Use of MC3-type lipids and lipid nanoparticles in preparation

Abstract

MC-3 type lipids and methods for preparing such lipids are provided. The lipids have the formula (A) (wherein each R is independently a C 12 ~C 16 where at least one of the double bonds is in the Z configuration; R' is an optional alkyl; W is O, NH, or NR", where R" is a C1-C3 alkyl group; X is O, NH, NR", or absent; Z is [-(CH2) m -NG 1 G 2 G 3 In the formula, m is 1 to 5; 1 and G 2 is independently 1~3 is alkyl, G 3 is hydrogen, C 1~3 Such lipids are useful in the preparation of delivery vehicles for siRNA and other pharmaceutical agents. In selected embodiments, such delivery vehicles take the form of lipid nanoparticles. JPEG2024521852000019.jpg34170

Description

Technical Field

[0001] This specification provides lipids that can be incorporated into delivery vehicles to facilitate the encapsulation of a wide range of therapeutic agents or prodrugs, such as, but not limited to, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceuticals, and salts thereof.

Background Art

[0002] Nucleic acid-based therapies have great potential in medicine. However, in order to realize this potential, nucleic acids must be delivered to the target site in the patient. This presents a challenge because nucleic acids are rapidly degraded by enzymes in the plasma after administration. Even if the nucleic acid is delivered to the disease site, the problem of intracellular delivery still remains. To address these problems, lipid nanoparticles have been developed that protect nucleic acids from such degradation and facilitate delivery through the cell membrane to intracellular compartments where the relevant translation machinery exists.

[0003] An important component of lipid nanoparticles (LNP) is ionizable lipid. Ionizable lipids are typically positively charged at low pH, which facilitates their association with negatively charged nucleic acids. However, ionizable lipids are neutral at physiological pH and are more biocompatible in biological systems. Furthermore, it has been suggested that the ability of these lipids to ionize at low pH after the lipid nanoparticles are taken up by cells via endocytosis enables endosomal escape. This, on the one hand, allows the nucleic acid to be released into the intracellular compartment. Most studies on cationic LNP have focused on the formulation of nucleic acids, but in addition to nucleic acids, the delivery of other therapeutic agents or prodrugs is also possible using the delivery platform.

[0004] Considerable research has been devoted to the identification of amino lipids with high efficacy. The ionizable lipid called DLin-MC3-DMA or "MC3" (dilinoleyl-methyl-4-dimethylaminobutyrate) constitutes the state-of-the-art ionizable lipid for siRNA formulations. This ionizable lipid is a key component of Onpattro (registered trademark), a lipid nanoparticle formulation incorporating siRNA that silences the gene causing a hereditary neurodegenerative disease called hereditary transthyretin-mediated amyloidosis. Such formulations containing MC3 constituted the first small interfering RNA (siRNA)-based therapy approved by the US Food and Drug Administration (FDA).

[0005] The MC3 ionizable lipid is widely recognized as an improved version of another amino lipid called KC2, which has approximately threefold the potency. In studies of over 50 amino lipids, MC3 was identified as having an ED 50 of 0.03, while the ED 50 of KC2 was 0.10 with respect to FVII gene silencing in mice using siRNA. (Jayaraman et al., 2012, Angew. Chem. Int. Ed., 51:8529 - 8533.) This means that formulations containing MC3 require approximately one-third the amount of siRNA to achieve the same final result as similar KC2-based formulations. Since nucleic acids are expensive, this represents a significant savings for the large-scale manufacture of ionizable lipids.

[0006] Both the MC3 and KC2 amino lipids (structures are as follows) have two carbon chains (designated herein as "R") that converge on a single carbon atom, which functions as the attachment point for an ionizable terminal "head" group.

[0007]

Chemical Structure

[0008] In both MC3 and KC2, the carbon chain is an unsaturated carbon obtained from linoleic acid or the corresponding ester. 18 These 18-carbon unsaturated chains are the best chains identified to date for the maximum potency of siRNA formulations. (Semple et al., 2010, Nat. Biotechnol. 28:172~176 and Heyes et al., 2005, J. Controlled Release, 107:276~287.) Furthermore, aminolipids with chains shorter than 18 carbon atoms are difficult to prepare using conventional synthetic routes. Shorter-chain lipids can be produced from esters of unsaturated fatty acids incorporating fewer than 18 carbon atoms, but such esters are not found in nature or are extremely expensive to synthesize using known methods. Therefore, little attention has been paid to studying aminolipids for nucleic acid delivery that have unsaturated chains with fewer than 18 carbon atoms.

[0009] In this technological field, there is a need for ionizable lipids for the delivery of therapeutic agents or prodrugs that have improved or equivalent efficacy to known lipids and can be manufactured conveniently and cost-effectively.

[0010] definition As used herein, “MC3-type lipid” refers to any lipid having the structure defined by formula A or its equivalent, including but not limited to ionizable lipids.

[0011] As used herein, the term "ionizable lipid" refers to a lipid that is electrostatically neutral at a given pH and can accept or donate protons, thereby becoming electrostatically charged, the electrostatically neutral form having a calculated logarithm greater than 8 of the partition coefficient between water and 1-octanol (i.e., cLogP). As used herein, the term "alkyl" in relation to the R group as described herein refers to a carbon-containing chain having various degrees of saturation, whether linear or branched.

[0012] As used herein, the term "C1-C3 alkyl" refers to a linear or branched carbon chain having a total of up to three carbon atoms, which is optionally unsaturated.

[0013] As used herein, the term "helper lipid" means sterols, e.g., cholesterol or its derivatives; diacylglycerols or their derivatives, glycerophospholipids including, e.g., phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), etc.; and sphingolipids, e.g., ceramides, sphingomyelin, cerebrosides, gangliosides, or their reduced analogs lacking double bonds in the sphingosine unit. This term encompasses natural or synthetic lipids.

[0014] As used herein, the term “delivery vehicle” includes, but is not limited to, any preparations in which the lipids described herein can be formulated, including a delivery vehicle containing a helper lipid.

[0015] As used herein, the term “nanoparticles” refers to any suitable particles that may be formulated from lipids and may contain one or more helper lipid components. The one or more lipid components may include ionizable lipids prepared by the methods described herein, and / or additional lipid components such as helper lipids. The term includes, but is not limited to, multilayer vesicles, monolayer vesicles, and bilayer vesicles, including vesicles having a high electron-density core. The term also includes polymer-lipid hybrids, which include particles in which lipids are bonded to a polymer.

[0016] As used herein, the term “encapsulation” in relation to the incorporation of cargo molecules into a delivery vehicle refers to any association between any component or section of the delivery vehicle, such as nanoparticles, and the cargo. [Prior art documents] [Patent Documents]

[0017] [Patent Document 1] U.S. Provisional Patent Application No. 63 / 194,471 [Patent Document 2] U.S. Provisional Patent Application No. 63 / 195,269 [Patent Document 3] U.S. Provisional Patent Application No. 63 / 202,210 [Non-patent literature]

[0018] [Non-Patent Document 1] Jayaraman et al., 2012, Angew. Chem. Int. Ed., 51:8529~8533 [Non-Patent Document 2] Semple et al., 2010, Nat. Biotechnol. 28:172~176 [Non-Patent Document 3] Heyes et al., 2005, J. Controlled Release, 107:276~287 [Non-Patent Document 4] Kulkarni et al., 2018, ACS Nano, 12:4787 [Non-Patent Document 5] Kulkarni et al., 2017, Nanoscale, 36:133347 [Non-Patent Document 6] Rebuffat et al., 2002, Faceb J. 16(11):1426-8 [Overview of the project] [Problems that the invention aims to solve]

[0019] This disclosure aims to address one or more limitations in the known field of the art, or to provide useful alternatives thereto. [Means for solving the problem]

[0020] The present disclosure is at least partially based on the surprising discovery that certain MC3-type amino lipids having an unsaturated chain with less than 18 carbon atoms have improved nucleic acid delivery efficacy when formulated within a delivery vehicle. The effectiveness of such short-chain amino lipids is better than or equivalent to that of the state-of-the-art amino lipid, MC3 having dilinoleyl chains (unsaturated C 18 ). For example, the inventors have found that short-chain MC3-type lipids (e.g., having an unsaturated C 17 moiety) have mRNA efficacy in vitro and in vivo that is superior to or equivalent to that of the longer-chain benchmark lipid MC3 when formulated within a delivery vehicle. The inventors have further found that siRNA-containing delivery vehicles comprising short-chain MC3-type lipids (e.g., having an unsaturated C 17 moiety) have efficacy that is equivalent to or somewhat better than MC3 in some cases, which is in contrast to studies suggesting that the dilinoleyl chains (unsaturated C 18 ) of MC3 lipids are optimal for activity.

[0021] Furthermore, the inventors have identified a class of short-chain (shorter than the unsaturated moiety) MC3-type lipids that are readily prepared by the methods described herein, in which longer-chain fatty acid esters (e.g., commercially available unsaturated C 18 ~C 22 ) are shortened and then converted to MC3-type lipids in a synthetic pathway that includes the Claisen condensation step described herein. Accordingly, the present disclosure further addresses previous drawbacks in the synthesis of MC3-type lipids having less than 18 carbon atoms and enables the preparation of a new class of short-chain lipids.

[0022] Other objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description and figures.

Brief Description of the Drawings

[0023] [Figure 1] ​​Figure 1A is a bar graph showing the capture percentage, particle size, and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) containing the ionizable lipid nor-MC3 or DLin-MC3-DMA (MC3). The LNPs were composed of 50 / 10 / 38.5 / 1.5 mol% ionizable lipid / DSPC / chol / PEG-DMG, with an amine-to-phosphate (N / P) ratio of 6. Figure 1B is a graph showing the luminescence intensity as a function of mRNA concentration after adding mRNA-containing LNPs containing the ionizable lipid nor-MC3 or MC3 to HuH7 cells. The LNPs were composed of 50 / 10 / 38.5 / 1.5 mol% ionizable lipid / DSPC / chol / PEG-DMG (N / P=6), and the mRNA encoded firefly luciferase. Figure 1C shows the luminescence intensity / mg in the liver (left graph) or spleen (right graph) of LNPs containing ionizable lipid nor-MC3 or mRNA containing MC3, 4 hours after intravenous administration to C57Bl / 6J mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% ionizable lipids / DSPC / chol / PEG-DMG (N / P=6). [Figure 2] Figure 2A is a bar graph showing the capture percentage, particle size, and PDI of siRNA-containing LNPs that include ionizable lipids nor-MC3 or MC3. The LNPs contained 50 / 10 / 38.5 / 1.5 mol% ionizable lipids / DSPC / chol / PEG-DMG, with an N / P ratio of 3. Figure 2B is a graph showing the normalized luminescence intensity (%) after adding siRNA-containing LNPs that include ionizable lipids nor-MC3 or MC3 to 22Rv1 cells modified to stably express luciferase. The LNPs contained 50 / 10 / 38.5 / 1.5 mol% ionizable lipids / DSPC / chol / PEG-DMG (N / P=3), and the siRNA encoded luciferase. [Modes for carrying out the invention]

[0024] MC3 type lipids This disclosure provides MC3-type lipids having the structure of formula A: Formula A:

[0025] [ka]

[0026] Each R is independent of C 12 ~C 16 The alkyl group has a carbon skeleton, and each alkyl group has 1 to 3 C=C double bonds, or 1 to 2 double bonds, or 2 double bonds, and at least one of the double bonds is in a Z configuration, most favorably each double bond of R is in a Z configuration; R' is C2~C 24 It is an optional alkyl group having a skeleton and containing 0 to 3 C=C double bonds; Each R, and any R' alkyl group, is optionally substituted with one or more C1-C3 alkyl groups at any given position; W is O, NH, or NR, where R is a C1-C3 alkyl group, such as methyl; X is either absent or present, and if present, is O, NH, or NR'', where R'' is a C1-C3 alkyl, such as methyl; Z is [-(CH2)] m -NG 1 G 2 G 3 It is an alkylamino chain defined as ], Here, m is 1-5, 2-4, or 2-3, G 1 and G 2 These are independently C1-C3 alkyl groups, such as methyl, and G 3 It is a hydrogen atom that does not exist (i.e., a lone pair of electrons), or a C1-C3 alkyl group, such as methyl.

[0027] In one embodiment, each R alkyl group has two double bonds in a Z configuration.

[0028] In one embodiment, the C1-C3 alkyl groups of formula A substituted on the R, R' alkyl skeleton or R' of "NR" are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl. If present, the C1-C3 alkyl groups typically replace hydrogen atoms on the R and / or R' carbon skeleton.

[0029] In another embodiment, the lipid of formula A has the structure of formula B below. Formula B:

[0030] [ka]

[0031] In the formula, the R alkyl group (see formula A) is R 1 -[CH2] n It is represented in parts, and this is C 12 ~C 17 It is a linear alkyl group, [CH2] n The n of the part is 2 to 7, and each R 1 C 15 The following is R 1 -[CH2] n The portion is optionally substituted with a C1-C3 alkyl group; Each R 1 Each independently has 1 to 3 double bonds, or 1 to 2 double bonds, or 2 double bonds, with at least one of the double bonds in a Z configuration, and most favorably R 1 Each double bond is in a Z configuration.

[0032] In another embodiment, X in formula B is absent, and the lipid has the structure of formula C described below. Formula C:

[0033] [ka]

[0034] In the formula, m is 1 to 5, 2 to 4, or 2 to 3; G 1and G 2 These are independently C1-C3 alkyl groups, and most advantageously, each G 1 and G 2 It is a methyl group; G 3 It is either nonexistent (i.e., a lone pair of electrons) or hydrogen (depending on pH).

[0035] In another embodiment, the lipid of formula B has the structure of formula D described below. Formula D:

[0036] [ka]

[0037] In the formula, n is between 2 and 7.

[0038] In another non-limiting example, the lipid of general formula B has the structure of formula E shown below. Formula E:

[0039] [ka]

[0040] In the formula, n is between 2 and 7.

[0041] In a further embodiment, the lipid of formula B has the structure of formula F below. Formula F:

[0042] [ka]

[0043] In another embodiment, the lipid has a nor-MC3 structure.

[0044] [ka]

[0045] C 17 Or a method for producing MC3 type lipids having a shorter R alkyl group. C 17 Alternatively, MC3-type lipids having shorter R-alkyl groups can be prepared using the method described below. Linoleic acid esters (e.g., methyl linoleate) are the starting materials in the synthesis scheme described below, but as will be understood by those skilled in the art, other fatty acids may also function as starting materials, and the scheme described below is merely an example of a selected embodiment.

[0046] As mentioned above, in both KC2 and MC3 ionizable aminolipids, the carbon chain is unsaturated C obtained from linoleic acid or the corresponding ester. 18 It is a part.

[0047] [ka]

[0048] MC3 1 and its analogues can be represented by general formula 3 (Scheme 2 below). The method described herein provides the synthesis of MC3-type lipid 4, the chain incorporating 17 or fewer carbon atoms.

[0049] [ka]

[0050] In the formula, n is between 2 and 7; W is O, NH, or NR" in the formula, where R" is a C1-C3 alkyl, such as methyl; X is either absent or present, and if present, is O, NH, or NR'', where R'' is a C1-C3 alkyl, such as methyl; Z is [-(CH2)] m -NG 1 G 2 G 3 An alkylamino chain defined as ], where m is 1-5, 2-4 or 2-3, G 1 and G2 These are independently C1-C3 alkyl groups, such as methyl, and G 3 It is either a non-existent (i.e., lone pair) hydrogen, or a C1-C3 alkyl, such as methyl.

[0051] A type 4 lipid with n=7 is described in concurrently pending and co-owned U.S. Provisional Patent Application No. 63 / 194,471, “Method for Producing an Ionizable Lipid,” which is incorporated herein by reference.

[0052] U.S. Provisional Patent Application No. 63 / 194,471 also describes a method for producing ionizable lipids from common fatty acid esters 5 (Scheme 3). As described therein, the fatty acid ester is subjected to Claisen condensation under Mukaiyama conditions to produce ketoester 6, which is optionally R 2 An alkyl group is added.

[0053] [ka]

[0054] The ketoester is subsequently hydrolyzed and decarboxylated to produce ketone 7 (or 7a), which can be further reduced to alcohol 8 (or 8a). As described in the co-pending U.S. Provisional Patent Application No. 63 / 194,471, ketalization of ketone 7 yields a KC2-type lipid, and esterification of alcohol 8 yields an MC3-type lipid.

[0055] The chemistry involves esters in which n carbon atoms are incorporated into the acidic portion, providing ionizable lipids having a hydrophobic chain with n-1 carbon atoms. For example, C 18 Methyl linoleate, a fatty acid ester, is C 17 This yields a nor-MC3 exhibiting a chain (Scheme 4). Such a nor-lipid structure can be represented as structure 4 above, with n=7.

[0056] [ka]

[0057] Type IV, shorter-chain lipids, can be obtained from esters of unsaturated fatty acids incorporating fewer than 18 carbon atoms.

[0058] Formulation of lipids within the delivery vehicle The MC3-type lipids of this disclosure can be formulated within a variety of drug delivery vehicles (also referred to herein as “delivery vehicles”) known to those skilled in the art. Examples of delivery vehicles include lipid nanoparticles, which include liposomes, lipoplexes, lipid-containing polymer nanoparticles, polymer-based nanoparticles, emulsions, and micelles.

[0059] In one embodiment, the MC3-type lipid of the present disclosure is formulated within a delivery vehicle by mixing it with additional lipids, which optionally include helper lipids such as vesicle-forming lipids and aggregation-inhibiting lipids such as hydrophilic polymer-lipid complexes (e.g., PEG-lipids).

[0060] As described above, helper lipids include sterols, diacylglycerols, ceramides, or their derivatives.

[0061] Examples of sterols include cholesterol or cholesterol derivatives, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, and fucosterol.

[0062] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dierydoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof. These lipids may be synthesized or obtained from natural sources such as eggs.

[0063] Suitable ceramide derivatives are egg sphingomyelin or dihydrosphingomyelin.

[0064] Delivery vehicles incorporating the MC3-type lipids of this disclosure can be prepared using a wide variety of well-described formulation methods known to those skilled in the art, including, but not limited to, extrusion, ethanol injection, and in-line mixing. In one embodiment, the preparation method is an in-line mixing technique in which an aqueous solution and an organic solution are mixed using a rapid mixing device, as described in whole herein by reference in Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni et al., 2017, Nanoscale, 36:133347, respectively.

[0065] The delivery vehicle may also be nanoparticles that are lipoplexes containing a lipid core stabilized by a surfactant. Vesicle-forming lipids may be used as stabilizers. In another embodiment, the lipid nanoparticles are polymer-lipid hybrid systems containing a polymer nanoparticle core surrounded by stabilized lipids.

[0066] The MC3-type lipid-containing nanoparticles of this disclosure may, alternatively, be prepared from lipid-free polymers. Such nanoparticles may comprise a concentrated core of a therapeutic agent surrounded by a polymer shell, or they may have a solid or liquid dispersed throughout the polymer matrix.

[0067] The MC3-type lipids described herein may also be incorporated into emulsions, which are drug delivery vehicles containing oil droplets or oil cores. The emulsions may be lipid-stabilized. For example, the emulsions may contain oil-filled cores stabilized with emulsifying components such as lipid monolayers or bilayers.

[0068] MC3-type lipids may be incorporated into micelles. Micelles are self-assembling particles composed of amphiphilic lipids or polymer components that are used for drug delivery within a hydrophobic core.

[0069] A further class of drug delivery vehicles known to those skilled in the art that may be used to formulate MC3-type lipids in this specification is carbon nanotubes.

[0070] Delivery of nucleic acids, genetic material, proteins, peptides, or other charged drugs The MC3-type lipids disclosed herein can facilitate the incorporation of compounds or molecules having a net negative or positive charge (also referred to herein as "cargo" or "cargo molecule") into a delivery vehicle and subsequent delivery to target cells in vitro or in vivo.

[0071] In one embodiment, the cargo molecule is genetic material such as nucleic acid. The nucleic acid may include, but is not limited to, RNA including small interfering RNA (siRNA), nuclear small RNA (snRNA), microRNA (miRNA), messenger RNA (mRNA), or DNA, such as vector DNA or linear DNA. The length of the nucleic acid can vary and may include nucleic acids ranging from 5 to 50,000 nucleotides in length. The nucleic acid may be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or hybrids thereof. Single-stranded nucleic acid may include antisense oligonucleotides.

[0072] In one embodiment, the cargo is mRNA comprising at least one peptide, polypeptide, or polynucleotide encoding a protein. The mRNA includes, but is not limited to, small activated RNA (saRNA) and trans-amplified RNA (taRNA), as described herein by reference in concurrently pending U.S. Provisional Patent Application No. 63 / 195,269, “mRNA Delivery Using Lipid Nanoparticles.”

[0073] The mRNA used herein includes both modified and unmodified RNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA may be purified from a natural source, produced using a recombinant expression system and optionally purified, or chemically synthesized.

[0074] In embodiments where mRNA is a chemically synthesized molecule, mRNA may include nucleoside analogs, such as chemically modified bases or sugars, and / or analogs having skeletal modifications. In some embodiments, mRNA may include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7- Azaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages), or comprising them.

[0075] The mRNAs of this disclosure may be synthesized according to any of a variety of known methods. For example, the mRNAs in a particular embodiment may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0076] In some embodiments, the mRNA synthesized in vitro may be purified before encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0077] This disclosure can be used to encapsulate mRNA of various lengths. In some embodiments, this disclosure can be used to encapsulate in vitro synthesized mRNA in lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0078] Typically, mRNA synthesis involves the addition of a "cap" at the 5' end and a "tail" at the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect mRNA from exonuclease degradation.

[0079] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron-responsive elements. In some embodiments, the 5' untranslated region may be about 50 to 500 nucleotides in length.

[0080] In some embodiments, the 3' untranslated region includes one or more of the following: a polyadenylation signal, a protein binding site that affects the stability of mRNA location within the cell, or one or more miRNA binding sites. In some embodiments, the 3' untranslated region may be 50 to 500 nucleotides or longer.

[0081] While mRNA generated from in vitro transcription reactions may be desirable in certain embodiments, mRNA from other mRNA sources, such as bacteria, fungi, plants, and / or animals, is also intended.

[0082] mRNA sequences may include reporter gene sequences, but the inclusion of reporter gene sequences in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA in in vitro or in vivo studies in animal models to assess their in vivo distribution.

[0083] In another embodiment, the cargo is siRNA. The siRNA is incorporated into an endogenous cellular mechanism to cause mRNA disruption, thereby preventing transcription. Since RNA is readily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to the target site.

[0084] siRNAs incorporated in embodiments of this disclosure can be used to specifically inhibit the expression of a wide variety of target polynucleotides. siRNA molecules targeting specific polynucleotides can be readily prepared according to procedures known in the art. siRNA target sites can be selected, and the corresponding siRNAs can be chemically synthesized, formed by in vitro transcription, or expressed from vectors or PCR products. A wide variety of different siRNA molecules can be used to target specific genes or transcripts. siRNAs may be double-stranded RNA, or hybrid molecules containing both RNA and DNA, e.g., one RNA strand and one DNA strand. siRNAs may vary in length, e.g., 15–30 nucleotides or 20–25 nucleotides. In certain embodiments, siRNAs are double-stranded and have a 3' overhang or a 5' overhang. In certain embodiments, the overhangs are UU or dTdT 3'. In certain embodiments, siRNAs have a stem-loop structure.

[0085] In further embodiments, the cargo molecule is a microRNA or nuclear small RNA. MicroRNAs (miRNAs) are short non-coding RNA molecules that are transcribed from genomic DNA but are not translated into proteins. These RNA molecules are thought to play a role in regulating gene expression by binding to a region of target mRNA. Binding of miRNAs to target mRNA can downregulate gene expression, for example, by inducing translational repression, deadenylation, or degradation of the target mRNA. Nuclear small RNAs (snRNAs) are typically longer non-coding RNA molecules involved in gene splicing. snRNA molecules may have therapeutic importance in diseases resulting from splicing defects.

[0086] In another embodiment, the cargo is a DNA vector described in the shared concurrently pending U.S. Provisional Patent Application No. 63 / 202,210, “DNA Vector Delivery Using Lipid Nanoparticles,” which is incorporated herein by reference. The DNA vector may be administered to a subject for the purpose of repairing, enhancing, blocking, or reducing the expression of cellular proteins or peptides. Thus, the nucleotide polymer may be a nucleotide sequence comprising genomic DNA, cDNA, or RNA.

[0087] As those skilled in the art will understand, a vector may encode a promoter region, an operator region, or a structural region. A DNA vector may contain double-stranded DNA or be composed of a DNA-RNA hybrid. Examples of double-stranded DNA, not limited to these, include structural genes, genes containing operator control and stop regions, and self-replicating systems such as vector DNA.

[0088] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triple-stranding oligonucleotides. To maintain long-term activity, single-stranded nucleic acids preferably have some or all of their nucleotide links substituted with stable non-phosphodiester links, such as phosphorothioates, phosphorodithioates, phosphoroselenates, or O-alkylphosphotriester links.

[0089] DNA vectors may contain nucleic acids modified at one or more sugar moieties and / or one or more pyrimidine or purine bases. Such sugar modifications may include substitution of one or more hydroxyl groups with halogen, alkyl, amine, or azide groups, or may be functionalized as ethers or esters. In another embodiment, the entire sugar may be substituted with sterically and electronically similar structures, including aza sugars and carbocyclic sugar analogs. Modifications at the purine or pyrimidine base moieties include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutions known to those skilled in the art.

[0090] In certain embodiments, DNA vectors may be modified with modifying molecules such as peptides, proteins, steroids, or sugar moieties. Modification of DNA vectors with such molecules can facilitate delivery to target sites. In some embodiments, such modifications cause the DNA vector to move across the nuclei of target cells. For example, modifying factors may bind to specific portions of the DNA vector (typically not encoding the target gene), but may also have nuclear homing properties, such as peptides or other modifying factors with nuclear localization signals. An unspecified example of a modifying factor is a steroid-peptide nucleic acid complex, as described by Rebuffat et al., 2002, Faceb J. 16(11):1426-8, incorporated herein by reference. DNA vectors may contain sequences encoding different proteins or peptides. Promoters, enhancers, stress or chemical control promoters, antibiotic-sensitive or nutrient-sensitive regions, and therapeutic protein-coding sequences may be included as needed. Non-coding sequences may also be present in the DNA vector.

[0091] The nucleic acids used in this method may be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by various liquid-phase or solid-phase methods. In general, solid-phase synthesis is preferred. Detailed descriptions of procedures for solid-phase synthesis of nucleic acids by phosphite-tryester, phosphotryester, and H-phosphonate chemistry are widely available.

[0092] In one embodiment, the DNA vector is double-stranded DNA and contains more than 700 base pairs, more than 800 base pairs, more than 900 base pairs, or more than 1000 base pairs.

[0093] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0094] Gene editing systems containing charged lipids can also be incorporated into the delivery vehicle. This includes Cas9-CRISPR, TALEN, and zinc finger nuclease gene editing systems. In the case of Cas9-CRISPR, a guide RNA (gRNA) may be incorporated into the delivery vehicle containing the MC3-type lipid described herein, together with a plasmid or mRNA encoding the Cas9 protein. Optionally, a ribonucleoprotein complex may be incorporated into the delivery vehicle containing the lipid described herein. Similarly, this disclosure includes embodiments in which genetic material encoding the DNA-binding and cleavage domains of a zinc finger nuclease or TALEN system is incorporated into the delivery vehicle together with the MC3-type lipid of this disclosure.

[0095] While various nucleic acid cargo molecules have been described above, it will be understood that the above examples are non-limiting, and this disclosure is not considered to be limited to specific cargo molecules encapsulated within a delivery vehicle.

[0096] For example, the MC3-type lipids described herein can also facilitate the incorporation of proteins and peptides into delivery vehicles containing ribonucleoproteins. These include both linear and non-linear peptides, proteins, or ribonucleoproteins.

[0097] Although pharmaceutical compositions have been described above, the MC3-type lipids described herein may also be components of any nutritional, cosmetic, cleansing, or food product.

[0098] Pharmaceutical preparations In some embodiments, a delivery vehicle containing cargo molecules is part of a pharmaceutical composition administered to treat and / or prevent a disease condition. The treatment may provide preventive, ameliorative, or therapeutic benefits. The pharmaceutical composition is administered in any preferred dosage.

[0099] In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intra-arterial, intravenous, subcutaneous, or intramuscularly. In a further embodiment, the pharmaceutical composition is for intratumoral or intrauterine administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreously, subretinally, subarachnoidally, or by other local routes.

[0100] The pharmaceutical composition contains pharmaceutically acceptable salts and / or excipients.

[0101] The compositions described herein may be administered to patients. The term "patient," as used herein, includes human or non-human subjects.

[0102] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. [Examples]

[0103] material The lipids 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and 10× phosphate-buffered saline (pH 7.4) were purchased from Sigma Aldrich (St. Louis, MO). The ionizable aminolipids were synthesized as previously described in U.S. Provisional Patent Application No. 63 / 194,471, "Method for Producing an Ionizable Lipid," which is incorporated herein by reference.

[0104] Luciferase activity was analyzed using mRNA encoding firefly luciferase, purchased from APExBIO Technology LLC (Houston, TX).

[0105] We evaluated the ability of LNPs to knock down firefly luciferase in cell lines using siRNA targeting firefly luciferase purchased from Integrated DNA Technologies (IDT, Coralville, IA).

[0106] method Preparation of lipid nanoparticles (LNPs) containing mRNA or siRNA Lipids used in formulation, nor-MC3 or MC3, DSPC, cholesterol, and PEG-DMG were dissolved in ethanol in appropriate ratios to a final lipid concentration of 10 mM. Nucleic acids (siRNA or mRNA) were dissolved in appropriate buffers such as 25 mM sodium acetate, pH 4 or sodium citrate, pH 4 to the concentration required to achieve the appropriate amine-to-phosphate ratio. The aqueous and organic solutions were mixed using a rapid mixing device at a flow rate ratio of 3:1 (v / v, respectively) and a total flow rate of 20 mL / min, as described in Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni et al., 2017, Nanoscale, 36:133347 (both incorporated herein by reference). The resulting mixtures were dialyzed directly against 1000 times the volume of PBS, pH 7.4. All formulations were concentrated using an Amicon centrifugation filter unit and analyzed using the method described below.

[0107] LNP Analysis Particle size analysis of LNPs in PBS was performed using backscatter measurements of dynamic light scattering with a Malvern Zetasizer (Worcestershire, UK). The reported particle size corresponds to the number-weighted average diameter (nm). Total lipid concentration was determined by extrapolation from cholesterol content measured according to the manufacturer's recommendations using the Cholesterol E-Total Cholesterol Assay (Wako Diagnostics, Richmond, VA). The encapsulation efficiency of the formulation was determined using the Quant-iT RiboGreen Assay kit (Invitrogen, Waltham, MA). Briefly, the total siRNA or mRNA content in the solution was measured by dissolving lipid nanoparticles in a TE solution containing 2% Triton Tx-100, and the free DNA vector in the solution (outside the LNPs) was measured based on RiboGreen fluorescence in a TE solution without Triton. The total siRNA or mRNA content in the formulation was determined using a modified Bligh-Dyer extraction procedure. In short, LNP preparations containing siRNA or mRNA were dissolved in a mixture of chloroform, methanol, and PBS to form a single phase, and the absorbance at 260 nm was measured using a spectrophotometer.

[0108] In vitro analysis of Huh7 cells Huh7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For cell processing, 10,000 cells were added to each well of a 96-well plate. After 24 hours, the medium was aspirated and replaced with medium containing diluted LNP at relevant concentrations ranging from 0.03 to 10 μg / mL mRNA. Expression analysis was performed after 24 hours, and luciferase levels were measured using the Steady-Glo Luciferase Kit (Promega). Cells were lysed using Glo Lysis Buffer (Promega).

[0109] In vivo analysis in C57Bl / 6 mice LNP-mRNA encoding firefly luciferase was injected intravenously (tail vein) into 6-8 week old C57BL / 6 mice. Four hours after injection, the animals were euthanized, and the liver and spleen were removed. The tissues were homogenized in Glo Lysis buffer, and a luciferase assay was performed using the Steady Glo Luciferase Assay Kit (following the manufacturer's recommendations).

[0110] Organic synthesis of nor-MC3 Unless otherwise specified, all reagents and solvents were commercially available and used without further purification, with the exception of THF (freshly distilled from Na / benzophenone under Ar) and CH2Cl2 (freshly distilled from CaH2 under Ar). Dry methanol was freshly distilled from magnesium pieces. All reactions were carried out under an argon atmosphere. The reaction mixture from the aqueous treatment was dried by passing it over a plug of anhydrous Na2SO4 held in a filter tube and then evaporated by rotation under reduced pressure. Thin-layer chromatography was performed on silica gel plates coated with silica gel (Merck 60 F254 plates), and column chromatography was performed on 230-400 mesh silica gel. The developed chromatograms were visualized by staining with I2 or potassium permanganate solution. Nuclear magnetic resonance spectroscopy, 1 H (300MHz) and 13 13C NMR (75 MHz) was recorded in CDCl3 solution at room temperature. 1 The 1H NMR spectrum is referenced to residual CHCl3 (7.26 ppm). 13 ¹³C NMR spectra were referenced to the CDCl3 triplet median (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on a delta scale. Multiplicity is reported as "s" (singlet), "d" (doublet), "t" (triplet), "q" (quadruplet), and "m" (multilet), and further classified as "app" (clear) and "br" (broad). Low-resolution and high-resolution mass spectra (m / z) were obtained in electrospray (ESI) and field desorption / field ionization (FD / FI) modes.

[0111] The synthesis of nor-MC3 from methyl linoleate was carried out as described below. As discussed, the synthesis of nor-MC3 involves subjecting the fatty acid ester to a Claisen condensation under Mukaiyama conditions as described in Scheme 3 above to produce ketoester 6. Ketoester 6 is then hydrolyzed and decarboxylated to produce ketone 7, which can be further reduced to alcohol 8 (or 8a). As described in the co-located concurrently pending U.S. Provisional Patent Application No. 63 / 194,471 (incorporated herein by reference) and as described below, esterification of alcohol 8 (or 8a) yields an MC3-type lipid.

[0112] (a) Synthesis of ketoesters from methyl linoleate:methyl(11Z,14Z)-2-((7Z,10Z)-hexadeca-7,10-dien-1-yl)-3-oxoicosa-11,14-dienoate.

[0113] [ka]

[0114] A solution of TiCl4 (9.6 g, 5.7 mL, 45.0 mmol) in toluene (12 mL) was added dropwise to a cold (0°C, ice bath) stirred solution of a suitable methyl ester (e.g., methyl linoleate; 30.0 mmol, 8.8 g) and tributylamine (Bu3N) (10.2 g, 12.9 mL, 54.0 mmol) in toluene (50.0 mL). After stirring at 0°C for 1.5 hours, TLC and 1The reaction was completed as determined by 1H NMR. The reaction solution was then diluted with hexane (60 mL), and water (60 mL) was carefully added. Since heat was generated by the addition of water, the temperature of the mixture was controlled by stirring and cooling in an ice bath. The organic phase was separated, and the aqueous phase was extracted with further hexane (2 × 40 mL). The combined organic extract was washed with water, passed over a plug of anhydrous Na₂SO₄, and concentrated under vacuum. Proton NMR analysis of the residue showed the presence of some residual toluene. Suspended inorganic substances (possibly TiO₂) may also be present. The crude product may be purified by column chromatography (3% diethyl ether in hexane) to produce a pure keto ester (96%), or the process may proceed directly to the next step. NMR showed that the product typically exists as a mixture of keto (major) and enol derivatives in a 2:1 ratio. 1 H NMR (keto form) δ 5.37 (m, 8H), 3.77 (s, 3H), 3.45 (t, 1 H), 2.79 (t, 4H), 2.40 (t, 2H), 2.07 (m, 8H), 1.85-1.20 (m, 32H), 0.91 (t, 6H). 13 ¹¹C NMR (keto form): δ 205.6, 170.6, 130.2, 130.1, 129.9, 129.8, 59.2, 52.4, 42.0, 32.1, 29.9, 29.8, 29.8, 29.7, 29.5, 29.4, 29.4, 29.3, 29.2, 29.1, 28.4, 27.6, 27.4, 27.3, 27.3, 23.6, 22.8, 14.3 (some peaks are doubled). LRMS: m / z 557 [M+H] + , 579 [M+Na] +

[0115] (b) Ketones: Hydrolysis and decarboxylation of ketoesters to produce (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-one.

[0116] [ka]

[0117] A 10% w / vol aqueous solution of NaOH (5 mL) was added to a solution of the crude ketoester (5.0 g) in 95% ethanol (25 mL). The mixture was stirred overnight at room temperature. Three to four drops of the reaction mixture were added to a 3N aqueous solution of HCl (0.5 mL), and the mixture was extracted with hexane. The combined extract was evaporated until dry, and the residue was collected. 1 The completion of the reaction was confirmed by 1H NMR. The disappearance of the OCH3 signal and the downward shift of the triplet 3.45 (ketoester) to 3.51 (keto acid) indicated that the reaction was complete. The reaction mixture was concentrated in a rotary evaporator to remove ethanol. The aqueous residue was cooled in an ice bath, diluted with hexane (60 mL), and vigorously stirred while carefully adding concentrated aqueous HCl dropwise (heat was generated). When the pH of the mixture reached approximately 1, the phases were separated, and the aqueous phase was extracted with further hexane (2 × 20 mL). The combined organic extracts were washed with DI water (30 mL), passed over a plug of anhydrous Na₂SO₄, and concentrated in a rotary evaporator. The NMR spectrum of the crude product was recorded to confirm the presence of the desired keto acid. The flask containing the residue from the rotary evaporator was stoppered with a septum and completely purged with argon (balloon; needle vent). The flask was heated with a heat gun until it was too hot to touch (100-130°C) (while still sealed under argon gas and vented with a needle), and decarboxylation began. As the decarboxylation reaction progressed, foaming of the residue became noticeable. After about 10 minutes, no further foaming was clearly observed. The flask was cooled to room temperature, and the residue was again... 1 Analysis by 1H NMR revealed that it was a nearly pure ketone. If desired, the crude ketone may be purified by column chromatography (gradient: 1 → 3% v / v ether in hexane; yield 97%). However, most advantageously, the crude ketone is introduced directly into the next step. 1 H NMR δ 5.32 (m, 8H), 2.74 (t, 4H), 2.35 (t, 4H), 2.02 (m, 8H), 1.55-1.20 (m, 28H), 0.87 (t, 6H). 13¹³C NMR δ 210.9, 130.0, 129.8, 128.0, 127.8, 42.6, 31.4, 29.5, 29.24, 29.22, 29.1, 29.0, 27.0 (two overlapping peaks), 25.5, 23.7, 22.5, 14.0. LRMS m / z 499 [M+H] + , 521 [M+Na] + .

[0118] (c) Reduction of ketones to alcohol: (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ol.

[0119] [ka]

[0120] Solid NaBH4 (2 mmol) was gradually added to a stirred solution of ketone (2 mmol) in 95% ethanol (10 mL) at 0°C (ice bath). After stirring at 0°C for 1 hour, the reaction mixture was extracted with TCL (5% ether in hexane), or more reliably, 3-4 drops were added to saturated NH4Cl aqueous solution (0.5 mL), and the mixture was extracted with hexane. The combined extract was evaporated until dry, and the residue was removed. 1 The completion of the reaction was confirmed by 1H NMR. Both methods indicated that the reaction was complete. The reactants were quenched by carefully adding saturated NH4Cl aqueous solution (care must be taken to avoid H2 generation and foaming), and the mixture was concentrated in a rotary evaporator to remove ethanol. The aqueous residue was extracted with hexane (3 × 10 mL). The combined extracts were concentrated by passing them through an anhydrous Na2SO4 plug to obtain crude alcohol, which was purified by silica gel column chromatography in hexane with 5 → 10% v / v ethyl acetate (yield 91%). 1 H NMR δ 5.36 (m, 8H), 3.58 (m, 1H), 2.78 (t, 4H), 2.1-1.9 (m, 8H), 1.6-1.2 (m, 36H), 0.89 (t, 6H). LRMS: m / z 501 [M+H]+ , 523 [M+Na] +

[0121] (d) Procedure for esterification of alcohol (4-dimethylamino)butanoylation to produce (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-yl 4-(dimethylamino)butanoate (=nor-MC3).

[0122] [ka]

[0123] The above alcohol (1 mmol, 1.0 equivalent), 4-dimethylaminobutyrate (1.2 mmol, 1.2 equivalents), diisopropylethylamine (1.5 mmol, 1.5 equivalents), and DMAP (0.1 mmol, 0.1 equivalent) were mixed in 3 mL of dry CH2Cl2 at room temperature for 5 minutes, and then EDCI (1.5 mmol, 1.5 equivalents) was added. The mixture was stirred overnight at room temperature under argon to obtain TLC (5% MeOH in CH2Cl2) and 1 ¹H NMR indicated that the reaction was complete. The solution was further diluted with CH₂Cl₂ (10 mL) and sequentially washed with saturated NaHCO₃ aqueous solution (5 mL) and water (10 mL). The organic phase was passed over an anhydrous Na₂SO₄ plug and concentrated under vacuum. The crude product residue was purified by flash column chromatography in 3% v / v MeOH in CH₂Cl₂ containing 0.1% NEt₃ (yield 78%). 1 H NMR δ 5.33 (m, 8H), 4.85 (m, 1H), 2.75 (t, 4H), 2.40 (m, 2H), 2.34 (t, 2H), 2.20 (s, 6H), 2.02 (m, 8H), 1.77 (m, 2H), 1.50 (m, 6H), 1.39-1.17 (m, 36H), 0.87 (t, 6H). 13C NMR δ 173.3, 130.1, 130.0, 127.9, 127.8, 74.1, 59.0, 45.4, 34.1, 32.4, 31.5, 29.7, 29.6, 29.5, 29.4, 29.33, 29.30, 29.2, 27.15, 27.14, 25.3, 23.1, 14.8. LRMS: m / z 614 [M+H] + .

[0124] (Example 1) mRNA-containing LNPs with nor-MC3 ionizable lipids exhibit superior transfection efficiency compared to the MC3 benchmark. LNP formulations containing ionizable lipids nor-MC3 or MC3, DSPC, cholesterol, and PEG-DMG, along with mRNA encoding luciferase, were prepared. The lipid nanoparticles contained 50 / 10 / 38.5 / 1.5 mol% ionizable lipids / DSPC / chol / PEG-DMG, with a nitrogen-to-phosphorus ratio (N / P) of 6.

[0125] Figure 1B shows the transfection efficiency of LNPs containing nor-MC3 versus MC3 benchmark ionizable lipids. The transfection efficiency of LNPs containing nor-MC3 was superior to that of those containing MC3 benchmark lipids at all mRNA concentrations (μg / mL mRNA) measured after the addition of Huh7, and significantly improved at the highest dose measured (10 μg / mL mRNA). MC3 is a state-of-the-art ionizable lipid for LNP formulations that encapsulate nucleic acids, and its carbon chain is an unsaturated C derived from linoleic acid. 18 These results are particularly surprising because this is a portion of the chain that has been reported to be the best chain identified to date for maximum potency in siRNA formulations. (Semple et al., 2010, Nat. Biotechnol. 28:172~176 and Heyes et al., 2005, J. Controlled Release, 107:276~287.) It was hypothesized that shorter-chain lipids would have reduced potency, but the opposite effect was observed.

[0126] Figure 1A shows the results of characterization studies on LNP diameter, PDI, and encapsulation efficiency of mRNA LNPs containing nor-MC3 and MC3. Advantageously, mRNA-LNPs containing nor-MC3 lipids exhibited similar diameter, PDI, and mRNA encapsulation efficiency (capture) to mRNA-LNPs containing MC3.

[0127] (Example 2) mRNA-containing LNPs, including nor-MC3, demonstrate superior in vivo delivery of mRNA to the liver and spleen compared to the MC3 benchmark. An LNP preparation containing 50 / 10 / 38.5 / 1.5 mol% of nor-MC3 or MC3-ionizable lipids / DSPC / chol / PEG-DMG and mRNA encoding luciferase was tested for in vivo distribution in the liver and spleen of C57BL / 6 mice after injection. The mRNA dose was 1 mg / kg. Luminescence intensity in the liver or spleen was measured 4 hours after injection.

[0128] The results shown in Figure 1C indicate that the luminescence intensity per 1 mg of liver was higher in nor-MC3 than in the MC3 benchmark. Similar results were observed for the luminescence intensity per 1 mg of spleen.

[0129] MC3 having a C18 unsaturated carbon chain (obtained from linoleic acid) 17 The results showing superior mRNA delivery to both the liver and spleen by nor-MC3-LNP are particularly surprising, as it was hypothesized that it would provide improved efficacy in nucleic acid delivery compared to shorter nor-MC3 chains with unsaturated chains.

[0130] (Example 3) LNPs containing nor-MC3 siRNA exhibit transfection efficiency comparable to that of the MC3 benchmark. As demonstrated in Examples 1 and 2, mRNA-encapsulated LNP formulations containing nor-MC3 exhibit superior or equivalent transfection efficiency and significantly improved in vivo distribution compared to MC3 formulations. In vitro physical characterization and transfection studies comparing nor-MC3 and MC3 were similarly performed using siRNA-containing LNPs. The results are discussed below.

[0131] An LNP formulation containing an ionizable lipid nor-MC3 or MC3, DSPC, cholesterol, and PEG-DMG, which contains an siRNA encoding luciferase, was prepared as described above. The lipid nanoparticles contained 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG, and the nitrogen-to-phosphorus ratio (N / P) was 3.

[0132] The transfection efficiency is shown in Figure 2B. The transfection efficiency of nor-MC3-containing siRNA LNPs was generally equivalent to that of MC3 benchmark lipids, but was better with 0.5 μg / mL siRNA than with MC3. The semi-maximal effective siRNA concentrations (EC) of nor-MC3 and MC3 LNP formulations. 50 ) were relatively equivalent (EC of nor-MC3) 50 =0.1644 μg / mL siRNA, EC3 50 =0.1308 μg / mL siRNA). C obtained from linoleic acid. 18 These results are also surprising, given that, considering previous studies reporting that the portion provides the highest efficacy for siRNA delivery, nor-MC3 was presumed to have lower efficacy than MC3 in in vitro transfection.

[0133] Figure 2A shows the results for nor-MC3 versus MC3 regarding LNP diameter, PDI, and siRNA encapsulation efficiency. Advantageously, LNPs containing nor-MC3 lipids showed similar diameter, PDI, and siRNA encapsulation efficiency (capture) to those of MC3.

[0134] The examples are intended to illustrate the preparation, formulation, and properties of short-chain MC3 type lipids, but are not intended to limit the scope of the present invention.

Claims

1. nor-MC3: 【Chemistry 1】 Lipids having the structure.

2. A drug delivery vehicle comprising the lipid described in claim 1.

3. The drug delivery vehicle according to claim 2, wherein the drug delivery vehicle is a lipid nanoparticle.

4. The drug delivery vehicle according to claim 3, comprising a helper lipid and a hydrophilic polymer-lipid complex.

5. The drug delivery vehicle according to claim 4, wherein the helper lipid is a sterol, diacylglycerol, or ceramide.

6. The drug delivery vehicle according to claim 5, wherein diacylglycerol is selected from dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, palmitoyloleoyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylethanolamine, palmitoyloleoyl-phosphatidylglycerol, dipalmitoylphosphatidylethanolamine, dimyristoyl-phosphatidylethanolamine, distearoylphosphatidylethanolamine, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielidoyl-phosphatidylethanolamine, stearoyloleoyl-phosphatidylethanolamine, egg phosphatidylcholine, and mixtures thereof.

7. A drug delivery vehicle according to any one of claims 2 to 6, comprising nucleic acid.

8. The drug delivery vehicle according to claim 7, wherein the nucleic acid is RNA selected from siRNA, snRNA, miRNA, and mRNA.

9. The drug delivery vehicle according to claim 7, wherein the nucleic acid is a single-stranded antisense oligonucleotide.

10. A method for producing nor-MC3 lipids according to claim 1, (i) A step of reacting methyl linoleate in a Claisen condensation reaction in the presence of a catalyst to produce the ketoester methyl(11Z,14Z)-2-((7Z,10Z)-hexadeca-7,10-dien-1-yl)-3-oxoicosa-11,14-dienoate; (ii) A step of reacting a ketoester via hydrolysis and decarboxylation to produce the ketone (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-one; (iii) A step of reducing the ketone to produce the alcohol (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ol; (iv) A step of preparing nor-MC3 lipids from the alcohol by esterifying the alcohol with 4-(dimethylamino)butyric acid to produce nor-MC3 lipids. A method that includes this.

Images