Compositions and methods

Abstract

A composition comprising: (i) a cationically ionizable lipid; (ii) a steroid; (iii) a phospholipid; wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile and (c) an inorganic polyphosphate; wherein the molar ratio between the cationically ionizable lipid and the cholesterol is between about 1.5 to about 2.25; is disclosed. Compositions containing these ingredients together with nucleic acids, and compositions containing these ingredients together with mono-oligomer-conjugated lipids and with lysophospholipids, are also disclosed.

Description

[0001] COMPOSITIONS AND METHODS

[0002] Technical Field

[0003] The present disclosure relates generally to the field of nucleic acid (such as DNA or RNA, in particular mRNA) compositions, particularly although not exclusively compositions containing lipid particles (such as lipid nanoparticles) without polymer-conjugated lipids or comprising lipids as alternative to polymer-conjugated lipids, and to the use of such compositions, in particular for delivering nucleic acids to cells of a subject or in therapy.

[0004] Background to the Invention

[0005] The use of a recombinant nucleic acid (such as DNA or RNA) for delivery of foreign genetic information into target cells is well known. A recombinant nucleic acid may be administered in naked form to a subject in need thereof; however, usually a recombinant nucleic acid is administered using a composition. For example, nucleic acid, such as RNA, may be delivered to a subject using different delivery vehicles, based mostly on cationic polymers or lipids which together with the nucleic acid form nanoparticles. The nanoparticles are intended to protect the nucleic acid, such as RNA, from degradation, enable delivery of the nucleic acid, such as RNA, to the target site and facilitate cellular uptake and processing by the target cells. The efficiency of the nucleic acid delivery depends, in part, on the molecular composition of the nanoparticle and can be influenced by numerous parameters, including particle size, formulation, and charge.

[0006] Lipid nanoparticles (LNP) are well established delivery vehicles for RNA. Typically, LNPs comprise a cationically ionisable lipid, a steroid (typically cholesterol), a neutral phospholipid (typically phosphatidylcholine or phosphatidylethanolamine), and a conjugated lipid which is often a PEG-lipid. The cationically ionizable lipid is a main component and serves the purpose of (i) binding and encapsulating the RNA cargo and (ii) binding to and eventually mixing with lipids of the endosomal membrane thereby facilitating an endosome escape. The cationically ionizable lipid together with the steroid and the neutral phospholipid form the body of the LNP which is a polycationic particle. Typically, the LNP bodies alone cannot be isolated or stored due to colloidal instability. This is understood as aggregation between polycationic particles in the presence of even small amounts of non-encapsulated polynucleotides such as RNA or DNA. A similar process of aggregate formation occurs whencontacting cationic particles with bodily fluids such as blood or lymph as biomaterials such as proteins (albumin, immunoglobulins etc), lipoproteins or cells are polyanions.

[0007] PEG lipids are one example of a wider class of lipids generally termed “stealth lipids” or “polymer-grafted lipids”. As described in Le, T.C. et al. Sci. Rep. 2019, 9, 265, according to “Whitesides’ rules”, stealth polymers or protein resistant surfaces should have the following characteristics: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge. Examples of stealth polymers previously incorporated into nanomedicine include poly(sarcosine) (pSar), poly(2-oxazoline) (pOx); poly(oxazine) (pOz), poly(vinyl pyrrolidone) (PVP); poly(7V-(2-hydroxypropyl)-methacrylamide) (pHPMA); poly(dehydroalanine) (pDha); poly(aminoethoxy ethoxy acetic acid) (pAEEA) and poly(2 -methylaminoethoxy ethoxy acetic acid) (pmAEEA). All of the above may be conjugated to lipids, the terms “stealth lipid” and “polymer-grafted lipids” being used generally to describe stealth polymers when conjugated to lipids.

[0008] The problem of colloidal instability and bioincompatibility of LNPs is addressed by PEG-conjugated lipids or other stealth polymers providing a steric barrier between the polycationic particle and the polyanionic surface of cells or proteins. PEG steric hindrance prevents interparticle fusion and promotes the formation of a homogeneous population of LNPs where diameters <100 nm can be achieved.

[0009] However, a number of problems are associated with the inclusion of PEG-conjugated lipids or other stealth polymers in LNPs as vehicles for nucleic acid delivery. PEG-conjugated lipids inhibit an interaction between the particles and cells which inhibits cellular uptake and reduces overall transfection efficiency. PEGylation has been associated with accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and / or complement activation as well as storage diseases (Bendele Ret al., Toxicological Sciences 1998, 42, 152-157; Young MA etal., 2007, Translational Research 149(6), 333-342; S.M. Moghimi, J. Szebeni, Progress in Lipid Research 200342:463-478). The presence of anti-PEG antibodies in the plasma induces a higher clearance of the particles by the Monophagocyte System (MPS) which at the end reduces the efficacy of the drug. Furthermore, due to broad use of PEGs as ingredients of food, cosmetic, hygienic products, and medicines, a certain percentage of the general population have "pre-existing" anti-PEG antibodies. Such anti-PEG antibodies are associated with decreased efficacy of PEGylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.Thus, the use of PEG-conjugated lipids is associated with hypersensitivity and generation of anti-PEG antibodies, which are limiting or even preventing the therapeutic efficacy of PEGylated LNP upon repeated administrations. Without wishing to be bound by theory, similar immune recognition and generation of antibodies may also occur in situations of recurrent treatment with nucleic acid therapeutics using LNPs containing other stealth polymers as a delivery vehicle.

[0010] The combined challenges therefore create a need for alternative designs of carrier for nucleic acids. It would therefore be desirable to provide carriers capable of acting as vehicles for delivery of active ingredients, in particular having colloidal stability and being biocompatible upon administration, while avoiding the use of PEG-conjugated lipids and other stealth polymers. Furthermore, there is a need for zwitterionic neutral LNPs that are free of polymer-grafted lipids.

[0011] W02023 / 036960 describes LNP compositions for nucleic acid delivery which include phosphatidylserine and polysorbate 20 (i.e., Tween20), which is a form of PEG-lipid having 20 repeating units of polyethylene glycol. Notably, the carriers described in all of these documents all also comprise PEG-lipid and therefore do not solve the above-described problems addressed by the present invention.

[0012] LNPs including other alternatives to PEG-lipids are also known in the art. W02020 / 069718 describes RNA particles comprising polysarcosine. WO2023 / 193892 describes LNP compositions for nucleic acid delivery which include an inorganic polyphosphate.

[0013] WO2024 / 028325 describes LNP compositions for nucleic acid delivery which include an amphiphilic oligo ethylene glycol (OEG)-conjugated compound. WO2024 / 133635 describes LNP compositions for nucleic acid delivery which include a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion. Berger el al., Journal of Controlled Release 2023, 361, 87-1001 describes DSPE-poly (vinyl pyrrolidone)-30 as a replacement for PEG-lipids in siRNA lipoplexes.

[0014] Summary of the Invention

[0015] In a first aspect, the present disclosure provides a composition comprising:

[0016] (i) a cationically ionizable lipid;

[0017] (ii) a steroid;

[0018] (iii) a phospholipid;wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate.

[0019] In a second aspect, the present disclosure provides a composition comprising:

[0020] (i) a cationically ionizable lipid;

[0021] (ii) a steroid;

[0022] (iii) a phospholipid;

[0023] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate;

[0024] wherein the molar ratio between the cationically ionizable lipid and the steroid is between about 1.5 to about 2.25.

[0025] In a third aspect, the present disclosure provides a composition comprising:

[0026] (i) a cationically ionizable lipid;

[0027] (ii) a steroid;

[0028] (iii) a phospholipid;

[0029] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate;

[0030] wherein the phospholipid is present in the composition in an amount of between about 10 and about 30 mol% of the total lipid present in the composition.

[0031] In a fourth aspect, the present disclosure provides a composition comprising:

[0032] (i) a cationically ionizable lipid;

[0033] (ii) a steroid;

[0034] (iii) a phospholipid; and

[0035] (iv) a lysophospholipid;wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0036] In a fifth aspect, the present disclosure provides a composition comprising:

[0037] (i) a cationically ionizable lipid;

[0038] (ii) a steroid;

[0039] (iii) a phospholipid; and

[0040] (iv) a monomer- or oligomer-grafted lipid and wherein the monomer- or oligomer- grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units;

[0041] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0042] In a sixth aspect, the present disclosure relates to a method for delivering a composition to a subject, the method comprising administering to a subject a composition of any of the first to fifth aspects. It is understood that any embodiment described herein in the context of the any of the first to fifth aspects may also apply to any embodiment of the sixth aspect.

[0043] In a seventh aspect, the present disclosure provides the composition of any of the first to fifth aspects for use in medicine, particularly although not exclusively for use in a prophylactic and / or therapeutic treatment of disease.

[0044] In an eighth aspect, the present disclosure provides processes for making the composition of the first aspect. In one embodiment of the eighth aspect, provided is a method of preparing the composition of the invention, wherein the composition comprises:

[0045] (i) a cationically ionizable lipid;

[0046] (ii) a steroid;

[0047] (iii) a phospholipid;

[0048] wherein the method comprises:

[0049] (a) preparing a lipid mixture comprising a cationically ionizable lipid, a steroid and a phospholipid;

[0050] (b) preparing an aqueous solution comprising an acidic buffer;(c) mixing the lipid mixture prepared in (a) with the aqueous solution prepared in (b); and (d) adjusting the pH of the mixture obtained in step (c) to a pH of between 6.5 and 8; to form the composition.

[0051] In such methods, the aqueous solution may further comprise a nucleic acid and the lipid mixture may further comprise an additional lipid with a packing parameter of < 0.5, for example as calculated by the method of example 11.

[0052] In a ninth aspect, the present disclosure provides a composition comprising:

[0053] (i) a cationically ionizable lipid;

[0054] (ii) a steroid;

[0055] (iii) a phospholipid;

[0056] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate;

[0057] wherein the molar ratio between the cationically ionizable lipid and the cholesterol is between about 1.5 to about 2.25.

[0058] Advantages and Surprising Findings

[0059] It has surprisingly been found by the present inventors that the compositions of the present invention, in particular lipid particle compositions, such as lipid nanoparticles comprising cationically ionizable lipid, a steroid and a phospholipid, have an excellent colloidal stability and cargo encapsulation efficiency even in the absence of PEG-lipids or other polymer-grafted lipids.

[0060] It has also been surprisingly found that the inclusion of an additional lipid with a critical packing parameter of < 0.5 (as defined herein) to the lipid nanoparticles above, resulted in particles with an excellent cargo encapsulation efficiency and that can be frozen and thawed without loss of colloidal stability. Without wishing to be bound by theory, polymer lipids with a critical packing parameter of < 0.5 may provide a combination of lipid geometries that lead to the effective stabilization of the particle surface and avoid aggregation after freezing and thawing. Examples of such additional lipids with a critical packing parameter of < 0.5 are lysophospholipids and monomer- or oligomer grafted lipids comprising between 1 and 5 consecutive monomer repeating units.It has also been surprisingly found that both the earlier named lipid compositions are biocompatible upon administration despite the absence of PEGylated lipids or other polymer-grafted lipids and specifically reach the gastrointestinal tract. It has also been surprisingly found that the earlier named lipid compositions, although presenting similar solid core structures, have a different internal lipid-RNA packing resulting in larger interlamellar spacings compared to the PEG-lipid containing formulation.

[0061] It has also been surprisingly found that the long-term stability of polymer-free LNPs is excellent over a time period of at least 6 months in frozen and liquid conditions, thereby not requiring a steric coating of polymer material. It has also been surprisingly found that polymer-free LNPs are potent at activating the immune response following intramuscular expression of an mRNA encoded antigen, allowing for their potential application as vaccine candidate formulations.

[0062] Brief Description of the Figures

[0063] Figure 1 - Graphs depicting the particle size in nm (A), encapsulation efficiency (EE%) (B) and delta size FT1 (particle size difference after one freeze-thaw cycle) (C) versus the DSPC% as described in example 2.

[0064] Figure 2 - Graphs depicting the particle size in nm (A), EE% (B) and delta size FT1 (particle size difference after one freeze-thaw cycle) (C) versus the ALC-0366 to cholesterol (Choi) ratio as described in example 2.

[0065] Figure 3 - Graphs depicting the delta size FT1 (particle size difference after one freeze-thaw cycle) for particles of compositions comprising 10% (w / v) and 20% (w / v) sucrose versus DSPC% (A) and ALC-0366 to cholesterol (Choi) ratio (B) as described in example 2.

[0066] Figure 4 - Schematic overview of the methods as described in example 4.

[0067] Figure 5 - Graphs depicting the particle the size in nm versus the N / P ratio (A), the particle size in nm versus the freeze-thaw (FT) cycle for multiple N / P ratios (B) and the EE% versus the N / P ratio (C) as described in example 5.

[0068] Figure 6 - Graphs depicting the particle size in nm and the poly-dispersity index (PDI) (A) and (B) and EE% (C) and (D) for compositions comprising ALC-0366 as cationically ionizable lipid and lyso-PC lipids with varying ALC-0366 to cholesterol (Choi) ratios and varying N / P ratios as described in example 6. The term “xLNP” refers to the composition comprising a cationically ionizable lipid, a steroid and a phospholipid as defined below. TheXX:O groups include the composition comprising a cationically ionizable lipid, a steroid, phospholipid and a lysophospholipid as defined below.

[0069] Figure 7 - Graphs depicting the delta size (nm) after one freeze-thaw cycle at -80°C for compositions with varying ALC-0366 to cholesterol (Choi) ratios at an N / P ratio of 6 (A) and 10 (B) as described in example 6. The term “xLNP” refers to the composition comprising a cationically ionizable lipid, a steroid and a phospholipid as defined below. The XX: 0 groups include the composition comprising a cationically ionizable lipid, a steroid, phospholipid and a lysophospholipid as defined below.

[0070] Figure 8 - Graphs depicting the particle size in nm and the poly-dispersity index (PDI) (A), EE% (B) and delta size (nm) after one freeze thaw cycle (C) for compositions with varying BHD to cholesterol (Choi) ratios as described in example 6. The term “xLNP” refers to the composition comprising a cationically ionizable lipid, a steroid and a phospholipid as defined below. The XX:0 groups include the composition comprising a cationically ionizable lipid, a steroid, phospholipid and a lysophospholipid as defined below.

[0071] Figure 9 - Graphs depicting the particle size in nm and the poly-dispersity index (PDI) (A), EE% (B) and delta size (nm) after one freeze thaw cycle (C) for compositions with varying cationically ionizable lipids and DSPC% as described in example 7.

[0072] Figure 10- Graphs depicting the particle size in nm (A), delta size (nm) after one freeze thaw cycle (B) and EE% (C) for compositions with varying oligomer-conjugated lipid (also referred to herein as “shortmer”) compositions and formulations as described in example 8.

[0073] Figure 11- Graphs depicting the particle size in nm and the poly-dispersity index (PDI (A) and EE% (B) for compositions with varying M0G% as described in example 8.

[0074] Figure 12 - Graphs depicting the particle size in nm and the poly-dispersity index (PDI (A), delta size (nm) after one freeze thaw cycle (B) and EE% (C) for compositions with varying neutral lipids as described in example 8.

[0075] Figure 13 - Graphical overview of the experimental set-up of example 9.

[0076] Figure 14 - In vivo bioluminescence imaging at 6 hours post-injection with mice receiving varying compositions as described in example 9. Scale shows Radiance in p / sec / cm2 / sr. Scale represents Min 3.2e7 (low) and Max 4.4e8 (high). Mice are shown in the dorsal (A) or ventral (B). In vivo bioluminescence imaging of mice at 24 hours post injection as described inexample 9. Mice are shown in dorsal (C) and ventral (D) orientation. Scale shows Radiance in p / sec / cm2 / sr. Scale represents Min le8 (low) and Max le9 (high).

[0077] Figure 15 - Bioluminescence imaging of organs and tissues at 24 hours post injection of mice receiving varying compositions as described in example 9. Each picture, top to bottom, heart, lung, liver, kidneys, spleen, gastrointestinal tract (duodenum to rectum), inguinal lymph nodes. Scale shows Radiance in p / sec / cm2 / sr. Scale represents Min 5e5 (low) and Max 2e7 (high).

[0078] Figure 16 - Representative cryo-EM image of xLNP as described in example 10.

[0079] Figure 17 - Representative cryo-EM image of shortmer-xLNP with Sari as described in example 10.

[0080] Figure 18 - Representative cryo-EM image of shortmer-xLNP with pSar3 as described in example 10.

[0081] Figure 19 - Representative cryo-EM image of shortmer-xLNP with pSar5 as described in example 10.

[0082] Figure 20 - Representative cryo-EM image of PEG-lipid containing benchmark formulation as described in example 10.

[0083] Figure 21 - Violin plot depicting the particle size (nm) of solid core and phase separated xLNPs and xLNPs + shortmers as described in example 10.

[0084] Figure 22 - SAXS profiles for xLNP, shormer-xLNPs (Sari, Sar3 and Sar5) and the PEG-lipid containing benchmark formulation as described in example 10.

[0085] Figure 23 - Graphical overview of the critical packing parameter.

[0086] Figure 24 - Graphical overview of the experimental set-up of example 9.

[0087] Figure 25 - Graph depicting the n vivo pharmacokinetic profile of encoded antibody on mRNAused for formulations described in example 12.

[0088] Figure 26 - Graphs depicting the anti-polymer (A) IgG and (B) IgM responses against the relevant lipids for formulations described in example 12.

[0089] Figure 27 - Graphs illustrating the physicochemical properties, including particle size (nm) and polydispersity (a), mRNA encapsulation efficiency (%) (b), and the increase in particlesize (nm) over five freeze-thaw cycles at -80 °C (c), for three-component xLNP formulations with varying ionizable lipids and DSPC content, as described in example 13.

[0090] Figure 28 - Graphs illustrating the physicochemical properties, including particle size (nm) and polydispersity, for four-component xLNP formulations. These formulations vary in ionizable lipid type, DSPC content, and the fourth component, which is either C14-Sar(l)-Ac (a) or 18:0 LysoPC (b), as described in example 14.

[0091] Figure 29 - Graphs illustrating the mRNA encapsulation efficiency (%) for four-component xLNP formulations. These formulations vary in ionizable lipid type, DSPC content, and the fourth component, which is either 18:0 LysoPC (a) or C14-Sar(l)-Ac (b), as described in example 14.

[0092] Figure 30 - Graphs illustrating the increase in particle size (nm) over five freeze-thaw cycles at -80 °C for four-component xLNP formulations. These formulations vary in DSPC content, ionizable lipid type, and the fourth component, which is either C14-Sar(l)-Ac (a) or 18:0 LysoPC (b), as described in example 14.

[0093] Figure 31 - Graphs illustrating the particle size (nm) and PDI during long-term stability at -80 °C for three and four-component xLNP formulations. These formulations vary in DSPC content, fourth component type, and ionizable lipid type, which is either ALC-0366 (a), BHD-C2C2-PipZ (b), BODD-MePyr (c) or BODD-DMA(d), as described in example 15. Figure 32 - Graphs illustrating the mRNA encapsulation efficiency (%) during long-term stability at -80 °C for three and four-component xLNP formulations. These formulations vary in DSPC content, fourth component type, and ionizable lipid type, which is either ALC-0366 (a), BHD-C2C2-PipZ (b), BODD-MePyr (c) or BODD-DMA (d), as described in example 15.

[0094] Figure 33 - Graphs illustrating the particle size (nm) and PDI during long-term stability at 5 °C for three and four-component xLNP formulations. These formulations vary in DSPC content, fourth component type, and ionizable lipid type, which is either ALC-0366 (a), BHD-C2C2-PipZ (b), BODD-MePyr (c) or BODD-DMA (d), as described in example 15. Figure 34 - Graphs illustrating the mRNA encapsulation efficiency (%) during long-term stability at 5 °C for three and four-component xLNP formulations. These formulations vary in DSPC content, fourth component type, and ionizable lipid type, which is either ALC-0366 (a), BHD-C2C2-PipZ (b), BODD-MePyr (c) or BODD-DMA (d), as described in example 15.Figure 35 - Graphs illustrating the mRNA integrity (%) during long-term stability at 5 °C for three and four-component xLNP formulations. These formulations vary in DSPC content, fourth component type, and ionizable lipid type, which is either ALC-0366 (a), BHD-C2C2-PipZ (b), BODD-MePyr (c) or BODD-DMA (d), as described in example 15.

[0095] Figure 36 - Schematic overview of the methods as described in example 16.

[0096] Figure 37 - Representative cryo-EM image of three-component xLNP with the ionizable lipid BHD-C2C2-PipZ as described in example 17.

[0097] Figure 38 - Representative cryo-EM image of four-component xLNP with the ionizable lipid BHD-C2C2-PipZ and the fourth component 18:0 LysoPC as described in example 17.

[0098] Figure 39 - Representative cryo-EM image of four-component xLNP with the ionizable lipid BHD-C2C2-PipZ and the fourth component C14-Sar(l)-Ac as described in example 17. Figure 40 - Representative cryo-EM image of PEG-LNP with the ionizable lipid BHD-C2C2-PipZ and DSPC content at 10 mol% as described in example 17.

[0099] Figure 41 - Representative cryo-EM image of PEG-LNP with the ionizable lipid BHD-C2C2-PipZ and DSPC content at 20 mol% as described in example 17.

[0100] Figure 42 - Violin plot depicting the particle size (nm) of solid core and phase separated particles from the relevant three- and four-component xLNP and PEG-LNP formulations with the ionizable lipid BHD-C2C2-PipZ as described in example 17.

[0101] Figure 43 - SAXS profiles from the relevant three- and four-component xLNP formulations with the ionizable lipid BHD-C2C2-PipZ, varying DSPC content and varying ionizable lipid to cholesterol ratio as described in example 17.

[0102] Figure 44 - SAXS profiles from the relevant four-component lyso-xLNP formulations with varying ionizable lipid types as described in example 17.

[0103] Figure 45 - Graphical overview of the experimental set-up of example 18

[0104] Figure 46 - Bioluminescence imaging of mice receiving intraveneous (i.v.) injections of varying compositions as described in example 18. Scale shows Radiance in p / sec / cm2 / sr. Scale represents Min 5e7 (low) and Max 5e8 (high) for (A), Min le6 (low) and Max l,7e7 (high) for (B) and Min 4e5 (low) and Max 4e7 (high) for (C). In vivo bioluminescence imaging of mice in ventral orientation at 6 hours post injection (A), ex vivo bioluminescence imaging of heart,lung, spleen, kidneys, inguinal lymph nodes and liver (top to bottom) (B) and gastrointestinal tract (stomach to rectum, top to bottom) 24 h post injection (C).

[0105] Figure 47 - Graphical overview of the experimental set-up of example 19.

[0106] Figure 48 - Bioluminescence imaging of mice receiving intraveneous (i.v.) injections of varying compositions as described in example 19. Scale shows Radiance in p / sec / cm2 / sr. Scale represents Min 9e6 (low) and Max le9 (high) for (A), Min l,6e6 (low) and Max l,7e7 (high) for (B) and Min 1,06 (low) and Max 4,4e6 (high) for (C). In vivo bioluminescence imaging of mice in ventral orientation at 6 hours post injection (A), ex vivo bioluminescence imaging of lung, spleen and liver (top to bottom) (B) and gastrointestinal tract (stomach to rectum, top to bottom) 24 h post injection (C).

[0107] Figure 49 - Graphs depicting reciprocal serum antibody titer (a) and serum antibody IgG concentration (b) against the encoded antigen at the indicated days after intramuscular application of Balb / c mice (5 mice per group) with 0.1 pg mRNA dose of the indicated formulation. Mice were treated a second time (boost) with 0.1 pg mRNA dose of the same formulation 21 days after the first immunization. Data are presented as mean ± SEM.

[0108] Figure 50 - Graph depicting interferon-y ELISpot using splenocytes of mice treated with the indicated formulation, sacrificed on d70.

[0109] Detailed Description

[0110] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements.

[0111] Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0112] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice ofthe present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are 25 explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988;

[0113] Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0114] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0115] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0116] Definitions

[0117] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0118] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group ofmembers, integers or steps. The term "consisting essentially of means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of which, in turn, encompasses the term "consisting of. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of or "consisting of. Likewise, at each occurrence in the present application, the term "consisting essentially of may be replaced with the term "consisting of.

[0119] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0120] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0121] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0122] "Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5. "Physiological conditions" as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and / or a temperature of about 37°C.As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0123] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of 10 moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like material.

[0124] The expression "substantially free of X", as used herein, means that a mixture (such as a composition described herein or an aqueous phase thereof) is free of X in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture.

[0125] For example, "substantially free of a polymer-conjugated lipid, wherein the polymer-conjugated lipid has more than 5 consecutive monomer repeating units" as used herein, means the composition is free of the stated polymer-conjugated lipid in such manner as it is practically and realistically feasible. For example, if the composition is substantially free of a lipid comprising the stated polymer-conjugated lipid, the amount of a lipid comprising the stated polymer-conjugated lipid in the composition may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, 35 less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Similar considerations apply to expressions containing the phrase "substantially free of (such as but not limited to, “a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion” and / or “substantially free of an inorganic polyphosphate”)The term “buffer” as used herein related to a buffer system is typically an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it.The buffer may be any suitable buffer known in the art. Suitable buffering agents include citrate buffers, acetate buffers, succinate buffers, glutarate buffers, adipate buffers, maleate buffers, malate buffers, tartrate buffers, lactate buffers, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) and MES (2-(N-morpholino)-ethanesulfonic acid), phthalate buffers and formate buffers.

[0126] The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are Ci-40 alkyl (such as Ce-40 alkyl, Ce-30 alkyl, C6-20 alkyl, or C10-20 alkyl), C2-40 alkenyl (such as C6-40 alkenyl, Ce-30 alkenyl, or C6-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(Ci-6 alkyl). In some embodiments, the hydrocarbyl group is optionally substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0127] The term "heterohydrocarbyl" means a hydrocarbyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the hydrocarbyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. In one embodiment, the heterohydrocarbyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0128] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, ndecyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, n-triacontyl, n-tetracontyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylgroup are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0129] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene i.e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3 -propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3 -butylene, 2,3 -butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3-iso-butylene), the pentylene isomers (e.g., 1,1 -pentylene, 1,2-pentylene, 1,3 -pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1 -isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3 -heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituent may be the same or different). In one embodiment, the alkylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carboncarbon double bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1 -undecenyl, 2-undecenyl, 3 -undecenyl, 4-undecenyl, 5 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like.. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to forty, such as six to thirty, typically six to twenty, such as six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon triple bonds. Generally, the maximal number of carboncarbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkynyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0130] The terms "cycloalkyl" and “cycloalkenyl” represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, and cyclodecenyl. The cycloalkyl or cycloalkenyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the cycloalkyl or cycloalkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 5 or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the aryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl).

[0131] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S.

[0132] Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotri azolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrol othi azolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl,pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0133] The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the 5 maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heterocyclyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0134] The term “alkylcycloalkyl” means a cycloalkyl group, as defined above, which is substituted with an alkyl group, as defined above, the cycloalkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylcycloalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0135] The term “cycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted cycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the cycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0136] The term “alkylcycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule and the cycloalkyl portion in turn being substituted with a further alkyl group. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylcycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0137] The term “alkylaryl” means an aryl group, as defined above, which is substituted with an alkyl group, as defined above, the aryl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylaryl" means that one or more (such as 1 to themaximum number of hydrogen atoms bound to an alkylaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0138] The term “arylalkyl” means an alkyl group, as defined above, which is substituted with an aryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted arylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a arylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the arylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0139] The term “alkylheteroaryl” means a heteroaryl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heteroaryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylheteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0140] The term “heteroaryl alkyl” means an alkyl group, as defined above, which is substituted with a heteroaryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heteroarylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylalkyl group, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heteroarylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0141] The term “alkylheterocyclyl” means a heterocyclyl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylheterocyclyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0142] The term “heterocyclylalkyl” means an alkyl group, as defined above, which is substituted with a heterocyclyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heterocyclylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heterocyclyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heterocyclylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0143] The term “organosulfuric acid” or “organosulfate” means a compound of formula R-OSO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, orheterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “organosulfate” is used when the group is deprotonated. Depending on the pH, the sulfate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0144] The term “organosulfonic acid” or “organosulfonate” means a compound of formula R-SO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfonate” is used when the group is deprotonated. Depending on the pH, the sulfonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonate group is typically deprotonated at physiological pH).

[0145] The term “carboxylic acid” or “carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “carboxylate” is used when the group is deprotonated. Depending on the pH, the carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0146] The term “dicarboxylic acid” means a compound of formula HChC-R’-CChH, wherein R’ is alkylene or alkenylene group (all as defined above, either in a broadest aspect or a preferred aspect).

[0147] The term "ester" as used herein means a compound having the structure R-C(O)O-R’ (including its isomerically arranged structure R-OC(O)-R’, unless it is specified to the contrary), wherein R and R’ are each independently hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). When the term denotes a substituent connected to the rest of a molecule, the ester moiety may have the structure R-C(O)O- or R-0C(0)-, where R is as defined above. In one embodiment, each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker).

[0148] The term “organophosphate” means a compound of formula R0-P(=0)(0H)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphate group is typically deprotonated at physiological pH).

[0149] The term “organophosphonate” means a compound of formula R-P(=0)(0H)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0150] “Halo” means fluoro (-F), chloro (-C1), bromo (-Br) or iodo (-1).

[0151] “Amine” means the group -NR2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. When both groups R are hydrogen, the amine group is a primary amine group. When one R is hydrogen and the other R is other than hydrogen, the amine group is a secondary amine group. When both groups R are other than hydrogen, the amine group is a tertiary amine group. Examples of preferred tertiary amine groups are defined and exemplified below in relation to cationically ionizable lipids.

[0152] “Hydroxyl” - means the group -OH. “Sulfhydryl” - means the group -SH. “Nitro” means the group -NO2.“Ether” means an oxygen atom to which two hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect) are attached. The ether may be a cyclic ether, wherein the two hydrocarbyl groups together form a ring, and may include dioxolane groups. “Thioether” means a sulfur atom to which two a hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect) are attached. The ether may be a cyclic thioether, wherein the two hydrocarbyl groups together form a ring, and may include dithiane groups. “Amide” means the group -C(=O)NR(R’), wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect) and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0153] “Sulfonamide” means the group -S(=0)2NRR’, wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0154] “Carbamate” means the group -O-C(=O)NRR’ wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0155] “Carbohydrate” means a compound having the empirical formula Cm(H20)n where m may or may not be different from n. The term “carbohydrate residue” or “carbohydrate moiety”defines a residue attached to another atom, where one hydrogen atom of the carbohydrate is replaced by a bond attached to the rest of the molecule. The carbohydrate moiety may be a monosaccharide moiety. The monosaccharide moiety may have the D- or L-configuration. Furthermore, the monosaccharide moiety may be an aldose or ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6, carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (ie it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0156] In another embodiment, the monosaccharide moiety is a pentose moiety (ie it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0157] In another embodiment, the carbohydrate may be a higher saccharide (ie a di-, or oligosaccharide) comprising more than one monosaccharide moiety joined together by glycoside bonds. When the monosaccharide moieties are hexose moieties, the glycoside bonds may be l-a,l'-a glycoside bonds, 1 ,2'-gly coside bonds (which maybe l-a2’ or 1 '-P-2' glycoside bonds), l,3'-glycoside bonds (which may be l-a-3' or 1- P-3 '-glycoside bonds), 1 ,4'-gly coside bonds (which may be l-a-4' or l-P-4'-glycoside bonds), 1 ,6'-gly coside bonds (which may be l-a-6' or 1 - -6'-gly coside bonds), or any combination thereof. In one embodiment, the higher saccharide comprises 2 monosaccharide units (i.e. is a disaccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose and trehalulose. In another embodiment, the higher saccharide comprises 3 to 10 monosaccharide units (ie is an oligosaccharide) in a chain, which may be branched or unbranched. Preferably, the oligosaccharide comprises 3 to 8, more preferably 3 to 6, monosaccharide units. Examples of suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose.

[0158] A “phosphatidylserine” is a compound of formula R-C(=O)-O-CH2-CH-[O-(C=O)-R’]-CH2-O-P(=O)(OH)O-CH2-CH(NH2)COOH, i.e. a compound having a glycerol backbone with an acyl group R-C(=O)-O- bonded to the carbon at the 1 -position, another acyl group R’C(=O)-O- bonded to the carbon at the 2-position, and a phosphate group bonded to the carbon at the 3-position, the phosphate also being esterified with the hydroxyl moiety of a serine residue. Typically, the phosphate group is deprotonated such that the group is anionic at physiological pH. The serine amino acid moiety may be in a zwitterionic form. The groups R and R’ may be the same or different and each is independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Preferably the group R is an alkyl group, such as a Ce-30 alkyl group. Preferably the group R’ is an alkenyl group, such as a Ce-30 alkenyl group.

[0159] A “phosphatidylglycerol” is a compound of formula R-C(=0)-0-CH2-CH-[-0-(C=0)-R’]-CH2-O-P(=O)(OH)O-CH2-CH(OH)CH2OH, i.e. a compound having a first glycerol backbone with an acyl group R-C(=O)-O- bonded to the carbon at the 1 -position, another acyl group R’C(=O)-O- bonded to the carbon at the 2-position, and a phosphate group bonded to the carbon at the 3-position, this phosphate group being esterified with a second glycerol moiety. Typically, the phosphate group is deprotonated such that the group is anionic at physiological pH. The groups R and R’ may be the same or different and each is independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Preferably the group R is an alkyl group, such as a Ce-30 alkyl group. Preferably the group R’ is an alkenyl group, such as a Ce-30 alkenyl group.

[0160] “List A” substituents are selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5-or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR’, -

[0161]

[0162] X1C(=X1)X1R’, wherein X1is independently selected from O, S, NH and N(CH3); and each R’ is independently selected from the group consisting of H, Ci-4 alkyl, C2-4 alkenyl, C2-4alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(Ci-3 alkyl), -S(Ci-3 alkyl), -NH2, -NH(CI-3 alkyl), -N(CI-3 alkyl)2, -NHS(O)2(CI-3alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(Cl-3alkyl), -C(=O)NH2-z(Ci-3alkyl)z, -NHC(=0)(Cl-3 alkyl), -NHC(=NH)NHZ-2(C1-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(Ci-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, propyl or isopropyl. In some embodiments, List A substituents are selected from List Al, consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, List A substituents are selected from List A2, consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3.

[0163] Particles

[0164] The present invention provides in one aspect a composition comprising particles as described herein. The particles are capable of delivering a nucleic acid payload to a target. In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, a particle is a nucleic acid-containing particle such as a particle comprising DNA, RNA, preferably mRNA, or a mixture thereof.

[0165] In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro-or nano-sized compact structure.According to the present disclosure, the term "particle" includes nanoparticles. The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationically ionizable lipid or a cationic polymer, wherein all three external dimensions of the particle are in the nanoscale, z.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

[0166] In some embodiments, the particles described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of about 10 to about 1000 nm, about 15 to about 500 nm, about 20 to about 400 nm, about 25 to about 300 nm, about 30 to about 250 nm, about 40 to about 200 nm, about 45 to about 150 nm, or about50 to about 100 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 150 nm, such as from about 60 nm to about 120 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 60 nm to about 150 nm.

[0167] Lipid Nanoparticles

[0168] In one embodiment of the present disclosure, the composition is a lipid nanoparticle (LNP) composition. In the present disclosure, LNPs may be understood as oil-in-water emulsions. LNPs thus typically comprise a central complex of lipid and optionally a nucleic acid (e.g., DNA or RNA, preferably non-interfering RNA, such as mRNA, or mixtures thereof) embedded in a disordered, non-lamellar phase made of lipid.Nucleic acid-lipid particles, such as nucleic acid-lipid nanoparticles (LNP) are obtainable from combining a nucleic acid with lipids as outlined in more detail below. The lipid mixtures used for LNP formation typically do not form lamellar (bilayer) phases in water under physiological conditions. The LNPs typically do not comprise or encapsulate an aqueous core. The LNPs typically comprise a lipidic (or oily) core.

[0169] In some embodiments, the lipid nanoparticles described herein have an average size (such as a diameter) that in some embodiments ranges from about 10 to about 1000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most about 900 nm (e.g., at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm, such as in the range of about 10 to about 1000 nm, about 15 to about 500 nm, about 20 to about 400 nm, about 25 toabout 300 nm, about 30 to about 250 nm, about 40 to about 200 nm, about 45 to about 150 nm, or about 50 to about 100 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 150 nm, such as from about 60 nm to about 120 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 60 nm to about 150 nm.

[0170] Aqueous dispersion

[0171] The composition of the present invention typically takes the form of an aqueous dispersion including lipid particles, such as LNPs. The skilled person understands an aqueous dispersion to have an aqueous mobile phase and a dispersed phase. In this specification the term “dispersion” in its broadest sense takes its usual meaning in chemistry as a system in which distributed particles of one material (the “dispersed phase”) are dispersed in a phase of another material (the “continuous phase” or the “mobile phase”).

[0172] In one embodiment, the dispersion is a solid-liquid dispersion, in which the dispersed phase is solid and the mobile phase is a liquid. In one embodiment, the dispersion is a liquid-liquid dispersion, in which the dispersed phase and the mobile phase are both liquids.

[0173] In one embodiment, the dispersion is a colloid. The term "colloid" as used herein describes a stable mixture in which the dispersed particles do not settle out. Typically, the dispersed particles have at least in one direction a dimension roughly between 1 nm and 1 pm, or in such a system discontinuities are found at distances of that order.

[0174] In one embodiment, the dispersion is a suspension. The term “suspension” as used herein is a heterogeneous dispersion of larger particles in a medium. Unlike solutions and colloids, if left undisturbed for a long period of time, the suspended particles may settle out of the mixture. The use of the terms “colloid” and “suspension” is sometimes overlapping or synonymous, with colloids in some instances being considered a sub-type of suspensions.

[0175] In one embodiment, the mobile phase is a solution. The term “solution” as used herein is a homogeneous mixture comprising a solvent which is typically water and solutes which can be salts, buffers, tonifiers and the like, as long as these materials are molecularly distributed within the solvent. The mobile phase may comprise solutes, as described further herein. The dispersed phase comprises a lipid mixture including a cationically ionisable lipid, as defined herein.Lipids

[0176] The compositions of the invention contain a mixture of lipids. The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and also one or more hydrophilic moieties or groups.

[0177] Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases.

[0178] Lipids may comprise a polar portion and an apolar (or non-polar) portion. The term “amphiphile” as used in this specification is broadly defined herein as a molecule comprising hydrophobic moieties and hydrophilic moieties and / or a polar and apolar portion. As both cationic and anionic lipids both contain such groups, they are therefore amphiphiles. In this specification the term “cationic lipid” is therefore synonymous with “cationic amphiphile” and the term “anionic lipid” is synonymous with “anionic amphiphile”.

[0179] Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and / or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophilic groups may comprise polar and / or charged groups and include at least one amine and optionally hydrophilic non-charged groups such as hydroxyl, carbohydrate, sulfhydryl, nitro or like groups and may further include anionic groups such as phosphate, phosphonate, carboxylic acid, sulfate, sulfonate (all as defined and exemplified above) and other like groups.

[0180] The term "hydrophobic" as used herein with respect to a compound, group or moiety means that said compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, excludes water molecules. In some embodiments, the term "hydrophobic" refers to any compound, group or moiety which is substantially immiscible or insoluble in aqueous solution. In some embodiments, a hydrophobic compound, group or moiety is substantially nonpolar.

[0181] Examples of hydrophobic groups are hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and / or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophobic group can have functional groups (e.g., ether, thioether, ester, di oxolane, halide, amide,sulfonamide, carbamate, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution. The hydrophobic moieties of a lipid may have between 24 and 60 carbon atoms and can be hydrocarbyls (as described and exemplified above, typically comprising alkyl, alkenyl or alkynyl groups as described and exemplified above). The 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, with each such moiety typically having at least 6 carbon atoms. An example for segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the DACA moiety as described in WO2011 / 003834 wherein each of the acyl or alkyl groups comprise between 12 and 20 carbon atoms. Another example are lipids wherein the hydrophobic moiety comprises a steroid moiety, such as a cholesteryl moiety.

[0182] The hydrophobic moieties of a lipid preferably have between 24 and 60 carbon atoms and can also be heterohydrocarbyls wherein the heteroatoms are selected from N, O or S forming one, two, three or four non-charged groups of ether, thioether, ester, amide, carbamate, sulfonamide and the like. The 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, provided that each such moiety has at least 6 carbon atoms. An example for segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the diacylglycerol or dialkylglycerol moiety wherein each of the acyl or alkyl comprise between 12 and 20 carbon atoms. An example for hydrophobic moieties wherein each segment is heterohydrocarbyl are the ester-branched moieties in lipids such as SM-102 or ALC-315, as defined and exemplified below.

[0183] Cationically ionizable lipids

[0184] The compositions of the present invention also contain a cationically ionizable lipid. In one embodiment the compositions of the present invention comprise a cationically ionizable lipid. Typically, although not exclusively, when the intermediate lipid particle is a lipid nanoparticle (either empty or loaded), the lipid is a cationically ionizable lipid.

[0185] As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which, depending on whether it is protonated or deprotonated, has a net positive charge or is neutral, z.e., a lipid which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral.In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is capable of being protonated, preferably under physiological or slightly acidic conditions.

[0186] The cationically ionizable lipid may be any suitable lipid typically used in the formation of lipid nanoparticles (LNPs). In one embodiment, the cationically ionizable lipid may be a lipid described in any one of WO 2025 / 026545, WO 2017 / 075531, WO 2018 / 081480,

[0187] WO 2017 / 049245, WO 2015 / 095340, WO 2018 / 087753, or WO 2022 / 081750, the contents of which are incorporated herein by reference.

[0188] In one embodiment, the cationically ionizable lipid is selected from the group consisting of:

[0189] [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-315);

[0190] ((3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2 -butyl octanoate) (ALC-0366);

[0191] 1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA);

[0192] 2.2-dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 1.2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0193] di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); bis-(2 -butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)-nonadecanedioate (A9); (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);

[0194] O-[N-{(9Z,12Z)-octadeca-9,12-dien-l-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (H Y 501);

[0195] 2-(di-((9Z, 12Z)-octadeca-9, 12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2); 4-((di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)oxy)-A,A-dimethyl-4-oxobutan-4-amine (HYAM-2);

[0196] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8, 1-diyl) bis(2-hexyldecanoate) (EA-405);

[0197] (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8, 1-diyl) bis(2-hexyldecanoate) (HY-405);

[0198] di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate

[0199]

[0200] described in US2022 / 0218622A1);

[0201] bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate

[0202]

[0203] described in US2022 / 0218622A1);

[0204] bis(2-octyldodecyl) 3,3’-((2-(pyrrolidin-l-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr);

[0205] bis(2-octyldodecyl) 3,3’-(((l-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-lMe-3PipD);

[0206] bis(2-octyldodecyl) 3,3’-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA);

[0207] bis(2-octyldodecyl) 3,3’-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ);

[0208] bis(2-octyldodecyl) 3,3’-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr);

[0209] bis(2-hexyldecyl) 3,3’-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ)

[0210] or a mixture of any thereof.

[0211] In one embodiment, the cationically ionizable lipid is selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, ALC-0315, ALC-0366, SM-102, HY-501, EA-405, HY-405, DODMA, and Dlin-MC3-DMA. In one embodiment, the cationically ionizable lipid is selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, ALC-0315, SM-102, HY-501, and DODMA. In one embodiment, the cationically ionizable lipid is selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, SM-102, HY-501, and DODMA.In one embodiment, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl]-di(hexane- 6.1 -diyl) bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationically ionizable lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2 -butyl octanoate) (ALC-0366). In one embodiment, the cationically ionizable lipid is l,2-dioleoyloxy-3-dimethylaminopropane (DODMA). In one embodiment, the cationically ionizable lipid is 2.2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA). In one embodiment, the cationically ionizable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA). In one embodiment, the cationically ionizable lipid is l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In one embodiment, the cationically ionizable lipid is di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In one embodiment, the cationically ionizable lipid is A / .s-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)-nonanamido)-nonadecanedioate (A9). In one embodiment, the cationically ionizable lipid is (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5). In one embodiment, the cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102). In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-lMe-Pyr. In one embodiment, the cationically ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-l-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY 501).

[0212] In one embodiment, the cationically ionizable lipid is represented by formula I:

[0213]

[0214] I

[0215] or a pharmaceutically acceptable salt thereof, wherein:

[0216] L1and L2are each independently an optionally substituted C1-C30 aliphatic group; L3is a bond or optionally substituted C1-C10 aliphatic group;

[0217] X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-;

[0218] T1and T2are each independently an optionally substituted C3-C30 aliphatic;G is -N(R1)C(S)N(R1)2, -OH, -N(R1)2, -N+(R2)3, -N(R4)C(O)R2, -N(R4)S(O)2R2, -N(R4)C(O)N(R2)2, -CH(N-R'), and -R3;

[0219] each R1is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic and OR2;

[0220] R2is selected from the group consisting of H and optionally substituted C1-C10 aliphatic;

[0221] R3is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 4- to 12 membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted C6-C12 aryl, or optionally substituted C3-C12 cycloaliphatic; and

[0222] each R4is independently selected from H and optionally substituted Ci-Ce aliphatic. In one embodiment, the cationically ionizable lipid is selected from the group consisting of: BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, BODD-C2C2-DMA, ALC-0315, ALC-0366, and SM-102.

[0223] In one embodiment, the cationically ionisable lipid is present in an amount of 20 to 70 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationically ionisable lipid is present in an amount of 30 to 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationically ionisable lipid is present in an amount of 40 to 50 mol% of the total lipids present in the lipid mixture. In one preferred embodiment, the cationically ionisable lipid is present in an amount of 45 to 55 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture component of both empty lipid particle compositions excluding the nucleic acid and loaded lipid particles including the nucleic acid.

[0224] Neutral Lipids - Phospholipids and Monoacylglycerol

[0225] The composition may also additionally comprise a phospholipid. In one embodiment, the phospholipid may be zwitterionic neutral lipid (i.e. it carries both a positive and a negative charge, so that it is neutral at a pH ranging around neutral).

[0226] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins. The hydrocarbyl portion of the acyl moieties of phospholipids is as defined above, but is preferably an alkylgroup (as defined above) having 6 to 40, preferably 8 to 24, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carboncarbon double bonds. The acyl parts of the phospholipids may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of lignoceroyl, behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties. In a specific embodiment, neutral phospholipids have a Tmof 30°C or higher and are selected from di-stearoyl or di-palmitoyl or stearoyl-palmitoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties having 14 to 22 carbon atoms (including the acyl carbon), preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.

[0227] Examples of such phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and phosphatidylethanolamines, in particular diacylphosphatidyl-ethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidyl ethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l '-rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further phosphatidyl-ethanolamine lipids with different hydrophobic chains.

[0228] In one embodiment, the phospholipid is DSPC and is present in the lipid mixture in an amount of about 15 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is DSPC and is present in the lipid mixture in an amount of about 20 mol % of the total lipids present in the lipid mixture.In some embodiments, the phospholipid is combined with a monoacylglycerol.

[0229] In some embodiments, the phospholipid is combined with a saturated monoacylglycerol. In some embodiments, the phospholipid is DSPC and is combined with a monoacylglycerol. In some embodiments, the phospholipid is DSPC and is combined with a saturated monoacylglycerol .

[0230] In some preferred embodiments, the phospholipid is DSPC and an additional neutral lipid selected from the group consisting of 1-oleoyl-rac-glycerol (MOG), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) and dimyristoylphosphatidylcholine (DMPC), is also present.

[0231] In some preferred embodiments, of the phospholipid, such as DSPC, and the additional neutral lipid is present in the compositions described herein in a total concentration ranging from about 10 mol % to about 65 mol %, such as from about 20 mol % to about 60 mol %, from about 30 mol % to about 50 mol %, from about 15 mol % to about 25 mol % or about 20% of the total lipids present in the compositions described herein.

[0232] In some preferred embodiments the phospholipid is DSPC and is present at a concentration of about 10 mol% of the total lipids present in the compositions described herein, and an additional neutral lipid selected from the group consisting of 1-oleoyl-rac-glycerol (MOG), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) and dimyristoylphosphatidylcholine (DMPC) is also present.

[0233] In some preferred embodiments the phospholipid DSPC and is present at a concentration of about 10 mol% of the total lipids present in the compositions described herein, and is combined with an additional neutral lipid at a concentration of about 5% to about 15%, such as about 10 mol% of the total lipids present in the compositions described herein.

[0234] In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of from about 5 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 10 mol % to about 30 mol % of the total lipids present inthe lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 15 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 20 mol % of the total lipids present in the lipid mixture.

[0235] In one embodiment, the neutral lipid is a monoacylglycerol and is present in the lipid mixture in an amount of about 1 mol % to about 20 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a monoacylglycerol and is present in the lipid mixture in an amount of about 2 mol % to about 15 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a monoacylglycerol and is present in the lipid mixture in an amount of from about 3 mol % to about 10 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a monoacylglycerol and is present in the lipid mixture in an amount of about 4 mol % to about 8 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a monoacylglycerol and is present in the lipid mixture in an amount of about 5 mol % to about 7 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid a monoacylglycerol is present in the lipid mixture in an amount of about 10 mol % of the total lipids present in the lipid mixture.

[0236] In one embodiment, the neutral lipid is selected from 1-oleoyl-rac-glycerol, 1,2-dipalmitoyl- w-glycero-3 -phosphocholine, l,2-dimyristoyl- w-glycero-3-phosphatidylcholine and 1,2-dioleoyl- w-glycero-3-phosphoethanolamine.

[0237] In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 1 mol % to about 20 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 2 mol % to about 15 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 3 mol % to about 10 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 4 mol % to about 8 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 5 mol % to about 7 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 6 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of up to about 5 mol % of the total lipids present inthe lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 5 mol % of the total lipids present in the lipid mixture.

[0238] In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol and is present in the lipid mixture in an amount of about 1 mol % to about 20 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol and is present in the lipid mixture in an amount of about 2 mol % to about 15 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol and is present in the lipid mixture in an amount of from about 3 mol % to about 10 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol and is present in the lipid mixture in an amount of about 4 mol % to about 8 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol and is present in the lipid mixture in an amount of about 5 mol % to about 7 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a 1-oleoyl-rac-glycerol is present in the lipid mixture in an amount of about 6 mol % of the total lipids present in the lipid mixture.

[0239] Steroid

[0240] The compositions of the present invention also comprise a steroid. In one embodiment, the steroid comprises a sterol. In one embodiment, the steroid is cholesterol.

[0241] Thus, in some embodiments, the compositions described herein comprise a cationically ionizable lipid (as defined herein) and cholesterol.

[0242] Thus, in some embodiments, the compositions described herein comprise a cationically ionizable lipid (as defined herein), a phospholipid and cholesterol.

[0243] In some embodiments, the steroid is present in the compositions described herein in a concentration ranging from about 10 mol % to about 65 mol %, such as from about 20 mol % to about 60 mol %, from about 30 mol % to about 50 mol %, or from about 20 mol % to about 40 mol % of the total lipids present in the compositions described herein. In some preferred embodiments, the steroid is present in the composition in an amount from about 24 to 36 mol% of the total lipids present in the composition. In some embodiments, the steroid is cholesterol and the cholesterol is present in the compositions described herein in a concentration ranging from about 10 mol % to about 65 mol %, such as from about 20 mol % to about 60 mol %, from about 30 mol % to about 50 mol %, or from about 20 mol % to about 40 mol % of the total lipids present in the compositions described herein. In somepreferred embodiments, the cholesterol is present in the composition in an amount from about 24 to 36 mol% of the total lipids present in the composition.

[0244] In certain preferred embodiments, the compositions described herein comprise a phospholipid, monoacylglycerol and cholesterol, preferably in the concentrations given above. In some embodiments, the lipid nanoparticle compositions comprise a phospholipid, a monoacylglycerol (such as 1-oleoyl-rac-glycerol) and cholesterol, preferably in the concentrations given above. In some embodiments, the compositions described herein comprise DSPC and cholesterol, preferably in the concentrations given above.

[0245] In certain preferred embodiments, the compositions described herein comprise a cationically ionizable lipid and cholesterol, preferably in the concentrations given above. In some embodiments, the compositions comprise a steroid and a cationically ionizable lipid selected from the group consisting of di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ), (4-Hydroxybutyl) azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) and 6-((9-((2-butyloctanoyl)oxy)nonyl)(3-hydroxypropyl)amino)hexyl 2-propyloctanoate (ALC-0366), preferably in the concentrations given below.

[0246] In some embodiments, the compositions comprise a cholesterol and a cationically ionizable lipid selected from the group consisting of di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ), (4-Hydroxybutyl) azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) and 6-((9-((2-butyloctanoyl)oxy)nonyl)(3-hydroxypropyl)amino)hexyl 2-propyloctanoate (ALC-0366), preferably in the ratios given below.

[0247] In some embodiments, the molar ratio between the cationically ionizable lipid and the steroid is ranging from about 0.75 to about 2.5, such as from about 1 to about 2.5, from about 1.5 to about 2.5, preferably from about 1.75 to about 2.25, or more preferably from about 1.75 to about 2.0.

[0248] In some embodiments, the molar ratio between the cationically ionizable lipid and the cholesterol is ranging from about 0.75 to about 2.5, such as from about 1 to about 2.5, from about 1.5 to about 2.5, preferably from about 1.75 to about 2.25, or more preferably from about 1.75 to about 2.0.Compounds preferably absent

[0249] Polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units

[0250] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid comprises more than 5 monomer repeating units. In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units. In this specification the term “polymer-conjugated” preferably means a polymer (as defined below), (optionally conjugated to a lipid) wherein the polymer has the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge.

[0251] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a polymer-conjugated lipid having more than 5 monomer repeating units.

[0252] Examples of such polymers which may be conjugated to a lipid include poly(sarcosine) (pSar) (which may be conjugated to a lipid - to produce a polysarcosinylated lipid), poly(oxazoline) (POX); poly(oxazine) (POZ), poly(vinyl pyrrolidone) (PVP); poly(N-(2-hydroxypropyl)-methaciylamide) (pHPMA); poly(dehydroalanine) (pDha); poly(aminoethoxy ethoxy acetic acid) (pAEEA) and poly(2 -methylaminoethoxy ethoxy acetic acid) (pmAEEA).

[0253] The compositions described herein are substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polyethylene gly col-conjugated lipid (also referred to herein as a PEGylated lipid or PEG-lipid) having more than 5 consecutive ethylene glycol repeating units. The term "PEGylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art. In one embodiment, the compositions described herein are substantially free (as defined above, either in its broadest aspect or a preferred aspect) of polyethylene glycol (PEG). The PEG may comprise more than 5 consecutive ethylene glycol repeating units. The PEG may be in any form, either lipid conjugated, or in the form of a surfactant. In one embodiment, thecomposition is substantially free from a polysorbate. In one embodiment, the composition is substantially free from polysorbate 20. In one embodiment, the composition is substantially free from polysorbate 40. In one embodiment, the composition is substantially free from polysorbate 60. In one embodiment, the composition is substantially free from polysorbate 80. Where the PEGylated lipid is part of an LNP composition, the PEGylated lipid may be associated with or bound to the LNP. The PEG or PEG-lipid may comprise more than 5 consecutive ethylene glycol repeating units. The PEG or PEG-lipid may comprise 6-1000, 6-500, 6-100, 6-50, 6-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100, or 10-50, ethylene glycol repeating units, which may be consecutive. The PEG or PEG-lipid may comprise 6-1000, optionally 8-100, ethylene glycol repeating units, which are preferably consecutive. A "polymer" as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0254] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a polysarcosine-conjugated lipid having more than 5 monomer repeating units, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion, the polysarcosine portion having the repeating unit shown below, where x represents the number of monomer repeating units.

[0255]

[0256] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a polyoxazoline (POX)-conjugated and / or a polyoxazine (POZ)-conjugated lipid and / or a POX / POZ-conjugated lipid having more than 5 monomer repeating units, also referred to herein as a conjugate of a POX and / or POZ polymer and one or more hydrophobic chains or as oxazolinylated and / or oxazinylated lipid or POX and / or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion, the polyoxazoline portion (pOx) having the repeating unit shown below. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion, the poly oxazine (pOz) portion having the repeating unit shown below. The term "oxazolinylated / oxazinylated lipid" or "POX / POZ-lipid" or "POXZ-lipid" refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and polyoxazine, i.e. a polymer having both the pOx and pOz repeating units shown below, where x represents the number of monomer repeating units.

[0257]

[0258] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) having more than 5 monomer repeating units. In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) conjugated to a lipid. The term “poly(vinyl pyrrolidone)” or “PVP” means a polymer having a vinyl pyrrolidine repeating unit, i.e. the repeating unit shown below, where x represents the number of monomer repeating units.

[0259]

[0260] pVP

[0261] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) having more than 5 monomer repeating units. In one embodiment, the compositionis substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid. The term “poly(N-(2-hydroxypropyl)-methacrylamide” or “pHPMA” means a polymer having the repeating unit shown below, swhere x represents the number of monomer repeating units.

[0262]

[0263] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(dehydroalanine) (pDha) having more than 5 monomer repeating units. In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(dehydroalanine) (pDha) conjugated to a lipid. The term “pDha” means a polymer having the repeating unit shown below, where x represents the number of monomer repeating units.

[0264]

[0265] pDha

[0266] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of amphiphilic oligoethylene glycol (OEG)-conjugated lipids having more than 5 monomer repeating units. Examples of amphiphilic oligoethylene glycol (OEG)-conjugated lipids include poly(aminoethyl-ethylene glycol acetyl) (pAEEA) and / or poly(methylaminoethyl-ethylene glycol acetyl) (pmAEEA). The terms “pAEEA” and “pmAEAA” means a polymer having the repeating unit shown below, where x represents the number of monomer repeating units:

[0267]

[0268] pAEEA pmAEEAMultivalent anions

[0269] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a multivalent anion. The multivalent anion may be understood to refer to an ion having multiple (i.e., more than one) negative charges. For example, the multivalent anion may be a dianion, i.e., having a charge of 2-, or having two negative charges. In another example the multivalent anion may be a trianion, i.e. having a charge of 3- or having three negative charges. In yet another example the multivalent anion may be a tetraanion, i.e. having a charge of 4- or having four negative charges. In further examples the multivalent anion may have a plurality of negative charges. Typically, the multivalent anion is not, or does not comprise, a nucleic acid, such as DNA or RNA. In some embodiments, the multivalent anion has no more than 20 negative charges (i.e., a charge of 20-), preferably no more than 10 negative charges (i.e., a charge of 10-), or most preferably no more than 5 negative charges (i.e., a charge of 5-). The multivalent anion may have 2-20, 2-15, 2-10, 2-8, 2-5, 3-20, 3-15, 3-10, 3-8, or 3-5 negative charges, optionally 2-10 negative charges, preferably 2-5 negative charges.

[0270] The multivalent anion may be selected from the group consisting of: an inorganic polyphosphate (as further defined herein), an inorganic phosphate (e.g., PO43-), sulfate, sulfite, pyrosulfate, dithionate, dithionite, metabisulfite, thiosulfate, trithionate or tetrathionate, a dicarboxylic acid (e.g., oxalic, malonic, succinic, glutaric, adipic, pimelic, sebacic, phthalic, isophthalic or terephthalic acid), a substituted dicarboxylic acid (e.g., tartronic, mesoxalic, malic, tartaric, aspartic, glutamic, hydroxyglutaric or saccharinic acid), a tricarboxylic acid (e.g., citric, isocitric, propane- 1,2, 3 -tricarboxylic or trimesic acid), or mixtures thereof. Typically, the multivalent anion in water is molecularly dissolved as a solute and does not form supramolecular assemblies such as micelles. Structurally, the multivalent anion is not, or does not comprise, a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion (e.g., the multivalent anion is not a negatively charged lipid).

[0271] Inorganic polyphosphate

[0272] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of an inorganic polyphosphate. The inorganic polyphosphate can be any linear, cyclic, or branched inorganic polyphosphate. In some embodiments, the inorganic polyphosphate is a linear inorganic polyphosphate (such as a linear inorganic triphosphate). In some embodiments, the inorganic polyphosphate comprisesthe formula [PxO(3x+i)]y‘, wherein x is an integer and is at least 3; and y is the anionic charge. For example, if x is 3, the inorganic polyphosphate is a linear inorganic triphosphate comprising the formula [PsOio]5'. Likewise, if x is 4, the inorganic polyphosphate is a linear or branched inorganic tetraphosphate comprising the formula [P4O13]6'. In some embodiments, the inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof. In some embodiments, the inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof. In some embodiments, the inorganic polyphosphate is triphosphate.

[0273] Negatively charged amphiphile

[0274] In one embodiment, the composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a negatively charged amphiphile.

[0275] In this specification the term “negatively charged amphiphile” is defined generally as a negatively charged molecule having both hydrophilic and lipophilic moieties. The negatively charged amphiphiles as specified are anisotropic and have a hydrophilic portion and a lipophilic portion. The negative charge is situated in the hydrophilic portion of the amphiphile. The negatively charged amphiphile may have one negatively charged group or multiple (e.g. 2, 3, 4, or 5) negatively charged groups.

[0276] As the skilled person will readily understand, depending on the pKaof the amphiphile and the pH, the amphiphile may be present in a protonated form (described in standard chemical nomenclature as the acid) or in a negatively charged, deprotonated form (described in standard chemical nomenclature by the name of the acid with the suffix “-ate” substituting for “acid”). Anionic amphiphiles may encompass both protonated and deprotonated forms, regardless of the form in which they are described in this specification.

[0277] The lipophilic moiety of the negatively charged amphiphile may be hydrocarbyl groups or heterohydrocarbyl groups, as defined above. Where the lipophilic moieties are hydrocarbyl groups, they may be selected from alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkylalkyl groups, alkylcycloalkyl groups, alkylcycloalkylalkyl groups, aryl groups, alkylaryl groups, arylalkyl groups, and alkylarylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect). Where the lipophilic moieties are heterohydrocarbyl groups, they may be alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either ina broadest aspect or a preferred aspect). The lipophilic moiety may comprise two or more groups from the aforementioned list.

[0278] Typical classes of amphiphiles having a constitutive negative charge include organosulfates, organosulfonates, organophosphates and organophosphonates.

[0279] Additional Lipids

[0280] Lysophospholipid

[0281] The compositions described herein may also contain a lysophospholipid.

[0282] A "lysophospholipid" as used herein, is given its ordinary meaning, z.e., a phospholipid comprising a glycerol backbone with a single fatty acid chain (typically at the 1 -position) and wherein a phosphate group is linked to the glycerol backbone (typically at the 3-position). The fatty acid chains of the lysophospholipids of the present invention may have from 12 to 22 carbon atoms, preferably from 14 to 20 carbon atoms. The fatty acid chain of the lysophospholipid may be saturated or unsaturated. Preferably the fatty acid chain of the lysophospholipid is saturated.

[0283] In one embodiment the lysophospholipid is a lysophosphatidylethanolamine. In one embodiment the lysophospholipid is a lysophosphatidylethanolamine wherein the fatty acid chain has from 12 to 22 carbon atoms, preferably from 14 to 20 carbon atoms. In one embodiment the lysophospholipid is a lysophosphatidylethanolamine wherein the fatty acid chain of the lysophosphatidylethanolamine is saturated.

[0284] In one preferred embodiment the lysophospholipid is a lysophosphatidylcholine. In one embodiment the lysophospholipid is a lysophosphatidylcholine wherein the fatty acid chain has from 12 to 22 carbon atoms, preferably from 14 to 20 carbon atoms. In one embodiment the lysophospholipid is a lysophosphatidylcholine wherein the fatty acid chain of the lysophosphatidylcholine is saturated.

[0285] In one embodiment the lysophospholipid is l-hexadecanoyl-sn-glycero-3-phosphocholine (16:0 Lyso PC).In one especially preferred embodiment, the lysophospholipid is l-heptadecanoyl-.w-glycero-3 -phosphocholine (17:0 lyso-PC), having the following structure:

[0286]

[0287] In one especially preferred embodiment, the lysophospholipid is l-octadecanoyl-.s / / -glycero-3 phosphocholine (18:0 lyso-PC), having the following structure:

[0288]

[0289] In one especially preferred embodiment, the lysophospholipid is l-tetradecanoyl-.s / z-glycero-3 -phosphocholine (14:0 lyso-PC), having the following structure:

[0290]

[0291] In one especially preferred embodiment, the lysophospholipid is l-eicosanoyl-.s / / -glycero-3-phosphocholine (20:0 lyso-PC), having the following structure:

[0292]

[0293] In one especially preferred embodiment, the lysophospholipid is l-oleoyl-.s / / -glycero-3 phosphocholine (18:1 lyso-PC), having the following structure:

[0294]

[0295] In one embodiment the lysophospholipid is present in the composition in an amount between about 1% and about 8 mol% of the total lipids present in the composition. In one embodiment the lysophospholipid is present in the composition in an amount of between about 2% and about 8 mol% of the total lipids present in the composition. In one especially preferredembodiment the lysophospholipid is present in the composition in an amount of about 3 mol% of the total lipids present in the composition.

[0296] In one embodiment the lysophospholipid is a saturated lysophosphatidylcholine and is present in the composition in an amount between about 1% and about 8 mol% of the total lipids present in the composition. In one embodiment the lysophospholipid is a saturated lysophosphatidylcholine and is present in the composition in an amount of between about 2% and about 8 mol% of the total lipids present in the composition. In one especially preferred embodiment the lysophospholipid is a saturated lysophosphatidylcholine and is present in the composition in an amount of about 3 mol% of the total lipids present in the composition. Monomer- or oligomer-grafted lipid

[0297] The compositions described herein may also contain a monomer- or oligomer-grafted lipid. In the present specification the term “monomer- or oligomer-grafted lipid” in its broadest sense means a lipid or lipid-like material, as defined above (either in a broadest aspect or a preferred aspect) conjugated to a monomer or oligomer, as defined below (either in a broadest aspect or a preferred aspect).

[0298] In this specification an “oligomer” is defined as a molecular structure comprising 2, 3, 4, or 5 repeat units (monomers) connected by covalent bonds. The monomers may be the same or different. In one embodiment, the monomers are all the same. The term “monomer-grafted lipid” has one monomer conjugated to the lipid or lipid-like material.

[0299] In one embodiment, the oligomer grafted lipid has 2, 3, or 4 repeat units. In one embodiment, the oligomer grafted lipid has 2 repeat units. In one embodiment, the oligomer grafted lipid has 3 repeat units. In one embodiment, the oligomer grafted lipid has 4 repeat units. In one embodiment, the oligomer grafted lipid has 5 repeat units.

[0300] In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo(ethylene-glycol) conjugated lipid. The mono- or oligo(ethylene-glycol) may comprise 1, 2, 3, 4 or 5 ethylene glycol repeating units, which are preferably consecutive. In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo(ethylene-glycol) conjugated lipid, comprising 1, 2, 3 or 4 ethylene glycol repeating units, which are preferably consecutive.

[0301] Other examples of monomer- or oligomer-grafted lipids include mono- or oligo(sarcosine) (oSar)-conjugated lipids, mono- or oligo(oxazoline)-conjugated lipids; mono- oroligo(oxazine)-conjugated lipids, mono- or oligo(vinyl pyrrolidone)-conjugated lipids; mono-or oligo(7V-(2-hydroxypropyl)-methacrylamide)-conjugated lipids; mono- or oligo(dehydroalanine)-conjugated lipids; mono- or oligo(aminoethoxy ethoxy acetic acid)-conjugated lipids and mono- or oligo(2 -methylaminoethoxy ethoxy acetic acid) conjugated lipids.

[0302] In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligosarcosine-conjugated lipid. The term "mono- or oligosarcosinylated lipid" as used herein refers to a molecule comprising both a lipid portion and a mono- or oligosarcosine, also known as a mono-oligo(N-methylglycine) portion, the mono- or oligosarcosine portion having the repeating unit shown below:

[0303]

[0304] wherein x refers to the number of sarcosine units. The mono- or oligosarcosine may have from 1 to 5, such as from 2 to 5, such as from 3 to 5 sarcosine units. The monosarcosine has 1 sarcosine unit. The oligosarcosine may have 2 sarcosine units. The oligosarcosine may have 3 sarcosine units. The oligosarcosine may have 4 sarcosine units. The oligosarcosine may have 5 sarcosine units.

[0305] In one embodiment, the monomer- or oligomer-grafted lipid comprises a mono- or oligosarcosine portion (as defined and exemplified above), the carbonyl terminus of which is bonded to a (Ce-30 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to a capping group. In one embodiment, the monomer- or oligomer-grafted lipid comprises a mono- or oligosarcosine portion (as defined and exemplified above), the carbonyl terminus of which is bonded to a (C12-22 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to a capping group. In one embodiment, the monomer- or oligomer-grafted lipid comprises a mono- or oligosarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (C14 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to a capping group.

[0306] The capping group may be any group suitable for capping the amino terminus of an amino acid. In one embodiment, the capping group is selected from the group consisting of acetyl,methoxy carbamate, methyl sulfonamide, isopropyl, tert-butyl, propyl, butyl, cyclopropyl, ethoxy carbamate, ethyl sulfonamide, methyl and ethyl urea, allyl (2-aminoethyl) carbamate, vinyl carbamate and sulfamide.

[0307] In one preferred embodiment, the capping group is an acetyl group.

[0308] In one especially preferred embodiment, the monomer- or oligomer-grafted lipid is N-methyl-N-(2-oxo-2-(tetradecylamino)ethyl)acetamide (C14-pSar(l)-Ac), having the following structure:

[0309]

[0310] In one especially preferred embodiment, the monomer- or oligomer-grafted lipid is N-methyl-N-(2-oxo-2-(tetradecylamino)ethyl)acetamide (C14-pSar(3)-Ac), having the following structure:

[0311]

[0312] In one especially preferred embodiment, the monomer- or oligomer-grafted lipid is N-methyl-N-(2-(methyl(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)-2-(N-methyl-2-(N-methylacetamido)acetamido)acetamide (C14-pSar(5)-Ac), having the following structure:

[0313]

[0314] In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo-oxazoline (oOX)-conjugated and / or an oligooxazine (oOZ)-conjugated lipid and / or a conjugate of a mono- or oligo-oxazolinylated / oxazinylated lipid. The term "mono- or oligo-oxazolinylated lipid" or "oOX-lipid" refers to a molecule comprising both a lipid portion and a mono- or oligooxazoline portion, the mono- or oligooxazoline portion (oOx) having the repeating unit shown below. The term "mono- or oligo-oxazinylated lipid" refers to a molecule comprising both a lipid portion and a mono- or oligooxazine portion, the mono- or oligooxazine (oOz) portion having the repeating unit shown below. The term "mono- or oligo-oxazolinylated / oxazinylated lipid" or "oOXZ-lipid" refers to a molecule comprising both a lipid portion and a portion of a cooligomer of oligooxazoline and oligooxazine, i.e. an oligomer having both the oOx and oOz repeating units shown below:

[0315]

[0316] wherein x refers to the number of Ox and / or Oz monomer repeating units. The total number of Ox and / or Oz monomer repeating units in the oligomer may comprise from 1 to 5, from 2 to 5, from 3 to 5 Ox and / or Oz monomer repeating units. The mono- Ox and / or mono- Oz has 1 oxazoline and / or oxazine unit. The oOx and / or oOz may have 2 oxazoline and / or oxazine units. The oOx and / or oOz may have 3 oxazoline and / or oxazine units. The oOx and / or oOz may have 4 oxazoline and / or oxazine units. The oOx and / or oOz may have 5 oxazoline and / or oxazine units.

[0317] In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo(vinyl pyrrolidone) (oVP)-conjugated lipid. The term “mono- oligo(vinyl pyrrolidone)” or “oVP” means a oligomer having a vinyl pyrrolidone repeating unit, i.e. the repeating unit shown below:

[0318]

[0319] wherein x refers to the number of VP monomer repeating units. The total number of VP monomer repeating units in the mono- or oligomer may be from 1 to 5, from 2 to 5, from 3 to 5 VP monomer repeating units. The mono(vinyl pyrrolidone) has 1 vinyl pyrrolidone unit. The oligo(vinyl pyrrolidone) may have 2 vinyl pyrrolidone units. The oligo(vinyl pyrrolidone) may have 3 vinyl pyrrolidone units. The oligo(vinyl pyrrolidone) may have 4 vinyl pyrrolidone units. The oligo(vinyl pyrrolidone) may have 5 vinyl pyrrolidone units. In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo(7V-(2-hydroxypropyl)methacrylamide) (oHPMA)-conjugated lipid. The term “mono- or oligo(7V-(2-hydroxypropyl)-methacrylamide)” or “oHPMA” means a mono- or oligomer having the repeating unit shown below:

[0320]

[0321] wherein x refers to the number of HPMA monomer repeating units. The total number of HPMA monomer repeating units in the oligomer may be from 1 to 5, such as from 2 to 5, such as from 3 to 5 HPMA monomer repeating units. The mono(A-(2-hydroxypropyl)-methacrylamide) has 1 A-(2-hydroxypropyl)methacrylamide unit. The oligo(A-(2-hydroxypropyl)methacrylamide) may have 2 A-(2-hydroxypropyl)methacrylamide units. The oligo(A-(2-hydroxypropyl)methacrylamide) may have 3 A-(2-hydroxypropyl)methacrylamide units. The oligo(A-(2-hydroxypropyl)methacrylamide) may have 4 A-(2-hydroxypropyl)-methacrylamide units. The oligo(A-(2-hydroxypropyl)methacrylamide) may have 5 N-(2-hydroxypropyl)methacrylamide units.

[0322] In one embodiment, the monomer- or oligomer-grafted lipid is a mono- or oligo(dehydroalanine) (oDha)-conjugated lipid. The term oligo(dehydroalanine) (“oDha”) means an oligomer having the repeating unit shown below:

[0323]

[0324] oDha

[0325] wherein x refers to the number of monomer repeating units. The total number of monomer repeating units in the oligomer may be from 1 to 5, such as from 2 to 5, such as from 3 to 5 Dha monomer repeating units. The mono(dehydroalanine) has 1 dehydroalanine unit. The oligo(dehydroalanine) may have 2 dehydroalanine units. The oligo(dehydroalanine) may have 3 dehydroalanine units. The oligo(dehydroalanine) may have 4 dehydroalanine units. The oligo(dehydroalanine) may have 5 dehydroalanine units.

[0326] In one embodiment, the monomer- or oligomer-grafted lipid is an amphiphilic oligoethylene glycol (OEG)-conjugated lipid. Examples of amphiphilic oligoethylene glycol (OEG)-conjugated lipids include mono- or oligo(aminoethyl-ethylene glycol acetyl) (oAEEA) and / or oligo(methylaminoethyl-ethylene glycol acetyl)(omAEEA). The terms “oAEEA” and “omAEAA” means a oligomer having the repeating unit shown below:

[0327]

[0328] oAEEA omAEEA

[0329] wherein x refers to the number of AEEA or mAEEA monomer repeating units. The total number of AEEA or mAEEA monomer repeating units in the oligomer may be from 1 to 5, such as from 2 to 5, such as from 3 to 5 AEEA or mAEEA monomer repeating units. The mono AEEA or mAEEA has 1 AEEA or mAEEA unit. The oAEEA or omAEEA may have 2 AEEA or mAEEA units. The oAEEA or omAEEA may have 3 AEEA or mAEEA units. The oAEEA or omAEEA may have 4 AEEA or mAEEA units. The oAEEA or omAEEA may have 5 AEEA or mAEEA units.

[0330] In one embodiment, the monomer- or oligomer grafted lipid is present in the lipid mixture in an amount of about 0.5 to about 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the monomer- or oligomer grafted lipid is present in the lipid mixture in an amount of about 1 to about 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the monomer- or oligomer grafted lipid is present in the lipid mixture in an amount of about 3 to about 4 mol% of the total lipids present in the lipid mixture.

[0331] Critical packing parameter

[0332] In some embodiments, the lipid particle compositions of the present invention also comprise an additional lipid with a critical packing parameter of < 0.5.

[0333] The skilled person understands the critical packing parameter (CPP) to be a theoretical framework for determining the type of assembly formed by surfactants.

[0334] The critical packing parameter (CPP) may be calculated using a formula comprising the volume of the hydrophobic part (V), the critical length of the hydrophobic part (lc) and the surface area of the hydrophilic head group (ao), as show in Equation 1 below:

[0335]

[0336] The critical length of the hydrophobic part (lc) may be calculated using Equation 2 below:lr< lmnx« (1.54 + 1.265 ■ n)

[0337] wherein n is the number of carbon atoms in the hydrophobic chain.

[0338] The hydrophobic part (V) may be calculated using Equation 3 below:

[0339] V = (2.74 + 26.9 ■ n) A3

[0340] wherein n is the number of carbon atoms in the hydrophobic chains.

[0341] The surface area of the head group (A) may be determined by molecular dynamics simulations as described in Kobierski et al. Colloids and Surfaces B: Biointerfaces 2022, 211, 112298.

[0342] The surface area of the head group (A) may be derived from small-angle X-ray scattering data as described in Yagmur et al. Langmuir 2010, 26(2), 1177-85.

[0343] Preferably, the surface area of the head group (ao, in A) is determined by calculating the topological polar surface area (TPSA) as described in Ertl et al. J. Med. Chem. 2000, 43, 3714-3717.

[0344]

[0345]

[0346] *: non-hydrogen atom; single bond; d: double bond; # triple bond; aromatic bond; atomic symbol in lowercase: atom is part of an aromatic system; b: nitro group; c: Middle nitrogen in azide group; d: atom in a three-membered ring; e: nitrogen in isocyano group; f: as in pyridine N-oxide.

[0347] As is commonly known in the art, the general shapes of molecules associated with the determined CPPs, and the preferred structures of their systems can be divided into four groups. A molecule with a CPP value < 1 / 3 adopts a cone shape and prefers the formation of micellar structures. A molecule with a CPP value ranging from 1 / 3 to 1 / 2 adopts a truncated cone shape and prefers the formation of vesicular or flexible layers. A molecule with a CPP value close to 1 adopts a cylindrical shape and prefers the formation of planar layers. An example of a molecule with a CPP value close to 1 is phosphatidylinositol. A molecule with a CPP value >1 adopts an inverted cone shape and prefers the formation of inverse vesicular or inverse micellar structures. An example of a molecule with a CPP value >1 is cholesterol. In a preferred embodiment of the present invention the critical packing parameter is calculated by the method used in example 11.

[0348] Nucleic acid

[0349] The compositions of the present application may contain a nucleic acid. The nucleic acid may exert a therapeutic effect, particularly although not exclusively when transfected into a cell. The nucleic acid is preferably RNA, such as mRNA.

[0350] In one embodiment, the composition comprises a nucleic acid. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA.

[0351] A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.The term "nucleoside" relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. Nucleic acids may include one or more modified nucleosides or nucleotides. Examples of modified nucleosides or nucleotides which may be incorporated into nucleic acids include N7-alkylguanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-C1-4 alkylguanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyluracil, and N(l)-Cl-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y), and Nl-methyl-pseudouri dine (ml ).

[0352] DNA

[0353] In some embodiments of all aspects of the disclosure, the nucleic acid is DNA. Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" represents "deoxy"). In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxy-ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (ie., naturally occurring) nucleotide residues oranalogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0354] RNA

[0355] In some embodiments of all aspects of the disclosure, the nucleic acid is RNA. According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acids adenosine (A), uridine (U), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides (z.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (ie., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), singlestranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. Thenucleic acid may be mRNA, saRNA, taRNA, or mixtures thereof. The nucleic acid is preferably mRNA. In some instances, the nucleic acid is not siRNA.

[0356] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may capable of or configured to express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and capable of or configured for expressing a pharmaceutically active peptide or protein. In some embodiments, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface. Alternatively, the RNA can be non-coding RNA such as antisense-RNA, micro RNA (miRNA) or siRNA.

[0357] mRNA

[0358] In preferred embodiments of all aspects of the disclosure, the nucleic acid is mRNA.

[0359] According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region and a 3' untranslated region (3'-UTR).

[0360] mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0361] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0362] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

[0363] In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The term "in vitro transcription" or "IVT" as used herein means that the transcription (z.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g, cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription. The RNA (such as mRNA) may be modified. The RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly). One or more uridine in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. The RNA may comprise a modified nucleoside in place of at least one uridine. Preferably, the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside). The modified nucleoside may be independently selected from pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (y) or Nl-methyl-pseudouridine (mh| / ).

[0364] In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcriptionmay be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0365] In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA").

[0366] Inhibitory RNA

[0367] In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA. The term "inhibitory RNA" as used herein means RNA which selectively hybridizes to and / or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and / or translation thereof. Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.

[0368] The term "antisense RNA" as used herein refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and / or translation of said mRNA.

[0369] The size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0370] By "small interfering RNA" or "siRNA" as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs. Typically siRNAs comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded"hairpin" area. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.

[0371] As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.

[0372] According to the present disclosure, siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide", revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. Further guidance with respect to the selection of target sequences and / or the design of siRNA can be found on the webpages of Protocol Online (www.protocol-online.com) using the keyword "siRNA". Thus, in some embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.

[0373] siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Selection of other suitable promoters is within the skill in the art. Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.

[0374] The term "miRNA" (microRNA) as used herein relates to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradationand / or prevent translation of target mRNAs. miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing.

[0375] miRNA can be obtained using a number of techniques known to those of skill in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.

[0376] Method of forming the composition

[0377] In a further aspect, the present disclosure provides methods for producing the composition of the invention.

[0378] Preferably, the compositions of the inventions are lipid particle compositions (as defined herein), especially lipid nanoparticle (LNP) compositions.

[0379] A number of methods of making LNPs are known in the art and the skilled person would be readily capable of selecting a suitable method and applying this to make the LNP compositions of the present invention. For example, LNP preparations can be produced by rapid mixing of an aqueous solution optionally comprising a nucleic acid (typically an RNA) and an organic phase comprising a lipid mixture, under conditions such that a sudden change in solubility of lipids is triggered, which drives the lipids towards self-assembly in the form of LNPs. Described herein are exemplary methods of preparing the LNP compositions of the invention.

[0380] Thus, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0381] (i) a cationically ionizable lipid;

[0382] (ii) a steroid; and

[0383] (iii) a phospholipid;

[0384] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5consecutive monomer repeating units, (b) a negatively charged amphiphile and (c) an inorganic polyphosphate;

[0385] wherein the method comprises:

[0386] (a) providing an aqueous phase;

[0387] (b) providing an organic phase comprising the lipid mixture; and

[0388] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0389] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0390] (i) a cationically ionizable lipid;

[0391] (ii) a steroid;

[0392] (iii) a phospholipid; and

[0393] (iv) a nucleic acid;

[0394] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile and (c) an inorganic polyphosphate;

[0395] wherein the method comprises:

[0396] (a) providing an aqueous phase comprising a nucleic acid;

[0397] (b) providing an organic phase comprising the lipid mixture; and

[0398] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0399] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0400] (i) a cationically ionizable lipid;

[0401] (ii) a steroid;

[0402] (iii) a phospholipid; and

[0403] (iv) a lipid with a critical packing parameter < 0.5;wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units,

[0404] wherein the method comprises:

[0405] (a) providing an aqueous phase;

[0406] (b) providing an organic phase comprising the lipid mixture; and

[0407] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0408] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0409] (i) a cationically ionizable lipid;

[0410] (ii) a steroid;

[0411] (iii) a phospholipid;

[0412] (iv) a lipid with a critical packing parameter < 0.5;

[0413] (v) a nucleic acid;

[0414] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units,

[0415] wherein the method comprises:

[0416] (a) providing an aqueous phase comprising a nucleic acid;

[0417] (b) providing an organic phase comprising the lipid mixture; and

[0418] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0419] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises: a lipid mixture comprising:

[0420] (i) a cationically ionisable lipid;

[0421] (ii) a steroid;

[0422] (iii) a phospholipid; and

[0423] (iv) a lipid having a critical packing parameter of < 0.5;

[0424] wherein the method comprises:(a) an aqueous phase;

[0425] (b) providing an organic phase comprising the lipid mixture; and

[0426] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0427] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0428] (a) a nucleic acid;

[0429] (b) a lipid mixture comprising:

[0430] (i) a cationically ionisable lipid;

[0431] (ii) a steroid;

[0432] (iii) a phospholipid; and

[0433] (iv) a lipid having a critical packing parameter of < 0.5;

[0434] wherein the method comprises:

[0435] (a) providing a nucleic acid in an aqueous phase;

[0436] (b) providing an organic phase comprising the lipid mixture; and

[0437] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition.

[0438] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0439] (a) optionally a nucleic acid;

[0440] (b) a lipid mixture comprising:

[0441] (i) a cationically ionisable lipid;

[0442] (ii) a steroid;

[0443] (iii) a phospholipid; and

[0444] (iv) optionally a lipid having a critical packing parameter of < 0.5, wherein the lipid comprises a lysophospholipid;

[0445] wherein the method comprises:

[0446] (a) providing an aqueous phase optionally comprising a nucleic acid;

[0447] (b) providing an organic phase comprising the lipid mixture;

[0448] (c) mixing the aqueous phase provided under (a) with the organic phase providedunder (b), to form the composition; and

[0449] (d) immediately or shortly after step (c), adjusting the pH of the intermediate composition; to form the composition.

[0450] In one embodiment, provided herein is a method of preparing the composition of the invention, wherein the composition comprises:

[0451] (a) optionally a nucleic acid; and

[0452] (b) a lipid mixture comprising:

[0453] (i) a cationically ionisable lipid;

[0454] (ii) a steroid;

[0455] (iii) a phospholipid; and

[0456] (iv) a lipid having a critical packing parameter of < 0.5, wherein the lipid comprises a monomer- or oligomer-grafted lipid molecule and wherein the monomer- or oligomer grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units;

[0457] wherein the method comprises:

[0458] (a) providing an aqueous phase, optionally comprising a nucleic acid;

[0459] (b) providing an organic phase comprising the lipid mixture;

[0460] (c) mixing the aqueous phase provided under (a) with the organic phase provided under (b), to form the composition; and

[0461] (d) immediately or shortly after step (c), adjusting the pH of the intermediate composition; to form the composition.

[0462] In the above-defined embodiments, step (d) is typically carried out without delay after step (c). In some embodiments, step (d) is carried out less than 1 minute after step (c). In some embodiments, step (d) is carried out less than 30 seconds after step (c). In some embodiments, step (d) is carried out less than 20 seconds after step (c). In some embodiments, step (d) is carried out less than 10 seconds after step (c). In some embodiments, step (d) is carried out less than 5 seconds after step (c). In some embodiments, step (d) is carried out less than 2 seconds after step (c). In some embodiments, step (d) is carried out less than 1 second after step (c).

[0463] The LNPs optionally comprise or encapsulate the nucleic acid. The nucleic acid may be a DNA or RNA, preferably RNA (such as mRNA). Additional and preferred features of thecomposition comprising LNPs, the nucleic acid, the lipid mixture, and each of components (i), (ii), (iii) and optionally (iv), are further described herein and that disclosure applies equally to the methods of the invention.

[0464] Step (a) an aqueous phase optionally comprising the nucleic acid may comprise mixing the nucleic acid with an aqueous phase to prepare a solution of the nucleic acid in a first aqueous phase. The aqueous phase may comprise or consist essentially of water. The aqueous phase may comprise or consist essentially of water and a salt (e.g., sodium chloride). The aqueous phase may be acidified (for example, using the acidic buffers below). The aqueous phase may have a pH below 7.0, such as a pH between about 4.0 and about 6.5, preferably between about 4.0 and about 6.0; in one embodiment, between about 4.5 and about 5.5; in specific embodiments about 4.5, in other specific embodiments about 5.0 and in other specific embodiments about pH 5.5. The aqueous phase may comprise water and a buffer system (as defined and exemplified below). The aqueous phase may comprise water and no buffer or essentially no buffer. For example, the aqueous phase may comprise 10 mM or less of a buffer substance. The aqueous phase may have between about 5 mM and about 100 mM of a buffer substance, preferably between about 10 mM and about 50 mM, in specific embodiments about 10 mM, in other specific embodiments about 30 mM, and in other specific embodiments about 30 mM of a buffer substance.

[0465] The aqueous phase may comprise a buffer system (as defined and exemplified above), e.g., a buffer system which has a pH below 7.0, such as a pH between about 4.0 and about 6.0. In one embodiment, the buffering agent is a malate buffer. In one embodiment, the buffering agent is an acetate buffer. In one embodiment, the buffering agent is a phosphate buffer. In one embodiment, the buffering agent is a 50 mM acetate buffer. In one embodiment, the buffering agent is a 10 mM phosphate buffer.

[0466] The organic phase may comprise an organic solvent selected from a lower alcohol, such as alcohols (in particular aliphatic alcohols) having up to 6 carbon atoms, and mixtures of two or more of these alcohols. The organic solvent is preferably completely miscible with water. The organic phase may be an alcohol. The organic phase may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, 1,2-propanediol, butanol, isobutanol, tert-butanol, acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and mixtures thereof. Preferred organic phases are ethanol, propanol, isopropanol, acetone, or mixtures thereof.The organic phase may be acidified, e.g. by providing between 0.5 and 1.5 equivalent of acid relative to the cationically ionizable lipid. The acid is not particularly limited provided it is capable of acting as such. In one embodiment, the acid is an organic acid. In one embodiment, the acid is a weak organic acid. In one embodiment, the acid is selected from the group consisting of hydrochloric acid, acetic acid, succinic acid, glutaric acid, malonic acid, adipic acid, malic acid, malonic acid, tartaric acid, citric acid, lactic acid, and the like. The aqueous phase and / or the organic phase may be acidified. Preferably, at least one of the aqueous phase or the organic phase is acidified. The aqueous phase or the organic phase may comprise an acid. The acid is not particularly limited provided it is capable of acting as such. In one embodiment the acid is an inorganic acid, in particular a monobasic inorganic acid, e.g. hydrochloric acid, hydrobromic acid, or nitric acid. In one embodiment the acid is an organic acid, which may be a mono-, di- or polybasic organic acid. Examples of the organic acid include a monocarboxylic acid such as acetic acid, propionic acid, or lactic acid, a dicarboxylic acid such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, tartaric acid, or malic acid, and a polycarboxylic acid such as citric acid, isocitric acid, or trimesic acid. Preferably, the acid is citric acid or acetic acid. The aqueous phase may have a pH of below 7.0, optionally at most about 6.5, such as at most about 6.0, at most about 5.9, at most about 5.8, at most about 5.7. For example, the aqueous phase may have a pH of between about 4.5 and about 6.5, such as between about 5.0 and about 6.0. In one embodiment, the aqueous phase may have a pH of between about 5.2 and about 5.8. In one embodiment, the aqueous phase may have a pH of between about 3.7 and about 4.3.

[0467] In one embodiment, the composition is subjected to one or more further steps following the mixing of the organic and aqueous phases in step (c). In one embodiment the composition is subjected to pH adjustment. In one embodiment the composition is subjected to dialysis. In one embodiment the composition is subjected to dilution. In one embodiment the composition is subjected to filtration. In one embodiment the composition is subjected to Tangential Flow Filtration (TFF).

[0468] In one embodiment, a pH adjustment step is used to adjust the pH of the composition. The target pH for the pH adjustment step may be between about 6.5 and about 8.0, between about 7.0 and about 8.0, preferably between about 7.4 and about 7.7. Typically, this is carried out by treating the composition with the required amount of an aqueous alkali so as to reach thetarget pH. Examples of suitable alkalis are well known to those skilled in the art, and include alkali metal hydroxides (such as lithium, sodium or potassium hydroxides), aqueous ammonia, buffers comprising one or more amine moieties such as tris(hydroxymethyl)-aminomethane (Tris), triethanolamine, 4-(2-hy droxy ethyl)- 1 -piperazine-ethanesulfonic acid (HEPES), histidine, or phosphate buffer. Preferably, the aqueous alkali is a buffer comprising an amine moiety.

[0469] After mixing the aqueous phase optionally comprising the nucleic acid with the organic phase comprising the lipid mixture, the pH may be adjusted to the target pH to form the composition. Such a pH adjustment step may be performed by addition of an aqueous buffer system, such as those described and exemplified above, and which is preferably selected from tris(hydroxymethyl)aminomethane (Tris), triethanolamine, 4-(2 -hydroxy ethyl)- 1-piperazineethanesulfonic acid (HEPES), histidine, or phosphate buffer. Typically the aqueous buffer system has a pH of above 6.5, optionally above 7.0, preferably above 7.5 (e.g., a pH of between 6.5-8.5, preferably between 7.4 and 8.0). Suitable examples and preferred concentrations include 10-lOOOmM Tris pH 7.4 - 7.7; these are added to the mixture obtained in step (c).

[0470] The pH adjustment step may be performed rapidly so that, for example, the buffer addition and mixing takes less than 5 minutes, preferably less than 1 minute, more preferred less than 30 seconds, more preferred less than 20 seconds, optionally less than 10 seconds, such as less than 5 seconds, such as less than 2 seconds, such as less than 1 second. In a preferred embodiment the buffer is added in a continuous flow mode.

[0471] In one embodiment, the composition is subjected to dialysis. As is known to the person skilled in the art, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing. The dialysis method may be diffusion dialysis, tangential flow dialysis, electrodialysis, Donnan dialysis, reverse electrodialysis or electro-electrodialysis.

[0472] Typically, the dialysis is carried out using one or more buffer systems. Examples of buffer systems are known to those skilled in the art, and are defined and exemplified above. In one embodiment, the buffer system is selected from Tris, triethanolamine, HEPES, histidine, or phosphate buffer.

[0473] In one embodiment, the composition is subjected to a diluting step. A diluting step may comprise adding a dilution solution (e.g., water) to an intermediate composition. Suchdilution solution may comprise one or more additional compounds (e.g., a cryoprotectant) and optionally a buffer system (as defined and exemplified above). The diluting step may comprise adding the final aqueous phase to an intermediate composition. A diluting step may be carried out to change the pH and / or to change the buffer system and / or to add one or more additional compounds (e.g., a cryoprotectant).

[0474] In one embodiment, the composition is subjected to filtration. As is known to the person skilled in the art, filtration typically comprises the removal of solid particles by passing the composition through a filter or membrane, such that particles (especially those of a set particle size exceeding that of the membrane or filter) are removed. Typical filtration methods include sterile filtration and tangential flow filtration.

[0475] In one embodiment, the composition is subjected to freezing. In one embodiment, the composition is frozen and stored at -80°C.

[0476] In one embodiment, provided herein is a method of preparing a composition comprising lipid nanoparticles (LNPs) dispersed in a final aqueous phase, wherein the composition comprises:

[0477] (i) a cationically ionizable lipid;

[0478] (ii) a steroid;

[0479] (iii) a phospholipid;

[0480] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile and (c) an inorganic polyphosphate; and

[0481] (iv) optionally further comprising a lipid, wherein the lipid has a critical packing parameter of < 0.5;

[0482] wherein the method comprises:

[0483] (a) providing a first aqueous phase;

[0484] (b) providing an organic phase comprising the lipid mixture;

[0485] (c) mixing the first aqueous phase provided under (a) with the organic phase provided under (b), thereby preparing a first intermediate composition comprising a lipid colloid dispersed in a second aqueous phase;

[0486] (d) optionally diluting and / or adjusting the pH of the second aqueous phase; and optionally

[0487] (e) dialysing and / or diluting (preferably dialysing) the first intermediate compositionprepared under (c) or (d) using the first, second or a final aqueous phase, thereby preparing the composition comprising the LNPs dispersed in the final aqueous phase.

[0488] In one embodiment, provided herein is a method of preparing a composition comprising lipid nanoparticles (LNPs) dispersed in a final aqueous phase, wherein the composition comprises:

[0489] (i) a cationically ionizable lipid;

[0490] (ii) a steroid;

[0491] (iii) a phospholipid;

[0492] (iv) a nucleic acid; and

[0493] (v) a lysophospholipid;

[0494] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units;

[0495] wherein the method comprises:

[0496] (a) providing the nucleic acid in a first aqueous phase;

[0497] (b) providing an organic phase comprising the lipid mixture;

[0498] (c) mixing the first aqueous phase provided under (a) with the organic phase provided under (b), thereby preparing a first intermediate composition comprising a lipid colloid dispersed in a second aqueous phase;

[0499] (d) optionally diluting and / or adjusting the pH of the second aqueous phase; and optionally

[0500] (e) dialysing and / or diluting (preferably dialysing) the first intermediate composition prepared under (c) or (d) using the first, second or a final aqueous phase,

[0501] thereby preparing the composition comprising the LNPs dispersed in the final aqueous phase.

[0502] In one embodiment, provided herein is a method of preparing a composition comprising lipid nanoparticles (LNPs) dispersed in a final aqueous phase, wherein the composition comprises:

[0503] (i) a cationically ionizable lipid;

[0504] (ii) a steroid;

[0505] (iii) a phospholipid;

[0506] (iv) optionally a nucleic acid;wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, a monomer- or oligomer-grafted lipid molecule and wherein the monomer- or oligomer grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units;

[0507] wherein the method comprises:

[0508] (a) providing a first aqueous phase, optionally comprising a nucleic acid;

[0509] (b) providing an organic phase comprising the lipid mixture;

[0510] (c) mixing the first aqueous phase provided under (a) with the organic phase provided under (b), thereby preparing a first intermediate composition comprising a lipid colloid dispersed in a second aqueous phase;

[0511] (d) optionally diluting and / or adjusting the pH of the second aqueous phase; and optionally

[0512] (e) dialysing and / or diluting (preferably dialysing) the first intermediate composition prepared under (c) or (d) using the first, second or a final aqueous phase,

[0513] thereby preparing the composition comprising the LNPs dispersed in the final aqueous phase.

[0514] Step (a) of providing a first aqueous phase may comprise mixing an aqueous solution optionally containing the nucleic with a first aqueous buffer solution to prepare a first aqueous phase optionally comprising a nucleic acid. The first aqueous phase may comprise or consist essentially of water. The first aqueous phase may comprise or consist essentially of water and salt (e.g., sodium chloride). The first aqueous phase may be acidified. The first aqueous phase may have a pH below 6.0, such as a pH between about 3.5 and about 5.9, preferably between about 4.5 and about 5.0.

[0515] The first aqueous phase may comprise water and a buffer system. The first aqueous phase may comprise water and no buffer or essentially no buffer. The first aqueous phase may comprise a buffer system, e.g., a buffer system which has a pH below 6.0, such as a pH between about 3.5 and about 5.9, preferably between about 4.5 and about 5.5. Examples of buffer systems are known to those skilled in the art, and are defined and exemplified above. Preferably, the buffer system is a phosphate buffer or an acetate buffer.

[0516] The organic phase provided in step (b) is as defined and exemplified above. Preferred organic phases are ethanol, propanol, isopropanol, acetone, or mixtures thereof. The organic phasemay be acidified so as to protonate the cationically ionizable lipid. Typically, between 0.5 and 1.5 equivalents of acid (relative to the cationically ionisable lipid) is added.

[0517] The first aqueous phase may be acidified, preferably at least one of the first aqueous phase or the organic phase is acidified. The first aqueous phase may have a pH of below 6.0, optionally at most about 5.5, such as at most about 5.0, at most about 4.9, at most about 4.8, at most about 4.7, at most about 4.6, or preferably at most about 4.5. For example, the first aqueous phase may have a pH of between about 3.5 and about 5.9, preferably between about 4.0 and about 5.5. Typically, in step (c) the first aqueous phase provided under (a) with the organic phase provided under (b) are mixed in a ratio (a:b) from 6:1 to 1:1 (v / v), preferably 4:1 to 2:1 (v / v), more preferably 3:1 (v / v).

[0518] Typically, in step (d) the second aqueous phase is diluted with water in a ratio (second aqueous phase: water) of from 1 : 1 to 1 :5 (v / v), preferably 1 :2 (v / v). Without wishing to be bound by theory, it is believed that the water addition is thought to “harden” the very soft and fragile particles obtained in the step (a) which still contain up to 25% of the organic solvent used in that step.

[0519] The dilution or adjustment of pH in step (d) can be done with water in case of a dilution or can be done with buffers. Typically, the buffers are selected from Tris, triethanolamine, HEPES, histidine, or phosphate buffer. Typically, the aqueous buffer system has a pH of above 6.5, optionally above 7.0, preferably above 7.5 (e.g., a pH of between 6.5-8.5, preferably between 7.4 and 8.0). In one embodiment, the aqueous buffer system is a 50 mM Tris buffer at pH 7.7.

[0520] In one embodiment, in step (d) the pH is adjusted to neutral conditions. In view of the absence of stealth lipids from the composition, pH adjustment preferably needs to be rapid so as to avoid the particles fusing at an intermediate pH between that of the step (a) and neutral pH of about 7 to 8. In one embodiment, pH adjustment step (d) may be performed within 5 seconds, within 10 seconds, within 30 seconds, within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes of mixing step (c). Typically, the pH adjustment is carried out using a buffer: preferably, the buffers are selected from Tris, triethanolamine, HEPES, histidine, or phosphate buffer.

[0521] The dialysis or dilution step (e) may be performed essentially immediately after dilution and / pH adjustment in step (d), dialysis or dilution step (e) may be performed at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 2 minutes, at least 5 minutes,at least 10 minutes, at least 20 minutes, at least 30 minutes, at least an hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours or more than 72 hours, after dilution / pH adjustment step (d).

[0522] The concentration of the optional nucleic acid in the composition comprising the LNPs dispersed in the final aqueous phase may be about 1 mg / 1 to about 2000 mg / 1, such as about 100 mg / 1 to about 800 mg / 1. The concentration of the optional nucleic acid in the composition may be about 5 mg / 1 to about 500 mg / 1, such as about 10 mg / 1 to about 400 mg / 1, about 10 mg / 1 to about 300 mg / 1, about 10 mg / 1 to about 200 mg / 1, about 10 mg / 1 to about 150 mg / 1, or about 10 mg / 1 to about 100 mg / 1, preferably about 10 mg / 1 to about 140 mg / 1, more preferably about 20 mg / 1 to about 130 mg / 1, more preferably about 30 mg / 1 to about 120 mg / 1. In some embodiments, the concentration of the optional nucleic acid (in particular RNA) in the composition is 1 mg / 1 to about 50 mg / 1 or about 10 mg / 1 to about 100 mg / 1.

[0523] The nitrogen to phosphate (N / P) ratio in the compositions of the present invention, such as lipid nanoparticle (LNP) compositions, refers to the molar ratio of positively charged nitrogen groups (N) in the cationically ionizable lipids to negatively charged phosphate groups (P) present in the nucleic acid molecules during the formulation of the composition. In one embodiment, the N / P ratio is between about 3 to about 15. In one embodiment, the N / P ratio is about 4 to about 12. In one embodiment, the N / P ratio is between about 6 and about 12.

[0524] Where the optional nucleic acid in the composition is RNA or mRNA, the ratio of nitrogen in the cationically ionizable lipid to phosphate groups in the RNA or mRNA (N / P ratio) may be between about 3 to about 15, such as about 4 to about 12, preferably between about 6 and about 12.

[0525] The methods may further comprise a step of freezing the composition comprising the LNPs dispersed in the final aqueous phase. The composition may be frozen to a temperature of -10°C or below (e.g., to -15°C or below, preferably to -20°C or below, in preferred embodiments to about -80°C). Thus, provided herein are also compositions of the invention in frozen form. The methods may further comprise a step of lyophilizing the composition comprising the LNPs dispersed in the final aqueous phase. Thus, provided herein are also compositions of the invention in lyophilized form.

[0526] In some embodiments, the lipid nanoparticles described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm. The LNPs described herein may have an average diameter of from about 40 nm to about 300 nm, preferably from about 40 nm to about 250 nm; most preferably from about 50 nm to about 200 nm and even more preferred from about 60nm to 120nm.

[0527] Cryoprotectants

[0528] In some embodiments, the composition of the present invention comprises a cryoprotectant. In this specification, the term “cryoprotectant” when used in its broadest sense means any substance capable of protecting a composition from damage caused by freezing temperatures. Examples of cryoprotectants include glycols (i.e. alcohols containing at least two hydroxy groups, such as glycerol and propylene glycol) and carbohydrates, as defined and exemplified herein.

[0529] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, lactose and glucose, or a mixture of any thereof. In one embodiment, the cryoprotectant is selected from the group consistingof sucrose, trehalose and glucose, or a mixture of any thereof. Preferably, the cryoprotectant is sucrose.

[0530] In one embodiment, the cryoprotectant is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 3% to about 25% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 8% to about 22% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the cryoprotectant is present in a concentration of 8% to about 12% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 10% (w / v). In one embodiment, the cryoprotectant present in a concentration of about 15% to about 25% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 18% to about 22% (w / v). In one embodiment, the cryoprotectant is present in a concentration of about 20% (w / v).

[0531] In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 3% to about 25% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 8% to about 22% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of 8% to about 12% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 10% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 15% to about 25% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 18% to about 22% (w / v). In one embodiment, the cryoprotectant is sucrose and is present in a concentration of about 20% (w / v).

[0532] Pharmaceutical compositions

[0533] The lipid nanoparticle compositions described herein are useful as or for preparing pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

[0534] The lipid nanoparticle compositions described herein may be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceuticalcomposition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. In some embodiments, the therapeutically effective agent is or comprises the nucleic acid, as described herein. In the context of the present disclosure, the pharmaceutical composition preferably comprises RNA (such as mRNA) as described herein. In some embodiments, the therapeutically effective agent is or comprises an RNA (preferably mRNA), as described in the present disclosure, which comprises a nucleic acid sequence (e.g., an ORF) encoding one or more polypeptides, e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein.

[0535] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions e.g., Freund’s adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF a, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0536] The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (i.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses.In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

[0537] In some embodiments, the pharmaceutical composition is in frozen form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature ranging from about -90°C to about -10°C, such as from about -80°C to about -40°C or from about -40°C to about -25°C, or from about -25°C to about -10°C, or a temperature of about -20°C.

[0538] In some embodiments of the pharmaceutical compositions in frozen form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at -80°C.

[0539] In some embodiments of the pharmaceutical compositions in frozen form, the nucleic acid (such as RNA) integrity after thawing the frozen pharmaceutical composition is at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100%, e.g., after thawing the frozen composition which has been stored (for at least 18 hours, such as for at least 24 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks) at -80°C.

[0540] In some embodiments of the pharmaceutical compositions in frozen form, the encapsulation efficiency (EE%), size (Zaverage) and / or size distribution and / or PDI of the particles after thawing the frozen pharmaceutical composition is essentially equal to the size (Zaverage) and / or size distribution and / or PDI of the particles before freezing. For example, if a ready-to-use pharmaceutical composition is prepared from a frozen pharmaceutical composition as described herein, it is preferred that the encapsulation efficiency (EE%), size (Zaverage) and / or size distribution and / or PDI of the particles contained in the ready-to-use pharmaceutical composition is essentially equal to the encapsulation efficiency (EE%), size (Zaverage) and / orsize distribution and / or PDI of the particles contained in the frozen pharmaceutical composition before freezing (such as contained in the formulation prepared in step (I) of the method of the second aspect).

[0541] In some embodiments of the pharmaceutical compositions in frozen form, the size (Zaverage) difference between the particles before one freeze-thaw cycle and after one freeze-thaw cycle (delta-FTl) is determined. For example, the size (Zaverage) difference (delta-FTl) is preferably between 0 and 50 nm, more preferably between 0 and 30 nm, most preferably substantially 0 nm.

[0542] In some embodiments of the pharmaceutical composition in liquid form, the encapsulation efficiency (EE%), size (Zaverage) and / or size distribution and / or PDI of the particles of the pharmaceutical composition, when stored, e.g., at -80°C or higher for at least 18 hours, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the encapsulation efficiency (EE%), size (Zaverage) (and / or size distribution and / or poly dispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at -80°C or higher for at least 18 hours, is essentially equal to the encapsulation efficiency (EE%), size (Zaverage) (and / or size distribution and / or PDI) of the particles of the initial pharmaceutical composition, z.e., before storage. In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at -80°C or higher for at least 18 hours is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm. In some embodiments, the PDI of the particles after storage of the pharmaceutical composition, e.g., at -80°C or higher for at least 18 hours is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at -80°C or higher for at least 18 hours is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least 18 hours is less than 0.3 (preferably less than 0.2, more preferably less than 0.1). In some embodiments, the encapsulation efficiency (EE%) of the particles after storage of the pharmaceutical composition, e.g., at -80°C or higher for at least 18 hours is between about 80 and 100%, preferably between about 90 and 100%, more preferably between 95 and 100%.The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0543] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0544] The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the particles or pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0545] In particular embodiments, a pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, z.e., a pharmaceutical composition which can be used for inducing an immune response) is formulated as a single-dose in a container, e.g., a vial. In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in the vial is equal in volume. In some embodiments, a first dose is a different volume than a subsequent dose.

[0546] A "stable" multi-dose formulation preferably exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biologicalmolecule component^ s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunologic response when administered to a subject.

[0547] The pharmaceutical compositions of the present disclosure may contain buffers (in particular, derived from the nucleic acid (such as RNA) compositions with which the pharmaceutical compositions have been prepared), preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure, in particular the ready-to-use pharmaceutical compositions, comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0548] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0549] The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient.

[0550] Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants

[0551] "Pharmaceutically acceptable salts" comprise, for example, acid addition salts which may, for example, be formed by using a pharmaceutically acceptable acid such as hydrochloric acid, acetic acid, lactic acid, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) or benzoic acid. Furthermore, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH4+); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations). Illustrative examples of pharmaceutically acceptable salts can be found in the prior art; see, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)). Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the present disclosure.The term "diluent" relates a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol and water.

[0552] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxy ethylene / polyoxy -propylene copolymers.

[0553] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0554] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0555] In one embodiment, the compositions described herein, such as the pharmaceutical compositions or ready-to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly or intratum orally. In certain embodiments, the (pharmaceutical) composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for intramuscular administration.Medical Uses and Methods of Treatment

[0556] The lipid nanoparticle compositions described herein may be used in the therapeutic or prophylactic treatment of disease.

[0557] The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviours, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories.

[0558] In the present context, the term "treatment" or "treating" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

[0559] Further aspects

[0560] Additional aspects of the present invention are described by way of the following numbered clauses:

[0561] 1. A composition comprising:

[0562] (i) a cationically ionizable lipid;

[0563] (ii) a steroid; and

[0564] (iii) a phospholipid;wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate. 2. The composition of clause 1, further comprising an additional lipid, wherein the additional lipid has a critical packing parameter of < 0.5 as calculated by the method of example 11. 3. The composition of clause 2, wherein the additional lipid comprises a monomer- or oligomer-grafted lipid and wherein the monomer- or oligomer-grafted lipid comprises between 1 and 5 consecutive monomer repeating units.

[0565] 4. The composition of clause 2, wherein the additional lipid comprises a lysophospholipid.

[0566] 5. The composition of clause 4, wherein the lysophospholipid is selected from the group consisting of a lysophosphatidylcholine and a lysophosphatidylethanolamine.

[0567] 6. The composition according to clause 4 or 5, wherein the lysophospholipid is present in the composition in an amount between about 1 mol% and about 8 mol% of the total lipid present in the composition, preferably about 3 mol% of the total lipid present in the composition. 7. The composition according to any of clauses 4 to 6, wherein the lysophospholipid is saturated.

[0568] 8. The composition according to any of clauses 4 to 7, wherein the lysophospholipid is a saturated lysophosphatidylcholine.

[0569] 9. The composition according to any of clauses 4 to 8, wherein the lysophospholipid fatty acid chain comprises between 12 and 22 carbon atoms, preferably between 14 and 20 carbon atoms.

[0570] 10. The composition according to any of clauses 4 to 9, wherein the lysophospholipid is selected from the group consisting of l-heptadecanoyl-.s / / -glycero-3 -phosphocholine, 1-octadecanoyl -s / / -glycero-3 phosphocholine, 1 -tetradecanoyl -s / / -glycero-3 -phosphocholine and I -eicosanoyl-.s / / -glycero-3 -phosphocholine.

[0571] 11. The composition of clause 4, wherein the monomer or oligomer-grafted lipid molecule comprises a monomer repeating unit selected from the group consisting of mono-or oligoethylene glycol, mono- or oligosarcosine, mono- or oligooxazoline, mono- or oligooxazine, mono- or oligo(vinyl pyrrolidone), mono- or oligo(N-(2-hydroxypropyl)-methacrylamide),mono- or oligo(dehydroalanine), mono- or oligo(aminoethoxy ethoxy acetic acid), and mono-or oligo(2 -methylaminoethoxy ethoxy acetic acid) and derivatives and combinations thereof.

[0572] 12. The composition of clause 11, wherein the monomer repeating unit is mono- or oligosarcosine (Sar).

[0573] 13. The composition of clause 12, wherein the monomer or polymer-grafted lipid molecule comprises a compound of formula 1

[0574] R’-NH-(Sar)n-R

[0575] wherein:

[0576] R’ is a hydrocarbyl group;

[0577] Sar is a sarcosine residue, the C-terminal group thereof being bonded to the NH group and the N-(CHs)- terminal group thereof being bonded to the capping group R;

[0578] n is a number between 1 and 5; and

[0579] R comprises a capping group.

[0580] 14. The composition of clause 14 wherein the group R’ is an alkyl or alkenyl group having from 12 to 22 carbon atoms.

[0581] 15. The composition of clause 13 or 14 wherein the capping group is selected from the group consisting of acetyl, methoxy carbamate, methyl sulfonamide, isopropyl, tert-butyl, propyl, butyl, cyclopropyl, ethoxy carbamate, ethyl sulfonamide, methyl and ethyl urea, allyl (2-aminoethyl) carbamate, vinyl carbamate and sulfamide.

[0582] 16. The composition of clause 15 wherein the capping group is acetyl.

[0583] 17. The composition of any of clauses 4 or 12 to 14, wherein the monomer or oligomer-grafted lipid is selected from the group consisting of N-methyl-N-(2-oxo-2-(tetradecylamino)ethyl)acetamide (C14-Sar(l)-Ac), N-methyl-N-(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2-oxoethyl)-2-(N-methylacetamido)acetamide (C14-Sar(3)-Ac) and N-methyl-N-(2-(methyl(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)-2-(N-methyl-2-(N-methylacetamido)acetamido)acetamide (C14-Sar(5)-Ac).

[0584] 18. The composition of clause 4 or 12 to 14 or 18, wherein the molar contribution of monomer or oligomer-grafted lipid is between about 1% and about 10%, preferably between about 3% and about 4%.19. The composition of any preceding clause, wherein the molar ratio between the cationically ionizable lipid and the steroid is between about 1 and about 2.5.

[0585] 20. The composition of clause 19, wherein the molar ratio between the cationically ionizable lipid and the steroid is or between about 1.5 to about 2.25.

[0586] 21. The composition of clause 20, wherein the molar ratio between the cationically ionizable lipid and the steroid is or preferably between about 1.75 and about 2.0.

[0587] 22. The composition of any preceding clause, wherein the cationically ionizable lipid is present in the composition in an amount of 40 to 60 mol% of the total lipid present in the composition.

[0588] 23. The composition of clause 22, wherein the cationically ionizable lipid is present in the composition in an amount of 45 to 55 mol% of the total lipid present in the composition. 24. The composition of any preceding clause, wherein the steroid is present in the composition in an amount of 20 to 40 mol%, preferably 24 to 36 mol% of the total lipid present in the composition.

[0589] 25. The composition of any preceding clause, wherein the phospholipid is present in the composition in an amount of 10 to 25 mol%, preferably 15 to 22 mol%, of the total lipid present in the composition.

[0590] 26. The composition of any preceding clause, wherein the phospholipid is present in the composition in an amount of about 20 mol% of the total lipid present in the composition. 27. The composition of any preceding clause, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) or dimyristoylphosphatidylcholine (DMPC) or a mixture thereof.

[0591] 28. The composition of any preceding clause, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

[0592] 29. The composition of clause 27, wherein the distearoylphosphatidylcholine (DSPC) is present in a molar ratio of at least 10% of the total lipids present in the composition, and the composition, further comprises an additional lipid, in a molar ratio of at least 10% of the total lipids present in the composition, wherein the additional lipid is selected from the groupconsisting of 1-oleoyl-rac -glycerol (MOG), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) or dimyristoylphosphatidylcholine (DMPC). 30. The composition according to any preceding clause, wherein the steroid is cholesterol.

[0593] 31. The composition of any preceding clause, further comprising a nucleic acid.

[0594] 32. The composition according to clause 31, wherein the nucleic acid is RNA.

[0595] 33. The composition according to clause 31, wherein the nucleic acid is DNA.

[0596] 34. The composition according to clause 32, wherein the nucleic acid is mRNA.

[0597] 35. The composition according to any preceding clause, wherein the cationically ionizable lipid is selected from the group consisting of:

[0598] [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)) (ALC-315);

[0599] 2.2-dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 1.2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA);

[0600] N,N-dimethyl-2, 3 -di oleyloxypropylamine (DODMA);

[0601] di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); bis-(2 -butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)-nonadecanedioate (A9); (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);

[0602] O-[N-{(9Z,12Z)-octadeca-9,12-dien-l-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4- (dimethylamino)butanoate (H Y 501);

[0603] di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD- C2C2-PipZ);

[0604] bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMe-Pyr);

[0605] bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-l-yl)ethyl)azanediyl)dipropionate (BODD-C2C2- Pyr);

[0606] bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMePyr);

[0607] bis(2-octyldodecyl) 3,3'-(((l-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-lMe-3PipD);bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA);

[0608] bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ);

[0609] bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr);

[0610] bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ);

[0611] di(nonadecan-9-yl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (DND-C2-C4-PipZ);

[0612] or a mixture of any thereof.

[0613] 36. The composition according to any preceding clause, wherein the cationically ionizable lipid is selected from the group consisting of:

[0614] di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ),

[0615] (4-Hydroxybutyl) azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315) and 6-((9-((2-butyloctanoyl)oxy)nonyl)(3-hydroxypropyl)amino)hexyl 2-propyloctanoate (ALC-0366).

[0616] 37. The composition of any preceding clause, wherein the N / P ratio is between about 3 and about 15, preferably between about 6 and about 12, more preferably about 10.

[0617] 38. The composition of any preceding clause, further comprising a cryoprotectant.

[0618] 39. The composition of clause 38, wherein the amount of cryoprotectant is between about 5% (w / v) and about 30% (w / v), preferably between about 10% and about 20% (w / v).

[0619] 40. The composition of clauses 38 or 39, wherein the cryoprotectant is selected from the group consisting of trehalose, sucrose and glucose, preferably wherein the cryoprotectant is sucrose.

[0620] 41. A method of preparing the composition according to any of clauses 1 to 40, the method comprising:

[0621] (a) preparing a lipid mixture comprising a cationically ionizable lipid, a steroid and a phospholipid;(b) preparing an aqueous solution comprising an acidic buffer;

[0622] (c) mixing the lipid mixture prepared in (a) with the aqueous solution prepared in (b); and (d) adjusting the pH of the mixture obtained in step (c) to a pH of between 6.5 and 8, to form the composition.

[0623] 42. The method according to clause 41, wherein the aqueous solution of step (b) further comprises a nucleic acid.

[0624] 43. The method according to clause 41 or 42, wherein the lipid mixture of step (a) further comprises a lipid having a critical packing parameter of < 0.5.

[0625] 44. The method according to any of clauses 41 to 43, wherein the acidic buffer is selected from the group consisting of acetate, phthalate, succinate, formate, malate, oxalate, citrate, phosphate, preferably selected from the group consisting of acetate and phosphate.

[0626] 45. The method according to any of clauses 41 to 44, wherein the concentration of acidic buffer is between about 10 and about 50 mM.

[0627] 46. The method according to any of clauses 44 to 45, wherein the acidic buffer is phosphate.

[0628] 47. The method according to clause 46, wherein the concentration of phosphate is 10 mM.

[0629] 48. The method according to any of clauses 41 to 45, wherein the acidic buffer is acetate. 49. The method according to clause 48, wherein the concentration of acetate is 50 mM. 50. A pharmaceutical composition, comprising the composition of any of clauses 1 to 40 together with a pharmaceutically acceptable carrier.

[0630] 51. The composition of any one of clauses 1 to 39, or the pharmaceutical composition of clause 50, for use in medicine.

[0631] 52. The composition of any one of clauses 1 to 39, or the pharmaceutical composition of clause 50 for use in treating gastrointestinal disease.

[0632] Preferred embodiments

[0633] In one embodiment the composition comprises,

[0634] (i) a cationically ionizable lipid;

[0635] (ii) cholesterol;

[0636] (iii) distearoylphosphatidylcholine; and(iv) optionally an mRNA;

[0637] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate; and wherein the molar ratio between the cationically ionizable lipid and the cholesterol is between about 1.75 and about 2.0.

[0638] In one embodiment the composition comprises,

[0639] (i) a cationically ionizable lipid;

[0640] (ii) cholesterol;

[0641] (iii) distearoylphosphatidylcholine; and

[0642] (iv) optionally an mRNA;

[0643] wherein the composition is substantially free of (a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units, (b) a negatively charged amphiphile having a hydrophilic portion and a lipophilic portion and (c) an inorganic polyphosphate; and wherein the molar ratio between the cationically ionizable lipid and the cholesterol is between about 1.75 and about 2.0 and / or wherein the distearoylphosphatidylcholine is present in the composition in an amount of about 20 mol% of the total lipid present in the composition.

[0644] In one embodiment the composition comprises,

[0645] (i) a cationically ionizable lipid;

[0646] (ii) cholesterol;

[0647] (iii) distearoylphosphatidylcholine;

[0648] (iv) optionally an mRNA; and

[0649] (v) a lysophospholipid;

[0650] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0651] In one embodiment the composition comprises,(i) a cationically ionizable lipid;

[0652] (ii) cholesterol;

[0653] (iii) distearoylphosphatidylcholine;

[0654] (iv) optionally an mRNA; and

[0655] (v) a lysophospholipid selected from l-heptadecanoyl-sw-glycero-3- phosphocholine (17:0 lyso-PC), l-octadecanoyl-sw-glycero-3 phosphocholine 18:0 lyso-PC), l-oleoyl- w-glycero-3 phosphocholine (18:1 lyso-PC), 1- tetradecanoyl-sw-glycero-3-phosphocholine (14:0 lyso-PC), and 1-eicosanoyl- w-glycero-3 -phosphocholine (20:0 lyso-PC);

[0656] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0657] In one embodiment the composition comprises,

[0658] (i) a cationically ionizable lipid selected from ALC-0366 and BHD-C2C2-PipZ; (ii) cholesterol;

[0659] (iii) distearoylphosphatidylcholine;

[0660] (iv) optionally an mRNA; and

[0661] (v) a lysophospholipid selected from l-heptadecanoyl-sw-glycero-3- phosphocholine (17:0 lyso-PC), l-octadecanoyl-sw-glycero-3 phosphocholine 18:0 lyso-PC), l-oleoyl- w-glycero-3 phosphocholine (18:1 lyso-PC), 1- tetradecanoyl-sw-glycero-3-phosphocholine (14:0 lyso-PC), and 1-eicosanoyl- w-glycero-3 -phosphocholine (20:0 lyso-PC);

[0662] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0663] Preferably the molar ratio between the cationically ionizable lipid and the cholesterol is between about 1.75 and about 2.0.

[0664] Preferably, the lysophospholipid is present in an amount of about 1 to about 8 mol%, or about 2 to about 8 mol%, or about 3 mol% of the total lipids present in the composition.

[0665] In one embodiment the composition comprises,(i) a cationically ionizable lipid;

[0666] (ii) cholesterol;

[0667] (iii) distearoylphosphatidylcholine;

[0668] (iv) optionally an mRNA; and

[0669] (v) a saturated lysophospholipid;

[0670] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0671] In one embodiment the composition comprises,

[0672] (i) a cationically ionizable lipid;

[0673] (ii) cholesterol;

[0674] (iii) a distearoylphosphatidylcholine;

[0675] (iv) optionally an mRNA; and

[0676] (v) a monomer- or polymer-grafted lipid molecule and wherein the monomer- or polymer grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units; and

[0677] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0678] In one embodiment the composition comprises,

[0679] (i) a cationically ionizable lipid;

[0680] (ii) cholesterol;

[0681] (iii) a distearoylphosphatidylcholine;

[0682] (iv) optionally an mRNA; and

[0683] (v) a monomer- or oligomer-grafted lipid molecule and wherein the monomer- or polymer grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units; and

[0684] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.In one embodiment the composition comprises:

[0685] (i) a cationically ionizable lipid;

[0686] (ii) cholesterol;

[0687] (iii) a distearoylphosphatidylcholine;

[0688] (iv) optionally an mRNA; and

[0689] (v) a monomer- or oligomer-grafted lipid molecule of formula 1

[0690] R’-NH-(Sar)n-R

[0691] wherein

[0692] R’ is a hydrocarbyl group;

[0693] Sar is a sarcosine residue, the C-terminal group of being bonded to the NH group and the N-(CHs)- terminal group being bonded to the capping group R;

[0694] N is a number between 1 and 5; and

[0695] R comprises a capping group as defined herein, preferably acetyl;

[0696] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0697] In one embodiment the composition comprises,

[0698] (i) a cationically ionizable lipid;

[0699] (ii) cholesterol;

[0700] (iii) a distearoylphosphatidylcholine;

[0701] (iv) optionally an mRNA; and

[0702] (v) a monomer- or oligomer-grafted lipid molecule selected from the following:

[0703] N-methyl-N-(2-oxo-2-(tetradecylamino)ethyl)acetamide (C14-Sar(l)Ac); N-methyl-N-(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2-oxoethyl)- 2-(N -methyl acetami do)-acetami de (C14-S ar(3 ) Ac) ; ;

[0704] N-methyl-N-(2-(methyl(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)-2-(N-methyl-2-(N-methylacetamido)- acetamido)-acetamide (C14-Sar(5)Ac); andwherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0705] In one embodiment the composition comprises,

[0706] (i) a cationically ionizable lipid selected from BHD-C2C2-PipZ and ALC-366; (ii) cholesterol;

[0707] (iii) a distearoylphosphatidylcholine;

[0708] (iv) optionally an mRNA; and

[0709] (v) a monomer- or oligomer-grafted lipid molecule selected from the following:

[0710] N-methyl-N-(2-oxo-2-(tetradecylamino)ethyl)acetamide (C14-Sar(l)Ac); N-methyl-N-(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2-oxoethyl)- 2-(N -methyl acetami do)-acetami de (C14-S ar(3 ) Ac) ;

[0711] N-methyl-N-(2-(methyl(2-(methyl(2-oxo-2-(tetradecylamino)ethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)-2-(N-methyl-2-(N-methylacetamido)- acetamido)-acetamide (C14-Sar(5)Ac); and

[0712] wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

[0713] In one embodiment, the monomer- or oligomer grafted lipid as defined above is present in the lipid mixture in an amount of about 0.5 to about 10 mol%, or about 1 to about 10 mol%, or about 3 to about 4 mol% of the total lipids present in the lipid mixture.

[0714] In some preferred embodiments, the phospholipid is DSPC and an additional neutral lipid selected from the group consisting of 1-oleoyl-rac-glycerol (MOG), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) and dimyristoylphosphatidylcholine (DMPC), is also present.

[0715] In some preferred embodiments, the cationically ionizable lipid is ALC-366, the phospholipid is DSPC and an additional neutral lipid selected from the group consisting of 1-oleoyl-rac-glycerol (MOG), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) and dimyristoylphosphatidylcholine (DMPC), is also present.In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 2 mol % to about 15 mol % of the total lipids present in the lipid mixture, or about 3 mol % to about 10 mol %, or about 4 mol % to about 8 mol %, or about 5 mol % to about 7 mol of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of up to about 5 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 5 mol % of the total lipids present in the lipid mixture.

[0716] In one embodiment the composition comprises ALC-0366, cholesterol and DSPC, optionally in a ratio of about 50.9:29.1 :20.

[0717] In one embodiment the composition comprises ALC-0366, cholesterol, DSPC and C14-Sarl-Ac, optionally in a ratio of about 49.4:27.6:20:3.

[0718] In one embodiment the composition comprises ALC-0366, cholesterol, DSPC and C14-Sar3-Ac, optionally in a ratio of about 49.4:27.6:20:3.

[0719] In one embodiment the composition comprises ALC-0366, cholesterol, DSPC and C14-Sar5-Ac, optionally in a ratio of about 49.4:27.6:20:3.

[0720] In one embodiment, the composition comprises:

[0721] a cationically ionizable lipid selected from ALC-0366, BHD-C2C2-PipZ, BODD-DMA and BODD-MePyr;

[0722] cholesterol; and

[0723] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0724] optionally in a ratio of about 50.9:29.1:20, or about 44.55:25.45:30, or about 38.2:21.8:40.

[0725] In one embodiment, the composition comprises:

[0726] a cationically ionizable lipid selected from ALC-0366, BHD-C2C2-PipZ, BODD-DMA and BODD-MePyr;

[0727] cholesterol;

[0728] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and

[0729] an additional component selected from Sari and lysoPC;

[0730] optionally in ratio of about 49:28:20:3 or about 42.64:24.36:30:3.In one embodiment, the composition comprises:

[0731] a cationically ionizable lipid selected from ALC-0366, BHD-C2C2-PipZ, BODD-DMA and BODD-MePyr;

[0732] cholesterol;

[0733] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and

[0734] an additional component selected from C14-Sar(l)-Ac andl8:0 LysoPC;

[0735] optionally in ratio of about 49:28:20:3 or about 42.64:24.36:30:3.

[0736] In one embodiment, the composition comprises BHD-C2C2-PipZ and cholesterol at a ratio of about 1.75, about 20 mol% DSPC and about 3 mol% of 18:0 LysoPC. In one embodiment, the composition comprises about 49 mol % BHD-C2C2-PipZ, about 28 mol % cholesterol, about 20 mol% DSPC and about 3 mol% of 18:0 LysoPC.

[0737] In one embodiment, the composition comprises BHD-C2C2-PipZ, cholesterol, DSPC and LysoPC 18:0, optionally in a ratio of about 49.4:27.6:20:3.

[0738] In one embodiment, the composition comprises BHD-C2C2-PipZ, cholesterol and DSPC, optionally in a ratio of about 50.9:29.1:20.

[0739] In one embodiment, the composition comprises BHD-C2C2-PipZ, cholesterol and DSPC optionally in a ratio of about 44.5:25.4:30.

[0740] In one embodiment, the composition comprises BHD-C2C2-PipZ, cholesterol, DSPC and lysoPC, optionally in a ratio of about 49.4:28:20:3.

[0741] In one embodiment, the composition comprises ALC-0366, cholesterol, DSPC and lysoPC, optionally in a ratio of about 49.4:28:20:3.

[0742] In one embodiment, the composition comprises BODD-MePyr, cholesterol, DSPC and lysoPC, optionally in a ratio of about 49.4:28:20:3.

[0743] In one embodiment, the composition comprises BODD-DMA, cholesterol, DSPC and lysoPC, optionally in a ratio of about 49.4:28:20:3.

[0744] In one embodiment, the composition comprises BHD-C2C2-PipZ, cholesterol and DSPC, optionally in a ratio of about 50.9:29.1:20.In one embodiment, the composition comprises:

[0745] a cationically ionizable lipid selected from ALC 366, ALC 0315 and BHD-C2C2-PipZ; cholesterol; and

[0746] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0747] optionally in a molar ratio of about 51.1:34.8:14.1.

[0748] In one embodiment, the composition comprises:

[0749] ALC 366;

[0750] cholesterol; and

[0751] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0752] optionally in a composition of about 53.3 %: 26.7 %: 20 %.

[0753] In one embodiment, the composition comprises:

[0754] ALC 366;

[0755] cholesterol; and

[0756] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0757] optionally in ratio of about 50.0:29.1:20 or about 53.3:26.7:20.

[0758] In one embodiment, the composition comprises:

[0759] ALC 366;

[0760] cholesterol;

[0761] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and

[0762] a further component selected from a monomer or oligomer-grafted lipid, lysoPC and a neutral lipid;

[0763] optionally in ratio of about 53.3-0.5x:26.7-0.5x:20:x.

[0764] In one embodiment, the composition comprises:

[0765] BHD-C2C2-PipZ;

[0766] cholesterol;

[0767] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and

[0768] LysoPC (18:0);optionally in ratio of about 49:28:20:3.

[0769] Examples

[0770] Materials and Methods

[0771] Analysis of Lipid Nanoparticles size and polydispersity

[0772] Mean particle size and size distribution of LNPs in a sample of drug product was measured by dynamic light scattering (DLS). The method employs a particle sizer that uses back-scatter at 173° to determine particle size. The results are reported as the Z-average size of the particles and the poly dispersity index. The poly dispersity values are used to describe the width of fitted log-normal distribution around the measured Z-average size and are generated using proprietary mathematical calculations within the particle sizing software. Results for size and polydispersity are reported as nm and poly dispersity index (PDI) value, respectively. pH determination

[0773] Sample pH values were measured using a Mettler-Toledo pH meter (SevenCompact S220). The pH standards pH 4, 7 and 10 were used to calibrate the pH meter within the expected pH range prior measurement. After calibration slope range 95 - 105% is achieved, pH 7 standard was measured again (7 ± 0.5). For sample measurement, the electrode of the pH meter was dipped into a 150 pL sample volume in a 500 pL Eppendorf tube to allow the membrane of the electrode to be in contact with the suspension. pH value was reported after two separate samples were measured.

[0774] Zeta potential

[0775] Zeta potential was determined by measuring the electrophoresis mobility using a Zetasizer Ultra (Malvern Panalytical, Kassel, Germany) The sample was diluted to 2 pg / mL final LNP in TRIS pH 7.4 without sucrose or O.lxPBS respective native buffer of the formulation. Zeta measurements were performed in triplicates at 150 V using the automatic acquirement mode with a maximum of 100 accumulated measurements per replicate.

[0776] Osmolality

[0777] Osmolality was determined using osmometer Osmomat 3000 or 030-D (Gonotec, USA). Calibration was performed with reference standards covering the expected range in 500 pL Gonotec tubes (used standards are as follows: 0, 100, and 500 mOsmol / kg), and the calibration is checked with a 290mOsmol / kg reference solution. After result within the rangeof 290 ±15 mOsmol / kg is achieved, a sample of 50 pL LNPs in a new 500 pL Gonotec tube was used to determine the osmolality value. Duplicates are made and the mOsmol / kg value is noted, or a printout is taken.

[0778] Analysis of mRNA encapsulation efficiency

[0779] The RNA content was determined by disrupting the LNPs with detergent Triton™ X-100 and subsequently measuring the total RNA content based on the signal of the RNA-binding fluorescent dye RiboGreen® using a spectrofluorophotometer. RNA encapsulation was calculated by comparing the RiboGreen® signals of LNP samples in the absence (free RNA) and presence (total RNA) of Triton™ X-100. Results for RNA content and encapsulation were reported as mg / ml and percentage, respectively.

[0780] RNA integrity

[0781] RNA integrity was determined by capillary electrophoresis. RNA-LNPs treated with Triton / Ethanol / Tween20 were applied to a gel matrix contained in a capillary. The RNA and its derivatives, degradants and impurities were separated according to their sizes. The gel matrix contains a fluorescence dye which binds specifically to the RNA components which allows detection by a laser-induced fluorescence (LIF) detector. The excitation wavelength was set to 495 nm, the emission wavelength was 537 nm. The data were evaluated using the software ProSize. The integrity of the RNA was verified by comparing with the reference standard.

[0782] Bioluminescence imaging (BLI)

[0783] At study start 16, 13 or 22 BALB / c_RJ mice (female, 9 weeks) were intravenously injected with either 100 pl 0.9% NaCl solution or test items (LNPs) corresponding to 1 pg RNA encoding for the reporter firefly Luciferase (Luc) and 9 pg mRNA encoding an antibody or green fluorescent protein (eGFP,) or Thy 1.1 (or cluster of differentiation 90, CD90.1) into the tail vein. Uptake and translation of Luc-RNA were evaluated by in vivo bioluminescence imaging using the Revvity IVIS™ imaging system at 6 and 24 h post injection. An aqueous solution of L-Luciferin (250 pL, 1.6 mg) was injected intraperitoneally. Emitted photons from live animals or extracted tissues (24 h after i.v. injection) were quantified 5 min later with an exposure time of 1 sec (in vivo) or 10 sec (ex vivo) or 2 sec (in vivo) or 60 sec (ex vivo). Pictures were processed using IVIS Living Image 4.0 software.Immunisation.

[0784] Female BALB / c mice ( 8-12 weeks) were randomly allocated to groups. LNP formulations were diluted in PBS or saline (0.9% NaCI) and injected intramasularly (i.m.) into the gastrocnemius muscle at a volume of 20 pL under isoflurane anaesthesia.

[0785] Tissue preparation.

[0786] Peripheral blood was collected from the vena facialis under isoflurane anaesthesia. Blood was centrifuged for 5 minutes at 16.000 x g, and the serum was immediately used for downstream assays or stored at -20 °C. Spleen single-cell suspensions were prepared in PBS by mashing tissue against the surface of a 70 pm cell strainer (BD Falcon) using the plunger of a 3-mL syringe (BD Biosciences). Erythrocytes were removed by hypotonic lysis.

[0787] Serum antibody and IgG determination:

[0788] MaxiSorp plates (Thermo Fisher Scientific) were coated with recombinant antigen (100 ng / 100 pL) in sodium carbonate buffer for antibody detection in mouse sera, and the bound IgG was identified using an HRP-conjugated secondary antibody along with a TMB substrate (Biotrend). Data collection was performed using a BioTek Epoch reader and Gen5 software. For concentration analysis, the signal of the specific samples was correlated to a standard curve of an isotype control.

[0789] T-cell responses:

[0790] ELISpot assays were performed with mouse IFNy ELISpotPLUSkits according to the manufacturer's instructions (Mabtech). A total of 2.5 x 105splenocytes was ex vivo restimulated with the full-length antigen peptide mix (0.1 pg / mL final concentration per peptide, JPT) or controls (gp70-AHl [SPSYVYHQF] (Slansky, J. E. et al., Immunity 13, 529-538, 2000), JPT, 4 pg / mL; Concanavalin A (ConA), Sigma, 2 pg / mL). Streptavidin-ALP substrate was added, and spots counted using an ELISpot plate reader (ImmunoSpot® S6 Core Analyzer, CTL). Spot numbers were evaluated using ImmunoCapture Image Aquision Software V7.0 and ImmunoSpot 7.0.17.0 Professional.

[0791] Anti-polymer antibody assay:

[0792] The plate was coating with 10g of lipid per well and incubate with the blocking buffer. Anti-PEG antibody standards are prepared by diluting anti-PEG IgM and IgG antibodies to 1 pg / mL in Dilution / Blocking Buffer. Serum samples are pre-diluted in a separate plate and they are transferred to blocked assay plates for IgG and IgM detection, alongside antibody standards in standard wells and Blocking / Dilution Buffer in blank wells. The Detectionantibody was added per well and the assay stops by adding stop

[0793] solution. The Absorbance was measured at 450 nm with optional correction at 590 nm.

[0794] Protein expression:

[0795] A MaxiSorp plate was cover with 100 pl / well of antiIMAB362 and incubate with the blocking buffer. The samples were added to the plate and incubate. After addition

[0796] stop solution the absorbance was measured at 405 nm and 492 nm.

[0797] Example 1 - three-component LNP manufacturing

[0798] In the composition of this example, the serendipitous manufacturing of 3-component LNPs without any polymer grafted lipid is described and are hereafter referred to as xLNPs. The xLNPs were produced with a compositional ratio of 51.1 mol% of cationically ionizable lipid, 34.8 mol% of cholesterol and 14.1 mol% of DSPC, according to Method A described in the Process Method section below. Here, three different cationically ionizable lipids were used in this composition together with modLuc-mRNA, to determine the broader suitability of manufacturing LNPs without polymer-grafted lipids (Ta b le 1).

[0799] The results listed in Table 2 demonstrate that LNPs without polymer-grafted lipids, manufactured with different cationically ionizable lipids, had a similar colloidal size (60-80 nm), poly dispersity (PDI < 0.2), and percent encapsulation efficiency (%EE) of the mRNA cargos (>80%). Furthermore, the surface charges were measured to be neutral with a potential in all cases ranging between -5 to +5 mV, revealing the zwitterionic neutral nature of the lipid nanoparticles at the final pH of 7.5. However, the results listed in Table 3 demonstrate that the particle size and their poly dispersity (PDI) increase when exposed to a single freeze thaw cycle (FTlAnm) at -80 °C.

[0800]

[0801]

[0802] Table 1 - Molecular structures of the lipids used in Example 1

[0803]

[0804] Table 2 - Size and PDI of xLNPs after manufacturing

[0805]

[0806] Table 3 - Size and PDI of xLNPs after a single freeze-thaw cycle at -80 °C Example 2 - variations to the composition

[0807] In the composition of this example, the lipid composition of xLNPs were systematically varied to determine the ideal range for physiochemical properties and resistance to freezethaw at -80 °C. Here, the cationically ionizable lipid was ALC-0366, the structural lipid wascholesterol, and the phospholipid was DSPC. The compositional evaluation was divided into two different experiments: a) A single ratio of ALC-0366 to cholesterol ratio with varying molar contributions of DSPC, b) A single molar percentage of DSPC and evaluating different ALC-0366 to cholesterol ratios. Additionally, the weight per volume contribution of sucrose in the final storage matrix on the freeze-thaw stability was determined. All xLNPs were manufactured using modLuc-mRNA according to Method A, described in the Process Method section.

[0808] Screening was performed where the ALC-0366 to cholesterol ratio was set to 2.0, followed by determination of particle size, encapsulation efficiency (EE%) and delta size over a singlefreeze cycle at -80°C (AFT1) were evaluated for DSPC range between 10-25 %. Figure 1 shows that particle size decrease with the percentage of DSPC (Figure la) from 100 to 70 nm, where the formulations with a DSPC content range between 15-25 mol% display particle sizes between 70 and 80 nm. Furthermore, the highest %EE is obtained for LNPs with 20 mol% of DSPC (Figure lb). Finally, the size increased over 1 freeze-thaw cycle at -80°C reduces with increase mol% of DSPC (Figure 1c). From these results, the optimal range of DSPC content for xLNPs with an ALC-0366 to cholesterol ratio of 2.0 lies between 15 and 25 mol%, yielding favourable particle sizes (70-80 nm), good encapsulation efficiencies (>80%) and limited size increase over a single freeze thaw cycle (<25 nm).

[0809] To further explore the contribution of the ALC-0366 to cholesterol ratio, xLNPs were formulated with a fixed amount of 20 mol% DSPC and the trend for size, %EE and delta size after one freeze-thaw at -80°C for different ALC-0366 to cholesterol ratios was determined. Figure 2 demonstrates that xLNPs with an ALC-0366 to cholesterol ratio between 1 and 2 generates similar particle sizes, varying between 70-85 nm (Figure 2a). Here, xLNPs with increasing ALC-0366 to cholesterol ratios showed a minor decrease in encapsulation efficiency (Figure 2b). However, all values were still within the range above 80%.

[0810] Furthermore, an increase of the ALC-0366 to cholesterol ratio increased the LNP stability over a single freeze thaw cycle at -80°C (Figure 2c). Here, the initial change from a ratio of 1.0 to 1.25 reduced the size increase from 260 nm to 49 nm. Further increase led to further reduction of the size increase, where xLNPs with a ratio of 1.75 or 2.0 showed a size increase in the favorable range <25 nm.

[0811] The difference between 10% or 20% weight per volume of sucrose showed a minor contribution to xLNPs with higher ALC-0366 to cholesterol ratios (>1.5), especially forxLNPs with a fixed ratio of 2.0 and a ranging DSPC content from 10 to 20 mol% (Figure 3a). No significant benefits for the use of a higher sucrose concentration were observed for lower ALC-0366 to cholesterol ratios at a fixed content of 20 mol% DSPC with ALC-0366 to cholesterol ratios between 1.0 and 2.5 (Figure 3b).

[0812] Example 3 - chemistry optimization

[0813] In this example variations to the acidifier were tested. To find a common descriptor for various acidifier molecules, their logD values were used, which is a measure of the lipophilicity of a molecule describing how well a molecule can dissolve in lipids or fats. The logD value is the logarithm of the ratio of the concentration of a molecule in a lipid phase to its concentration in an aqueous phase. Table 4 presents acidifiers and their logD values determined in silico at respectively pH 5.5 and 7.5. All xLNPs were manufactured using modLuc-mRNA according to Method B, described in the Process Method section below. Table 5 shows the results for the three selected acidifiers; acetate (highest logD), malate (intermediate logD) and phosphate (lowest logD) at different concentrations and pHs. Here, it is shown that acetate 50 mM and phosphate 10 mM leads to better results compared to malate. The majority of LNPs with malate as acidifier present aggregation and encapsulation efficiency lower than 65%. Table 6 shows the evaluation for two ALC-0366 to cholesterol ratio, 1.75 and 2, where phosphate 10 mM, yields smaller particle size, higher encapsulation efficiency and better frozen stability (AFT1, -80°C) compared to 50 mM acetate.

[0814]

[0815] Table 4. Predicted values of logP and logD

[0816]

[0817] Table 5. Results for the different acidifiers

[0818]

[0819] Example 4 - upscaling and tangential flow filtration of xLNP

[0820] In the composition of this study, the upscale manufacturing of xLNP and purification using tangential flow filtration (TFF) was evaluated. Here, xLNPs were manufactured using modLuc-mRNA according to Method D described in the Process Methods section. The composition of xLNPs in this example was with an ALC-0366 to cholesterol ratio of 2.0 and a 20% molar contribution of DSPC. Figure 4 presents a detailed scheme of the manufacturing process, where the xLNP manufactured using pumping devices and loaded into the TFF system. Herein, xLNPs were up concentrated two times, followed by two volumetric volumes of diafiltration buffer, a second concentration step followed by a final diafiltration step consisting of ten volumetric volumes of diafiltration buffer.Table 7 shows the physicochemical parameters of the final product demonstrating that LNPs were successfully manufactured and further purified by TFF with size result below 100 nm and %EE > 90%. These data demonstrate that xLNP can be upscaled from a standard lab scale manufacturing employing dialysis for purification towards a GMP-compatible manufacturing employing TFF or other pump-based purification.

[0821]

[0822] Example 5 - changes to the N / P ratio

[0823] The nitrogen to phosphate ratio in lipid nanoparticles (LNPs) refers to the molar ratio of positively charged nitrogen groups (N) in (ionizable)-cationic lipids (e.g., ALC-0366) to negatively charged phosphate groups (P) present in RNA or DNA molecules during the LNP formulation. In this example, changes to the N / P ratio were assessed. The composition of ALC-0366 to cholesterol was set to 1.75 and 20% molar contribution of DSPC was selected. The N / P ratios evaluated were 6, 8, 10, and 12. The manufacturing process was performed with modLuc-mRNA according to Method C described in the Process Methods sections below.

[0824] Figure 5 demonstrates that for the range of N / P ratio studied, the size of xLNP shows particle size values < 85 nm. As the N / P ratio increased, the particle size decreased, remaining constant with an N / P ratio higher than 10 (Figure 5a). In addition, there is an increase of the particle size over multiple freeze-thaw cycles with same trend for all N / P ratios (Figure 5b). Finally, the encapsulation efficiency was % EE > 90 for all the N / P ratios evaluated (Figure 5c). These results demonstrate that xLNPs can be formulated using a range of NP ratios. Example 6 - LysoPC

[0825] In the composition of this example, the feasibility of manufacturing lyso-xLNPs with different lysophospholids (LysoPC) to improve stability was studied. A list of lysoPC molecules used in this example are shown in Table 8.

[0826] The tested formulations included one out of two cationically ionizable lipids (ALC-0366 or BHD-C2C2-PipZ) and with a cationically ionizable lipid to cholesterol ratio of 1.75 or 2. In all cases, the molar contribution of DSPC was set to 20 mol% of the total lipids present in thecomposition. As a fourth component, the molar contribution of the lysoPC molecules was set to 3%. Furthermore, both N / P ratios of 6 and 10 were evaluated. The manufacturing of these lyso-xLNPs was performed using modLuc-mRNA according to Method B, described in the Process Method section.

[0827] Figure 6 presents the results for lyso-xLNP with the different LysoPC variants and the cationically ionizable lipid ALC-0366. For all the lyso-xLNP the size was lower than 100 nm with smaller size around 85 nm for ALC-0366 to cholesterol 1.75 (Figure 6a and b). The variant 18:1 lysoPC with an N / P ratio of 6 led to aggregation during formation and is therefore not shown in the figure. In the case of N / P ratio of 10, these lyso-xLNPs also showed higher particle size (>100 nm) compared to the fully saturated variants and the encapsulation efficiency could not be determined properly due to a high amount of free RNA. For all other lysoPC variants, the encapsulation efficiencies are higher than 90% (Figures 6c and d). Furthermore, it is shown that with the inclusion of the saturated lysoPC lipids, the particle size increase over one freeze-thaw cycle at -80°C is improved compared to the xLNPs and lower than 10 nm for ALC-0366 to cholesterol molar ratio of 1.75 and N / P ratio of 6 (Figure 7). Also, when using the BHD-C2C2-PipZ cationically ionizable lipid, lower particle sizes (<65 nm) and high encapsulation efficiencies (>95%) have been observed, with a similar improvement over a single freeze thaw cycle at -80 °C compared with the xLNPs by the inclusion of lysoPC lipids (Figures 8a and b).

[0828]

[0829]

[0830] Table 8. Structure of the lysoPC variants used

[0831] Example 7 - Oligomer-grafted lipids

[0832] In this example oligomer-grafted lipids (alternatively named “shortmer lipids”) were investigated. Here, these three sarcosine molecules were conjugated to a single carbon chain (Cl 4) and capped with an acetyl moiety to preserve its neutral charge, these structures are shown (Table 9). In the composition of this example, the addition of mono- or oligosarcosine based shortmer lipids to the xLNP is described and hereafter referred to as shortmer-LNPs. Shortmer-LNPs were formulated with two different cationically ionizable lipids, BHD-C2C2-PipZ and ALC-0366, and with shortmer lipids Sari and Sar3 at molar contributions of 4 and 8% using modLuc-mRNA as according to Method C as describe d in the Process Method

[0833] section (Table 10). Furthermore, the influence of the acidifier on the physicochemical properties was assessed, as well as resistance over one freeze-thaw cycle at -80 °C (AFT1,

[0834] nm) for the shortmer-xLNP s with Sari, Sar3 and Sar5 at molar contributions o f" 2, 3 and 4% (Table 11).

[0835] Figure 9 demonstrates that shortmer-xLNP can be formulated with different cationically ionizable lipids, BHD-C2C2-PipZ as well as ALC-0366, using Sari and Sar3 at molar contributions of 4 and 8% with high encapsulation efficiencies (>80%) and small particle sizes (< 80 nm). By using the manufacturing process involving the phosphate acidifier (10 mM) instead of the acetate acidifier (50 mM), as well as lower concentrations of Sari, 3 and 5 at molar contributions of 2, 3 and 4%, particles with formed with small sizes (< 80 nm), high encapsulation efficiencies (>90%) and low size increase over a single freeze thaw cycle at -80 °C (< 6 nm) in all cases.

[0836]

[0837]

[0838] Table 9. Structure of the oligomer-graftec lipids (shortmer lipids)

[0839]

[0840] Table 10. Composition for the different shortmers for both cationically ionizable lipids

[0841]

[0842] Table 11. Composition for the different shortmers for acetate and phosphate acidifiers Example 8 - variations of phospholipid

[0843] In the composition of this example, the influence of phospholipid variation on the formation and stability of xLNPs was evaluated. The detailed list of evaluated phospholipids is provided in Table 12. Different percentages of DSPC were substituted by neutral lipid with composition ALC-0366 50.9%, cholesterol 29.1%, DSPC 20-x% and the neutral lipid x% with N / P6 and / or N / P 10 and compared with their respective xLNP. The formulation of all LNPs was performed using modLuc-RNA according to Method C as described in the Process Method section.

[0844] A tested range of substitutions of DSPC in the xLNP composition led to direct aggregation during the formation of the xLNPs as shown in Table 13. For a contribution of 10 mol% of DSPC and 10 mol% of another neutral lipid only led to successful formation and encapsulation in the case of DPPC and MOG, albeit it is the case that the particle size and its poly dispersity were highly affected in the case of MOG. Here, molar contributions of MOG and DOPE lower than 10 mol% had a more positive impact on the physiochemical properties of the xLNPs (Table 13). A neutral lipid percentage of 5 mol% presented a proper balance between size and PDI and chosen as optimum. Figure 12 demonstrates that the addition of 5 mol% of neutral lipids yield xLNPs with size < 100 nm, %EE above 80% in the case of a N / P ratio of 6 and above 95% in the case of a N / P ratio of 10. Furthermore, the stability of these xLNP variants with neutral lipids DPPC and DMPC show similar particle size increase over a single freeze-thaw cycle -80°C at a N / P ratio of 6 compared to the xLNPs with 20 mol% of DSPC. For these variants, a significant improvement for freeze-thaw stability was observed at a N / P ratio of 10.

[0845]

[0846]

[0847] Table 12. structure of the phospholipids (and monoacylglycerol) studied

[0848]

[0849] Table 13. Size, PDI and %EE of xLNP and different neutral lipids. Example 9 - in vivo experiments

[0850] In this example the biodistribution of cellular transfection of xLNP and shortmer-xLNPs formulations was determined. Here, four formulations with the cationically ionizable lipid ALC-0366, the structural lipid cholesterol, the phospholipid DSPC and in some cases a supplemental shortmer-sarcosine lipid (C14-Sarl-Ac, C15-Sar3-Ac or C14-Sar5-Ac), as displayed in Table 14 were tested. Furthermore, a benchmark LNP formulation including a polyethylene glycol (PEG)-grafted lipid (ALC-0159) was included. All xLNPs were formulated using a 1 :9 ratio of luciferase: and an mRNA encoding an antibody, at an N / P ratio of six according to method C described in the Process Methods section below. The benchmark LNP formulation was formulated according to method E described in the Process Methods section below. The formulations were administered intravenously (tail vein injection) in 15 female BALB / c (9-week old) mice at a total dose of 10 pg RNAin triplicate. As a control, a single mouse was injected with saline solution. The animals were treated for bioluminescence imaging (BLI) as described in the methods section, at 6- and 24-hours post injection (Figure 13). Hereafter, animals were sacrificed and the liver, spleen, gastrointestinaltract (Gl-tract), lymph nodes, kidneys, lung and heart were harvested for ex vivo BLI imaging.

[0851] The in vivo BLI imaging after 6 hours post injection revealed a localized and intense transfection for BM and more diffuse for xLNP and shortmer-xLNPs (Figure 14). At 24 hours, a similar but lower intense signal was observed for all groups. For the negative control (saline), no signal was observed at both the 6- and 24-hour time point. The ex vivo BLI imaging after 24 hours post injection showed localized and strong transfection in the liver and spleen for BM, as well as minor expression in the lymph nodes, in agreement with the locations observed in in vivo BLI imaging (Figure 15). However, for xLNP and shortmer-xLNPs, significant BLI signal was observed in multiple organs such as the liver, spleen, lymph nodes and Gl-tract. Here, the signal compared to the benchmark was lower in the liver and spleen, but the signal in the Gl-tract was surprisingly and significantly enhanced. These results are in-line with the more diffuse signal observed by in vivo imaging. Altogether, these results indicate a reduced targeting of the main organs which take up LNPs (liver, spleen), but a higher and novel targeting towards the Gl-tract, exclusive to the xLNP and shortmer-xLNP formulations.

[0852]

[0853] Table 14. Physicochemical results for the compositions evaluated in vivo Example 10 - structure of the particles

[0854] In this example, the structure and morphology of xLNP and shortmer-xLNPs was studied using cryogenic electron microscopy (cryo-EM) and small-angle x-ray scattering (SAXS). Here, the xLNP, shortmer-xLNPs and PEG-lipid benchmark control were employed for the in vivo study described in Example 9 were evaluated for structural properties and morphology with cryo-EM and SAXS.Using cryo-EM, representative images of xLNP, shortmer-LNP and the benchmark were collected (Figure 16-20) in all cases showing similar solid core structures. For xLNP and shortmer-xLNPs, violin plots from multiple cryo-EM images were constructed to show the amount of solid core particles (>90%) and phase-separated particles (<10%) by particle counting (Table 15, Figure 21). Furthermore, the particle size derived from cryo-EM provides an average of 50-54 nm for xLNP + Sar3 and Sar5, while for Sari and xLNP is around 60 nm. For the xLNP and xLNP-shortmers a different lamellar core structure compared to the benchmark could be observed by cryo-EM, which was further studied using SAXS. SAXS profiles for xLNP and shortmer-xLNPs possess a similar Bragg peak around q (nm ) = 1.0 (Figure 22). In the case of the benchmark formulation, the Bragg peak is shifted more to the higher q range at q (nm4) = 1.16. Given the observed lamellar structure, Equitation 1 was used to determine the lamellar spacing. Here, since only a single Bragg peak is observed, the n value is set to 1 for the n [1,0] lamellar lattice. This calculation shows that the interlayer distance for shortmer-xLNPs is around 6.5 nm, for xLNP around 7.8 nm and for the benchmark formulation around 5.6 nm (Table 16).

[0855] 2nn

[0856] dlamellar = - < With n = n [1,0] = 1

[0857] q

[0858] >

[0859]

[0860] Table 15. Descriptive statistics of xLNP and shortmer-xLNPs morphology and size

[0861]

[0862]

[0863] Table 16. SAXS parameters for xLNP and xLNP-shortmers Example 11 - critical packing parameter

[0864] In this example demonstrates the critical packing parameter (CPP) values for the molecules exemplified in the present invention. The CCP influences the spatial organization of the formed membranes (Figure 23).

[0865] Table 18 summarizes the molecules which have been added to with three-component xLNPs, as described in the previous examples. Here, their molar contribution to the composition is given along with an allocation to CPP range, the preferred shape of the individual molecule and the final effect on stabilization of the xLNPs. The CPP values for MOG, DPPC, DOPE and DMPC were taken as previously reported by Kobierski et al. Colloids and Surfaces B: Biointerfaces 2022, 211, 112298, Qiu et al. Biomaterials 2000, 21, 223-234, Kulkarni et al. Chemistry and physics of lipids 2019, 281, 16-21 and Vant Hag et al. Chem. Soc. Rev., 2017.

[0866] 46, 2795. The CPP values for lipids with polyethylene glycol (PEG) conjugated are typically reported to be < 1 / 3 and prefer the formation of micellar structures according to Hashizaki et al. Chem. Pharm. Bull. 200654(4) 561-563. The CPP values for the lyso-PC and C14-Sarx-Ac variants were calculated using Equation 1 :

[0867]

[0868] The calculation of the critical length of the hydrophobic part (Lc) and the volume of the hydrophobic part (V) for all molecules were done using Equations 2 and 3 according to the relevant hydrocarbon chain lengths for the molecule.

[0869] Equation 2: lc< lmax« (1.54 + 1.265 ■ ri)

[0870] Equation 3: V = (2.74 + 26.9 ■ ) A3

[0871] wherein n is the number of carbon atoms in the hydrophobic chains.

[0872] The CPP values for the lyso-PC and C14-Sarx-Ac variants are listed in Table 19. For the lyso-PC variants, the previously reported surface area of the head group (aO) by Camie et al. Biochemica et Biophysica Acta 1979554, 340-357 was used. For the C14-Sarx-Ac variants,the surface area of the head group (aO) was determined according to the topological polar surface area (TPSA) method as described by Ertl et al. J. Med. Chem. 2000, 43, 3714-3717. In summary, the surface area of the head group (aO) was calculated by taking the sum of the atom types of the head group as defined Table 17 below.

[0873] >

[0874]

[0875] *: non-hydrogen atom; single bond; d: double bond; #: triple bond; aromatic bond; atomic symbol in lowercase: atom is part of an aromatic system; b: nitro group; c: Middle nitrogen in azide group; d: atom in a three-membered ring; e: nitrogen in isocyano group; f: as in pyridine N-oxide.

[0876] Table 17. Atomic contributions to PSA as described by Ertl et al. J. Med. Chem. 2000, 43,

[0877] 3714-3717.

[0878] > >

[0879]

[0880]

[0881] Table 18. Summary of CPP values and the effect on xLNP stabilization

[0882]

[0883] Table 19. CPP values for lyso-PC and C14-Sarx-Ac molecules Example 12 - Absence of polymer antibody response against shortmer-xLNPs

[0884] In the composition of this example, the in vivo pharmacokinetics (PK) for an antibody encoded by mRNA, delivered through LNP formulations, and the determination of polymer antibody formation against the polymers or oligomers in these LNP formulation compositions was determined. Here, the same formulations as from Example 9 were administered intravenously (tail vein injection) in 8-week-old BALB / c mice at a total dose of 10 pg RNA, and multiple blood sampling points were planned over the period of 2 weeks (Figure 24). As a control, a single mouse was injected with saline solution. Here, each group consisted of 8 mice, which were subdivided into subgroups of 4 mice. From all mice, pre-bleeds were taken 6 days prior to the experiment start to serve as null values for antibody PK and polymer-antibody read-outs. All mice were injected with the respective formulation or control, after which blood was withdrawn for antibody PK determination from all four mice of the first subgroup after 3 hours and the from all four mice of other subgroup after 6 hours. From two out of four mice from each group, an additional blood sampling for antibody PK analysis was performed at respectively 24 hours and 72 hours after the injection, after which these micewere sacrificed. The final blood sampling point was after 7 days post injection, serving for antibody PK and anti-polymer antibody determination, after which the mice were sacrificed, and the experiment was ended.

[0885] From all blood collections, a PK profile of the encoded antibody was made (Figure 25). Here, the BM-366 formulation has a higher antibody expression across the timeframe compared to the xLNP and Sari, 3 and 5 formulations. This is in correlation with the lower protein expression observed for luciferase in the liver from the previous experiment. Notably, the antibody expression is highly similar for xLNP, Sari, Sar3 and Sar5 formulations, proving that inclusion of the fourth component does not have a detrimental effect on the potency of the formulation. The blood samples collected in the pre-bleed and after 7 days post injection were included in a generic ELISA anti-polymer assay testing for the generation of IgG and IgM. Here, plates were coated with the respective polymer or shortmer grafted lipid included in the formulation, followed by rinsing and incubation with the according sera. For the saline control, the PEG lipid was used for incubation on the plates. The xLNP formulation was excluded from this assay as it does not contain a polymer or shortmer grafted lipid. It can be observed that the PEG formulation leads to a 2.9-fold increase in IgG and a 8.3 in IgM from pre-bleed compared to day 7 (Figure 26). For the sarcosine shortmer containing formulations (Sari, Sar3 and Sar5), only a minor increase is observed from pre-bleed compared to day 7 for Sar (1.3-fold) and Sar5 (1.5-fold) for IgG (Figure 26A). Within the accuracy of this experiment, these increases can be considered negligible. For IgM, no increase is observed for all sarcosine oligomer containing formulations (Figure 26B). These results underline that the shortmer sarcosine lipids do not trigger polymer-antibody generation and that shortmer-LNPs thereby behave essentially like polymer-free LNPs, such as three-component xLNP or four-component lyso-xLNPs.

[0886] Example 13 - Freeze-thaw stability of three-component xLNPs

[0887] In the composition of this example, the influence of different ionizable lipids on the formation and stability of 3 -component xLNPs was evaluated. xLNP formulations were constituted with four different cationically ionizable lipids, ALC-0366, BHD-C2C2-Pipz, BODD-C2C2-DMA and BODD-C2C2-MePyrl using modLuc-mRNA as according to Method F as described in the Process Method section. The list and structure of the ionizable lipids are provided in Table 1 and Table 20. Furthermore, the influence of the ionizable lipidand the percentage of DSPC on the physicochemical properties was assessed, as well as resistance over five freeze-thaw cycles at -80 °C (AFT5, nm) (Figure 27)

[0888] The results demonstrated that 3 -component xLNPs without polymer grafted lipid can be successfully manufactured using various cationically ionizable lipids. The particle size of all xLNPs ranged between 60 and 95 nm. At a concentration of 40% DSPC, the particles obtained from the manufacturing immediately aggregated, regardless of the ionizable lipid. The encapsulation efficiency was above 88% for all the formulations with DSPC concentration of 20 mol% and 30 mol%. Additionally, the delta size after 5FT at -80°C was smaller for all the ionizable lipids when the DSPC concentration was 30 mol% compared to 20 mol%. In all the cases, the delta size remained below 15 nm except for BODD-C2C2-MePyr at a 20 mol% DSPC concentration. These findings highlight that three-component xLNPs can be successfully manufactured using a range of DSPC concentrations (< 40 mol%), and that the exact amount of DSPC in combination with the type of ionizable lipid can have an influence on particle size, stability, and encap sulation efficiency.

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[0890]

[0891] Table 20. Structure of the ionizable lipids studied

[0892] Example 14 - Freeze-thaw stability of four-component xLNPs

[0893] In the composition of this example, the influence of different ionizable lipids on the formation and stability of 4-components xLNP was evaluated. The xLNP were formulated using four different cationically ionizable lipids, being ALC-0366, BHD-C2C2-Pipz, BODD-DMAor BODD-MePyr. Both C14-Sar(l)-Ac or 18:0 LysoPC were used as an additional fourth component. All xLNPs were formulated using modLuc-mRNA following Method F as described in the Process Method section. Furthermore, the impact of the ionizable lipid and the DSPC percentage on the physicochemical properties of 4-component xLNP was assessed, as well as their stability over five freeze-thaw cycles at -80 °C (AFT5, nm).The results demonstrated that 4-component xLNPs can be manufactured with different cationically ionizable lipids (Figure 28). In some cases, the particle size increased as the DSPC concentration was increased from 20 mol% to 30 mol%. This increase is particularly pronounced for the combination of the ionizable lipid ALC-0366 with 18:0 LysoPC or Cl 4-Sar(l)-Ac resulting in particle size exceeding 100 nm. Nevertheless, the encapsulation efficiency for all formulations was 84% or higher, with the majority being over 90% (Figure 29). This demonstrates that despite particle size increase, effective RNA-xLNP formation was achieved regardless of the fourth component. Furthermore, the delta size over 5 freeze-thaw cycles at -80°C for 4-component xLNPs using C14-Sar(l)-Ac was below 20 nm for the all the ionizable lipids (Figure 30). Notably, 4-component xLNPs with 18:0 LysoPC provide the highest stability over freeze-thaw cycles at -80°C. In addition, as was observed for 3-components xLNP in example 13, a DSPC concentration of 30% promoted a smaller delta size after 5FT, further supporting the role of DSPC in enhancing the stability of xLNP formulations. Together, these results demonstrate that the addition of a fourth component, such as 18:0 LysoPC or C14-Sar(l)-Ac, can have stabilizing benefits for xLNPs.

[0894] Example 15 - Long-term frozen and liquid stability of three- and four-component xLNPs. In the composition of this example, the long-term stability of both 3- and 4-component xLNPs formulated with different ionizable lipids, was assessed at storage temperatures of 5 °C ± 3 °C and -80°C ± 10 °C over a period of 6 months. Here, the xLNPs formulated for freeze-thaw stability studies in Examples 13 and 14 were used. Only the formulation combining the ionizable lipid ALC-0366 with 18:0 LysoPC and a DSPC content of 30 mol% was excluded from the stability study, due to the large particle size at tO.

[0895] For the stability at frozen conditions (-80 °C), the particle size of all formulations remained below 100 nm, and the PDI below 0.3, over a period of 6 months (Figure 31). Furthermore, the encapsulation efficiency of all formulations remained above 80% in all cases (Figure 32). For three component xLNPs, the size and PDI increase over time is lower for the formulations containing 30 mol% DSPC in comparison to 20 mol%, regardless of the ionizable lipid. Furthermore, the stability over time can be improved by including a fourth component (18:0- LysoPC or C14-Sar(l)-Ac) to the xLNPs with 20 mol% DSPC. The combination of the fourth component as well as increase of DSPC content to 30 mol% can also lead to a further improvement to the colloidal properties compared to the individual applications, for example for xLNPs with BODD-MePyr and 18:0 LysoPC.For the stability in liquid conditions (5 °C), similar observations can be made compared to the long-term stability in frozen conditions. Here, both the increase of the DSPC content from 20 to 30 mol%, as well as the introduction of a fourth component can reduce the particle size and PDI increase over time, either reflected at the 3- or 6-month timepoint (Figure 33). Most notable are the excellent stability profiles of the xLNPs with ionizable lipids ALC-0366, BHD-C2C2-PipZ and BODD-DMA. In the case of BODD-MePyr, the increase of DSPC content has a higher impact compared to the introduction of the fourth component.

[0896] Nevertheless, the encapsulation efficiency of all formulations remains above 80%, with the majority being over 90%, with nearly no change over the duration of the stability study (Figure 34).

[0897] For liquid conditions, the preservation of RNA integrity is an additional critical quality attribute at risk, due to the possibility of active hydrolysis occurring at these conditions. It can be observed that the loss of RNA integrity over time for 3 component xLNPs with a DSPC content of 20 mol% is in the order of 15% over 3 months, regardless of the ionizable lipid (Figure 35). Here, the increase of the DSPC content to 30 mol% does not significantly reduce this RNA integrity loss. However, the addition of the fourth component does reduce the RNA integrity loss to 5 - 10% over the same period, regardless of the ionizable lipid. At the 6-month timepoint, a less significant difference between groups is observed, but overall having a higher or similar RNA integrity for four-component xLNPs compared to their 3-component counterparts.

[0898] Overall, the long-term stability data shown in this example confirm the preliminary results from freeze-thaw cycling in examples 13 and 14 and provide additional information. Both the increase of the DSPC content as the introduction of fourth component, or both simultaneously, are shown to improve the preservation of the critical quality attributes particle size, PDI, encapsulation efficiency and RNA integrity in liquid or frozen conditions over time.

[0899] Example 16 - Upscaling and tangential flow filtration of four-component xLNPs.

[0900] In the composition of this example, the upscale manufacturing of xLNP and purification using tangential flow filtration (TFF) was evaluated. Here, four-component lyso-xLNPs were manufactured using modLuc-mRNA according to Method G described in the Process Methods section. The xLNP were comprised of BHD-C2C2-PipZ and cholesterol at a ratio of 1.75, along with 20 mol% DSPC and 3 mol% of 18:0 LysoPC. Figure 36 presents a schematicof the manufacturing process, where the 4-components xLNP manufactures using four piston pumps and the intermediate xLNP is loaded into the TFF system. Herein, xLNP were up concentrated 1.5 times, followed by two volumetric volumes of diafiltration buffer, a second concentration step followed by a final diafiltration step consisting of eight volumetric volumes of diafiltration buffer. Table 21 shows the physicochemical parameters of the final product demonstrating that LNPs were successfully manufactured and further purified by TFF with size result of 66 nm and %EE > 95%. These data demonstrate that four-components xLNP can be upscaled from a standard lab scale manufacturing employing syringe pump and dialysis for purification towards a GMP-compatible manufacturing employing 4-pump Knauer system for manufacturing and TFF or other pump-based purification.

[0901]

[0902] Table 21. Physicochemical parameters of 4-components xLNP using Knauer pump + TFF Example 17 - Additional information on structure of xLNP particles

[0903] In the composition of this example, further structural analysis of xLNP and PEG-LNP formulations was performed. Here, analysis of cryo-EM images and SAXS profiles was performed as described in Example 10.

[0904] In the first experiment, relevant formulations with the ionizable lipid BHD-C2C2-PipZ from examples 15 and 19 were selected for cryo-EM analysis. Here, the representative structures of three-component xLNP (Figure 37), four component lyso-xLNP (Figure 38), four component Sarl-xLNP (Figure 39), 10 mol% DSPC containing PEG-LNPs (Figure 40) and 20 mol% DSPC containing PEG-LNPs (Figure 41) were revealed. All xLNP formulations showed a high occurrence of solid core structures, whereas PEG-LNPs had a more frequent display of phase-separated structures. From multiple cryo-EM images, violin plots were constructed (Figure 42), and descriptive statistical analysis was performed (Table 22) to confirm the representative images, showing that the amount of solid core particles for xLNP formulations was > 94%, whereas the PEG-LNPs with 10 and 20 mol% DSPC showed 59% and 33% of solid core particles respectively. These results support the findings demonstrated in Example 10, showing that the general morphology of xLNP formulations primarilyconsists of solid core particles, irrespective of ionizable lipid type, fourth component selection or composition alterations.

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[0910] Table 22. Descriptive statistical analysis of xLNP and PEG-LNPs morphology and size In the second experiment, SAXS was used to provide more information on structural similarity of various xLNP compositions. Here, relevant formulations from examples 15 and 18 were selected. The initial comparison was of three- and four-component xLNPs, with an ionizable lipid to cholesterol ratio of 1.75, containing either 20 or 30 mol% DSPC, the ionizable lipid BHD-C2C2-PipZ and the fourth component 18:0 LysoPC (Figure 43). For xLNPs with 20 mol% of DSPC, also the additional ratio of ionizable lipid to cholesterol of 2.0 was investigated. The SAXS profiles demonstrate similarity for all formulations with a Bragg peak around 1 nm'1(Figure 43). Analysis of all curves validates this observation, showing a high similarity of radii of gyration (13.8 - 15.1 nm), Bragg peak positions (0.92 - 0.99 nm’1) and derived interlamellar spacings (6.3 - 6.8 nm) (Table 23). These results support previous findings that there is a high structural similarity between three and four-component xLNPs, as

[0911] for xLNPs with varying DSPC content and ionizable lipid to cholesterol ratios.

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[0913]

[0914]

[0915] Table 23. SAXS parameters for 3-components and 4-components xLNPs In the third experiment, further SAXS analysis was performed to determine the impact of ionizable lipid type on xLNP structure. Here, the formulations from Example 18 were selected. In this case, the data from lyso-xLNP with the ionizable lipid BHD-C2C2-PipZ is the same as previously displayed in this example but added for clarity. Like the previous experiment, The SAXS profiles demonstrate similarity for all formulations with a Bragg peak around 1 nm'1(Figure 44). Analysis of all curves validates this observation, showing a high similarity of radii of gyration (13.9 - 15.1 nm), Bragg peak positions (0.89 - 0.96 nm’1) and derived interlamellar spacings (6.5 - 7.0 nm) (Table 24). These results support previous findings that there is a high structural similarity for four-component xLNPs with varying ionizable lipid types.

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[0917]

[0918] Table 24. SAXS parameters for 4-components lyso-xLNP and with different ionizable lipids Example 18 - Comparability of xLNPs with different ionizable lipids in vivo.

[0919] In this example, the potency and biodistribution of cellular transfection of 4-component xLNPs with various cationically ionizable lipids was determined. Here, four formulations with the cationically ionizable lipid BHD-C2C2-PipZ, ALC-0366, BODD-MePyr and BODD-DMA as displayed in Table 25 were tested. All the formulations were formulated using a 1:9 ratio of luciferase: and an mRNA encoding a green fluorescent protein (eGFP), at an N / P ratio of six according to Method F described in the Process Methods section below. The formulations were administered intravenously (tail vein injection) in 129-week-old female BALB / c mice at a total dose of 10 pg mRNA in triplicate. As a control, a single mouse was injected with saline (0.9% NaCl) solution. The animals were treated for and subjected to bioluminescence imaging (BLI) as described in the methods section, at 6- (invivo) and 24-hours (ex vivo) post injection (Figure 45). Animals were sacrificed 24 hours after administration of test items and the heart, lung, spleen, kidneys, inguinal lymph nodes, liver and gastrointestinal tract (Gl-tract) were harvested for ex vivo BLI imaging.

[0920] The in vivo BL imaging after 6 hours post injection revealed a well detectable luminescence signal at the anatomical location of liver tissue for all administred formulations with highest intensity for BHD-C2C2-PipZ-LysoPC18:0 containing xLNP. Additionally, luminescence signal was detected across the abdominal region of the mice administered with all four test items. The signal was most intense in mice injected with ALC-0366. The ex vivo BL imaging at 24 hours post injection showed liver and spleen as main (major) source of luminescence signal and less intense but real transfection of mRNA formulations in structures of the GL tract as well as the inguinal (draining) lymph nodes. Intensities and distribution were in agreement with the pattern observed in in vivo BL imaging at 6h post injection (Figure 46). For the negative control (saline), no signal was observed at both the 6- and 24-hour time point. Similarly to previous experiments, the results indicate a reduced targeting on the main organs which take up LNPs (liver, spleen), but a higher and novel targeting towards the GL tract. When comparing the four ionizable lipids, the bioluminescence signal is most pronounced in the abdominal region for ALC-0366 formulation. Together, it is shown that different ionizable lipids follow same biodistribution and transfection patterns for 4-component lyso-xLNPs. Furthermore, the biodistribution for 4-component lyso-xLNPs is comparable to the previously observed for 3- and 4-component shortmer-xLNPs (including C14-Sar(l)-Ac) with the ionizable lipid ALC-0366.

[0921]

[0922] Table 25. Physicochemical results for the compositions evaluated in vivo Example 19 - Impact of PEG-removal and DSPC content in vivo.

[0923] In this example, the impact of PEG-removal and the DSPC content on the potency and biodistribution of cellular transfection was evaluated. Here, four PEG-LNP formulations with the cationically ionizable lipid BHD-C2C2-PipZ, the structural lipid cholesterol, thephospholipid DSPC and DMG-PEG2000 with different ionizable lipid to cholesterol ratio and DSPC content were evaluated (Table 26). Furthermore, two 3-component xLNP formulations with different DSPC content and one lyso-xLNP were included. All LNPs were formulated using a 1:9 ratio of luciferase: and an mRNA encoding for Thy 1.1 (Cluster of Differentiation, CD, 90.1) protein, at an N / P ratio of six according to Method F for xLNPs (LNP25, LNP27 and LNP55) or Method H for PEG-LNPs (LNP23, LNP29, LNP53 and LNP54) as described in the Process Methods section. The formulations were administered intravenously (tail vein injection) in 21 8-week-old female BALB / c mice at a total dose of 10 pg RNAin triplicate. As a control, a single mouse was injected with saline (0.9% NaCl) solution. The animals were treated for and subjected to bioluminescence imaging (BLI) as described in the methods section, at 6- and 24-hours post injection (Figure 47). Animals were sacrificed 24 hours after administration of test items and the lung, spleen, liver, and gastrointestinal tract (Gl-tract), were harvested for ex vivo BLI imaging.

[0924] The BL imaging in vivo (6h) and ex vivo (24h) shows the main source of bioluminescence signal for all mice injected with test items liver, followed by spleen organ. In addition, in vivo imaging shows a diffusely distributed bioluminescence signal in de abdominal region which can be allocated to GI tract during ex vivo imaging. Furthermore, lymph nodes in can also be identified as targeted (detectable BL signal) by administered test items. The results show, that increase in DSPC content does promote the shift of transfection (BL signal) from liver towards spleen and lymph nodes, which is independent of ionizable lipid to cholesterol ratio (Figure 48).

[0925]

[0926]

[0927] Table 26. Composition and physicochemical properties for the compositions evaluated in vivo Example 20 - In vivo intramuscular application of xLNPs

[0928] In the composition of this example, three- and four-component xLNPs were tested in vivo through intramuscular application to evaluate their application as infectious disease vaccines. Here, three-component xLNP and four-component lyso-xLNP were chosen and compared to a polymer-grafted lipid (Ac-AEEAw-VitE) containing formulation. (Table 27). Each LNP was formulated using an mRNA encoding for an antigen. Each formulation was administered to Balb / c mice (n = 5) intramuscularly with a 0.1 pg mRNA dose, followed by a second treatment (boost) with the same dose after 21 days. Serum antibody titers and IgG concentrations were determined at days 7, 14, 21, 28, 36, 42, 56 and 70 days after the initial administration. Furthermore, T cell-responses induced after intramuscular application of the formulations were determined using an interferon-y ELISpot assay after sacrifice of the animals on day 70, where splenocytes were stimulated ex vivo with a peptide pool of the antigen. Serum antibody titers, IgG concentrations and T-cell responses were shown to be comparable for all formulations across all timepoints (Figure 49 and Figure 50). These results demonstrate that polymer-free three- and four-component xLNP formulations can be used as effective ways of delivering an antigen through intramuscular application and serve as future vaccine candidates.

[0929]

[0930] Process Methods

[0931] Method A: Acetate based downscale manufacturing process

[0932] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA using syringe pumps (T-piece mixer). For example, the lipid mixture was composed of a cationically ionizable lipid (ALC 366, ALC 0315 or BHD-C2C2-PipZ) (see Example 1), cholesterol, and l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC) dissolved in ethanol at molar ratios of 51.1 :34.8: 14.1. The aqueous phase contained mRNA diluted in the acetate buffer (pH 5.5) was mixed with the ethanol phase at a volume ratio of 3:1 (aqueous: ethanol), followed by in-line dilution with TRIS buffer (pH 7.7) (T-piece mixer). The obtained formulation was purified with dialysis. After manufacturing, intermediate LNPs were dialyzed overnight (16-20 hours) against the respective buffer 10 mM Tris pH 7.4 at room temperature. After buffer exchange the drug product was adjusted with the buffer to an RNA concentration of 0.1 mg / mL and diluted with sucrose solution as cryoprotectant to the final RNA concentration. Thereafter, the adjusted drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -80°C.

[0933] Method B: Process optimization

[0934] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA as described in Method A. In this method, lipid mix is composed of the cationically ionizable lipid ALC-0366, cholesterol, and DSPC in a composition 53.3%:26.7:20% dissolved in ethanol. The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6. The aqueous phase contained mRNA diluted in different acidifier buffers was mixed with the ethanol phase at a volume ratio of 3 : 1 (aqueous: ethanol), followed by in-line dilution with TRIS buffer (pH 7.7) via (T-piece mixer). In this method there were used different acidifiers (phosphate, acetate, and malate) at several concentrations (10, 30 and 50 mM) and pH 4.5, 5 and 5.5. The process parameters, dialysis and storage were the same as Method A.

[0935] Method C: Phosphate based manufacturing process

[0936] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA as described in Method A. The lipid mixture was composed of the cationically ionizable lipid ALC-0366, cholesterol and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at different composition 50.0:29.1:20 or 53.3:26.7:20. Additionally, this method was also used in a lipid mixture composed of lipid ALC-0366, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and other component (i.e. shortmer, lysoPC or neutral lipid) in a composition (53.3-0.5x:26.7-0.5x:20:x). The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6 and 10. The aqueous phase contained mRNA diluted in phosphate buffer (pH 4.5). The process parameters, dialysis and storage were the same as Method A.

[0937] Method D: Up scaled manufacturing process

[0938] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA similar to Method A: The lipid mixture was composed of an cationically ionizable lipid ALC 366, cholesterol and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), at molar ratios 53.3:26.7:20 as referred in example 4. The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6. Tangential flow filtration (TFF) was used to replace the LNPs buffer with Tris buffer at pH 7.4. The TFF process was done at room temperature using 100 kDa molecular weight cut-off (MWCO) hollow fiber. Thereafter, the adjusted drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -80°C.

[0939] Method E: Benchmark PEG containing LNP manufacturing process

[0940] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA as described in Method A. In this method, lipid mix is composed of the cationically ionizable lipid ALC-0366, cholesterol, DSPC and ALC-0159 with a composition (47.5:40.7:10:1.8) dissolved in ethanol. The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6. The aqueous phase containing mRNA diluted in acetate buffer pH 4 was mixed with the ethanol phase at a volume ratio of 3 : 1 (aqueous: ethanol), followed by in-line dilution with acetate buffer pH 4 (T-piece mixer). After manufacturing, dialysis was performed as described in Method A against the respective buffer IxPBS, pH 7.4 at room temperature. Samples were up concentrated. The drug product was adjusted with the buffer to an RNA concentration of 1 mg / mL and diluted with sucrose solution as cryoprotectant to the final RNA concentration. Thereafter, the adjusted drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -80°C.

[0941] Method F: Composition optimization

[0942] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA as described in Method A. The lipid mixture was composedof an cationically ionizable lipid (ALC-0366, BHD-C2C2-PipZ, BODD-DMA or BODD-MePyr) cholesterol and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at different compositions, 50.9:29.1:20, 44.55:25.45:30 or 38.2:21.8:40. Additionally, this method was also used in a lipid mixture composed of an ionizable lipid (ALC-0366, BHD-C2C2-PipZ, BODD-DMA or BODD-MePyr), cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and other component (Sari or lysoPC) in a composition (49:28:20:3 or 42.64:24.36:30:3 ). The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6 and 10. The aqueous phase contained mRNA diluted in phosphate buffer (pH 4.5). The process parameters, dialysis and storage were the same as Method A.

[0943] Method G: Upscale manufacturing process Knauer pump + TFF

[0944] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA similar to Method A: The lipid mixture was composed of an cationically ionizable lipid BHD-C2C2-PipZ, cholesterol and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and LysoPC 18:0 at molar ratios 49:28:20:3. The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6. The manufacturing process was performed using 4-pump Knauer system with a total flow rate of 360 mL / min. Tangential flow filtration (TFF) was used to replace the LNPs buffer with Tris buffer at pH 7.4. The TFF process was done at room temperature using 100 kDa molecular weight cut-off (MWCO) hollow fiber. Thereafter, the adjusted drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -80°C.

[0945] Method H: PEG containing LNP manufacturing process

[0946] LNPs were formulated by mixing an organic phase containing dissolved lipids with an aqueous phase containing RNA as described in Method A. In this method, lipid mix is composed of the cationically ionizable lipid BHD-C2C2-PipZ, cholesterol, DSPC and PEG-DMG200 with BHD-C2C2-PipZ to cholesterol ratio of 1.17 with a composition (47.5:40.7:10:1.8 or 42.5:35.7:20:1.8) dissolved in ethanol. Additionally, a composition of BHD-C2C2-PipZ to cholesterol ratio of 1.75 was manufactured using same method with a composition (49.7:28.4:20:1.8 or 43.4:24.8:30:1.8). The ratio of nitrogen present in the cationically ionizable lipid to phosphate groups in the mRNA (N / P) was set to 6. The aqueous phase containing mRNA diluted in citrate buffer pH 4 was mixed with the ethanol phase at a volume ratio of 3 : 1 (aqueous: ethanol), followed by in-line dilution with citrate buffer pH 4.After manufacturing, dialysis was performed as described in Method A against the respective buffer Phosphate 10 mM, pH 7.4 at room temperature. Samples were up concentrated. The drug product was adjusted with the buffer to an RNA concentration of 0.3 mg / mL and diluted with sucrose solution as cryoprotectant to the final RNA concentration. Thereafter, the adjusted drug product was filtered through a 0.22 pm polyethersulfone (PES) filter and stored at -80°C.

[0947] Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A composition comprising:(i) a cationically ionizable lipid;(ii) a steroid;(iii) a phospholipid;wherein the composition is substantially free of:(a) a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units,(b) a negatively charged amphiphile; and(c) an inorganic polyphosphate; andwherein the molar ratio between the cationically ionizable lipid and the steroid is between about 1.5 to about 2.25.

2. The composition of claim 1, wherein the molar ratio between the cationically ionizable lipid and the steroid is between about 1.75 and about 2.0.

3. A composition comprising:(i) a cationically ionizable lipid;(ii) a steroid;(iii) a phospholipid; and(iv) a monomer- or oligomer-grafted lipid and wherein the monomer- or oligomer-grafted lipid molecule comprises between 1 and 5 consecutive monomer repeating units; wherein the composition is substantially free of a polymer-conjugated lipid wherein the polymer moiety of the polymer-conjugated lipid has more than 5 consecutive monomer repeating units.

4. The composition of any one of claims 1 to 3, further comprising a lysophospholipid.

5. The composition of claim 4, wherein the lysophospholipid is present in the composition in an amount between about 1 mol% and about 8 mol% of the total lipid present in the composition, preferably about 3 mol% of the total lipid present in the composition.

6. The composition of claim 4 or 5, wherein the lysophospholipid is a lysophosphatidylcholine.

7. The composition of any one of claims 4 to 6, wherein the lysophospholipid is selected from the group consisting of l-heptadecanoyl-sw-glycero-3 -phosphocholine, 1- octadecanoyl-sw-glycero-3 phosphocholine, 1 -tetradecanoyl-sw-glycero-3 -phosphocholine and l-eicosanoyl-sw-glycero-3 -phosphocholine.

8. The composition of any preceding claim, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

9. The composition of any preceding claim, wherein the phospholipid is present in an amount of between 10 and 30 mol%, preferably between 15 and 25 mol%, of the total lipids present in the composition.

10. The composition of claim 9, wherein the phospholipid is DSPC and the molar contribution of DSPC is at least 10%, and the composition further comprises an additional neutral lipid, wherein the additional neutral lipid is selected from the group consisting of a monoacylglycerol, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE) or dimyristoylphosphatidylcholine (DMPC) and the molar contribution of the additional neutral lipid is at least 10%.

11. The composition according to any preceding claim, wherein the steroid is cholesterol.

12. The composition according to any preceding claim, further comprising a nucleic acid.

13. The composition according to claim 12, wherein the nucleic acid is mRNA.

14. A method of preparing the composition according to any one of claims 1 to 13, the method comprising:(a) preparing a lipid mixture comprising a cationically ionizable lipid, a steroid and a phospholipid;(b) preparing an aqueous solution comprising an acidic buffer;(c) mixing the lipid mixture prepared in (a) with the aqueous solution prepared in (b); and (d) adjusting the pH of the mixture obtained in step (c) to a pH of between 6.5 and 8;to form the composition.

15. The method according to claim 14, wherein the acidic buffer is selected from the group consisting of acetate and phosphate, optionally wherein the concentration of acidic buffer is between about 10 and about 50 mM.

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