Method of determining nucleic acid concentration in aqueous solution or dispersion

Abstract

A method of determining the nucleic acid concentration in an aqueous dispersion, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and optionally ii) a mobile phase selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy; is provided. A method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and / or ii) a solvent selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy; is also provided.

Description

[0001] METHOD OF DETERMINING NUCLEIC ACID CONCENTRATION IN AQUEOUS SOLUTION OR DISPERSION

[0002] Field of the Invention

[0003] This invention relates to a method of determining the nucleic acid concentration in an aqueous solution or dispersion, particularly although not exclusively in a nucleic acid- lipid particle formulation.

[0004] Background to the Invention

[0005] The reliable quantification of nucleic acids (e.g. RNA, DNA) is an essential quality parameter for many experiments and manufacturing steps. Standard methods to determine the nucleic acids quantity in aqueous solution are mainly based on fluorescence dyes interacting with the nucleic acids. These assays have major drawbacks, based on the fact that the dye properties and binding to nucleic acids are highly sensitive towards components which are intrinsic in the solutions (e.g. salts, high or low pH, lipids, impurities). Furthermore, a reference nucleic acid is obligatory, against which samples are quantified.

[0006] Another method used in the art is ultraviolet / visible (UV) absorption spectroscopy, which employs the characteristic absorption maximum (at ~260nm) of the nucleic acids. Absorption signals are converted into a mass or molar concentration by using the Beer-Lambert law (logy = A = sic).

[0007] However, two major problems have to be overcome to make the UV-Visible method applicable for use for quantification of nucleic acid in aqueous solutions. Firstly, the chemicals used must be suitable for UV-Vis spectroscopy. In particular, they should not interfere with UV-Vis measurements and not impact the absorption spectra of the nucleic acid. Secondly, the extinction coefficient (e) of nucleic acids is highly dependent on the chemical environment, such as the buffer matrix, thereby posing a risk for underestimation of the nucleic acid concentrations. This therefore has a direct, major impact on the measured absorption and thereby measured nucleic acid content (c = — ). Since even small traces of ions (e.g. the presence of salts, such as sodium chloride in the buffer component) have a considerable influence on the measurement (matrix effect), all nucleic acid quantitations in buffered solutions are potentially affected, leading to lower concentration values.

[0008] Nucleic acids used for therapeutic indications are often delivered in the form of nucleic acid containing particles, generally referred to herein as “nucleic acid-lipid particles”. Examples of formulations including nucleic acid-lipid particles include lipid nanoparticle (LNP) or lipoplex (LPX) formulations. The nucleic acids used in such formulations may be RNA, DNA or a mixture of the two.

[0009] For such formulations, in addition to the parameters described above, a reliable nucleic acid content method for release, stability testing, formulation development and for in-process control is required. In all these steps, the buffer composition usually changes so that a direct effect on the nucleic acid content is to be expected. Moreover, for the UV spectroscopy measurement, it is necessary to completely disrupt the lipid nanoparticles in order to reliably and correctly measure the nucleic acid content without any impact of particulate scattering. Commonly used solubilization agents like sodium dodecyl sulfate (SDS), however, do not work for e.g. formulations employing complex lipid architectures (e.g. LPX with an excess of lipids resulting in high nitrogen / phosphate (N / P) ratios). Therefore, a generally applicable solubilization method which is applicable for a wide range of lipid-based formulations is needed.

[0010] WO202 1 / 061594 describes a method for measuring the RNA concentration of a suspension of one specific RNA-lipid nanoparticle (RNA-LNPs) wherein the RNA- LNPs comprise ionizable cationic lipids, the method comprising: (a) mixing a suspension of RNA-LNPs with an assay diluent comprising a surfactant and an alkylamine to provide a diluted sample solution; (b) measuring absorbance of the diluted sample solution at lambda maximum of the RNA ( max) to provide a Net Absorbance or measuring absorbance of the diluted sample solution at kmax and at 400 nm and subtracting the absorbance at 400 nm from the absorbance at kmax to provide an adjusted Net Absorbance; and (c) using the Net Absorbance or the adjusted Net Absorbance to determine the RNA concentration in the suspension of the RNA-LNPs. However, the use of an alkylamine means that the method is carried out at high pH (typically around pH 12). At such a high pH, many nucleic acid molecules are degraded, in particular hydrolysed, meaning that the method is unreliable or cannot be used with base-sensitive nucleic acid molecules.

[0011] Ferrari Marilyn E. et al; (Analytical Methods for the Characterization of Cationic Lipid-Nucleic Acid Complexes; Human Gene Therapy (1998) vol. 9, No. 3, pages 341-351) describes a method of determining the concentration of DNA released from lipoplexes and DNA controls, where the method comprises mixing the sample with a zwitterionic surfactant (Zwittergent® 3-14) and sodium acetate at a pH value of 6. DNA concentration is determined by ultraviolet-visible spectroscopy. However, there is no disclosure of a medium containing both a zwitterionic surfactant and a mobile phase selected from an alcohol, acetone, dimethyl sulfoxide, or a mixture of any thereof.

[0012] Alison O. Nwokeoji et al. (Accurate Quantification of Nucleic Acids Using Hypochromicity Measurements in Conjunction with UV Spectrophotometry; Analytical Chemistry (2017) vol. 89, No 24, pages 13567-13574) describes a method of determining nucleic acid concentration in an aqueous solution, using ultraviolet- visible spectroscopy, the aqueous solution comprising a nucleic acid sample and dimethyl sulfoxide. However, there is no disclosure of a medium containing a zwitterionic surfactant and / or a solvent selected from an alcohol, acetone, or a mixture thereof.

[0013] Summary of the Invention

[0014] According to a first aspect of the invention, there is provided a method of determining the nucleic acid concentration in an aqueous dispersion, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and optionally ii) a mobile phase selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

[0015] According to a second aspect of the invention, there is provided a method of determining the nucleic acid concentration in an aqueous dispersion, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and ii) a mobile phase selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

[0016] According to a third aspect of the invention, there is provided a method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and / or ii) a solvent selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

[0017] According to a fourth aspect of the invention, there is provided a method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; or ii) a solvent selected from the group consisting of an alcohol, acetone, or a mixture thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

[0018] According to a fifth aspect of the invention, there is provided a method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and ii) a solvent selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

[0019] Advantages and Surprising Findings

[0020] The inventors have surprisingly found that the use of the zwitterionic surfactants, optionally together with a mobile phase as described below, in the method of the invention when applied to aqueous dispersions, solves the problem of solubilizing a wide range of lipid-based nanoparticles, in particular lipoplexes (LPX) and lipid nanoparticles (LNP) to enable their nucleic acid concentration to be reliably determined by UV-Vis spectroscopy. The surfactants and, where used, the mobile phases are suitable for UV-Vis spectroscopy. In particular, they do not interfere with UV measurements and do not disturb the absorption spectra of the nucleic acid.

[0021] Similarly, the inventors have surprisingly found that the use of the zwitterionic surfactants and / or the solvents as described below, in the method of the invention when applied to aqueous solutions, solves the problem of solubilizing a wide range of lipid-based nanoparticles, in particular lipoplexes (LPX) and lipid nanoparticles (LNP) to enable their nucleic acid concentration to be reliably determined by UV-Vis spectroscopy. The surfactants and / or solvents are suitable for UV-Vis spectroscopy. In particular, they do not interfere with UV measurements and do not shift the absorption spectra of the nucleic acid. Furthermore, the inventors have surprisingly found that the use of the zwitterionic surfactants, either together with a mobile phase as described below (for aqueous dispersion) or as an alternative to the solvents described below (for aqueous solutions), in the method of the invention, eliminates the problem of the extinction coefficient varying with the conditions (in particular, the buffer matrix, the concentration of ions present, such as sodium and chloride ions, and the pH). The applicability of a general extinction coefficient for the measurements opens up the use of UV-Vis spectroscopy to determine the concentration of the nucleic acid in the formulation in a reliable direct comparable and more correct manner. Thus, both nucleic acid alone and in formulation can be analyzed with the identical method, which significantly increases the comparability and standardization of the manufacturing processes (e.g. in-process controls).

[0022] Furthermore, the inventors have surprisingly found that the use of the zwitterionic surfactants, either together with a mobile phase as described below (for aqueous dispersion) or with the solvents described below (for aqueous solutions), in the method of the invention, improved sensitivity of the UV / Vis spectrum analysis. For example, the combination of zwitterionic surfactants and a mobile phase or solvent comprising an alcohol were found to have positive effects on the detection of different nucleic acids of diverse chemical composition (mRNA, nucleoside-modified RNA, uRNA, saRNA, DNA), particularly mRNA.

[0023] In addition, and in contrast to the method described in WO2021 / 061594, the use of a zwitterionic surfactant and / or a solvent avoids the use of alkylamines, thereby enabling the method to be carried out at acidic / neutral pH, and therefore can be used on a wider range of nucleic acid-containing solutions while avoiding the issue of the degradation of the nucleic acid.

[0024] In addition, and in contrast to the method described in WO2021 / 061594, the assay is highly specific, high-throughput capable, robust, easy and quick to apply. The assay can be performed in complex matrices including a number of ingredients (e.g. buffer, salt, lipids and sugars). In addition, the solubilization of the nanoparticle can be monitored to a certain extent by a UV / Vis spectrum analysis. Finally, the method can deal with different nucleic acids of diverse chemical composition (mRNA, nucleoside-modified RNA, uRNA, saRNA, DNA) and also a wide nucleic acid concentration range. Furthermore, in contrast to this prior art method, hold-time studies show that the assay produces a stable signal over hours, which simplifies much for the applicability in the GMP area. Finally, the method has a wide linear range and can therefore also deal with highly diluted nucleic acid or nanoparticle solutions (e.g. samples from in-use studies), independent of the chemical composition of the nucleic acid (mRNA, nucleoside-modified RNA, or DNA).

[0025] Brief Description of the Figures

[0026] Figure 1 illustrates the matrix effect of sodium ions on the UV absorption of nucleic acid (RNA). (A) Shown are representative absorption spectra of the identical concentrated RNA with increasing levels of sodium chloride (dark to light grey). (B) Normalized and averaged absorption signal of different RNAs with increasing sodium chloride levels (dark to light grey), including an exponential fit of the resulting data. Within all titration experiments, RNA overall dilution and buffer conditions (except sodium) were kept always constant. Error bars represent standard deviations (n= 6 different RNAs measured in triplicates).

[0027] Figure 2 illustrates the absorbance values of different, representative RNAs (A) short (-1,000 nts) and B) long (-2,000 nts) at five different RNA concentrations and three conditions (water (black) or 28mM sodium chloride (light grey) or 28mM sodium chloride plus zwitterionic surfactant and ethanol (grey)) that were used for the dilution. Inset in B shows a second RNA in the presence of lOOmM sodium chloride with and without zwitterionic surfactant and ethanol. C) Measurement of three additional RNAs with the method of the invention using a zwitterionic surfactant (n- tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and ethanol in a dose range of 5 to 30 pg / mL. For all conditions, the data points were fitted by a linear regression. Within all titration experiments, RNA overall dilution and buffer conditions were kept always constant. Error bars represent standard deviations (n= 3).

[0028] Figure 3 illustrates the impact of additional sodium chloride on the nucleic acid (RNA) UV absorption of lipid-based RNA nanoparticles (NP) solubilized by sodium dodecyl sulfate. Shown data points are the mean and s.d. (error bars) of three technical replicates. Figure 4 shows UV spectra of an untreated (grey) complex, lipid-based nucleic acid (RNA) containing NP (LPX) or treated with SDS (black) or with zwitterionic surfactant (n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and alcohol (“NP+ZWG+alcohol”- light grey). Inset shows a magnification around 260nm wavelength.

[0029] Figure 5 shows UV spectra of an untreated (grey) complex, lipid-based nucleic acid (RNA) containing NP (LNP) or treated with zwitterionic surfactant (n-tetradecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and alcohol (“NP+ZWG+alcohol”- black). Inset shows a magnification around 260nm wavelength.

[0030] Figure 6 illustrates the recovery of matrix-effects by the combined use of zwitterionic detergents and organic solvents. Shown are the normalized differences in absorption values at 260 nm for non-nucleoside-modified mRNA (dark grey) and Nl- methylpseudouridine modified mRNA (light grey) in response to saturating levels of mono- and bivalent ions, compared to an ion-reduced environment (w / o addition; left). Note that also without salt addition, a minor base level of ions was present. (Right) For a mixture of both zwitterionic detergent and organic solvents, recoveries of the RNA absorption are seen for all tested mono- and bivalent ions. Errors bars represent standard deviations (n=3 replicates).

[0031] Figure 7 illustrates the recovery of matrix-effects by the use of zwitterionic detergents. Shown are the normalized differences in absorption values at 260 nm for non-nucleoside-modified mRNA (dark grey) and N1 -methylpseudouridine modified mRNA (light grey) in response to saturating levels of sodium chloride (“NaCl”), compared to a ion-reduced environment (“w / o addition”). Note that also without salt addition, a minor base level of ions was present. For different zwitterionic detergents, a recovery of the RNA absorption are seen for all tested mono- and bivalent ions. Errors bars represent standard deviations (n=3 replicates).

[0032] Figure 8 illustrates the recovery of matrix-effects by the use of zwitterionic detergents and / or organic solvents. Shown are the normalized differences in absorption values at 260 nm for nonnucleoside-modified mRNA (dark grey) and N1 -methylpseudouridine modified mRNA (light grey) in response to organic solvents (second and third bars; “EtOH” and “IP A”) or the combination of organic solvents with zwitterionic detergents (fourth and fifth bars; “EtOH + mix” and “IP A + mix”). In direct comparison, the absorption in presence of saturating levels of sodium chloride are shown (first bar; “NaCl”). Normalization was performed against the RNA absorption in low concentrated buffered condition. Unperturbed recovery of the RNA’s absorption could be observed in different combinations of zwitterionic detergents and organic solvents (cf “EtOH + mix” and “IP A + mix”). Errors bars represent standard deviations (n=3 replicates).

[0033] Figure 9 shows solubilization and linearity of lipid-based nanoparticles (NP) by the use of the new method. A) Shown are the averaged, blank-corrected spectra (n=3 technical replicates) for each dilution of the NP after solubilization with a zwitterionic surfactant (n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and alcohol. B) Linearity of the new method tested by parallel dilution (2 pg / mL to 27.5 pg / mL). Shown are the mean and s.d. (n=3 technical triplicates) for each dilution for the NP (#1 : triangle; #2: diamond and #3 : box) including a linear fit indicated by the dotted lines. C) analogue to B an alternative NP with a different composition is tested for linearity. Exemplary for the NP batch 4 the respective dilutions were considered for the plotted values.

[0034] Figure 10 shows the impact on incubation time on the nucleic acid (RNA) UV- absorption of RNA samples (A) or lipid-based RNA nanoparticles (B) solubilized by the method of the invention. The absorption of differently diluted samples was either measured directly (black) and after the indicated incubation time at room temperature (grey). Shown are mean values (n=3 technical replicates), standard deviation (error bars) and linear fits of the data.

[0035] Figure 11 illustrates harmonized RNA content methods employing zwitterionic detergents and / or organic solvents offer superior process control compared to conventional UV methods. Shown are the normalized mass balances in % (RNA output divided by RNA input) of three separate LPX manufacturing processes based on RNA content quantifications of drug substances and drug products. Compared to non-aligned methods that either use dilution in buffer and / or an anionic surfactants (dark grey), use of the developed method employing zwitterionic surfactants (n- tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and organic solvent (grey) yield a process mass balance close to 100% (dotted line). Figure 12 is a UV spectrum of (A) a sample buffer (24 pl) without LNPs / RNA ( = blank) and (B) a sample buffer (24 pl) with LNPs containing 100 pg / mL RNA as used in Example 6. Detailed Description

[0036] 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.

[0037] 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; Bey er / W alter, "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.

[0038] Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Definitions

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are Ci-40 alkyl (such as Ce-40 alkyl, Ce-30 alkyl, C6-20 alkyl, or 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.

[0048] 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.

[0049] 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 halo or hydroxy.

[0050] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1 -ethylene, 1,2-ethylene), propylene (i.e., 1,1- propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3- propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3- butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1-iso- butylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1- pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso- pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1- isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3 -heptylene, 1,4-heptylene, 1,5 -heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5- octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2- butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituent may be the same or different).

[0051] 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 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 carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1 -propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5- heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7- nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6- decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10- undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6- dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different).

[0052] 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 halogen or hydroxy.

[0053] 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 halogen or hydroxy.

[0054] 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 halogen, hydroxy or nitro.

[0055] 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, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotri azolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrol othi azolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from halogen or hydroxy. 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 halogen or hydroxy.

[0056] 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 halogen or hydroxy. 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.

[0057] 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 halogen or hydroxy.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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,

[0063] 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.

[0064] 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,

[0065] 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 (in the anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0066] 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 (in the zwitterionic surfactants as defined below, the sulfonate group is typically deprotonated at neutral or alkaline pH).

[0067] The term “carboxylic acid” or “carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “carboxylate” is used when the group is deprotonated. Depending on the pH, the carboxylic acid may be protonated or deprotonated (in the zwitterionic surfactants as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0068] “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 Ci-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.

[0069] A “quaternary ammonium” salt is a compound containing a group -N+R3, 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 Ci-6 alkyl group. In contrast to some amines as defined above which are protonated only at certain pH, a quaternary ammonium salt carries a constitutive positive charge (as defined herein) at all pH.

[0070] “Hydroxyl” - means the group -OH. “Sulfhydryl” - means the group -SH. “Nitro” means the group -NO2.

[0071] A “ready -to-use” formulation, as used herein, refers to a formulation that does not require constitution or dilution with a prescribed amount of diluent for injection, or other suitable diluent, before use by the designated route. For example, a formulation in a vial, of the desired concentration, that need only be drawn into a syringe.

[0072] Nucleic Acid

[0073] The method of the present invention are carried out on an aqueous solution (as described below) including a nucleic acid. The method is applicable to different nucleic acids formats, including RNA (e.g. uRNA, modRNA, saRNA) and DNA, and with a wide nucleic acid concentration range. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises cDNA and mRNA, which are 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.

[0074] 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.

[0075] 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-pseudouri dine (ml'P).

[0076] RNA

[0077] 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 analogs 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.

[0078] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may be 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

[0079] 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). 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.

[0080] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0081] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

[0082] 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.

[0083] In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The term "in vitro transcription" or "IVT" as used herein means that the transcription (z.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g, cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™).

[0084] For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription. The RNA (such as mRNA) may be modified. The RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly). One or more uridine in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. The RNA may comprise a modified nucleoside in place of at least one uridine. Preferably, the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside). The modified nucleoside may be independently selected from pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (y) or Nl-methyl-pseudouridine (mly).

[0085] 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.

[0086] 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.

[0087] Inhibitory RNA

[0088] In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] DNA

[0094] 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 analogues 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 analogues 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.

[0095] Pharmaceutically active peptides or polypeptides

[0096] "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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD 19), antigen receptor accessory molecules (e.g., CD-3y, CD3-6, CD-3s, 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.

[0101] Aqueous Systems, Solutions and Dispersions

[0102] The method of the present invention is carried out in an aqueous system. The aqueous system may be homogeneous, having a single phase, or heterogeneous, having more than one (preferably only two) phases. The system contains a liquid which is or comprises water - the liquid forming the mobile phase when the system is a dispersion and the solvent when the system is a solution.

[0103] In one aspect, the aqueous system is an aqueous solution. The term “aqueous solution” as used herein is a homogeneous mixture of one component (the solute) in another (the solvent). The solvent is or comprises water. The aqueous solution may contain other solutes which can be salts, buffers, sugars, tonifiers and the like, as long as these materials are molecularly distributed within the solvent.

[0104] In another embodiment, the aqueous system is an aqueous dispersion having an aqueous mobile phase and a dispersed phase. In this specification the term “dispersion” in its broadest sense takes its usual meaning in chemistry as a heterogeneous system in which distributed particles of one material (the “dispersed phase”) are dispersed in a phase of another material (the “continuous phase” or the “mobile phase”). The mobile phase is or comprises water. In one embodiment, the mobile phase contains an organic solvent, examples of which are described below in relation to solvents.

[0105] In one embodiment, the dispersion is a solid-liquid dispersion, in which the dispersed phase is solid and the mobile phase is a liquid. In one embodiment, the dispersion is a liquid-liquid dispersion, in which the dispersed phase and the mobile phase are both liquids.

[0106] In one embodiment, the aqueous dispersion is a colloid. The term "colloid" as used herein describes a stable mixture in which the dispersed particles do not settle out. Typically, the dispersed particles have at least in one direction a dimension roughly between 1 nm and 1 pm, or in such a system discontinuities are found at distances of that order.

[0107] In one embodiment, the aqueous dispersion is a suspension. The term “suspension” as used herein is a heterogeneous dispersion of larger particles in a medium. Unlike solutions and colloids, if left undisturbed for a long periods of time, the suspended particles may settle out of the mixture. The use of the terms “colloid” and “suspension” is sometimes overlapping or synonymous, with colloids in some instances being considered a sub-type of suspensions.

[0108] In one embodiment, the aqueous solution or the aqueous mobile phase may comprise a buffer system. As is known to the person skilled in the art, a buffer system is typically an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it. The buffer may be any suitable buffer known in the art. Suitable buffering agents include citrate buffers, acetate buffers, phosphate buffers, HEPES (4- (2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid), TRIS (2-amino-2- (hydroxymethyl)propane- 1,3 -diol) and MES (2-(N-morpholino)-ethanesulfonic acid) buffers.

[0109] In one embodiment, the aqueous solution or the aqueous mobile phase may comprise one or more salts. In one embodiment, the cationic moiety of the salt may be monovalent. In one embodiment, the cationic moiety of the salt may be divalent. In one embodiment, the cationic moiety of the salt may be trivalent. In one embodiment, the anionic moiety of the salt may be monovalent. In one embodiment, the anionic moiety of the salt may be divalent. In one embodiment, the anionic moiety of the salt may be trivalent. Suitable salts may include mono- / bi / tri-valent anions / cations (e.g. NaCl, KC1, RbCl, LiCl, MgCh, CaCh).

[0110] When it contains only monovalent ions, the salt, when present, is typically present in the aqueous solution or the aqueous mobile phase in a concentration of 5 to 250 mM. When the salt alternatively or additionally contains divalent ions, the salt is typically present in the aqueous solution or the aqueous mobile phase in a concentration of 0.5 to 50 mM.

[0111] In one embodiment, the aqueous solution or the aqueous mobile phase may comprise one or more sugars. Suitable sugars include sucrose, trehalose and glucose.

[0112] When present, the sugar is typically present in the aqueous solution or the aqueous mobile phase in a concentration of 5 to 30% (w / w).

[0113] Nucleic Acid-Lipid Particle

[0114] In one embodiment, the method of the present invention is carried out on a nucleic acid which is present in the form of a nucleic acid-lipid particle formulation. As described in more detail herein, the method can be applied on nucleic acid-lipid particles such as lipoplexes (LPX) or lipid-nanoparticle systems (LNP) in different buffer matrices.

[0115] In one embodiment, the method of the present invention is carried out on a lipid particle comprising a lipid or lipid mixture, as defined herein, and a nucleic acid. 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”. When the nucleic acid is DNA, such particles are also referred to herein as “DNA-lipid particles”. When the nucleic acid is a mixture of RNA and DNA, such particles are also referred to herein as “RNA-DNA-lipid particles”. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA, saRNA, uRNA, modRNA, 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 a mixture of RNA and DNA. In one embodiment, the nucleic acid is RNA which encodes for one or more personalized cancer antigens.

[0116] 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, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and / or amphiphilic proteins / polypeptides). 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 and / or additional polymers) 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, amphiphilic polymers, and / or amphiphilic proteins / polypeptides) optionally in combination with additional substances (e.g., additional lipids and / or additional polymers) 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. In this respect, the term "microsized" means that all three external dimensions of the particle are in the microscale, i.e., between 1 and 5 pm. According to the present disclosure, the term "particle" includes lipoplex particles (LPXs), lipid nanoparticles (LNPs), lipopolyplex particles, virus-like particles (VLPs), and mixtures thereof (e.g., a mixture of two or more of particle types, such as a mixture of LPXs and VLPs or a mixture of LNPs and VLPs).

[0117] In one embodiment, the method of the present invention is carried out on a nucleic acid-lipid particle which 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 in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. 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.

[0118] Lipid nanoparticles (LNP) are obtainable from combining a nucleic acid with lipids. The lipids 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.

[0119] In some embodiments, the lipid nanoparticles described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to 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.

[0120] In one embodiment, the method of the present invention is carried out on a nucleic acid-lipid particle which is a lipoplex (LPX). In general, lipoplexes (LPXs) are electrostatic complexes which are obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid and a phase comprising liposomes. Thus, in some embodiments, the LPXs may be formed by mixing preformed cationic lipid liposomes with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid-lipoplexes.

[0121] In the context of the present disclosure, the term "nucleic acid-lipoplex particle" or “nucleic acid-LPX” relates to a particle that comprises lipid, in particular cationic lipid or cationically ionizable lipid, and nucleic acid. Electrostatic interactions between positively charged liposomes and negatively charged nucleic acid results in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may be generally formed using a cationic lipid or cationically ionizable lipid, optionally in combination with additional lipids, as defined in more detail below.

[0122] Lipids and Amphiphiles

[0123] The nucleic acid-lipid particles on which the method of the invention is carried out may 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.

[0124] 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”.

[0125] 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.

[0126] 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.

[0127] Cationic and Cationically Ionizable Lipids

[0128] The aqueous solutions and nucleic acid-lipid particles on which the method of the present invention is carried out may also contain a cationic lipid or cationically ionizable lipid, or a mixture of any thereof.

[0129] 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 (as defined above) 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).

[0130] In one embodiment, the cationic lipid is a monovalent cationic lipid.

[0131] In one embodiment, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

[0132] Examples of cationic lipids include, but are not limited to l,2-dialkyloxy-3- dimethylammonium propanes and l,2-dialkenyloxy-3 -dimethylammonium propanes (each alkyl or alkenyl portion being preferably having 12 to 20 carbon atoms), such as l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), l,2-diacyloxy-3- dimethylammonium propanes (the alkyl or alkenyl part of each acyl preferably having 12 to 20 carbon atoms), such as l,2-dioleoyl-3 -trimethylammonium propane (DOTAP) or l,2-dioleoyl-3 -dimethylammonium -propane (DODAP); dimethyldioctadecylammonium (DDAB); dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE); l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2- dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2- (spermine carboxamide)ethyl]-N,N-dimethyl- 1 -propanamium trifluoroacetate (DOSPA).

[0133] The structures of DODMA and DODAP are shown below.

[0134]

[0135] The structures of DOTMA, DOTAP and analogues thereof are shown below.

[0136] The structures of DOTAP and further suitable homologues are shown below.

[0137] The structures of DOTMA, DORIE and further suitable homologues are shown below. Further suitable cationic lipids are described in Sun and Lu, Pharmaceutical Research, 2023, https: / / doi.org / 10.1007 / sl l095-022-03460-2.

[0138] In one embodiment, the lipid is a cationically ionizable lipid. 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.

[0139] In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is capable of being protonated, preferably under physiological or slightly acidic conditions.

[0140] In one embodiment, the cationic or cationically ionizable lipid is selected from the group consisting of:

[0141] [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-315);

[0142] 1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA);

[0143] 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);

[0144] 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);

[0145] (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);

[0146] O- [N- { (9Z, 12Z)-octadeca-9, 12-dien- 1 -yl) } -N- { 7 -pentadecylcarbonyloxy octyl } - amino]4-(dimethylamino)butanoate (HY501 );

[0147] 2-(di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2);

[0148] 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- hexyl decanoate) (EA-405);

[0149] (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2- hexyldecanoate) (HY-405); palmitoyl-oleoyl-nor-arginine (PONA); guanidino-di[(heptadecyl)methyl]carboxylic acid (GUADACA); 4-methylpyridinium-di(heptadecyl)methylcarboxylic acid (MPDACA);

[0150] 1.2-dioleoyl-3 trimethylammonium propane (DOTAP);

[0151] 1.2-dioleoyl-3-dimethylammomium propane (DODAP); and

[0152] 1.2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA); or a mixture of any thereof.

[0153] In one embodiment, the cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA). In one embodiment, the cationic lipid is 4-methylpyridinium-di(heptadecyl)- methylcarboxylic acid (MPDACA). In one embodiment, the cationic lipid is 1,2- dioleoyloxy-3 -trimethylammonium propane (DOTAP). In one embodiment, the cationic lipid is l,2-dioleoyl-3 -dimethylammonium -propane (DODAP).

[0154] 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 l,2-dioleoyloxy-3 -dimethylaminopropane (DODMA). In one embodiment, the cationically ionizable lipid is 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA). In one embodiment, the cationically ionizable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (D-Lin-MC3-DMA). In one embodiment, the cationically ionizable lipid is l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In one embodiment, the cationically ionizable lipid is di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In one embodiment, the cationically ionizable lipid is 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-9,12- dien-l-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).

[0155] In some embodiments, the cationically ionizable lipid is selected from those described generally and specifically in WO 2018 / 087753.

[0156] In some embodiments, the cationically ionizable lipid is selected from the group consisting of:

[0157] Hy 501 : m.w: 761 .26

[0158] 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).

[0159] In one embodiment, the cationic or cationically ionisable lipid is present in an amount of 20 to 70 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic or cationically ionisable lipid is present in an amount of 30 to 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the cationic or cationically ionisable lipid is present in an amount of 40 to 50 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture component of both the aqueous dispersion and the nucleic acid-lipid particle.

[0160] Additional Lipids

[0161] The lipid mixture in the aqueous solution and nucleic acid-lipid particles on which the method of the present invention is carried out may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise a neutral or zwitterionic lipid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a steroid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid, as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid (such as a steroid), as defined and exemplified below.

[0162] Neutral Lipid

[0163] The aqueous solution and nucleic acid-lipid particles on which the method of the present invention is carried out may also additionally comprise 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).

[0164] 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 having 6 to 40, preferably 8 to 24, carbon atoms or an alkenyl group having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carboncarbon double bonds. The acyl parts of the phospholipids may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of lignoceroyl, behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties. In a specific embodiment, neutral phospholipids have a Tmof 30°C or higher and are selected from di-stearoyl or dipalmitoyl 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.

[0165] Examples of such phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero- 3 -phosphocholine (OChemsPC), l-hexadecyl-sn-10-glycero-3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)- sn-glycero-3 -phosphocholine (DOPG), 1 ,2-dipalmitoyl-sn-glycero-3 -phospho-( 1 ' - rac-glycerol) (DPPG), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further phosphatidyl-ethanolamine lipids with different hydrophobic chains. 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.

[0166] Thus, in some embodiments, the lipid nanoparticle compositions described herein comprise a cationic or cationically ionizable lipid (as defined herein) and a phospholipid. In some embodiments, the lipid nanoparticle compositions described herein comprise a cationic or cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any thereof.

[0167] In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.

[0168] In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.

[0169] In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phosphatidylcholine and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 1 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 2 mol % to about 25 mol % of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the lipid mixture.

[0170] In each of the above embodiments, the term “lipid mixture” in this context applies to the lipid mixture component of both the aqueous dispersion and the nucleic acid-lipid particle.

[0171] Steroid

[0172] The aqueous solution and nucleic acid-lipid particles on which the method of the present invention is carried out may also include a steroid.

[0173] In this specification the term “steroid” takes its normal meaning in the art as meaning a compound having a fused tetracyclic core structure typically composed of seventeen carbon atoms, bonded in four "fused" rings: three six-member cyclohexane rings (referred to in the illustration below as rings A, B and C) and one five-member cyclopentane ring (the D ring). The steroids may vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. In one embodiment, the steroid comprises a sterol. These comprise a hydroxyl group at position 3 on ring A, and the hydroxyl group may be etherified or esterified. In one embodiment, the steroid is cholesterol.

[0174] In one embodiment, the steroid is present in an amount ranging from about 10 mol % to about 65 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 20 mol % to about 60 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 30 mol % to about 50 mol % of the total lipids present in the lipid mixture. pH and avoiding alkylamines

[0175] In one embodiment, the method is carried out at a pH from 6.0 to 8.0. In one embodiment, the method is carried out at a pH from 6.2 to 7.5. As described in more detail above, carrying out the method at a pH around neutral, allows the method to can be used on a wider range of nucleic acid-containing solutions while avoiding the degradation of the nucleic acid.

[0176] In one embodiment, the method is carried out in a medium substantially free of an alkylamine. As described in more detail above, avoiding the use of an alkylamine means that the method can be carried out at neutral pH enables the method to be more reliably used across a wider range of nucleic acid molecules, in particular with basesensitive nucleic acid molecules.

[0177] Step a) - Providing Sample of Nucleic Acid

[0178] The first step of the method of the present invention is the preparation of a sample containing the nucleic acid.

[0179] In one embodiment, the nucleic acid is provided alone, in the absence of other formulation ingredients. In one embodiment, the nucleic acid is provided in a formulation with other formulation ingredients. Typical such formulation ingredients may include salts, buffers, and sugars, each as defined and exemplified above. In one embodiment, the nucleic acid is provided in a nucleic acid-lipid particle formulation, as described and exemplified above.

[0180] In one embodiment, step a) of the method of the invention comprises the step of purifying the sample. In this specification the terms "purification", "purified" or "purifying", takes its general meaning in the art as referring to removal of undesired substances from the sample, such that the sample which is used in the subsequent steps contains a lower amount of these undesired substances. In one embodiment, purification results in the undesired substances being eliminated from the sample.

[0181] Zwitterionic Surfactant

[0182] In one embodiment, the method of the present invention employs a zwitterionic surfactant. In this specification, the term “zwitterion” takes its usual meaning in chemistry as a molecule that contains an equal number of positively- and negatively- charged functional groups. It is therefore be understood that, for some zwitterionic compounds, in solution a chemical equilibrium may be established between the molecule in its neutral form and in its zwitterionic form. Amino acids are an example of a class of such zwitterionic molecules.

[0183] In one embodiment, the zwitterionic compound is a betaine. As is known to those skilled in the art, betaines are zwitterions that cannot isomerize to an all-neutral form, as there is no proton which can be transferred from the positively charged to the negatively charged functional group. Examples of such betaines are those when the positive charge is located on a quaternary ammonium or quaternary phosphonium group.

[0184] In one embodiment, the zwitterionic surfactant is a quaternary ammonium compound (as defined and exemplified above). In one embodiment, the zwitterionic surfactant contains a quaternary ammonium group -N+R3, wherein each R is independently a Ci- 6 alkyl group. In one embodiment, the zwitterionic surfactant contains a quaternary ammonium group -N R.3, wherein each R is independently a Ci-4 alkyl group. In one embodiment, the zwitterionic surfactant contains a quaternary ammonium group - N+R3, wherein each R is independently a methyl or ethyl group. In one embodiment, the zwitterionic surfactant contains a trimethylammonium group -N+(CH3)3.

[0185] In one embodiment, the zwitterionic surfactant has a carboxylate group (as defined and exemplified above). In one embodiment, the zwitterionic surfactant contains an alkylenecarboxylate group, where the alkylene group is as defined and exemplified above. In one embodiment, the zwitterionic surfactant contains an alkylenecarboxylate group, where the alkylene group is as defined and exemplified above, and a quaternary ammonium group, as defined and exemplified above.

[0186] In one embodiment, the zwitterionic surfactant has a sulfonate functional group (as defined and exemplified above). In one embodiment, the zwitterionic surfactant contains an alkylenesulfonate group, where the alkylene moiety is as defined and exemplified above and is preferably a C1-20, more preferably C1-10, even more preferably C2-5 alkylene group). In one embodiment, the zwitterionic surfactant contains an alkylenesulfonate group, where the alkylene moiety of the group is as defined and exemplified above, and a quaternary ammonium group, as defined and exemplified above.

[0187] In one embodiment, the zwitterionic surfactant is of formula (I):

[0188] R1-N+(R2)(R3)-R4(I) wherein

[0189] R1is C6-24 alkyl or A1-NH-C(=O)-R5;

[0190] A1is C1-6 alkylene;

[0191] R2and R3are each independently C1-3 alkyl;

[0192] R4is -A2-SO3- or -A2-CO ;

[0193] A2is Ci -20 alkylene optionally substituted with hydroxy;

[0194] R5is C6-24 alkyl or -A3-St;

[0195] A3is Ci-4 alkylene; and St is a steroid moiety. In one embodiment, R1is C6-24 alkyl. In one embodiment, R1is C8-22 alkyl. In one embodiment, R1is Cio-18 alkyl. In one embodiment, R1is C12-18 alkyl. In one embodiment, R1is C14-16 alkyl.

[0196] In one embodiment, A1is Ci-6 alkylene. In one embodiment, A1is C2-5 alkylene. In one embodiment, A1is C3-4 alkylene.

[0197] In one embodiment, R5is C8-22 alkyl. In one embodiment, R5is Cio-18 alkyl. In one embodiment, R5is C12-18 alkyl. In one embodiment, R5is C14-16 alkyl.

[0198] In one embodiment, R5is -A3-St; wherein A3is C2-3 alkylene; and St is a steroid moiety.

[0199] The steroid moiety St may be any compound which includes the structural unit referred to above in the general definition of steroids. It may include any functional groups commonly present on steroid structures.

[0200] The steroid moiety St may be attached to the rest of the molecule at any position on the steroid skeleton. Typically, the moiety is attached to the rest of the molecule at the 3-position or 17-position of the steroid skeleton.

[0201] In one embodiment, the steroid moiety St is a cholic acid derivative, the pentanoic acid side-chain of the cholic acid moiety forming an amide group with the AJ-NH- group on the rest of the molecule.

[0202] In one embodiment, R1is A1-NH-C(=O)-R5wherein A1is as defined above (in its broadest aspect or a preferred aspect, and R5is C8-22 alkyl. In one embodiment, R5is C10-18 alkyl. In one embodiment, R5is C12-18 alkyl. In one embodiment, R5is C14-16 alkyl.

[0203] In one embodiment, R2is methyl or ethyl. In one embodiment, R2is methyl.

[0204] In one embodiment, R3is methyl or ethyl. In one embodiment, R3is methyl. In one embodiment, A2is C1-10 alkylene optionally substituted with hydroxy. In one embodiment, A2is C2-6 alkylene optionally substituted with hydroxy. In one embodiment, A2is C3-4 alkylene.

[0205] In one embodiment, R4is -A2-SO3‘, wherein A2is Ci-io alkylene optionally substituted with hydroxy. In one embodiment, R4is -A2-SO3‘, wherein A2is C2-6 alkylene optionally substituted with hydroxy. In one embodiment, R4is -A2-SO3‘, wherein A2is C3-4 alkylene.

[0206] In one embodiment, R4is -A2-CO2‘, wherein A2is C1-20 alkylene optionally substituted with hydroxy. In one embodiment, R4is -A2-CO2‘, wherein A2is C2-6 alkylene optionally substituted with hydroxy. In one embodiment, R4is -A2-CO2‘, wherein A2is C3-4 alkylene.

[0207] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate;

[0208] 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate,

[0209] (N-dodecy 1 -N,N -dimethyl ammoni o)buty rate,

[0210] 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof.

[0211] All of the above zwitterionic surfactants are commercially available.

[0212] It is particularly preferred that the zwitterionic surfactant is n-tetradecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate. This is also known as Zwittergent® 3-14 and is commercially available from a number of sources.

[0213] The zwitterionic surfactant may be present in any concentration which is sufficient to enable it to perform the function of solubilizing the nucleic acid. In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous mobile phase is between 0.1% and 20% (w / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous mobile phase is between 0.5% and 15% (w / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous mobile phase is between 2% and 10% (w / v).

[0214] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate; 3-[N,N-dimethyl(3-myristoylaminopropyl)- ammonio]propanesulfonate, (N-dodecyl-N,N-dimethylammonio)butyrate, 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof, and is present in a concentration of between 0.1% and 20%, preferably between 0.5% and 15%, more preferably between 2% and 10%, even more preferably between 5% and 8%, and most preferably between 6% and 7%.

[0215] In one embodiment, the zwitterionic surfactant is n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate and is present in a concentration of between 0.1% and 20%, preferably between 0.5% and 15%, more preferably between 2% and 10%, even more preferably between 5% and 8%, and most preferably between 6% and 7%.

[0216] Solvent

[0217] In one embodiment of the aspect of the invention, where the nucleic acid is present in the form of an aqueous solution, the method of the present invention employs a solvent to solubilise the nucleic acid. Typically, such a solvent is an organic solvent.

[0218] The solvent is not particularly restricted provided it is not immiscible with water and capable of dissolving the nucleic acid to at least some extent. Polar organic solvents are preferred. Examples of suitable solvents include alcohols (such as Ci-io alcohols, preferably Ci-6 alcohols, more preferably Ci-4 alcohols), ketones (preferably C3-5 ketones, such as acetone), ethers (such as tetrahydrofuran), esters (wherein the alkyl and the acyl part are typically Ci-4, such as ethyl acetate), nitriles (typically C2-4 nitriles such as acetonitrile), sulfoxides (typically di(Ci-3) alkyl sulfoxides, such as dimethyl sulfoxides), amides (typically N,N-di(Ci-3) alkyl formamides such as dimethylformamide), and mixtures thereof. In one embodiment, the solvent is selected from the group consisting of an alcohol, acetone, or a mixture thereof.

[0219] In one embodiment, the solvent is selected from the group consisting of a Ci-io alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof. In one embodiment, the solvent is selected from the group consisting of a Ci-io alcohol, acetone, or a mixture thereof.

[0220] The solvent is selected from the group consisting of a Ci-4 alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof. In another embodiment, the solvent is selected from the group consisting of a Ci-4 alcohol, acetone, or a mixture thereof.

[0221] In one embodiment, the solvent is a Ci-4 alcohol. In one embodiment, the solvent is a C3-4 alcohol. In one embodiment, the solvent is a C3 alcohol. In one embodiment, the solvent is selected from the group consisting of methanol, ethanol, and isopropanol, or a mixture of any thereof. In one embodiment, the solvent is methanol. In one embodiment, the solvent is ethanol. In one embodiment, the solvent is isopropanol.

[0222] In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 10% and 50% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 15% and 45% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 20% and 40% (v / v).

[0223] In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is a C3-4 alcohol, preferably isopropanol, in an amount between 10% and 50% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is a C3-4 alcohol, preferably isopropanol, in an amount between 15% and 45% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is a C3-4 alcohol, preferably isopropanol, in an amount between 20% and 40% (v / v).

[0224] The solvent may typically be present in a mixture with water. In this aspect, the solvent and water may be present in any proportions in which they are miscible. In one embodiment, the solvent is a mixture of a Ci-4 alcohol and water. In one embodiment, the solvent is a mixture of a methanol and water. In one embodiment, the solvent is a mixture of ethanol and water. In one embodiment, the solvent is a mixture of isopropanol and water.

[0225] When the solvent is a mixture of a Ci-4 alcohol and water, the Ci-4 alcohol and water are typically present in the mixture in proportions of 1-99% (v / v) Ci-4 alcohol and 1- 99% water. In one embodiment, the mixture contains 10-70% (v / v) Ci-4 alcohol and 30-90% water. In one embodiment, the mixture contains 15-50% (v / v) Ci-4 alcohol and 50-85% water. In one embodiment, the mixture contains 20-40% (v / v) Ci-4 alcohol and 60-80% water. In one embodiment, the mixture contains 25-35% (v / v) Ci-4 alcohol and 65-75% water. In one embodiment, the mixture contains 30% (v / v) Ci-4 alcohol and 70% water.

[0226] When the solvent is a mixture of ethanol and water, the Ci-4 alcohol and water are typically present in the mixture in proportions of 1-99% (v / v) ethanol and 1-99% water. In one embodiment, the mixture contains 10-70% (v / v) ethanol and 30-90% water. In one embodiment, the mixture contains 15-50% (v / v) ethanol and 50-85% water. In one embodiment, the mixture contains 20-40% (v / v) ethanol and 60-80% water. In one embodiment, the mixture contains 25-35% (v / v) ethanol and 65-75% water. In one embodiment, the mixture contains 30% (v / v) ethanol and 70% water.

[0227] Combinations of Zwitterionic Surfactant and solvent

[0228] In one embodiment, the method of the present invention employs a combination of a zwitterionic surfactant and a solvent to solubilise the nucleic acid. In this regard, any combination of any of the zwitterionic surfactants defined generally and specifically above, and any of the solvents defined generally and specifically above, may be adopted.

[0229] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate;

[0230] 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate, (N-dodecy 1 -N,N -dimethyl ammoni o)buty rate,

[0231] 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof; and the solvent is selected from the group consisting of a Ci-4 alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof.

[0232] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N-dimethyl-3 -ammoni o-l -propanesulfonate;

[0233] 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate,

[0234] (N-dodecy 1 -N,N -dimethyl ammoni o)buty rate,

[0235] 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof; and the solvent is selected from the group consisting of a Ci-4 alcohol, acetone, or a mixture thereof.

[0236] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N-dimethyl-3 -ammoni o-l -propanesulfonate;

[0237] 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate,

[0238] (N-dodecy 1 -N,N -dimethyl ammoni o)buty rate,

[0239] 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof; and the solvent is a Ci-4 alcohol or a mixture of any thereof.

[0240] In one embodiment, the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate; n-hexadecyl-N,N-dimethyl-3 -ammoni o-l -propanesulfonate;

[0241] 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate,

[0242] (N-dodecy 1 -N,N -dimethyl ammoni o)buty rate,

[0243] 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof; and the solvent is selected from the group consisting of methanol, ethanol, and isopropanol, or a mixture of any thereof. In one embodiment, the zwitterionic surfactant is n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate; and the solvent is ethanol.

[0244] In one embodiment, the zwitterionic surfactant is n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate; and the solvent is isopropanol.

[0245] According to one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate, is between 0.1% and 20% (w / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, is between 0.5% and 15% (w / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n- tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, is between 2% and 10% (w / v).

[0246] According to one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 10% and 50% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 15% and 45% (v / v). In one embodiment, the concentration of the solvent in the aqueous solution or aqueous dispersion is between 20% and 40% (v / v).

[0247] According to one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate, is between 0.1% and 20% (w / v) and the concentration of the solvent in the aqueous solution or aqueous dispersion is between 10% and 50% (v / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate, is between 0.5% and 15% (w / v) and the concentration of the solvent in the aqueous solution or aqueous dispersion is between 15% and 45% (v / v). In one embodiment, the concentration of the zwitterionic surfactant in the aqueous solution or aqueous dispersion, preferably n-tetradecyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate, is between 2% and 10% (w / v) and the concentration of the solvent in the aqueous solution or aqueous dispersion is between 20% and 40% (v / v).

[0248] Step b) - Mixing with Zwitterionic Surfactant and / or solvent

[0249] Step b) of the method of the present invention comprises mixing the sample with a medium containing the zwitterionic surfactant and / or the solvent (when the medium is an aqueous solution), or the zwitterionic surfactant (when the medium is an aqueous dispersion). The mixing can be carried out using techniques well known to those skilled in the art.

[0250] In one embodiment, the aqueous solution and / or the aqueous dispersion is a ready-to- use formulation.

[0251] When the medium is an aqueous solution, simple mixing of the components may suffice. When the medium is an aqueous dispersion, further means may be required in order to achieve thorough mixing. Examples of techniques which may be used include homogenization, centrifugation and vortexing, all of which are well known to those skilled in the art.

[0252] Step b’) - Reference Sample

[0253] In one embodiment, the method of the present invention comprises the additional step of providing a reference sample. The reference sample contains the zwitterionic surfactant, and / or the solvent, and the buffer component in which the sample is provided which is used to solubilise the nucleic acid which is to be determined in the subsequent step c) but not containing a nucleic acid.

[0254] The step b’) is carried out after step a) but before step c). It may be carried out before, simultaneously with, or after step b). Step c) - Measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy

[0255] Step c) of the method according to the invention comprises analysis of the sample containing the nucleic acid and the zwitterionic surfactant and / or the solvent by ultraviolet-visible (UV / Vis) spectroscopy. In one embodiment of the method according to the invention, step c) comprises determination of the concentration of the nucleic acid by ultraviolet-visible (UV / Vis) spectroscopy.

[0256] In one embodiment, the method is carried out using a total sample volume size of from 1 pl to 2000 pl. In one embodiment, the method is carried out using a total sample volume size of from 10 pl to 1500 pl. In one embodiment, the method is carried out using a total sample volume size of from 10 pl to 1000 pl. In one embodiment, the method is carried out using a total sample volume size of from 10 pl to 750 pl. In one embodiment, the method is carried out using a total sample volume size of from 10 pl to 500 pl. In one embodiment, the method is carried out using a total sample volume size of from 10 pl to 250 pl. In one embodiment, the method is carried out using a total sample volume size of from 50 pl to 150 pl.

[0257] In these embodiments, the solvent is preferably a C3 alcohol, such as isopropanol.

[0258] As is known to the person skilled in the art, UV / Vis spectroscopy is an absorption spectroscopic method comprising passing ultraviolet and / or visible light through a sample and measuring the absorbance of the ultraviolet and / or visible light after it has passed through to determine the concentration of the substances.

[0259] Typically, this method involves use of a UV / Vis spectrophotometer for detection. The presence of an analyte gives a response which is proportional to the concentration of the analyte.

[0260] The method may be used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer-Lambert law:

[0261] A = logio (7o / 7) = zcL wherein A is the measured absorbance, Io is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the path length through the sample, and c the concentration of the absorbing species. For each species and wavelength, a is a constant known as the molar absorptivity or extinction coefficient.

[0262] By using the Beer-Lambert law, the absorption is directly converted into a concentration by using a generic (derived from literature or experimentally determined) nucleic acid extinction coefficient (e.g. 25 mL / mg*cm for mRNA). If necessary, the absorbance can be corrected against the value obtained for the reference sample (particularly if the aqueous solution or dispersion contains buffers which have an absorbance in the UV range).

[0263] In contrast to the methods of the prior art, solubilising the nucleic acid sample using the surfactant and / or the solvent prior to carrying out the analysis using UV / Vis spectroscopy results in the extinction coefficient 8 being much less dependent on the conditions (in particular, the buffer matrix, the concentration of ions present, such as sodium and chloride ions, and the pH), thus enabling the concentration of the nucleic acid in the sample to be determined more reliably and consistently than was previously possible in the art.

[0264] Typically, the wavelength of the ultraviolet and / or visible light to which the sample is subjected may be between 100 nm and 750 nm. Preferably, the wavelength of the ultraviolet light to which the sample is subjected is between 200 nm and 300 nm. More preferably, the wavelength of the ultraviolet light to which the sample is subjected is between 240 nm and 280 nm. A particularly preferred wavelength for nucleic acid detection is 260 nm.

[0265] In one embodiment, the instrument's response to the analyte in the unknown is compared with the response to the reference standard. This is very similar to the use of calibration curves. The response (e.g., peak area) for a particular concentration is known as the response factor. The method may be used for quantification of the nucleic acid in the sample.

[0266] Quantification may be defined as concentration or amount.

[0267] In one embodiment, the quantification is relative quantification (i.e. measuring the quantities of one or more nucleic acids relative to one another, and to the target RNA). The relative concentration can be determined via the relative peak areas of the nucleic acids (after blank subtraction) and the summed peak areas of the nucleic acids.

[0268] In one embodiment, the quantification is absolute quantification (i.e. measuring the absolute quantities of the nucleic acid in the sample). The absolute concentration can be calculated using the total peak area of the nucleic acids (after blank subtraction) and the extinction coefficient corresponding to a specific nucleic acid with the mean substances’ length. The relative concentration can then here be determined by the ratio of the absolute nucleic acids concentration and the total RNA concentration of the sample. The total nucleic acids concentration of the sample can be determined with a spectrophotometer using ultraviolet absorption spectroscopy at 260 nm.

[0269] The solubilization of the nanoparticle can be monitored via the UV / Vis spectra and also by normalization to the absorption values at 340nm.

[0270] Examples

[0271] General Procedure:

[0272] The nucleic acid in aqueous solution (buffered or not) or the nucleic acid-lipid particle (in buffer) is diluted into the linear working range of the spectrophotometric readout with appropriate buffers, followed by addition of the zwitterionic detergent and / or organic solvent. Homogenization and / or solubilization of the particle is afterwards achieved by thorough mixing by vortexing, followed by transfer into appropriate cuvettes (UV-transparent polystyrene / quartz) and the UV measurement.

[0273] The spectrophotometric measurement is based on the decreased power of a radiant beam in relation to the distance that it travels through an absorbing medium and in relation to the concentration of absorbing molecules encountered in that medium. These two factors determine the proportion of the total incident energy that reaches the detector. The decrease in power of the monochromatic radiation passing through a homogeneous absorbing medium is stated quantitatively by the Beer-Lambert law:

[0274] A = c ■ £ -d where A is the mean nucleic acid absorption at 260 nm, c is the concentration (pg / mL), s is the specific mass extinction coefficient of the nucleic acid (unit: mL / pg cm) and d is the path length of the cuvette (1 cm). If appropriate, a corresponding blank measurement (e.g. buffer only) is performed and the sample absorbance is corrected against the reference absorbance.

[0275] UV spectra are acquired over the full UV and visible range between 210 to 700 nm with a spectral resolution of 0.5 nm. Each obtained spectrum is baseline-corrected for the absorbance at 340 nm.

[0276] Example 1 - UV absorption measurements of nucleic acid as a function of ionic strength and buffer composition

[0277] RNA (-2,000 nucleotides) in buffered conditions at pH 6.2 was incubated with various sodium chloride concentrations ranging from 0-250 mM (Figure 1 A), followed by acquisition of UV spectra under identical conditions with a spectrophotometer. We demonstrated a direct, dynamic change in RNA UV absorption behaviour as function of the chemical environment. The decrease which was strongest at the A260 absorption maximum (Figure 1 A) commonly used for RNA quantification by the Beer-Lambert law, thereby heavily impacting such kind of measurement.

[0278] UV absorption spectra of six representative RNAs (Figure IB) were collected in trace levels of sodium chloride (dark grey) or with increasing sodium chloride levels up to 250 mM (light grey), showing the characteristic decrease of the overall UV absorption signal. The initial drop in UV absorption was characterized by sharp exponential decrease with low sodium chloride concentrations. These findings in turn indicated that the use of a universal literature-derived RNA extinction coefficient is highly susceptible to the exact buffer conditions, as changes and differences in the chemical composition result in the less accurate and precise RNA quantification.

[0279] Similar experiments were conducted with RNA solutions in the presence (28 mM or 100 mM) or absence of sodium chloride (Figure 2A / B). As expected, all samples with sodium chloride show at all tested concentrations a significantly lower absorbance in comparison to the identical samples without sodium chloride. By comparison, in presence of the identical sodium chloride levels after adding 6.75% zwitterionic surfactant (n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14) and 30% ethanol, absorbance values were obtained, which were highly similar to the identical RNA samples without sodium chloride. The higher absorbance of the samples in the presence of zwitterionic surfactants and organic solvent confirmed the “elimination or nullification” of the matrix effect by the newly developed protocol. In addition, the new developed protocol showed a direct linear relationship between RNA concentration and A260 absorbance (Figure 2). For three RNAs, the linearity over a broad concentration range could be shown (Figure 2C), in the presence of zwitterionic detergent and alcohol. The root mean square of the linear fits for all analyzed RNAs showed excellent agreement (R2~ 1), highlighting that the new method allows for reliable quantification of the RNA content for the complete range of dose variations (24-fold differences).

[0280] In order to test the impact of sodium chloride on formulated nucleic acid, three different lipid-based RNA nanoparticles (NP) were analyzed in combination with 0.5% w / v SDS (standard method to disrupt the NP) at increasing concentrations of sodium chloride up to 200 mM. The NP of the latter type showed a general decrease of the UV signal in response to increasing sodium levels (Figure 3), similar to unformulated nucleic acid (Figure 1). At higher sodium chloride concentrations, the UV signal of the NP went into saturation. This demonstrated that UV absorbance measurements of nucleic acid and nucleic acid lipid-based NP samples were in general highly influenced by ions (e.g. sodium chloride) in the matrix. Example 2 - Solubilization of lipid-based NP (LPX) according to the method of the invention

[0281] This Example demonstrates that the method of the invention can solubilize lipid- based NP (LPX), which cannot completely solubilized by use of conventional means (e.g. by anionic detergents like sodium dodecyl sulfate, SDS) and other types ofNP (LNP).

[0282] Complex, lipid-based nucleic acid containing samples (e.g. LPX with excess of cationic lipid over the RNA, N / P charge ratio of 4) were treated with anionic detergent (0.5% SDS) or with the method of the invention (6.75% n-tetradecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14; 30% ethanol). As expected, the non-treated samples shows the typical UV spectra largely influenced by light scattering (Figure 4). This demonstrated that NP must be properly disrupted before analyzing the nucleic acid content by UV spectroscopy. The conventional anionic detergent is able to disrupt the NP only partially, which can be seen by the UV spectrum still influenced by scattered light (Figure 4, inset). This effect is not visible in NP samples treated with the method of the invention (zwitterionic surfactant + organic solvent), thereby demonstrating that the NPs are completely disrupted with the method of the invention.

[0283] In a further experiment, lipid-based nucleic acid containing NPs (LNPs) of different composition and molecular architecture were treated with the method of the invention. As expected, non-treated samples show the typical UV spectra, which is largely influenced by light scattering (Figure 5). This demonstrates again that NP must be disrupted before analyzing the nucleic acid (RNA) content by UV spectroscopy. This effect is not visible anymore in LNP samples treated with the method of the invention. This demonstrates that a broad range of lipid-based particles can be completely disrupted, and the nucleic acid content can be measured by UV spectroscopy. Example 3 - UV absorption measurements of nucleic acids in the presence of mono- and bivalent ions vis-a-vis an ion-reduced environment

[0284] UV absorption measurements of nucleic acids are generally biased by monovalent and divalent ions (Figure 6, left part; cf LiCl, NaCl, KC1, RbCl, MgCh and CaCh). By applying the method of the present invention, this general matrix effect can be eliminated, and a more accurate measurement can be achieved.

[0285] The performance of the method of the present invention was assessed with different chemical composition of the nucleobases of the RNA in the presence of mono- and bivalent ions, with and without the method of the present invention. In Figure 6 (first row of bars), non-nucleoside-modified mRNA and Nl-methylpseudouridine-modified mRNA (mlTRNA) were diluted into buffer matrices containing saturating levels of ions (monovalent: 150 mM; bivalent: 10 mM). Compared with RNA solely diluted in low-salt buffered conditions (Figure 6, first bar; “w / o addition”), a systematic decrease in the RNA’s absorption at 260 nm could be observed for all ions, in line with data shown in Figure 1. The matrix effects were nullified in mixtures of zwitterionic detergent and organic solvent (Figure 6, right part; cf LiCl, NaCl, KC1, RbCl, MgCh and CaCh with “+ mix”), independent of the chemical identity of the RNA (mRNA or mlTRNA) or salt present, confirming the general applicability of the method of the present invention.

[0286] Matrix mitigation was additionally tested for different zwitterionic detergents, showing the general applicability of the developed method. Compared to conditions under saturating sodium chloride levels, addition of the zwitterionic detergents Zwittergent® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, “ZWG”), ABS-14 (3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]- propanesulfonate; “ABS-14”), DDMAB ((N-dodecyl-N,N-dimethylammonio)- butyrate; “DDMAB”) or CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate; “CHAPS”) led to recovery of the measured absorption at 260 nm (Figure 7). The same was tested for different organic solvents, in form of ethanol (“EtOH”) and isopropyl alcohol (“IP A”) (Figure 8). Compared to the absorption signal at 260 nm in presence of 150 mM sodium chloride, mRNA and ml'PRNA both showed a recovery in absorption alone in presence of organic solvent, which was however exceeded in presence of a zwitterionic detergent.

[0287] Example 4 - Proof-of-concept for lipid-based NP (LPX) solubilization with zwitterionic surfactant and organic solvent

[0288] In Figure 9A, different concentrations of lipid-based nucleic acid containing samples (LPX at a negative charge ratio) were treated with the method of the invention (6.75% n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14; 30% ethanol). As for each of the tested DP RNA concentrations, absorption at longer wavelengths was identical, with no observable light scattering signal from potentially non-solubilized NPs. This proved that for the tested DP concentration range, complete solubilization of the NP by the use of the method of the invention is achieved.

[0289] In order to test the applicability of the method of the invention for variations of the dose, individually prepared dilutions of three independent NP batches were tested in triplicates in the concentration range from 2 to 27.5 pg / mL (Figure 9B) or in the range of 1 to 60 pg / mL for an alternative NP (Figure 9C). The obtained absorption values at 260 nm and resulting determined RNA concentrations can be seen in Figure 9. In general, the obtained absorption values increased linearly with the RNA concentration of the NP. The concentration doses showed absorption values that followed a direct linear -dose relationship with R2values close to 1. In consequence, the determined NP stock concentrations from each NP dose dilution were in excellent agreement with the expected nominal concentration. This data demonstrated that the method is applicable and linear for lipid-based NP in a wide dose range (up to 60-fold differences).

[0290] To investigate the impact of the hold-times on the nucleic acid content by the new method, nucleic acid (RNA) alone (Fig. 10A) and NP (Fig. 10B) were prepared at different dilutions and measured directly after mixing (with 6.75% n-tetradecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate, Zwittergent® 3-14; and 30% ethanol) and the identical samples were then re-measured after an incubation period of 3 to 4 hours at 4°C. Both series of measurements were in very good agreement. Therefore, it can be assumed that the hold-time has no major impact in the tested time period. Example 5 - Superior analytical and process control by elimination of matrixdependent changes on nucleic acid absorption and extinction coefficients

[0291] As shown in Figure 11, the method of the invention allows for a precise analytical and process control by elimination of matrix effects that impact nucleic acid absorption. In the aforementioned figure, RNA mass balances (quantification of RNA output divided by RNA inputs) for two different kinds of RNA content methods of a typical NP (LPX) manufacturing are shown: (1) RNA content testing by conventional means employing drug substance testing in buffer and absence of detergent and drug product testing employing an anionic detergent for LPX solubilization and (2) RNA content testing of the new method by simultaneous use of a zwitterionic surfactant in combination with an organic solvent.

[0292] As the data highlight, a more reliable and robust process control with the latter is possible with a mass balance close to 100%, whereas unaligned RNA content testing showed a positive RNA content offset. The reason for this are changes in the RNA extinction coefficient between the different chemical matrices (c.f. Figure 1), while the new UV RNA content method guarantees a constant chemical environment by elimination of matrix effects.

[0293] Example 6 - UV absorption measurements using NanoDrop 2000 Spectrophotometer

[0294] The method of the invention was carried out using a NanoDrop 2000 Spectrophotometer (Thermo Fisher) employing Nanodrop 2000 / 2000c software (Absorption = 260nm; Base line correction = 340nm; Factor = 40).

[0295] Two comparative preparations were formulated. The first, a sample buffer (24 pl) without LNPs / RNA (Fig. 12A, = blank). The second, a sample buffer (24 pl) with LNPs containing 100 pg / mL RNA (Fig. 12B). The samples were combined with a mixture of an 30% (v / v) aqueous solution of n-tetradecyl-N,N-dimethyl-3-ammonio- 1 -propanesulfonate (24 pl; Zwittergent® 3-14), 99% (v / v) isopropanol (36 pl) and HEPES buffer (36 pl; pH 6.0). Thus, the final mixtures comprised 6% (v / v) n- tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate and 30% (v / v) isopropanol. Absorbance was measured in 60 pl samples of the final mixture. The measurement of the RNA concentration resulted in determination of 100 pg / mL RNA (± 2 pg / mL RNA).

[0296] 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

Claims1. A method of determining the nucleic acid concentration in an aqueous dispersion, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and ii) a mobile phase selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

2. A method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; or ii) a solvent selected from the group consisting of an alcohol, acetone, or a mixture thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

3. A method of determining the nucleic acid concentration in an aqueous solution, the method comprising the steps a) to c): a) providing a sample of the nucleic acid; b) mixing the sample with a medium containing: i) a zwitterionic surfactant; and ii) a solvent selected from the group consisting of an alcohol, acetone, and dimethyl sulfoxide, or a mixture of any thereof; and c) measuring the concentration of the nucleic acid using ultraviolet-visible spectroscopy.

4. A method according to any one of claims 1 to 3, wherein the nucleic acid is present in the form of a nucleic acid-lipid particle formulation.

5. A method according to any one of claims 1 to 3, further comprising the additional step b’): b’) providing a reference sample containing the zwitterionic surfactant but not containing a nucleic acid.

6. A method according to any one of claims 1 to 3, wherein step c) further comprises using the net absorbance to determine the concentration of the nucleic acid in the sample of the nucleic acid-lipid particle formulation.

7. A method according to any preceding claim, wherein the zwitterionic surfactant, where present, is a quaternary ammonium compound having a carboxylate or sulfonate functional group.

8. A method according to claim 7, wherein the zwitterionic surfactant is of formula (I):R!-N+(R2)(R3)-R4(I) whereinR1is C6-24 alkyl or A1-NH-C(=O)-R5;A1is Ci-6 alkylene;R2and R3are each independently C1-3 alkyl;R4is -A2-SO3- or -A2-CO2’;A2is Ci -20 alkylene optionally substituted with hydroxy;R5is C6-24 alkyl or -A3-St;A3is Ci-4 alkylene; and St is a steroid moiety.

9. A method according to claim 7 or claim 8, wherein the zwitterionic surfactant is selected from the group consisting of: n-tetradecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate,n-hexadecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate, 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate, (N-dodecyl-N,N-dimethylammonio)butyrate, and 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, or a mixture of any thereof.

10. A method according to any preceding claim, wherein the concentration of the zwitterionic surfactant in the aqueous solution or aqueous mobile phase is between 0.1% and 20% (w / v).

11. A method according to any preceding claim, wherein the solvent and / or the mobile phase is selected from the group consisting of a Ci-4 alcohol and a mixture of any thereof.

12. A method according to claim 9, wherein the Ci-4 alcohol is selected from the group consisting of methanol, ethanol, and isopropanol, or a mixture of any thereof.

13. A method according to any preceding claim, wherein the solvent and / or the mobile phase is a mixture of a Ci-4 alcohol and water, the mixture containing 20- 40% (v / v) Ci-4 alcohol and 60-80% water.

14. A method according to any preceding claim, wherein the method is carried out at a pH from 6.0 to 8.0.

15. A method according to claim 13, wherein the method is carried out at a pH from 6.2 to 7.5.

16. A method according to any preceding claim, wherein the method is carried out in a medium substantially free of an alkylamine.

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