WO2025133105A1 - Compositions and methods - Google Patents

Abstract

A composition comprising: (a) a cationically ionizable lipid; (b) cholesterol; (c) a cholesterol ester; and (d) a neutral lipid; wherein the cholesterol ester (c) is present in an amount of up to about 75 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition, is disclosed. Methods of preparing the composition, nucleic acid-lipid particle compositions and methods of preparing them using the composition, and their use in medicine, are also disclosed.

Description

COMPOSITIONS AND METHODS

Technical Field

The present disclosure relates to compositions, in particular lipid mixture compositions and nucleic acid-lipid particle compositions, such as lipid nanoparticles (LNP), to methods for producing them, and to their use in medicine.

Background to the Invention

Lipid nanoparticles (LNPs) have demonstrated huge potential as delivery technology for nucleic acid vaccines for treating a wide range of conditions, such as in cancer immunotherapy, gene therapy, and the treatment of infectious diseases.

WO2023/194508 and WO2023/193892 describe a composition, such as an LNP composition, comprising (i) a nucleic acid; (ii) a cationically ionizable lipid; (iii) a steroid; (iv) a neutral lipid; and (v) an inorganic polyphosphate, particularly a triphosphate or higher-order polyphosphate.

However, while the LNP compositions prepared according to the above publications were found to be active and tolerable, it was also found that they lack colloidal stability, specifically when exposed to freezing conditions. Without wishing to be bound by theory, it is hypothesized that a high proportion of cholesterol in the lipid mixture was the cause of this colloidal instability. In particular, lack of solubility of cholesterol is thought to create structural imperfections within the LNP disordered phase.

Use of cholesterol esters to completely replace cholesterol for the transfection of mRNA was investigated by Patel et al., Nat. Comm. (2020) 11:983; https://doi.org/10.1038/s41467-020-14527-2) and resulted in a complete loss of transfection. Patel et al. concluded that modifications of the cholesterol core region or esterification of its 3 ’-hydroxyl group are not tolerated. Paunovska et al. (2018) ^CS o 12(8) 8341-8349; doi 10.1021/acsnano.8b03640) demonstrate in vivo delivery for particles comprising cholesterol, oxidized cholesterol or esterified cholesterol. Any reported changes were modest and for hepatocytes - a central target cell type -unmodified cholesterol was found in the best performing group, so no particular advantage is ascribed to cholesterol esters.

Moreover, Patel et al. and Paunovska et al. references both carried out a complete substitution of cholesterol by its analogues. Neither document discloses that a partial substitution of cholesterol by cholesterol esters would be advantageous or result in a biologically acceptable product.

US2010/0297242 describes a low density lipoprotein (LDL)-like cationic nanoparticle for delivering a nucleic acid gene, and a method for delivering nucleic acid genes using the same. The nanoparticle comprises a lipid core part containing cholesteryl ester and triglyceride, and a cationic surface lipid part containing cholesterol, phospholipids and a cationic lipid (as defined herein), which forms a cationic surface of the lipid core part via hydrophobic interaction. The process for forming the nanoparticles involves first isolating the particles having a positive charge, which are then contacted with siRNA-PEG in a subsequent step. The siRNA is understood to be present on the surface of the particles and is accessible from the mobile phase.

Summary of the Invention

In a first aspect, the invention provides a composition, particularly although not exclusively a lipid particle composition, such as a lipid nanoparticle (LNP) composition, the composition comprising:

(a) a cationically ionizable lipid;

(b) cholesterol; and

(c) a cholesterol ester; and

(d) a neutral lipid; wherein the cholesterol ester (c) is present in an amount of up to about 75 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition. In one embodiment of the first aspect, the invention provides a composition, particularly although not exclusively a lipid particle composition, such as a lipid nanoparticle (LNP) composition, the composition comprising:

(a) a cationically ionizable lipid;

(b) cholesterol; and

(c) a cholesterol ester; and

(d) a neutral lipid; wherein the cholesterol ester (c) is present in an amount of about 5 to about 75 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition.

In one embodiment of the first aspect, the invention provides a composition, particularly although not exclusively a lipid particle composition, such as a lipid nanoparticle (LNP) composition, the composition comprising:

(a) a cationically ionizable lipid;

(b) cholesterol; and

(c) a cholesterol ester; and

(d) a neutral lipid; wherein the cholesterol ester (c) is present in an amount of about 20 to about 70 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition.

In one embodiment of the first aspect, the cholesterol ester (c) is present in an amount of about 5 to about 50 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition.

In one embodiment of the first aspect, the cholesterol ester (c) is present in an amount of about 25 to about 75 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition.

In one embodiment, the cholesterol (b) is present in an amount of at least about 15 mol% of the total lipids present in the composition.

In one embodiment, the cholesterol ester (c) is present in an amount of about 1 to about 40 mol% of the total of the lipids present in the composition. In one embodiment, the total amount of cholesterol (b) and cholesterol ester (c) is present in an amount of about 20 to about 60 mol% of the total of the lipids present in the composition.

In one embodiment, the composition is substantially free (as defined herein) of triglycerides. In one embodiment, the composition does not contain a triglyceride.

In one embodiment, the composition is substantially free (as defined herein) of a cationic lipid (as defined herein). In one embodiment, the composition does not contain a cationic lipid.

In one embodiment, the composition is substantially free (as defined herein) of dioleoylphosphatidylethanolamine (DOPE). In one embodiment, the composition does not contain DOPE.

In one embodiment, the invention provides a composition of the first aspect, further comprising a multivalent anion, for example an inorganic polyphosphate.

In one embodiment, the invention provides a composition of the first aspect, further comprising an anionic amphiphile.

In one embodiment, the invention provides a composition of the first aspect, further comprising a stealth lipid.

In a second aspect, the invention provides a nucleic acid-lipid particle composition, such as an LNP composition, comprising:

(i) the composition according to the first aspect; and

(ii) a nucleic acid.

In a third aspect, the invention provides a method of producing the nucleic acid-lipid particle composition according to the second aspect, the method comprising mixing:

(a) a cationically ionizable lipid;

(b) cholesterol;

(c) a cholesterol ester; (d) a neutral lipid; and

(e) the nucleic acid; to form the nucleic acid-lipid particle composition.

In one embodiment of the second aspect, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a stealth moiety. In one embodiment of the second aspect, the nucleic acid-lipid particle composition does not contain a stealth moiety. In one embodiment of the second aspect, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a poly(alkylene glycol)- conjugated lipid. In one embodiment of the second aspect, the nucleic acid-lipid particle composition does not contain a poly(alkylene glycol)-conjugated lipid. In one embodiment of the second aspect, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a poly(ethylene glycol)-conjugated lipid. In one embodiment of the second aspect, the nucleic acid-lipid particle composition does not contain a poly(ethylene glycol)-conjugated lipid. In one embodiment of the second aspect, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a poly(ethylene glycol) conjugated to the nucleic acid. In one embodiment of the second aspect, the nucleic acid-lipid particle composition does not contain a polyethylene glycol) conjugated to the nucleic acid.

In a fourth aspect, the invention provides a method of producing the nucleic acid- lipid particles according to the second aspect, the method comprising mixing:

(a) an organic phase comprising cationically ionizable lipid, cholesterol, cholesterol ester, and neutral lipid; and,

(b) an aqueous phase comprising a nucleic acid; to form the nucleic acid-lipid particle composition.

In a fifth aspect, the invention provides a pharmaceutical composition comprising the nucleic acid-lipid particle according to the second aspect, and a pharmaceutically acceptable carrier.

In a sixth aspect, the invention provides the nucleic acid-lipid particle according to the second aspect, for use in medicine, for example for use in treating or preventing a viral infection.

Advantages and Surprising Findings

It has surprisingly been found by the present inventors that partial replacement of cholesterol in lipid compositions, in particular LNP compositions, by a cholesterol ester, is possible and advantageous.

In particular, in contrast to the teaching of Patel et al. and Paunovska et al. which teach total replacement of cholesterol by cholesterol esters, it has surprisingly been found by the present inventors that when cholesterol in lipid mixture compositions, in particular LNP compositions, is partially replaced by a cholesterol ester, biological activity is retained.

Furthermore, it has been found that the resulting compositions, while retaining at least equivalent biological efficacy (both in the presence and absence of serum), compared with prior art compositions not containing cholesterol esters, have a much-improved colloidal stability and can be stored in liquid state for at least 28 weeks without a notable growth of the particle size or polydispersity. The compositions can also be stored in a frozen state without notable alterations of particles size of polydispersity and have an improved stability upon freeze/thaw. This represents an improvement compared with prior art compositions not containing cholesterol esters.

Furthermore, it has been found that nucleic acid-lipid particle compositions comprising cholesterol esters have a much-improved stability of the nucleic acid being encapsulated, and can be stored in liquid state for at least 28 weeks without a notable reduction in nucleic acid integrity. The materials can also be stored in frozen state without a reduction in nucleic acid integrity. In contrast, prior art compositions which do not contain cholesterol esters display a loss of nucleic acid integrity over time as is known in the art. The compositions of the present invention, such as LNP compositions, comprising cholesterol esters, are therefore particularly suitable as pharmaceutical compositions for nucleic acid therapeutics and can be expected to have a long shelf life. It has also been found that, when the cholesterol is partially replaced with cholesterol ester, the lipid particles have both a higher efficacy and a higher stability when compared with particles not containing a cholesterol ester.

Furthermore, in contrast to the compositions as described in US2010/0297242, the compositions of the present invention do not require the presence of either triglycerides or cationic lipids (as defined herein). Without being bound by theory, in contrast to the process as defined therein, which results in the nucleic acid being present on the surface of the particle, the process of the present invention results in the nucleic acid (such as mRNA) being encapsulated into the interior of the nucleic acid- lipid particle (such as a lipid nanoparticle) and not therefore accessible from the mobile phase (see e.g., Example 4, Table 14). A nucleic acid adsorbed on the surface of particles as in US2010/0297242 is prone to degradation by nucleases, can be detached from such surface by polyanions such as proteins or heparin and, upon cellular contact, exposes the nucleic acid to receptors of the innate immune systems, specifically to Toll-like-receptors, thereby provoking an immune response. The use of constitutively charged cationic lipids is associated with aggregation of such particles with blood components and resulting toxicity or a biodistribution to endothelia. See Santel A et al. (2006) Gene Therapy 13:1222-1234 or Reinsch C (2008) Strategies for the delivery of oligonucleotides in vivo. In: Kurreck, J. Therapeutic Oligonucleotides.

Brief Description of the Figures

Figure 1 is a flow diagram illustrating the method as used in Example 2 of the present invention (using cholesterol acetate as an example of a cholesterol ester), this illustrates an exemplary process by which compositions of the invention may be manufactured;

Figure 2 is a flow diagram illustrating the method as used in Example 3 of the present invention this illustrates an exemplary process by which compositions of the invention may be manufactured (using cholesterol acetate as an example of a cholesterol ester);

Figure 3 illustrates the stability of LNP compositions of the present invention comprising cholesterol acetate; Figure 4 shows the particle size of the LNPs of the matrix of formulations tested at 25%, 50%, 75% and 100% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate, with different ionizable lipids, as described in Examples 6 and 7=;

Figure 5 shows the size increase in % after 2 weeks storage at 4 °C of the LNPs of the matrix of formulations (see Example 6) tested at 25%, 50%, 75% and 100% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate;

Figure 6 shows the expression, relative to 100% cholesterol, of the matrix of formulations (see Example 6) tested at 25%, 50%, 75% and 100% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate;

Figure 7 shows the particle size of the LNPs of formulations (see Example 11) tested at 0% to 60% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate;

Figure 8 shows the size increase in % after 1 day, 2 weeks, and 4 weeks storage at 4 °C, of the LNPs of formulations (see Example 11) tested at 0% to 60% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate;

Figure 9 shows the size increase in % of the LNPs of formulations (see Example 11) tested after 1 , 2 and 3 freeze-thaw (FT) cycles at 0% to 60% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate; and

Figure 10 shows the potency, relative to % benchmark (BM) of the LNPs of formulations (see Example 11) (the benchmark formulation comprising 47.5 mol% BHD-C2C2-PipZ, 10 mol% DSPC, and 42.5% cholesterol) tested at 0% to 60% cholesterol replacement by cholesterol acetate, laurate, linoleate and oleate.

Detailed Description

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. 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.

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 of the 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; 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.

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.

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.

Definitions

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.

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 of members, 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'.

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.

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. 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%. For example, with respect to a pH value, the term “about” may in preferred instances indicate deviation from the indicated numerical value by up to 0.3. 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.

The expression "substantially free of X", as used herein, means that the composition described herein 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. Specific meanings of the term “substantially free” in relation to certain components of the composition are defined herein.

"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.

As used in the present disclosure, "mol % of the lipid mixture composition" is defined as the ratio of the number of moles of that particular lipid component to the total number of moles of all lipids in the lipid mixture composition, multiplied by 100. In this context, in some embodiments, the term "total lipid" and/or “total lipid mixture” includes lipids and lipid-like material.

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 C1-40 alkyl (such as Ce-40 alkyl, Ce-30 alkyl, C6-20 alkyl, or C 10-20 alkyl), C2-40 alkenyl (such as Ce-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.

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.

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, neo-pentyl, 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 alkyl 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 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.

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 -isobutylene, 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), i.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 carboncarbon 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 carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1 -propenyl, 2-propenyl (i.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 "alkenylene" refers to a diradical 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 alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 5 it 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 (A) configuration. Exemplary alkenylene groups include ethen-l,2-diyl, vinylidene (also called ethenylidene), 1- propen-l,2-diyl, l-propen-l,3-diyl, 1 -propen-2, 3-diyl, allylidene, l-buten-l,2-diyl, 1- buten-1, 3-diyl, l-buten-l,4-diyl, l-buten-2, 3-diyl, l-buten-2,4-diyl, l-buten-3,4-diyl, 2-buten-l,2-diyl, 2-buten- 1,3 -diyl, 2-buten-l,4-diyl, 2-buten-2, 3-diyl, 2-buten-2,4- diyl, 2-buten-3,4-diyl, and the like. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 15 up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene 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 alkenylene 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 carbon-carbon 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.

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 terms "cycloalkylene" and “cycloalkenylene” represents cyclic non-aromatic versions of "alkylene" and "alkenylene" 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 cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Exemplary cycloalkylenene groups include cyclopentenylene and cyclohexenylene.

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 (/.< ., 2-, 3-, or 4-methoxypheny 1) .

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. 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, isoindo lyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimi dinyl, 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. 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.

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 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 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. 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.

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.

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 the maximum 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.

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.

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.

The term “heteroarylalkyl” 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.

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.

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. The term “organosulfuric acid” or “sulfate” 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, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfate” is used when the group is deprotonated. Depending on the pH, the sulfate group may be protonated or deprotonated.

The term “sulfonic acid” or “sulfonate” 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.

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.

The term “dicarboxylic acid” or “dicarboxylate” 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). The term “dicarboxylate” is used when the group is deprotonated. Depending on the pH, the dicarboxylic acid may be protonated or deprotonated. The term “hydroxy carboxylic acid” or “hydroxy 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), which is substituted by one or more (preferably 1 to 5, such as 1, 2 or 3) hydroxy groups. The term “hydroxy carboxylate” is used when the group is deprotonated. Depending on the pH, the hydroxy carboxylic acid may be protonated or deprotonated.

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-OC(O)-, 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).

“Alkanoyl” means R-C(=O)- wherein R is alkyl, as defined herein.

“Alkenoyl” means R-C(=O)- wherein R is alkenyl, as defined herein.

The term “phosphate” 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. The term “phosphonate” means a compound of formula R-P(=O)(OH)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.

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

“Amine” means the group -NR2, wherein each 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.

“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.

“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.

“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 C 1-6 alkyl group.

“Amidine” means the group -C(=NR)NR’R” wherein R, 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.

A “phosphatidic acid” is a compound of formula R-C(=0)-0-CH2-CH-[0-C(=0)-R’]- CH2-O-P(=O)(OH)2, 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. 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.

A “phosphatidylserine” is a compound of formula R-C(=0)-0-CH2-CH-[0-C(=0)- 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.

A “phosphatidylglycerol” is a compound of formula R-C(=O)-O-CH2-CH-[O-C(=O)- 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.

“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 (i.e. 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.

In another embodiment, the monosaccharide moiety is a pentose moiety (i.e. it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety. In another embodiment, the carbohydrate may be a higher saccharide (i.e. 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, l,2'-glycoside bonds (which maybe l-a2’ or 1 ’-|3-2’ glycoside bonds), 1,3 '-glycoside bonds (which may be l-a-3' or 1-P- 3'-glycoside bonds), l,4'-glycoside bonds (which may be l-a-4' or l-P-4'-glycoside bonds), l,6'-glycoside bonds (which may be l-a-6' or l-P-6'-glycoside 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 (i.e. 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.

“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’, -N(R’)2, -S(0)o-2R’, -S(O)I-2OR’,

wherein X^! is independently selected from O, S, NH and N(CHs); and each R’ is independently selected from the group consisting of H, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, 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-3 alkyl), -S(O)₂NH₂-Z(CI-3 alkyl)_z, -C(=O)OH, -C(=O)O(Cl-3 alkyl), -C(=O)NH₂-Z(CI-3 alkyl)_z, -NHC(=O)(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, or propyl. 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.

Nucleic Acid

The lipid particle compositions of the present application contain an active ingredient. The active ingredient is a nucleic acid. Preferably, the lipid particle compositions of the present application contain RNA, such as mRNA. Typically, the lipid particle compositions described herein comprise lipid particles that encapsulate the 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. In one embodiment, the nucleic acid is DNA.

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 alkyl-uracil, 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-pseudouridine (mI ).

RNA

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 analogues 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 analogues 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 (z.e., naturally occurring) nucleotide residues or analogues thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded 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. The active ingredient may be mRNA, saRNA, taRNA, or mixtures thereof. The active ingredient is preferably mRNA. In some instances, the active ingredient is not siRNA.

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. mRNA

In preferred embodiments of all aspects of the disclosure, the nucleic acid is mRNA. 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). Typically the mRNA comprises: a 5 ’cap, a 5’UTR, a peptide/polypeptide/protein coding region, a 3’UTR and a poly-A tail. 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. According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

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

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 (i.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 (m h|/). 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 (mly).

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 transcription may 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.

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"). The lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. The lipid particles containing RNA as described herein may contain an mRNA encoding a replicase protein, and one or more RNA molecules capable of being replicated or amplified by the replicase. Inhibitory RNA

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.

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. 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.

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. 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.

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. 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.

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 degradation and/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. 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.

DNA

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 (z.e., naturally occurring) nucleotide residues or analogs 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.

Pharmaceutically active peptides or polypeptides

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (preferably mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system. Similarly, an RNA (such as mRNA) encodes a protein if translation of that RNA (e.g., in a cell) produces that protein.

In some embodiments, the active ingredient is 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. In some embodiments, the RNA (preferably mRNA) described in the present disclosure is capable of expressing said peptide or protein, in particular if transferred into a cell or subject. Thus, in some embodiments, the RNA (preferably mRNA) described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein. In this respect, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon. Such RNA (preferably mRNA) encoding a pharmaceutically active peptide or protein is also referred to herein as "pharmaceutically active RNA" (or "pharmaceutically active mRNA"). In some embodiments, RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides. In some embodiments, RNA (preferably mRNA) described in the present disclosure comprises a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, or more) patient-specific antigens suitable for personalized cancer therapy. In some embodiments, the lipid particle compositions comprising RNA may comprise one or more species of RNA, wherein each RNA encodes a different peptide or protein.

Preferably, the RNA (i) contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified for optimized efficacy of the RNA (e.g., increased translation efficacy, decreased immunogenicity, and/or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml\|/), and 5-methyl-uridine); and/or codon-optimization), or (iii) both (i) and (ii).

The term "pharmaceutically active peptide or protein" may be understood to mean a peptide or protein that can be used in the treatment of an individual where the expression of the peptide or protein would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or to lessen the severity of such disease or disorder.

Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, interferons, such as interferon-alpha (IFN-a), interferon beta (IFNP) or interferon-gamma (IFN-y), interleukins, such as interleukin 2 (IL2), IL-4, IL7, IL-10, IL-11, IL12, IL15, IL-21 and IL23, colony stimulating factors, such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic protein (BMP); immunoglobulin superfamily members including antibodies (e.g., IgG), T cell receptors (TCRs), chimeric antigen receptors (CARs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3y, CD3-6, CD-3c, CD79a, CD79b), co-stimulatory or inhibitory molecules e.g., CD28, CD80, CD86); other immunologically active compounds such as tumor-associated antigens, pathogen- associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.

Particles

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 or a mixture thereof.

In some embodiments, the particle contains an envelope (e.g., one or more layers or lame lias) 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 cationic or cationically ionizable lipid or a cationic polymer, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

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 45 nm to about 120 nm, from about 50 nm to about 110 nm, from about 55 nm to about 100 nm or from about 60 nm to about 90 nm.

In some embodiments, nucleic acid may be non-covalently associated with a particle. In some embodiments, the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and/or may be contained in the particle (encapsulated nucleic acid). In preferred embodiments the nucleic acid is not accessible to small molecule probes such as dyes. A standard test for encapsulation uses the RiboGreen® assay protocol, which uses the RiboGreen® dye which is a fluorescent nucleic acid stain for quantitating intact nucleic acid. Under this assay protocol, in one embodiment the nucleic acids are non-accessible to RiboGreen® by 70% or more, preferably 80% or more, more preferably 90% or more and even more preferably 95% or more.

The N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.

Lipid Nanoparticles

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., RNA (such as mRNA), DNA 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.

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 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 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 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 45 nm to about 120 nm, from about 50 nm to about 110 nm, from about 55 nm to about 100 nm or from about 60 nm to about 90 nm. Lipid Mixture Composition

The present invention provides in one aspect a composition as defined herein. The composition comprises a cationically ionizable lipid, cholesterol; a cholesterol ester; and a neutral lipid.

In one embodiment, the composition takes the form of a mixture of lipids including a cationically ionizable lipid, cholesterol; a cholesterol ester; and a neutral lipid, as defined herein. The composition may also include additional lipids, such as grafted lipids or anionic amphiphiles or further compounds, such as multivalent anions, as described in more detail below.

This composition, in the absence of any nucleic acid, is also referred to herein as “the lipid mixture composition”.

In one embodiment, the lipid mixture composition is substantially free (as defined herein) of triglycerides (as defined below). In one embodiment, the lipid mixture composition does not contain a triglyceride.

In one embodiment, the lipid mixture composition is substantially free (as defined herein) of cationic lipids (as defined herein). In one embodiment, the lipid mixture composition does not contain a cationic lipid.

Method of Forming Lipid Mixture Composition

In a further aspect, the present invention provides methods for producing the lipid mixture composition of the invention.

In one aspect, there is provided a method of producing the lipid mixture composition as defined herein, the method comprising mixing:

(a) a cationically ionizable lipid;

(b) cholesterol;

(c) cholesterol ester; and (d) a neutral lipid; to form the lipid mixture composition.

The mixing can be carried out using methods well known to those skilled in the art.

In one embodiment, the method is carried out as a continuous process. As is known to the person skilled in the art, a continuous process (also known as a continuous flow process) typically involves the materials that are being processed are continuously undergoing a procedure, either undergoing mixing (to form the lipid mixture) or undergoing any of the processing steps as defined herein. Continuous processing is typically contrasted with batch production, which, as is known to the person skilled in the art is a method of manufacturing where the products are typically made as specified groups or amounts, within a time frame.

Typically, a continuous process means that the method operates 24 hours per day, seven days per week. The continuous process may undergo occasional shutdowns, such as for maintenance.

Nucleic Acid-Lipid Particle Composition

The present disclosure further provides a lipid particle comprising the composition as defined herein and a nucleic acid. In one embodiment, there is provided a lipid particle obtained or obtainable by the methods defined herein. Such particles are also referred to herein as “nucleic acid-lipid particles”. When the nucleic acid is RNA, such particles are also referred to herein as “RNA-lipid particles”.

In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA, saRNA, taRNA, or mixtures thereof. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA which encodes for one or more personalized (i.e., patientspecific) cancer antigens. In one embodiment, the nucleic acid is RNA which encodes for one or more immunologically active proteins, such as a tumour-associated antigen, or a pathogen-associated antigen (such as a bacterial, parasitic, or viral antigen). In the present disclosure, it is preferred that the nucleic acid-lipid particle is a lipid nanoparticle (LNP). The function of the LNP is to stabilise and encapsulate the nucleic acid to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol. The LNPs and/or their lipid components may have adjuvant activity.

In the present disclosure, LNPs may be understood as oil-in-water emulsions. LNPs thus typically comprise a central complex of mRNA and lipid embedded in a disordered, non-lamellar phase made of lipid. This is in contrast to the structure of a liposome which comprises unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers surrounding an encapsulated aqueous lumen. In some instances, the nucleic acid-lipid particles described herein are not liposomes. In some instances, the nucleic acid-lipid particles described herein are not lipoplexes.

Lipid nanoparticles (LNP) are obtainable from combining a nucleic acid with lipids, such as the lipid mixture composition of the present invention. 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. The LNP typically comprise an electron-dense core.

The nucleic acid-lipid particle composition may also include one or more stabilising ingredients, the function of which is to further stabilise the nucleic acid-lipid particle. In one embodiment, the stabilising ingredient is an anionic amphiphile, as defined and exemplified below. In one embodiment, the stabilising ingredient is a grafted lipid, as defined and exemplified below. In one embodiment, the stabilising ingredient is a multivalent anion, such as an inorganic polyphosphate, as defined and exemplified below.

In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter that in some embodiments ranges from about 40 nm to about 1000 nm, from about 40 nm to about 800 nm, from about 40 nm to about 700 nm, from about 40 nm to about 600 nm, from about 40 nm to about 500 nm, from about 40 nm to about 450 nm, from about 40 nm to about 400 nm, from about 40 nm to about 350 nm, from about 40 nm to about 300 nm, from about 40 nm to about 250 nm, from about 40 nm to about 200 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of less than lOOnm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 40 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 50 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 10 to about 1000 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 15 to about 500 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 20 to about 400 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 25 to about 300 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 30 to about 250 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 40 to about 200 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 45 to about 150 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 50 to about 100 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 45 nm to about 120 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 50 nm to about 110 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 55 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (such as lipid nanoparticles) as described herein have an average diameter of from about 60 nm to about 90 nm. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined herein) of triglycerides (as defined below). In one embodiment, the nucleic acid-lipid particle composition does not contain a triglyceride.

In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a cationic lipid (as defined herein). In one embodiment, the nucleic acid-lipid particle composition does not contain a cationic lipid.

In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a stealth moiety. In one embodiment, the nucleic acid-lipid particle composition does not contain a stealth moiety. In one embodiment, the nucleic acid- lipid particle composition is substantially free (as defined herein) of a compound containing a poly(alkylene glycol) moiety. In one embodiment, the nucleic acid-lipid particle composition does not contain a compound containing a poly(alkylene glycol) moiety. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined herein) of a compound containing a poly(ethylene glycol) moiety. In one embodiment, the nucleic acid-lipid particle composition does not contain a compound containing a poly(ethylene glycol) moiety.

Method of Forming Nucleic Acid-Lipid Particle Composition

In a further aspect, the present disclosure provides methods for producing the nucleic acid-lipid particle compositions as disclosed herein.

Thus, in one embodiment, the method comprises mixing:

(a) a cationically ionizable lipid;

(b) cholesterol;

(c) a cholesterol ester;

(d) a neutral lipid; and

(e) a nucleic acid; to form the nucleic acid-lipid particle composition.

In some embodiments, lipids, such as the lipid mixture composition, are introduced into the mixture in solution in an organic solvent. Examples of suitable organic solvents include alcohols (in particular aliphatic alcohols) having up to 6 carbon atoms, ketones having up to 6 carbon atoms (such as acetone), and mixtures thereof. In preferred embodiments, the organic solvent is completely miscible with water. In some embodiments, the organic solvent is selected from the group consisting of ethanol, propanol, isopropanol, 1 ,2-propanediol, and mixtures of two or more of these alcohols. Preferably, the organic solvent is ethanol.

In some embodiments, the nucleic acid is introduced into the mixture in aqueous solution.

In some embodiments, the methods of forming the nucleic acid-lipid particle composition employ an aqueous acid. In one embodiment, the aqueous solution containing the nucleic acid is acidified.

Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2018/081480, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, W02011/141705, and WO 01/07548, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

For example, in some embodiments, lipids that are useful for delivery of nucleic acids are solubilized in ethanol, at a pre-determined weight or molar ratios/percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles (LNP) are prepared at a total lipid to nucleic acid (e.g. RNA or DNA) molar ratio of approximately 6:1 to 30:1. In some embodiments, such RNA or DNA can be diluted to 0.2 mg/mL in acetate buffer.

In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising a nucleic acid (e.g., RNA or DNA) can be formed as follows: an ethanol solution comprising lipids, comprising a cationically ionizable lipid, cholesterol, a cholesterol ester, a neutral lipid, and, optionally, other ingredients such as a grafted lipid or an anionic amphiphile), is injected into an aqueous solution comprising a nucleic acid (e.g., RNA or DNA).

In some embodiments, lipid and nucleic acid solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationically ionizable lipid, cholesterol, a cholesterol ester, a neutral lipid, and, optionally, other ingredients such as a grafted lipids or an anionic amphiphile) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid.

In some embodiments, the method comprises further subjecting the nucleic acid-lipid particle to one or more further processing steps.

In some embodiments, the method further comprises the addition of water immediately after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 1 minute after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 30 seconds after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 20 seconds after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 10 seconds after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 5 seconds after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 2 seconds after mixing the lipids with the nucleic acid. In some embodiments, water is added less than 1 second after mixing the lipids with the nucleic acid.

In some embodiments, the method further comprises the addition of a neutralizing buffer, such as Tris or HEPES or phosphate buffer, immediately after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 1 minute after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 30 seconds after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 20 seconds after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 10 seconds after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 5 seconds after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 2 seconds after mixing the lipids with the nucleic acid. In some embodiments, the neutralizing buffer is added less than 1 second after mixing the lipids with the nucleic acid. In some embodiments, the resulting pH of the composition following addition of the neutralizing buffer is between pH 7.0 and pH 8.0.

In some embodiments, the method further comprises the step of addition of a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) immediately after mixing the lipids with the nucleic acid. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) has a concentration of 1 mM to 100 mM. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) has a concentration of 5 mM to 20 mM. In one embodiment, the solution containing the multivalent anion (such as an inorganic polyphosphate) has a pH of 7.0 to 9.0. In one embodiment, the solution containing the multivalent anion (such as an inorganic polyphosphate) has a pH of 7.5 to 8.5. In some embodiments, the resulting pH of the composition following addition of the solution containing the multivalent anion (such as an inorganic polyphosphate) is between pH 7.0 and pH 8.0. Typically, prior to the further processing steps, the nucleic acid-lipid particle composition is in the form of a raw colloid. In some embodiments, the method further comprises the step of addition of a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) to the raw colloid. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) has a concentration of 25 mM to 2500 mM. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) has a concentration of 100 mM to 500 mM. This step is preferred when water is added immediately after the lipids and the nucleic acid are mixed.

In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 1 minute after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 30 seconds after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 20 seconds after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 10 seconds after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 5 seconds after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 2 seconds after mixing the lipids with the nucleic acid. In some embodiments, a solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is added less than 1 second after mixing the lipids with the nucleic acid. In some embodiments, the resulting pH of the composition following addition of the solution containing a multivalent anion (as defined below, either in its broadest aspect or a preferred aspect, such as an inorganic polyphosphate) is between pH 7.0 and pH 8.0. In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle composition to one or more purification and/or concentration steps. In one embodiment, the purification step comprises a dialysis or filtration step. In one embodiment, the dialysis or filtration step comprises tangential flow filtration. In one embodiment, the concentration step comprises tangential flow filtration or pressured ultrafiltration. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particle composition to a filtration or dialysis step. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particle composition to a tangential flow filtration step.

In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle composition to one or more dilution steps. In one embodiment, the one or more dilution steps comprise addition of storage matrix.

In one embodiment, the method further comprises the step of sterile filtration of the nucleic acid-lipid particle.

In one embodiment, the nucleic acid-lipid particles are not subjected to any further purification steps.

In one embodiment, the method of producing the nucleic acid-lipid particle is carried out as a continuous process, as described generally herein.

Lipids and Amphiphiles

The lipid mixture compositions and nucleic acid-lipid particle 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.

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. 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”.

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.

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.

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, dioxolane, 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.

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

5 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.

In one embodiment, the compositions of the present invention do not comprise triglycerides. As used herein, the term “triglyceride” takes its normal meaning in the art of an ester derived from glycerol and three fatty acids, the fatty acyl moieties providing the hydrophobic moieties of the lipid. As is known to the person skilled in the art, the fatty acyl moieties which esterify the glycerol moiety may be the same or different. Typically, the acyl moieties which esterify the hydroxyl moieties of the glycerol backbone may be alkanoyl groups (as defined herein) having 4 to 40, such as

6 to 30, such as 12 to 20 carbon atoms, or alkenoyl groups (as defined herein) having 4 to 40, such as 6 to 30, such as 12 to 20 carbon atoms.

Cationically Ionizable Lipids

The lipid mixture compositions and nucleic acid-lipid particle compositions of the present invention also contain a cationically ionizable lipid, or a mixture of any thereof. 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. When present in lipid nanoparticles, a substantial portion, e.g. at least half, of the cationically ionizable lipid is protonated at a pH below 7. Conversely, at least half of the cationically ionizable lipid or less is not protonated at a pH above 7. In some embodiments, the cationically ionizable lipid comprises 1, 2 or 3, such as 1 or 2, such as 1, nitrogen atom (N) which is capable of being protonated below pH 7.

A large number of different types of cationically ionizable lipids are known in the art and can be prepared according to techniques well established to the skilled person.

In one embodiment, the cationically ionizable lipid is a compound represented by formula (TL-I):

/L¹— X¹- T¹

G- L³- N 2- X²- T²

TL-I or a pharmaceutically acceptable salt thereof, wherein:

L¹ and L² are each independently an optionally substituted C1-C30 aliphatic group;

L³ is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10-membered heteroaliphatic group;

X¹ and X² are each independently selected from a bond, -OC(O)-, -C(O)O-, - S(O)₂N(R^!)-, -N(R^!)S(O)₂, -S(O)-, -S(O)₂-, -S(O)₂C(R^!)₂-, -OC(S)C(R^!)₂-, - C(R¹)2C(S)O-, and -S-, wherein one or both of X¹ or X² is selected from - S(O)₂N(R¹ )-, -N(R^!)S(O)₂, -S(O)-, -S(O)₂-, -S(O)₂C(R¹)₂-, -OC(S)C(R^!)₂-, - C(R¹)₂C(S)O-, and -S-; each R¹ is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

T¹ and T² are each independently an optionally substituted C3-C30 aliphatic; G is -N(R²)C(S)N(R²)₂, -N⁺(R³)₃, -OH, -N(R²)₂, -N(R⁵)C(O)R³, -N(R⁵)S(O)₂R³, - N(R⁵)C(O)N(R³)₂, -CH(N-R²), or-R⁴; each R² is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic or OR³; or two instances of R² come together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring or an optionally substituted 4- to 12-membered heteroaryl ring; each R³ is, independently, at each instance, selected from the group consisting of H and optionally substituted C1-C10 aliphatic; and

R⁴ is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, Ce-Ci₂ aryl substituted with one or more of-(CH₂)o- 6-OH or -(CH₂)o-6-N(R⁵)₂, or C3-Ci₂ cycloaliphatic substituted with one or more of oxo, -(CH₂)O-6-OH, or -(CH₂)o-6-N(R⁵)₂; each R⁵ is independently selected from H and optionally substituted Ci-Ce aliphatic.

In some embodiments of formula (TL-I), L¹ and L² are each independently -(CH₂)e- io-.

In some embodiments of formula (TL-I), X^! and X² are each independently selected from a -S(0)₂N(R^!)-, and -S(0)₂

In some embodiments of formula (TL-I), X¹ and X² are each -S(0)₂N(R^!)-, where each R¹ is independently R¹ is Ci-Cio aliphatic.

In some embodiments of formula (TL-I), X¹ and X² are each -S(0)₂.

In some embodiments of formula (TL-I), T^! and T² are each independently selected from optionally substituted C.3-C₂o alkyl.

In some embodiments of formula (TL-I), G is -N(H)C(S)N(R²)₂, where each R² is selected from optionally substituted Ci-Ce aliphatic and OH.

In some embodiments of formula (TL-I), G is -OH. In some embodiments of formula (TL-I), -L³-G is selected from:

In some embodiments of formula (TL-I), the compound is 7,7 ’-((4- hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane- 1 -sulfonamide)

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (TL-I), the compound is 7 ,7’-((4-(3,3- dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane- 1 -sulfonamide)

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (TL-I), the compound is

or a pharmaceutically acceptable salt thereof.

Thiolipid compounds of formula (TL-I) can be prepared according to PCT/EP2023/071270, the contents of which are incorporated herein by reference. In one embodiment, the cationically ionizable lipid is selected from the group consisting of:

7,7 ’ -((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane- 1 -sulfonamide)

7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-l-

the compound having the structure

[(4-hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ALC-315);

((3-hydroxypropyl)azanediyl)bis(nonane-9, 1 -diyl) bis(2 -butyloctanoate) (ALC-366);

1.2-dioleoyloxy-3-dimethylaminopropane (DODMA);

2.2-dilinoleyl-4-dimethylaminoethyl-[ 1 ,3]-dioxolane (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); di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); bis-( -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);

O-[N- {(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)} -N- {7-pentadecylcarbonyloxyoctyl} - amino]4-(dimethylamino)butanoate (HY501 );

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);

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

(2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2- hexyldecanoate) (HY-405); di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate

described in

US2022/0218622A1); bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate

described in US2022/0218622A1); or a mixture of any thereof.

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

7,7 ’ -((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylheptane- 1 -sulfonamide) (BNT -

51); 7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-l- sulfonamide) (BNT-52); the compound having the structure

[(4-hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ALC-315);

((3-hydroxypropyl)azanediyl)bis(nonane-9, 1 -diyl) bis(2 -butyloctanoate) (ALC-366);

1.2-dioleoyloxy-3-dimethylaminopropane (DODMA);

2.2-dilinoleyl-4-dimethylaminoethyl-[ 1 ,3]-dioxolane (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); 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);

O-[N- {(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)} -N- {7-pentadecylcarbonyloxyoctyl} - amino]4-(dimethylamino)butanoate (HY 501);

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

(2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2- hexyldecanoate) (HY-405); di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMe-Pyr); bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-l-yl)ethyl)azanediyl)dipropionate (BODD- C2C2-Pyr); bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-lMePyr); bis(2-octyldodecyl) 3,3'-(((l-methylpiperidin-3-yl)methyl)azanediyl)dipropionate

(BODD-C2C2- 1 Me-3PipD); bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD- C2C2-DMA); bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-

C2C4-Pyr); bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate

(BHD-C2C4-PipZ); di(nonadecan-9-yl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate

(DND-C2-C4-PipZ); or a mixture of any thereof.

In one preferred embodiment, the cationically ionizable lipid is selected from the group consisting of:

7,7 ’ -((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylheptane- 1 -sulfonamide) (BNT - 51);

7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-l- sulfonamide) (BNT-52);

[(4-hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2 -hexyldecanoate) (ALC-315);

1.2-dioleoyloxy-3-dimethylaminopropane (DODMA);

2.2-dilinoleyl-4-dimethylaminoethyl-[ 1 ,3]-dioxolane (DLin-KC2-DMA); heptatriaconta-6 ,9,28,31 -tetraen- 19-y l-4-(dimethy lamino)butanoate (D-Lin-MC 3 - DMA);

1.2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319);

A/.s-(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);

O-[N- {(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)} -N- {7-pentadecylcarbonyloxyoctyl} - amino]4-(dimethylamino)butanoate (HY501 ); di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)-dipropionate (BODD-C2C2-lMe-Pyr); bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD- C2C2-DMA); or a mixture of any thereof.

In one embodiment, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl]- di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationically ionizable lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2- butyloctanoate) (ALC-366). In one embodiment, the cationically ionizable lipid is 1 ,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-y l-4-(dimethy lamino)butanoate (D-Lin-MC 3 - 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- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In one embodiment, the cationically ionizable lipid is bis-(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 O-[N-{(9Z,12Z)-octadeca-9,l 2-dien- 1- yl)} -N- {7-pentadecylcarbonyloxyoctyl} -amino]4-(dimethylamino)butanoate (HY501). In one embodiment, the cationically ionizable lipid is 2-(di-((9Z,12Z)- octadeca-9,12-dien- l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2). In one embodiment, the cationically ionizable lipid is 7,7’-((4-hydroxybutyl)azanediyl)- bis(N-hexyl-N-octylheptane-l -sulfonamide) (BNT-51). In one embodiment, the cationically ionizable lipid is 7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N- hexyl-N-octylheptane-1 -sulfonamide) (BNT-52). In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2- 1 Me-Pyr.

In one embodiment, the cationically ionizable lipid is the compound having the structure

In some embodiments, the cationically ionizable lipid is selected from those described generally and specifically in WO 2018/087753.

In some embodiments, the cationically ionizable lipid is selected from the group consisting of:

Hy 501: m.w: 761.26 In one embodiment, the cationically ionizable lipid is 4-((di-((9Z,12Z)-octadeca-9,12- dien-l-yl)amino)oxy)-A,A-dimethyl-4-oxobutan-4-amine (HYAM-2). In one embodiment, the cationically ionizable lipid is ((2-(4-(dimethylamino)butanoyl)- oxy)ethyl)-azanediylbis(octane 8,1 -diyl) bis(2-hexyldecanoate) (EA-405). In one embodiment, the cationically ionizable lipid is (2-(4-(dimethylamino)butanoyl)- oxy)azanediylbis-(octane 8,1 -diyl) bis(2 -hexyldecanoate) (HY-405). 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 (HY501).

In one embodiment of the lipid mixture composition, the cationically ionizable lipid is present in an amount of about 10 to about 70 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cationically ionizable lipid is present in an amount of about 20 to about 70 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cationically ionizable lipid is present in an amount of about 30 to about 60 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cationically ionizable lipid is present in an amount of about 40 to about 50 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the nucleic acid-lipid particle composition, the cationically ionizable lipid is present in an amount of about 10 to about 70 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cationically ionizable lipid is present in an amount of about 20 to about 70 mol% of the total lipids present in the nucleic acid- lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cationically ionizable lipid is present in an amount of about 30 to about 60 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cationically ionizable lipid is present in an amount of about 40 to about 50 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the lipid mixture compositions of the invention are substantially free (as defined herein) of cationic lipids. In one embodiment, the lipid mixture compositions of the invention do not contain a cationic lipid.

In one embodiment, the nucleic acid-lipid particle compositions of the invention are substantially free (as defined herein) of cationic lipids. In one embodiment, the nucleic acid-lipid particle compositions of the invention do not contain a cationic lipid.

As used herein, the term “cationic lipid” means a lipid or lipid-like material, as defined herein, having a constitutive positive charge. In this context a “constitutive charge” means that the cationic lipid carries the positive charge at all physiological pH. The cationic lipids carrying constitutive charged cationic moieties are typically quaternary ammonium salts or salts of organic bases, such as nitrogen-containing bases. Typically, such organic bases are strong bases (i.e. bases which are completely protonated when dissolved in a solvent, such as but not limited to an aqueous solvent, such that the concentration of the unprotonated species is too low to be measured).

Cholesterol

The lipid mixture compositions and the nucleic acid-lipid particle compositions of the present invention also comprise a steroid. The steroid is cholesterol. As also detailed further herein, the lipid mixture compositions and the nucleic acid-lipid particle compositions of the present invention also comprise a cholesterol ester, which also has the cholesterol steroid ring structure.

In one embodiment of the lipid mixture compositions, the cholesterol is present in an amount of at least about 15 mol% of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 50 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 20 mol % to about 35 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 6 mol % to about 48 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 13 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 32 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 23 mol % to about 48 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 19 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 32 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 13 mol % to about 26 mol % of the total lipids present in the lipid mixture composition. In one embodiment, the cholesterol is present in an amount ranging from about 6 mol % to about 13 mol % of the total lipids present in the lipid mixture composition.

In one embodiment of the nucleic acid-lipid particle compositions, the cholesterol is present in an amount of at least about 15 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 50 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 20 mol % to about 35 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 6 mol % to about 48 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 13 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 32 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 23 mol % to about 48 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 19 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 15 mol % to about 32 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 13 mol % to about 26 mol % of the total lipids present in the nucleic acid- lipid particle composition. In one embodiment, the cholesterol is present in an amount ranging from about 6 mol % to about 13 mol % of the total lipids present in the nucleic acid-lipid particle composition.

Cholesterol Ester

The lipid mixture compositions and the nucleic acid-lipid particle compositions of the present invention also comprise a cholesterol ester (also known as a cholesteryl ester).

In this specification, the term “cholesterol ester” takes its normal meaning in the art as meaning an ester of general formula R-C(=O)-O-Chol, where Choi is a cholesteryl residue (the oxygen atom being present at the 3 -position of the sterol residue) and R is an organic group.

The organic group R may be a hydrocarbyl group or a heterohydrocarbyl group, as defined above. Where the organic group R is a hydrocarbyl group, it 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 organic group R is a heterohydrocarbyl group, it may be alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect).

In one embodiment, the organic group R is an aliphatic group. In one embodiment, the organic group R is an alkyl group. In one embodiment, the organic group R is a straight-chain alkyl group. In one embodiment, the organic group R is an alkenyl group. In one embodiment, the organic group R is a straight-chain alkenyl group. In one embodiment, the organic group R has from 1 to 40 carbon atoms. In one embodiment, the organic group R has from 1 to 30 carbon atoms. In one embodiment, the organic group R has from 1 to 20 carbon atoms. In one embodiment, the organic group R has from 2 to 20 carbon atoms.

In one embodiment, the organic group R is an alkyl group having from 1 to 40 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 40 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 30 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 30 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 27 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 27 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 23 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 23 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 20 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 20 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 12 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 12 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 10 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 10 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 8 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 8 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 6 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 6 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 5 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 5 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 1 to 4 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 1 to 4 carbon atoms. In one embodiment, the organic group R is a alkyl group having from 9 to 23 carbon atoms. In one embodiment, the organic group R is a straightchain alkyl group having from 9 to 23 carbon atoms. In one embodiment, the organic group R is an alkyl group having from 11 to 17 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having from 11 to 17 carbon atoms. In one embodiment, the organic group R is an alkyl group having 11 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having 11 carbon atoms. In one embodiment, the organic group R is an alkyl group having 15 carbon atoms. In one embodiment, the organic group R is a straight-chain alkyl group having 15 carbon atoms. In one embodiment, the organic group R is an alkyl group having 17 carbon atoms. In one embodiment, the organic group R is a straightchain alkyl group having 17 carbon atoms. In one embodiment, the organic group R is a methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1 ,2-dimethylpropyl, or iso-amyl group. Preferably, the organic group R is a methyl group. In one embodiment, the organic group R is an n- undecyl, n-tridecyl, n-pentadecyl, or n-heptadecyl group. Preferably, the organic group R is an n-pentadecyl or n-heptadecyl group.

In one embodiment, the organic group R is an alkenyl group having from 2 to 40 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 2 to 40 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 2 to 30 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 2 to 30 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 2 to 20 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 2 to 20 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 2 to 10 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 2 to 10 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 2 to 6 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 2 to 6 carbon atoms. In the above embodiments, the alkenyl group may typically have 1 to 6, such as 1 to 4, such as 1, 2 or 3 carbon-carbon double bonds (provided that the total number of carbons permit this).

In one embodiment, the organic group R is an alkenyl group having from 11 to 23 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 11 to 23 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 13 to 21 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 13 to 21 carbon atoms. In one embodiment, the organic group R is an alkenyl group having from 15 to 19 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having from 15 to 19 carbon atoms. In one embodiment, the organic group R is an alkenyl group having 17 carbon atoms. In one embodiment, the organic group R is a straight-chain alkenyl group having 17 carbon atoms. In the above embodiments, the alkenyl group may typically have 1, 2 or 3 carbon-carbon double bonds. In one embodiment, the organic group R is a straight-chain alkenyl group having 17 carbon atoms and one carbon-carbon double bond. In one embodiment, the organic group R is a straight-chain alkenyl group having 17 carbon atoms and two carbon-carbon double bonds. The carbon-carbon double bonds may be in the cis (Z)- or trans (£)- stereochemistry. Preferably the carbon-carbon double bonds are in the cis (Z)- stereochemistry.

In one embodiment, the organic group R is an alkynyl group having from 2 to 40 carbon atoms. In one embodiment, the organic group R is an alkynyl group having from 2 to 30 carbon atoms. In one embodiment, the organic group R is an alkynyl group having from 2 to 20 carbon atoms. In one embodiment, the organic group R is an alkynyl group having from 2 to 10 carbon atoms. In one embodiment, the organic group R is an alkynyl group having from 2 to 6 carbon atoms.

In one embodiment, the organic group R is a cycloalkyl group having from 3 to 40 carbon atoms. In one embodiment, the organic group R is a cycloalkyl group having from 3 to 30 carbon atoms. In one embodiment, the organic group R is a cycloalkyl group having from 3 to 20 carbon atoms. In one embodiment, the organic group R is a cycloalkyl group having from 3 to 10 carbon atoms. In one embodiment, the organic group R is a cycloalkyl group having from 3 to 6 carbon atoms.

In one embodiment, the organic group R is a cycloalkenyl group having from 3 to 40 carbon atoms. In one embodiment, the organic group R is a cycloalkenyl group having from 3 to 30 carbon atoms. In one embodiment, the organic group R is a cycloalkenyl group having from 3 to 20 carbon atoms. In one embodiment, the organic group R is a cycloalkenyl group having from 3 to 10 carbon atoms. In one embodiment, the organic group R is a cycloalkenyl group having from 3 to 6 carbon atoms.

In one embodiment, the organic group R is an alkylcycloalkyl group having a total of from 4 to 40 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkyl group having a total of from 4 to 30 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkyl group having a total of from 4 to 20 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkyl group having a total of from 4 to 10 carbon atoms.

In one embodiment, the organic group R is an alkylcycloalkylalkyl group having a total of from 5 to 40 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkylalkyl group having a total of from 5 to 30 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkylalkyl group having a total of from 5 to 20 carbon atoms. In one embodiment, the organic group R is an alkylcycloalkylalkyl group having a total of from 5 to 10 carbon atoms.

In one embodiment, the organic group R is an aryl group having from 5 to 40 carbon atoms. In one embodiment, the organic group R is an aryl group having from 5 to 30 carbon atoms. In one embodiment, the organic group R is an aryl group having from 6 to 18 carbon atoms. In one embodiment, the organic group R is an aryl group having from 6 to 14 carbon atoms. In one embodiment, the organic group R is an aryl group having from 6 to 10 carbon atoms.

In one embodiment, the organic group R is an alkylaryl group having a total of from 6 to 40 carbon atoms. In one embodiment, the organic group R is an alkylaryl group having a total of from 6 to 30 carbon atoms. In one embodiment, the organic group R is an alkylaryl group having a total of from 6 to 20 carbon atoms. In one embodiment, the organic group R is an alkylaryl group having a total of from 6 to 10 carbon atoms.

In one embodiment, the organic group R is an alkylaryl group having a total of from 6 to 20 carbon atoms. In one embodiment, the organic group R is an arylalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the organic group R is an arylalkyl group having a total of from 6 to 30 carbon atoms. In one embodiment, the organic group R is an arylalkyl group having a total of from 6 to 20 carbon atoms. In one embodiment, the organic group R is an arylalkyl group having a total of from 6 to 10 carbon atoms.

In one embodiment, the organic group R is an alkylarylalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the organic group R is an alkylarylalkyl group having a total of from 8 to 30 carbon atoms. In one embodiment, the organic group R is an alkylarylalkyl group having a total of from 8 to 20 carbon atoms.

In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester (i.e. the organic group R is an alkyl group), the alkanoyl part having from 2 to 30 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 30 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 28 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 28 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 20 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 20 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 10 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 10 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 6 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 2 to 6 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 10 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 10 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 12 to 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 12 to 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having 12 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having 12 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having 16 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having 16 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having 18 carbon atoms. Examples of such cholesterol esters include cholesterol acetate, cholesterol propionate, cholesterol butyrate, cholesterol valerate, cholesterol caproate, cholesterol enanthate, cholesterol caprylate, cholesterol pelargonate, cholesterol caprate, cholesterol undecanoate, cholesterol laurate, cholesterol myristate, cholesterol palmitate, cholesterol stearate, and cholesterol arachidate. In one preferred embodiment, the cholesterol ester is cholesterol acetate. In one preferred embodiment, the cholesterol ester is cholesterol laurate. In one preferred embodiment, the cholesterol ester is cholesterol palmitate. In one preferred embodiment, the cholesterol ester is cholesterol stearate.

In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester (i.e. the organic group R is an alkenyl group), the alkenoyl part having from 3 to 30 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part being straight-chain and having from 3 to 30 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part having from 3 to 28 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part being straight-chain and having from 3 to 28 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkanoyl part having from 12 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part being straight-chain and having from 12 to 24 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part having from 14 to 20 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkanoyl part being straight-chain and having from 14 to 20 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part having 16 or 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part being straight-chain and having 16 or 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part having 16 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part being straight-chain and having 16 carbon atoms. . In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkenoyl part having 18 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkenoyl ester, the alkanoyl part being straight-chain and having 18 carbon atoms. In each of the above embodiments, the alkenoyl groups may have 1 to 6, such as 1 to 4, preferably 1, 2 or 3, more preferably 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in the cis (Z)- or trans (E)- stereochemistry. Preferably the carbon-carbon double bonds are in the cis (Z)-stereochemistry. Examples of such cholesterol esters include cholesterol oleate, cholesterol linoleate, cholesterol ricinoleate, cholesterol linolenate, cholesterol arachidonate, cholesterol linolelaidate, cholesterol elaidate, cholesterol erucate, cholesterol myristoleate, cholesterol palmitoleate, cholesterol vaccenate, and cholesterol sapienate. In one preferred embodiment, the cholesterol ester is cholesterol oleate. In one preferred embodiment, the cholesterol ester is cholesterol linoleate. In one embodiment, the cholesterol ester is not cholesterol oleate.

In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 1 to about 40 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 3 to about 40 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 4 to about 30 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 6 to about 26 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 10 to about 20 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 10 to about 15 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 15 to about 20 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of 3 to 5 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 6 to about 13 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 10 to about 21 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 13 to about 26 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 19 to about 40 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 1 to about 40 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 3 to about 40 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 4 to about 30 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 6 to about 26 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 10 to about 20 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 10 to about 15 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 15 to about 20 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 3 to about 5 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 6 to about 13 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 10 to about 21 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 13 to about 26 mol% of the total lipids present in the nucleic acid lipid particle composition. In one embodiment of the nucleic acid lipid particle composition, the cholesterol ester is present in an amount of about 19 to about 40 mol% of the total lipids present in the nucleic acid lipid particle composition.

In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 5 to about 50 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 10 to about 30 % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 20 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol linoleate and is present in an amount of about 5 to about 20 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol linoleate and is present in an amount of about 8 to about 12 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol linoleate and is present in an amount of about 10 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol laurate and is present in an amount of about 5 to about 20 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol laurate and is present in an amount of about 8 to about 12 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol laurate and is present in an amount of about 10 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of up to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition (i.e. the cholesterol ester replaces up to about 75 mol% of the cholesterol that would normally be present in the lipid mixture composition). In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 5 to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 5 to about 50 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 10 to about 30 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 5 to about 20 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 23 to about 27 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 25 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 20 to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition (i.e. the cholesterol ester replaces about 20 to about 75 mol% of the cholesterol that would normally be present in the lipid mixture composition). In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 20 to about 70 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 25 to about 65 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 30 to about 60 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 25 to about 45 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 30 to about 40 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 30 to about 50 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 35 to about 45 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 38 to about 42 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is present in an amount of about 40 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In any of these embodiments, in one embodiment the cholesterol ester is cholesterol acetate. In one embodiment the cholesterol ester is cholesterol laurate. In one embodiment the cholesterol ester is cholesterol oleate. In one embodiment the cholesterol ester is cholesterol linoleate.

In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 20 to about 70 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 25 to about 65 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 30 to about 60 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 25 to about 45 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the cholesterol ester is cholesterol acetate and is present in an amount of about 30 to about 40 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of up to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition (i.e. the cholesterol ester replaces up to about 75 mol% of the cholesterol that would normally be present in the nucleic acid-lipid particle composition). In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 5 to about 50 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 10 to about 30 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 5 to about 20 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 23 to about 27 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 25 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition.

In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 20 to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition (i.e. the cholesterol ester replaces about 20 to about 75 mol% of the cholesterol that would normally be present in the nucleic acid-lipid particle composition). In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 20 to about 70 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 25 to about 65 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 30 to about 60 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 25 to about 45 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 30 to about 40 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 30 to about 50 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 35 to about 45 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 38 to about 42 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the cholesterol ester is present in an amount of about 40 mol% of the total of cholesterol and cholesterol ester present in the nucleic acid-lipid particle composition. In any of these embodiments, in one embodiment the cholesterol ester is cholesterol acetate. In embodiment the cholesterol ester is cholesterol laurate. In one embodiment the cholesterol ester is cholesterol oleate. In one embodiment the cholesterol ester is cholesterol linoleate.

In one embodiment of the lipid mixture composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 20 to about 60 mol% of the total of the lipids in the lipid mixture composition. In one embodiment of the lipid mixture composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 35 to about 55 mol% of the total of the lipids in the lipid mixture composition. In one embodiment of the lipid mixture composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 45 to about 55 mol% of the total of the lipids in the lipid mixture composition. In one embodiment of the lipid mixture composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 40 mol% of the total of the lipids in the lipid mixture composition. In one embodiment of the lipid mixture composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 50 mol% of the total of the lipids in the lipid mixture composition. In any of these embodiments, in one embodiment the cholesterol ester is cholesterol acetate. In embodiment the cholesterol ester is cholesterol laurate. In one embodiment the cholesterol ester is cholesterol oleate. In one embodiment the cholesterol ester is cholesterol linoleate.

In one embodiment of the nucleic acid-lipid particle composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 20 to about 60 mol% of the total of the lipids in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 35 to about 55 mol% of the total of the lipids in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 45 to about 55 mol% of the total of the lipids in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 40 mol% of the total of the lipids in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the total amount of cholesterol and cholesterol ester is present in an amount of about 50 mol% of the total of the lipids in the nucleic acid-lipid particle composition. In any of these embodiments, in one embodiment the cholesterol ester is cholesterol acetate. In embodiment the cholesterol ester is cholesterol laurate. In one embodiment the cholesterol ester is cholesterol oleate. In one embodiment the cholesterol ester is cholesterol linoleate. Neutral Lipid

The lipid mixture in the lipid mixture compositions and nucleic acid-lipid particle compositions of the present invention also comprises a neutral lipid. The neutral lipid is preferably a neutral phospholipid. In one embodiment, the phospholipid may be zwitterionic (i.e. it carries both a positive and a negative charge, so that it is neutral at a pH ranging around neutral).

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 alkyl group (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 carbon-carbon 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 T_m of 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.

Examples of such phospholipids include phosphatidylcholines, in particular 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), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and further phosphatidylcholines with different hydrophobic chains; lysophosphatidylcholines, such as l-hexadecyl-sn-10-glycero-3 -phosphocholine (Cl 6 Lyso PC); phosphatidylethanolamines, in particular diacylphosphatidyl-ethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1 ,2-di-(9Z-octadecenoyl)- sn-glycero-3 -phosphocholine (DOPG), 1 ,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 phosphatidylethanolamine lipids with different hydrophobic chains.

In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof. In one preferred embodiment, the neutral lipid is DSPC. In another preferred embodiment, the neutral lipid is DPPC. In one embodiment, the neutral lipid comprises DSPC. In one embodiment, the neutral lipid is a mixture of DSPC and DPPC. In one embodiment, the neutral lipid is not DOPE.

In one embodiment of the lipid mixture composition, the neutral lipid is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the neutral lipid is a phospholipid and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phospholipid and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phospholipid and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phospholipid and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the neutral lipid is a phosphatidylcholine and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phosphatidylcholine and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phosphatidylcholine and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is a phosphatidylcholine and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the neutral lipid is DSPC and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is DSPC and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is DSPC and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the neutral lipid is DSPC and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the lipid mixture composition.

In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid- lipid particle composition, the neutral lipid is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the nucleic acid-lipid particle composition.

In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phospholipid and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phospholipid and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid- lipid particle composition, the neutral lipid is a phospholipid and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phospholipid and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in nucleic acid-lipid particle composition.

In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phosphatidylcholine and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phosphatidylcholine and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in nucleic acid-lipid particle composition. In one embodiment of nucleic acid-lipid particle composition, the neutral lipid is a phosphatidylcholine and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is a phosphatidylcholine and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is DSPC and is present in an amount of about 1 mol % to about 40 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the lipid mixture composition, the neutral lipid is DSPC and is present in an amount of about 2 mol % to about 25 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is DSPC and is present in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the neutral lipid is DSPC and is present in an amount of from about 8 mol % to about 12 mol % of the total lipids present in the nucleic acid-lipid particle composition.

Additional Lipids

The lipid mixture in the lipid mixture compositions and nucleic acid-lipid particles of the present invention may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise an anionic amphiphile, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a grafted lipid, as defined and exemplified below.

Negatively Charged Amphiphile

The lipid mixture compositions and nucleic acid-lipid particles of the present invention may also include a negatively charged amphiphile (also referred to herein as an “anionic amphiphile”). In this specification the term “amphiphile” is defined generally as a molecule having both hydrophilic and lipophilic moieties (as defined above). The amphiphiles useful in the compositions of the present invention 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.

As indicated above, the function of the negatively charged amphiphile is to stabilise the nucleic acid-lipid particles, especially in the absence of a grafted lipid. In one embodiment, the nucleic acid-lipid particle contains a negatively charged amphiphile and is substantially free (as defined herein) of a grafted lipid, as defined below. In one embodiment, the nucleic acid-lipid particle contains a negatively charged amphiphile and is substantially free (as defined herein) of a PEG-conjugated lipid, as defined below.

As the skilled person will readily understand, depending on the pKa of 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”). The present invention encompasses anionic amphiphiles in both protonated and deprotonated forms, regardless of the form in which they are described in this specification.

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 in a broadest aspect or a preferred aspect). The lipophilic moiety may comprise two or more groups from the aforementioned list.

In one embodiment, the lipophilic portion of the negatively charged amphiphile has from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile has from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile has from 6 to 20 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile has from 6 to 20 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkyl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkyl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkyl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkenyl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkenyl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkenyl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkynyl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkynyl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkynyl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkyl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkyl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkyl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkenyl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkenyl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is a cycloalkenyl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkyl group having a total of from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkyl group having a total of from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkylalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkylalkyl group having a total of from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylcycloalkylalkyl group having a total of from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an aryl group having from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an aryl group having from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an aryl group having from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylaryl group having a total of from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylaryl group having a total of from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylaryl group having a total of from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an arylalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an arylalkyl group having a total of from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an arylalkyl group having a total of from 6 to 20 carbon atoms.

In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylarylalkyl group having a total of from 6 to 40 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylarylalkyl group having a total of from 6 to 30 carbon atoms. In one embodiment, the lipophilic portion of the negatively charged amphiphile is an alkylarylalkyl group having a total of from 6 to 20 carbon atoms.

In one embodiment, the negatively charged amphiphile has a constitutive negative charge. In this context a “constitutive negative charge” means that the amphiphile carries the negative charge at all physiological pH. Amphiphiles carrying constitutive charged anionic moieties are typically salts of organic strong acids (i.e. organic acids of formula HA which dissociates when dissolved in a solvent S that the proton is transferred completely to the solvent molecule, such that the concentration of the undissociated species HA is too low to be measured).

Typical classes of amphiphiles having a constitutive negative charge include sulfates, sulfonates, phosphates and phosphonates.

In one embodiment, the negatively charged amphiphile is a sulfate (as defined above, either in its broadest aspect or a preferred aspect). In one embodiment, the sulfate is an alkyl sulfate (i.e. in which the group R in the general definition above is an alkyl group) having 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms. Typical examples of sulfates include sodium lauryl sulfate.

In one embodiment, the negatively charged amphiphile is a sulfonate (as defined above, either in its broadest aspect or a preferred aspect). In one embodiment, the sulfonate is an alkyl sulfonate (i.e. in which the group R in the general definition above is an alkyl group) having 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms. In one embodiment, the sulfonate is an alkylaryl sulfonate (i.e. in which the group R in the general definition above is an aryl group substituted with an alkyl group) having a total of 10 to 40, preferably 12 to 30, more preferably 16 to 24 carbon atoms. Typical examples of sulfonates include sodium hexadecane sulfonate (sodium cetyl sulfonate) and sodium dodecylbenzene sulfonate.

In one embodiment, the negatively charged amphiphile is a phosphate (as defined above, either in its broadest aspect or a preferred aspect). In one embodiment, the phosphate is an alkyl phosphate (i.e. in which the group R in the general definition above is an alkyl group) having 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms. Typical examples of phosphonates include octadecylphosphoric acid and dodecylphosphoric acid.

In one embodiment, the negatively charged amphiphile is a phosphonate (as defined above, either in its broadest aspect or a preferred aspect). In one embodiment, the phosphonate is an alkyl phosphonate (i.e. in which the group R in the general definition above is an alkyl group) having 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms. Typical examples of phosphonates include octadecylphosphonic acid and dodecylphosphonic acid.

In one embodiment, the negatively charged amphiphile has a pH-sensitive charge. In this context a “pH-sensitive charge” means that the amphiphile carries the negative charge at alkaline pH, but may be neutral at neutral or acidic pH. Amphiphiles carrying constitutive charged anionic moieties are typically salts of organic weak acids (i.e. organic acids of formula HA which remains largely undissociated when dissolved in a solvent S so that the proton is only partially transferred completely to the solvent molecule).

In one embodiment, the negatively charged amphiphile is a carboxylic acid or carboxylate (as defined above, either in its broadest aspect or a preferred aspect). Typical examples of carboxylic acids include hexanoic acid, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, icosanoic acid, tricosanic acid, 2-hydroxytetradecanoic acid, 2-methyloctadecanoic acid, 2-bromohexadecanoic acid, 2-propylpentanoic acid, 2-butyloctanoic acid, 2-hexyldecanoic acid, 9-hydroxy- stearic-acid, traws-2-decenoic acid, (9Z)-9-hexadecenoic acid, linolic acid, linolenic acid, oleic acid, elaidic acid, arachidonic acid, cyclododecanoic acid, adamantylacetic acid, dicyclohexylacetic acid, traws-4-pentylcyclohexane-carboxylic acid, 4- (decyloxy)benzoic acid, 4-octylbenzoic acid, cholic acid, lithocholic acid, or a mixture of any thereof.

In one embodiment, the carboxylic acid is selected from the group consisting of alkylcarboxylic acids (i.e. where the lipophilic portion of the carboxylic acid is an alkyl group) having a total of 6 to 40 carbon atoms, optionally substituted by a hydroxyl group; alkenylcarboxylic acids (i.e. where the lipophilic portion of the carboxylic acid is an alkenyl group) having a total of 6 to 40 carbon atoms; cycloalkylcarboxylic acids (i.e. where the lipophilic portion of the carboxylic acid is a cycloalkyl group) having a total of 6 to 40 carbon atoms; alkylcycloalkylcarboxylic acids (i.e. where the lipophilic portion of the carboxylic acid is an alkylcycloalkyl group) having a total of 6 to 40 carbon atoms; alkylarylcarboxylic acids (i.e. where the lipophilic portion of the carboxylic acid is an alkylaryl group) having a total of 6 to 40 carbon atoms; dicarboxylic acids having 4 to 10 carbon atoms in the dicarboxyl moiety, optionally esterified with an alkyl group having 6 to 40 carbon atoms or an alkenyl group having 6 to 40 carbon atoms; or a mixture of any thereof.

In one embodiment, the carboxylic acid is selected from the group consisting of , octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, 2-butyloctanoic acid, 2-hexyldecanoic acid, 2-hydroxypalmitic acid, 2- methyloctadecanoic acid, hexadecenylsuccinic acid, neodecanoic acid, cyclohexanepentanoic acid, 1 -adamantaneacetic acid, 4-pentylcyclohexanecarboxylic acid, cyclododecanecarboxylic acid, p-nonylbenzoic acid, 2-decenoic acid, 3 -decenoic acid, palmitoleic acid, linolenic acid, linoleic acid, oleic acid, elaidic acid, arachidonic acid, lithocholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and cholic acid, or a mixture of any thereof.

In one embodiment, the negatively charged amphiphile has both constitutively and pH-sensitive negatively charged groups. Examples of negatively charged amphiphiles having both such groups include phosphatidylserines.

In one embodiment, the negatively charged amphiphile has a pH sensitive charge and pH sensitive anionic moiety is a carboxylic acid. One or more charged groups can be present in the amphiphile and in preferred embodiments a single charged moiety is present in an amphiphile. The polar region of the negatively charged amphiphile may comprise additional uncharged polar moieties. Preferred uncharged polar moieties are hydroxyl or amide groups and one or more uncharged polar moieties can be present in the negatively charged amphiphile.

In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid with diacylglycerol. The hydrocarbyl portion of the acyl moieties of the diacylglycerol portion is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 8 to 18, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 18, carbon atoms. Typically, the acyl moieties are present on the 1- and 2-positions of the glycerol moiety. The acyl parts of the diacylglycerol moiety may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties, preferably selected from the group consisting of stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties, preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties. The dicarboxylic acid moiety is as defined above, and preferably has 2 to 20 carbon atoms, more preferably 2 to 10, even more preferably 2 to 8 carbon atoms. Examples of the dicarboxylic acid moiety include oxalate, malonate, succinate, glutarate, adipate, pimelate and suberate. In one embodiment, the negatively charged amphiphile is a hemiester of succinic acid with diacylglycerol (i.e. the dicarboxylic acid moiety is a succinic acid moiety) - also referred to herein as a “diacylglycerol hemisuccinate”. Typical examples of such negatively charged amphiphiles include 1,2- dilauroylglyceryl hemisuccinate (DLGS), 1 ,2-dimyristoylglyceryl hemisuccinate (DMGS), 1 ,2-dipalmitoylglyceryl hemisuccinate (DPGS), l-palmitoyl-2- stearoylglyceryl hemisuccinate (PSGS), distearoylglyceryl hemisuccinate (DSGS), 1 ,2-dioleoylglyceryl hemisuccinate (DOGS), 1 -stearoyl, 2-myristoyl- glycerylhemisuccinate (SMGS), l-palmitoyl-2-oleoylglyceryl hemisuccinate (POGS) and analogues of any of the above thereof wherein the dicarboxylic acid portion is oxalate, malonate, succinate, glutarate, adipate, pimelate or suberate.

Dimyristoylglyceryl hemisuccinate, dipalmitoylglyceryl hemisuccinate or distearoylglyceryl hemisuccinate are preferred. In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid with a steroid. The dicarboxylic acid moiety is as defined and exemplified above, and typically contains a total (including the acyl carbons) of 2 to 10, preferably 3 to 6, carbon atoms. The ester group may esterify any free hydroxyl group on the steroid molecule.

In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid with cholesterol. The dicarboxylic acid moiety is as defined above, and preferably has 2 to 20 carbon atoms, more preferably 2 to 10, even more preferably 2 to 8 carbon atoms. Examples of the dicarboxylic acid moiety include oxalate, malonate, succinate, glutarate, adipate, pimelate and suberate. In one embodiment, the negatively charged amphiphile is a hemiester of succinic acid with cholesterol (i.e. the dicarboxylic acid moiety is a succinic acid moiety) - also referred to herein as “cholesteryl hemisuccinate”. Typical examples of such negatively charged amphiphiles include those listed in Table 1 below.

Table 1

In one embodiment, the negatively charged amphiphile is a monoester or diester of a phosphoric acid, wherein one of the phosphoric acid hydroxyl groups is esterified with diacylglycerol. The hydrocarbyl portion of the acyl moieties of the diacylglycerol portion is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 8 to 18, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 18, carbon atoms. The acyl parts of the diacylglycerol moiety may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties, preferably selected from the group consisting of stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties, preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties. When the negatively charged amphiphile is a diester of a phosphoric acid, the second hydroxyl group may be esterified with an alkyl group (as defined above) having 1 to 6 carbon atoms, a glyceryl group, or an O-serinyl group.

In one embodiment, the negatively charged amphiphile is an anionic phospholipid. Typical examples of such anionic phospholipids suitable as negatively charged amphiphiles include phosphatidylserines, phosphatidylglycerols or phosphatidic acids (all as defined above, either in its broadest aspect or a preferred aspect). The hydrocarbyl portion of the acyl moieties of such anionic phospholipids is as defined above, but is preferably an alkyl group (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 carbon-carbon double bonds. The acyl parts of the phospholipids may be the same or different. In one embodiment, the acyl moieties are present on the 1- and 2-positions of the phospholipid. In one embodiment, the acyl moieties are present on the 1- and 3-positions of the phospholipid. 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 T_m of 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.

In one embodiment, the negatively charged amphiphile is a phosphatidylserine, the acyl parts of which can be any of those defined and exemplified above. In one embodiment the negatively charged amphiphile is 1 ,2-dioleoylphosphatidylserine (DOPS).

In one embodiment, the negatively charged amphiphile is a phosphatidic acid, the acyl parts of which can be any of those defined and exemplified above. In one embodiment the negatively charged amphiphile is 1 ,2-dioleoylphosphatidic acid (DOPA).

In one embodiment, the negatively charged amphiphile is a phosphatidyl glycerol, the acyl parts of which can be any of those defined and exemplified above. In one embodiment the negatively charged amphiphile is 1 ,2-palmitoyloleoylphosphatidyl glycerol (POPG).

In yet other embodiments, the negatively charged amphiphile is comprising a transfection enhancer element as described in W02008/074487. Suitable examples of negatively charged amphiphiles are listed in Table 2 below.

The anionic amphiphile can further be characterized by its molecular volume and the shape factor K. The anionic amphiphile is typically non-protonated and in its charged state. The anionic amphiphile typically adsorbs a counterion from the mobile phase. For the purposes of describing the shape factor K, the counterion is modelled as a sodium ion including its shell of hydration, having a molecular volume of 93 A³ according to Siepi et al (2011). Thus, in one embodiment the anionic amphiphile (including a hydrated sodium ion) has a shape factor K between 0.25 and 2, preferably between 0.4 and 1.0.

In one embodiment the partial molecular volume of the polar head group of the anionic amphiphile itself is between 40 and 120A³, preferably between 50 and 80A³. In one embodiment the partial molecular volume of the apolar tail group is between 120 and 600A³, preferably between 200 and 400A³. Values of K and partial molecular volumes for certain anionic amphiphiles are provided in Table 3 below:

Table 3

In one embodiment, the negatively charged amphiphile is selected from the group consisting of: a carboxylic acid; a phosphonic acid; a sulfate; a sulfonate; a hemiester of a dicarboxylic acid with diacylglycerol; a hemiester of a dicarboxylic acid with cholesterol; a phosphatidylserine, a phosphatidic acid or a phosphatidylglycerol; or a mixture of any thereof.

In one embodiment, the negatively charged amphiphile is selected from the group consisting of: a carboxylic acid selected from the group consisting of alkylcarboxylic acids having a total of 6 to 40 carbon atoms, optionally substituted by a hydroxyl group; alkenylcarboxylic acids having a total of 6 to 40 carbon atoms; cycloalkylcarboxylic acids having a total of 6 to 40 carbon atoms; alkylcycloalkylcarboxylic acids having a total of 6 to 40 carbon atoms; and alkylarylcarboxylic acids having a total of 6 to 40 carbon atoms; or a mixture of any thereof; an alkyl sulfate having 6 to 30 carbon atoms; an alkyl sulfonate having 6 to 30 carbon atoms; an alkylaryl sulfonate having a total of 12 to 40 carbon atoms; an alkyl phosphonate having 6 to 30 carbon atoms; a hemiester of a dicarboxylic acid having 2 to 10 carbon atoms with diacylglycerol; a hemiester of a dicarboxylic acid having 2 to 10 carbon atoms with cholesterol; a diacylphosphatidylserine, wherein the hydrocarbyl portion of each of the acyl moieties thereof is an alkenyl group having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carbon-carbon double bonds; or a mixture of any thereof.

In one embodiment, the negatively charged amphiphile is selected from the group consisting of:

(i) an aliphatic carboxylic acid having between 12 and 24 carbon atoms;

(ii) a hemiester of a dicarboxylic acid with diacylglycerol, wherein each acyl part may be the same or different and each has 6 to 18 carbon atoms, preferably selected from the group consisting of 1 ,2-dilauroylglyceryl hemisuccinate, 1 ,2-dimyristoylglyceryl hemisuccinate, 1 ,2-dipalmitoylglyceryl hemisuccinate 1 ,2-distearoylglyceryl hemisuccinate;

(iii) a hemiester of a dicarboxylic acid with cholesterol, wherein the dicarboxylic acid moiety preferably has 2 to 20 carbon atoms, more preferably 2 to 10 carob atoms, and is even more preferably selected from the group consisting of cholesterol hemisuccinate, cholesterol hemimalonate and cholesterol hemiadipate;

(iv) an organic sulfate or sulfonate, preferably an alkyl sulfate or alkyl sulfonate wherein the alkyl part has 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms, and is more preferably selected from the group consisting of sodium lauryl sulfate, sodium hexadecane sulfonate and sodium dodecylbenzene sulfonate;

(v) an organic phosphonate, preferably an alkyl phosphonate in which the alkyl part has 6 to 30, preferably 8 to 24, more preferably 12 to 18 carbon atoms, more preferably selected from the group consisting of octadecylphosphonic acid and dodecylphosphonic acid; and

(vi) an anionic phospholipid, wherein the hydrocarbyl portion of the acyl moieties of such anionic phospholipids is preferably an alkyl group having 6 to 40, carbon atoms or an alkenyl group having 6 to 40 carbon atoms and 1 to 6 carbon-carbon double bonds, and more preferably selected from the group consisting of phosphatidylserine, phosphatidylglycerol and phosphatidic acid; or a mixture of any thereof.

In one embodiment, the negatively charged amphiphile is selected from the group consisting of: a carboxylic acid selected from the group consisting of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, 2-butyloctanoic acid, 2-hexyldecanoic acid, 2-hydroxypalmitic acid, 2-methyloctadecanoic acid, hexadecenylsuccinic acid, neodecanoic acid, cyclohexanepentanoic acid, 1- adamantaneacetic acid, 4-pentylcyclohexanecarboxylic acid, cyclododecanecarboxylic acid, p-nonylbenzoic acid, 2-decenoic acid, 3-decenoic acid, palmitoleic acid, linolenic acid, linoleic acid, oleic acid, elaidic acid, arachidonic acid, lithocholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and cholic acid; sodium dodecyl sulfate; a sulfonate selected from the group consisting of sodium cetyl sulfonate and decyl benzenesulfonate; a phosphonate selected from the group consisting of dodecyl phosphonate and octadecyl phosphonate; cholesteryl hemisuccinate; a diacylglycerol hemisuccinate selected from the group consisting of 1,2- dilauroylglyceryl hemisuccinate (DLGS), 1 ,2-dimyristoylglyceryl hemisuccinate (DMGS), 1 ,2-dipalmitoylglyceryl hemisuccinate (DPGS), l-palmitoyl-2- stearoylglyceryl hemisuccinate (PSGS), 1 ,2-distearoylglyceryl hemisuccinate (DSGS), 1 -stearoyl, 2-myristoyl-glycerylhemisuccinate (SMGS), and l-palmitoyl-2- oleoylglyceryl hemisuccinate (POGS); or a mixture of any thereof.

In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 20 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 15 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 14 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 13 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 12 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 11 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 10 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 9 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 8 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 7 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 6 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of up to about 5 mol% of the total lipid present in the lipid mixture composition.

In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 0.1 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 0.2 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 0.5 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 1 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 2 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 3 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 4 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 5 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 6 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of at least about 7 mol% of the total lipid present in the lipid mixture composition.

In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.1 to about 20 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.2 to about 20 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.5 to about 20 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 1 to about 20 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 2 to about 15 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 4 to 12 about mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 6 to about 10 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 7 to about 9 mol% of the total lipid present in the lipid mixture composition.

In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.1 to about 10 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.2 to about 7.5 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 0.5 to about 7 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 1 to about 6 mol% of the total lipid present in the lipid mixture composition. In one embodiment, the negatively charged amphiphile is present in the lipid mixture composition in an amount of about 2 to about 5 mol% of the total lipid present in the lipid mixture composition.

In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of up to about 20 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 15 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 14 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 13 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 12 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 11 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 10 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 9 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 8 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 7 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 6 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of up to about 5 mol% of the total lipid present in the nucleic acid-lipid particle composition.

In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of at least about 0.1 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 0.2 mol% of the total lipid present in the nucleic acid- lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 0.5 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of at least about 1 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 2 mol% of the total lipid present in the nucleic acid- lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 3 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 4 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 5 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 6 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of at least about 7 mol% of the total lipid present in the nucleic acid-lipid particle composition.

In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of about 0.1 to about 20 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 0.2 to about 20 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 0.5 to about 20 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 1 to about 20 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 2 to about 15 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 4 to 12 about mol% of the total lipid present in the nucleic acid- lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 6 to about 10 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of about 7 to about 9 mol% of the total lipid present in the nucleic acid-lipid particle composition.

In one embodiment, the negatively charged amphiphile is present in the nucleic acid- lipid particle composition in an amount of about 0.1 to about 10 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 0.2 to about 7.5 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 0.5 to about 7 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 1 to about 6 mol% of the total lipid present in the nucleic acid-lipid particle composition. In one embodiment, the negatively charged amphiphile is present in the nucleic acid-lipid particle composition in an amount of about 2 to about 5 mol% of the total lipid present in the nucleic acid-lipid particle composition.

Grafted Lipids

The lipid mixture compositions and nucleic acid-lipid particle compositions of the present invention may also contain a grafted lipid. As indicated above, the function of the grafted lipid is to stabilise the nucleic acid-lipid particle composition.

In the present specification the term “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 polymer, as defined below (either in a broadest aspect or a preferred aspect).

A "polymer" as used herein, is given its ordinary meaning, z.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, z.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, z.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.

In one embodiment, the grafted lipid is capable of acting as a stealth lipid. In this specification the term “stealth lipid” means a stealth polymer (as defined below) conjugated to a lipid (as defined herein). In this specification the term “stealth polymer” means a polymer (as defined above) having the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge. In some embodiments, a stealth polymer is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a stealth polymer can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

In one embodiment, the grafted lipid is a poly(alkylene glycol) conjugated lipid, such as a poly( ethylene glycol)conjugated lipid (also known as a PEG-lipid or PEGylated lipid). 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. The PEG-lipid may comprise 5-1000, 5-500, 5-100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10- 1000, 10-500, 10-100, or 10-50, ethylene glycol repeating units, which may be consecutive.

In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein each of R¹² and R¹³ is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In some embodiments of this formula, each of R¹² and R¹³ is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

In some embodiments of this formula, R¹² and R¹³ are identical. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 16 carbon atoms.

In some embodiments of this formula, R¹² and R¹³ are different. In some embodiments, one of R¹² and R¹³ is a straight alkyl chain containing 12 carbon atoms and the other of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms.

In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45. In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)- 2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG- PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '- di(tetradecanoyloxy)propyl- 1 -0-(o -methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co -methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(co methoxy(polyethoxy)ethyl)carbamate, and the like. In some embodiments of this formula, the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 1700 to about 3000 g/mol, or from about 1800 to about 2200 g/mol. In one embodiment, the PEG portion of the pegylated lipid has an average molecular weight of about 2000 g/mol.

In one embodiment, the grafted lipid is l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG) wherein the PEG portion of the pegylated lipid has an average molecular weight of from about 1800 to about 2200 g/mol, preferably about 2000 g/mol.

In one embodiment, the grafted lipid is 2- [(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159). In some embodiments, the pegylated lipid has the following structure:

Other examples of grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids, poly(vinyl pyrrolidone) (PVP)-conjugated lipids; poly(.V-(2-hydroxypropyl)- methacrylamide) (pHPMA)-conjugated lipids; poly(dehydroalanine) (pDha)- conjugated lipids; poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipids and poly(2-methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipids.

In one embodiment, the grafted lipid is a polysarcosine-conjugated lipid, 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:

wherein x refers to the number of sarcosine units. The polysarcosine may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 sarcosine units.

In one embodiment, the grafted lipid is a polyoxazoline (POX)-conjugated and/or a polyoxazine (POZ)-conjugated lipid and/or a POX/POZ-conjugated lipid, 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 polyoxazine (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:

wherein x refers to the number of pOx and/or pOz units. The total number of pOx and/or pOz repeating units in the polymer may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 pOx and/or pOz units.

In one embodiment, the grafted lipid is a poly(vinyl pyrrolidone) (PVP)-conjugated lipid. In one embodiment, the lipid nanoparticle 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.

pVP

In one embodiment, the grafted lipid is a poly(V-(2-hydroxypropyl)methacrylamide) (pHPMA)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(7V-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid The term “poly(A-(2-hydroxypropyl)-methaciylamide” or “pHPMA” means a polymer having the repeating unit shown below.

In one embodiment, the grafted lipid is a poly(dehydroalanine) (pDha)-conjugated lipid. The term “pDha” means a polymer having the repeating unit shown below.

pDha

In one embodiment, the grafted lipid is an amphiphilic oligoethylene glycol (OEG)- conjugated lipid. 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:

pAEEA pmAEEA wherein x refers to the total number of pAEEA and/or pmAEEA units in the polymer. The total number of pAEEA and/or pmAEEA repeating units in the polymer may comprise from 1 to 100, from 5 to 50, from 5 to 25, preferably from 7 to 14.

In one embodiment the grafted lipid is selected from the group consisting of: a poly(alkylene glycol)-conjugated lipid; a poly(sarcosinate)-conjugated lipid; a poly(oxazoline) (POX)-conjugated lipid; a poly(oxazine) (POZ)-conjugated lipid; a poly(vinyl pyrrolidone) (PVP)-conjugated lipid; a poly( V-(2-hydroxypropyl)-methacrylamide) (pHPMA)-conjugated lipid; a poly(dehydroalanine) (pDha)-conjugated lipid; a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2-methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipid; or a mixture of any thereof. In one embodiment, the grafted lipid is selected from the group consisting of: a poly(ethylene glycol)-conjugated lipid; a poly(sarcosinate)-conjugated lipid; a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; or a mixture of any thereof

In one embodiment of the lipid mixture composition, the grafted lipid is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the grafted lipid is a poly(ethylene glycol)-conjugated lipid and is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is a polyethylene glycol)-conjugated lipid and is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is a poly(ethylene glycol)-conjugated lipid and is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of the lipid mixture composition, the grafted lipid is ALC-0159 and is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is ALC-0159 and is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the lipid mixture composition, the grafted lipid is ALC-0159 and is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the lipid mixture composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the nucleic acid-lipid particle composition.

In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is a poly(ethylene glycol)-conjugated lipid and is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is a poly( ethylene glycol)-conjugated lipid and is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is a poly(ethylene glycol)-conjugated lipid and is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the nucleic acid-lipid particle composition.

In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is ALC-0159 and is present in an amount of about 0.5 to about 10 mol% of the total lipids present in the nucleic acid-lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is ALC-0159 and is present in an amount of about 0.2 to about 5 mol% of the total lipids present in the nucleic acid- lipid particle composition. In one embodiment of the nucleic acid-lipid particle composition, the grafted lipid is ALC-0159 and is present in an amount of about 1 to about 2.5 mol% of the total lipids present in the nucleic acid-lipid particle composition.

In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a stealth polymer. In this specification the term “stealth polymer” means a polymer (as defined above) (optionally conjugated to a lipid) having the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, (c) no hydrogen bond donor groups; and (d) no net charge. In some embodiments, a stealth polymer is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a stealth polymer can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a stealth polymer- conjugated lipid. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly(alkylene glycol)-conjugated lipid, such as a poly(ethylene glycol)-conjugated lipid, as defined and exemplified above. In one embodiment, the lipid mixture particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polysarcosine-conjugated lipid, as defined and exemplified above. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polyoxazoline (POX)-conjugated and/or a polyoxazine (POZ)-conjugated lipid and/or a POX/POZ-conjugated lipid, as defined and exemplified above. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) conjugated to a lipid, as defined and exemplified above. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(A-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid, as defined and exemplified above. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of poly(dehydroalanine) (pDha) conjugated to a lipid, as defined and exemplified above. In one embodiment, the lipid mixture composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of amphiphilic oligoethylene glycol (OEG)-conjugated lipids, such as poly(aminoethyl- ethylene glycol acetyl) (pAEEA)-conjugated lipids and/or poly(methylaminoethyl- ethylene glycol acetyl) (pmAEEA)-conjugated lipids, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a stealth polymer- conjugated lipid. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly(alkylene glycol)-conjugated lipid, such as a poly( ethylene glycol)- conjugated lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polysarcosine-conjugated lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a polyoxazoline (POX)-conjugated and/or a polyoxazine (POZ)- conjugated lipid and/or a POX/POZ-conjugated lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) conjugated to a lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(/V- (2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of poly(dehydroalanine) (pDha) conjugated to a lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of amphiphilic oligoethylene glycol (OEG)-conjugated lipids, such as poly(aminoethyl- ethylene glycol acetyl) (pAEEA)-conjugated lipids and/or poly(methylaminoethyl- ethylene glycol acetyl) (pmAEEA)-conjugated lipids, as defined and exemplified above.

In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a stealth polymer conjugated to the nucleic acid. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly(alkylene glycol) conjugated to the nucleic acid, such as a poly(ethylene glycol)-conjugated siRNA, mRNA or DNA, as defined and exemplified above.

In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a stealth polymer. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a poly( alkylene glycol)-conjugated lipid, such as a polyethylene glycol)-conjugated lipid, as defined and exemplified above.

Multivalent Anion, such as Inorganic Polyphosphate

The lipid mixture compositions and nucleic acid-lipid particles of the present invention may also further include a multivalent anion, such as an inorganic polyphosphate. As indicated above, the function of the multivalent anion is to stabilise the nucleic acid-lipid particle composition. In particular, using a multivalent anion, such as an inorganic polyphosphate, makes it possible to prepare compositions which are stable (in particular with respect to the colloidal size of the particles contained in said compositions), which can be stored in liquid form, which can repeatably be frozen and thawed, which contain nucleic acid that is in a stable form, and which maintain high biological efficacy, even if the composition does not comprise a PEG lipid or any other stealth lipid (or grafted lipid).

In some embodiments, the multivalent anion (such as inorganic polyphosphate, e.g., a linear inorganic polyphosphate, such as triphosphate) forms the surface of the particles (such as LNPs), along with phospholipids. In some embodiments, after formation of the particles, a multivalent anion (such as an inorganic polyphosphate) as disclosed herein is added to the particles resulting in the association of the multivalent anion (such as inorganic polyphosphate) to the surface of the particles due to electrostatic attraction between the positively charged surface of the particles and the anionic multivalent anion (such as anionic inorganic polyphosphate). This association (or decoration) leads to neutralization of the surface charge on the particle or even to the formation of stable anionic particles. The term "multivalent anion" as used herein 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.

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).

In some embodiments, the multivalent anion is an inorganic polyphosphate. The inorganic polyphosphate can be any linear, cyclic, or branched inorganic polyphosphate. In some embodiments of the first aspect, the inorganic polyphosphate is a linear inorganic polyphosphate (such as a linear inorganic triphosphate).

In some embodiments, the inorganic polyphosphate comprises the formula [PxO(3x+i)]^y, wherein x is an integer and is at least 2, preferably 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 diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof, such as from the group consisting of triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof. In some preferred embodiments, the inorganic polyphosphate is selected from the group consisting of triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof. In some preferred embodiments, the inorganic polyphosphate is triphosphate.

In some embodiments, the molar ratio of the multivalent anion (such as the inorganic polyphosphate) to the cationically ionizable lipid is at least about 1:2. For example, the molar ratio of the multivalent anion (such as the inorganic polyphosphate) to the cationically ionizable lipid may be at least about 0.55, at least about 0.60, at least about 0.65, at least about 2:3, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, at least about 1.00, at least about 1.10, at least about 1.20, at least about 1.30, at least about 4:3, at least about 1.40, at least about 1.50, at least about 1.60, at least about 1.70, at least about 1.80, at least about 1.90, or at least about 2.0.

In some preferred embodiments, the molar ratio of the multivalent anion (such as the inorganic polyphosphate) to the cationically ionizable lipid is at least about 2:3. In some preferred embodiments, the molar ratio of the multivalent anion (such as the inorganic polyphosphate) to the cationically ionizable lipid is at least about 4:3.

In some embodiments of the nucleic acid-lipid particle composition, the composition comprises particles dispersed in an aqueous phase, the particles comprise at least a portion of the nucleic acid, at least a portion of the cationically ionizable lipid, at least a portion of the cholesterol, at least a portion of the cholesterol ester, and at least a portion of the neutral lipid; and at least a portion of the multivalent anion (such as the inorganic polyphosphate) is associated with the particles. In some embodiments of the nucleic acid-lipid particle composition, the composition comprises particles dispersed in an aqueous phase, and at least 10% (such as at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%) of the multivalent anion (such as the polyphosphate) present in the composition is associated with the particles. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) is present in the final nucleic acid-lipid particle composition at a concentration of about 0.5 mM to about 50 rnM. In one embodiment, the multivalent anion (such as an inorganic polyphosphate) is present in the final nucleic acid-lipid particle composition at a concentration of about 1 mM to about 10 mM.

In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of a multivalent anion. In one embodiment, the nucleic acid-lipid particle composition is substantially free (as defined above, either in its broadest aspect or a preferred aspect) of an inorganic polyphosphate.

In one embodiment, the nucleic acid-lipid particle composition includes an anionic amphiphile, as defined and exemplified above, and a grafted lipid, as defined and exemplified above. In one embodiment, the nucleic acid-lipid particle composition includes a grafted lipid, as defined and exemplified above, and a multivalent anion, such as an inorganic polyphosphate, as defined and exemplified above.

Preferred Combinations

The lipid mixture compositions and nucleic acid-lipid particle compositions of the present invention may contain a variety of specific lipids in combination.

In one embodiment, the ionizable lipid is [(4-hydroxybutyl)azanediyl]di(hexane-6,l- diyl) bis(2-hexyldecanoate) (ALC-315) and the cholesterol ester is cholesterol acetate. In one embodiment, the ionizable lipid is [(4-hydroxybutyl)azanediyl]di(hexane-6,l- diyl) bis(2 -hexyldecanoate) (ALC-315) and the cholesterol ester is selected from the group consisting of cholesterol acetate, cholesterol laurate, cholesterol oleate and cholesterol linoleate.

In one embodiment, the ionizable lipid is selected from the group consisting of BHD- C2C2-PipZ, BODD-C2C2-lMe-Pyr, and BODD-C2C2-DMA, and the cholesterol ester is selected from the group consisting of cholesterol laurate, cholesterol oleate and cholesterol linoleate. In one embodiment, the ionizable lipid is selected from the group consisting of BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, and BODD-C2C2- DMA, and the cholesterol ester is selected from the group consisting of cholesterol oleate and cholesterol linoleate.

In one embodiment, the ionizable lipid is BHD-C2C2-PipZ and the cholesterol ester is selected cholesterol oleate. In one embodiment, the ionizable lipid is the group consisting of BHD-C2C2-PipZ and the cholesterol ester is cholesterol linoleate. In one embodiment, the ionizable lipid is the group consisting of BHD-C2C2-PipZ and the cholesterol ester is cholesterol laurate.

In one embodiment, the ionizable lipid is BODD-C2C2-lMe-Pyr and the cholesterol ester is cholesterol oleate. In one embodiment, the ionizable lipid is BODD-C2C2- IMe-Pyr and the cholesterol ester is cholesterol linoleate. In one embodiment, the ionizable lipid is BODD-C2C2-lMe-Pyr and the cholesterol ester is cholesterol laurate.

In one embodiment, the ionizable lipid is BODD-C2C2-DMA and the cholesterol ester is cholesterol oleate. In one embodiment, the ionizable lipid is BODD-C2C2- DMA and the cholesterol ester is cholesterol linoleate. In one embodiment, the ionizable lipid is BODD-C2C2-DMA and the cholesterol ester is cholesterol laurate.

Pharmaceutical Compositions

The nucleic acid-lipid particle compositions described herein are useful as or for preparing pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

The nucleic acid-lipid particle 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 pharmaceutical composition 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 active ingredient, as described herein. In the context of the present disclosure, the pharmaceutical composition comprises a nucleic acid as described herein. In some embodiments, the therapeutically effective agent is or comprises a nucleic acid, 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.

In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the initial pharmaceutical composition (z.e., after its preparation, but before freezing, or storing) is at least 50%, preferably at least 60%, more preferred at least 70%, and most preferred at least 80%, such as at least 90%.

In some embodiments, the size (Zaverage) of the particles of the initial pharmaceutical composition (z.e., after its preparation, but before freezing, or storing) is between about 50 nm and about 500 nm, preferably between about 40 run and about 200 nm, more preferably between about 40 nm and about 120 nm.

In some embodiments, the polydispersity index (PDI) of the particles of the initial pharmaceutical composition (z.e., after preparation, but before freezing, or storing) is less than 0.3, preferably less than 0.2, more preferably less than 0.1.

The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (z.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 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 -90°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.

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 -20°C.

In some embodiments of the pharmaceutical compositions in frozen form, when the nucleic acid is mRNA, the mRNA integrity after thawing the frozen pharmaceutical composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100% of the initial mRNA integrity, e.g., after thawing the frozen composition which has been 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) at -20°C.

In some embodiments of the pharmaceutical compositions in frozen form, the 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 of the initial pharmaceutical composition 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 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 initial size (Zaverage) and/or size distribution and/or PDI of the particles contained in the frozen pharmaceutical composition before freezing. In some embodiments, when the nucleic acid is mRNA, the size of the mRNA particles and the mRNA integrity of the pharmaceutical composition after one freeze/thaw cycle, preferably after two freeze/thaw cycles, more preferably after three freeze/thaw cycles, more preferably after four freeze/thaw cycles, more preferably after five freeze/thaw cycles or more, are essentially equal to the size of the mRNA particles and the mRNA integrity of the initial pharmaceutical composition (z.e., before the pharmaceutical composition has been frozen for the first time).

In some embodiments, the pharmaceutical composition is in liquid form and can be stored at a temperature ranging from about 0°C to about 20°C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature ranging from about 1°C to about 15°C, such as from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C.

In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the pharmaceutical composition when stored is at least 70%, preferably at least 80%, more preferably at least 90%, of the initial mRNA integrity (z.e., the mRNA integrity of the initial pharmaceutical composition).

In some embodiments of the pharmaceutical compositions in liquid 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, or at least 24 months, preferably at least 4 weeks. For example, the liquid 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 5°C.

In some embodiments of the pharmaceutical composition in liquid form, when the nucleic acid is mRNA, the mRNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the mRNA integrity of the liquid composition, when stored, e.g., at 0°C or higher for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least 4 months, or at least 6 months), may be at least 90%, compared to the mRNA integrity of the initial composition, z.e., the mRNA integrity before the composition has been stored. In some embodiments, the mRNA integrity of the composition after storage for at least four weeks (e.g., for at least three months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, is at least 90%, compared to the mRNA integrity before storage.

In some embodiments, when the nucleic acid is mRNA, the initial mRNA integrity of the pharmaceutical composition (z.e., after its preparation but before storage) is at least 50% and the mRNA integrity of the pharmaceutical composition after storage for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, or at least 3 months), preferably at a temperature of 0°C or higher, such as about 2°C to about 8°C, is at least 90% of the initial mRNA integrity.

In some embodiments of the pharmaceutical composition in liquid form, the size (Zaverage) (and/or size distribution and/or poly dispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0°C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the size (Zaverage) (and/or size distribution and/or polydispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0°C or higher for at least one week, is essentially equal to the 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 0°C or higher for at least one week 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 0°C or higher for at least one week 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 0°C or higher for at least one week 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 size (Zaverage) (and/or size distribution and/or PDI) of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week is essentially equal to the size (Zaverage) (and/or size distribution and/or PDI) of the particles before storage. In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0°C or higher for at least one week 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 one week is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

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.

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 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.

In particular embodiments, a pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, i.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.

A "stable" multi-dose formulation preferably exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule 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.

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.

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

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. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavouring agents, or colorants.

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.

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 polyoxyethylene/polyoxy-propylene copolymers.

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).

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

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 intratumorally. 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

The nucleic acid-lipid particles and pharmaceutical compositions comprising them as described herein may be used in the therapeutic or prophylactic treatment of various diseases, in particular diseases in which provision of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, provision of an antigen or epitope which is derived from a virus may be useful in the treatment or prevention of a viral disease caused by said virus. Provision of a tumour antigen or epitope may be useful in the treatment of a cancer disease wherein cancer cells express said tumour antigen. Provision of a functional protein or enzyme may be useful in the treatment of genetic disorder characterized by a dysfunctional protein, for example in lysosomal storage diseases (e.g. mucopolysaccharidoses) or factor deficiencies. Provision of a cytokine or a cytokine-fusion may be useful to modulate tumour microenvironment.

Therefore, in one aspect there is disclosed the nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in medicine.

In one embodiment, there is provided a nucleic acid-lipid particle, or pharmaceutical composition as defined herein for use in delivery of a nucleic acid (such as an mRNA) to a cell. In one embodiment, there is provided a nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in transfecting a cell with a nucleic acid (such as an mRNA). In one embodiment, there is provided use of a nucleic acid-lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for delivery of a nucleic acid (such as an mRNA) to a cell. In one embodiment, there is provided use of a nucleic acid-lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for transfecting a cell with a nucleic acid (such as an mRNA). In one embodiment, there is provided a method of delivery of a nucleic acid (such as an mRNA) to a cell, the method comprising administering to the cell the nucleic acid-lipid particle, or pharmaceutical composition as defined herein. In one embodiment, there is provided a method of transfecting a cell with a nucleic acid (such as an mRNA), the method comprising adding to the cell the nucleic acid-lipid particle, or pharmaceutical composition as defined herein; and incubating the mixture of the composition and cells for a sufficient amount of time. In some embodiments, in particular those where the nucleic acid (such as an mRNA) encodes a pharmaceutically active protein, the mixture of the composition and cells is incubated for a time sufficient to allow the expression of the pharmaceutically active protein. In some embodiments, the sufficient amount of time is at least one hour (such at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and/or up to about 48 hours (such as up to about 36 or up to about 24 hours). In some embodiments, incubating the mixture of the composition and cells is conducted in the presence of serum (such as human serum).

The cell may be any cell capable of receiving nucleic acid (such as an mRNA) to produce a therapeutic effect. In one embodiment, the cell is a liver cell. In one embodiment, the cell is a spleen cell. In one embodiment, the cell is a lung cell.

In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, for use in treating a disease treatable by a nucleic acid (such as an mRNA). In one embodiment, there is provided use of a composition as defined herein, in the manufacture of a medicament for treating a disease treatable by a nucleic acid (such as an mRNA). In one embodiment, there is provided a method of treating a disease treatable by a nucleic acid (such as an mRNA) in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in a prophylactic and/or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein in the manufacture of a medicament for a prophylactic and/or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided a method of prophylactic and/or therapeutic treatment of a disease involving an antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in inducing an immune response. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for inducing an immune response.

In one embodiment, there is provided a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, for use in treating cancer. In one embodiment, there is provided use of a nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for treating cancer. In one embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

The term "disease" (also referred to as "disorder" herein) 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, such as autoimmune diseases. 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. The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, sexually transmitted diseases (e.g., chlamydia, gonorrhoea, or syphilis), SARS, coronavirus diseases (e.g., COVID-19), acquired immune deficiency syndrome (AIDS), measles, chicken pox, cytomegalovirus infections, herpes simplex virus (e.g., HSV-1, HSV-2), hepatitis (such as hepatitis B or C), influenza (flu, such as human flu, swine flu, dog flu, horse flu, and avian flu), HPV infection, shingles, rabies, common cold, gastroenteritis, rubella, mumps, anthrax, cholera, diphtheria, foodbome illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, pertussis, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dientamoebiasis, giardiasis, amoebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiasis.

In some embodiments, the nucleic acid-lipid particle or a pharmaceutical composition described herein may be used in the therapeutic or prophylactic treatment of an infectious disease.

In the present context, the term "treatment", "treating" or "therapeutic intervention" 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. The term "therapeutic treatment" relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.

The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer, infectious diseases) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination.

In some embodiments of the disclosure, the aim is to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, to a subject, in particular a subject in need thereof. In some embodiments of the disclosure, the aim is to provide a protein replacement therapy, such as production of erythropoietin, Factor VII, Von Willebrand factor, P- galactosidase, Alpha-N-acetylglucosaminidase, to a subject, in particular a subject in need thereof.

In some embodiments of the disclosure, the aim is to modulate/reprogram immune cells in the blood.

In some embodiments, the compositions described herein, which contain mRNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof (in the following simply "SARS-CoV-2 S nucleic acid compositions" which explicitly include SARS-CoV-2 S RNA compositions), following administration to a subject, induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a panel of different S protein variants such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants. In some embodiments, the panel of different S protein variants comprises at least 5, at least 10, at least 15, or even more S protein variants. In some embodiments, such S protein variants comprise variants having amino acid modifications in the RBD domain and/or variants having amino acid modifications outside the RBD domain. In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets VOC-202012/01.

In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets 501. V2.

In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "Cluster 5". In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "B.1.1.28".

In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "B.1.1.248".

In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets the Omicron (B.1.1.529) variant.

A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Pharmaceutical compositions described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.

The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons. Examples

Example 1 - Partial replacement of cholesterol by cholesterol ester mRNA having a concentration of 0.4 mg/mL was provided in 50 mM sodium acetate pH 5.5. An organic phase was provided 47.4 mM of total lipids in ethanol having the composition as listed in Table 4. The formulation does not comprise a stealth lipid. Quench buffer was water for injection. A raw colloid was produced by continuous mixing of the acidified mRNA with the organic phase, immediately followed by quenching. Directly after production, 250 mM of sodium triphosphate was added to the raw colloid to arrive at a final concentration of 5 mM sodium triphosphate. At this point, the pH of the resulting colloid is about neutral.

The resulting colloid was dialyzed against 50 mM HEPES, 5 mM sodium triphosphate, and 300mM sucrose pH 7.4 and adjusted to a concentration of RNA=0.1mg/mL. See also the process flow diagram in Figure 1, in which “3P” means sodium triphosphate and “MPES” means modified polyether sulfone. Although this Example and the Figure exemplifies cholesterol acetate, the process is equally applicable to compositions containing other cholesterol esters.

Table 4

The resulting products had the following particles size at the time of release and particle growth was monitored over time in liquid or frozen state. The frozen state was reached twice (2 x freeze/thaw (F/T) cycles).

Table 5

The materials displayed the following activities when tested for expression of a model cargo in cultured cells. For the test HepG2 cells were cultivated. Materials as provided in this example were diluted with the same volume buffer with full human serum and incubated for 30min at room temperature. 50 ng of the materials were added per well of a 96 well plate. Luciferase expression was monitored after 24 hours. Expression levels are provided in Table 6 below.

Table 6

The data demonstrate that a partial replacement of cholesterol by a cholesterol ester, specifically cholesterol acetate, is possible and advantageous. The resulting material is non- inferior to the starting material in terms of biological efficacy both in the presence and absence of serum. The resulting material, however, has a much- improved colloidal stability, more specifically an improved resistance to freeze/thaw conditions. Particles not comprising cholesterol show a size increase of >200nm (2x - 80°C) or > than 400nm (2x -20°C) whereas particles comprising a mixture of cholesterol and cholesterol acetate, particularly those having at least 20% cholesterol acetate have an improved stability upon freeze/thaw. A particular advantage is observed for materials comprising mixture of 40 or more% cholesterol acetate (based on total cholesterol). Example 2 - LNPs comprising cholesterol ester display very high stability

Cholesterol ester LNPs were prepared using an aqueous-ethanol mixing protocol in a volume part ratio of 3:1:2 (Acidified mRNA : organic phase : quench buffer). For that, mRNA having a concentration of 0.4 mg/mL was provided in 50 mM sodium acetate pH 5.5. The organic phase comprising 47.4 mM of total lipids (45.7 mol% ionizable lipid, 9.5 mol% DSPC and 44.8 mol% as sum of cholesterol and cholesterol acetate) was provided in ethanol. The different cholesterol acetate shares to total cholesterol and resulting mol% are listed in Table 4. Quench buffer was water for injection. A raw colloid was produced by continuous mixing of the acidified mRNA with the organic phase, immediately followed by quenching directly after production. 250 mM sodium triphosphate stock solution was added to the raw colloid to arrive at 5 mM sodium triphosphate.

The neutralized colloid was dialyzed against 50mM HEPES, 5 mM sodium triphosphate pH 7.4 followed by up-concentration using a cross-flow membrane (MikroKros 20 cm 100K modified polyether sulfone (MPES) 0.5 mm (C02-E100- 05)).

Eventually, solutions having 50mM HEPES 5mM sodium triphosphate (3P) solution pH7.4 and 50mM HEPES 5mM sodium triphosphate (3P), 1200mM sucrose pH7.4 were added to arrive at a final product having 0.3 mg/mL of RNA in 50 mM HEPES, 5 mM 3P, 300 mM sucrose, pH 7.4. See also the process flow diagram in figure 1.

The resulting products had the following particles size at the time of release and particle growth was monitored over time in liquid or frozen state. See Table 7 below.

able 7 The materials of this example had the following polydispersity at the time of release and over time in liquid or frozen state. See Table 8 below.

able 8

The materials of this example had the following RNA integrity at the time of release and over time in liquid or frozen state. See Table 9 below.

able 9

The materials of this example had the following content of cholesterol acetate at the time of release and over time in liquid or frozen state. See Table 10 below.

Table 10 The data demonstrate that a partial replacement of cholesterol by a cholesterol ester, specifically cholesterol acetate, is possible and advantageous. The resulting material has a much-improved colloidal stability and can be stored in liquid state for at least 28 weeks without a notable growth of the particle size or polydispersity. The materials can also be stored in frozen state without notable alterations of particles size of polydispersity. This is different and favourably compares to the material not comprising cholesterol acetate.

The materials of this example comprising cholesterol acetate have a much-improved stability of the RNA being encapsulated and can be stored in liquid state for at least 28 weeks without a notable reduction in RNA integrity. The materials can also be stored in frozen state without a reduction in RNA integrity. In contrast, the material not having cholesterol acetate displays a loss of RNA integrity over time as is known in the art. The LNP comprising cholesterol acetate are therefore preferred for RNA therapeutics having a long shelf life.

Example 3 - Method of continuous production mRNA having a concentration of 0.4 mg/ml was provided in 50mM sodium acetate pH 5.5. An organic phase was provided 47.4 mM of total lipids in ethanol. The formulation does not comprise a stealth lipid. 3 volumes of the acidified RNA was combined with 1 volume of the organic phase in a continuous mixing process using a mixing T. A quench solution having lOmM sodium triphosphate pH 8 was added to the product stream in a continuous mixing process using a second mixing T. The total flow rate in this process was 112.5 ml/min. A raw colloid having about neutral pH is obtained and collected.

The raw colloid was dialyzed against 10 mM HEPES, 5 mM sodium triphosphate pH 8.0 and concentrated using a MikroKros hollow fibre (20cm, 100k MPES, 0.5mm inner diameter). Buffer having 10 mM HEPES, 5 mM sodium triphosphate pH 8.0 and buffer comprising 1.2M sucrose are added to arrive at a final strength of the product of 0.3mg/mL RNA in a buffer having 10 mM HEPES, 5 mM sodium triphosphate 300mM sucrose pH 8.0. See also the process flow diagram in figure 2. Release specification of materials obtained in Example 3 comprising composition D (LNP001) and E (LNP002) as listed in Table 11.

Table 11

Table 12 below shows the material specification upon storage in liquid state at 5°C or 25°C for 2 weeks.

Table 12

This Example demonstrates that LNP comprising cholesterol acetate but no stealth lipid can be manufactured when the pH of the primary colloid obtained after mixing of the acidified RNA and the organic phase is adjusted instantaneously. This is in contrast to the state of the art where water is used to reduce the content of ethanol at this mixing step, the so-called quenching and a transition of the pH in subsequent processing steps is typically achieved in minutes or hours. Here, the pH transition is immediate within the time of mixing. Since the process is carried out in a continuous mode the mixing time is below one second. Quenching and adjustment of pH is combined into a single step whereas the known process is using two distinct steps. The materials obtained have a low polydispersity substantially below PDI of 0.1 thus representing a homogeneous dispersed phase. The encapsulation of RNA is essentially complete.

Example 4 - Lipid nanoparticles comprising cholesterol ester and stealth lipid

An RNA phase was prepared by mixing 2.0 mg/mL vaccine mRNA with 50 mM citrate buffer pH 4.0 to a final concentration of 0.4 mg/mL mRNA. An organic phase was prepared by dissolving the lipids according to ratios described in Table 13 to final concentration of 45.5 mM in absolute ethanol, corresponding to a nitrogen: phosphate (N:P) ratio of 6.

Table 13. Percentages refer to molar ratios (mol%). “CE30” represents replacement of 30 mol% of the cholesterol by cholesterol acetate; CE40” represents replacement of 40 mol% cholesterol by cholesterol acetate.

LNP formation was performed by rapid mixing of RNA phase, organic phase, and dilution buffer (50 mM citrate pH 4.0) at a volumetric ratio of 3: 1 :2 respectively, with a total flow rate of 150 mL/min. Here the initial rapid mixing of the RNA and organic phase occurred through a stainless-steel mixer with an internal diameter of 0.5 mm, where dilution was subsequently performed through a stainless-steel mixer with an internal diameter of 2.0 mm. The obtained intermediate LNPs were purified by dialysis against 10 mM Tris pH 7.4 overnight at room temperature. Following purification, the LNPs were filtered using a 0.22 pm PES filter and diluted to a final concentration of 0.1 mg/mL RNA with the final buffer matrix consisting of 10 mM TRIS pH 7.4 and 10% w/v sucrose as cryoprotectant. The product specifications at release were measured and shown in Table 14.

The LNP formulations containing cholesterol acetate showed superior stability over 5 freeze-thaw cycles at -20 °C compared with the formulation without cholesterol acetate. In Figure 3 it is shown that the particle size of cholesterol acetate containing LNPs remains unchanged during freeze-thaw cycles and the polydispersity index (PDI) remains below 0.1 , whereas the formulation without cholesterol shows an upward trend in both particle size and PDI as a function of freeze-thaw.

Example 5 - General Method - preparation of LNP by a liquid-dispensing system

Ethanolic solutions of a cationically ionisable lipid (50mM), cholesterol (40mM), a cholesterol ester selected from cholesteryl acetate, cholesteryl laurate, cholesteryl linoleate and cholesteryl oleate (40mM), DSPC (33mM), and DMG-PEG (8mM) were prepared. The different lipid solutions were dispensed using a liquid-dispensing system in predefined ratios into a 96-well plate in the following order: cationically ionisable lipid, cholesterol, cholesterol ester, phospholipid, and DMG-PEG. Enough ethanol was then added to complete a final volume of 33 pL.

A solution containing mRNA (0.2 mg/mL) was added to each lipid solution until a final volume of 200 pL was reached, resulting in a final nucleic acid concentration of 55 ng/pL. Aliquots were taken and further diluted stepwise using buffer at neutral pH, generating LNP with a final nucleic acid concentration of 5 ng/pL. Typically, the obtained LNPs were used without further purification.

Example 6 - Formulations prepared using a liquid-dispensing system

Applying the method described generally in Example 5, samples having the lipid composition shown in T able 15 were prepared using three ionizable lipids with different structures selected from BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, and BODD-C2C2- DMA. All LNP comprising PEG lipid contained luciferase-mRNA as cargo.

To demonstrate the impact of replacing cholesterol by diverse cholesterol esters, each formulation in Table 15 was formulated again, replacing a portion of cholesterol (25, 50, 75 or 100%) with different cholesterol esters, selected from cholesteryl acetate, cholesteryl laurate, cholesteryl oleate, and cholesteryl linoleate. A replacement of 100% implied that no cholesterol was left in the formulation. The matrix of formulations tested is shown in Figure 4. For example, for Sample ID 1-1, 51 different formulations were prepared: containing 37.5 mol% ionizable lipid (BHD-C2C2-PipZ, BODD-C2C2-lMe-Pyr, or BODD- C2C2-DMA); 10 mol% DSPC; 1.8 mol% DMG-PEG, and:

(i) 50.7 mol% cholesterol + 0 mol% cholesterol ester,

(ii) 38.02 mol% cholesterol + 12.68 mol% cholesterol ester (25% replacement),

(iii) 25.35 mol% cholesterol + 25.35 mol% cholesterol ester (50% replacement), or

(iv) 12.68 mol% cholesterol + 38.02 mol% cholesterol ester (75% replacement), where the cholesterol ester was cholesteryl acetate, cholesteryl laurate, cholesteryl oleate or cholesteryl linoleate.

Table 15

Example 7 - Impact of cholesterol ester content on the formation of LNP comprising PEG lipid

After processing, the particle size of the formulations prepared in Example 6 are illustrated in Figure 4. The figure shows that LNP comprising a PEG lipid (PEG2000- DMG) with diameters between 50 and 200 nm can be formed upon replacing cholesterol with different cholesterol esters up to 75% replacement. Generally, a total replacement of cholesterol with cholesterol ester (100% replacement) did not result in stable particles. These results are based on screening of a large compositional space, as shown in Table 15 and Figure 4, demonstrating the findings are generally applicable. Example 8 - Impact of cholesterol ester content on liquid stability of LNP comprising PEG lipid

Particle growth of the formulations of Example 6 was monitored after 2 weeks storage at 4 °C (Figure 5). The figure shows that LNP comprising PEG lipid (PEG2000-DMG) with diameters between 50 and 200 nm, as shown in Example 7, can be formed and are stable for 2 weeks after preparation. Generally, particles formed with 100% replacement of cholesterol with cholesterol ester showed poor stability over the storage period.

Example 9 - Impact of cholesterol ester content on the transfection potency of LNP comprising PEG lipid

For transfection studies, 10 pL of the formulations prepared in Example 6 were prediluted in 10 pL human AB serum in a 96-well plate and incubated at room temperature for 30 min. After incubation, the formulations were added to 100 pL of cell culture media DMEM containing 0.22e5 Hek293 cells. After 24 h of incubation (37 °C, 5 % CO2), the cells were analyzed for transfection potency using a luciferase assay.

Figure 6 introduces the results of in vitro testing on Hek293 cells. The potency of each formulation in Example 6 is presented relative to the respective formulations containing only cholesterol (no cholesterol esters, Table 16). The figure shows that LNP comprising PEG lipid with diameters between 50 and 200 nm, as shown in Example 7, are generally active and able to transfect, even when up to 75% of the cholesterol content was replaced by cholesterol esters. For the different ionizable lipids, many LNP formulations having up to 50% of the cholesterol content replaced by cholesterol esters were found to be active and showed good transfection potencies. The most potent LNP formulations in this assay were generally observed to have about 25% of the cholesterol content replaced by cholesterol esters.

Example 10 - General Method - preparation of PEG-free LNP by microfluidic mixing

Ethanolic solutions of a cationically ionisable lipid (50mM), cholesterol (50mM), various cholesterol esters (15mM), and DSPC (33mM) were prepared. The different lipid solutions were mixed manually using a programmable multistep pipette. Predefined volumes were pipetted into a 96-well plate in the following order: ethanol, cationically ionisable lipid, cholesterol, cholesterol ester, and phospholipid. The final volume of each lipid mix was 800 pL. Separately, a mRNA stock solution (0.3 mg/mL) was prepared in acetate buffer (50mM acetate, pH 5.5).

The solutions containing lipids and mRNA were mixed in a T-junction, producing raw colloid material. In a second T-junction, the initial raw colloid was diluted with water to decrease the ethanol content. Next, 250 mM of sodium triphosphate was added to the raw colloid to arrive at a final concentration of 5 mM sodium triphosphate. Particles were then dialyzed overnight against 50 mM HEPES, 5 mM sodium triphosphate, pH 7.4. After determination of the total mRNA concentration, the samples were diluted in two consecutive steps. First, with a 50 mM HEPES, 5 mM sodium triphosphate, 1200 mM sucrose, pH 7.4 solution, to reach a sucrose concentration of 300 mM. The second step with 50 mM HEPES, 5 mM sodium triphosphate, 300mM sucrose, pH 7.4, to bring each sample to a final cargo concentration of 0.05 g/L. Other suitable manufacturing methods for preparing polyphosphate LNP are described, for example in WO2023/194508A1.

Example 11 - Formulations prepared using microfluidic mixing

Applying the method described generally in Example 10, samples having the lipid composition shown in Table 16 were prepared using BHD-C2C2-PipZ as ionizable lipid, a cholesterol ester selected from cholesteryl acetate, cholesteryl laurate, cholesteryl oleate and cholesteryl linoleate, and luciferase-mRNA as cargo. The cholesterol replacement level ranged from 0 to 60% in 10% increments.

Table 16 Example 12 - Impact of cholesterol ester content on the size of PEG-free particles

After processing, the size of the fully processed LNP prepared in Example 11 is illustrated in Figure 7. The figure shows that PEG-free LNP with diameters between 50 and 120 nm can be formed upon replacing cholesterol with different cholesterol esters up to 60% replacement.

Example 13 - Impact of cholesterol ester content on liquid and freeze/thaw stability of PEG-free particles

Particle growth of the formulations in Example 11 was measured after 1 day, 2 weeks, and 4 weeks storage at 4 °C (Figure 8). The figure shows that the liquid stability improves with the cholesterol ester content. Compositions having between 30% and 60%, and more preferred between 40% and 50%, were selected for liquid stability.

In addition to liquid stability, particle growth was measured after 1, 2 and 3 freeze-thaw cycles at -80 °C (Figure 9). Freeze-thaw stability improves with the cholesterol ester content. Compositions having between 30% and 60%, and even more preferred between 40% and 50%, were selected for freeze-thaw stability. For freeze-thaw stability, esters from cholesterol and unsaturated acids were preferred. More preferred were acids containing 1 or 2 unsaturated bonds. In particular, the two best performing specific lipids for freeze-thaw stability were cholesteryl oleate and cholesteryl linoleate. Cholesterol laurate formulations were found to be more suited to liquid storage.

Example 14 Impact of the cholesterol ester content on the transfection potency of PEG-free particles

Applying the method described generally in Example 9, transfection studies were performed for the formulations in Example 11. Figure 10 introduces the results of in- vitro testing on Hek293 cells. The potency of each formulation in Example 11 is presented relative to a benchmark formulation (BHD-C2C2-PipZ/DSPC/Cholesterol) containing no cholesterol esters. The figure shows that the LNP formulated in Example 11 still show sufficient activity even up to 60% cholesterol replacement. Overall, the data demonstrate that a good balance between improved particle stability and high transfection activity can be achieved by replacing between 20% to 60% of cholesterol in the lipid mixture composition (and resulting nucleic acid-lipid particle composition with cholesterol esters, with the most optimum range across the tested formulations being found at about 40% replacement of cholesterol by cholesterol ester.

The findings were found to apply to both LNP having and not having a stealth lipid. A variety of aliphatic cholesterol esters can be used, and esters with unsaturated fatty acids were found to be particularly effective. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present 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 for carrying out the invention which are obvious to those skilled in chemistry, biochemistry, molecular biology, biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A composition comprising:

(a) a cationically ionizable lipid;

(b) cholesterol; and

(c) a cholesterol ester; and

(d) a neutral lipid; wherein the cholesterol ester (c) is present in an amount of up to about 75 mol% of the total of cholesterol (b) and cholesterol ester (c) present in the composition.

2. The composition according to claim 1, wherein the cholesterol (b) is present in an amount of about 6 mol% to about 48 mol% of the total of the lipids present in the composition.

3. The composition according to claim 2, wherein the cholesterol (b) is present in an amount of about 13 mol% to about 40 mol% of the total of the lipids present in the composition.

4. The composition according to claim 3, wherein the cholesterol (b) is present in an amount of about 15 mol% to about 32 mol% of the total of the lipids present in the composition.

5. The composition according to any one of claims 1 to 4, wherein the cholesterol ester (c) is present in an amount of about 3 to about 40 mol% of the total of the lipids present in the composition.

6. The composition according to claim 5, wherein the cholesterol ester (c) is present in an amount of about 6 to about 26 mol% of the total of the lipids present in the composition.

7. The composition according to claim 6, wherein the cholesterol ester (c) is present in an amount of about 10 to about 21 mol% of the total of the lipids present in the composition.

8. The composition according to any one of claims 1 to 7, wherein the total amount of cholesterol (b) and cholesterol ester (c) is present in an amount of about 20 to about 60 mol% of the total of the lipids in the composition.

9. The composition according to any one of claims 1 to 8, which is a lipid particle composition.

10. The composition according to any one of claims 1 to 9, wherein the cholesterol ester (c) is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 40 carbon atoms.

11. The composition according to claim 10, wherein the cholesterol ester (c) is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 20 carbon atoms.

12. The composition according to claim 11, wherein the cholesterol ester (c) is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 6 carbon atoms.

13. The composition according to claim 12, wherein the cholesterol ester (c) is cholesterol acetate.

14. The composition according to claim 11, wherein the cholesterol ester (c) is a cholesterol alkanoyl ester, the alkanoyl part having from 12 to 18 carbon atoms.

15. The composition according to claim 14, wherein the cholesterol ester (c) is cholesterol laurate.

16. The composition according to any one of claims 1 to 9, wherein the cholesterol ester (c) is a cholesterol alkenoyl ester, the alkenoyl part having from 12 to 18 carbon atoms.

17. The composition according to claim 16, wherein the cholesterol ester (c) is cholesterol oleate or cholesterol linoleate.

18. The composition according to any one of claims 1 to 17, wherein the cholesterol ester is present in an amount of about 5 to about 75 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

19. The composition according to any one of claims 1 to 17, wherein the cholesterol ester is present in an amount of about 20 to about 70 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

20. The composition according to any one of claims 1 to 19, wherein the cholesterol ester is present in an amount of about 30 to about 50 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

21. The composition of claim 20, wherein the cholesterol ester is present in an amount of about 38 to about 42 mol% of the total of cholesterol and cholesterol ester present in the lipid mixture composition.

22. The composition according to any preceding claim, wherein the cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylheptane-l-sulfonamide) (BNT-51);

7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-l- sulfonamide) (BNT-52);

1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA);

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); di((Z)-non-2-en-l-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);

O- [N- { (9Z, 12Z)-octadeca-9, 12-dien- 1 -yl) } -N- { 7 -pentadecylcarbonyloxy octyl } - amino]4-(dimethylamino)butanoate (HY501 ); di(heptadecan-9-yl) 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(l-methylpyrrolidin-2-yl)ethyl)azanediyl)- dipropionate (BODD-C2C2-lMe-Pyr); bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); or a mixture of any thereof.

23. The composition according to any preceding claim, wherein the cationically ionizable lipid (a) is present in an amount of about 10 to about 50 mol% of the total lipid composition.

24. The composition according to any preceding claim, further comprising a multivalent anion.

25. The composition according to claim 24, wherein the multivalent anion comprises an inorganic polyphosphate.

26. The composition according to claim 25, wherein the inorganic polyphosphate is selected from the group consisting of diphosphate, triphosphate, tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate, and mixtures thereof.

27. The composition according to claim 26, wherein the inorganic polyphosphate is selected from the group consisting of diphosphate, triphosphate, tetraphosphate, pentaphosphate, and mixtures thereof.

28. The composition according to claim 27, wherein the inorganic polyphosphate is triphosphate.

29. The composition according to any one of claims 25 to 28, wherein the molar ratio of the inorganic polyphosphate to the cationically ionizable lipid is at least about 1 :2.

30. The composition according to claim 29, wherein the molar ratio of the inorganic polyphosphate to the cationically ionizable lipid is at least about 2:3.

31. The composition according to claim 30, wherein the molar ratio of the inorganic polyphosphate to the cationically ionizable lipid is, at least about 4:3.

32. The composition according to any preceding claim, wherein the neutral lipid is a neutral or zwitterionic phospholipid.

33. The composition according to claim 32, wherein the neutral or zwitterionic phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); di ol eoy Iphosphati dy 1 ethanol amine (DOPE) ; l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG); N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); or a mixture of any thereof.

34. The composition according to claim 33, wherein the neutral or zwitterionic phospholipid is distearoylphosphatidylcholine (DSPC).

35. The composition according to claim 33, wherein the neutral or zwitterionic phospholipid is dipalmitoylphosphatidylcholine (DPPC).

36. The composition according to any preceding claim, further comprising a grafted lipid.

37. The composition according to claim 36, wherein the grafted lipid is selected from the group consisting of: a polyethylene glycol) conjugated lipid (PEG-lipid); a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2-methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipid; or a mixture of any thereof.

38. The composition according to claim 37, wherein the grafted lipid is a poly(ethylene glycol) conjugated lipid (PEG-lipid);

39. The composition according to claim 37 or claim 38, wherein the grafted lipid is 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) wherein the PEG portion of the pegylated lipid has an average molecular weight of about 2000 g/mol.

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

41. The composition according to claim 40 wherein the nucleic acid is mRNA.

42. A nucleic acid-lipid particle composition comprising:

(i) the composition according to any preceding claim; and

(ii) a nucleic acid.

43. The nucleic acid-lipid particle composition according to claim 42, wherein the nucleic acid is mRNA.

44. The nucleic acid-lipid particle composition according to claim 42 or claim 43, for use in medicine.

45. The nucleic acid-lipid particle composition according to claim 44, for use in treating or preventing a viral infection.

46. A method of producing the composition as defined in any one of claims 1 to 39, the method comprising mixing: (a) a cationically ionizable lipid;

(b) cholesterol;

(c) cholesterol ester; and

(d) a neutral lipid; to form the composition.

47. A method of producing the nucleic acid-lipid particle composition according to claim 42 or claim 43, the method comprising mixing the composition as defined in any one of claims 1 to 39 with a nucleic acid to form the nucleic acid-lipid particle composition.

48. A method of producing the nucleic acid-lipid particle composition according to claim 42 or claim 43, the method comprising mixing:

(a) a cationically ionizable lipid;

(b) cholesterol;

(c) a cholesterol ester;

(d) a neutral lipid; and

(e) a nucleic acid; to form the nucleic acid-lipid particle composition.

49. A method of producing the nucleic acid-lipid particle composition according to claim 42 or claim 43, the method comprising mixing:

(a) an organic phase comprising cationically ionizable lipid, cholesterol, cholesterol ester, and neutral lipid; and,

(b) an aqueous phase comprising a nucleic acid; to form the nucleic acid-lipid particle composition.