efficacy of the LNPs comprising vitamin A propionate to deliver the luciferase mRNA into human cells was measured as described in Example 3.1 and 3.2 above, using 0%, 3% and 5% vitamin A propionate instead of vitamin A palmitate.
  • Lipid nanoparticle compositions were prepared as described in Example 1 .1/ Table 4 above.
  • the efficacy of the LNPs comprising vitamin A to deliver the luciferase mRNA into human cells was measured as described in Example 3.1 and 3.2 above, using 0%, 3% and 5% vitamin A instead of vitamin A palmitate.
  • LNP-based formulations often suffer from broad particle size distributions which can impact consistency, efficacy, and scalability in manufacturing. As such, optimizing LNP composition and processing conditions to produce particles with narrower size distributions is desirable. 4.1 Preparation of the LNPs
  • Lipid nanoparticle compositions were prepared as described in Example 1.1.
  • compositions were prepared and tested:
  • LNP composition comprising vitamin A palmitate (as described in Table 2 above). The results are shown in FIG. 5 A, B and C. • LNP composition comprising vitamin A propionate (as described in Table 3 above).
  • LNP composition comprising vitamin A palmitate (as described in Table 5 below). The results are shown in FIG. 7 A, B and C.
  • LNP composition comprising vitamin A propionate (as described in Table 6 below). The results are shown in FIG. 8 A, B and C.
  • Table 5 mRNA-loaded LNPs comprising vitamin A palmitate
  • Table 6 mRNA-loaded LNPs comprising vitamin A propionate
  • PPI polydispersity index
  • PDI polydispersity index

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Abstract

La présente invention concerne des nanoparticules lipidiques (NPL) comprenant de la vitamine A ou un dérivé de vitamine A. Les NPL selon la présente invention présentent une stabilité et une efficacité de délivrance d'acide nucléique améliorées.
PCT/EP2025/069780 2024-07-12 2025-07-10 Nanoparticules lipidiques améliorées Pending WO2026013203A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2998289A1 (fr) * 2011-06-08 2016-03-23 Nitto Denko Corporation Composés pour cibler l'administration de médicaments et renforcer l'activité arnsi
CN117257761A (zh) * 2023-09-20 2023-12-22 莎穆(上海)生物科技有限公司 一种包载核酸的抗氧化脂质纳米粒、其制备方法及应用
WO2024078193A1 (fr) * 2022-10-11 2024-04-18 中国科学院化学研究所 Nanoparticule lipidique, son procédé de préparation et son utilisation

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EP2998289A1 (fr) * 2011-06-08 2016-03-23 Nitto Denko Corporation Composés pour cibler l'administration de médicaments et renforcer l'activité arnsi
WO2024078193A1 (fr) * 2022-10-11 2024-04-18 中国科学院化学研究所 Nanoparticule lipidique, son procédé de préparation et son utilisation
CN117257761A (zh) * 2023-09-20 2023-12-22 莎穆(上海)生物科技有限公司 一种包载核酸的抗氧化脂质纳米粒、其制备方法及应用

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AKINC, A. ET AL.: "A combinatorial library of lipid-like materials for delivery of RNAi therapeutics", NATURE BIOTECHNOLOGY, vol. 26, no. 5, 2008, pages 561 - 569
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JIA, Y. ET AL.: "Lipid nanoparticles optimized for targeting and release of nucleic acid", ADVANCED MATERIALS, vol. 36, 2024, pages 2305300
MAEKIMASATOSHI ET AL.: "Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery", JOURNAL OF CONTROLLED RELEASE, vol. 344, 2022, pages 80 - 96
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