Lipid nanoparticles for oligonucleotide delivery

Abstract

The present invention relates to a compound of formula (I): [Formula 1] JPEG2024540108000003.jpg3566 or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof. The present invention also provides lipid nanoparticles comprising an ionizable lipid-like compound according to formula I and one or more RNA molecules, as well as pharmaceutical compositions or vaccines comprising such lipid nanoparticles.

Description

[Technical field]

[0001] The present invention relates to novel lipids and lipid nanoparticles (LNPs) comprising these novel lipids / lipidoids in combination with other lipid components that can be used for the delivery of oligonucleotides, especially for the delivery of RNA such as self-amplifying RNA (saRNA). The present invention also relates to therapeutic products and their uses. [Background technology]

[0002] There are many challenges associated with the delivery of nucleic acids to effect desired responses in biological systems. The COVID-19 pandemic and the vaccines developed in response to it have shown that nucleic acid-based prophylactic vaccines have enormous potential. mRNAs and saRNAs used as vaccines or even in therapeutic contexts, however, face the problem of being susceptible to nuclease degradation in plasma, interstitium and lymph. Moreover, free RNAs have a limited ability to gain access to intracellular compartments where the relevant translational machinery resides.

[0003] Lipid nanoparticles (LNPs) formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, and PEGylated lipids have been used to prevent RNA degradation in plasma and facilitate cellular uptake of oligonucleotides. LNPs currently known in the art are specifically designed and optimized for delivery of conventional mRNA or siRNA. Meanwhile, next-generation oligonucleotide-based therapeutics are being developed that focus on the use of larger RNA constructs such as self-amplifying RNA (saRNA). SaRNA has the advantage that RNA is equipped with a mechanism that allows self-amplification, thereby allowing the use of lower concentrations of RNA in therapy. saRNA is typically a long and negatively charged molecule, and therefore requires a good delivery system. These LNPs must be able to protect the oligonucleotide from the action of nucleases and deliver it into the cell by interacting with the negatively charged cell membrane.

[0004] The encapsulation of negatively charged RNA in LNPs is highly dependent on the interaction with positively charged amino lipids. The selection of lipids for LNPs in this way is very important as it helps to capture RNA molecules and promote endosomal escape. As some studies have shown, some cationic lipids with permanent positive charges appear to be less efficient and more toxic, so the use of other lipids is recommended. The lipid or lipid-like compounds according to the invention are protonated at low pH but show a relatively neutral surface charge at physiological pH. This has two advantages: it prevents non-specific lipid-protein interactions such as the binding of a large amount of charged (macro)molecules in biological fluids (e.g., albumin) and it promotes endosomal escape of RNA by protonation of the compound in the acidic environment of the endosome. It has been shown that acidification disrupts the membrane and allows RNA to escape from the endosome. Thus, these compounds contribute to efficient encapsulation and delivery of RNA.

[0005] LNPs are generally formulated with two or more additional excipients: (i) sterols, which increase the stability of the LNP bilayer and promote membrane fusion; (ii) optionally, phospholipids, which reinforce the LNP bilayer structure and aid in endosomal escape; and (iii) lipid-polyethylene glycol (PEG) conjugates, which intercalate into the LNP bilayer and provide a PEG coating that reduces LNP aggregation, reduces nonspecific binding of proteins by steric hindrance, and reduces nonspecific endocytosis by immune cells.

[0006] US 9 439 968 discloses compositions and methods for the preparation, manufacture and therapeutic use of LNPs containing lipidoids prepared from the conjugate addition of alkylamines to acrylates. Some of the lipidoids are -CH2CH2C(=O)OR B This allows the specific lipidoid R B Each of these is a linear alkyl chain. These lipids were designed for delivery of small interfering RNA constructs, i.e., short strands of RNA.

[0007] Blakney et al. (2019) discusses suitable LNP formulations for saRNA. Nevertheless, there remains a need for other improved ionizable lipids and lipid nanoparticles for delivery of oligonucleotides such as RNA, particularly saRNA or other large RNA constructs. Preferably, these lipid nanoparticles provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic delivery, and provide intracellular delivery of the nucleic acid. Furthermore, these lipid-nucleic acid particles should be well tolerated and provide an appropriate prophylactic / therapeutic index, such that subject administration / treatment of a patient with an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention provides these and / or related advantages. Summary of the Invention

[0008] The present invention and its embodiments help provide a solution to one or more of the problems mentioned above and meet one or more of the desired properties. To this end, the present invention relates to an ionizable lipid-like compound according to formula (I), or a pharma-ceutically acceptable salt, tautomer, or stereoisomer thereof. [ka] Here, m, R 1 , R 2 , R 3 and R 4 is as defined herein.

[0009] In a second aspect, the present invention also relates to lipid nanoparticles encapsulating oligonucleotides, in particular RNA, and comprising at least one ionizable lipid according to formula I or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0010] In a further embodiment, a pharmaceutical composition, including a vaccine, is provided comprising at least one lipid nanoparticle carrying at least one nucleic acid according to the second embodiment. Said pharmaceutical composition or vaccine can be used to treat or prevent a disease, such as an infectious disease. Such use includes, for example, administering to a subject an effective amount of an RNA construct encoding a gene of interest encapsulated or formulated in a lipid nanoparticle as described herein, in the formation of a self-replicating RNA molecule, and / or using a composition according to the invention. For example, the invention provides the use of an encapsulated self-replicating RNA molecule of the invention encoding an antigen for inducing an immune response in a subject, or the use of the encapsulated RNA in RNA-based protein replacement therapy. The pharmaceutical composition or vaccine can further comprise a pharma- ceutically acceptable carrier.

[0011] In another aspect, the present invention relates to a compound suitable for delivery of oligonucleotides such as RNA, said compound being an ionizable lipid-like compound according to formula (I) as defined in any of the embodiments of the first aspect, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0012] Further embodiments of aspects of the invention are provided in the detailed description and claims.

[0013] definition As used herein, the following terms have the following meanings:

[0014] As used herein, "a," "a," and "the" refer to both singular and plural references unless the context clearly dictates otherwise. As an example, "a compartment" refers to one or more compartments.

[0015] As used herein, "about" refers to a measurable value, such as a parameter, amount, time duration, etc., and is meant to encompass a variation from the stated value of no more than + / -20%, preferably no more than + / -10%, more preferably no more than + / -5%, even more preferably no more than + / -1%, and even more preferably no more than + / -0.1%, as appropriate for practicing the inventions disclosed hereinbefore. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0016] As used herein, "Comprise," "comprising," "comprises," and "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," or "contains," and are inclusive or open-ended terms specifying the presence of what follows, such as a component, and do not exclude or preclude the presence of additional, unrecited components, features, elements, materials, or steps that are known in the art or disclosed herein.

[0017] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the range as well as the recited endpoints.

[0018] The expression "mol%" in the specification means the relative amount in moles of each component based on the overall formulation, unless otherwise defined. A mole is defined as exactly 6.02214076 x 10^23 particles, which may be atoms, molecules, ions, or electrons.

[0019] The terms "one or more" or "at least one" are themselves clear, such as one or more or at least one member of a group of members, but by way of further illustration, the terms specifically include reference to any one of said members, or any two or more of said members (e.g., ≧3, ≧4, ≧5, ≧6 or ≧7 of said members, etc.), up to and including all of these members.

[0020] References throughout this specification to "one embodiment" or "an embodiment" mean that at least one embodiment of the invention includes a particular feature or structure or characteristic described in connection with this embodiment. Thus, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, particular features, structures or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features that are included in other embodiments but not in others, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the embodiments described in the claims may be used in any combination.

[0021] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning commonly understood by those skilled in the art to which the invention belongs. For further guidance, definitions of terms used in the specification are included to better understand the teachings of the present invention. Terms or definitions used in this specification are provided solely to aid in the understanding of the present invention.

[0022] The term "lipid" refers to, but is not limited to, a group of organic compounds that contain esters of branched or unbranched fatty acids and are generally characterized by poor solubility in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids or glycolipids; and (3) "derived lipids," such as steroids.

[0023] In the context of the present invention, the term "sterols", also known as steroid alcohols, is a subgroup of steroids that occur naturally in plants, animals and fungi or can be produced by some bacteria.

[0024] The term "neutral lipid" refers to any of a number of lipid species that exist in either uncharged or neutral amphoteric form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholine, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamine, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramide; steroids, such as sterols and their derivatives. The neutral lipids may be synthetic or naturally occurring.

[0025] The term "charged lipid" refers to any of a number of lipid species that exist in either positively charged form, i.e., "cationic lipids," or negatively charged form, i.e., "anionic lipids," at all pH values ​​from pH 3 to pH 9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl sterol (e.g., DC-Chol).

[0026] The term "ionizable" as used herein, for example "ionizable lipid", "ionizable lipid-like structure" or "ionizable amino lipid" or "ionizable compound", refers to the property that a compound is neutral or charged depending on pH. Typically, in the context of the present invention, an ionizable lipid is an ionizable cationic lipid, which contains one or more primary, secondary or tertiary amino groups that are protonated only when exposed to a pH below a certain value. Different nitrogens within a single ionizable amino lipid according to formula I can be protonated at different pHs (i.e., different nitrogens can have different pKa). Depending on the high number of ionizable nitrogens in such ionizable cationic lipids, such lipids can have a higher cationic charge available for complexing RNA.

[0027] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., 1-1000 nm) and containing multiple lipid molecules physically bound to each other by intermolecular forces. Lipid nanoparticles can be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles or the internal phase in a suspension. An active or therapeutic agent, e.g., a nucleic acid, is encapsulated in the lipid portion of the lipid nanoparticle, or in the aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cells, e.g., a deleterious immune response.

[0028] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide active or therapeutic agents, such as nucleic acids (e.g., mRNA), with complete or partial encapsulation, and the nucleic acid may be fully incorporated within the nanoparticle or (partially) associated with the nanoparticle surface. According to one embodiment, the nucleic acid (e.g., saRNA or mRNA) is fully encapsulated in the lipid nanoparticle.

[0029] The term "oligonucleotide" or "polynucleotide" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded formation, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of self-amplifying RNA (saRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, dicer substrate RNA or viral RNA (vRNA), guide RNA (gRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally derived, and have similar binding properties as the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence may also include its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and / or deoxyinosine residues. A "nucleotide" includes the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Base" further includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications which place new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0030] As used herein, "buffering agents" include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, hydroxide calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, dibasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0031] The term "N / P ratio" (or N:P ratio) as used herein is the molar ratio of nitrogen atoms in the complexed lipid / phosphate group in RNA. This ratio represents the interaction between the cationic charge of the ionized amino (N+) group of the ionizable amino-lipid and the anionic ionic charge of the phosphate (PO4-) group of the backbone of the nucleic acid, and is the basis for the complexation of RNA with the ionizable amino-lipid. To determine the N / P ratio of lipid nanoparticles containing the ionizable lipids such as the lipid of formula I, the number of positively charged nitrogen atoms at the pH at which the LNP is formulated is considered. The N / P ratio of a lipid / nucleic acid complex (e.g., lipid / RNA) can potentially affect other properties such as its net surface charge, the size and stability of the LNP containing the lipid / nucleic acid complex.

[0032] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by a governmental agency such as the EMA and / or the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0033] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0034] "Pharmaceutical composition" refers to a formulation of a compound of the invention and a vehicle generally accepted in the art for delivering a biologically active compound to a mammal, e.g., a human. Such a vehicle includes any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0035] "Effective amount" or "therapeutically effective amount" refers to the amount of the compound of the present invention or lipid nanoparticles containing it, which is sufficient to perform treatment in a mammal, preferably a human, when administered to an animal, preferably a mammal, more preferably a human. The amount of lipid nanoparticles of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, its symptoms and severity, the method of administration, and the age of the animal to be treated, but can be routinely determined by those skilled in the art in light of their own knowledge and this disclosure.

[0036] As used herein, the terms "self-replicating" and "self-amplifying" are used interchangeably and refer to molecules such as RNA that contain within them sequence-specific signals or signature sequences that allow for the self-replication or self-amplification of the molecule.

[0037] As used herein, "treating" or "treatment" includes the treatment of a disease or disorder of interest in a mammal, preferably a human, having the disease or disorder of interest; (i) preventing the onset of, or reducing the likelihood or severity of, a disease or condition in a mammal, particularly where the mammal is susceptible to, but has not yet been diagnosed with, the disease or condition; (ii) inhibiting the disease or illness, i.e. slowing or stopping its development; (iii) alleviating the disease or illness, i.e., causing regression of the disease or illness; (iv) Relieving symptoms caused by disease or illness, such as relieving pain without addressing the underlying disease or illness. Thus, preventive treatments such as vaccination are included, whereby disease mediated by infection with pathogens, such as viruses or bacteria, is prevented or reduced in incidence and / or severity. As used herein, the terms "disease" and "disease" may be used interchangeably, or a particular disease or illness may not have a known causative agent (so the etiology has not yet been elucidated), and therefore it is not yet recognized as a disease, but only as an undesirable symptom or syndrome, where a more or less specific set of symptoms has been identified by clinicians.

[0038] "Stereoisomer" refers to a compound consisting of the same atoms linked by the same bonds but having different, incompatible three-dimensional structures. Embodiments of the present invention contemplate various stereoisomers and mixtures thereof, and include "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0039] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. Embodiments of the invention include tautomers of any of the above compounds.

[0040] "Alkyl" refers to an unbranched (also called straight-chain or linear) or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which may be saturated or unsaturated (i.e., containing one or more double and / or triple bonds), such as 1-4 carbon atoms (C1-C4 alkyl), 4-20 carbon atoms (C4-C20 alkyl), 6-16 carbon atoms (C6-C16 alkyl), 6-9 carbon atoms (C6-C9 alkyl), 11-20 carbon atoms (C11-C20 alkyl), 1-12 carbon atoms (C1-C2 ... C12 alkyl), 2-6 carbon atoms (C2-C6 alkyl) or 1-6 carbon atoms (C1-C6 alkyl) which are attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless stated otherwise in the specification, alkyl groups are optionally substituted.

[0041] "Branched alkyl" is given its ordinary meaning in the art and typically refers to an otherwise straight chain alkyl group having one or more alkyl substituents. Such alkyl groups may thus contain a secondary carbon radical (a secondary carbon radical is a carbon radical bonded to two other carbon atoms), a tertiary carbon (a tertiary carbon is a carbon atom bonded to three other carbon atoms), or a quaternary carbon (a quaternary carbon is a carbon atom bonded to four other carbon atoms). In a branched alkyl chain having a secondary carbon radical, the point of attachment of the alkyl chain to the remainder of the molecule is not through a carbon atom that is covalently bonded to only one other carbon atom. A branched alkyl, as used herein, can have, for example, from 4 to 30 carbon atoms (C4-C30 alkyl), where the point of attachment to the remainder of the molecule is through the second (second carbon atom), third, fourth, sixth, seventh, eighth, ninth, tenth, eleventh, fifteenth, thirteenth, fourteenth, or fifteenth carbon atom, e.g., heptadecane-8-yl. A branched alkyl can have, for example, 4 to 30 carbon atoms (C4-C30 alkyl), including a carbon atom bonded to three other carbon atoms. A branched alkyl can have, for example, 4 to 30 carbon atoms (C4-C30 alkyl), including a carbon atom bonded to four other carbon atoms. For example, 3,5,5 trimethylhexylphenyl is an alkyl group (hexyl) with three methyl branches (i.e., one tertiary and one quaternary carbon), and is therefore a branched alkyl bonded to a phenyl group. Unless otherwise specified, branched alkyl includes all isomers thereof.

[0042] "Alkylene" or "alkylene chain" refers to an unbranched or branched divalent hydrocarbon chain that is saturated or unsaturated (i.e., containing one or more double and / or triple bonds) and has, for example, 1 to 24 carbon atoms (C1-C24 alkylene), 1 to 15 carbon atoms (C1-C15 alkylene), 1 to 12 carbon atoms (C1-C12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 2 to 6 carbon atoms (C2-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), connecting the remainder of the molecule to a radical group consisting only of carbon and hydrogen, such as, for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise in the specification, an alkylene chain can be optionally substituted.

[0043] As used herein, the term "substituted" refers to a group in which at least one hydrogen atom (e.g., one, two, three, or all hydrogen atoms) has been replaced with, but is not limited to, a halogen atom, such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a C1-C12 alkyl group; a cycloalkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR' or; -S(O)xR'; -S-SR'; -C(=O)SR'; -SC(=O)R'; -NR' R'; -NR'C(=O)R'; -C(=O)R'; -C(=O)NR'R'; -NR'C(=O)NR'R'; -OC(=O)NR'R'; -NR'C(=O)OR'; -NR'S(O)xNR'R'; -NR'S(O)xR'; and -S(O)xNR'R', where each R' is independently H, C1-C20 alkyl or cycloalkyl, and x is 0, 1, or 2, meaning any of the above groups (e.g., alkyl, alkylene, or heterocyclyl).

[0044] An ester or amide functional group is defined herein as -C(=O)O- or -OC(=O)- or -NHC(=O)- or -C(=O)NH-. As used in claim 1, an ester or amide functional group is defined as: -C(=O)OR A Or -OC(=O)R A or -NHC(=O)R A or -C(=O)NHR A According to R A Next to the base, or -C(=O)OR B Or -OC(=O)R B or -NHC(=O)R B or -C(=O)NHR B According to R B Next to the base, or -C(=O)OR C Or -OC(=O)R C or -NHC(=O)R C or -C(=O)NHR C According to R C Next to the base, or -C(=O)OR D or -OC(=O)R D or -NHC(=O)R D or -C(=O)NHR D According to R D It can be placed next to the base. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows the in vivo expression over time of luciferase encoding saRNA administered to mice in Example 2 using an exemplary compound of the invention (triangle graph) compared to the negative control (pie graph), C12-200 (square graph), and comparison compounds C1 or C2 (diamond graph). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In a first aspect, the present invention provides a compound of formula (I): [ka] 1. An ionizable lipid-like compound according to claim 1, each m is independently 1, 2, 3, 4, or 5; R 1 ,-L 1 N[L 2 F 1 R A ]2 or -L 3 F 2 R B and Each R 2 , R 3 , and R 4 ,-L 4 F 3 R C and Each L 1 , L 2 , L 3 , and L 4 are independently C2-C10 alkylene; Each F 1 , F 2 , and F 3 is independently an ester or amide functional group, and Each R A , R B , and R C is independently a branched C4-C30 alkyl; The present invention relates to an ionizable lipid-like compound, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0047] The compounds according to formula I are characterized, among other compounds, by a large number of ionizable nitrogens in combination with branched lipid moieties, allowing efficient encapsulation of RNA molecules in lipid nanoparticles, especially large RNA constructs (e.g., having 5000 nt or more), such as self-amplifying RNA constructs, which have been found to be proficient in delivering said RNA cargo to cells.

[0048] According to one embodiment, for compounds of formula I, F 1, F 2 and F 3 Each of -F is an ester. In one embodiment, for a compound of formula I, each -F 1 R A , -F 2 R B and-F 3 R C R is each R A , R B and R C It has the structure -O-C(=O)-R, where

[0049] In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, comprises at least one amide functional group. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, comprises at least two amide functional groups. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, comprises at least three amide functional groups. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, comprises at least four amide functional groups.

[0050] In one embodiment, the ionizable lipid-like compound according to formula (I) or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof has at least one ester functional group, —CHC(═O)OR A where R A is an alkyl chain. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, comprises at least one ester functional group, —CH2OC(═O)R A where R A is an alkyl chain. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, has at least two ester functional groups, —CHC(═O)OR A where RA is an alkyl chain. In one embodiment, the ionizable lipid-like compound according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, has at least two ester functional groups, —CH2OC(═O)R A where R A is an alkyl chain. In one embodiment, the ionizable lipid-like compound according to formula (I) or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof comprises at least three ester functional groups. In one embodiment, the ionizable lipid-like compound according to formula (I) or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof comprises at least four ester functional groups.

[0051] In one embodiment, each branched alkyl side chain R A , R B and R C is a C8-C30 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C8-C23 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C8-C20 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C11-C30 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C11-C23 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C11-C20 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is a C13-C30 alkyl chain, a C13-C23 alkyl chain, or a C13-C20 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and RC is a C15-C30 alkyl chain, a C15-C23 alkyl chain, or a C15-C20 alkyl chain. In one embodiment, each branched alkyl side chain R A , R B and R C is selected from C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20 alkyl. In one embodiment, each branched alkyl side chain R A , R B and R C is selected from C11, C12, C13, C14 and C15 alkyl. In one embodiment, each R A , R B and R C is the same. In one embodiment, R A , R B and R C is an unsubstituted branched alkyl group.

[0052] In one embodiment, each branched alkyl side chain R A , R Bare independently selected from the following: henicosan-2-yl, docosan-2-yl, tricosan-2-yl, tetracosan-2-yl, pentacosan-2-yl, hexacosan-2-yl, heptacosane-2-yl, octacosan-2-yl, nonacosane-2-yl, triacontan-2-yl, henicosan-2-yl, docosan-2-yl, tricosan-2-yl, tetracosan-2-yl, pentacosan-2-yl, hexacosan-2-yl, heptacosane-2-yl, octacosan-2-yl, nonacosane-2-yl, triacontan-2-yl, Eicosan-3-yl, docosan-3-yl, tricosan-3-yl, tetracosan-3-yl, pentacosan-3-yl, hexacosan-3-yl, heptacosan-3-yl, octacosan-3-yl, nonacosan-3-yl, triacontan-3-yl, henicosan-4-yl, docosan-4-yl, tricosan-4-yl, tetracosan-4-yl, pentacosan-4-yl, hexacosan-4-yl, heptacosan-4-yl, octacosan-4-yl, nonacosan-4-yl, triacontan-4-yl, henicosan-5-yl, docosan -5-yl, tricosan-5-yl, tetracosan-5-yl, pentacosan-5-yl, hexacosan-5-yl, heptacosan-5-yl, octacosan-5-yl, nonacosan-5-yl, triacontan-5-yl, heneicosan-6-yl, docosan-6-yl, tricosan-6-yl, tetracosan-6-yl, pentacosan-6-yl, hexacosan-6-yl, heptacosan-6-yl, octacosan-6-yl, nonacosan-6-yl, triacontan-6-yl, heneicosan-7-yl, docosan-7-yl, tricosan-7-yl yl, tetracosan-7-yl, pentacosan-7-yl, hexacosan-7-yl, heptacosan-7-yl, octacosan-7-yl, nonacosan-7-yl, triacontan-7-yl, henocosan-8-yl, docosan-8-yl, tricosane-8-yl, tetracosan-8-yl, pentacosan-8-yl, hexacosan-8-yl, heptacosan-8-yl, octacosan-8-yl, nonacosan-8-yl, triacontan-8-yl, henocosan-9-yl, docosan-9-yl, tricosane-9-yl, tetracosan-9-yl,Pentacosan-9-yl, hexacosan-9-yl, heptacosan-9-yl, octacosan-9-yl, nonacosan-9-yl, triacontan-9-yl, henicosan-10-yl, docosan-10-yl, tricosan-10-yl, tetracosan-10-yl, pentacosan-10-yl, hexacosan-10-yl, heptacosan-10-yl, octacosan-10-yl, nonacosan-10-yl, triacontan-10-yl, docosan-11-yl, tricosan-11-yl, tetracosan-11-yl, pentacosan-11-yl, hexacosan-10-yl Saccosan-11-yl, heptacosan-11-yl, octacosan-11-yl, nonacosan-11-yl, triacontan-11-yl, tetracosan-12-yl, pentacosan-12-yl, hexacosan-12-yl, heptacosan-12-yl, octacosan-12-yl, nonacosan-12-yl, triacontan-12-yl, hexacosan-13-yl, heptacosan-13-yl, octacosan-13-yl, nonacosan-13-yl, triacontan-13-yl, octacosan-14-yl, nonacosan-14-yl, triacontan 14-yl, triacontan-15-yl, butan-2-yl, pentan-2-yl, hexan-2-yl, heptan-2-yl, octan-2-yl, nonan-2-yl, decan-2-yl, hexan-3-yl, heptan-3-yl, octan-3-yl, nonan-3-yl, decan-3-yl, octan-4-yl, nonan-4-yl, decan-4-yl, decan-5-yl, undecan-2-yl, dodecane ( dodecan-2-yl, tridecan-2-yl, tetradecan-2-yl, pentadecane-2-yl, hexadecan-2-yl, heptadecane-2-yl, octadecane-2-yl, nonadecan-2-yl, eicosan-2-yl, undecan-3-yl, dodecan-3-yl, tridecan-3-yl, tetradecan-3-yl, pentadecane-3-yl,Hexadecan-3-yl, heptadecan-3-yl, octadecan-3-yl, nonadecan-3-yl, eicosan-3-yl, undecane-4-yl, dodecane-4-yl, tridecane-4-yl, tetradecane-4-yl, pentadecane-4-yl, hexadecan-4-yl, heptadecan-4-yl, octadecan-4-yl, nonadecan-4-yl, eicosan-4-yl, undecane-5-yl, dodecane-5-yl, tridecane-5-yl, tetradecane-5-yl, pentadecane-5-yl, hexadecan-5-yl, heptadecan-5-yl, octadecan-5-yl, nonadecan-5-yl, eicosan-5-yl yl, dodecan-6-yl, tridecan-6-yl, tetradecan-6-yl, pentadecane-6-yl, hexadecan-6-yl, heptadecane-6-yl, octadecane-6-yl, nonadecan-6-yl, eicosan-6-yl, tetradecan-7-yl, pentadecane-7-yl, hexadecan-7-yl, heptadecane-7-yl, octadecane-7-yl, nonadecan-7-yl, eicosan-7-yl, hexadecan-8-yl, heptadecane-8-yl, octadecane-8-yl, nonadecan-8-yl, eicosan-8-yl, octadecane-9-yl, nonadecan-9-yl, and eicosan-9-yl. In one embodiment, the C4-C30 alkyl chain (R, A , R B and R C The point of attachment of R to the remainder of the molecule may be via the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th or 15th carbon atom in the alkyl chain. In one embodiment, the lipid nanoparticle comprises at least one ionizable lipid-like structure according to formula (I), or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof, each R A , R B and R C is independently selected from undecane-5-yl, tridecane-6-yl, pentadecan-7-yl, heptadecan-8-yl, and nonadecan-9-yl. In one embodiment, each branched alkyl side chain R A , R B and R Cis selected from heptadecan-8-yl and nonadecan-9-yl. In one embodiment, each R A , R B and R C is pentadecan-7-yl. In one embodiment, the branched alkyl side chain R A , R B and R C is substituted with one or more alkyl groups. In one embodiment, the branched alkyl side chain R A , R B and R C is substituted with one or more methyl or ethyl groups. In one embodiment, the branched alkyl side chain R A , R B and R C is substituted with any isomer of propyl, butyl or pentyl. In one embodiment, the branched alkyl side chain R A , R B and R C is substituted with either the hexyl, heptyl, octyl or nonyl isomers. These alkyl chains provide a large apolar zone and good globular positioning of the lipid-like compound for encapsulation of large oligonucleotides.

[0053] In one embodiment, each R A , R B and R C is independently selected from undecane-5-yl, tridecane-6-yl, pentadecan-7-yl, heptadecan-8-yl, and nonadecan-9-yl. A , R B and R C is selected from undecane-5-yl, tridecane-6-yl, pentadecan-7-yl, heptadecan-8-yl, and nonadecan-9-yl. A , R B and R C is pentadecan-7-yl. In one embodiment, each R A , R B and R Cis undecane-5-yl. In one embodiment, each R A , R B and R C is tridecan-6-yl. In one embodiment, each R A , R B and R C is pentadecan-7-yl.

[0054] In one embodiment, each L 1 , L 2 , L 3 and L 4 is independently C2-C8 alkylene. 1 , L 2 , L 3 and L 4 is independently a C2-C6 alkylene. 1 , L 2 , L 3 and L 4 is independently selected from: ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene. 1 , L 2 , L 3 and / or L 4 is unsubstituted. In a further embodiment, L 1 , L 2 , L 3 and / or L 4 is an unsubstituted linear alkylene. In one embodiment, L 1 , L 2 , L 3 and / or L 4 includes cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, or cyclooctane. 1 , L 2 , L 3 and / or L 4is a halogen atom such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a C1-C8 alkyl group; a cycloalkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';‐S‐SR';‐C(=O)SR';‐SC(=O)R';‐NR'R';‐NR'C(=O)R';‐C(=O)R';‐C(=O)NR'R';‐NR'C(=O)NR'R';‐OC(=O)NR'R';‐NR'C(=O)OR';‐NR'S(O) x NR'R';‐NR'S(O) x R'; and / or -S(O) x It's NR'R'.

[0055] In one embodiment, -R 2 , -R 3 , -R 4 is the structure L 4 F 3 R C where L 4 is C2-C6 alkylene, F 3 is an ester or amide functional group, and R C is selected from undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, and nonadecan-9-yl. An example of the structure is -CH2CH2C(=O)NHR C , -CH2CH2CH2C(=O)NHR C , -CH2CH2CH2CH2C(=O)NHR C , -CH2CH2CH2CH2CH2C(=O)NHR C , -CH2CH2CH2CH2CH2CH2C(=O)NHR C , -CH2CH2NHC(=O)R C , -CH2CH2CH2NHC(=O)R C , -CH2CH2CH2CH2NHC(=O)R C , -CH2CH2CH2CH2CH2NHC(=O)R C , -CH2CH2CH2CH2CH2CH2NHC(=O)R C , -CH2CH2OC(=O)R C, -CH2CH2CH2OC(=O)R C , -CH2CH2CH2CH2OC(=O)R C , -CH2CH2CH2CH2CH2OC(=O)R C , -CH2CH2CH2CH2CH2CH2OC(=O)R C , -CH2CH2C(=O)OR C , -CH2CH2CH2C(=O)OR C , -CH2CH2CH2CH2C(=O)OR C , -CH2CH2CH2CH2CH2C(=O)OR C , -CH2CH2CH2CH2CH2CH2CH2C(=O)OR C where -R C is undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, or nonadecan-9-yl.

[0056] In one embodiment, -R 1 Ha-L 3 F 2 R B where L 3 is C2-C6 alkylene, F 2 is an acid ester or amide functional group, and R B is selected from undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, or nonadecan-9-yl. An example of the structure is -CH2CH2C(=O)NHR B , -CH2CH2CH2C(=O)NHR B , -CH2CH2CH2CH2C(=O)NHR B , -CH2CH2CH2CH2CH2C(=O)NHR B , -CH2CH2CH2CH2CH2CH2C(=O)NHR B , -CH2CH2NHC(=O)R B , -CH2CH2CH2NHC(=O)R B , -CH2CH2CH2CH2NHC(=O)R B , -CH2CH2CH2CH2CH2NHC(=O)R B , -CH2CH2CH2CH2CH2CH2NHC(=O)RB , -CH2CH2OC(=O)R B , -CH2CH2CH2OC(=O)R B , -CH2CH2CH2CH2OC(=O)R B , -CH2CH2CH2CH2CH2OC(=O)R B , -CH2CH2CH2CH2CH2CH2OC(=O)R B , -CH2CH2C(=O)OR B , -CH2CH2CH2C(=O)OR B , -CH2CH2CH2CH2C(=O)OR B , -CH2CH2CH2CH2CH2C(=O)OR B , -CH2CH2CH2CH2CH2CH2CH2C(=O)OR B where -R B is undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, or nonadecan-9-yl.

[0057] In one embodiment, -R 1 Ha-L 1 N[L 2 F 1 R A ]2, where L 1 is C2-C6 alkylene, L 2 is C2-C6 alkylene, F 1 is an acid ester or amide functional group, and R A is selected from undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, or nonadecan-9-yl. 2 F 1 R A An example of the structure is -CH2CH2C(=O)NHR A , -CH2CH2CH2C(=O)NHR A , -CH2CH2CH2CH2C(=O)NHR A , -CH2CH2CH2CH2CH2C(=O)NHR A , -CH2CH2CH2CH2CH2CH2C(=O)NHR A , -CH2CH2NHC(=O)R A, -CH2CH2CH2NHC(=O)R A , -CH2CH2CH2CH2NHC(=O)R A , -CH2CH2CH2CH2CH2NHC(=O)R A , -CH2CH2CH2CH2CH2CH2NHC(=O)R A , -CH2CH2OC(=O)R A , -CH2CH2CH2OC(=O)R A , -CH2CH2CH2CH2OC(=O)R A , -CH2CH2CH2CH2CH2OC(=O)R A , -CH2CH2CH2CH2CH2CH2OC(=O)R A , -CH2CH2C(=O)OR A , -CH2CH2CH2C(=O)OR A , -CH2CH2CH2CH2C(=O)OR A , -CH2CH2CH2CH2CH2C(=O)OR A , -CH2CH2CH2CH2CH2CH2CH2C(=O)OR A where -R A is undecane-5-yl, tridecane-6-yl, pentadecane-7-yl, heptadecan-8-yl, or nonadecan-9-yl. 2 F 1 R A In addition to the structural examples, L 1 is ethylene, propylene or butylene, in particular ethylene.

[0058] In one embodiment, L 1 , L 2 , L 3 and L 4 is ethylene. In one embodiment, L 1 , L 2 , L 3 and L 4 is butylene. In one embodiment, L 1 , L 2 , L 3 and L 4 is hexene. In one embodiment, L 1 is ethylene, and L 2 , L3 and L 4 is butylene. In one embodiment, L 1 is ethylene, and L 2 , L 3 and L 4 is hexylene.

[0059] In one embodiment, L 1 is ethylene. In a further embodiment, L 1 is ethylene, L 2 and L 4 is butylene, pentylene or hexylene, in particular L 2 and L 4 is butylene.

[0060] In one embodiment, m is 1, 2, 3, 4 or 5, or m is selected from 1, 3 and 5. In a further embodiment, m is 1, 2 or 3. In yet another embodiment, m is 1.

[0061] In a further embodiment, the ionizable lipid-like structure or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof is as shown in Table 1.

[0062] Table 1: Examples of structures according to embodiments of the invention, wherein each m is independently 1, 2, 3, 4 or 5; each n is independently 1, 2, 3, 4 or 5; and each o is independently 1, 2, 3, 4 or 5. [Table 1-1] [Table 1-2]

[0063] It will be understood that the present invention also applies to compounds according to the structures disclosed in Table 1, further including pharma- ceutically acceptable salts, tautomers, or stereoisomers thereof. Within the scope of this embodiment, the structures of the final compounds provided in the schemes of Section 1.1 of the Examples are equally included. Some lipid-like compounds and their corresponding LNPs respond to pH changes and can therefore be protonated or deprotonated. At low pH, the nitrogen groups of these compounds can be protonated.

[0064] In one embodiment of the invention, n in the structures shown in Table 1 is an integer from 1 to 5, such as 1, 3 or 5. In one embodiment, m in the structures shown in Table 1 is an integer from 1 to 5, such as 1, 3 or 5. In one embodiment, o in the structures shown in Table 1 is an integer between 1 and 5, e.g., 3, 4 or 5.

[0065] In one embodiment of the invention, the ionizable lipid-like compound or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof is as shown in Table 2. Compounds set forth in the examples falling within the scope of the compounds of Formula I are also considered representative examples.

[0066] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0067] In one embodiment, the compound according to formula I, 1 Ga-L 3 F 2 R B and R 2 , R 3 and R 4 are -L 4 F 3 R C and each L 3 and L 4 is ethylene (C2 alkylene), and each R A , R B and R C are independently branched C15-C30 alkyl. Alternatively, the compound according to formula I, 1 Ga-L 3 F 2 R B and R 2 , R 3 and R 4 are -L 4 F 3 R C And each L 3 and L 4 is ethylene (C2 alkylene), -F 2 R B ‐O‐C(=O)‐R B , -C(=O)-N-R B and -NH-C(=O)-R B , selected from -F 3 R C ‐O‐C(=O)‐R C , -C(=O)-NH-R C , -N-C(=O)-R C In a further embodiment, -F2 R B ‐O‐C(=O)‐R B and each ‐F 3 R C ‐O‐C(=O)‐R C It is.

[0068] In one embodiment, the compound according to formula I, 1 Ga-L 1 N[L 2 F 1 R A ]2, and R 2 , R 3 , R 4 are -L 4 F 3 R C And each R A , R B and R C is independently a branched C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22 alkyl.

[0069] In one embodiment, the compound according to formula I, 1 , L 2 , L 3 and / or L 4 each is a C alkyl; A , R B and R C is independently a branched C15, C16, C17, C18, C19, C20, C21 or C22 alkyl.

[0070] In one embodiment, R 1 Ha-L 3 F 2 R B It is.

[0071] In one embodiment, the compound according to formula I, 1 is ethylene and L 2 , L 3 and L 4 is butylene or hexylene, and R A , R B and R Cis defined as C11-C15 branched alkyl. In yet another embodiment, the compound according to formula I is 1 is ethylene, and L 2 , L 3 and L 4 is butylene, and R A , R B and R C is further defined as undecane-5-yl, tridecane-6-yl, pentadecan-7-yl. In a further embodiment, F 1 , F 2 and F 3 has the structure -O-C(=O)-R.

[0072] In certain embodiments, m=1 and L 1 is ethylene and L 2 , L 3 and L 4 is butylene or hexylene, and R A , R B , and R C is undecane-5-yl, tridecane-6-yl, pentadecan-7-yl.

[0073] In a second aspect, the present invention relates to one or more lipid nanoparticles (LNPs) comprising at least one compound according to formula I, in particular for use in a method of treatment as defined in the embodiments herein for the formulation of an oligonucleotide.

[0074] Thus, lipid nanoparticles (LNPs) comprise one or more oligonucleotides. In one embodiment, said oligonucleotides are oligonucleotides such as RNA or DNA, preferably self-amplifying RNA (saRNA) having a length of at least 1000bp, more preferably 2000bp, 3000bp, 4000bp, 5000bp or more. In one embodiment, one or more saRNA molecules or one or more other oligonucleotides have a minimum length of at least 1000bp, more preferably 2000bp, 3000bp, 4000bp, 5000bp or more. The length of an oligonucleotide can be expressed in a number of base pairs (bp) or a number of nucleotides (nt). In the context of the present invention, the terms "base pairs" and "nucleotides" are used interchangeably when used in the context of the length of an oligonucleotide (e.g., DNA or RNA), where one base pair (bp) is considered to be the equivalent of one nucleotide (nt).

[0075] In one embodiment, the oligonucleotide is an RNA molecule, such as an mRNA. In a particular embodiment, the RNA has a minimum length of at least 1000 bp, more preferably 2000 bp, 3000 bp, 4000 bp, 5000 bp or more. In a particularly preferred embodiment, the RNA is a self-amplifying RNA. It has been found that the lipid nanoparticles provided herein are particularly suitable for use in combination with RNA of a particular size, i.e. length, preferably an RNA of a self-amplifying RNA (saRNA). Since saRNA is typically larger than conventional mRNA, lipid nanoparticles described in the state of the art often result in poor encapsulation or suboptimal in vivo delivery and thus expression of the gene of interest encoded by the RNA. As shown in the examples, it has now been found that compounds according to formula I allow efficient encapsulation of large RNA molecules (such as saRNA constructs), while the resulting lipid nanoparticles allow good functionality (intracellular delivery and expression) of the RNA. In one embodiment, the lipid nanoparticle composition comprises an RNA oligonucleotide, such as an saRNA, specifically an mRNA having a length corresponding to 5000 bp or more. The size of the (sa)RNA may be 500-50000bp, preferably 1000-40000bp, more preferably 5000-30000nt, or 8000-16000bp. More preferably, the size of said RNA, such as saRNA, may be 5000-20000bp, preferably 6000-19000bp, preferably 7000-18000bp, preferably 8000-17000bp, more preferably 8000-16000bp. According to certain embodiments, the RNA encapsulated in the LNP is at least 8000, at least 9000, or at least 10000nt. Furthermore, according to said embodiments, the RNA does not exceed 20000, 18000 or 16000nt.

[0076] The self-replicating nature of the mRNA constructs in saRNA is based on the genomic RNA of the RNA virus, but lacks the genes encoding one or more structural proteins. The self-replicating RNA molecules can be translated to produce the non-structural proteins of the RNA virus or heterologous proteins encoded by the self-replicating RNA.

[0077] Self-replicating RNA molecules are designed such that they cannot induce the production of infectious viral particles. One suitable system to achieve self-replication is to use RNA replicons based on alphaviruses. These +-strand replicons are translated after delivery to the cell to give a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein and self-cleaves to give a replication complex to create a genomic -strand copy of the +-strand delivered RNA. These -strand transcripts can themselves be transcribed to give further copies of the +-strand parent RNA and also to give subgenomic transcripts that code for the desired gene product. Thus, translation of the subgenomic transcripts results in in situ expression of the desired gene product by the cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest Virus, eastern equine encephalitis virus, Venezuelan Equine Encephalitis Virus, and the like.

[0078] Preferred self-replicating RNA molecules encode (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) a protein / peptide as described herein. The polymerase may be an alphavirus replicator, such as the alphavirus protein nsP4.

[0079] Although naturally occurring alphavirus genomes encode structural virion proteins in addition to the nonstructural replicase polyproteins, the alphavirus-based self-replicating RNA molecules of the invention preferably do not encode alphavirus structural proteins. Thus, the self-replicating RNA can make genomic RNA copies of itself within the cell, but cannot make RNA-containing alphavirus virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules cannot perpetuate themselves in infectious formations. The alphavirus structural proteins required for wild-type virus persistence are absent from the self-replicating RNA of the invention, and their location is determined by genes encoding the desired gene products, such that the subgenomic transcript encodes the desired gene product rather than the structural alphavirus virion proteins. Thus, in certain embodiments, the self-replicating RNA molecules of the invention comprise sequences encoding the nonstructural alphavirus proteins and sequences encoding a protein / peptide of interest (e.g., an antigen for a vaccine). More specifically, the self-replicating RNA molecules of the invention comprise sequences encoding the four nonstructural alphavirus proteins and sequences encoding a protein / peptide of interest (e.g., an antigen for a vaccine). Preferably, the self-replicating RNA molecule is derived from an alphavirus that has been engineered to lack the ability to produce at least one structural alphavirus protein, more preferably, the self-replicating RNA molecule is derived from an alphavirus that has been engineered to lack the ability to produce at least two, and more preferably, all, structural alphavirus proteins.In a particular embodiment, the self-replicating RNA molecule of the present invention comprises, in 5' to 3' order, (i) a 5' sequence required for nonstructural protein-mediated amplification, (ii) a nucleotide sequence encoding an alphavirus, in particular Venezuelan equine encephalitis virus, nonstructural proteins nsP1, nsP2, nsP3, and nsP4, (iii) a promoter operably linked to a heterologous nucleic acid sequence encoding a protein / peptide of interest (e.g., an antibody for a vaccine), wherein the heterologous nucleic acid sequence replaces one or all of the alphavirus structural protein genes, (iv) a 3' sequence required for nonstructural protein-mediated amplification, and (v) a polyadenylate tract.

[0080] Recently, saRNA (RNA replicon) vaccination has been recognized as an innovative nanotechnology-based vaccination strategy. As mentioned above, unlike viral replicon particles (i.e., RNA encapsulated in viral capsid proteins), sa-mRNA can only be produced by in vitro transcription. Thus, the entire manufacturing process is completely cell-free, resulting in a therapeutic drug with a precisely defined composition. sa-mRNA vaccines have several attractive features, such as extended duration (approximately 2 months) and magnitude of expression, compared to their non-replicating counterparts. In addition, the intracellular replication of sa-mRNA is transient, and double-stranded RNA (dsRNA) may induce interferon-mediated host defense mechanisms by triggering pattern recognition receptors. This results in a strong antigen-specific immune response against the inserted target molecule. Thus, the sa-mRNA vector system is ideally suited for vaccine development, as it offers high transient transgene expression and an inherent adjuvant effect.

[0081] Alternatively, saRNA constructs are contemplated for use in the LNPs described herein that are useful for the delivery of therapeutic proteins or peptides, for example, in protein replacement therapy, particularly due to the typically long expression profile of saRNA compared to conventional mRNA when delivered to cells. The repetitive nature of protein replacement therapy requires LNPs with efficient encapsulation, good cellular delivery and high systemic tolerability (including, for example, low toxicity). The compounds and LNPs disclosed herein exhibit favorable properties to address this need.

[0082] In some embodiments, the self-amplifying RNA molecule is based on the genomic RNA of an RNA virus, but lacks a gene encoding one or more structural proteins. The self-amplifying RNA molecule can be translated to produce the nonstructural proteins of the RNA virus and the heterologous protein encoded by the self-amplifying RNA.

[0083] In some embodiments, the self-amplifying RNA molecule can be designed so that it cannot induce the production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes that code for structural proteins required to produce viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semliki Forest Virus, or Venezuelan Equine Encephalitis Virus (VEEV), one or more genes that code for viral structural proteins, such as capsid and / or envelope glycoproteins, can be omitted. If desired, the self-amplifying RNA molecule of the present invention can be designed to induce the production of attenuated or virulent infectious viral particles, or to produce viral particles that are capable of a single subsequent infection.

[0084] In some embodiments, the self-amplifying RNA molecules described herein can be engineered to express multiple nucleotide sequences from two or more open reading frames, thereby allowing co-expression of proteins, such as two or more antigens, together with cytokines or other immune regulators, which can enhance the generation of an immune response. Such self-replicating RNA molecules can be particularly useful in producing multiple gene products (e.g., proteins) simultaneously, for example, as bivalent or multivalent vaccines or in gene therapy applications.

[0085] In one embodiment, the molar ratio of lipid to oligonucleotide in the lipid nanoparticle is 1:1 to 100:1. In some embodiments, the lipid to mRNA ratio in the liposome can be about 5:1 to about 80:1, about 10:1 to about 75:1, about 15:1 to about 60:1, about 15:1 to about 50:1, and / or at least 40:1. These ratios ensure optimal RNA adsorption to the lipid nanoparticle.

[0086] In some embodiments, the lipid nanoparticles are 30 nm to 250 nm, 40 nm to 250 nm, 50 nm to 250 nm, 50 nm to 150 nm, 50 nm to 130 nm, 60 nm to 230 nm, 70 nm to 210 nm, 70 nm to 200 nm, 80 nm to 200 nm, 90 nm to 200 nm, 70 nm to 190 nm, 80 nm to 190 nm, 70 nm to 180 nm, 70 nm to 150 nm, 70 nm to 130 nm, 70 nm to 110 nm, 80 nm to 150 nm, 80 nm to 130 nm, 80 nm to 120 nm, 90 nm to 110 nm, or , having an average diameter (also referred to as average particle size) of about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, and are substantially non-toxic.

[0087] In certain embodiments, the diameter of the particles ranges from 1 nm to 1000 nm. In certain embodiments, the diameter of the particles ranges from 200 nm to 300 nm. In certain embodiments, the diameter of the particles ranges from 300 nm to 400 nm. In certain embodiments, the diameter of the particles ranges from 400 nm to 500 nm. In certain embodiments, the diameter of the particles ranges from 600 nm to 700 nm. In certain embodiments, the diameter of the particles ranges from 700 nm to 800 nm. In certain embodiments, the diameter of the particles ranges from 800 nm to 900 nm. In certain embodiments, the diameter of the particles ranges from 100 nm to 1000 nm. In certain embodiments, the diameter of the particles ranges from 20 nm to 2000 nm.

[0088] In one embodiment, the N / P ratio between the ionizable cationic lipid and the oligonucleotide (such as saRNA) in the lipid nanoparticle according to the present invention is 5:1 to 50:1. In some embodiments, the N / P ratio in the LNP can be about 5:1 to about 45:1, about 10:1 to about 45:1, about 15:1 to about 40:1, about 20:1 to about 40:1, about 30:1 to about 50:1. These ratios ensure optimal RNA adsorption to lipid nanoparticles, especially when the RNA is of a certain length, such as more than 5000nt, more than 6000, more than 7000nt, more than 8000nt, more than 9000nt, etc.

[0089] In one embodiment, the lipid nanoparticle composition according to the present invention has a zeta potential ranging from -30mV to +30mV. Zeta potential is an indicator of the charge of the particle surface, with a negative zeta potential indicating that the particle surface is mostly covered with RNA, and a positive zeta potential indicating that the complex lipids are not saturated by RNA adsorption. The zeta potential is higher for the composition described in the state of the art, which indicates less binding of oligonucleotides on the surface of the lipid nanoparticles. There is no significant difference in particle size between the present invention and the state of the art.

[0090] Despite the fact that the LNPs of the present invention are particularly useful for use in the context of saRNA (or other large RNA molecules), the latter can also be used to complex other oligonucleotides, such as DNA or RNA. In some embodiments, the LNPs encapsulate long RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), trans-amplified mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA (gRNA), riboswitches, immunostimulatory (immunostimulatory) RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0091] In some embodiments, the LNP encapsulates a modified RNA molecule. In some embodiments, the modification of the RNA molecule comprises a chemical modification, including a backbone modification as well as a sugar modification or a base modification. In this context, the modified RNA molecule as defined herein comprises a nucleotide analog / modification, such as a backbone modification, a sugar modification or a base modification. A backbone modification in the context of the present disclosure is a modification in which the phosphate of the backbone of the nucleotide contained in the RNA molecule is chemically modified. A sugar modification in the context of the present disclosure is a chemical modification of the sugar of the nucleotide of the RNA molecule. Furthermore, a base modification in the context of the present disclosure is a chemical modification of the base portion of the nucleotide of the RNA molecule. In this context, the nucleotide analog or modification is selected from nucleotide analogs applicable to transcription and / or translation. In further embodiments, the modified RNA is 6-aza-cytidine, 2-thio-cytidine, α-thiocytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine. , inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deza-adenosine.

[0092] The lipid-like compound according to formula (I) or a pharma- ceutically acceptable salt is preferably present in the LNP formulation at a concentration of about 5-60 mol%, preferably 12.5-60 mol%, and more preferably 20-45 mol%.

[0093] LNPs are typically formulated with two or more excipients having: (i) a sterol, which enhances LNP bilayer stability and promotes membrane fusion; (ii) optionally, a phospholipid, which reinforces LNP bilayer structure and aids in endosomal escape; and (iii) a lipid-polyethylene glycol (PEG) conjugate, which inserts into the LNP bilayer and provides a PEG coating that reduces LNP aggregation, reduces nonspecific binding of proteins by steric hindrance, and reduces nonspecific endocytosis by immune cells. In further embodiments, the LNPs may further comprise one or more buffering agents.

[0094] In one embodiment, in addition to the lipid-like structures of formula (I), the LNPs according to the invention further comprise: at least PEG or a PEG conjugate, at least one sterol, and - optionally at least one phospholipid and / or at least one second ionizable lipid.

[0095] In one embodiment, the second ionizable lipid is a compound of formula (I). In one embodiment, the second ionizable lipid is not a compound of formula (I).

[0096] In one embodiment, the PEG or PEG conjugate is present in the LNP formulation according to the invention at a concentration of 0.2-10 mol%, preferably 0.5-5 mol%. The PEG compound is preferably selected from PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, and DSPE carboxyPEG. For example, in certain embodiments, the PEG compound is C14 PEG2000 DMG, C15 PEG2000 DMG, C16 PEG2000 DMG, C18 PEG2000 DMG, C14 PEG 2000 ceramide, C15 PEG2000 ceramide, C16 PEG2000 ceramide, C18 PEG2000 ceramide, C14 PEG2000 PE, C15 PEG2000 PE, C16 PEG2000 PE, C18 PEG2000 PE, C14 PEG350 PE, C14 PEG5000 PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, or DSPE carboxy PEG.In a particularly preferred embodiment, the PEG conjugate is DMG-PEG.

[0097] In one embodiment, the sterol compound is present in the LNP formulation according to the invention at a concentration of 30-60 mol%, preferably 30-50 mol%, more preferably 40-50 mol%. The sterol is preferably selected from the group of ergosterol, campesterol, oxysterol, anthrosterol, desmosterol, nicasterol, sitosterol, stigmasterol, cholesterol or a derivative thereof, such as 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol). In a preferred embodiment, the sterol is cholesterol.

[0098] In one embodiment, the LNP formulation according to the invention comprises at least one phospholipid, non-limiting examples of which include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexanediamine (DCPE ... Phospholipids include phosphatidylethanolamine (DPPE), dipalmitoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPS (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, and 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE). Phospholipids reinforce the LNP bilayer structure and aid in endosomal escape. In one embodiment, the phospholipid in the nanoparticle composition is DOPE. In one embodiment, the LNP comprises a phospholipid, the phospholipid being present in said LNP at a concentration of 0.2-45 mol %, preferably 0.5-35 mol %, and said phospholipid is preferably DOPE.

[0099] In one embodiment, the LNP formulation according to the invention comprises at least one second ionizable lipid other than the lipid-like structure described in formula (I). Non-limiting possible second ionizable lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); N-( 1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), N,N-dimethyl-2,3-dioleoyloxy)propyl dimethylaminopropane (DLinDMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA ... 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylamine butyrate (DLin-MC3-DMA), Di((Z)-non-2-en-1-yl)9-propane (DLin-MPZ), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylamine butyrate (DLin-MC3-DMA), Di((Z)-non-2-en-1-yl)9-propane (DLin-MC3-DMA), The second ionizable lipid is present in the LNP formulation of the present invention at a concentration of 0.5-40 mol%, preferably 0.5-30 mol%. In a more preferred embodiment, the second ionizable lipid is C12-200 or DLin-KC2-DMA, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof. .

[0100] In one embodiment, the lipid nanoparticle comprises at least one second ionizable lipid, wherein the total concentration of said ionizable lipid-like structure according to formula (I) or a pharma-ceutically acceptable salt, tautomer, or stereoisomer thereof and said second ionizable lipid is present in said LNP at a concentration of 12.5-60 mol%, preferably 25-50 mol%, and more preferably 30-40 mol%.

[0101] In one embodiment, LNP formulations according to the invention include many commercially available preparations of lipids. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE), GIBCO / BRL, Grand Island, NY), LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3 dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), GIBCO / BRL), and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, Promega Corp., Madison, Wis.).

[0102] In one embodiment, an LNP formulation according to the invention comprises at least two lipids or lipid-like compounds according to formula (I) or selected from Table 1. The presence of at least two ionizable lipids or lipid-like compounds has been found to positively affect the encapsulation of oligonucleotides such as saRNA, as well as the in vivo delivery of the LNP.

[0103] In one embodiment, the lipid nanoparticles comprise at least one ionizable lipid-like structure according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, in a concentration of 12.5-60 mol %; DOPE in a concentration of 0.5-35 mol %; cholesterol in a concentration of 30-50 mol %; and DMG-PEG in a concentration of 0.5-5 mol %, wherein the sum of the concentrations does not exceed 100%.

[0104] In one embodiment, the lipid nanoparticle comprises at least two compounds: one ionizable lipid-like structure according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, and a second ionizable lipid-like compound. In yet another embodiment, the first compound is an ionizable lipid-like structure according to formula (I), and the second compound is another ionizable lipid-like compound, present in a ratio of 2:1 to 1:1. In one embodiment, the first ionizable lipid-like structure according to formula (I), or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, and the second ionizable lipid or lipid-like compound are present in the LNP composition in an equimolar ratio. Thus, the first and second lipid or lipid-like compounds are present in the composition in a ratio of 1:1. In one embodiment, the overall contribution of the composition does not exceed 50 mol% in the overall lipid nanoparticle composition, preferably, the overall contribution of the composition is at least 25 mol%, and more preferably, the overall contribution of the composition is about 35 mol%. In certain embodiments, the two compounds are present in the LNP in an equimolar ratio, whereby the first ionizable lipid-like compound is present between 10-30 mol% and the second ionizable lipid-like compound is present between 10-30 mol%. In another particular embodiment, the ratio of the first ionizable lipid to the second ionizable lipid is greater than 1:1, and both lipids are present at 10-30 mol%.

[0105] LNPs can be prepared using any method known in the art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods known to those skilled in the art. In some embodiments, the method of preparing particles is double emulsion process and spray drying. The conditions used to prepare particles can be altered to obtain particles of desired size or characteristics (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The method of preparing particles and conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) can also depend on the drug to be encapsulated. Methods developed to create particles for delivery of encapsulated drugs are described in the literature.

[0106] The lipid nanoparticles according to the invention can be prepared. More generally, LNPs can be prepared using methods including: - preparing a first alcoholic composition comprising one or more ionizable lipids according to formula (I), a phospholipid different from formula (I), a sterol, a PEG lipid, and a suitable alcoholic solvent; - preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent; - mixing said first and second compositions in a microfluidic mixing device.

[0107] In a further step, the lipid components are mixed at an appropriate concentration in an alcoholic vehicle such as ethanol to which an aqueous composition containing nucleic acids is added and subsequently loaded into a microfluidic mixing device.

[0108] The goal of microfluidic mixing is to achieve complete and rapid mixing of multiple samples (i.e., lipid and nucleic acid phases) in a low-pressure precision mixer. Such sample mixing is typically achieved by enhancing diffusion effects between the flows of different species. Multiple low-pressure precision mixers can be used.

[0109] Other techniques suitable for preparing the LNPs of the invention include dispersing the components in a suitable dispersion medium, such as aqueous and alcoholic solvents, and applying one or more of the following methods: ethanol dilution, simplified hydration, sonication, heating, vortexing, ether injection, French press, cholic acid, Ca 2+ Melting method, freeze-thaw method, reverse phase evaporation method, T-junction mixing, Microfluidic Hydrodynamic Focusing, Staggered Herringbone mixing, etc.

[0110] If the particles prepared by any of the above methods have a particle size range outside the desired range, the particles can be sized, for example, using a sieve. The particles can also be coated. In some embodiments, the particles are coated with a targeting agent. In other embodiments, the particles are coated to achieve desired surface properties (e.g., a specific charge).

[0111] In another aspect of the present invention, a pharmaceutical composition or (RNA) vaccine is disclosed comprising one or more lipid nanoparticles as defined above. Said composition or vaccine is particularly useful for veterinary and human use.

[0112] The pharmaceutical composition or vaccine can be formulated in an aqueous liquid containing one of several buffers. Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, hydroxide calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, dihydrogen potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0113] Suitable routes of administration include parenteral administration. Formulations suitable for parenteral administration, such as intraarticular, intravenous, intraperitoneal, intramuscular, intradermal or subcutaneous injection, include aqueous and non-aqueous, isotonic sterile injection solutions or suspensions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. In the practice of the present invention, the composition is preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.

[0114] The compositions of the (self-replicating) RNA molecules can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Cells transfected with the (self-replicating) RNA molecules can also be administered intravenously or parenterally.

[0115] The composition or vaccine may be administered as a single dose or multiple doses requiring a series of two or more doses administered within a predetermined period of time, which may be from 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks to 1 year.

[0116] In one embodiment, the composition or vaccine is administered periodically, such as annually or biennially. A suitable dose is 0.05 to 1 ml, more preferably 0.25 to 0.75 ml, for example 0.5 ml.

[0117] The pharmaceutical composition or vaccine is preferably sterile and can be sterilized by conventional sterilization techniques. The vaccine or composition can contain pharma- ceutically acceptable auxiliary substances such as pH adjusting and / or buffering agents and tonicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., to approximate physiological conditions.

[0118] The osmolarity (osmotic tension) of the composition or vaccine must be adjusted with sodium salts, such as sodium chloride. The osmolarity of pharmaceutical compositions for parenteral administration is typically 0.9% or 9 mg / ml NaCl.

[0119] Vaccines of the invention may have an osmolality (osmolality) between 200 mOsm / kg and 400 mOsm / kg, for example between 240-360 mOsm / kg or 290-310 mOsm / kg.

[0120] Preservative-free vaccines are desirable. However, if desired, the vaccines of the invention can contain one or more preservatives, such as phenol and 2-phenoxyethanol. Thiomersal, a mercury-containing preservative, should be avoided in favor of mercury-free compositions.

[0121] The vaccines of the invention are preferably non-pyrogenic, for example containing <1 EU (endotoxin unit, a standard measure) per dose, preferably <0.1 EU per dose. The vaccines are preferably gluten-free.

[0122] In a preferred embodiment, the composition or RNA vaccine comprises saRNA molecules, each of which comprises a sequence encoding a nonstructural alphavirus protein and one or more sequences encoding an antigen.

[0123] The self-replicating RNA content of the composition or RNA vaccine of the present invention is generally expressed in RNA amount per dose. Preferred doses are 0.1-100μg self-replicating RNA, preferably 0.5-90μg self-replicating RNA, preferably 0.1-75μg self-replicating RNA, preferably 0.1-50μg self-replicating RNA, preferably 0.5-50μg self-replicating RNA, preferably 0.5-25μg self-replicating RNA, more preferably 0.5-10μg self-replicating RNA, more preferably 1-10μg, even more preferably 1-5μg self-replicating RNA, and can be seen at much lower levels of expression (e.g., 0.05μg self-replicating RNA / dose during in vitro use).

[0124] Preferably, the alphavirus is a Venezuelan Equine Encephalitis Virus (VEEV). In a more particular implementation, the alphavirus is an attenuated Venezuelan Equine Encephalitis Virus (VEEV), such as strain TC-83, or a strain with at least 90% sequence identity, preferably at least 95%, more preferably at least 97%, and even more preferably at least 99% sequence identity. Strain TC-83 is publicly available and its genome is in Genbank under accession number L01443.1.

[0125] For example, various genetically modified mutants (variants) of alphaviruses have been produced that improve the generation of self-replicating RNA molecules and their use for vaccination, as disclosed in US2015299728, WO1999018226 and US7332322, all of which are incorporated herein by reference. In particular, it has been found to be beneficial to have a guanine as the third nucleotide in the 5'UTR of the replicon and / or to have a Q739L mutation in nonstructural protein 2 (nsP2). Thus, in certain embodiments of the present invention, the self-replicating RNA molecule comprises an A3G mutation in the 5'UTR region. In another specific embodiment, the self-replicating RNA molecule comprises a Q739L mutation in nonstructural protein 2 (nsP2). In a preferred embodiment, the self-replicating RNA molecule comprises sequences encoding the nonstructural proteins of an alphavirus, in particular VEEV, in particular VEEV TC-83, wherein the self-replicating RNA molecule comprises an A3G mutation in the 5'UTR and a Q739L mutation in nsP2. In a further preferred embodiment, the self-replicating RNA molecule encodes the nonstructural proteins nsP1, nsP2, nsP3 and nsP4 of VEEV TC-83, wherein preferably the Q739L mutation is present in nsP2.

[0126] In another aspect, the present invention relates to a pharmaceutical composition or vaccine for use in the treatment and / or prevention of disease, both for human and / or veterinary disorders. The present invention provides a vaccine comprising one or more LNPs according to the present invention. The vaccine of the present invention can be used to induce an immune response, in particular an immune response against a disease-associated antigen, or against a cell expressing the disease-associated antigen, such as cancer. Thus, the vaccine can be used for the prophylactic and / or therapeutic treatment of disease, including disease-associated antigens or cells expressing the disease-associated antigen, such as cancer. Preferably, said immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen or an antigen linked to an infectious disease. The antigen encoded by the RNA comprised in the LNPs described herein is preferably a disease-associated antigen or elicits an immune response against a disease-associated antigen or a cell expressing the disease-associated antigen.

[0127] In another embodiment, the LNPs described herein can be used within the framework of protein replacement therapy in subjects in which a particular protein is defective or missing. In a further embodiment, the LNPs and formulations as described herein can be used in protein replacement therapy for rare (genetic) diseases.

[0128] The vaccine or pharmaceutical composition according to the invention may be used for the prevention and / or treatment of infectious diseases caused by viruses, bacteria, fungi and / or parasites.

[0129] Without wishing to be limiting, the viruses include Coronaviruses, Orthomyxoviruses, Paramyxoviridae viruses, Pneumoviruses, Rubulaviruses, Paramyxoviruses, Metapneumoviruses and Morbilliviruses, Poxviridae, Orthopoxviruses, e.g., Variola virus, vera, Picornaviruses, Enteroviruses, Rhinoviruses, Heparnaviruses, Cardioviruses, Aphthoviruses, Bunyaviruses, Heparnaviruses, Filoviruses, Togaviruses, Flaviviruses, Pestiviruses, Hepadnaviruses, other hepatitis viruses, Rhabdoviruses, Caliciviridae, Retroviruses, Reoviruses, Parvoviruses, Herpesviruses, Papovaviruses, Adenoviruses.

[0130] Fungi include, but are not limited to, Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton nigricans ... megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton fabiform. from the Dermatophytes, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis, and Enterocytozoon bieneusi.bieneusi, and less commonly Brachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp., Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisae, Saccharomyces boulardii, and the like. boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp. spp.), Mucor spp., Absidia spp., Mortierella spp., Cunninghamella spp., Saksenaea spp., Alternaria spp., Curvularia spp.spp.), Helminthosporium spp., Fusarium spp., Aspergillus spp., Penicillium spp., Monolinia spp., Rhizoctonia spp., Paecilomyces spp., Pithomyces spp. and Cladosporium spp.

[0131] Without being limited thereto, the parasite may be selected from the Plasmodium genus, such as P. falciparum, P. vivax, P. malariae, or P. ovale. Thus, the present invention may be used for immunization against malaria. In some embodiments, the immunogen is It induces an immune response against parasites from the Caligidae family, in particular those from the genera Lepeophtheirus and Caligus, e.g., Lepeophtheirus salmonis or sea lice such as Caligus rogercresseyi.

[0132] Bacteria include, but are not limited to, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani, Cornynebacterium diphtheriae, Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus agalactiae, and others. agalactiae, Chlamydia trachomatis, Chlamydia pneumoniae, Helicobacter pylori, Escherichia coli, Bacillus anthracis, Yersinia pestis, Staphylococcus epidermis, Clostridium perfringens or Clostridium botulinum, Legionella pneumophila, Coxiella burnetii burnetiid, Brucella, Francisella, Neisseria gonorrhoeae, Treponema pallidum, Haemophilus ducreyiducreyi, Enterococcus faecalis or Enterococcus faecium, Staphylococcus saprophyticus, Yersinia enterocolitica, Mycobacterium tuberculosis, Rickettsia, Listeria monocytogenes, Vibrio cholerae, Salmonella typhi, Borrelia burgdorferi, Porphyromonas gingivalis, Klebsiella.

[0133] In one embodiment, treating cancer comprises administering to a subject in need thereof an effective amount of a vaccine or pharmaceutical composition according to an embodiment of the invention. In some embodiments, the method further comprises administering an anti-cancer agent.

[0134] Without being limited thereto, said tumor-antigens include cancer-testis antigens, such as NY-ESO-1, SSX2, SCP1, and polypeptides of the RAGE, BAGE, GAGE, and MAGE families, such as GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MAGE-12 (which can be used to address, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, bladder tumors; (b) mutant antigens, such as p53 (associated with various solid tumors such as colorectal cancer, lung cancer, head and neck cancer), p21 / Ras (associated with melanoma, pancreatic cancer, colorectal cancer, etc.), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (associated with head and neck cancer, etc.), CIA 0205 (associated with bladder cancer, etc.), HLA-A2-R1701, beta-catenin (associated with melanoma, etc.), TCR (associated with T-cell non-Hodgkin's lymphoma, etc.), BCR-ab1 (associated with chronic myeloid leukemia, etc.), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) overexpressed antigens, such as galectin 4 (associated with colorectal cancer, etc.), galectin 9 (associated with Hodgkin's disease, etc.), proteinase 3 (associated with chronic myeloid leukemia, etc.), WT1 (associated with various leukemias, etc.), carbonic anhydrase (associated with renal cancer, etc.), aldolase A (associated with lung cancer, etc.), PRAME (associated with melanoma, etc.), HER-2 / neu (associated with breast, colon, lung, ovarian cancer, etc.), mammaglobin, alpha-fetoprotein (associated with liver cancer, etc.), KSA (associated with colorectal cancer, etc.), gastrin (associated with pancreatic and gastric cancer, etc.), telomerase catalytic protein, MUC-1 (associated with breast and ovarian cancer, etc.), G-250 (associated with renal cell carcinoma, etc.), p53 (associated with breast and colon cancer, etc.), and carcinoembryonic antigen (associated with cancers of the digestive tract, such as breast, lung, and colorectal cancer, etc.); (d) shared antigens, e.g., melanoma-melanocyte differentiation antigens such as MART-1 / Melan A, gp100, MC1R, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and tyrosinase-related protein-2 / TRP2 (associated with melanoma, etc.); (e) prostate-associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, etc. associated with prostate cancer; (f) immunoglobulin idiotypes (eg, associated with myeloma and B cell lymphoma). In certain embodiments, the tumor immunogens include p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA125, CA 15-3 (CA 27.29 / BCAA), CA195, CA242, CA-50, CAM43, CD68 / KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

[0135] The present invention is further described by the following non-limiting examples which further illustrate the present invention and are not intended, nor should they be construed, to limit the scope of the invention.

[0136] Working Example 1. Synthesis of Compounds According to Formula I 1.1. General scheme 1.1.1 Synthetic route to compound 9-4 [ka]

[0137] 1.1.2. Synthetic Route to Compounds 9-19 [ka]

[0138] 1.1.3 Synthetic routes to compounds 9-23 and 9-25 [ka]

[0139] 1.1.4. Synthetic Routes to Compounds 9-38 and 9-39 [ka]

[0140] 1.1.5 Synthetic routes to compounds 9-46 [ka]

[0141] 1.1.6 Synthetic routes to compounds 9-50 [ka]

[0142] 1.1.7 Synthetic routes to compounds 9-51 and 9-52 [ka]

[0143] 1.1.8 Synthetic routes to compounds 9-53 and 9-54 [ka]

[0144] 1.2 General Experimental Protocol for ZIPH009 Abbreviations used AcOH Acetic acid DCM Dichloromethane DIPEA Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMP Dess-Martin Periodinane EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ESI Electrospray Ionization EtOAc Ethyl acetate HATU Azabenzotriazole tetramethyluronium hexafluorophosphate MeOH Methanol MS mass spectrometry NMR nuclear magnetic resonance TBAF Tetra-n-butylammonium fluoride THF Tetrahydrofuran

[0145] General Considerations Unless otherwise stated, all chemicals and solvents were used as received from their respective suppliers. Acetone (99+%), dichloromethane (99.8+%), ethyl acetate (99.5+%), ethanol (99.8%), toluene (99.85%), 1,2-dichloroethane (99.8+%), methanol (99.8+%), ammonia (7N in methanol) and heptane (99+%) were purchased commercially. Millipore water (MILLI-Q IQ 7005 purification system) was used.

[0146] Chromatographic purification was performed using an automated flash chromatography NextGen300+ system with ELSD and UV detectors using commercially available normal phase silica flash cartridges (12, 40, or 80 g) at a flow rate of 20-30 mL / min. Thin layer chromatography (TLC) analysis was performed using precoated TLC aluminum sheets (DC Kleselgel 60 F254) / UV254 (layer: 0.20 mm silica gel with fluorescent indicator UV254). Spots were detected with UV at 254 nm.

[0147] Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 at 303 K using Bruker Model Avance II 400 (Deimos) and Bruker Model Avance II 700 (Hera) Fourier transform NMR spectrometers (unless otherwise stated). Samples were prepared using approximately 10–30 mg of compound dissolved in 0.6–1.0 mL of deuterated solvent (CDCl3) using ca. All spectra were analyzed using either the TMS reference peak or solvent residual peaks (e.g., 1 For H, δ = 0.00 ppm in CDCl3 13 The NMR spectra were referenced to either the NMR spectrum (δ = 77.16 ppm for C), ...

[0148] Liquid chromatography-mass spectrometry (LC-MS) (Agilent LC / MSD and 1260 Infinity II LC System + Open LAB CDS ChemStation Edition and Thermo Scientific Charged Aerosol Detectors) samples were prepared by dissolving 0.1–1 mg of compound in an acetonitrile:isopropanol (1:1) mixture (~0.5 mL). A reversed-phase column (CSH phenylhexyl column, 130 Å, 2.5 μm, 2.1 mm × 50 mm) was used. Two different methods were used. Method 1: The eluents used were 50% A (0.1% formic acid in H2O), 12.5% ​​B (0.1% formic acid in CH3CN) and 37.5.5% c (0.1% formic acid in isopropanol) with a flow rate of 0.350 mL / min for 33 min. Method 2: The eluent used was 100% A (0.1% formic acid in H2O) to 100% B (0.1% formic acid in CH3CN) over 13.5 min at a flow rate of 0.400 mL / min.

[0149] 1.2.1. Preparation of Compounds 9-11, 9-12, 9-13, 9-14, 9-15, and 9-16 Step 1: Under Ar atmosphere, diol 9-2 (3 equiv.) was dissolved in DCM. To this solution, carboxylic acid 9-1 (1 equiv.), EDC·HCl (1.1 equiv.) and DMAP (0.8–1 equiv.) were added sequentially. The solution was stirred at room temperature overnight. The reaction mixture was then washed twice with (0.2 M–0.5 M) HCl, dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated in vacuo. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% heptane to 20% / 80% heptane / EtOAc) to give the ester intermediates 9-5, 9-6, 9-7, 9-8, 9-9 and 9-10.

[0150] Step 2: Alcohol 9-3 (1 equiv.) was dissolved in DCM under Ar atmosphere. This solution was treated portionwise with Dess-Martin periodinane ts (1.2-1.3 equiv.) at 0-10 °C. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was then treated with saturated Na2S2O3 solution. The aqueous phase was discarded and the organic phase was washed twice with saturated NaHCO3 solution. The combined organic phase was dried over anhydrous magnesium sulfate and evaporated in vacuo. The crude residue was then suspended in heptane and the white solid was removed by filtration. The solvent was evaporated in vacuo and the residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% heptane to 60% / 40% heptane / EtOAc) to give the aldehyde intermediates 9-11, 9-12, 9-13, 9-14, 9-15 and 9-16.

[0151] 1.2.2. Preparation of Compounds 9-21 Step 1: Under Ar atmosphere, amino alcohol 9-17 (1 eq.) was dissolved in DCM. To this solution, carboxylic acid 9-1 (1 eq.), HATU (1.1 eq.) and DIPEA (5 eq.) were added sequentially. The solution was stirred at room temperature overnight. The reaction mixture was then washed twice with brine solution, dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated under vacuum. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% heptane to 35% / 75% heptane / EtOAc) to give amide intermediate 9-20.

[0152] Step 2: Alcohol 9-18 (1 equiv.) was dissolved in DCM under Ar atmosphere. This solution was treated portionwise with Dess-Martin periodinane TS (1.55 equiv.) at 0-10 °C. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was then treated with saturated Na2S2O3 solution. The aqueous phase was discarded and the organic phase was washed twice with saturated NaHCO3 solution. The combined organic phase was dried over anhydrous magnesium sulfate and evaporated in vacuo. The crude residue was then suspended in heptane and the white solid was removed by filtration. The solvent was evaporated in vacuo and the residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% heptane to 60% / 40% heptane / EtOAc) to give the aldehyde intermediate 9-21.

[0153] 1.2.3. Preparation of Compounds 9-26, 9-27, 9-28, 9-29, 9-30, and 9-31 Step 1: Aldehyde intermediate 9-4 (5.5-7.5 equiv.) was dissolved in MeOH under Ar atmosphere and the solution was heated to 30 °C. AcOH (10-30 equiv., 0.17-0.68 vol%) was then added to the solution followed by N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (1 equiv.). After 1 min, sodium cyanoborohydride (6.5-10 equiv.) was added and then stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% DCM to 95% / 3.8% / 1.2% DCM / MeOH / MeOH with NH37N) to give compounds 9-26, 9-27, 9-28, 9-29, 9-30 and 9-31.

[0154] 1.2.4. Preparation of Compounds 9-32, 9-33, 9-34, 9-35, 9-36, and 9-37 Step 1: Aldehyde intermediate 9-4 (4.6-7 equiv.) was dissolved in MeOH under Ar atmosphere and the solution was heated to 30 °C. AcOH (10-30 equiv., 0.17-0.68 vol%) was then added to the solution followed by 2,2'-(piperazine-1,4-diyl)diethanamine (1 equiv.). After 1 min, sodium cyanoborohydride (6.5-9 equiv.) was added and then stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% DCM to 95% / 3.8% / 1.2% DCM / MeOH / MeOH with NH37N) to give compounds 9-32, 9-33, 9-34, 9-35, 9-36 and 9-37.

[0155] 1.2.5. Preparation of Compounds 9-40 Step 1: Aldehyde intermediate 9-19 (5.6 equiv.) was dissolved in MeOH under Ar atmosphere and the solution was heated to 30° C. Then AcOH (30 equiv., 0.17 vol%) was added to the solution followed by N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (1 equiv.). After 1 min, sodium cyanoborohydride (10 equiv.) was added and then stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% DCM to 95% / 3.5% / 1.5% DCM / MeOH / MeOH with NH37N) to give compound 9-40.

[0156] 1.2.6. Preparation of Compounds 9-41 Step 1: Under Ar atmosphere, the aldehyde intermediate 9-19 (5 equiv.) was dissolved in MeOH and the solution was heated to 30° C. Then AcOH (25 equiv., 0.12 vol%) was added to the solution followed by 2,2′-(piperazine-1,4-diyl)diethanamine (1 equiv.). After 1 min, sodium cyanoborohydride (9 equiv.) was added and then stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was then purified by normal phase on a NextGen 300+ flash chromatography system (gradient from 100% DCM to 95% / 3.5% / 1.5% DCM / MeOH / MeOH with NH37N) to give compound 9-41.

[0157] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17] [Table 3-18] [Table 3-19]

[0158] 2. Preparation and testing of LNPs 2.1 LNP formulation Cationic lipids, DOPE, cholesterol, and DMG-PEG lipids were dissolved in ethanol or DMSO at a molar ratio of 35:20:43.5:1.5 and concentrations of 100:25:20:25 μg / μL (for C12-200) or 170:25:20:25 μg / μL (for comparison lipids). Lipid nanoparticles (LNPs) were prepared by targeting saRNA with an N / P ratio of approximately 40 / 1. Briefly, firefly luciferase saRNA of approximately 9659 nucleotides was diluted to 0.5 mg / mL in 5 mM citrate buffer at pH 4.5. The lipid solution and the aqueous saRNA solution were mixed at a ratio of approximately 1:3 (vol / vol) using a microfluidic mixer (Ignite from Precision Nanosystems, California, USA) with a total flow rate of 10 ml / min or higher. Then, the ethanol was removed and the external buffer was replaced with 10 mM TRIS-HCL, pH 7.4 by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with 0.2 μm pores. The particle size of the lipid nanoparticles was 50-150 nm and the polydispersity index (PI) was 0.1-0.4, measured by Zeta Sizer (Malvern Panalytical, UK). The charge of the formulations was also measured by Zeta Sizer and ranged from 20 to -20 mV.

[0159] See Table 3 below for the results obtained. Comparator compounds 1 and 2 have the following structures C1 and C2, respectively: [ka]

[0160] 2.2 Encapsulation saRNA loading in LNP formulations was quantified using the Quant-iT RiboGreen assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as previously described. Fourteen samples were diluted 10-fold in 1× Tris HCL-EDTA (TE) buffer (10 mM Tris-HCL, 1 mM EDTA, pH 7.5) with or without 2% (v / v) Triton X-100 (Sigma-Aldrich, Saint Louis, Missouri, USA). Standard solutions were also prepared in 1× TE with or without 2% (v / v) Triton X-100 to account for fluorescence variations. The assay was performed according to the manufacturer's protocol. Samples were loaded into a black 96-well plate and fluorescence was analyzed at 485 nm excitation and 528 nm emission on a microplate reader (Tecan Infinite® 200 PRO). Encapsulation efficiency was calculated to be between 80 and 100%. See Table 3 below for the results obtained.

[0161] 2.3 In vivo testing Female Swiss mice (6 weeks old) were purchased from Janvier Laboratories (Paris, France) and housed in individually ventilated cages with free access to food and water. Mice were anesthetized with isoflurane (Zoetis, Louvain-La-Neuve, Belgium) (induction 5%, maintenance 2%) and injected intramuscularly with a total of 1 μg of LNP-formulated luciferase saRNA in 100 μL Tris-HCL (50 μL / leg) (n=7 per group). Expression of luciferase-induced bioluminescence was measured via non-invasive bioluminescence imaging (In vivo Imaging System (IVIS) Lumina III, Perkin Elmer, Waltham, Massachusetts, USA) 10 min after subcutaneous injection of 200 μL D-luciferin (GoldBio, Saint Louis, Missouri, USA, #LUCK-1G) on days 0 (before injection), 1, 3, 5, 7, 10, 15 and 20.

[0162] See Table 4 and Figure 1 for results showing higher expression of saRNA when administered in LNPs containing compounds of the present invention compared to prior art compounds. Table 5 supports the good tolerability of compounds according to Formula I.

[0163] [Table 4] [Table 5] [Table 6-1] [Table 6-2]

[0164] Example 3. Formulation of LNPs according to one embodiment of the present invention Several LNPs are obtained that contain saRNA and at least one compound shown in Table 1. These LNPs have been tested and shown to efficiently encapsulate RNA and deliver RNA in vivo. Examples of LNP in action: [Table 7]

Claims

1. Formula (I): 【Chemistry 1】 The compound according to each m is independently 1, 2, 3, 4, or 5; and R １ Ha, -L １ N [L ２ F １ R Ａ ] ２ Or -L ３ F ２ R Ｂ and each R ２ , R ３ , and R ４ Ha, -L ４ F ３ R Ｃ and Each L １ , L ２ , L ３ , and L ４ are independently C2-C10 alkylene; Each F １ , F ２ , and F ３ is independently an ester or amide functional group, and Each R Ａ , R Ｂ , and R Ｃ are independently branched C4-C30 alkyl; A compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

2. Each R Ａ , R Ｂ , and R Ｃ is independently selected from undecane-5-yl, tridecane-6-yl, pentadecan-7-yl, heptadecan-8-yl, and nonadecan-9-yl.

3. Each L ２ , L ３ , and L ４ is independently selected from C4-C10 alkylene, or from C4-C8 alkylene, or from C4, C5, or C6 alkylene; or Each L ２ , L ３ , and L ４ The compound of claim 1 , wherein is butylene or hexylene.

4. -L ２ F １ R Ａ , -L ３ F ２ R Ｂ , and -L ４ F ３ R Ｃ is independently an ester group.

5. F １ , F ２ , and F ３ is an ester group positioned according to -L-O-C(=O)-R, -L is L ２ , L ３ , and L ４ represents one of the following: -R is R Ａ , R Ｂ , and R Ｃ 2. The compound of claim 1, wherein:

6. A lipid nanoparticle (LNP) comprising at least one compound according to any one of claims 1 to 5 and one or more oligonucleotides.

7. The lipid nanoparticle of claim 6, wherein the oligonucleotide is RNA or DNA, preferably an RNA-like oligonucleotide having a length of at least 5000 nt.

8. The lipid nanoparticles are at least one PEG or PEG conjugate; at least one sterol, and at least one phospholipid and / or at least one second ionizable lipid; The lipid nanoparticle of claim 6, further comprising:

9. The lipid nanoparticle of claim 6, wherein the compound according to formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is present in the LNP at a concentration of 12.5 to 60 mol%.

10. The lipid nanoparticle of claim 6, wherein the lipid nanoparticle comprises at least one second ionizable lipid, and the total concentration of the ionizable lipid or lipid-like compound is 12.5 to 60 mol%.

11. The LNP comprises a phospholipid; The phospholipid is present in the LNP at a concentration of 0.5 to 35 mol %, The lipid nanoparticle of claim 6, wherein the phospholipid is preferably DOPE.

12. the sterol is present at a concentration of 30 to 50 mol %, The lipid nanoparticle of claim 6, wherein the sterol is preferably cholesterol.

13. containing PEG conjugates at a concentration of 0.5-5 mol %; The PEG conjugate is preferably DMG-PEG. The lipid nanoparticle of claim 6.

14. A pharmaceutical composition or vaccine comprising one or more lipid nanoparticles according to claim 6 and, optionally, a pharmaceutically acceptable carrier.

15. 15. A pharmaceutical composition or vaccine according to claim 14 for use in the treatment and / or prevention of a disease.

16. A compound according to any one of claims 1 to 5 for use in the preparation of a pharmaceutical composition for therapeutic use, comprising RNA encoding a gene of interest.

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