Formulations for use in methods of treating and / or preventing disease

Abstract

The present invention relates to a formulation for use in a method of treatment comprising local administration of the formulation to a subject, the local administration being selected from the group consisting of intramuscular administration, subcutaneous administration, intravitreal administration, intrathecal administration, intratumoral administration, intracerebral administration, and intradermal administration, the formulation comprising lipid nanoparticles, the lipid nanoparticles comprising a lipid composition comprising a first lipid and a second lipid, and a therapeutically effective nucleic acid molecule, the lipid nanoparticles being amphoteric, overall neutrally charged lipid nanoparticles.

Description

[Technical field]

[0001] The present invention relates to formulations comprising a payload molecule and a lipid composition, formulations for localized delivery of a payload, formulations for use in methods for treating and / or preventing a disease, formulations for use in methods for localized delivery of a payload, pharmaceutical compositions comprising said formulations, pharmaceutical compositions for localized delivery of a payload, pharmaceutical compositions for use in methods for treating and / or preventing a disease, and methods of making said formulations. [Background technology]

[0002] Currently, lipid nanoparticles (LNPs) applied for intramuscular, subcutaneous, and intradermal administration of RNA are based on strong cationic lipids, more often pH-sensitive cationic lipids (also called ionizable cationic lipids).

[0003] Strongly cationic LNPs are made of lipid systems that contain strong cationic lipids in addition to neutral co-lipids and PEGylated lipids. The pKa of the strong cationic lipids is usually above 9, which means that the cationic lipids are positively charged over a wide pH range. Thus, such strong cationic lipids are positively charged at acidic (pH 3-6.7) and neutral (pH 6.8-7.4) pH values, and even show persistent cationic character at weakly basic (pH 7.5-8) and moderately basic (pH 8.1-10) pH values. This property allows strong complexation of the overall negatively charged ribonucleic acid (RNA) and strong interaction with the overall negatively charged cell membranes, such as endosomal membranes, that are intended to be transfected by LNPs containing such strong cationic lipids. However, the strong cationic charge of this type of lipid interacts with other negatively charged components, such as extracellular substances such as proteins; then the complex containing the RNA and lipids dissociates rapidly and the RNA thus released is rapidly degraded. Interaction with such negatively charged extracellular material may also cause aggregation of lipid nanoparticles and therefore inhibit functional uptake of LNPs into target cells.

[0004] On the other hand, pH-sensitive cationic LNPs are made with neutral co-lipids, PEGylated lipids, and ionizable cationic lipids that feature typical pKa values ​​of 8-9.5. Due to the relatively low pKa value, such cationic lipids are positively charged at acidic pH values ​​(typically pH 4.5-5.5); at these acidic pH values, it can electrostatically interact with RNA, i.e., complex the RNA accordingly to form RNA-encapsulated LNPs. Once the LNPs are formed and the pH of the suspension medium is adjusted to a neutral pH value, the pH-sensitive lipids at the surface of the LNPs have lost their positive charge, e.g., due to deprotonation of amino groups, while the RNA remains trapped inside the LNP particle. As a result, the LNP surface is rather neutral and hydrophobic. These LNPs were originally designed as hepatocyte-targeting LNPs for intravenous application, and are known for their strong interactions with hydrophobic substances such as ApoE. Summary of the Invention

[0005] The problem underlying the present invention is to provide a means for delivering a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably said payload is a nucleic acid molecule, most preferably an RNA molecule.

[0006] Another problem underlying the present invention is to provide a means suitable for the local administration of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably said payload is a nucleic acid molecule, most preferably an RNA molecule.

[0007] Another problem underlying the present invention is to provide a means for the local administration of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably said payload is a nucleic acid molecule, most preferably an RNA molecule.

[0008] Another problem underlying the present invention is to provide a means suitable for the local delivery of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably this payload is a nucleic acid molecule, most preferably an RNA molecule.

[0009] Another problem underlying the present invention is to provide a means for the localized delivery of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably said payload is a nucleic acid molecule, most preferably an RNA molecule.

[0010] Another problem underlying the present invention is to provide means suitable for the treatment and / or prevention of a disease, which comprises the local administration of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably this payload is a nucleic acid molecule, most preferably an RNA molecule.

[0011] Another problem underlying the present invention is to provide a means for treating and / or preventing a disease, wherein the treatment and / or prevention of a disease comprises the local administration of a payload, preferably a payload that is negatively charged at neutral or physiological pH values, more preferably said payload is a nucleic acid molecule, most preferably an RNA molecule.

[0012] A further problem underlying the present invention is to provide a method for producing such a means.

[0013] These and other problems are solved by the subject matter of the attached independent claims; preferred embodiments can be taken from the attached dependent claims.

[0014] Furthermore, these and other problems are also solved by the subject matter of the following aspects and embodiments thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The underlying problem of the present invention is solved, in a first aspect (which is also the first embodiment of the first aspect), by a formulation comprising a payload molecule and a lipid composition, wherein the lipid composition comprises a first lipid and a second lipid, the first lipid is an anionic lipid having at least one molecular moiety with a pKa value of 4 to 7, and the second lipid is (a) a cationic lipid having at least one molecular moiety with a pKa value of 9.5 to 13.5, which is greater, or (b) a cationic lipid that is positively charged independently of pH.

[0016] In a second embodiment of the first aspect (which is also an embodiment of the first embodiment of the first aspect), the pKa value of the first lipid is 5 to 7.

[0017] In a third embodiment of the first aspect (which is also an embodiment of the first and second embodiments of the first aspect), the pKa value of the first lipid is 5.5 to 6.5.

[0018] In a fourth embodiment of the first aspect (which is also an embodiment of the first, second, and third embodiments of the first aspect), the pKa value of at least one molecular moiety of the second lipid is 9.5 < pKa ≤ 13.5, and preferably, the pKa value is 9.8 to 13.2.

[0019] In a fifth embodiment of the first aspect (which is also an embodiment of the first, second, third, and fourth embodiments of the first aspect), the pKa value of at least one molecular moiety of the second lipid is 10 to 13.

[0020] In a sixth embodiment of the first aspect (which is also an embodiment of the first, second, and third embodiments of the first aspect), the second lipid is a lipid that is positively charged independently of pH.

[0021] In a seventh embodiment of the first aspect (which is also an embodiment of the first, second, third, fourth, fifth, and sixth embodiments of the first aspect), the first lipid has at least one negative charge.

[0022] In an eighth embodiment of the first aspect (which is also an embodiment of the seventh embodiment of the first aspect) the first lipid carries at least one negative charge at neutral pH value.

[0023] In a ninth embodiment of the first aspect (which is also an embodiment of the eighth embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably a pH value of about 6.9 to about 7.5, more preferably a pH value of about 7.2 to about 7.5.

[0024] In a tenth embodiment of the first aspect (which is also an embodiment of any one of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the first aspect), the second lipid carries at least one positive charge.

[0025] In an eleventh embodiment of the first aspect (which is also an embodiment of the tenth embodiment of the first aspect) the second lipid carries at least one positive charge at neutral pH values.

[0026] In a twelfth embodiment of the first aspect (which is also an embodiment of the eleventh embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably a pH value of about 6.9 to about 7.5, more preferably a pH value of about 7.2 to about 7.5.

[0027] In a thirteenth embodiment of the first aspect (which is also an embodiment of any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the first aspect), the first lipid has at least one carboxylic acid group.

[0028] In a fourteenth aspect of the first aspect (which is also an embodiment of any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth aspects of the first aspect, preferably of the thirteenth aspect of the first aspect), the second lipid bears at least one group selected from the group comprising an amino group, an imino group, a guanidino group, an amino group substituted with an alkyl group, an imino group substituted with an alkyl group, and a guanidino group substituted with an alkyl group.

[0029] In a fifteenth embodiment of the first aspect (which is also an embodiment of the fourteenth embodiment of the first aspect), the alkyl group is a straight and / or branched C1, C2, C3, or C4 hydrocarbon group.

[0030] In a sixteenth embodiment of the first aspect (which is also an embodiment of any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth aspects of the first aspect), the lipid composition comprises a third lipid.

[0031] In a seventeenth embodiment of the first aspect (which is also an embodiment of the sixteenth embodiment of the first aspect) the third lipid is a neutral lipid.

[0032] In an 18th embodiment of the first aspect (which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th and 17th aspects of the first aspect), the lipid composition comprises a fourth lipid.

[0033] In a nineteenth embodiment of the first aspect (which is also an embodiment of the eighteenth embodiment of the first aspect) the fourth lipid is a PEGylated lipid.

[0034] In a twentieth embodiment of the first aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth aspects of the first aspect), the lipid composition forms lipid nanoparticles.

[0035] In a twenty-first embodiment of the first aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth aspects of the first aspect), the lipid composition constitutes lipid nanoparticles.

[0036] In a twenty-second embodiment of the first aspect (which is also an embodiment of the twentieth and twenty-first aspects of the first aspect), the surface of the lipid nanoparticle provides an approximately equal number of positive and negative charges.

[0037] In a twenty-third embodiment of the first aspect (which is also an embodiment of the twenty-second embodiment of the first aspect), the surface of the lipid nanoparticle is the outer surface of the lipid nanoparticle.

[0038] In a twenty-fourth embodiment of the first aspect (which is also an embodiment of the twentieth and twenty-first aspects of the first aspect), approximately equal numbers of positive and negative charges are present on the surface of the lipid nanoparticle.

[0039] In a twenty-fifth embodiment of the first aspect (which is also an embodiment of the twenty-fourth embodiment of the first aspect), the surface of the lipid nanoparticle is the outer surface of the lipid nanoparticle.

[0040] In a 26th embodiment of the first aspect (which is also an embodiment of the 22nd, 23rd, 24th and 25th aspects of the first aspect), the number of positive and negative charges is the number of positive (cationic) and negative (anionic) charges, respectively, at a neutral pH value.

[0041] In a 27th embodiment of the first aspect (which is also an embodiment of the 26th embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably about 6.9 to about 7.5, more preferably about 7.2 to about 7.5.

[0042] In a 28th embodiment of the first aspect (which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th and 27th aspects of the first aspect), the lipid composition comprises a first lipid and a second lipid in a dispersion medium.

[0043] In a twenty-ninth embodiment of the first aspect (which is also an embodiment of the twenty-eighth embodiment of the first aspect) the dispersion medium comprises lipid nanoparticles.

[0044] In a thirtieth embodiment of the first aspect (which is also an embodiment of the twenty-eighth and twenty-ninth aspects of the first aspect), the dispersion medium constitutes the continuous phase.

[0045] In a thirty-first embodiment of the first aspect (which is also an embodiment of the thirtieth embodiment of the first aspect), the continuous phase comprises a pharma- ceutically acceptable aqueous buffer system, preferably the buffer system has a physiological pH value and isosmolar strength and further comprises a cryoprotectant, more preferably the buffer system has a pH value of 7.0-7.4 and an osmolality of 250-330 mosmol / kg and comprises a cryoprotectant selected from the group comprising sucrose and trehalose, even more preferably the buffer system is a 10 mM phosphate buffer or a 10 mM TRIS / HCl buffer at pH 7.4 and comprises 270 mM sucrose as cryoprotectant.

[0046] In a thirty-second embodiment of the first aspect (which is also an embodiment of the twenty-eighth, twenty-ninth, thirtieth and thirty-first aspects of the first aspect), the pH value of the dispersion medium is a neutral pH value.

[0047] In a 33rd embodiment of the first aspect (which is also an embodiment of the 32nd embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably about 6.9 to about 7.5, more preferably about 7.2 to about 7.5.

[0048] In a thirty-fourth embodiment of the first aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second and thirty-third embodiments of the first aspect), the molar ratio of the first lipid to the second lipid is such that the number of negative (anionic) charges provided by the lipid composition and the number of positive (cationic) charges provided by the lipid composition are approximately the same.

[0049] In a thirty-fifth embodiment of the first aspect (which is also an embodiment of the thirty-fourth embodiment of the first aspect), the molar ratio of the first lipid of the lipid composition to the second lipid of the lipid composition is such that the number of negative charges provided by the lipid composition on the surface of the lipid nanoparticle is approximately the same as the number of positive charges provided by the lipid composition on the surface of the lipid nanoparticle.

[0050] In a thirty-sixth embodiment of the first aspect (which is also an embodiment of the thirty-fifth embodiment of the first aspect), the surface of the lipid nanoparticle is the outer surface of the lipid nanoparticle.

[0051] In a thirty-seventh embodiment of the first aspect (which is also an embodiment of the thirty-fifth and thirty-sixth embodiments of the first aspect), the number of negative charges provided by the lipid composition on the surface of the lipid nanoparticle is approximately the same as the number of positive charges provided by the lipid composition on the surface of the lipid nanoparticle at a neutral pH value.

[0052] In a 38th embodiment of the first aspect (which is also an embodiment of the 37th embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably about 6.9 to about 7.5, more preferably about 7.2 to about 7.5.

[0053] In a thirty-ninth embodiment of the first aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second and thirty-third embodiments of the first aspect), the molar ratio of the first lipid to the second lipid is such that the number of negative (anionic) charges in the lipid composition and the number of positive (cationic) charges in the lipid composition are approximately the same.

[0054] In a fortieth embodiment of the first aspect (which is also an embodiment of the thirty-ninth embodiment of the first aspect), the molar ratio of the first lipid of the lipid composition to the second lipid of the lipid composition is such that the number of negative charges of the lipid composition on the surface of the lipid nanoparticle is approximately the same as the number of positive charges of the lipid composition on the surface of the lipid nanoparticle.

[0055] In a forty-first embodiment of the first aspect (which is also an embodiment of the fortieth embodiment of the first aspect), the surface of the lipid nanoparticle is the outer surface of the lipid nanoparticle.

[0056] In a 42nd embodiment of the first aspect (which is also an embodiment of the 40th and 41st embodiments of the first aspect), the number of negative charges of the lipid composition on the surface of the lipid nanoparticle is approximately the same as the number of positive charges of the lipid composition on the surface of the lipid nanoparticle at neutral pH values.

[0057] In a 43rd embodiment of the first aspect (which is also an embodiment of the 42nd embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably about 6.9 to about 7.5, more preferably about 7.2 to about 7.5.

[0058] In a 44th embodiment of the first aspect (which is also an embodiment of the 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd and 43rd aspects of the first aspect), the lipid nanoparticles are amphoteric lipid nanoparticles.

[0059] In a 45th embodiment of the first aspect (which is also an embodiment of the 44th embodiment of the first aspect), the lipid nanoparticles are amphoteric lipid nanoparticles at a pH value of about 7 to about 8, preferably at a pH of about 7.4.

[0060] In a 46th embodiment of the first aspect (which is also an embodiment of the 44th and 45th embodiments of the first aspect), the amphoteric lipid nanoparticles are overall neutrally charged lipid nanoparticles.

[0061] In a 47th embodiment of the first aspect (which is also an embodiment of the 46th embodiment of the first aspect), the amphoteric lipid nanoparticles are lipid nanoparticles that are overall neutrally charged at a pH value of about 7 to about 8, preferably at a pH of about 7.4.

[0062] In a 48th embodiment of the first aspect (which is also an embodiment of the 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th and 47th embodiments of the first aspect), the first lipid has a carboxylic acid group that is deprotonated in a pH-dependent manner and the second lipid has three functional groups that are protonated in a pH-dependent manner, wherein the lipid nanoparticle has a pH-dependent overall surface charge according to diagram (I) shown in FIG.

[0063] In a 49th embodiment of the first aspect (which is also an embodiment of the 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th and 47th embodiments of the first aspect), the first lipid has a carboxylic acid group that is deprotonated in a pH-dependent manner and the second lipid has two functional groups that are protonated in a pH-dependent manner, wherein the lipid nanoparticle has a pH-dependent overall surface charge according to diagram (II) shown in FIG.

[0064] In a fiftieth embodiment of the first aspect (which is also an embodiment of the twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third, thirty-fourth, thirty-fifth, thirty-sixth, thirty-seventh, thirty-eighth, thirty-ninth, fortieth ...

[0065] In a 51st embodiment of the first aspect (which is also an embodiment of the 48th, 49th and 50th aspects of the first aspect), the pH-dependent overall surface charge of the lipid nanoparticles is due to a negative charge provided by a first lipid of the lipid composition and a positive charge provided by a second lipid of the lipid composition.

[0066] In a 52nd embodiment of the first aspect (which is also an embodiment of the 48th, 49th, 50th and 51st aspects of the first aspect), the overall surface charge of the lipid nanoparticles is the overall surface charge of the lipid particles present in the dispersion medium.

[0067] In a 53rd embodiment of the first aspect (which is also an embodiment of the 52nd embodiment of the first aspect), the overall surface charge of the lipid nanoparticle is the overall charge of the outer surface of the lipid nanoparticle.

[0068] In a 54th embodiment of the first aspect (which is also an embodiment of the 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd and 53rd aspects of the first aspect), the lipid nanoparticles are hydrophilic nanoparticles.

[0069] A fifty-fifth embodiment of the first aspect (the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third, thirty-fourth, thirty-fifth, thirty-sixth, thirty-seventh, thirty-eighth, thirty-ninth, fortieth, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54), the formulation comprises lipid nanoparticles, wherein the lipid nanoparticles comprise a lipid composition and optionally a therapeutically active agent, and the nanoparticles are amphoteric, overall neutrally charged lipid nanoparticles at a pH of 7-8; wherein the lipid composition comprises a first lipid and a second lipid; wherein the first lipid is selected from the group consisting of a monoester of cholesterol and a dicarboxylic acid, an alkyl carboxylic acid, an alkenyl carboxylic acid, a monoester of a diacylglycerol ester and a dicarboxylic acid, and a diacylphosphatidyl alcohol; wherein the second lipid is a lipid having multiple cationic charges; Preferably, the therapeutically active substance herein is a therapeutically effective nucleic acid molecule.

[0070] A fifty-sixth aspect of the first aspect (the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third, thirty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third, thirty-fourth, twenty-six ... In each and any one of the embodiments of the 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th and 55th aspects), the first lipid is a monoester of cholesterol and a dicarboxylic acid.

[0071] In a 57th embodiment of the first aspect (which is also an embodiment of the 56th embodiment of the first aspect), the dicarboxylic acid of the monoester of cholesterol and a dicarboxylic acid is selected from the group consisting of succinic acid, glutaric acid, and adipic acid.

[0072] In a fifty-eighth embodiment of the first aspect (which is also an embodiment of the fifty-seventh embodiment of the first aspect), the monoester of cholesterol and a dicarboxylic acid is a hemisuccinic acid cholesterol ester.

[0073] In a fifty-ninth embodiment of the first aspect (which is also an embodiment of the fifty-seventh embodiment of the first aspect) the monoester of cholesterol and a dicarboxylic acid is cholesterol hemiglutarate.

[0074] In a sixtieth embodiment of the first aspect (which is also an embodiment of the fifty-seventh embodiment of the first aspect), the monoester of cholesterol and a dicarboxylic acid is a hemiadipate cholesterol ester.

[0075] A sixty-first aspect of the first aspect (the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third ... In each and any one of the third, thirty-fourth, thirty-fifth, thirty-sixth, thirty-seventh, thirty-eighth, thirty-ninth, fortieth, forty-first, forty-second, forty-third, forty-fourth, forty-fifth, forty-sixth, forty-seventh, forty-eighth, forty-ninth, fifty-first, fifty-second, fifty-third, fifty-fourth and fifty-fifth aspects, the first lipid is an alkyl carboxylic acid.

[0076] In a 62nd embodiment of the first aspect (which is also an embodiment of the 61st embodiment of the first aspect) the alkyl carboxylic acid is an alkyl carboxylic acid having 12, 13, 14, 15, 16, 17, 18, 19 or 20 C atoms.

[0077] In a 63rd embodiment of the first aspect (which is also an embodiment of the 62nd embodiment of the first aspect) the alkyl carboxylic acid is an alkyl carboxylic acid having 14, 15, 16, 17 or 18 C atoms.

[0078] In a sixty-fourth embodiment of the first aspect (which is also an embodiment of the sixty-second and sixty-third aspects of the first aspect), the alkyl carboxylic acid is a straight chain carboxylic acid.

[0079] In a sixty-fifth embodiment of the first aspect (which is also each and any one embodiment of the sixty-second, sixty-third and sixty-fourth embodiments of the first aspect), the alkyl carboxylic acid is a saturated alkyl carboxylic acid.

[0080] 66th aspect of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd aspects of the first aspect) In each and any one of the embodiments of the 3rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th and 55th aspects), the first lipid is an alkenyl carboxylic acid.

[0081] In a 67th embodiment of the first aspect (which is also an embodiment of the 66th embodiment of the first aspect) the alkenyl carboxylic acid is an alkenyl carboxylic acid having 12, 13, 14, 15, 16, 17, 18, 19 or 20 C atoms.

[0082] In a 68th embodiment of the first aspect (which is also an embodiment of the 67th embodiment of the first aspect) the alkenyl carboxylic acid is an alkenyl carboxylic acid having 14, 15, 16, 17 or 18 C atoms.

[0083] In a sixty-ninth embodiment of the first aspect (which is also each and any one embodiment of the sixty-sixth, sixty-seventh and sixty-eighth embodiments of the first aspect), the alkenyl carboxylic acid is a straight chain carboxylic acid.

[0084] In a 70th embodiment of the first aspect (which is also each and any one of the 66th, 67th, 68th and 69th embodiments of the first aspect), the alkenyl carboxylic acid is an unsaturated alkyl carboxylic acid, preferably the alkenyl carboxylic acid has 1, 2 or 3 double bonds.

[0085] In a seventy-first embodiment of the first aspect (which is also an embodiment of the seventieth embodiment of the first aspect) at least one double bond is in a cis configuration.

[0086] In a 72nd embodiment of the first aspect (which is also an embodiment of the 71st embodiment of the first aspect), the double bonds are all in the cis configuration.

[0087] In a 73rd embodiment of the first aspect (which is also each and any one of the 66th, 67th, 68th, 69th, 70th, 71st and 72nd aspects of the first aspect), the alkenyl carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, and linolenic acid.

[0088] In a 74th embodiment of the first aspect (which is also each and any one embodiment of the 66th, 67th, 68th, 69th, 70th, 71st, 72nd and 73rd embodiments of the first aspect), the alkenyl carboxylic acid is oleic acid.

[0089] In a 75th embodiment of the first aspect (which is also each and any one of the 66th, 67th, 68th, 69th, 70th, 71st, 72nd and 73rd embodiments of the first aspect), the alkenyl carboxylic acid is linoleic acid.

[0090] In a 76th embodiment of the first aspect (which is also each and any one of the 66th, 67th, 68th, 69th, 70th, 71st, 72nd and 73rd embodiments of the first aspect), the alkenyl carboxylic acid is linoleic acid.

[0091] 77th aspect of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, In each and any one of the thirty-sixth, thirty-seventh, thirty-eighth, thirty-ninth, fortieth, fortieth, fortieth, fortieth, second, third, fourth, fourth, fifth, sixth, sixth, seventh, eighth, sixth, seventh ...

[0092] In a 78th embodiment of the first aspect (which is also an embodiment of the 77th embodiment of the first aspect), the dicarboxylic acid of the monoester of the diacylglycerol ester and the dicarboxylic acid is selected from the group consisting of succinic acid, glutaric acid, and adipic acid.

[0093] In a seventy-ninth embodiment of the first aspect (which is also an embodiment of the seventy-eighth embodiment of the first aspect), the monoester of a diacylglycerol ester and a dicarboxylic acid is a hemisuccinic acid diacylglycerol ester.

[0094] In an 80th embodiment of the first aspect (which is also an embodiment of the 78th embodiment of the first aspect), the monoester of a diacylglycerol ester and a dicarboxylic acid is a hemiglutaric acid diacylglycerol ester.

[0095] In an 81st embodiment of the first aspect (which is also an embodiment of the 78th embodiment of the first aspect), the monoester of a diacylglycerol ester and a dicarboxylic acid is a hemiadipic acid diacylglycerol ester.

[0096] In an 82nd embodiment of the first aspect (which is also an embodiment of each and any one of the 77th, 78th, 79th, 80th and 81st embodiments of the first aspect, preferably the 79th embodiment of the first aspect), each acyl group of the diacylglycerol is independently a residue of a carboxylic acid, wherein the carboxylic acid is independently a carboxylic acid having 12, 13, 14, 15, 16, 17, 18, 19 or 20 C atoms.

[0097] In an 83rd embodiment of the first aspect (which is also an embodiment of each and any one of the 77th, 78th, 79th, 80th, 81st and 82nd aspects of the first aspect, preferably the 79th aspect of the first aspect), each acyl group of the diacylglycerol is each independently a residue of a carboxylic acid, wherein the carboxylic acid is each individually a carboxylic acid having 14, 15, 16, 17 or 18 C atoms.

[0098] In an 84th embodiment of the first aspect (which is also an embodiment of each and any one of the 77th, 78th, 79th, 80th, 81st, 82nd and 83rd aspects of the first aspect, preferably of any one of the 79th and 82nd and 83rd aspects of the first aspect), at least one of the acyl groups of the diacylglycerol is a residue of a carboxylic acid, wherein the carboxylic acid is a straight-chain carboxylic acid.

[0099] In an 85th embodiment of the first aspect (which is also an embodiment of each and any one of the 77th, 78th, 79th, 80th, 81st, 82nd, 83rd and 84th embodiments of the first aspect, preferably of any one of the 79th and 82nd, 83rd and 84th embodiments of the first aspect), at least one of the acyl groups of the diacylglycerol is a residue of a carboxylic acid, wherein the carboxylic acid is a saturated carboxylic acid.

[0100] In an 86th embodiment of the first aspect (which is also an embodiment of each and any one of the 77th, 78th, 79th, 80th, 81st, 82nd, 83rd and 84th aspects of the first aspect, preferably of any one of the 79th and 82nd, 83rd and 84th aspects of the first aspect), at least one of the acyl groups of the diacylglycerol is a residue of a carboxylic acid, wherein the carboxylic acid is an unsaturated carboxylic acid.

[0101] In an eighty-seventh embodiment of the first aspect (which is also an embodiment of each and any one of the eighty-sixth and any one of the first aspect), the unsaturated carboxylic acid has at least one double bond.

[0102] In an 88th embodiment of the first aspect (which is also an embodiment of each of the 86th and 87th embodiments of the first aspect), the unsaturated carboxylic acid has 1, 2 or 3 double bonds.

[0103] In an 89th embodiment of the first aspect (which is also an embodiment of each and any one of the 86th, 87th and 88th embodiments of the first aspect) at least one double bond is in a cis configuration.

[0104] In a 90th embodiment of the first aspect (which is also each and any one embodiment of the 87th, 88th and 89th embodiments of the first aspect), the double bonds are all in a cis configuration.

[0105] In a 91st embodiment of the first aspect (which is also each and any one embodiment of the 87th, 88th, 89th and 90th embodiments of the first aspect), the unsaturated carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, and linolenic acid.

[0106] In a 92nd embodiment of the first aspect (which is also an embodiment of the 91st embodiment of the first aspect) the unsaturated carboxylic acid is oleic acid.

[0107] In a 93rd embodiment of the first aspect (which is also an embodiment of the 91st embodiment of the first aspect) the unsaturated carboxylic acid is linoleic acid.

[0108] In a 94th embodiment of the first aspect (which is also an embodiment of the 91st embodiment of the first aspect) the unsaturated carboxylic acid is linoleic acid.

[0109] A ninety-fifth embodiment of the first aspect (the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth, thirty-first, thirty-second, thirty-third, thirty-fourth, thirty-fifth, thirty-sixth, thirty-seventh, thirty-eighth, thirty-eighth, thirty-sixth, thirty-seventh, thirty-eighth, thirty-sixth ... In each and any one of aspects 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55), the first lipid is a diacyl-phosphatidyl ester with an alcohol, wherein the alcohol has at least one negative charge at a neutral pH value.

[0110] In a 96th embodiment of the first aspect (which is also an embodiment of the 95th embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably a pH value of about 6.0 to about 7.6, more preferably a pH value of about 7.2 to about 7.5, and even more preferably a pH value of about 7.4.

[0111] In a 97th embodiment of the first aspect (which is also an embodiment of the 95th and 96th embodiments of the first aspect), the alcohol is selected from the group consisting of an OH-bearing amino acid and an OH-bearing alcohol, preferably the OH-bearing alcohol is an OH-bearing carboxylic acid.

[0112] In a 98th embodiment of the first aspect (which is also an embodiment of the 97th embodiment of the first aspect) the amino acid bearing an OH is an α-amino acid.

[0113] In a 99th embodiment of the first aspect (which is also an embodiment of the 97th embodiment of the first aspect) the amino acid bearing an OH is an L-amino acid.

[0114] In a 100th embodiment of the first aspect (which is also an embodiment of the 97th embodiment of the first aspect) the amino acid having an OH is an L-α-amino acid.

[0115] In a 101st embodiment of the first aspect (which is also each and any one embodiment of the 97th, 98th, 99th and 100th embodiments of the first aspect), the amino acid bearing an OH is selected from the group consisting of serine and threonine.

[0116] In a hundred and second embodiment of the first aspect (which is also an embodiment of each and any one of the ninety-seventh, ninety-eighth, ninety-ninth and one hundredth aspects of the first aspect) the first lipid is diacylphosphatidylserine.

[0117] In a 103rd embodiment of the first aspect (which is also each and any one embodiment of the 95th, 96th, 97th, 98th, 99th, 100th, 101st and 102nd embodiments of the first aspect, preferably the 102nd embodiment of the first aspect), each acyl group of the diacyl-phosphatidyl ester with an alcohol is each independently a residue of a carboxylic acid, wherein the carboxylic acid is each individually a carboxylic acid having 12, 13, 14, 15, 16, 17, 18, 19 or 20 C atoms.

[0118] In a 104th embodiment of the first aspect (which is also each and any one embodiment of the 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd and 103rd embodiments of the first aspect, preferably the 102nd and 103rd embodiments of the first aspect), each acyl group of the diacyl-phosphatidyl ester with an alcohol is each independently a residue of a carboxylic acid, wherein the carboxylic acid is each individually a carboxylic acid having 14, 15, 16, 17 or 18 C atoms.

[0119] In a 105th embodiment of the first aspect (which is also each and any one embodiment of the 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd and 104th embodiments of the first aspect, preferably the 102nd, 103rd and 104th embodiments of the first aspect), at least one of the acyl groups of the diacyl-phosphatidyl ester with an alcohol is a residue of a carboxylic acid, wherein the carboxylic acid is a straight-chain carboxylic acid.

[0120] In a 106th embodiment of the first aspect (which is also each and any one embodiment of the 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th and 105th embodiments of the first aspect, preferably the 102nd, 103rd, 104th and 105th embodiments of the first aspect), at least one of the acyl groups of the diacyl-phosphatidyl ester with an alcohol is a residue of a carboxylic acid, wherein the carboxylic acid is a saturated carboxylic acid.

[0121] In a 107th embodiment of the first aspect (which is also each and any one embodiment of the 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th and 105th embodiments of the first aspect, preferably the 102nd, 103rd, 104th and 105th embodiments of the first aspect), at least one of the acyl groups of the diacyl-phosphatidyl ester with an alcohol is a residue of a carboxylic acid, wherein the carboxylic acid is an unsaturated carboxylic acid.

[0122] In a hundred eighth embodiment of the first aspect (which is also an embodiment of the hundred seventh embodiment of the first aspect), the unsaturated carboxylic acid has at least one double bond.

[0123] In a 109th embodiment of the first aspect (which is also an embodiment of the 107th and 108th aspects of the first aspect), the unsaturated carboxylic acid has 1, 2 or 3 double bonds.

[0124] In a 110th embodiment of the first aspect (which is also an embodiment of the 108th and 109th embodiments of the first aspect) at least one double bond is in a cis configuration.

[0125] In a 111th embodiment of the first aspect (which is also an embodiment of the 108th, 109th and 110th embodiments of the first aspect), the double bonds are all in a cis configuration.

[0126] In a 112th embodiment of the first aspect (which is also an embodiment of the 107th, 108th, 109th, 110th and 111th aspects of the first aspect), the unsaturated carboxylic acid is selected from the group consisting of oleic acid, linoleic acid, and linolenic acid.

[0127] In a 113th embodiment of the first aspect (which is also an embodiment of the 112th embodiment of the first aspect) the unsaturated carboxylic acid is oleic acid.

[0128] In a 114th embodiment of the first aspect (which is also an embodiment of the 112th embodiment of the first aspect) the unsaturated carboxylic acid is linoleic acid.

[0129] In a 115th embodiment of the first aspect (which is also an embodiment of the 112th embodiment of the first aspect) the unsaturated carboxylic acid is linoleic acid.

[0130] A 116th embodiment of the first aspect (the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 9, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 and 115 of the embodiment, preferably the 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, In each and any one of the embodiments of the 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th and 115th aspects), the second lipid is a lipid having multiple cationic charges.

[0131] In a 117th embodiment of the first aspect (which is also an embodiment of the 116th embodiment of the first aspect) the lipid having multiple cationic charges is a lipid having two or more cationic charges.

[0132] In a 118th embodiment of the first aspect (which is also an embodiment of the 117th embodiment of the first aspect), the lipid having multiple cationic charges is a lipid having two or three cationic charges.

[0133] In a 119th embodiment of the first aspect (which is also an embodiment of the 116th, 117th and 118th embodiments of the first aspect), the cationic charge comprises multiple cationic charges at neutral pH values.

[0134] In a 120th embodiment of the first aspect (which is also an embodiment of the 119th embodiment of the first aspect), the neutral pH value is a pH value of about 6.7 to about 7.7, preferably a pH value of about 6.0 to about 7.6, more preferably a pH value of about 7.2 to about 7.5, and even more preferably a pH value of about 7.4.

[0135] In a 121st embodiment of the first aspect (which is also an embodiment of the 116th, 117th, 118th, 119th and 120th aspects of the first aspect), the second lipid is selected from the group consisting of β-(L-arginyl)-L-2,3-diaminopropionic acid-N,N-dialkylamide and L-arginyl-β-alanine-N,N-dialkylamide.

[0136] In a 122nd embodiment of the first aspect (which is also an embodiment of the 121st embodiment of the first aspect), the second lipid is β-(L-arginyl)-L-2,3-diaminopropionic acid-N,N-dialkylamide, wherein the dialkyl comprises a first alkyl and a second alkyl, wherein the first alkyl and the second alkyl are each independently selected from saturated alkyl, unsaturated alkyl, straight chain alkyl and branched chain alkyl.

[0137] In a 123rd embodiment of the first aspect (which is also an embodiment of the 122nd embodiment of the first aspect), the first alkyl and the second alkyl are each independently selected from saturated straight chain alkyl, unsaturated straight chain alkyl, saturated branched chain alkyl, and unsaturated branched chain alkyl.

[0138] In a 124th embodiment of the first aspect (which is also an embodiment of the 123rd embodiment of the first aspect) alkyl has 12, 13, 14, 15, 16, 17, 18, 19 or 20 C atoms.

[0139] In a 125th embodiment of the first aspect (which is also an embodiment of the 123rd and 124th embodiments of the first aspect), the alkyl has 14, 15, 16, 17 or 18 C atoms, preferably the alkyl has 16 or 18 C atoms.

[0140] In a 126th embodiment of the first aspect (which is also an embodiment of the 123rd, 124th and 125th aspects of the first aspect) the alkyl is saturated alkyl.

[0141] In a 127th embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th and 126th aspects of the first aspect) the alkyl is a saturated linear alkyl.

[0142] In a 128th embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th, 126th and 127th aspects of the first aspect), alkyl is palmityl.

[0143] In a 129th embodiment of the first aspect (which is also an embodiment of the 123rd, 124th and 125th aspects of the first aspect) the alkyl is an unsaturated alkyl.

[0144] In a 130th embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th and 129th aspects of the first aspect), the alkyl is an unsaturated linear alkyl.

[0145] In a 131st embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th, 129th and 130th aspects of the first aspect), the unsaturated alkyl comprises at least one double bond, and preferably at least one double bond is in a cis configuration.

[0146] In a 132nd embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th, 129th, 130th and 131st aspects of the first aspect), alkyl is oleyl.

[0147] In a 133rd embodiment of the first aspect (which is also an embodiment of the 123rd, 124th, 125th, 129th, 130th, 131st and 132nd aspects of the first aspect), one of the first alkyl and the second alkyl is a saturated alkyl, preferably a saturated linear alkyl, and the other of the first alkyl and the second alkyl is an unsaturated alkyl, preferably an unsaturated linear alkyl.

[0148] 134th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, , 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd , 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 and 133), in which the second lipid is β-(L-arginyl)-L-2,3-diaminopropionic acid-N -palmityl-N-oleylamide, L-arginyl-β-alanine-N-palmityl-N-oleylamide, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), and DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride).

[0149] In a 135th embodiment of the first aspect (which is also an embodiment of the 134th embodiment of the first aspect), DOTAP is (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) or other salt such as the mesylate, although the chloride salt is preferred.

[0150] In a 136th embodiment of the first aspect (which is also an embodiment of the 134th embodiment of the first aspect), DOTMA is 1,2-di-O-octadecenyl-3-trimethylammonium propane as the chloride salt, or other salts such as the mesylate salt, although the chloride salt is preferred.

[0151] In a 137th embodiment of the first aspect (which is also an embodiment of the 58th embodiment of the 134th embodiment of the first aspect), the DC-cholesterol is 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride or other salts such as the mesylate salt, although the chloride salt is preferred.

[0152] In a 138th embodiment of the first aspect (which is also an embodiment of the 134th embodiment of the first aspect) the second lipid is selected from the group comprising β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide and L-arginyl-β-alanine-N-palmityl-N-oleylamide, preferably the second lipid is β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide.

[0153] A 139th embodiment of the first aspect (the 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 8, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113rd, 114th, 115th, 116th, 117th, 118th, In another embodiment (which is also an embodiment of the aspects 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 and 138), the third lipid is a zwitterionic phospholipid or an uncharged sterol lipid.

[0154] In a 140th embodiment of the first aspect (which is also an embodiment of the 139th embodiment of the first aspect) the zwitterionic phospholipid is selected from the group comprising phosphatidylethanolamine and phosphatidylcholine.

[0155] In a 141 st embodiment of the first aspect (which is also an embodiment of the 139 th embodiment of the first aspect) the uncharged sterol lipid is selected from the group comprising animal sterols and plant sterols.

[0156] In a 142nd embodiment of the first aspect (which is also an embodiment of the 139th and 140th aspects of the first aspect), the zwitterionic phospholipid is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero -3-phosphoethanolamine), DLPE (1,2-lauroyl-sn-glycero-3-phosphoethanolamine), DPhyPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0157] In a 143rd embodiment of the first aspect (which is also an embodiment of the 139th embodiment of the first aspect) the uncharged sterol lipid is selected from the group comprising cholesterol and stigmasterol.

[0158] 144th embodiment of the first aspect (18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, , 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th , 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th , 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 1st 21, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 and 143), the fourth lipid is a shielding lipid.

[0159] In a 145th embodiment of the first aspect (which is also an embodiment of the 144th embodiment of the first aspect), the shield lipid is a lipid comprising a moiety selected from the group comprising PEG (polyethylene glycol), HES (hydroxyethyl starch), PG (polyglycine) and polySarc (poly-sarcosine (poly-N-methylglycine)).

[0160] In a 146th embodiment of the first aspect (which is also an embodiment of the 145th embodiment of the first aspect) the shield lipid is a PEGylated lipid.

[0161] In a 147th embodiment of the first aspect (which is also an embodiment of the 146th embodiment of the first aspect), the PEGylated lipid is selected from the group comprising methoxyPEG-DSPE, methoxyPEG-DMPE, methoxyPEG-DMG, methoxyPEG-DPG, methoxyPEG-DSG, methoxyPEG-c-DMA, 2(methoxyPEG)-N,N-dioctadecylacetamide, 2(methoxyPEG)-N,N-ditetradecylacetamide, methoxyPEG-C8-ceramide, and methoxyPEG-C16-ceramide.

[0162] A 148th embodiment of the first aspect (the 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 11 5, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 1st 14, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, In any one of embodiments of aspects 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 and 147), the PEG portion of the PEGylated lipid has a molecular weight of about 750 Da to about 5000 Da, preferably about 1500 Da to about 3000 Da.

[0163] In a 149th embodiment of the first aspect (which is also an embodiment of the 148th embodiment of the first aspect) the PEG is linear PEG or branched PEG.

[0164] In a 150th embodiment of the first aspect (which is also an embodiment of the 149th embodiment of the first aspect) the PEG is a linear PEG.

[0165] 151st embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 10 Nos. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 and 74 of the embodiment, preferably of the first aspect 5, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th , 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 and 150 aspects), the mole percentage of lipids in the lipid composition is - 20 to 80 mol% of a first lipid, - 20 to 80 mol% of a second lipid, - 10 to 50 mol% of a third lipid, and - 1-10 mol% of a fourth lipid and the total lipid content of the lipid composition is 100 mol%.

[0166] In a 152nd embodiment of the first aspect (which is also an embodiment of the 151st embodiment of the first aspect), the molar proportion of the lipid composition is - 20 to 60 mol% of a first lipid, - 20-60 mol% of a second lipid, - 10 to 40 mol% of a third lipid, and - 1-5 mol% of a fourth lipid and the total lipid content of the lipid composition is 100 mol%.

[0167] 153rd embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, Nos. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76 of the embodiment, preferably of the first aspect. 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, In any one of the embodiments of the aspects 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 and 152, the lipid composition comprises: - CHEMS as the first lipid, - β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid, - Cholesterol as the third lipid -Methoxy PEG2000-DMPE as the fourth lipid.

[0168] In a 154th embodiment of the first aspect (which is also an embodiment of the 153rd embodiment of the first aspect), the lipid composition comprises: - 49 mol% CHEMS as the first lipid, - 29 mol% of β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid, - 20 mol% cholesterol as a third lipid, and - 2 mol% mPEG2000-DMPE as the fourth lipid Includes.

[0169] 155th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 5 Nos. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 of the 8th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th and 76th embodiments, preferably of the first aspect , 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th , 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th , 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 and 154), the lipid composition comprises - CHEMS as the first lipid, - β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid, - Cholesterol as the third lipid, and - Methoxy PEG2000-DMPE as the fourth lipid Includes.

[0170] In a 156th embodiment of the first aspect (which is also an embodiment of the 155th embodiment of the first aspect), the lipid composition comprises: - 48 mol% CHEMS as the first lipid, - 29 mol% of β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid, - 18 mol% cholesterol as the third lipid, and - 5 mol% methoxy PEG2000-DMPE as the fourth lipid Includes.

[0171] 157th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, , 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 8th 7, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th , 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th , 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 and 152), the lipid composition is dissolved in a water-miscible solvent.

[0172] In a 158th embodiment of the first aspect (which is also an embodiment of the 157th embodiment of the first aspect), the solvent is selected from the group comprising ethanol, acetone, 1-butanol, 2-butanol, tert-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, 2-propanol, dimethylsulfoxide, preferably selected from the group comprising ethanol, tert-butanol and 1-butanol.

[0173] 159th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 10 5, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd , 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, In any one of embodiments of the present invention (particularly embodiments of aspects 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 and 158), the average particle size of the lipid nanoparticles is about 15 nm to about 400 nm.

[0174] In a 160th embodiment of the first aspect (which is also an embodiment of the 159th embodiment of the first aspect), the lipid nanoparticles have an average particle size of about 25 nm to about 200 nm.

[0175] In a 161st embodiment of the first aspect (which is also an embodiment of any one of the 159th and 160th embodiments of the first aspect), the average particle size of the lipid nanoparticles is from about 40 nm to about 100 nm.

[0176] In a 162nd embodiment of the first aspect (which is also an embodiment of any one of the 159th, 160th and 161st embodiments of the first aspect), the average particle size of the lipid nanoparticles is measured by dynamic light scattering (DLS).

[0177] A 163rd embodiment of the first aspect (the 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 1st 24, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, In each and any one of aspects 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 and 162), the lipid nanoparticles have a zeta potential or exhibit a zeta potential, wherein the zeta potential is from about -20 mV to about +20 mV.

[0178] In a 164th embodiment of the first aspect (which is also an embodiment of the 163rd embodiment of the first aspect), the zeta potential is from about −10 mV to about +10 mV, preferably from about −5 mV to about +5 mV.

[0179] 165th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th , 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 1st 01, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113rd, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd , 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 and 164), the payload molecule is selected from the group comprising a nucleic acid molecule, or a polypeptide molecule comprising 50 to 1000 amino acids and having an isoelectric point (pI) greater than 6.

[0180] In a 166th embodiment of the first aspect (which is also an embodiment of the 165th embodiment of the first aspect) the payload molecule is a nucleic acid molecule.

[0181] In a 167th embodiment of the first aspect (which is also an embodiment of each and any one of the 165th and 166th embodiments of the first aspect), the nucleic acid is selected from the group comprising a ribonucleic acid molecule, a deoxyribonucleic acid molecule, or a combination thereof.

[0182] In a 168th embodiment of the first aspect (which is also each and any one embodiment of the 165th, 166th and 167th embodiments of the first aspect), the nucleic acid molecule is a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, or a partially double-stranded nucleic acid molecule.

[0183] In a 169th embodiment of the first aspect (which is also an embodiment of each and any one of the 165th, 166th, 167th and 168th embodiments of the first aspect), the nucleic acid comprises about 15 to about 20,000 nucleotides, preferably the nucleic acid molecule comprises about 21 to about 10,000 nucleotides, more preferably the nucleic acid molecule comprises about 100 to about 8,000 nucleotides, and most preferably the nucleic acid molecule comprises about 200 to about 7,000 nucleotides.

[0184] In a 170th embodiment of the first aspect (which is also an embodiment of each and any one of the 165th, 166th, 167th, 168th and 169th aspects of the first aspect), the nucleic acid molecule is a functional nucleic acid molecule.

[0185] In a 171 st embodiment of the first aspect (which is also an embodiment of the 170 th embodiment of the first aspect), the functional nucleic acid molecule is selected from the group comprising mRNA, mRNA encoding Cas (CRISPR associated endonuclease protein), plasmid DNA, ssDNA (single stranded DNA), aptamer, Spiegelmer, siRNA molecule, antisense molecule, miRNA (microRNA) molecule, sgRNA (single guide RNA) molecule, saRNA (self-amplifying RNA) molecule, and combinations thereof.

[0186] In a 172nd embodiment of the first aspect (which is also an embodiment of the 165th, 166th, 167th, 168th, 169th, 170th and 171st embodiments of the first aspect), the mass ratio of lipids to nucleic acid in the formulation is 5-40, preferably 10-35, more preferably the mass ratio of lipids to nucleic acid in the formulation is 15-30, and most preferably the mass ratio of lipids to nucleic acid in the formulation is about 28.

[0187] In a 173rd embodiment of the first aspect (which is also an embodiment of the 165th, 166th, 167th, 168th, 169th, 170th, 171st and 172nd aspects of the first aspect), the nucleic acid molecule is comprised within a lipid nanoparticle.

[0188] In a 174th embodiment of the first aspect (which is also an embodiment of the 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd and 173rd embodiments of the first aspect), the mass ratio of lipid to nucleic acid in the lipid formulation is 5-40, preferably 10-35, more preferably the mass ratio of lipid to nucleic acid in the lipid formulation is 15-30, and most preferably the mass ratio of lipid to nucleic acid in the lipid formulation is about 28.

[0189] In a 175th embodiment of the first aspect (which is also an embodiment of the 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd and 174th embodiments of the first aspect), the mass ratio of lipid to nucleic acid in the lipid nanoparticle is 5-40, preferably 10-35, more preferably the mass ratio of lipid to nucleic acid in the lipid nanoparticle is 15-30, and most preferably the mass ratio of lipid to nucleic acid in the lipid nanoparticle is about 28.

[0190] In a 176th embodiment of the first aspect (which is also an embodiment of the 165th embodiment of the first aspect), the payload molecule is a polypeptide molecule comprising 10 to 1000 amino acid residues, preferably 50 to 900 amino acid residues, more preferably 80 to 800 amino acid residues.

[0191] In a 177th embodiment of the first aspect (which is also an embodiment of the 165th and 176th aspects of the first aspect) the polypeptide comprises or has an isoelectric point (pI) greater than 6.

[0192] 178th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 10 1, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd , 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, 145th, 1st In each and any one of the embodiments of the aspects 46, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175 and 176), the payload molecule is a therapeutically active substance or a precursor thereof.

[0193] 179th embodiment of the first aspect (1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 7th 7, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 1st 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 1 24, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, 14th 5, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178), wherein the formulation is suitable for topical administration.

[0194] In a 180th embodiment of the first aspect (which is also an embodiment of the 179th embodiment of the first aspect), the local administration is intramuscular administration.

[0195] In a 181st embodiment of the first aspect (which is also an embodiment of the 179th embodiment of the first aspect), the local administration is subcutaneous administration.

[0196] In a 182nd embodiment of the first aspect (which is also an embodiment of the 179th embodiment of the first aspect), the topical administration is intradermal administration.

[0197] The problem underlying the present invention is solved in a second aspect (which is also a first embodiment of the second aspect) by a formulation according to the first aspect (including any embodiment thereof) for use in a method for the treatment and / or prevention of a disease in a subject; the method comprising local administration of a payload molecule to the subject, preferably comprising local administration of the formulation.

[0198] In a second embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the local administration is intramuscular administration.

[0199] In a third embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the local administration is subcutaneous administration.

[0200] In a fourth embodiment of the second aspect (which is also an embodiment of the first embodiment of the second aspect), the local administration is intravitreal administration.

[0201] In a fifth embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the local administration is intrathecal administration.

[0202] In a sixth embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the local administration is intratumoral administration.

[0203] In a seventh embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the local administration is intracerebral administration.

[0204] In an eighth embodiment of the second aspect (which is also an embodiment of the first aspect of the second aspect), the topical administration is intradermal administration.

[0205] In a ninth embodiment of the second aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the second aspect), the disease is a disease that can be treated and / or prevented by a payload molecule.

[0206] The problem underlying the present invention is solved in a third aspect by a pharmaceutical composition comprising a formulation as defined in the first aspect of the invention (including any embodiment thereof) and a pharma- ceutically acceptable excipient.

[0207] The problem underlying the present invention is solved in a fourth aspect (which is also a first aspect of the fourth aspect) by a pharmaceutical composition according to the third aspect (including any aspect thereof) for use in a method for the treatment and / or prevention of a disease in a subject; the method comprising local administration of a payload molecule to the subject, preferably comprising local administration of a pharmaceutical composition.

[0208] In a second embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the local administration is intramuscular administration.

[0209] In a third embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the local administration is subcutaneous administration.

[0210] In a fourth embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the local administration is intravitreal administration.

[0211] In a fifth embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the local administration is intrathecal administration.

[0212] In a sixth embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the local administration is intratumoral administration.

[0213] In a seventh embodiment of the fourth aspect (which is also an embodiment of the first embodiment of the fourth aspect), the local administration is intracerebral administration.

[0214] In an eighth embodiment of the fourth aspect (which is also an embodiment of the first aspect of the fourth aspect), the topical administration is intradermal administration.

[0215] In a ninth embodiment of the fourth aspect (which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the fourth aspect), the disease is a disease that can be treated and / or prevented by a payload molecule.

[0216] The problem underlying the present invention is solved in a sixth aspect by the use of a first lipid in the preparation of a formulation for use according to the second aspect of the invention (including any embodiment thereof), wherein the first lipid is a first lipid as defined in the first aspect of the invention (including any embodiment thereof).

[0217] The problem underlying the present invention is solved in a seventh aspect by the use of a second lipid in the preparation of a formulation for use according to the second aspect of the invention (including any embodiment thereof), wherein the second lipid is a second lipid as defined in the first aspect of the invention (including any embodiment thereof).

[0218] The problem underlying the present invention is solved in an eighth aspect by the use of a third lipid in the preparation of a formulation for use according to the second aspect of the invention (including any embodiment thereof), wherein the third lipid is a third lipid as defined in the first aspect of the invention (including any embodiment thereof).

[0219] The problem underlying the present invention is solved in a ninth aspect by the use of a fourth lipid in the preparation of a formulation for use according to the second aspect of the invention (including any embodiment thereof), wherein the fourth lipid is a fourth lipid as defined in the first aspect of the invention (including any embodiment thereof).

[0220] Without wishing to be bound by any theory, the inventors have surprisingly found that, compared to the strong cationic LNPs and LNPs comprising ionizable cationic lipids used in the prior art for intramuscular, subcutaneous and intradermal administration of RNA, the lipid compositions used in the formulations according to the invention and the LNPs formed by such lipid compositions are superior to these strong cationic LNPs and pH-sensitive cationic LNPs comprising ionizable cationic lipids. The LNPs formed by the lipid compositions used in the formulations according to the invention are also referred to herein as the LNPs of the invention.

[0221] The lipid mixture of the present invention is preferably strongly positively charged at low pH values, since the cationic lipids are positively charged at acidic pH values, while the anionic lipids are no longer charged due to protonation of their carboxylic acid groups.These strongly positively charged lipid systems can electrostatically interact with RNA and accordingly complex the RNA to form LNPs.Once the LNPs of the present invention are formed and the pH of the suspension medium is adjusted to a neutral pH value, the cationic lipids on the surface of the LNPs are still positively charged, while at the same time the negative charges of the anionic lipids are regenerated by deprotonation of their carboxylic acid groups; meanwhile, the RNA remains trapped within the LNP particles.The molar ratio of cationic lipids to anionic lipids in these amphoteric LNP systems is preferably selected such that at neutral pH values, the number of cationic charges exhibited by the cationic lipids exceeds the number of anionic charges exhibited by the anionic lipids.As a result, the LNP surface is decorated with approximately the same number of positive and negative charges. Thus, the surface of the resulting LNP can be described as overall neutral, and in contrast to LNPs containing prior art ionizable cationic lipids, it is hydrophilic rather than hydrophobic.

[0222] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, a payload molecule, as preferably used herein, is a compound active at a target. The activity is preferably an activity selected from the group including biological activity, chemical activity, biochemical activity, physiological activity, pharmacological activity and physical activity. As preferably used herein, a biological activity is an activity that produces a biological effect at a target. Preferably, such a biological effect is an increase or decrease in the activity of a protein, polypeptide or peptide, wherein the protein or polypeptide is an enzyme, a structural protein, a scaffolding protein, a transmembrane protein, a channel protein, a secretory protein, a hormone, a mitochondrial protein, a receptor, a ligand, an epitope / neoepitope / antigen peptide (qualified for MHC-I binding as a 9-mer or MHC-II binding as a 13-25-mer) (see, e.g., Andreatta M et al., Bioinformatics 2016; 32:511-7), or a nucleic acid binding protein. Alternatively, such biological effect is an increase or decrease in the activity or half-life of a nucleic acid molecule, or the localization, positioning, processing, transport, or packaging of a nucleic acid molecule. Such nucleic acids can be deoxyribonucleic acid or ribonucleic acid (see, for example, Jurtz V et al., J Immunol 2017; 199:3360-8).

[0223] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, a target is selected from the group including molecules, intracellular structures, cells, tissues and organs. Preferably, the target is targeted by the formulation of the present invention. When it is desired to replace, substitute or supplement defective, abnormal, missing or low protein expression in a given cell by the target, the target function is assumed to result in a gain-of-function phenotype. Also, when it is desired to inhibit or eliminate overactive, dominant-active, abnormal or high protein expression in a given cell by the target, the target function is assumed to result in a loss-of-function phenotype. For example, in muscle cells, gene mutations are intended to be repaired at the DNA level, and defective mutant protein variants are replaced (transient forced expression of active protein) or repaired (mRNA exon skipping). Transient forced protein expression is also intended to be secreted, taken up into the cell and presented extracellularly by any type of antigen presenting cell present or attracted to the site of administration.

[0224] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, local administration is an administration selected from the group including intramuscular administration, subcutaneous administration, intravitreal administration, intrathecal administration, intratumoral administration, intracerebral administration, intramyocardial administration, intracoronary administration and intradermal administration.

[0225] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, a pH-independently positively charged lipid is a lipid that has an overall positive charge regardless of the pH to which such lipid is exposed, preferably any pH between 0 and 14. More preferably, the pH-independently positively charged lipid is selected from the group including DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane chloride).

[0226] In an alternative embodiment of each and any aspect of the present invention, including any embodiment thereof, as preferably used herein, the overall pKa value of the LNP is measured by using a TNS assay (6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt assay). In a first step, a 4x TNS master buffer is prepared, which consists of 100 mM citrate, 80 mM sodium phosphate, 80 mM ammonium acetate, and 600 mM sodium chloride, and a stock solution of 1 mg / ml TNS in a mixture of dimethylformamide and water (2 / 8 v / v). To make a series of 2x TNS assay buffers with different pH values ​​(e.g., ranging from pH 3 to pH 10), 20 ml of 4x TNS master buffer for each pH value is placed in a tube, such as a Falcon tube, the pH is adjusted to the target pH value with HCl or NaOH, and an appropriate amount of water is added to obtain 40 ml of 2x TNS assay buffer. Subsequently, a working solution of the LNPs for measuring the pKa is prepared by adding 8 μl of TNS stock solution (dimethylformamide and water (2 / 8 v / v)) and 8 μl of the formulation containing the LNPs (10 mM TRIS pH 7.5) (lipid concentration 2-8 mg / ml) to 2 ml of water. The measurements are carried out in black 96-well plates. For this purpose, 100 μl of 2× TNS assay buffer is placed in each well for each pH value and 100 μl of the working solution is added to each well to a final volume of 200 μl per well. The final mixture thus obtained is incubated for 30 min at 37 °C and then centrifuged for 1 min at 1000 rpm. The fluorescence intensity of the supernatant is read with a plate reader such as a Tecan M1000 plate reader at excitation 322 nm and emission 431 nm. After the measurements, the background fluorescence value of the empty wells of the 96-well plate is subtracted from each probe / lipid / buffer mixture. The fluorescence intensity values ​​are then normalized to the value at the lowest pH. Then, plot the normalized fluorescence intensity versus pH to obtain a line of best fit. Determine the point on the line of best fit where the normalized fluorescence intensity is equal to 0.5, and consider the corresponding pH value as the pKa of the LNP.

[0227] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, an amphoteric, overall neutrally charged lipid nanoparticle is a lipid nanoparticle in which the sum of the negative and positive charges borne by the lipid nanoparticle is zero. Preferably, the sum is zero when the lipid nanoparticle is exposed to a pH value of 7-8, more preferably when the lipid nanoparticle is exposed to a pH value of 7.4. As a result, the zeta potential of the lipid nanoparticle is zero ± 5 mV.

[0228] In an embodiment of each and any aspect (including any embodiment thereof) of the present invention, as preferably used herein, a straight and / or branched C1-C4 hydrocarbon group each and individually means any of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0229] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, a neutral lipid, as preferably used herein, is a lipid that has no charged molecular moieties or is a zwitterionic lipid molecule that contains equal amounts of positively and negatively charged molecular moieties. Preferably, the neutral lipid is a sterol, phosphatidylethanolamine, or phosphatidylcholine.

[0230] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, a PEGylated lipid is a compound comprising a lipid moiety and a PEG moiety bound to the lipid moiety.Preferably, the PEG moiety is covalently bound to the lipid moiety.The lipid moiety is preferably a sterol, a phosphatidylethanolamine or a diacylglycerol, and more preferably, the lipid moiety is selected from the group comprising 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-distearoyl-rac-glycerol, 1,2-dipalmitoyl-rac-glycerol and 1,2-dimyristoyl-rac-glycerol.

[0231] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, the outer surface of the lipid nanoparticle is the surface of the lipid nanoparticle that faces the medium surrounding the lipid nanoparticle. Preferably, the outer surface of the lipid nanoparticle faces the internal structure of the lipid nanoparticle.

[0232] In each and every embodiment of any aspect (including any embodiment thereof) of the present invention, as preferably used herein, a neutral pH value is a pH value of from about 6.7 to about 7.7, preferably from about 6.9 to about 7.5, more preferably from about 7.2 to about 7.5.

[0233] In an embodiment of each and any aspect (including any embodiment thereof) of the present invention, the negative charge of the lipid composition is provided by the first lipid.

[0234] In an embodiment of each and any aspect (including any embodiment thereof) of the present invention, the positive charge of the lipid composition is provided by a second lipid.

[0235] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the carboxylic acid group of the first lipid is present in a protonated or deprotonated form. When present in a deprotonated form, the carboxylic acid group confers a negative charge to the first lipid.

[0236] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), the pKa value of the first lipid is determined by the carboxylic acid group of the first lipid.Preferably, the pKa value of the first lipid is determined by the pKa value of the carboxylic acid group.More preferably, the pKa value of the carboxylic acid group is the pKa value of the first lipid.

[0237] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the functional group of the second lipid is present in a protonated or unprotonated form. When present in a protonated form, the functional group confers a positive charge on the second lipid.

[0238] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), the second lipid is (a) a cationic lipid having at least one molecular moiety with a pKa value of 9.5 or more and 13.5 or less, or (b) a cationic lipid that is positively charged in a pH-independent manner.

[0239] In an embodiment of each and any aspect (including any embodiment thereof) of the present invention, as preferably used herein, the term 12-20 hydrocarbon alkyl group includes 12 hydrocarbon alkyl group, 13 hydrocarbon alkyl group, 14 hydrocarbon alkyl group, 15 hydrocarbon alkyl group, 16 hydrocarbon alkyl group, 17 hydrocarbon alkyl group, 18 hydrocarbon alkyl group, 19 hydrocarbon alkyl group, and 20 hydrocarbon alkyl group.

[0240] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the term branched chain alkyl group, as preferably used herein, is an alkyl group having a backbone of hydrocarbon atoms, with at least one further hydrocarbon covalently bonded to a carbon atom of the backbone hydrocarbon at a position different from the hydrocarbons at either end of the backbone.

[0241] In the embodiment of each and any aspect of the present invention, including any embodiment thereof, the term unsaturated alkyl group, as preferably used herein, is an alkyl group that comprises at least two hydrocarbons and at least one double bond that covalently connects the two hydrocarbons. Those skilled in the art will understand that the unsaturated alkyl group comprises one or more double bonds. In a preferred embodiment, the unsaturated alkyl group comprises one, two, three, or four double bonds. Those skilled in the art will also understand that the double bond may be located between any two adjacent hydrocarbons of the alkyl group. Preferably, when the unsaturated alkyl group comprises more than one double bond, such double bond is arranged as a double bond separated by two single bonds. Those skilled in the art will also understand that the double bond is either a cis double bond or a trans double bond. Preferably, the double bond is a cis double bond.

[0242] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the compound CHEMS (hemisuccinic acid cholesterol ester) is a compound of the formula: The file is TIFF2025505258000001.tif37128. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000002.tif37128.

[0243] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the compound alkyl carboxylic acid is a compound of the formula: TIFF2025505258000003.tif16128 [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11] It is.

[0244] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the compound hemisuccinic acid diacylglycerol ester is a compound of the formula: TIFF2025505258000004.tif31128 [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11] It is.

[0245] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), the compound phosphatidylserine is a compound of the formula: TIFF2025505258000005.tif37134 [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11] It is. Preferably, the stereocenter has the formula: TIFF2025505258000006.tif37134 [wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11] It is defined as shown below.

[0246] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide is a compound of the formula: The file is TIFF2025505258000007.tif25159. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000008.tif25159.

[0247] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound L-arginyl-β-alanine-N-palmityl-N-oleylamide is a compound of the formula: The file is TIFF2025505258000009.tif25159. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000010.tif25159.

[0248] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl-chloride) is a compound of the formula: The file is TIFF2025505258000011.tif32142.

[0249] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane, preferably as the chloride salt) is a compound of the formula: The file is TIFF2025505258000012.tif18142.

[0250] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DC-cholesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) is a compound of the formula: The file is TIFF2025505258000013.tif31128. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000014.tif31128.

[0251] In an embodiment of each and any aspect of the present invention, including its embodiments, a zoosterol, as preferably used herein, is a sterol of animal origin.

[0252] In an embodiment of each and any aspect of the present invention, including its embodiments, a plant sterol, as preferably used herein, is a sterol derived from a plant.

[0253] In an embodiment of each and any aspect of the present invention, including embodiments thereof, a zwitterionic phospholipid, as preferably used herein, is a phospholipid that contains an equal number of positively charged and negatively charged functional groups.

[0254] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DSPC (1,2-distearoyl-sn-glycero-3-phosphoethanolcholine) is a compound of the formula: The file is TIFF2025505258000015.tif31170. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000016.tif31170.

[0255] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DMPC (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula:

[0256] TIFF2025505258000017.tif32150Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000018.tif32150.

[0257] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000019.tif31170. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000020.tif31170.

[0258] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000021.tif32150. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000022.tif32150.

[0259] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000023.tif32138. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000024.tif32138.

[0260] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DPhyPE (1,2-(7R,11R) diphytanoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000025.tif32165. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000026.tif32165.

[0261] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000027.tif31170. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000028.tif31170.

[0262] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) is a compound of the formula: The file is TIFF2025505258000029.tif31170. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000030.tif31170.

[0263] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound cholesterol is a compound of the formula: The file is TIFF2025505258000031.tif31128. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000032.tif31128.

[0264] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound stigmasterol (stigmasta-5,22-dien-3-ol) is a compound of the formula: The file is TIFF2025505258000033.tif40128. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000034.tif40128.

[0265] In an embodiment of each and any aspect of the present invention, including its embodiments, as preferably used herein, a shielding lipid is a lipid that provides steric stabilization of lipid nanoparticles and also reduces unintended interactions of the lipid nanoparticle surface with proteinaceous body fluids in vivo, which may otherwise cause aggregation or degradation of the LNP. This allows for sustained in vivo delivery of the lipid nanoparticle payload to target cells, thus allowing for more efficient delivery and prolonged functional bioactivity of the LNP payload. The term shielding also means that elements of the immune system or other defense or removal mechanisms of the organism to which such lipid composition is administered do not immediately interact with the lipid composition, again enhancing its functional bioavailability in the organism. To that extent, the shielding lipid acts as an anti-opsonizing compound. Without wishing to be bound by any mechanism of action or theory, it appears that the shielding lipids form a cover or coat that reduces the surface area of ​​the lipid composition available for interaction with the environment; otherwise, the lipid composition would fuse with other lipids or become bound by factors of the human and animal body, respectively, at a time too early for such interactions; however, it should be recognized that at a later stage, i.e., long after administration of the lipid composition, such interactions are usually preferred or desirable, at least to some extent, to provide for delivery of the payload of the lipid compositions and formulations of the present invention, respectively.

[0266] The shielding compound is preferably a biologically inert molecular moiety covalently attached to the lipid moiety. Depending on the hydrophobicity of the lipid moiety, e.g., the alkyl chain length of the lipid moiety from C12 to C18 carbons (i.e., 12, 13, 14, 15, 16, 17, 18, 19, and 20 hydrocarbons), the shielding compound can be designed to dissociate from the lipid nanoparticle over time, thereby making the nanoparticle surface more accessible for the intended interaction with the membrane of the target cell.

[0267] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-500-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-500]) is a compound of the formula: The file is TIFF2025505258000035.tif26162. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000036.tif26160.

[0268] Preferably, the compound mPEG-500-DSPE is present and used as the respective ammonium salt.

[0269] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-1000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]) is a compound of the formula: The file is TIFF2025505258000037.tif26165. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000038.tif26163.

[0270] Preferably, the compound mPEG-1000-DSPE is present and used as the respective ammonium salt.

[0271] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) is a compound of the formula: The file is TIFF2025505258000039.tif27169. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000040.tif27170.

[0272] Preferably, the compound mPEG-2000-DSPE is present and used as the respective ammonium salt.

[0273] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-5000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]) is a compound of the formula: The file is TIFF2025505258000041.tif25165. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000042.tif24163.

[0274] Preferably, the compound mPEG-5000-DSPE is present and used as the respective ammonium salt.

[0275] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]) is a compound of the formula: The file is TIFF2025505258000043.tif26166. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000044.tif26166.

[0276] Preferably, the compound mPEG-DSPE is present and used as the respective ammonium salt.

[0277] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) is a compound of the formula: The file is TIFF2025505258000045.tif29164. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000046.tif30170.

[0278] Preferably, the compound mPEG-2000-DMPE is present and used as the respective ammonium salt.

[0279] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) is a compound of the formula: The file is TIFF2025505258000047.tif27128.

[0280] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-DPG (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000) is a compound of the formula: The file is TIFF2025505258000048.tif27128.

[0281] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-DSG (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000) is a compound of the formula: The file is TIFF2025505258000049.tif32162. In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-C-DMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristoyloxypropyl-3-amine) is a compound of the formula: The file is TIFF2025505258000050.tif27128.

[0282] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound 2-(methoxyPEG-2000)-N,N-dioctadecylacetamide is a compound of the formula: The file is TIFF2025505258000051.tif23159.

[0283] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound 2-(methoxyPEG-2000)-N,N-ditetradecylacetamide is a compound of the formula: The file is TIFF2025505258000052.tif23129.

[0284] In an embodiment of each and any aspect of the present invention, including embodiments thereof, the compound mPEG-2000-C8-ceramide (N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}) is a compound of the formula: The file is TIFF2025505258000053.tif27143. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000054.tif27143.

[0285] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the compound mPEG-2000-C16-ceramide (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}) is a compound of the formula: The file is TIFF2025505258000055.tif27144. Preferably, the stereocenter has the formula: Defined as shown in TIFF2025505258000056.tif27144.

[0286] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the average particle size of the lipid nanoparticles is measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern Panalytical Ltd, Malvern, UK). All measurements are preferably performed in an aqueous buffer, with 270 mM sucrose solution buffered with 10 mM TRIS at pH 7.4 as the dispersion medium. DLS is disclosed in detail, for example, in Stetefeld, J., McKenna, SA & Patel, TR "Dynamic light scattering: a practical guide and applications in biomedical sciences" Biophys Rev 8, 409-427 (2016); and Thomas, JC "The determination of log normal particle size distributions by dynamic light scattering" Journal of Colloid and Interface Science 117, 187-192 (1987), the disclosures of which are incorporated herein by reference.

[0287] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the zeta potential, e.g., the zeta potential of lipid nanoparticles, is measured by Laser Doppler Electrophoresis using a Zetasizer Ultra (Malvern Panalytical Ltd, Malvern, UK). All measurements are preferably performed in an aqueous buffer, a 270 mM sucrose solution buffered with 10 mM TRIS at pH 7.4 as the carrier medium. Laser Doppler electrophoresis is described in detail, for example, in Clogston, JD & Patri, AK in "Characterization of Nanoparticles Intended for Drug Delivery" 697, 63-70 (Humana Press, 2011); and Sze, A., Erickson, D., Ren, L. & Li, D. "Zeta-potential measurement using the Smoluchowski equation and the slope of the current-time relationship in electroosmotic flow" Journal of Colloid and Interface Science 261, 402-410 (2003), the disclosures of which are incorporated herein by reference.

[0288] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), the overall charge of the lipid nanoparticles is about ±10 mV, preferably about ±5 mV.

[0289] It will be appreciated by those of skill in the art that the zeta potential of a lipid nanoparticle reflects the overall charge of such lipid nanoparticle.

[0290] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the nucleic acid molecule is a polymer of building blocks. The building blocks are nucleotides. In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the ribonucleic acid molecule is a polymer of building blocks, wherein the building blocks are ribonucleotides. In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the deoxyribonucleic acid molecule is a polymer of building blocks, wherein the building blocks are deoxyribonucleotides, preferably 2'deoxyribonucleotides. In an embodiment of each and any aspect of the present invention, including any embodiment thereof, the nucleotide building blocks are selected from the group comprising 2'-fluoro, 2'-methoxy modified ribonucleotides.

[0291] In an embodiment of each and any aspect of the present invention, including any of its embodiments, the nucleic acid molecule is a D-nucleic acid molecule. In one embodiment, as preferably used herein, a D-nucleic acid molecule is a polymer of building blocks, where the building blocks are D-nucleotides. In a preferred embodiment of each and any aspect of the present invention, including any of its embodiments, the D-nucleic acid molecule is a D-ribonucleic acid molecule. In one embodiment, as preferably used herein, a D-ribonucleic acid molecule is a polymer of building blocks, where the building blocks are D-ribonucleotides. In an alternative preferred embodiment of each and any aspect of the present invention, including any of its embodiments, the D-nucleic acid molecule is a D-deoxyribonucleic acid molecule. In one embodiment, as preferably used herein, a D-deoxyribonucleic acid molecule is a polymer of building blocks, where the building blocks are D-deoxyribonucleotides.

[0292] In an embodiment of each and any aspect of the present invention, including any of its embodiments, the nucleic acid molecule is a D,L-nucleic acid molecule. In one embodiment, as preferably used herein, a D,L-nucleic acid molecule is a polymer of building blocks, where the building blocks are both D-nucleotides and L-nucleotides. In such a D,L-nucleic acid molecule, it will be understood by those skilled in the art that any relative ratio of D-nucleotides and L-nucleotides can be realized. Furthermore, in such a D,L-nucleic acid molecule, it will be understood by those skilled in the art that any arrangement of D-nucleotides and L-nucleotides can be realized. In a preferred embodiment of each and any aspect of the present invention, including any of its embodiments, the D,L-nucleic acid molecule is a D,L-ribonucleic acid molecule. In one embodiment, as preferably used herein, a D,L-ribonucleic acid molecule is a polymer of building blocks, where the building blocks are both D-ribonucleotides and L-ribonucleotides. In alternative preferred embodiments of each and any aspect of the present invention, including any embodiment thereof, the D,L-nucleic acid molecule is a D,L-deoxyribonucleic acid molecule. In one embodiment, as preferably used herein, a D,L-deoxyribonucleic acid molecule is a polymer of building blocks, where the building blocks are both D-deoxyribonucleotides and L-deoxyribonucleotides.

[0293] In an embodiment of each and any aspect of the present invention, including any embodiment thereof, as preferably used herein, a single-stranded nucleic acid molecule is a nucleic acid molecule consisting of a single strand of nucleotides covalently linked to each other, preferably by a bond or linkage selected from the group consisting of a phosphodiester bond or a phosphothioether linkage. Preferably, such linkage covalently connects the OH group of the 3' C atom of the sugar moiety of a first nucleoside to the first OH group of a phosphate, and the OH group of the 5' C atom of the sugar moiety of a second nucleoside to the second OH group of said phosphate.

[0294] In an embodiment of each and any aspect of the present invention (including any embodiment thereof), as preferably used herein, a double-stranded nucleic acid molecule is a nucleic acid molecule that comprises a double-stranded structure. A double-stranded structure is a structure in which the nucleotides of the first strand and the nucleotides of the second strand are bound to each other. Such binding is preferably selected from the group including non-covalent and covalent binding. In one embodiment, the non-covalent binding is a binding through one or more hydrogen bonds or caused by one or more hydrogen bonds; preferably, the non-covalent binding is a binding through Watson-Crick base pairing or Hoogsteen base pairing between one or more nucleotides forming the first nucleotide strand and one or more nucleotides forming the second nucleotide strand or caused by such base pairing. More preferably, all nucleotides of the first nucleotide strand and all nucleotides of the second nucleotide strand are involved in such base pairing, whereby one nucleotide of the first strand is base paired with one nucleotide of the second strand.

[0295] In an alternative embodiment, the covalent bond is a bond via one or more covalent bonds between at least one nucleotide of a first nucleotide strand that forms a double-stranded structure and at least one nucleotide of a second nucleotide strand that forms the double-stranded structure.

[0296] In one embodiment, as preferably used herein, a functional nucleic acid molecule is a nucleic acid molecule that has a function different from a structural nucleic acid molecule. In a preferred embodiment, a functional nucleic acid molecule is different from a ribosomal ribonucleic acid molecule, different from a 7SL-RNA, and different from a ribozyme.

[0297] In one embodiment, as preferably used herein, an mRNA is a nucleic acid molecule that includes, in a 5'→3' direction, a cap structure, a 5' untranslated region (5'UTR), a coding sequence that usually starts with an AUG codon and ends with a stop codon linked to a coding sequence (CDS), a 3' untranslated region (3'UTR) and a polyA tail. Preferably, an mRNA is composed of ribonucleotides, more preferably D-ribonucleotides.

[0298] In one aspect, as preferably used herein, Cas (CRISPR-associated endonuclease protein)-encoding mRNA is an mRNA that encodes a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated endonuclease protein. Cas proteins and derivatives thereof include, for example, Cas9 DNA nuclease (NCBI Reference Sequence (mRNA): NC_002737.2; NCBI Reference Sequence (protein): NP_269215.1), or Cas13a-d RNA nucleases (e.g., LwaCas13a Leptotrichia wadeii (NCBI Reference Sequence: WP_021746003.1); PspCas13b Prevotella sp. P5-125; RfxCas13d Ruminococcus flavefaciens (NCBI Reference Sequence: WP_075424065.1)).

[0299] In one aspect, as preferably used herein, aptamer is a single-stranded nucleic acid molecule that can form a well-defined and stable three-dimensional structure, and can specifically bind to target molecule, and preferably, aptamer is composed of D-nucleotide.Aptamer can be identified against some target molecules (e.g., small molecules, proteins, nucleic acids), and even against cells, tissues, and organisms, and can inhibit the in vitro and / or in vivo function of specific target molecule. Aptamers are typically identified by a target-directed selection process called in vitro selection or Systematic Evolution of Ligands by Exponential Enrichment (SELEX) (Bock LC et al. (1992), Nature. 355(6360):564-6; Ellington & Szostak (1990) Nature, 346(6287):818-22; Tuerk C. and Gold L. (1990), Science. 249(4968):505-10). Unmodified aptamers are rapidly cleared from the bloodstream with half-lives ranging from minutes to hours, primarily due to degradation by nucleases and clearance from the body by the kidneys (a consequence of the inherent low molecular weight of aptamers). Therefore, in order for aptamers to be used therapeutically, they need to be modified at the 2' position of the sugar. More preferably, binding of the aptamer to the target molecule is by a non-covalent bond, even more preferably by a bond other than Watson-Crick or Hoogsteen base pairing.

[0300] In one embodiment, as preferably used herein, a spiegelmer is a single-stranded nucleic acid molecule that can form a well-defined and stable three-dimensional structure and can specifically bind to a target molecule, preferably, the spiegelmer is composed of L-nucleotides. Spiegelmers can be identified against some target molecules (e.g., small molecules, proteins, nucleic acids), and even against cells, tissues, and organisms, and can inhibit the in vitro and / or in vivo function of a specific target molecule. Spiegelmers are identified by mirror-image selection, which was first published in 1996 (Klussmann S. et al. (1996), Nat Biotechnol. 14(9):1112-5; Nolte A. et al. (1996), Nat Biotechnol. 14(9):1116-9). More preferably, binding of the aptamer to the target molecule is by a non-covalent bond, even more preferably by a bond other than Watson-Crick or Hoogsteen base pairing.

[0301] In one embodiment, as used herein, plasmid DNA is a recombinant circular double-stranded deoxyribonucleic acid molecule that contains a nucleotide sequence encoding, for example, a pharmacologically active polypeptide or protein, in particular an open reading frame under the control of a eukaryotic promoter sequence. Alternatively, the eukaryotic promoter system is used to express a pharmacologically active nucleotide sequence encoding a long non-coding RNA, tRNA, and / or shRNA / siRNA.

[0302] In one aspect, as preferably used herein, siRNA, also referred to in the art as short interfering RNA or silencing RNA, is a type of double-stranded RNA non-coding RNA molecule that is usually 20-24 base pairs in length and functions within the RNA interference (RNAi) pathway. It disrupts the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription and preventing translation. siRNA molecules can be catalytically generated by Dicer enzymes from long double-stranded RNA molecules and small hairpin RNA molecules. Post-transcriptional gene silencing induced by siRNA begins with the assembly of an RNA-induced silencing complex (RISC). This complex silences specific gene expression by cleaving mRNA molecules that code for target genes. To initiate this process, one of the two siRNA strands, the guide strand (antisense strand), is incorporated into RISC, while the other strand, the passenger strand (sense strand), is degraded. Certain Dicer enzymes may be involved in incorporating the guide strand into RISC. The siRNA then scans for an - essentially - perfectly complementary sequence on the mRNA molecule and guides the RISC to that complementary sequence. The cleavage of the mRNA molecule is thought to be catalyzed by the Piwi domain of the Argonaute protein of the RISC. The mRNA molecule is then precisely cleaved by cleaving the phosphodiester bond between the target nucleotides paired to the siRNA residues 10 and 11 counting from the 5' end. This cleavage results in mRNA fragments that are further degraded by cellular exonucleases. The 5' fragments are degraded from their 3' end by exosomes, while the 3' fragments are degraded from their 5' end by 5'-3' exoribonuclease 1 (XRN1). After cleavage, the target mRNA strand dissociates from the RISC, allowing more mRNA to be silenced. This dissociation process appears to be driven by external factors guided by ATP hydrolysis.Alternatively, siRNAs can be incorporated into the RNA-induced transcriptional silencing (RITS) complex, where the active RITS complex induces the formation of heterochromatin around the DNA that matches the siRNA, effectively silencing the gene in that region of DNA.

[0303] In one aspect, as preferably used herein, an antisense molecule or antisense oligonucleotide is a single-stranded deoxyribonucleic acid or a single-stranded ribonucleic acid.

[0304] In one aspect, as preferably used herein, natural or synthetic microRNA (miRNA, antagomir) is a small single-stranded non-coding RNA molecule containing about 22 nucleotides, which functions in gene silencing by post-transcriptional regulation of mRNA expression. miRNA functions through base pairing with complementary sequences in mRNA molecules. As a result, these mRNA molecules are silenced by one or more of the following processes: (1) the mRNA strand is cleaved into two fragments, (2) the mRNA is destabilized by shortening the poly(A) tail and degrading the mRNA, and (3) the translation of the mRNA is blocked.

[0305] In one embodiment, as preferably used herein, single guide RNA (sgRNA) is a specific RNA sequence that recognizes the target DNA region of interest and guides Cas nuclease there for editing. sgRNA is composed of two parts: crispr RNA (crRNA), which is a nucleotide sequence of 17, 18, 19 or 20 nucleotides that is complementary to the target DNA, and tracr RNA, which serves as a binding scaffold for Cas nuclease. sgRNA is a redesign of the naturally occurring two-piece guide RNA complex into a single continuous sequence. Simplified single guide RNA is used to guide Cas9 protein to bind and cut specific DNA sequence for genome editing.

[0306] In one embodiment, as preferably used herein, a self-amplifying RNA (saRNA) encodes a 5' and 3' CSE sequence, an alphavirus nsP1-4 gene, a subgenomic promoter, and a protein coding region of a therapeutic protein or antigen. After in situ translation, the nsP1-4 protein forms an RdRP complex that recognizes the adjacent CSE sequence and amplifies the protein-encoding transcript.

[0307] In one aspect, as preferably used herein, the mass ratio of lipid to nucleic acid is the ratio of the mass of the lipid composition of the formulation to the mass of the nucleic acid of the formulation.More preferably, the mass of the nucleic acid of the formulation is the total mass of the nucleic acid molecules contained in the formulation.

[0308] In one embodiment, as preferably used herein, the nucleic acid molecule is contained within a lipid nanoparticle. The term "contained within a lipid nanoparticle" preferably means that the majority of the individual nucleic acid molecules contained in the formulation are contained within the lipid nanoparticle, as will be understood by those skilled in the art. However, it is also within the scope of the present invention that at least a certain percentage of the individual nucleic acid molecules are attached to the lipid nanoparticle. It is also within the scope of the present invention that the majority of the individual nucleic acid molecules are associated with the lipid composition of the formulation comprising a payload molecule and a lipid composition, preferably the payload molecule and lipid composition are disclosed herein. It will be equally understood by those skilled in the art that the disclosures specifically provided herein regarding nucleic acid molecules as payloads of the formulations are equally applicable to polypeptides as payloads of the formulations.

[0309] In one aspect, as preferably used herein, intramuscular administration refers to injecting a substance into muscle (e.g., striated muscle, smooth muscle, skeletal muscle, cardiac muscle, sphincter muscle).This is one of several ways to administer a drug parenterally.Intramuscular injection may be preferred because muscle has many blood vessels that are larger than subcutaneous tissue, resulting in faster absorption than subcutaneous or intradermal injection.Drugs administered via intramuscular injection do not undergo the first-pass metabolic effect that affects oral drugs.

[0310] Common sites for intramuscular injection include the deltoid muscle in the upper arm and the gluteal muscle in the buttocks. In young children, the vastus lateralis muscle in the thigh is often used. Intramuscular administration following injection into skeletal muscle results in the delivery of large amounts of material to muscle tissue beneath the dermis and subcutaneous layers. Muscle is connected to a large network of blood and lymphatic vessels, providing depot effects of the material and distinct exposure to antigen-presenting cells.

[0311] Subcutaneous administration refers to delivery to the subcutaneous tissue area beneath the dermis and epidermis, while intradermal administration refers to the direct delivery of a substance to the dermis. Both routes provide some degree of depot effect. Intramyocardial administration refers to the direct injection of a substance into the heart muscle / myocardium. Additionally, non-skeletal muscles can be reached by direct injection of a substance into certain tissues, e.g., the urinary sphincter in the case of stress urinary incontinence.

[0312] In one embodiment, as preferably used herein, the disease for treatment and / or prevention that the formulation of the present invention can be used for is a disease that can be treated and / or prevented by a payload molecule.Diseases include, but are not limited to, peripheral arterial disease (mRNA payload: VEGF165 / COMP-Ang1, Follistatin-like 1), myositis (mRNA payload: anti-inflammatory cytokines), muscle hypertrophy (mRNA payload: myostatin), diet-induced obesity (mRNA payload: IL-6, BDNF, FGF21), ischemia-reperfusion injury (mRNA payload: myonectin), LAMA2-deficient muscular dystrophy (mRNA payload: αLNNd, Mini-agrin), depression (payload: kynurenine aminotransferase). Myopathies and neuromuscular disorders include, for example, the following diseases: myopathies, congenital myopathies, mitochondrial myopathies (payload tRNA), muscle degeneration, muscular dystrophies, myositis, cancer (e.g., soft tissue sarcoma), atrophy (payload mRNA of various growth factors), hypertrophy, pseudohypertrophy, muscle hypotonia, muscle spasms, cachexia, glycogen storage disease of muscle, muscle weakness (e.g., myasthenia gravis), and cardiomyopathies and myocardial infarction.

[0313] In one aspect, as preferably used herein, the prevention and / or treatment of disease using the basic formulation of the present invention includes vaccination.In the context of the present invention, intramuscular delivery of payload, such as protein or mRNA / nucleic acid encoding protein or polypeptide, which acts as epitope or neoepitope (antigen) for antigen-presenting cells, can be used as a vaccine to elicit antigen-specific immune response.Vaccination in the context of the present invention is intended to prevent or treat disease, such as infectious disease, cancer, allergy, autoimmune disease, etc.

[0314] In one embodiment, as preferably used herein, the pharma- ceutically acceptable excipient is a pharma-ceutically acceptable aqueous buffer system, preferably having a physiological pH value and isosmolar strength, and further comprising a cryoprotectant; more preferably, the buffer system has a pH value of 7.0-7.4 and an osmolality of 250-330 mosmol / kg, and comprises a cryoprotectant selected from the group comprising sucrose and trehalose; even more preferably, the buffer system is a 10 mM phosphate buffer or a 10 mM TRIS / HCl buffer at pH 7.4, and comprises 270 mM sucrose as a cryoprotectant. Other pharma-ceutically acceptable excipients are known to those skilled in the art.

[0315] In one aspect, as preferably used herein, the subject is a mammal selected from the group including human, monkey, rat and mouse, preferably the subject is a human.

[0316] In one aspect, as preferably used herein, a payload molecule is a therapeutically active substance.

[0317] In one aspect, as preferably used herein, therapy is a method for treating and / or preventing a disease in a subject, preferably the method comprises administering to the subject a substance suitable or effective for treating and / or preventing the disease.

[0318] In one embodiment, as preferably used herein, a therapeutic method is a method for treating and / or preventing a disease.More preferably, in such a method for treating and / or preventing a disease, a therapeutically active substance is administered to a subject, preferably to a subject in need of such treatment and / or prevention.In one embodiment, the subject is a human being.

[0319] It will be appreciated by those skilled in the art that the formulations disclosed herein, preferably those disclosed for use in therapy, can also be used in diagnosis, preferably in vivo or in situ diagnosis.

[0320] In one embodiment, as preferably used herein, diagnosis is a method for diagnosing a disease. More preferably, in such a method for diagnosing a disease, a diagnostic active substance is administered to a subject, preferably a subject in need of such a diagnosis. In one embodiment thereof, the subject is a human being.

[0321] In one aspect, as preferably used herein, the monoester of cholesterol and dicarboxylic acid is the ester formed between the OH group of cholesterol and one of the carboxy groups of dicarboxylic acid.Accordingly, when the dicarboxylic acid is succinic acid, the monoester of cholesterol and dicarboxylic acid is hemisuccinic acid cholesterol ester; when the dicarboxylic acid is glutaric acid, the monoester of cholesterol and dicarboxylic acid is hemiglutaric acid cholesterol ester; and when the dicarboxylic acid is adipic acid, the monoester of cholesterol and dicarboxylic acid is hemiadipic acid cholesterol ester.

[0322] In one embodiment, as preferably used herein, when a compound is said to be negatively charged at a designated pH value, this means that the compound has a negative charge in a medium having the designated pH value. In a preferred embodiment, the medium is a liquid that forms the liquid portion of the formulation comprising lipid nanoparticles, where the lipid nanoparticles comprise a lipid composition, and the lipid composition comprises a first lipid and a second lipid.

[0323] In one embodiment, as preferably used herein, when a compound is said to be positively charged at a designated pH value, this means that the compound has a positive charge in a medium having the designated pH value. In a preferred embodiment, the medium is a liquid that forms the liquid portion of the formulation comprising lipid nanoparticles, where the lipid nanoparticles comprise a lipid composition, and the lipid composition comprises a first lipid and a second lipid.

[0324] In one aspect, as preferably used herein, a lipid is a molecule that includes a hydrophobic portion and a hydrophilic portion, where the hydrophilic portion is a chemical group that attracts water, preferably a chemical group that carries at least one negative or positive charge at pH 7.2-7.5.

[0325] The formulations of the present invention, particularly those disclosed in relation to the first aspect, rely on the mixing of a solution containing the lipid components of the lipid composition with a solution containing mRNA, where the mixing is in-line mixing. The mixing step can be carried out by application of a microfluidic mixing device, in particular by using either a staggered herringbone mixer device, a Dean Vortex bifurcating mixing device, or a microfluidic mixing device. These devices allow a rapid, turbulence-free, diffusion-based mixing process due to specially designed microchannels. Zigzag herringbone mixers and their use in making particles preferably included in the compositions of the present invention are described, for example, in Zhigaltsev, IV et al. "Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing" Langmuir 28, 3633-3640 (2012), the disclosure of which is incorporated herein by reference. Microfluidic mixing and its use in making particles preferably included in the compositions of the present invention are described, for example, in Krzyszton, R. et al. "Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles" Nanoscale 9, 7442-7453 (2017), the disclosure of which is incorporated herein by reference.Finally, Dean vortex bifurcated mixers and their use in making particles preferably included in the compositions of the present invention are described, for example, in Chen, JJ, Chen, CH & Shie, SR "Optimal designs of staggered dean vortex micromixers" Int J Mol Sci 12, 3500-3524 (2011), the disclosure of which is incorporated herein by reference. In addition to microfluidic mixing methods, the mixture of the solution containing the lipid components of the lipid composition with the solution containing mRNA can also be performed using a confined impingement jet mixer [Knauer GmbH, Berlin, Germany]. [Brief description of the drawings]

[0326] The present invention is further illustrated by the following examples from which additional features, aspects and advantages may be obtained. [Figure 1] FIG. 1 shows the study design for the in vivo evaluation of luciferase expression following intramuscular injection into mice of FLuc-mRNA, FLuc-mRNA encapsulated in cationic LNPs, FLuc-mRNA encapsulated in generally neutral LNPs according to the present invention, and FLuc-mRNA encapsulated in ionizable cationic lipid-based neutral LNPs according to the prior art. [Diagram 2] FIG. 2 is a diagram showing the relative light units, which are an indication of the luciferase activity of various formulations; more specifically, FIG. 2 shows the luciferase expression results obtained after intramuscular injection into mice of FLuc-mRNA, FLuc-mRNA encapsulated in cationic LNPs, FLuc-mRNA encapsulated in generally neutral LNPs according to the present invention, and FLuc-mRNA encapsulated in ionizable cationic lipid-based neutral LNPs according to the prior art. [Diagram 3]FIG. 3 is a diagram I showing the pH-dependent total surface charge of lipid nanoparticles of one embodiment of a formulation according to the present invention, in which a first lipid of the lipid composition has a carboxylic acid group that is deprotonated in a pH-dependent manner and a second lipid of the lipid composition has three functional groups that are protonated in a pH-dependent manner. [Figure 4] FIG. 4 is Diagram II showing the pH-dependent total surface charge of lipid nanoparticles of one embodiment of a formulation according to the present invention, in which a first lipid of the lipid composition has a carboxylic acid group that is deprotonated in a pH-dependent manner and a second lipid of the lipid composition has two functional groups that are protonated in a pH-dependent manner. [Diagram 5] FIG. 5 is Diagram III showing the pH-dependent total surface charge of lipid nanoparticles of one embodiment of a formulation according to the present invention, in which a first lipid of the lipid composition has a carboxylic acid group that is deprotonated in a pH-dependent manner and a second lipid of the lipid composition has one functional group that is protonated in a pH-dependent manner. EXAMPLES

[0327] Example 1: Formulation of FLuc mRNA in lipid nanoparticles (PTX_LNP#1) for in vivo application by intramuscular, subcutaneous or intradermal administration For in vivo experiments, mRNA-LNPs were prepared using β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the cationic lipid in the formulation process. Alternatively, similar mRNA-LNPs can be prepared using other cationic lipids such as L-arginyl-β-alanine-N-palmityl-N-oleylamide in the same procedure.

[0328] A lipid solution was prepared by dissolving lipid component 2 β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide:DPhyPE:mPEG-2000-DSPE in ethanol at a molar ratio of 50:49:1. This lipid solution was then mixed with an aqueous solution of CleanCap® FLuc mRNA (5moU (=5-methoxy-uridine modified); Trilink Biotechnologies, San Diego) at a volume ratio (RNA solution:lipid solution) of 2:1 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) at a total flow rate of 18 ml / min. The concentrations of the applied lipids and mRNA were adjusted so that the total lipid to mRNA mass ratio of the thus obtained mRNA formulation was 28. After the mixing process, the formulation was dialyzed against 10 mM TRIS-buffered 270 mM sucrose solution using a 3.5 kDa MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature with gentle agitation of the dialysis buffer; the dialysis buffer was exchanged and dialyzed for another 2 hours at room temperature. Again, the dialysis buffer was exchanged and dialyzed overnight at 4°C. A total dialysis buffer of at least 300 times the volume of the sample was used during the dialysis procedure. Instead of sucrose, other sugars such as trehalose and glucose can be used in the formulation process as well.

[0329] The resulting formulation was then tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK)), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit, following manufacturer's protocol (Thermo Fisher Scientific)), and endotoxin content. The particle size was found to be 60-90 nm (Z average) with a zeta potential of 35 mV ± 10 mV. The formulation showed more than 90% encapsulation of mRNA. At this point, the final mRNA concentration of the formulation was adjusted to 100 μg / ml. The resulting mRNA-LNP formulation was stored at -80 °C until further in vitro or in vivo use.

[0330] Example 2: Formulation of FLuc mRNA in lipid nanoparticles (PTX_LNP#2) for in vivo application by intramuscular, subcutaneous or intradermal administration For in vivo experiments, mRNA-LNPs were prepared using β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the cationic lipid in the formulation process. Alternatively, similar mRNA-LNPs can be prepared using other cationic lipids such as L-arginyl-β-alanine-N-palmityl-N-oleylamide in the same procedure.

[0331] A lipid solution was prepared by dissolving lipid component 2 β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide:cholesterol:mPEG-2000-DSPE in ethanol at a molar ratio of 70:29:1. This lipid solution was then mixed with an aqueous solution of CleanCap® FLuc mRNA (5moU; Trilink Biotechnologies, San Diego) at a volume ratio (RNA solution:lipid solution) of 3:1 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) with a total flow rate of 18 ml / min. The concentrations of the applied lipids and mRNA were adjusted so that the total lipid to mRNA mass ratio of the resulting mRNA formulation was 14. After the mixing process, the formulation was dialyzed against 10 mM TRIS-buffered 270 mM sucrose solution using a 3.5 kDa MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature with gentle agitation of the dialysis buffer; the dialysis buffer was exchanged and dialyzed for another 2 hours at room temperature. Again, the dialysis buffer was exchanged and dialyzed overnight at 4°C. A total dialysis buffer of at least 300 times the volume of the sample was used during the dialysis procedure. Instead of sucrose, other sugars such as trehalose and glucose can be used in the formulation process as well.

[0332] The resulting formulation was then tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK)), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit, following manufacturer's protocol (Thermo Fisher Scientific)), and endotoxin content. The particle size was found to be 60-90 nm (Z average) with a zeta potential of 35 mV ± 10 mV. The formulation showed more than 90% encapsulation of mRNA. At this point, the final mRNA concentration of the formulation was adjusted to 100 μg / ml. The resulting mRNA-LNP formulation was stored at -80 °C until further in vitro or in vivo use.

[0333] Example 3: Formulation of FLuc mRNA in neutral lipid nanoparticles (PTX_LNP#3) for in vivo application by intramuscular, subcutaneous or intradermal administration For in vivo experiments, mRNA-LNPs were made using β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the cationic lipid in the formulation process. Alternatively, similar mRNA-LNPs can be prepared using other cationic lipids, such as L-arginyl-β-alanine-N-palmityl-N-oleylamide, in the same procedure.

[0334] A lipid solution was prepared by dissolving lipid component 2 β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide:CHEMS:cholesterol:mPEG-2000-DMPE in ethanol at a molar ratio of 29:49:20:2. This lipid solution was then mixed with a solution of CleanCap® FLuc mRNA (5moU; Trilink Biotechnologies, San Diego) in acetate buffer (50mM, pH4) at a volume ratio (RNA solution:lipid solution) of 2:1 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) with a total flow rate of 18ml / min. The concentrations of the applied lipids and mRNA were adjusted so that the total lipid to mRNA mass ratio of the resulting mRNA formulation was 28. After the mixing process, the formulation was dialyzed against 10 mM TRIS-buffered 270 mM sucrose solution using a 3.5 kDa MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature with gentle agitation of the dialysis buffer; the dialysis buffer was exchanged and dialyzed for another 2 hours at room temperature. Again, the dialysis buffer was exchanged and dialyzed overnight at 4°C. A total dialysis buffer of at least 300 times the volume of the sample was used during the dialysis procedure. Instead of sucrose, other sugars such as trehalose and glucose can be used in the formulation process as well.

[0335] The resulting formulation was then tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK)), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit, following manufacturer's protocol (Thermo Fisher Scientific)), and endotoxin content. Particle size was found to be 60-90 nm (Z average) with a zeta potential of 0 mV ± 10 mV. The formulation showed more than 90% encapsulation of mRNA. At this point, the final mRNA concentration of the formulation was adjusted to 100 μg / ml. The resulting mRNA-LNP formulation was stored at -80 °C until further in vitro or in vivo use.

[0336] Example 4: Formulation of FLuc mRNA in lipid nanoparticles (PTX_LNP#4) for in vivo application by intramuscular, subcutaneous or intradermal administration For in vivo experiments, mRNA-LNPs were made using β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the cationic lipid in the formulation process. Alternatively, similar mRNA-LNPs can be prepared using other cationic lipids, such as L-arginyl-β-alanine-N-palmityl-N-oleylamide, in the same procedure.

[0337] A lipid solution was prepared by dissolving lipid component 2 β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide:CHEMS:cholesterol:mPEG-2000-DMPE in ethanol in a molar ratio of 29:48:18:5. This lipid solution was then mixed with a solution of CleanCap® FLuc mRNA (5moU; Trilink Biotechnologies, San Diego) in acetate buffer (50mM, pH4) in a volume ratio (RNA solution:lipid solution) of 2:1 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) with a total flow rate of 18ml / min. The concentrations of applied lipids and mRNA were adjusted so that the total lipid to mRNA mass ratio of the thus obtained mRNA formulation was 32. After the mixing process, the formulation was dialyzed against 10 mM TRIS-buffered 270 mM sucrose solution using a 3.5 kDa MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature with gentle agitation of the dialysis buffer; the dialysis buffer was exchanged and dialyzed for another 2 hours at room temperature. Again, the dialysis buffer was exchanged and dialyzed overnight at 4°C. A total dialysis buffer of at least 300 times the volume of the sample was used during the dialysis procedure. Instead of sucrose, other sugars such as trehalose and glucose can be used in the formulation process as well.

[0338] The resulting formulation was then tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK)), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit, following manufacturer's protocol (Thermo Fisher Scientific)), and endotoxin content. Particle size was found to be 40-90 nm (Z average) with a zeta potential of 0 mV ± 10 mV. The formulation showed more than 90% encapsulation of mRNA. At this point, the final mRNA concentration of the formulation was adjusted to 100 μg / ml. The resulting mRNA-LNP formulation was stored at -80 °C until further in vitro or in vivo use.

[0339] Example 5: In vivo evaluation of luciferase expression after intramuscular injection of various PTX_LNPs in mice To evaluate the expression of firefly luciferase (FLuc) after intramuscular injection of various PTX_LNPs, a mouse study was designed as shown in the schematic diagram of FIG. 1. The group designations are shown in the table below; Group A was a saline group without FLuc-mRNA and was used as a vehicle control. Group B was simply FLuc-mRNA without lipid and was used as a control. Group C was an overall positively charged FLuc-mRNA-LNPs prepared as described in Example 1. Group D was an overall positively charged FLuc-mRNA-LNPs prepared as described in Example 2. Group E was an overall neutral FLuc-mRNA-LNPs according to the underlying invention, containing 2 mol% mPEG2000-DMPE, prepared as described in Example 3. Group F was an overall neutral FLuc-mRNA-LNPs according to the underlying invention, containing 5 mol% mPEG2000-DMPE, prepared as described in Example 4. Group G was a prior art ionizable cationic lipid-based neutral FLuc-mRNA-LNP. For these groups, luciferase expression was evaluated 24 hours after injection, whereas for groups H-M, which contained the same samples as groups B-G, luciferase expression was evaluated 72 hours after injection. Each group consisted of three C57BL / 6 mice aged 8-10 weeks.

[0340] For treatment, 6 x 10 μl of each test substance outlined in Table 1 below was administered intramuscularly to each thigh (left and right hind limbs) of the mice using an Omnican 50U-100 syringe with a 30G needle (0.30 mm x 12 mm). Therefore, prior to injection, the hair on the thighs of the mice was shaved, a cannula was inserted 4-5 mm deep into the muscle tissue, and 6 x 10 μl injections were administered per thigh muscle, spaced approximately 2-3 mm apart.

[0341] After 24 or 72 hours, mice were sacrificed and whole thigh muscle tissue was flash frozen in liquid nitrogen. For luciferase assay, frozen muscle tissue was ground in a mortar pre-cooled with dry ice, and 60–90 mg of tissue was homogenized in PROMEGA Luciferase Cell Culture Lysis lysis buffer (10 μl / mg tissue powder) using a bead crusher (QIAGEN TissueLyser LT) at 50 Hz for 5 min.

[0342] The homogenates were centrifuged and expressed luciferase in the supernatant was measured using the PROMEGA Nano-Glo® Dual-Luciferase® Reporter Assay System kit. Briefly, undiluted tissue lysates were mixed with an equal volume of luciferin-containing assay buffer and luminescence was immediately measured using a TECAN infinite 200 Pro luminometer.

[0343] The results are shown in Figure 2. Compared to the overall cationic LNPs (PTX_LNP#2 and PTX_LNP#3) and plain FLuc-mRNA, the overall charge-neutral LNPs (PTX_LNP#3 and PTX_LNP#4) showed significantly higher luciferase expression after intramuscular injection. Moreover, compared to the overall charge-neutral LNPs of the prior art (based on ionizable cationic lipids), the overall charge-neutral LNPs according to the underlying invention (PTX_LNP#3 and PTX_LNP#4) showed significantly higher luciferase expression after intramuscular injection. Interestingly, PTX_LNP#4, which contains more mPEG-DMPE lipids (5% vs. 2% in PTX_LNP#3), showed significantly higher luciferase expression after 72 hours than after 24 hours, indicating a sustained biological activity of PTX_LNP#4.

[0344] (Table 1) Group designation (dose / time point) of mouse experiments for luciferase expression after intramuscular injection. TIFF2025505258000057.tif123134* "Naked" CleanCap® FLuc mRNA (5moU) was dissolved in lipid-free 0.9% saline at a concentration of 0.100mg / ml.

[0345] The features of the invention disclosed in the specification, the claims and / or the drawings may, both individually or in any combination thereof, be important for realizing the invention in diverse forms thereof.

Claims

1. 1. A formulation for use in therapy comprising: the method of treatment comprises local administration of the formulation to the subject, the local administration being selected from the group consisting of intramuscular administration, subcutaneous administration, and intradermal administration; The formulation comprises lipid nanoparticles, the lipid nanoparticles comprising a lipid composition and a therapeutically active agent, and being amphoteric and overall neutrally charged at a pH of 7 to 8; the lipid composition comprises a first lipid and a second lipid; the first lipid is selected from the group consisting of CHEMS (hemisuccinic acid cholesterol ester), alkyl carboxylic acid, hemisuccinic acid diacylglycerol ester, and diacylphosphatidylserine, preferably the alkyl carboxylic acid is a saturated or unsaturated branched or straight chain alkyl carboxylic acid of 12-20 hydrocarbons, preferably the acyl group is a saturated or unsaturated branched or straight chain alkyl group of 12-20 hydrocarbons; The second lipid has a plurality of cationic charges at a pH of about 7.2 to about 7.5, preferably at a pH of about 7.4, and the second lipid is preferably selected from the group consisting of β-(L-arginyl)-L-2,3-diaminopropionic acid-N,N-dialkylamide and L-arginyl-β-alanine-N,N-dialkylamide, the alkyl chains being, independently of each other, 12 to 20 hydrocarbon saturated or unsaturated branched or linear alkyl groups; the therapeutically active agent is a therapeutically effective nucleic acid molecule; Formulations for use.

2. 2. The formulation for use according to claim 1, wherein the lipid composition comprises a third lipid and / or a fourth lipid, wherein the third lipid is a neutral lipid and the fourth lipid is a PEGylated lipid.

3. 3. The formulation for use according to any one of claims 1 to 2, wherein the surface of the lipid nanoparticle, preferably the outer surface of the lipid nanoparticle, provides an approximately equal number of positive (cationic) and negative (anionic) charges at a neutral pH value of 6.7 to 7.

7.

4. 4. The formulation for use according to any one of claims 1 to 3, wherein the molar ratio of the first lipid to the second lipid is such that the number of negative (anionic) charges of the lipid composition is approximately the same as the number of positive (cationic) charges of the lipid composition at a pH value of 6.7 to 7.

7.

5. 5. The formulation for use according to any one of claims 2 to 4, wherein the third lipid is (a) a zwitterionic phospholipid selected from the group consisting of phosphatidylethanolamine and phosphatidylcholine, or (b) an uncharged sterol lipid selected from the group comprising animal sterols and plant sterols.

6. 6. The formulation for use according to claim 5, wherein the zwitterionic phospholipid is selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-lauroyl-sn-glycero-3-phosphoethanolamine), DPhyPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

7. 7. The formulation for use according to any one of claims 2 to 6, wherein the fourth lipid is selected from the group comprising methoxy PEG-DSPE, methoxy PEG-DMPE, methoxy PEG-DMG, methoxy PEG-DPG, methoxy PEG-DSG, methoxy PEG-c-DMA, 2(methoxy PEG)-N,N-dioctadecylacetamide, 2(methoxy PEG)-N,N-ditetradecylacetamide, methoxy PEG-C8-ceramide, and methoxy PEG-C16-ceramide.

8. The lipid composition comprises: CHEMS as said first lipid; - β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid, - cholesterol as the third lipid; - methoxy PEG2000-DMPE as the fourth lipid A formulation for use according to any one of claims 2 to 7, comprising:

9. The molar ratio of the lipid composition is （a）： - 20 to 80 mol % of said first lipid, - 20 to 80 mol % of said second lipid, - 10 to 50 mol % of said third lipid, and - 1 to 10 mol % of said fourth lipid and the total lipid content of the lipid composition is 100 mol %; or （b）： - 20 to 60 mol % of said first lipid, - 20 to 60 mol % of said second lipid, - 10 to 40 mol % of said third lipid, and - 1 to 5 mol % of said fourth lipid and the total lipid content of the lipid composition is 100 mol%; A formulation for use according to any one of claims 2 to 8.

10. The lipid composition comprises: - 49 mol% of CHEMS as the first lipid, - 29 mol% of β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid; - 20 mol% cholesterol as said third lipid, and 2 mol% mPEG2000-DMPE as the fourth lipid Contains or The lipid composition comprises: - 48 mol% of CHEMS as the first lipid, - 29 mol% of β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleylamide as the second lipid; - 18 mol% cholesterol as the third lipid, and - 5 mol% methoxy PEG2000-DMPE as the fourth lipid 10. The formulation for use according to claim 9, comprising:

11. The formulation for use according to any one of claims 1 to 10, wherein the lipid nanoparticles have an average particle size of about 15 nm to about 400 nm, preferably about 25 nm to about 200 nm, more preferably about 40 nm to about 100 nm, as measured by dynamic light scattering (DLS).

12. The formulation for use according to any one of claims 1 to 11, wherein the zeta potential of the lipid nanoparticles is from about -10 mV to about +10 mV, preferably from about -5 mV to about +5 mV.

13. 13. The formulation for use according to any one of claims 1 to 12, wherein said therapeutically effective nucleic acid molecule is selected from the group consisting of mRNA, mRNA encoding Cas (CRISPR associated endonuclease protein), plasmid DNA, ssDNA (single stranded DNA), Aptamer, Spiegelmer, siRNA molecule, antisense molecule, miRNA (microRNA) molecule, sgRNA (single guide RNA) molecule, saRNA (self-amplifying RNA) molecule, and combinations thereof.

14. The formulation for use according to any one of claims 1 to 13, wherein the therapeutic method comprises a method for the treatment and / or prevention of a disease in a subject, said method comprising local administration of said formulation to said subject, preferably wherein local administration is selected from the group consisting of intramuscular administration, subcutaneous administration and intradermal administration.

Images