LNP COMPOSITIONS COMPRISING RNA AND METHODS FOR PREPARING, STORING AND USING THEM

Description

LNP COMPOSITIONS COMPRISING RNA AND METHODS FOR PREPARING, STORING AND USING THEM Divided from patent application BR 11 2023 008166 1, filed on November 15, 2021. FIELD OF THE INVENTION

[001] This disclosure relates generally to the field of lipid nanoparticle (LNP) compositions comprising RNA, methods for preparing and storing such compositions, and the use of such compositions in therapy. BACKGROUND OF THE INVENTION

[002] The use of recombinant nucleic acid (such as DNA or RNA) to deliver foreign genetic information into target cells is well known. The advantages of using RNA include transient expression and a non-transformative character. RNA does not need to enter the nucleus to be expressed and, moreover, it cannot integrate into the host genome, thus eliminating several risks, such as oncogenesis.

[003] A recombinant nucleic acid can be administered in pure form to a subject who needs it; however, recombinant nucleic acid is usually administered using a composition. For example, RNA can be delivered by so-called nanoparticle formulations containing RNA and a nanoparticle-forming carrier, for example, a cationic lipid (such as a permanently charged cationic lipid), a mixture of a cationic lipid and one or more additional lipids, or a cationic polymer. The fate of such nanoparticle formulations is controlled by several key factors (e.g., Petition 870260076800, dated 07 / 31 / 2026, page 14 / 442 2 / 377 size and size distribution of nanoparticles; etc.). These factors are, for example, referred to in the 2018 FDA Liposome Drug Products Guidance as specific attributes that should be analyzed and specified. Limitations to the clinical application of current nanoparticle formulations may lie in the lack of homogeneous, pure, and well-characterized nanoparticle formulations.

[004] LNPs comprising ionizable lipids may present advantages in terms of targeting and efficacy compared to other RNA nanoparticle products. However, obtaining sufficient shelf life as required for regular pharmaceutical use is a challenge. It is said that, for stabilization, LNPs comprising ionizable lipids need to be frozen at much lower temperatures, such as -80°C, which presents substantial challenges in the cold chain, or can only be stored above freezing temperature, for example, 5°C, where only limited stability can be achieved.

[005] RNA in solution or in LNPs is known to undergo slow fragmentation. Furthermore, in the presence of phosphate-buffered saline (PBS), RNA tends to adopt a very stable folded form that is difficult to access for translation. Both mechanisms, i.e., fragmentation and the formation of this stable RNA fold, are temperature-dependent and result in the loss of intact and accessible RNA, thus limiting the stability of the liquid product; however, they are essentially absent in the frozen state.

[006] Thus, a need remains in the state of Petition 870260076800, dated 07 / 31 / 2026, page 15 / 442 3 / 377 technique of (i) compositions comprising LNPs comprising ionizable lipids and RNA that are stable and can be stored in a temperature range compatible with regular technologies in pharmaceutical practice, in particular at a temperature of about 25°C or even in liquid form at temperatures between +2 and +20°C; (ii) ready-to-use compositions; (iii) compositions that can preferably be repeatedly frozen and thawed; and (iv) methods for preparing and storing such compositions. This disclosure addresses these and other requirements.

[007] The inventors have surprisingly found that the compositions and methods described herein meet the aforementioned requirements. In particular, it is demonstrated that, using a specific buffer substance, in particular tris(hydroxymethyl)aminomethane (Tris) and its protonated form, at low concentration (e.g., at most about 25 mM) and excluding inorganic phosphate anions, as well as citrate anions and EDTA anions, it is possible to prepare compositions that are stable and can be stored at about 25°C or even in liquid form. SUMMARY OF THE INVENTION

[008] In a first aspect, the present disclosure provides a composition comprising lipid nanoparticles (LNPs) dispersed in an aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the aqueous phase comprises a buffer system comprising a buffer substance selected from the group consisting of Tris and its protonated form, bis(2-hydroxyethyl)amino Petition 870260076800, dated 07 / 31 / 2026, page 16 / 442 4 / 377 tris(hydroxymethyl)methane (Bis-Tris-methane) and its protonated form and triethanolamine (TEA) and its protonated form, the monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the morpholinoethanesulfonic acid (MES) anion, the 3-(N-morpholino)propanesulfonic acid (MOPS) anion and the 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) anion; the concentration of the buffer substance in the composition is at most about 25 mM; and the aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions and substantially free of ethylenediaminetetraacetic acid (EDTA) anions.

[009] As demonstrated in the present application, the use of a buffer system based on the specific buffer substances specified above, in particular Tris and its protonated form, instead of PBS in a composition comprising LNPs inhibits the formation of a very stable folded form of RNA. Furthermore, the present application demonstrates that, surprisingly, by simply decreasing the concentration of the buffer substance in a composition comprising LNPs and a buffer system, wherein the LNPs comprise a cationically ionizable lipid and RNA, it is possible to obtain an RNA LNP composition having improved RNA integrity after a freeze-thaw cycle compared to a composition comprising the same buffer substance at a concentration of 50 mM. Thus, the claimed composition provides enhanced stability and can be stored in a temperature range compatible with regular technologies in practice. Petition 870260076800, dated 07 / 31 / 2026, page 17 / 442 5 / 377 pharmaceutical and provides a ready-to-use composition.

[0010] In a preferred embodiment of the first aspect, the buffer substance is Tris and its protonated form, that is, a mixture of Tris and its protonated form.

[0011] In one embodiment, the monovalent anion is selected from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 3-(N-morpholino)propanesulfonate, or from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate, preferably from the group consisting of chloride, acetate, lactate and morpholinoethanesulfonate, more preferably from the group consisting of chloride, acetate and morpholinoethanesulfonate, or from the group consisting of chloride, acetate and lactate, such as chloride or acetate.

[0012] In one embodiment, the buffer substance is Tris and its protonated form and the monovalent anion is selected from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 3-(N-morpholino)propanesulfonate, or from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate, preferably from the group consisting of chloride, acetate, lactate and morpholinoethanesulfonate, more preferably from the group consisting of chloride, acetate and morpholinoethanesulfonate, or from the group consisting of chloride, acetate and lactate, such as chloride or acetate. Petition 870260076800, dated 07 / 31 / 2026, page 18 / 442 6 / 377

[0013] In one embodiment of the first aspect, the concentration of the buffer substance, in particular the total concentration of Tris and its protonated form, in the composition is at most about 20 mM, such as at most about 19 mM, at most about 18 mM, at most about 17 mM, at most about 16 mM, at most about 15 mM, at most about 14 mM, at most about 13 mM, at most about 12 mM, at most about 11 mM or at most about 10 mM. In one embodiment, the lower limit of the buffer substance, in particular Tris and its protonated form, in the composition is at least about 1 mM, preferably at least about 2 mM, such as at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM or at least about 9 mM.For example, the concentration of the buffer substance, in particular the total concentration of Tris and its protonated form, in the composition may be between about 1 mM and about 20 mM, such as between about 2 mM and about 15 mM, between about 5 mM and about 14 mM, between about 7 mM and about 13 mM, between about 8 mM and about 12 mM, between about 9 mM and about 11 mM, such as about 10 mM.

[0014] In an embodiment of the first aspect, the aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or dibasic organic acid anions and / or polybasic organic acid anions. In a first subgroup, at least one of these criteria applies. For example, in an embodiment of this first subgroup, the aqueous phase is substantially free of Petition 870260076800, dated 07 / 31 / 2026, p. 19 / 442 7 / 377 inorganic sulfate anions. In another embodiment of this first subgroup, the aqueous phase is substantially free of carbonate anions. In another embodiment of this first subgroup, the aqueous phase is substantially free of dibasic organic acid anions. In another embodiment of this first subgroup, the aqueous phase is substantially free of polybasic organic acid anions.

[0015] In a second subgroup of the first aspect, at least two of the above criteria apply. For example, in one embodiment of this second subgroup, the aqueous phase is substantially free of inorganic sulfate anions and substantially free of carbonate anions. In another embodiment of this second subgroup, the aqueous phase is substantially free of inorganic sulfate anions and substantially free of dibasic organic acid anions. In another embodiment of this second subgroup, the aqueous phase is substantially free of inorganic sulfate anions and substantially free of polybasic organic acid anions. In another embodiment of this second subgroup, the aqueous phase is substantially free of carbonate anions and substantially free of dibasic organic acid anions. In another embodiment of this second subgroup, the aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions.In another embodiment of this second subgroup, the aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions.

[0016] In a third subgroup of the first aspect, Petition 870260076800, dated 07 / 31 / 2026, p. 20 / 442 8 / 377 at least three of the above criteria apply. For example, in one embodiment of this third subgroup, the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, and substantially free of dibasic organic acid anions. In another embodiment of this third subgroup, the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, and substantially free of polybasic organic acid anions. In yet another embodiment of this third subgroup, the aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions.In another embodiment of this third subgroup, the aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions.

[0017] In a fourth subgroup of the first aspect, at least four of the above criteria apply. That is, in this fourth subgroup, the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions.

[0018] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect), the composition comprises a cryoprotectant. In an embodiment Petition 870260076800, dated 07 / 31 / 2026, p. 21 / 442 9 / 377 alternative to the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect), the composition is substantially free of a cryoprotectant. Thus, particular examples of such embodiments are as follows: (1) the aqueous phase is substantially free of inorganic sulfate anions and the composition includes a cryoprotectant; (2) the aqueous phase is substantially free of carbonate anions and the composition includes a cryoprotectant; (3) the aqueous phase is substantially free of dibasic organic acid anions and the composition includes a cryoprotectant; (4) the aqueous phase is substantially free of polybasic organic acid anions and the composition includes a cryoprotectant; (5) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of carbonate anions, and the composition comprises a cryoprotectant; (6) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of dibasic organic acid anions, and the composition comprises a cryoprotectant; (7) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (8) the aqueous phase is substantially free of carbonate anions and substantially free of acid anions Petition 870260076800, dated 07 / 31 / 2026, page 22 / 442 10 / 377 organic dibasic, and the composition includes a cryoprotectant; (9) the aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (10) the aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (11) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the composition comprises a cryoprotectant; (12) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (13) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (14) the aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition comprises a Petition 870260076800, dated 07 / 31 / 2026, page 23 / 442 11 / 377 cryoprotectant; (15) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition comprises a cryoprotectant; (16) the aqueous phase is substantially free of inorganic sulfate anions and the composition is substantially free of a cryoprotectant; (17) the aqueous phase is substantially free of carbonate anions and the composition is substantially free of a cryoprotectant; (18) the aqueous phase is substantially free of dibasic organic acid anions and the composition is substantially free of a cryoprotectant; (19) the aqueous phase is substantially free of polybasic organic acid anions and the composition is substantially free of a cryoprotectant; (20) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of carbonate anions, and the composition is substantially free of a cryoprotectant; (21) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of dibasic organic acid anions, and the composition is substantially free of a cryoprotectant; (22) the aqueous phase is substantially free of inorganic sulfate anions and substantially free of anions of Petition 870260076800, dated 07 / 31 / 2026, page 24 / 442 12 / 377 polybasic organic acid, and the composition is substantially free of a cryoprotectant; (23) the aqueous phase is substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the composition is substantially free of a cryoprotectant; (24) the aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant; (25) the aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant; (26) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the composition is substantially free of a cryoprotectant; (27) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant; (28) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant; Petition 870260076800, dated 07 / 31 / 2026, page 25 / 442 13 / 377 (29) the aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant; (30) the aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the composition is substantially free of a cryoprotectant.

[0019] In an embodiment of the first aspect (in particular, in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the composition comprises a cryoprotectant, said cryoprotectant comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols. For example, the cryoprotectant may be selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof. In a preferred embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the composition comprises sucrose and / or glycerol as a cryoprotectant.

[0020] In a concretization of the first aspect (in particular in a concretization of the first, second, third or fourth subgroup above the first aspect, such Petition 870260076800, dated 07 / 31 / 2026, p. 26 / 442 14 / 377 as in any of the embodiments (1) to (30) listed above), wherein the composition comprises a cryoprotectant, the concentration of the cryoprotectant in the composition is at least 1% w / v, such as at least 2% w / v, at least 3% w / v, at least 4% w / v, at least 5% w / v, at least 6% w / v, at least 7% w / v, at least 8% w / v, or at least 9% w / v. In one embodiment, the concentration of the cryoprotectant in the composition is up to 25% w / v, as well as up to 20% w / v, up to 19% w / v, up to 18% w / v, up to 17% w / v, up to 16% w / v, up to 15% w / v, up to 14% w / v, up to 13% w / v, up to 12% w / v or up to 11% w / v. In one embodiment, the concentration of the cryoprotectant in the composition is 1% w / v 20% w / v, such as 2% w / v 19% w / v, 3% w / v 18% w / v, 4% w / v 17% w / v, 5% w / v 16% w / v, 5% w / v 15% w / v, 6% w / v 14% w / v, 7% w / v 13% w / v, 8% w / v 12% w / v, 9% w / v 11% w / v, or approximately 10% w / v.In one embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the composition comprises a cryoprotectant (in particular, sucrose and / or glycerol) at a concentration of 5% w / v 15% w / v, such as 6% w / v 14% w / v, 7% w / v 13% w / v, 8% w / v 12% w / v, or 9% w / v 11% w / v, or at a concentration of about 10% w / v.

[0021] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the composition comprises a cryoprotectant, the cryoprotectant is present in a concentration resulting in Petition 870260076800, dated 07 / 31 / 2026, p. 27 / 442 15 / 377 an osmolality of the composition in the range of approximately 50 x 10-3osmol / kg to about 400 x 10-3osmol / kg (such as, from about 50 x 10-3osmol / kg to about 390 x 10-3osmol / kg, from about 60 x 10-3osmol / kg to about 380 x 10-3osmol / kg, xos from about 70 10-3osmol / kg to about 370 x 10-3osmol / kg, from about 80 x 10-3osmol / kg to about 360 x 10-3osmol / kg, from about 90 x 10-3osmol / kg to about 350 x 10-3osmol / kg, from about 100 x 10-3osmol / kg to about 340 x 10-3osmol / kg, from about 120 x 10-3osmol / kg to about 330 x 10-3osmol / kg, from about 140 x 10-3osmol / kg to about 320 x 10-3osmol / kg, about 160 xmol 10-3osmol / kg to about 310 x 10-3osmol / kg, from about 180 x 10-3osmol / kg to about 300 x 10-3osmol / kg, or from about 200 x 10-3osmol / kg to about 300 x 10-3osmol / kg, based on total composition.

[0022] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the first aspect where the buffer substance is Tris and its protonated form, the monovalent anion is selected from the group consisting of chloride, acetate, glycolate and lactate, and the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition is at most equal to the concentration of the buffer substance in the composition. For example, the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition may be less than the concentration of the buffer substance in the composition. Thus, in those Petition 870260076800, dated 07 / 31 / 2026, p. 28 / 442 16 / 377 embodiments of the first aspect, where the concentration of the buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition is at most equal to about 20 mM, for example, less than 20 mM. Generally, the concentration of the monovalent anion, such as chloride and / or acetate (in particular the total concentration of all monovalent anions) in the composition may be less than about 15 mM, such as less than about 14 mM, less than about 13 mM, less than about 12 mM, less than about 11 mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM or less than about 5 mM. In one embodiment, the chloride concentration in the composition is as defined above (e.g., less than about 15 mM, etc.).) and the composition does not comprise acetate. In an alternative embodiment, the concentration of acetate in the composition is as defined above (e.g., less than about 15 mM, etc.) and the composition does not comprise chloride.

[0023] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the first aspect where the buffer substance is Tris and its protonated form, the sodium concentration in the aqueous phase and / or composition is less than 20 mM, such as less than about 15 mM, for example, less than about 14 mM, less than Petition 870260076800, dated 07 / 31 / 2026, p. 29 / 442 17 / 377 about 13 mM, less than about 12 mM, less than about mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, or less than about 5 mM.

[0024] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the first aspect where the buffer substance is Tris and its protonated form, the monovalent anion is selected from the group consisting of the MES, MOPS and HEPES anions, and the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition is at least equal to the concentration of the buffer substance in the composition. For example, the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition may be greater than the concentration of the buffer substance in the composition.Thus, in those embodiments of the first aspect, where the concentration of the buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition is at least equal to about 20 mM, for example, greater than 20 mM. Generally, the concentration of the monovalent anion (in particular the total concentration of all monovalent anions) in the composition may be greater than about 20 mM, such as greater than about 21 mM, greater than about 22 mM, greater than about 23 mM, greater than about. Petition 870260076800, dated 07 / 31 / 2026, p. 30 / 442 18 / 377 of 24 mM, greater than about 25 mM, greater than about 26 mM, greater than about 27 mM, greater than about 28 mM, greater than about 29 mM or greater than about 30 mM and preferably at most 50 mM, such as at most 45 mM, at most 40 mM or at most 35 mM.

[0025] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the pH of the composition is between about 6.5 and about 8.0. For example, the pH of the composition may be between about 6.9 and about 7.9, as between about 7.0 and about 7.9, between about 7.1 and about 7.8, between about 7.2 and about 7.7, between about 7.3 and about 7.6, between about 7.4 and about 7.6, or about 7.5.

[0026] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the composition comprises water as the principal component and / or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v / v). For example, the amount of water contained in the composition may be at least 50% (w / w), as at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). In particular, if the composition comprises a cryoprotectant, the amount of water contained in the composition may be at least Petition 870260076800, dated 07 / 31 / 2026, p. 31 / 442 19 / 377 less than 50% (w / w), such as at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition may be at least 95% (w / w). Additionally or alternatively, the total amount of solvent(s) other than water contained in the composition may be less than about 1.0% (v / v), such as less than about 0.9% (v / v), less than about 0.8% (v / v), less than about 0.7% (v / v), less than about 0.6% (v / v), less than about 0.5% (v / v), less than about 0.4% (v / v), less than about 0.3% (v / v), less than about 0.2% (v / v), less than about 0.1% (v / v), less than about 0.05% (v / v), or less than about 0.01% (v / v). In this respect, a cryoprotectant that is liquid under normal conditions will not be considered a solvent other than water, but rather a cryoprotectant. In other words, the optional limitation above that the total amount of solvent(s) other than water contained in the composition may be less than about 1.0% (v / v) does not apply to cryoprotectants that are liquid under normal conditions.

[0027] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the osmolarity of the composition is at most about 400 x 10-3 osmol / kg, such as at most about 390 x 10-3 osmol / kg, at most about 380 x 10-3 osmol / kg, at most Petition 870260076800, dated 07 / 31 / 2026, p. 32 / 442 20 / 377 about 370 x 10-3osmol / kg, maximum about 360 x 10-3osmol / kg, maximum about 350 x 10-3osmol / kg, maximum about 340 x 10-3osmol / kg, maximum about 330 x 10-3osmol / kg, maximum about 320 x 10-3osmol / kg, maximum about 310 x 10-3osmol / kg, or maximum about 300 x 10-3osmol / kg. If the composition does not include a cryoprotectant, the osmolality of the composition may be less than 300 x 10⁻³ osmol / kg, such as a maximum of about 250 x 10⁻³ osmol / kg, a maximum of about 200 x 10⁻³ osmol / kg, a maximum of about 150 x 10⁻³ osmol / kg, a maximum of about 100 x 10⁻³ osmol / kg, a maximum of about 50 x 10⁻³ osmol / kg, a maximum of about 40 x 10⁻³ osmol / kg, or a maximum of about 30 x 10⁻³ osmol / kg. If the composition includes a cryoprotectant, it is preferable that the main part of the osmolality of the composition be provided by the cryoprotectant.For example, the cryoprotectant can provide at least 50%, as well as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the composition's osmolality.

[0028] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the RNA concentration in the composition is about 5 mg / l about 150 mg / l. For example, the RNA concentration in the composition may be about 10 mg / l about 140 mg / l, such as about 20 mg / l about 130 mg / l, about 25 mg / l about 125 mg / l, about 30 mg / l about 120 mg / l, about 35 mg / l about 115 mg / l, about 40 Petition 870260076800, dated 07 / 31 / 2026, p. 33 / 442 21 / 377 mg / search of 110 mg / l, search of 45 mg / search of 105 mg / l, or search of 50 mg / search of 100 mg / l.

[0029] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the buffer substance is Tris and its protonated form, the pH of the composition is between about 6.5 and about 8.0, and the RNA concentration in the composition is about 5 mg / l about 150 mg / l. In this embodiment, it is preferable that the pH of the composition is between about 6.9 and about 7.9 and the RNA concentration in the composition is about 25 mg / l about 125 mg / l, such as about 30 mg / l about 120 mg / l.In a particularly preferred embodiment of the claimed composition, the buffer substance is Tris in its protonated form; the pH of the composition is between about 6.9 and about 7.9; the RNA concentration in the composition is about 30 mg / l to about 120 mg / l; the aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acids, and substantially free of polybasic organic acids; and the composition comprises a cryoprotectant. In a particularly preferred alternative embodiment of the claimed composition, the buffer substance is Tris in its protonated form; the pH of the composition is between about 6.9 and about 7.9; the RNA concentration in the composition is about 30 mg / l to about 120 mg / l; the aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acids, and substantially free of polybasic organic acids. Petition 870260076800, dated 07 / 31 / 2026, p. 34 / 442 22 / 377 of polybasic organic acids; and the composition is substantially free of a cryoprotectant.

[0030] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom that is capable of being protonated under physiological conditions. For example, the cationically ionizable lipid may have the structure of Formula (I): r3\ ^G3 / Ü / N. .Λ2. R1^G1^G2^R2(I) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, where L1, L2, G1, G2, G3, R1, R2 and R3 are as defined in this descriptive report. Preferably, the cationically ionizable lipid is selected from the following: structures I-1 to I-36 (shown here); and / or structures A to F (shown here); and / or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA) and 4((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14). In a particularly preferred embodiment, the cationically ionizable lipid is the lipid with structure I-3. Petition 870260076800, dated 07 / 31 / 2026, p. 35 / 442 23 / 377

[0031] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the LNPs further comprise one or more additional lipids. Preferably, one or more additional lipids are selected from the group consisting of lipids conjugated with polymers, neutral lipids, steroids and combinations thereof.In a preferred embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the LNPs comprise the cationically ionizable lipid as described in this descriptive report, a polymer-conjugated lipid (for example, a pegylated lipid or a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material), a neutral lipid (for example, DSPC) and a steroid (for example, cholesterol).

[0032] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the LNPs further comprise a lipid-conjugated polymer as one or more additional lipids, the lipid-conjugated polymer is a pegylated lipid. For example, the pegylated lipid may have the following structure: Petition 870260076800, dated 07 / 31 / 2026, p. 36 / 442 24 / 377 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, where R12, R13 and w are as defined herein.

[0033] In an embodiment of the first aspect (in particular, in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the LNPs further comprise a lipid-conjugated polymer as one of one or more additional lipids, the lipid-conjugated polymer is a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material. For example, the polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like material may be a selected member of the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate and a mixture thereof.

[0034] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the LNPs further comprise a neutral lipid as one or more additional lipids, the neutral lipid Petition 870260076800, dated 07 / 31 / 2026, p. 37 / 442 25 / 377 is a phospholipid. This phospholipid is preferably selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, and sphingomyelins.Particular examples of phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibeene ylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero3-phosphocholine (18:0 Dieter PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dileoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and dipitanoylphosphatidylethanolamine (DPyPE). In a particularly preferred embodiment, the neutral lipid is DSPC.

[0035] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), wherein the LNPs further comprise a steroid as a Petition 870260076800, dated 07 / 31 / 2026, p. 38 / 442 26 / 377 of one or more additional lipids, the steroid is a sterol, just like cholesterol.

[0036] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the phase does not comprise a chelating agent.

[0037] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the LNPs comprise at least about 75% of the RNA comprised in the composition. For example, LNPs may comprise at least about 76%, such as at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% of the RNA comprised in the composition.

[0038] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), RNA (as mRNA) is encapsulated within or associated with LNPs. Petition 870260076800, dated 07 / 31 / 2026, p. 39 / 442 27 / 377

[0039] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises a modified nucleoside in place of uridine. For example, the modified nucleoside may be selected from pseudouridine (ψ), N1-methyl-pseudouridine (η1ψ) and 5-methyl-uridine (m5U).

[0040] In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises one or more of the following: (a) a 5' cap, such as a cap1 or cap2 structure; (b) a 5' UTR; (c) a 3' UTR; and (d) a poly-A sequence, such as a poly-A sequence comprising at least 100 nucleotides, wherein the poly-A sequence is preferably a broken sequence of A nucleotides.

[0041] In a preferred embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) encodes one or more polypeptides. For example, one or more polypeptides may comprise an epitope to induce an immune response against an antigen in a subject. In a preferred embodiment, the RNA (such as mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, a Petition 870260076800, dated 07 / 31 / 2026, p. 40 / 442 28 / 377 immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or the immunogenic variant thereof. In an embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises an ORF encoding a full-length variant of the SARS-CoV2S protein with proline residue substitutions at positions 986 and 987 of the SEQ ID NO: 1. For example, the SARS-CoV-2 S protein variant may have at least 80% identity to SEQ ID NO: 7.

[0042] In a preferred embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the composition is in frozen form. Preferably, the RNA integrity after thawing the frozen composition is at least 50%, such as at least 52%, at least 54%, at least 55%, at least 56%, at least 58% or at least 60%, for example, after thawing the frozen composition that was stored at 20°C. Additionally or alternatively, the size (Z-mean) (and / or size distribution and / or polydispersity index (PDI)) of the LNPs after thawing the frozen composition is equal to the size (Z-mean) (and / or size distribution and / or PDI) of the LNPs before the composition was frozen.In one embodiment, the average size (Zaverage) of LNPs after thawing from the frozen composition is between approximately 50 nm and approximately 500 nm, preferably in between. Petition 870260076800, dated 07 / 31 / 2026, p. 41 / 442 29 / 377 approximately 40 nm and approximately 200 nm, more preferably between approximately 40 nm and approximately 120 nm. In one embodiment, the PDI of the LNPs after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In one embodiment, the average size (Z-size) of the LNPs after thawing the frozen composition is between approximately 50 nm and approximately 500 nm, preferably between approximately 40 nm and approximately 200 nm, more preferably between approximately 40 nm and approximately 120 nm, and the average size (Z-size) (and / or size distribution and / or PDI) of the LNPs after thawing the frozen composition is equal to the average size (Z-size) (and / or size distribution and / or PDI) of the LNPs before freezing.In one embodiment, the average size (Zaverage) of the LNPs after thawing the frozen composition is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the LNPs after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0043] In a preferred alternative embodiment of the first aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the composition is in liquid form. Preferably, the RNA integrity of the liquid composition, when stored, for example, at 0°C or more for at least one week, is sufficient to produce the effect. Petition 870260076800, dated 07 / 31 / 2026, p. 42 / 442 30 / 377 desired, for example, to induce an immune response. For example, the RNA integrity of the liquid composition, when stored, for example, at 0°C or higher for at least one week, can be at least 50%, as well as at least 52%, at least 54%, at least 55%, at least 56%, at least 58% or at least 60%. Additionally or alternatively, the size (Z-average) (and / or size distribution and / or polydispersity index (PDI)) of the LNPs in the liquid composition, when stored, for example, at 0°C or higher for at least one week, is sufficient to produce the desired effect, for example, to induce an immune response. For example, the size (Z-average) (and / or size distribution and / or polydispersity index (PDI)) of the LNPs in the liquid composition, when stored, for example, at 0°C or higher for at least one week, is equal to the size (Z-average) (and / or size distribution and / or PDI) of the LNPs in the initial composition, i.e., before storage.In one embodiment, the average size (Zaverage) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm. In one embodiment, the PDI of the LNPs after storage of the liquid composition, for example, a. The average size (Zaverage) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is less than 0.3, preferably less than 0.2, and more preferably less than 0.1. Petition 870260076800, dated 07 / 31 / 2026, page 43 / 442 31 / 377 of 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the size (Z-average) (and / or size distribution and / or PDI) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week is equal to the size (Z-average) (and / or size distribution and / or PDI) of the LNPs before storage. In one embodiment, the average size (Zaverage) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0044] In a second aspect, the present disclosure provides a method for preparing a composition comprising LNPs dispersed in a final aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the final aqueous phase comprises a buffer system comprising a final buffer substance and a final monovalent anion, the final buffer substance being selected from the group consisting of Tris and its protonated form, Bis-Tris-methane and its protonated form and TEA and its protonated form, the final monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the MES anion, the MOPS anion and the anion Petition 870260076800, dated 07 / 31 / 2026, pp. 44 / 442 32 / 377 of HEPES; the concentration of the final buffer substance in the composition is at most about 25 mM; and the final aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions, and substantially free of EDTA anions; wherein the method comprises: (I) preparing a formulation comprising LNPs dispersed in the final aqueous phase, wherein the LNPs comprise cationically ionizable lipid and RNA; and (II) optionally freezing the formulation at about 10°C or below, thereby obtaining the composition, wherein step (I) comprises: (a) prepare an RNA solution containing water and a first buffer system; (b) prepare an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids; (c) mixing the RNA solution prepared in (a) with the ethanolic solution prepared in (b), thereby preparing an intermediate formulation comprising the LNPs dispersed in an intermediate aqueous phase comprising the first buffer system; and (d) filtering the first intermediate formulation prepared in (c) using a final aqueous buffer solution comprising the final buffer system, thereby preparing the formulation comprising the LNPs dispersed in the final aqueous phase. Petition 870260076800, dated 07 / 31 / 2026, page 45 / 442 33 / 377

[0045] As demonstrated in the present application, the use of a specific buffer system based on the buffer substances specified above, in particular Tris and its protonated form, instead of PBS in a composition comprising LNPs inhibits the formation of a very stable folded form of RNA. Furthermore, the present application demonstrates that, surprisingly, simply decreasing the concentration of the buffer substance in a composition comprising LNPs and a buffer system, wherein the LNPs comprise a cationically ionizable lipid and RNA, it is possible to obtain an RNA LNP composition having improved RNA integrity after a freeze / thaw cycle compared to a composition comprising the same buffer substance at a concentration of 50 mM.Thus, the composition prepared by the claimed method provides enhanced stability, can be stored within a temperature range compatible with standard technologies in pharmaceutical practice, and provides a ready-to-use preparation.

[0046] In a particularly preferred embodiment of the second aspect, the final buffer substance is Tris and its protonated form, that is, a mixture of Tris and its protonated form.

[0047] In an embodiment of the second aspect, the final monovalent anion is selected from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 3-(N-morpholino)propanesulfonate, or from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 2-[4-(2-hydroxyethyl)piperazine-1 Petition 870260076800, dated 07 / 31 / 2026, page 46 / 442 34 / 377 il]ethanesulfonate, preferably from the group consisting of chloride, acetate, lactate and morpholinoethanesulfonate, more preferably from the group consisting of chloride, acetate, and morpholinoethanesulfonate, or from the group consisting of chloride, acetate and lactate, as chloride or acetate.

[0048] In an embodiment of the second aspect, the final buffer substance is Tris and its protonated form and the final monovalent anion is selected from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 3-(N-morpholino)propanesulfonate, or from the group consisting of chloride, acetate, glycolate, lactate, morpholinoethanesulfonate and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate, preferably from the group consisting of chloride, acetate, lactate and morpholinoethanesulfonate, more preferably from the group consisting of chloride, acetate, lactate and morpholinoethanesulfonate, more preferably from the group consisting of chloride, acetate and morpholinoethanesulfonate, as chloride or acetate.

[0049] In one embodiment, in particular if it is desired to prepare a composition in frozen form, the method of the second aspect comprises (II) freezing the formulation at about 10°C or below. Thus, in this embodiment, conducting the method of the second aspect results in a composition in frozen form.

[0050] In an alternative embodiment, in particular, if it is desired to prepare a composition in liquid form, the method of the second aspect does not include step (II). Thus, in this embodiment, conducting the method of Petition 870260076800, dated 07 / 31 / 2026, page 47 / 442 35 / 377 second aspect results in a composition in liquid form.

[0051] In an embodiment of the second aspect, step (I) further comprises one or more selected dilution and filtration steps, such as tangential flow filtration and diafiltration, after step (c). For example, a dilution step may comprise the addition of a dilution solution to an intermediate formulation. This dilution solution may comprise one or more additional compounds and, optionally, the final buffer system, wherein one or more additional compounds may comprise a cryoprotectant. One or more filtration steps (including steps (d), (f'), (g') and (h')) may be used to remove unwanted compounds (e.g., ethanol and / or one or more di- and / or polybasic organic acids) from the intermediate formulation and / or to increase the RNA concentration of the intermediate formulation and / or to alter the pH and / or the buffer system of the intermediate formulation.For this purpose, an aqueous buffer solution may be used that does not contain the undesirable compounds (so that the undesirable compounds are removed from the intermediate formulation into the aqueous buffer solution) and / or that is hypertonic compared to the aqueous buffer solution (so that water flows from the intermediate formulation into the aqueous buffer solution) and / or that has a pH and / or buffer system different from the pH and / or buffer system of the intermediate formulation.

[0052] In a preferred embodiment of the second aspect, stage (I) comprises: (a') provide an aqueous solution of RNA; (b') provide a first aqueous buffer solution Petition 870260076800, dated 07 / 31 / 2026, pp. 48 / 442 36 / 377 comprising a first buffer system; (c') mix the aqueous RNA solution provided in (a') with the first aqueous buffer solution provided in (b'), thus preparing an RNA solution containing water and the first buffer system; (d') prepare an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids; (e') mix the RNA solution prepared in (c') with the ethanolic solution prepared in (d'), thereby preparing a first intermediate formulation comprising LNPs dispersed in a first aqueous phase comprising the first buffer system; (f') optionally filter the first intermediate formulation prepared in (e') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising the LNPs dispersed in an additional aqueous phase comprising the additional buffer system, wherein the other aqueous buffer solution may be identical to or different from the first aqueous buffer solution; (g') optionally repeating step (f') one or two or more times, wherein the additional intermediate formulation comprising the LNPs dispersed in the additional aqueous phase comprising the additional buffer system obtained after step (f') of one cycle is used as the first intermediate formulation of the next cycle, wherein in each cycle the additional aqueous buffer solution may be identical to or different from the first aqueous buffer solution; Petition 870260076800, dated 07 / 31 / 2026, page 49 / 442 37 / 377 (h') filter the first intermediate formulation obtained in step (e'), if step (f') is absent, or the additional intermediate formulation obtained in step (f'), if step (f') is present and step (g') is not present, or the additional intermediate formulation obtained after step (g'), if steps (f') and (g') are present, using a final aqueous buffer solution comprising the final buffer system and having a pH of at least 6.0; and (i') optionally dilute the formulation obtained in step (h') with a diluting solution; Preparing, therefore, the formulation comprising the LNPs dispersed in the final aqueous phase.

[0053] In one embodiment of the second aspect, the concentration of the final buffer substance, in particular the total concentration of Tris and its protonated form, in the composition is at most about 20 mM, such as at most about 19 mM, at most about 18 mM, at most about mM, at most about 16 mM, at most about 15 mM, at most about 14 mM, at most about 13 mM, at most about 12 mM, at most about 11 mM or at most about 10 mM. In one embodiment, the lower limit of the final buffer substance, in particular Tris and its protonated form, in the composition is at least about 1 mM, preferably at least about 2 mM, such as at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about mM, at least about 8 mM or at least about 9 mM. For example, the concentration of the final buffer substance, in Petition 870260076800, dated 07 / 31 / 2026, p. 50 / 442 38 / 377 In particular, the total concentration of Tris and its protonated form in the composition may be between about 1 mM and about 20 mM, such as between about 2 mM and about 15 mM, between about 5 mM and about 14 mM, between about 7 mM and about 13 mM, between about 8 mM and about 12 mM, between about 9 mM and about 11 mM, such as about 10 mM.

[0054] In one embodiment of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or dibasic organic acid anions and / or polybasic organic acid anions. In a first subgroup of the second aspect, at least one of these criteria applies. For example, in one embodiment of this first subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions. In another embodiment of this first subgroup of the second aspect, the final aqueous phase is substantially free of carbonate anions. In another embodiment of this first subgroup of the second aspect, the final aqueous phase is substantially free of dibasic organic acid anions. In another embodiment of this first subgroup of the second aspect, the final aqueous phase is substantially free of polybasic organic acid anions.

[0055] In a second subgroup of the second aspect, at least two of the above criteria apply. For example, in an embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of Petition 870260076800, dated 07 / 31 / 2026, page 51 / 442 39 / 377 carbonate anions. In another embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of dibasic organic acid anions. In another embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of polybasic organic acid anions. In another embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of carbonate anions and substantially free of dibasic organic acid anions. In another embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions.In another embodiment of this second subgroup of the second aspect, the final aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions.

[0056] In a third subgroup of the second aspect, at least three of the above criteria apply. For example, in one embodiment of this third subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, and substantially free of dibasic organic acid anions. In another embodiment of this third subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions, Petition 870260076800, dated 07 / 31 / 2026, page 52 / 442 40 / 377 substantially free of carbonate anions and substantially free of polybasic organic acid anions. In another embodiment of this third subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions. In another embodiment of this third subgroup of the second aspect, the final aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions.

[0057] In a fourth subgroup of the second aspect, at least four of the above criteria apply. That is, in this fourth subgroup of the second aspect, the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions, and substantially free of polybasic organic acid anions.

[0058] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect), the formulation obtained in step (I) and / or the composition comprises a cryoprotectant. In an alternative embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect), the formulation obtained in step (I) and / or the composition is substantially free of a Petition 870260076800, dated 07 / 31 / 2026, page 53 / 442 41 / 377 cryoprotectant. Thus, specific examples of these embodiments are as follows: (1) the final aqueous phase is substantially free of inorganic sulfate anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (2) the final aqueous phase is substantially free of carbonate anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (3) the final aqueous phase is substantially free of dibasic organic acid anions, the formulation obtained in step (I) and / or the composition and comprises a cryoprotectant; (4) the final aqueous phase is substantially free of polybasic organic acid anions and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (5) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of carbonate anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (6) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (7) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (8) the final aqueous phase is substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the formulation obtained in step (I) Petition 870260076800, dated 07 / 31 / 2026, page 54 / 442 42 / 377 and / or the composition includes a cryoprotectant; (9) the final aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (10) the final aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (11) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (12) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (13) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (14) the final aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; Petition 870260076800, dated 07 / 31 / 2026, page 55 / 442 43 / 377 (15) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition comprises a cryoprotectant; (16) the final aqueous phase is substantially free of inorganic sulfate anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (17) the final aqueous phase is substantially free of carbonate anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (18) the final aqueous phase is substantially free of dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (19) the final aqueous phase is substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (20) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of carbonate anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (21) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of Petition 870260076800, dated 07 / 31 / 2026, page 56 / 442 44 / 377 dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (22) the final aqueous phase is substantially free of inorganic sulfate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (23) the final aqueous phase is substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (24) the final aqueous phase is substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (25) the final aqueous phase is substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (26) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of dibasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; Petition 870260076800, dated 07 / 31 / 2026, page 57 / 442 45 / 377 (27) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (28) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (29) the final aqueous phase is substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant; (30) the final aqueous phase is substantially free of inorganic sulfate anions, substantially free of carbonate anions, substantially free of dibasic organic acid anions and substantially free of polybasic organic acid anions, and the formulation obtained in step (I) and / or the composition is / are substantially free of a cryoprotectant.

[0059] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed Petition 870260076800, dated 07 / 31 / 2026, page 58 / 442 46 / 377 above), wherein the formulation obtained in step (I) and / or the composition comprises a cryoprotectant, said cryoprotectant comprising one or more compounds selected from the group consisting of carbohydrates and sugar alcohols. For example, the cryoprotectant may be selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof. In a preferred embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the formulation obtained in step (I) and / or the composition comprises sucrose and / or glycerol as a cryoprotectant.

[0060] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein the formulation obtained in step (I) and / or the composition comprises a cryoprotectant, the concentration of the cryoprotectant in the formulation and / or composition is at least 1% w / v, such as at least 2% w / v, at least 3% w / v, at least 4% w / v, at least 5% w / v, at least 6% w / v, at least 7% w / v, at least 8% w / v or at least 9% w / v. In one embodiment, the concentration of the cryoprotectant in the formulation and / or composition is up to 25% w / v, such as up to 20% w / v, up to 19% p / v, up to 18% p / v, up to 17% p / v, up to 16% p / v, up to 15% p / v, up to 14% p / v, up to 13% p / v, up to 12% p / v, or up to 11% p / v. In one embodiment, the concentration of the cryoprotectant in the formulation and / or composition is 1% w / v to 20% w / v, as well as Petition 870260076800, dated 07 / 31 / 2026, p. 59 / 442 47 / 377 2% p / v 19% p / v, 3% p / v 18% p / v, 4% p / v 17% p / v, 5% p / v 16% p / v, 5% p / v 15% p / v, 6% p / v 14% p / v, 7% p / v 13% p / v, 8% p / v 12% p / v, 9% w / v 11% w / v, or about 10% w / v. In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the formulation and / or composition comprises a cryoprotectant (in particular, sucrose and / or glycerol) at a concentration of 5% w / v 15% w / v, such as 6% w / v 14% w / v, 8% w / v 13% w / v, or 9% w / v 11% w / v, or at a concentration of about 10% w / v. For example, the method of the second aspect may comprise a dilution step using a dilution solution, wherein the dilution solution comprises a sufficient amount of a cryoprotectant to achieve the above cryoprotectant concentrations in the formulation obtained in step (I) and / or the composition.

[0061] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein the formulation obtained in step (I) and / or the composition comprises a cryoprotectant, the cryoprotectant is present in a concentration resulting in an osmolarity of the composition in the range of about 50 x 10-3 osmol / kg to about 400 x 10-3 osmol / kg (such as from about 50 x 10-3 osmol / kg to about 390 x 10-3 osmol / kg, from about 60 x 10-3 osmol / kg to about 380 x 10-3 osmol / kg, from Petition 870260076800, dated 07 / 31 / 2026, p. 60 / 442 48 / 377 from about 70 x 10-3osmol / kg to about 370 x 10-3osmol / kg, from about 80 x 10-3osmol / kg to about 360 x 10-3osmol / kg, from about 90 x 10-3osmol / kg to about 350 x 10-3osmol / kg, from about 100 x 10-3osmol / kg to about 340 x 10-3osmol / kg, from about 120 x 10-3osmol / kg to about 330 x 10-3osmol / kg, from about 140 x 10-3osmol / kg to about 320 x 10-3osmol / kg, from about 160 x 10-3osmol / kg to about 310x 10-3 osmol / kg, from about 180 x 10-3 osmol / kg to about 300 x 10-3 osmol / kg, or from about 200 x 10-3 osmol / kg to about 300 x 10-3 osmol / kg), based on the total weight of the formulation / composition. For example, the method of the second aspect may comprise a dilution step using a dilution solution, wherein the dilution solution comprises a sufficient amount of a cryoprotectant to achieve the above osmolarity values ​​in the formulation obtained in step (I) and / or the composition.

[0062] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the second aspect where the final buffer substance is Tris and its protonated form, the final monovalent anion is selected from the group consisting of chloride, acetate, glycolate and lactate, and the concentration of the final monovalent anion (in particular the total concentration of all final monovalent anions) in the composition is at most equal to the concentration of the final buffer substance in the composition. For example, the concentration of the final monovalent anion (in particular, the total concentration of all final monovalent anions) in the composition may Petition 870260076800, dated 07 / 31 / 2026, p. 61 / 442 49 / 377 being less than the concentration of the final buffer substance in the composition. Thus, in those embodiments of the second aspect, where the concentration of the final buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, the concentration of the final monovalent anion (in particular the total concentration of all final monovalent anions) in the composition is at most equal to about 20 mM, for example, less than 20 mM. Generally, the concentration of the monovalent anion, such as chloride and / or acetate (in particular the total concentration of all monovalent anions) in the composition may be less than about 15 mM, such as less than about 14 mM, less than about 13 mM, less than about 12 mM, less than about 11 mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM or less than about 5 mM.In one embodiment, the chloride concentration in the composition is as defined above (e.g., less than about 15 mM, etc.) and the composition does not comprise acetate. In an alternative embodiment, the acetate concentration in the composition is as defined above (e.g., less than about 15 mM, etc.) and the composition does not comprise chloride.

[0063] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the second aspect where the final buffer substance is Tris and its protonated form, the sodium concentration in the aqueous phase and / or Petition 870260076800, dated 07 / 31 / 2026, page 62 / 442 50 / 377 composition is less than 20 mM, such as less than about 15 mM, for example, less than about 14 mM, less than about 13 mM, less than about 12 mM, less than about mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, less than about 6 mM, or less than about 5 mM.

[0064] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), in particular in those embodiments of the second aspect where the final buffer substance is Tris and its protonated form, the final monovalent anion is selected from the group consisting of the MES, MOPS and HEPES anions, and the concentration of the final monovalent anion (in particular the total concentration of all final monovalent anions) in the composition is at least equal to the concentration of the final buffer substance in the composition. For example, the concentration of the final monovalent anion (in particular the total concentration of all final monovalent anions) in the composition may be greater than the concentration of the final buffer substance in the composition.Thus, in those embodiments of the second aspect, where the concentration of the final buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, the concentration of the final monovalent anion (in particular the total concentration of all final monovalent anions) in the composition is at least equal to about 20 mM, for example, greater than 20 mM. Generally, the concentration of the final monovalent anion (in particular the... Petition 870260076800, dated 07 / 31 / 2026, p. 63 / 442 51 / 377 total concentration of all final monovalent anions) in the composition may be greater than about 20 mM, such as greater than about 21 mM, greater than about 22 mM, greater than about 23 mM, greater than about 24 mM, greater than about 25 mM, greater than about 26 mM, greater than about 27 mM, greater than about 28 mM, greater than about 29 mM or greater than about 30 mM and preferably at most 50 mM, such as at most 45 mM, at most 40 mM or at most 35 mM.

[0065] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the pH of the final buffer system (and the pH of the composition) is between about 6.5 and about 8.0. For example, the pH of the composition may be between about 6.9 and about 7.9, as between about 7.0 and about 7.9, between about 7.1 and about 7.8, between about 7.2 and about 7.7, between about 7.3 and about 7.6, between about 7.4 and about 7.6, or about 7.5.

[0066] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the first buffer system (and the pH of the RNA solution obtained in step (a)) has a pH below 6.0, preferably at most about 5.5, at most about 5.0, at most about 4.9, at most about 4.8, at most about 4.7, at most about 4.6 or at most about 4.5. By Petition 870260076800, dated 07 / 31 / 2026, p. 64 / 442 52 / 377 For example, the pH of the first buffer system (and the pH of the RNA solution obtained in step (a)) may be between about 3.5 and about 5.9, or between about 4.0 and about 5.5, or between about 4.5 and about 5.0. To this end, the RNA solution obtained in step (a) may further comprise one or more di- and / or polybasic organic acids (e.g., citrate anions and / or EDTA anions). In this embodiment, it is preferable that step (d) be conducted under conditions that remove one or more di- and / or polybasic organic acids resulting in the formulation comprising the LNPs dispersed in the final aqueous phase with the final aqueous phase being substantially free of one or more di- and / or polybasic organic acids.For example, such conditions may include subjecting the intermediate formulation comprising the LNPs dispersed in the intermediate aqueous phase obtained in step (c) to at least one filtration step, such as tangential flow filtration or diafiltration, using a final buffer solution comprising the final buffer system (i.e., the final buffer substance and the final monovalent anion), wherein the final buffer solution does not contain one or more di- and / or polybasic organic acids (and preferably does not contain ethanol). Alternatively, such conditions may include (i) subjecting the intermediate formulation comprising the LNPs dispersed in the intermediate aqueous phase obtained in step (c) (i.e., a first intermediate formulation) to at least one filtration step, such as tangential flow filtration or diafiltration, using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising the LNPs. Petition 870260076800, dated 07 / 31 / 2026, p. 65 / 442 53 / 377 dispersed in an additional aqueous phase comprising the additional buffer system, wherein the additional buffer system of the additional aqueous buffer solution may be identical to or different from the buffer system used in step (a); (ii) optionally repeat step (i) one or two or more times, wherein the additional intermediate formulation comprising the LNPs dispersed in the additional aqueous phase obtained after step (i) of one cycle is used as the first intermediate formulation of the next cycle, wherein in each cycle the additional buffer system of the additional aqueous buffer solution may be identical to or different from the first buffer system used in step (a);and (iii) subject the intermediate formulation obtained in step (i) (if step (ii) is not present), or the intermediate formulation obtained in step (ii) (if step (ii) is present) to at least one filtration step, such as tangential flow filtration or diafiltration, using the final aqueous buffer solution, wherein at least one of the intermediate and final aqueous buffer solutions (preferably all intermediate and final aqueous buffer solutions) does not contain one or more di- and / or polybasic organic acids (and preferably does not contain ethanol).

[0067] Similarly, in an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, as in any of the embodiments (1) to (30) listed above), where step (I) comprises steps (a') to (e') and (h') (and optionally one or more of steps (f'), (g') and (i')), the first aqueous buffer solution (and the pH of the RNA solution obtained in step (c')) has a pH below Petition 870260076800, dated 07 / 31 / 2026, p. 66 / 442 54 / 377 6.0, preferably at most about 5.5, at most about 5.0, at most about 4.9, at most about 4.8, at most about 4.7, at most about 4.6, or at most about 4.5. For example, the pH of the first aqueous buffer solution (and the pH of the RNA solution obtained in step (c')) may be between about 3.5 and about 5.9, as well as between about 4.0 and about 5.5, or between about 4.5 and about 5.0. For this purpose, the first aqueous buffer solution provided in (b') (and the first aqueous phase) may further comprise one or more di- and / or polybasic organic acids (e.g., citrate anions and / or EDTA anions). In this embodiment, it is preferable that at least one of the steps (f') to (h') be carried out under conditions that remove one or more di- and / or polybasic organic acids from the first intermediate formulation and / or the other intermediate formulation resulting in a further interformulation comprising the LNPs dispersed in an additional aqueous phase or in the final aqueous phase with the additional and / or final aqueous phase being substantially free of one or more di- and / or polybasic organic acids.For example, such conditions may include the use of an additional aqueous buffer solution and / or a final buffer solution, wherein at least one of the additional aqueous buffer solutions and the final buffer solution (preferably all the additional aqueous buffer solutions and the final buffer solution) does not contain one or more die- or polybasic organic acids (and preferably does not contain ethanol). In one embodiment, the filtration steps may be tangential flow filtration or diafiltration, preferably tangential flow filtration. Petition 870260076800, dated 07 / 31 / 2026, page 67 / 442 55 / 377

[0068] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the first buffer system used in step (a) comprises the final buffer substance and the final monovalent anion used in step (d), preferably the buffer system and pH of the first buffer system used in step (a) are identical to the buffer system and pH of the final aqueous buffer solution used in step (d). For example, only an aqueous buffer solution is used in this embodiment of the second aspect.

[0069] Similarly, in an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, as in any of the embodiments (1) to (30) listed above), wherein step (I) comprises steps (a') to (e') and (h') (and optionally one or more of steps (f'), (g') and (i')), each of the first system and each other buffer system used in steps (b'), (f') and (g') comprises the final buffer substance and the final monovalent anion used in step (h'), preferably the buffer system and the pH of each of the first aqueous buffer solutions and of each additional aqueous buffer solution used in steps (b'), (f') and (g') are identical to the buffer system and the pH of the final aqueous buffer solution. For example, the aqueous buffer solutions used in steps (b'), (f'), if present, (g'), if present, and (h') of this embodiment of the second aspect are identical. Petition 870260076800, dated 07 / 31 / 2026, p. 68 / 442 56 / 377

[0070] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the formulation and / or composition comprises water as the principal component and / or the total amount of solvent(s) other than water contained in the composition is less than about 1.0% (v / v). For example, the amount of water contained in the formulation and / or composition may be at least 50% (w / w), as at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w). In particular, if the formulation and / or composition comprises a cryoprotectant, the amount of water contained in the formulation and / or composition may be at least 50% (w / w), as well as at least 55% (w / w), at least 60% (w / w), at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), or at least 90% (w / w). If the formulation and / or composition is / are substantially free of a cryoprotectant, the amount of water contained in the formulation and / or composition may be at least 95% (w / w). Additionally or alternatively, the total amount of solvent(s) other than water contained in the composition may be less than about 1.0% (v / v), such as less than about 0.9% (v / v), less than about 0.8% (v / v), less than about 0.7% (v / v), less than about 0.6% (v / v), less than about 0.5% (v / v), less than about 0.4% (v / v), less than about 0.3% (v / v), less than Petition 870260076800, dated 07 / 31 / 2026, p. 69 / 442 57 / 377 approximately 0.2% (v / v), less than approximately 0.1% (v / v), less than approximately 0.05% (v / v), or less than approximately 0.01% (v / v). In this respect, a cryoprotectant that is liquid under normal conditions will not be considered a solvent other than water, but rather a cryoprotectant. In other words, the optional limitation above that the total amount of solvent(s) other than water contained in the composition may be less than approximately 1.0% (v / v) does not apply to cryoprotectants that are liquid under normal conditions.

[0071] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the osmolarity of the composition is at most about 400 x 10-3 osmol / kg, such as at most about 390 x 10-3 osmol / kg, at most about 380 x 10-3 osmol / kg, at most about 370 x 10-3 osmol / kg, at most about 360 x 10-3 osmol / kg, at most about 350 x 10-3 osmol / kg, at most about 340 x 10-3 osmol / kg, at most about 330 x 10-3 osmol / kg, at most about 320 x 10-3 osmol / kg, at most about 310 x 10-3 osmol / kg, or at most about 300 x 10-3 osmol / kg.If the composition does not include a cryoprotectant, the osmolality of the composition may be less than 300 x 10⁻³ osmol / kg, such as a maximum of about 250 x 10⁻³ osmol / kg, a maximum of about 200 x 10⁻³ osmol / kg, a maximum of about 150 x 10⁻³ osmol / kg, a maximum of about 100 x 10⁻³ osmol / kg, a maximum of about 50 x 10⁻³ osmol / kg, a maximum of about 40 x 10⁻³ osmol / kg, or a maximum of about 30 x 10⁻³ osmol / kg. If the composition includes a cryoprotectant... Petition 870260076800, dated 07 / 31 / 2026, p. 70 / 442 58 / 377 it is preferable that the main part of the composition's osmolarity be provided by the cryoprotectant. For example, the cryoprotectant may provide at least 50%, as well as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the composition's osmolality.

[0072] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the RNA concentration in the composition is about 5 mg / l about 150 mg / l. For example, the RNA concentration in the composition may be about 10 mg / l about 140 mg / l, such as about 20 mg / l about 130 mg / l, about 25 mg / l about 125 mg / l, about 30 mg / l about 120 mg / l, about 35 mg / l about 115 mg / l, about 40 mg / l about 110 mg / l, about 45 mg / l about 105 mg / l, or about 50 mg / l about 100 mg / l.

[0073] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the final buffer substance is Tris and its protonated form, the pH of the composition is between about 6.5 and about 8.0, and the RNA concentration in the composition is about 5 mg / l to about 150 mg / l. In this embodiment, it is preferable that the pH of the composition is between about 6.9 and about 7.9 and the RNA concentration in the composition is about 25 mg / l to about 125 mg / l, such as about 30 mg / l. Petition 870260076800, dated 07 / 31 / 2026, p. 71 / 442 59 / 377 approximately 120 mg / l. In a particularly preferred embodiment of the second aspect, the buffer substance is Tris and its protonated form; the pH of the composition is between approximately 6.9 and approximately 7.9; the RNA concentration in the composition is approximately 25 mg / l approximately 125 mg / l, such as approximately 30 mg / l approximately 120 mg / l; the final aqueous phase is substantially free of sulfate anions, substantially free of dibasic organic acids and substantially free of polybasic organic acids; and the composition comprises a cryoprotectant.In a particularly preferred alternative embodiment of the second aspect, the buffer substance is Tris and its protonated form; the pH of the composition is between about 6.9 and about 7.9; the RNA concentration in the composition is from about 25 mg / l to about 125 mg / l, such as from about 30 mg / l to about 120 mg / l; the final aqueous phase is substantially free of sulfate anions, substantially free of dibasic organic acids, and substantially free of polybasic organic acids; and the composition is substantially free of a cryoprotectant.

[0074] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom that is capable of being protonated under physiological conditions. For example, the cationically ionizable lipid may have the structure of Formula (I): Petition 870260076800, dated 07 / 31 / 2026, p. 72 / 442 60 / 377 R3^G3 Λ1. / N.XL2R1Ί31Ό2R2(I) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L1, L2, G1, G2, G3, R1, R2 and R3 are as defined in this descriptive report. Preferably, the cationically ionizable lipid is selected from the following: structures I-1 to I-36 (presented herein); and / or structures A to F (shown herein); and / or N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA) and 4((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14). In a particularly preferred embodiment, the cationically ionizable lipid is the lipid with structure I-3.

[0075] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the ethanol solution prepared in step (b) or (d') further comprises one or more additional lipids and the LNPs further comprise one or more additional lipids. Preferably, one or more additional lipids are selected from the group consisting of lipids conjugated with polymers, neutral lipids, steroids and combinations thereof. In a preferred embodiment of the second aspect (in particular Petition 870260076800, dated 07 / 31 / 2026, p. 73 / 442 61 / 377 in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the one or more additional lipids comprise a polymer-conjugated lipid (for example, a pegylated lipid or a polysarcosine-lipid conjugate or a polysarcosine and lipid-like material conjugate), a neutral lipid (for example, DSPC) and a steroid (for example, cholesterol), such that the LNPs comprise the cationically ionizable lipid as described in this descriptive report, a polymer-conjugated lipid (for example, a pegylated lipid or a polysarcosine-lipid conjugate or a polysarcosine and lipid-like material conjugate), a neutral lipid (for example, DSPC) and a steroid (for example, cholesterol).

[0076] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein one or more additional lipids comprise a lipid conjugated with a polymer, the lipid conjugated with a polymer is a pegylated lipid. For example, the pegylated lipid may have the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, where R12, R13 and w are as herein. Petition 870260076800, dated 07 / 31 / 2026, p. 74 / 442 62 / 311 defined.

[0011] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein one or more additional lipids comprise a lipid conjugate with a polymer, the lipid conjugate with a polymer is a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material. For example, the polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like material may be a selected member of the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate and a mixture thereof.

[0018] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein one or more additional lipids comprise a neutral lipid, the neutral lipid is a phospholipid. Such a phospholipid is preferably selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Particular examples of phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), Petition 870260076800, dated 07 / 31 / 2026, page 75 / 442 63 / 377 dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibeeneylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl -phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero3-phosphocholine (18:0 Dieter PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and dipitanoylphosphatidylethanolamine (DPyPE). In a particularly preferred embodiment, the neutral lipid is DSPC.

[0079] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), wherein one or more additional lipids comprise a steroid, the steroid is a sterol, such as cholesterol.

[0080] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the ethanol solution comprises the cationically ionizable lipid, the polymer-conjugated lipid, the lipid Petition 870260076800, dated 07 / 31 / 2026, p. 76 / 442 64 / 377 neutral and the steroid in a molar ratio of 20% to 60% cationically ionizable lipid, 0.5% to 15% polymer-conjugated lipid, 5% to 25% neutral lipid and 25% to 55% steroid, based on the total molar amount of lipids in the ethanolic solution. For example, the molar ratio can be 40% to 55% cationically ionizable lipid, 1.0% to 10% polymer-conjugated lipid, 5% to 15% neutral lipid, and 30% to 50% steroid, as well as 45% to 55% cationically ionizable lipid, 1.0% to 5% polymer-conjugated lipid, 8% to 12% neutral lipid, and 35% to 45% steroid, based on the total molar amount of lipids in the ethanolic solution.

[0081] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the first aspect, such as in any of the embodiments (1) to (30) listed above), the final aqueous phase does not contain a chelating agent.

[0082] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the LNPs comprise at least about 75% of the RNA comprised in the composition. For example, the LNPs may comprise at least about 76%, such as at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least Petition 870260076800, dated 07 / 31 / 2026, p. 77 / 442 65 / 377 less about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% of the RNA comprised in the composition.

[0083] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), RNA (as mRNA) is encapsulated within or associated with LNPs.

[0084] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises a modified nucleoside in place of uridine. For example, the modified nucleoside may be selected from pseudouridine (ψ), N1-methyl-pseudouridine (η1ψ) and 5-methyl-uridine (m5U).

[0085] In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises one or more of the following: (a) a 5' cap, such as a cap1 or cap2 structure; (b) a 5' UTR; (c) a 3' UTR; and (d) a poly-A sequence, such as a poly-A sequence comprising at least 100 nucleotides, wherein the poly-A sequence is preferably a broken sequence of A nucleotides.

[0086] In a preferred embodiment of the second Petition 870260076800, dated 07 / 31 / 2026, p. 78 / 442 66 / 377 aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) encodes one or more polypeptides. For example, one or more polypeptides may comprise an epitope to induce an immune response against an antigen in a subject. In a preferred embodiment, the RNA (such as mRNA) comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.In an embodiment of the second aspect (in particular in an embodiment of the first, second, third or fourth subgroup above the second aspect, such as in any of the embodiments (1) to (30) listed above), the RNA (such as mRNA) comprises an ORF encoding a full-length SARS-CoV2 S protein variant with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1. For example, the SARS-CoV-2 S protein variant may have at least 80% identity with SEQ ID NO:7.

[0087] In a third aspect, the present disclosure provides a method for storing a composition, comprising preparing a composition according to the method of the second aspect and storing the composition at a temperature ranging from about -90°C to about -10°C, such as from about -90°C to about -40°C or from about -40°C to about -25°C or from about -25°C to about -10°C, or a temperature of about -20°C. In an embodiment of Petition 870260076800, dated 07 / 31 / 2026, p. 79 / 442 67 / 377 third aspect, the storage of the frozen composition is at least 1 week, as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months or at least 36 months, preferably at least 4 weeks. In an embodiment of the third aspect, the storage of the frozen composition is at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at 20°C. In an embodiment of the third aspect, the composition may be stored at -70°C.

[0088] In an embodiment of the third aspect, the method of storing a composition comprises preparing a composition in accordance with the method of the second aspect comprising step (II) (i.e., freezing the formulation at about -10°C or below); storing the frozen composition at a temperature ranging from about -90°C to about -10°C for a certain period of time (e.g., at least one week); and storing the frozen composition at a temperature ranging from about 0°C to about 20°C for a certain period of time (e.g., at least one week).

[0089] It is understood that any realization described here in the context of the first or second aspect (in particular, any realization of the first, second, third or fourth subgroup above the first aspect, such as any of the realizations (1) to (30) of the first aspect listed above or any realization of the first, Petition 870260076800, dated 07 / 31 / 2026, p. 80 / 442 68 / 377 second, third or fourth subgroup above the second aspect, such as any of the embodiments (1) to (30) of the second aspect listed above) may also apply to any embodiment of the third aspect.

[0090] In a fourth aspect, the present disclosure provides a method for storing a composition comprising preparing a liquid composition according to the method of the second aspect and storing the liquid composition at a temperature ranging from about 0°C to about 20°C, such as from about 1°C to about 15°C, from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C. In an embodiment of the fourth aspect, the storage of the liquid composition is at least 1 week, such as at least 2 weeks, at least weeks, at least 4 weeks, at least 1 month, at least months, at least 3 months, or at least 6 months, preferably at least 4 weeks.In implementing the fourth aspect, the liquid composition should be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, and most preferably at least 6 months at 5°C.

[0091] In an embodiment of the fourth aspect, the method of storing a composition comprises preparing a composition in accordance with the method of the second aspect comprising step (II) (i.e., freezing the formulation at about -10°C or below); and storing the frozen composition at a temperature ranging from about 0°C to about 20°C for a certain period of time (for Petition 870260076800, dated 07 / 31 / 2026, p. 81 / 442 69 / 377 example, at least one week).

[0092] It is understood that any embodiment described herein in the context of the first, second or third aspect (in particular, any embodiment of the first, second, third or fourth subgroup above the first aspect, such as any of the embodiments (1) to (30) of the first aspect listed above or any embodiment of the first, second, third or fourth subgroup above the second aspect, such as any of the embodiments (1) to (30) of the second aspect listed above) may also apply to any embodiment of the fourth aspect.

[0093] In a fifth aspect, the present disclosure provides a composition preparable by the method of the second, third or fourth aspect. In an embodiment of the fifth aspect, the composition may be in frozen form which, preferably, can be stored at a temperature of about -90°C or higher, such as from about -90°C to about -10°C. For example, the frozen composition of the fifth aspect may be stored at a temperature ranging from about -90°C to about -40°C or from about -40°C to about -25°C or from about -25°C to about -10°C, or a temperature of about -20°C. In an embodiment of the fifth aspect, the composition can be stored for at least 1 week, as well as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks.For example, the frozen composition can be stored for at least 4. Petition 870260076800, dated 07 / 31 / 2026, page 82 / 442 70 / 377 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more Ideally, at least 6 months at 20°C.

[0094] In an embodiment of the fifth aspect, where the composition is in frozen form, the RNA integrity after thawing the frozen composition is at least 50%, such as at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, or at least 60%, for example, after thawing the frozen composition that was stored at -20°C.

[0095] Additionally or alternatively, in an embodiment of the fifth aspect, where the composition is in frozen form, the (Z-average) size (and / or size distribution and / or polydispersity index (PDI)) of the LNPs after thawing the frozen composition is equal to the (Z-average) size (and / or size distribution and / or PDI) of the LNPs before the composition was frozen. In one embodiment, the (Z-average) size of the LNPs after thawing the frozen composition is between about nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about nm and about 120 nm. In one embodiment, the PDI of the LNPs after thawing the frozen composition is less than 0.3, preferably less than 0.2, more preferably less than 0.1. In one embodiment, the average size (Zaverage) of the LNPs after thawing from the frozen composition is between approximately 50 nm and approximately 500 nm, preferably between about 40 nm and about Petition 870260076800, dated 07 / 31 / 2026, p. 83 / 442 71 / 377 200 nm, more preferably between about 40 nm and about 120 nm, and the size (Z-average) (and / or size distribution and / or PDI) of the LNPs after thawing the frozen composition is equal to the size (Z-average) (and / or size distribution and / or PDI) of the LNPs before freezing. In one embodiment, the size (Z-average) of the LNPs after thawing the frozen composition is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the LNPs after thawing the frozen composition is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0096] In an alternative embodiment of the fifth aspect, the composition is in liquid form.

[0097] In an embodiment of the fifth aspect, where the composition is in liquid form, the RNA integrity of the liquid composition, when stored, for example, at 0°C or higher for at least one week, is sufficient to produce the desired effect, for example, to induce an immune response. For example, the RNA integrity of the liquid composition, when stored, for example, at 0°C or higher for at least one week, may be at least 50%, as well as at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, or at least 60%.

[0098] Additionally or alternatively, in an embodiment of the fifth aspect, where the composition is in liquid form, the size (Zaverage) (and / or size distribution and / or polydispersity index (PDI)) of the LNPs of Petition 870260076800, dated 07 / 31 / 2026, page 84 / 442 72 / 377 liquid composition, when stored, for example, at 0°C or higher for at least one week, is sufficient to produce the desired effect, for example, to induce an immune response. For example, the size (Z-average) (and / or size distribution and / or polydispersity index (PDI)) of the LNPs in the liquid composition, when stored, for example, at 0°C or higher for at least one week, is equal to the size (Z-average) (and / or size distribution and / or PDI) of the LNPs in the initial composition, i.e., before storage. In one embodiment, the size (Z-average) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm. In one concrete example, the PDI of LNPs after storage of the liquid composition, for example, the The average size (Zaverage) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is less than 0.3, preferably less than 0.2, and more preferably less than 0.1. In one embodiment, the average size (Zaverage) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, and more preferably between about 40 nm and about 120 nm. The average size (Zaverage) (and / or size distribution and / or PDI) of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is equal to the average size (Zaverage) (and / or size distribution and / or PDI) of the LNPs before storage. In one embodiment, the average size (Zaverage) of the LNPs Petition 870260076800, dated 07 / 31 / 2026, page 85 / 442 73 / 377 LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, are between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the LNPs after storage of the liquid composition, for example, at 0°C or higher for at least one week, is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0099] It is understood that any embodiment described here in the context of the first, second, third or fourth aspect (in particular, any embodiment of the first, second, third or fourth subgroup above the first aspect, such as any of the embodiments (1) to (30) of the first aspect listed above or any embodiment of the first, second, third or fourth subgroup above the second aspect, such as any of the embodiments (1) to (30) of the second aspect listed above) may also apply to any embodiment of the fifth aspect.

[00100] In a sixth aspect, the present disclosure provides a method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a frozen composition prepared by the method of the second or third aspect and thawing the frozen composition, thereby obtaining the ready-to-use pharmaceutical composition.

[00101] It is understood that any achievement described here in the context of the first, second, third, fourth or fifth aspect (in particular, any achievement of the first, second, third or fourth subgroup above) Petition 870260076800, dated 07 / 31 / 2026, p. 86 / 442 74 / 377 first aspect, such as any of the embodiments (1) to (30) of the first aspect listed above or any embodiment of the first, second, third or fourth subgroup above of the second aspect, such as any of the embodiments (1) to (30) of the second aspect listed above) may also apply to any embodiment of the sixth aspect. In a seventh aspect, the present disclosure provides a method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a liquid composition prepared by the method of the second or fourth aspect, thereby obtaining the ready-to-use pharmaceutical composition.

[00102] It is understood that any embodiment described herein in the context of the first, second, third, fourth, fifth or sixth aspect (in particular, any embodiment of the first, second, third or fourth subgroup above the first aspect, such as any of the embodiments (1) to (30) of the first aspect listed above or any embodiment of the first, second, third or fourth subgroup above the second aspect, such as any of the embodiments (1) to (30) of the second aspect listed above) may also apply to any embodiment of the seventh aspect.

[00103] In an eighth aspect, the present disclosure provides a ready-to-use pharmaceutical composition, preparable by the method of the sixth or seventh aspect.

[00104] It is understood that any achievement described here in the context of the first, second, third, fourth, fifth, sixth or seventh aspect (in particular, any Petition 870260076800, dated 07 / 31 / 2026, p. 87 / 442 75 / 377 concretization of the first, second, third or fourth subgroup above the first aspect, such as any of the concretizations (1) to (30) of the first aspect listed above or any concretization of the first, second, third or fourth subgroup above the second aspect, such as any of the concretizations (1) to (30) of the second aspect listed above) may also apply to any concretization of the eighth aspect.

[00105] In a ninth aspect, the present disclosure provides a combination of any one of the first, fifth, and eighth aspects for use in therapy.

[00106] It is understood that any concretization described here in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect may also apply to any concretization of the ninth aspect.

[00107] In a tenth aspect, the present disclosure provides a composition of any one of the first, fifth, and eighth aspects for use in inducing an immune response.

[00108] It is understood that any concretization described here in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect may also apply to any concretization of the tenth aspect.

[00109] Other detailed achievements are as follows:

[00110] 1. Composition comprising lipid nanoparticles (LNPs) dispersed in an aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the aqueous phase comprises a buffer system comprising a buffer substance and a monovalent anion, the substance being Petition 870260076800, dated 07 / 31 / 2026, p. 88 / 442 76 / 377 buffer selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris) and its protonated form, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BisTris-methane) and its protonated form, and triethanolamine (TEA) and its protonated form, the monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the morpholinoethanesulfonic acid (MES) anion, the 3-(N-morpholino)propanesulfonic acid (MOPS) anion and the 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) anion; the concentration of the buffer substance in the composition is at most about 25 mM; and the aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions, and substantially free of ethylenediaminetetraacetic acid (EDTA) anions.

[00111] 2. The composition of item 1, in which the buffer substance is Tris and its protonated form.

[00112] 3. The composition of item 1 or 2, wherein the concentration of the buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, preferably at most about 15 mM, more preferably at most about 10 mM, such as about 10 mM.

[00113] 4. The composition of any of items 1 to 3, wherein the aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or dibasic organic acid anions and / or polybasic organic acid anions, in particular substantially free of inorganic sulfate anions, carbonate anions, and organic acid anions. Petition 870260076800, dated 07 / 31 / 2026, page 89 / 442 77 / 377 dibasic and polybasic organic acid anions.

[00114] 5. The composition of any of items 1 to 4, wherein the monovalent anion is selected from the group consisting of chloride, acetate, glycolate and lactate, and the concentration of the monovalent anion in the composition is at most equal to, preferably less than, the concentration of the buffer substance in the composition, such as less than about 9 mM.

[00115] 6. The composition of any of items 1 to 4, wherein the monovalent anion is selected from the group consisting of MES, MOPS and HEPES anions, and the concentration of the monovalent anion in the composition is at least equal to, preferably greater than, the concentration of the buffer substance in the composition.

[00116] 7. The composition of any of items 1 to 6, wherein the pH of the composition is between about 6.5 and about 8.0, preferably between about 6.9 and about 7.9, as well as between about 7.0 and about 7.8.

[00117] 8. The composition of any of items 1 to 7, where water is the main component in the composition and / or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v / v).

[00118] 9. The composition of any of items 1 to 8, where the osmolality of the composition is at most about 400 x 10-3 osmol / kg.

[00119] 10. The composition of any of items 1 to 9, wherein the concentration of RNA in the composition is about 5 mg / l to about 150 mg / l, preferably about 10 mg / l to about 130 mg / l, more preferably about 30 mg / l to about 120 mg / l. Petition 870260076800, dated 07 / 31 / 2026, p. 90 / 442 78 / 377

[00120] 11. The composition of any of items 1 to 10, wherein the composition comprises a cryoprotectant, preferably at a concentration of at least about 1% w / v, wherein the cryoprotectant preferably comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols, more preferably the cryoprotectant is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof, more preferably the cryoprotectant comprises sucrose and / or glycerol.

[00121] 12. The composition of any of items 1 to 10, in which the composition is substantially free of a cryoprotectant.

[00122] 13. The composition of any of items 1 to 12, wherein the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom that is capable of being protonated under physiological conditions.

[00123] 14. The composition of any of items 1 to 13, wherein the cationically ionizable lipid has the structure of Formula (I): R3^G3 Λ1. / N. / L2\ R1Ό1Ό2R2(I) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, where: One of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- oo -NRaC(=O)O-, and the other from L1ou Petition 870260076800, dated 07 / 31 / 2026, p. 91 / 442 79 / 377 L2é -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, OC(=O)NRa- oo -NRaC(=O)O- or a direct link; G1 and G2 are, each independently, either unsubstituted C1-C12 alkylene or C2-C12 alkenylene; G3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Rae H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; ex is 0, 1 or 2.

[00124] 15. The composition of any of the items 1a 13, where:

[00125] (α) the cationically ionizable lipid is selected from structures I-1 to I-36 shown here; or

[00126] (β) the cationically ionizable lipid is selected from structures A to F shown here; or

[00127] (γ) the cationically ionizable lipid is the lipid with the I-3 structure shown here.

[00128] 16. The composition of any of the items 1a 15, wherein the LNPs further comprise one or more additional lipids, preferably selected from the group consisting of lipids conjugated with polymers, neutral lipids, steroids and combinations thereof, more preferably the LNPs comprise the cationically ionizable lipid, a lipid conjugated with polymer, a Petition 870260076800, dated 07 / 31 / 2026, page 92 / 442 80 / 377 neutral lipid and a steroid.

[00129] 17. The composition of item 16, in which the lipid conjugated with polymer comprises a pegylated lipid, in which the pegylated lipid preferably has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R12 and R13 are each independently a linear or branched alkyl chain, saturated or unsaturated, containing from 10 to 30 carbon atoms, in which the alkyl chain is optionally interrupted by one or more ester linkages; ew has an average value ranging from 30 to 60.

[00130] 18. The composition of item 16, wherein the lipid conjugate with polymer comprises a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material, wherein the polysarcosine-lipid conjugate or conjugate of polysarcosine and a lipid-like material is preferably a selected member of the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate and a mixture thereof.

[00131] 19. The composition of any of items 16 to 18, in which the neutral lipid is a phospholipid, Petition 870260076800, dated 07 / 31 / 2026, page 93 / 442 81 / 377 preferably selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diaraquidoylphosphatidylcholine (DAPC), dibeenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoioleoyl-phosphatidylcholine (POPC), 1,2-di-Ooctadecenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE),dilauroylphosphatidylethanolamine (DLPE) and dipitanoylphosphatidylethanolamine (DPyPE)).

[00132] 20. The composition of any of items 16 to 19, wherein the steroid comprises a sterol such as cholesterol.

[00133] 21. The composition of any of items 1 to, 20, in which the aqueous phase does not include a chelating agent.

[00134] 22. The composition of any of items 1 to 21, in which LNPs comprise at least about 75%, Petition 870260076800, dated 07 / 31 / 2026, page 94 / 442 82 / 377 preferably at least about 80% of the RNA comprised in the composition.

[00135] 23. The composition of any of items 1 to 22, in which RNA is encapsulated within or associated with LNPs.

[00136] 24. The composition of any of items 1 to 23, wherein the RNA comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m^) and 5-methyl-uridine (m5U).

[00137] 25. The composition of any of items 1 to 24, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence.

[00138] 26. The composition of item 25, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted sequence of A nucleotides.

[00139] 27. The composition of item 25 or 26, where the cap 5' is a cap1 or cap2 structure.

[00140] 28. The composition of any of items 1 to 27, in which the RNA encodes one or more polypeptides, wherein one or more polypeptides preferably comprise an epitope to induce an immune response against an antigen in a subject.

[00141] 29. The composition of item 28, wherein the RNA comprises an open reading frame (ORF) encoding an amino acid sequence comprising an S protein of SARS-CoV-2, an immunogenic variant thereof, or an immunogenic fragment of the S protein of SARS-CoV-2 or its variant. Petition 870260076800, dated 07 / 31 / 2026, page 95 / 442 83 / 377 immunogenic.

[00142] 30. The composition of item 28 or 29, wherein the RNA comprises an ORF encoding a full-length SARS-CoV2 S protein variant with proline residue substitutions at positions 986 and 987 of SEQ ID NO: 1.

[00143] 31. The composition of item 29 or 30, in which the SARS-CoV2 S protein variant has at least 80% identity with SEQ ID NO: 7.

[00144] 32. The composition of any of items 1 to 31, where the composition is in frozen form.

[00145] 33. The composition of item 32, in which the RNA integrity after thawing the frozen composition is at least 50% compared to the RNA integrity before the composition was frozen.

[00146] 34. The composition of item 32 or 33, in which the size (Z-average) and / or size distribution and / or polydispersity index (PDI) of the LNPs after thawing the frozen composition is equal to the size (Z-average) and / or size distribution and / or PDI of the LNPs before the composition was frozen.

[00147] 35. The composition of any of items 1 to 31, where the composition is in liquid form.

[00148] 36. A method for preparing a composition comprising LNPs dispersed in a final aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the final aqueous phase comprises a final buffer system comprising a final buffer substance and a final monovalent anion, the final buffer substance being selected from the group consisting of Tris and its form Petition 870260076800, dated 07 / 31 / 2026, page 96 / 442 84 / 377 protonated, Bis-Tris-methane and its protonated form, and TEA and its protonated form, and the final monovalent anion being selected from the group consisting of chloride, acetate, glycolate, lactate, the MES anion, the MOPS anion, and the HEPES anion; the concentration of the final buffer substance in the composition is at most about 25 mM; and the final aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions, and substantially free of EDTA anions; where the method includes: (I) preparing a formulation comprising LNPs dispersed in the final aqueous phase, wherein the LNPs comprise cationically ionizable lipid and RNA; and (II) optionally freezing the formulation at about 10°C or below, thus obtaining the composition, wherein step (I) comprises: (a) prepare an RNA solution containing water and a first buffer system; (b) prepare an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids; (c) mix the RNA solution prepared in (a) with the ethanolic solution prepared in (b), thereby preparing a first intermediate formulation comprising the LNPs dispersed in a first aqueous phase comprising the first buffer system; and (d) filter the first intermediate formulation prepared in (c) using a final aqueous buffer solution. Petition 870260076800, dated 07 / 31 / 2026, page 97 / 442 85 / 377 comprising the final buffer system, Preparing, therefore, the formulation comprising the LNPs dispersed in the final aqueous phase.

[00149] 37. The method in item 36, wherein step (I) further comprises one or more selected dilution and filtration steps.

[00150] 38. The method in item 36 or 37, wherein step (I) comprises: (a') provide an aqueous solution of RNA; (b') provide a first aqueous buffer solution comprising a first buffer system; (c') mix the aqueous RNA solution provided in (a') with the first aqueous buffer solution provided in (b'), thus preparing an RNA solution containing water and the first buffer system; (d') prepare an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids; (e') mix the RNA solution prepared in (c') with the ethanolic solution prepared in (d'), thereby preparing a first intermediate formulation comprising LNPs dispersed in a first aqueous phase comprising the first buffer system; (f') optionally filter the first intermediate formulation prepared in (e') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising the LNPs dispersed in an additional aqueous phase comprising the additional buffer system, wherein the other Petition 870260076800, dated 07 / 31 / 2026, page 98 / 442 86 / 377 aqueous buffer solution may be identical to or different from the first aqueous buffer solution; (g') optionally repeating step (f') one or two or more times, wherein the additional intermediate formulation comprising the LNPs dispersed in the additional aqueous phase comprising the additional buffer system obtained after step (f') of one cycle is used as the first intermediate formulation of the next cycle, wherein in each cycle the additional aqueous buffer solution may be identical to or different from the first aqueous buffer solution; (h') filter the first intermediate formulation obtained in step (e'), if step (f') is absent, or the additional intermediate formulation obtained in step (f'), if step (f') is present and step (g') is not present, or the additional intermediate formulation obtained after step (g'), if steps (f') and (g') are present, using a final aqueous buffer solution comprising the final buffer system and having a pH of at least 6.0; and (i') optionally dilute the formulation obtained in step (h') with a diluting solution; Preparing, therefore, the formulation comprising the LNPs dispersed in the final aqueous phase.

[00151] 39. The method of any of items 36 to 38, wherein the filtration is tangential flow filtration or diafiltration, preferably tangential flow filtration.

[00152] 40. The method of any of items 36 to 39, which comprises (II) freezing the formulation at approximately Petition 870260076800, dated 07 / 31 / 2026, p. 99 / 442 87 / 377 10°C or less.

[00153] 41. The method of any of items 36 to 40, wherein the final buffer substance is Tris and its protonated form.

[00154] 42. The method of any of items 36 to 41, wherein the concentration of the final buffer substance, in particular Tris and its protonated form, in the composition is at most about 20 mM, preferably at most about 15 mM, more preferably by about 10 mM, such as about 10 mM.

[00155] 43. The method of any of items 36 to 42, wherein the final aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or dibasic organic acid anions and / or polybasic organic acid anions, in particular substantially free of inorganic sulfate anions, carbonate anions, dibasic organic acid anions and polybasic organic acid anions.

[00156] 44. The method of any of items 36 to 43, wherein (i) the RNA solution prepared in step (a) further comprises one or more di- and / or polybasic organic acid anions, and step (d) is conducted under conditions that remove one or more di- and / or polybasic organic acid anions resulting in the formulation comprising the LNPs dispersed in the final aqueous phase with the final aqueous phase being substantially free of one or more di- and / or polybasic organic acid anions present in the RNA solution prepared in step (a); or (ii) the first aqueous buffer solution and the first aqueous phase comprise one or more di- and / or polybasic organic acid anions. Petition 870260076800, dated 07 / 31 / 2026, page 100 / 442 88 / 377 organic di- and / or polybasic and at least one of the steps (f') to (h') is conducted under conditions that remove one or more anions of organic di- and / or polybasic acids from the first intermediate formulation and / or the additional intermediate formulation.

[00157] 45. The method of any of items 36 to 44, wherein (i) the RNA solution obtained in step (a) has a pH below 6.0, preferably at most about 5.0, more preferably at most about 4.5; or (ii) the first aqueous buffer solution has a pH below 6.0, preferably at most about 5.0, more preferably at most about 4.5.

[00158] 46. The method of item 44 or 45, wherein one or more anions of di- and / or polybasic organic acids comprise citrate anions and / or EDTA anions.

[00159] 47. The method of any of items 36 to 43, wherein (i) the first buffer system used in step (a) comprises the final buffer substance and the final monovalent anion used in step (d), preferably the buffer system and pH of the first buffer system used in step (a) are identical to the buffer system and pH of the final aqueous buffer solution used in step (d); or (ii) each of the first buffer systems and each additional buffer system used in steps (b'), (f') and (g') comprises the final buffer substance and the final monovalent anion used in step (h'), preferably the buffer system and pH of each of the first aqueous buffer solutions and each additional aqueous buffer solution used in steps (b'), (f') and (g') are identical to the buffer system and pH of the final aqueous buffer solution. Petition 870260076800, dated 07 / 31 / 2026, page 101 / 442 89 / 377

[00160] 48. The method of any of items 36 to 47, wherein the final monovalent anion is selected from the group consisting of chloride, acetate, glycolate and lactate, and the concentration of the final monovalent anion in the composition is at most equal to, preferably less than, the concentration of the final buffer substance in the composition, such as less than about 9 mM.

[00161] 49. The method of any of items 36 to 48, wherein the final monovalent anion is selected from the group consisting of MES, MOPS and HEPES anions, and the concentration of the final monovalent anion in the composition is at least equal to, preferably greater than, the concentration of the final buffer substance in the composition.

[00162] 50. The method of any of items 36 to 49, wherein the pH of the composition is between about 6.5 and about 8.0, preferably between about 6.9 and about 7.9, as between about 7.0 and about 7.8.

[00163] 51. The method of any of items 36 to 50, where water is the main component in the formulation and / or composition and / or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v / v).

[00164] 52. The method of any of items 36 to 51, where the osmolality of the composition is at most about 400 x 10-3 osmol / kg.

[00165] 53. The method of any of items 36 to 52, wherein the concentration of RNA in the composition is about 5 mg / l about 150 mg / l, preferably about 10 mg / l about 130 mg / l, more preferably about 30 mg / l about 120 mg / l. Petition 870260076800, dated 07 / 31 / 2026, p. 102 / 442 90 / 377

[00166] 54. The method of any of items 36 to 53, wherein (i) step (I) further comprises dilution of the formulation prepared in (d) with a dilution solution, or step (i') is present, wherein the dilution solution comprises a cryoprotectant; and / or (ii) the formulation obtained in step (I) and the composition comprise a cryoprotectant, preferably at a concentration of at least about 1% w / v, wherein the cryoprotectant preferably comprises one or more selected from the group consisting of carbohydrates and sugar alcohols, more preferably the cryoprotectant is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof, more preferably the cryoprotectant comprises sucrose and / or glycerol.

[00167] 55. The method of any of items 36 to 53, wherein the formulation obtained in step (I) and the composition are substantially free of a cryoprotectant.

[00168] 56. The method of any of items 36 to 55, wherein the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom that is capable of being protonated under physiological conditions.

[00169] 57. The method of any of items 36 to 56, in which the cationically ionizable lipid has the structure of Formula (I): R3^G3,L1. / N. ^L2 R1Ό1^G2R2(I) or a salt, tautomer, prodrug, or stereoisomer Petition 870260076800, dated 07 / 31 / 2026, p. 103 / 442 91 / 377 pharmaceutically acceptable of the same, in which: One of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, OC(=O)NRa- or -NRaC(=O)O- or a direct connection; G1 and G2 are, each independently, either unsubstituted C1-C12 alkylene or unsubstituted C2-C12 alkenylene; G3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Rae H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; ex is 0, 1 or 2.

[00170] 58. The method of any of items 36 to 56, where: (α) the cationically ionizable lipid is selected from structures I-1 to I-36 shown here; or (β) the cationically ionizable lipid is selected from structures A to F shown here; or (γ) the cationically ionizable lipid is the lipid with structure I-3 shown here.

[00171] 59. The method of any of items 36 to 58, wherein the ethanolic solution prepared in step (b) or (d') further comprises one or more additional lipids and the Petition 870260076800, dated 07 / 31 / 2026, page 104 / 442 92 / 377 LNPs further comprise one or more additional lipids, wherein the one or more additional lipids are preferably selected from the group consisting of polymer-conjugated lipids, neutral lipids, steroids and combinations thereof, more preferably one or more additional lipids comprise a polymer-conjugated lipid, a neutral lipid and a steroid.

[00172] 60. The method of item 59, wherein the lipid conjugated with polymer comprises a pegylated lipid, wherein the pegylated lipid preferably has the following structure: R13 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R12 and R13 are each independently a linear or branched alkyl chain, saturated or unsaturated, containing from 10 to 30 carbon atoms, in which the alkyl chain is optionally interrupted by one or more ester linkages; ew has an average value ranging from 30 to 60.

[00173] 61. The method of item 59, wherein the lipid-polymer conjugate comprises a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material, wherein the polysarcosine-lipid conjugate or conjugate of polysarcosine and a lipid-like material is preferably a selected member of the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-Petition 870260076800, dated 07 / 31 / 2026, page 105 / 442 93 / 377 dialkyloxypropyl, a polysarcosine phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof.

[00174] 62. The method of any of items 59 to 61, wherein the neutral lipid is a phospholipid, preferably selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diaraquidoylphosphatidylcholine (DAPC), dibeenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoioleoyl-phosphatidylcholine (POPC), 1,2-di-Ooctadecenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE),dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE) and diphytanoylphosphatidylethanolamine (DPyPE).

[00175] 63. The method of any of items 59 to 62, wherein the steroid comprises a sterol such as Petition 870260076800, dated 07 / 31 / 2026, page 106 / 442 94 / 377 cholesterol.

[00176] 64. The method of any of items 36 to 63, wherein the cationically ionizable lipid, the polymer-conjugated lipid, the neutral lipid and the steroid are present in the ethanolic solution in a molar ratio of 20% to 60% of the cationically ionizable lipid, 0.5% to 15% of the polymer-conjugated lipid, 5% to 25% of the neutral lipid and 25% to 55% of the steroid, preferably in a molar ratio of 45% to 55% of the cationically ionizable lipid, 1.0% to 5% of the polymer-conjugated lipid, 8% to 12% of the neutral lipid and 35% to 45% of the steroid.

[00177] 65. The method of any of items 36 to 64, wherein the final aqueous phase does not comprise a chelating agent.

[00178] 66. The method of any of items 36 to 65, wherein the LNPs comprise at least about 75%, preferably at least about 80% of the RNA comprised in the composition.

[00179] 67. The method of any of items 36 to 66, in which RNA is encapsulated within or associated with LNPs.

[00180] 68. The method of any of items 36 to 67, wherein the RNA comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m^) and 5-methyl-uridine (m5U).

[00181] 69. The method of any of items 36 to 68, wherein the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence. Petition 870260076800, dated 07 / 31 / 2026, p. 107 / 442 95 / 377

[00182] 70. The method in item 69, wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence is preferably an interrupted sequence of A nucleotides.

[00183] 71. The method of item 69 or 70, where cap 5' is a cap1 or cap2 structure.

[00184] 72. The method of any of items 36 to 71, in which the RNA encodes one or more polypeptides, wherein one or more polypeptides preferably comprise an epitope to induce an immune response against an antigen in a subject.

[00185] 73. The method in item 72, wherein the RNA comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant.

[00186] 74. The method of item 72 or 73, wherein the RNA comprises an ORF encoding a full-length SARS-CoV2 S protein variant with proline residue substitutions at positions 986 and 987 of SEQ ID NO: 1.

[00187] 75. The method of item 73 or 74, where the SARS-CoV2 S protein variant has at least 80% identity with SEQ ID NO: 7.

[00188] 76. The method of any of items 36 to 39 and 41 to 75, which does not include step (II).

[00189] 77. A method of storing a composition comprising preparing a composition in accordance with the method of any of items 36 to 75 and storing the Petition 870260076800, dated 07 / 31 / 2026, p. 108 / 442 96 / 377 composition at a temperature ranging from about -90°C to about -10°C, such as from about -90°C to about -40°C or from about -25°C to about -10°C.

[00190] 78. The method of item 77, where the storage of the composition is at least 1 week, as well as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months or at least 36 months.

[00191] 79. A method of storing a composition, comprising preparing a composition according to the method of any of items 36 to 78 and storing the composition at a temperature ranging from about 0°C to about 20°C, such as from about 1°C to about 15°C, from about 2°C to about 10°C, or from about 2°C to about 8°C, or at a temperature of about 5°C.

[00192] 80. The method of item 79, where the storage of the composition is at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months or at least 6 months.

[00193] 81. A composition that can be prepared by the method of any of items 36 to 80.

[00194] 82. The composition of item 81, which is in frozen form.

[00195] 83. The composition of item 82, in which the RNA integrity after thawing of the frozen composition is at least 50% compared to the RNA integrity of the composition before the composition was frozen. Petition 870260076800, dated 07 / 31 / 2026, page 109 / 442 97 / 377

[00196] 84. The composition of item 82 or 83, in which the size (Z-average) and / or size distribution and / or polydispersity index (PDI) of the LNPs after thawing the frozen composition is equal to the size (Z-average) and / or size distribution and / or PDI of the LNPs before the composition was frozen.

[00197] 85. The composition of item 81, which is in liquid form.

[00198] 86. The composition of item 85, in which the RNA integrity after storage of the composition for at least 1 week is at least 50% compared to the RNA integrity before storage.

[00199] 87. The composition of item 85 or 86, in which the size (Z-average) and / or size distribution and / or polydispersity index (PDI) of the LNPs after storage of the composition for at least one week is equal to the size (Z-average) and / or size distribution and / or PDI of the LNPs before storage.

[00200] 88. Method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a frozen composition prepared by the method of any of items 36 to 75, 77 and 78, and thawing the frozen composition thereby obtaining the ready-to-use pharmaceutical composition.

[00201] 89. Method for preparing a ready-to-use pharmaceutical composition, the method comprising the step of providing a liquid composition prepared by the method of any of items 36 to 39, 41 to 76, 79 and 80, thereby obtaining the ready-to-use pharmaceutical composition. Petition 870260076800, dated 07 / 31 / 2026, page 110 / 442 98 / 377

[00202] 90. Ready-to-use pharmaceutical composition, preparable by the method of item 88 or 89.

[00203] 91. A combination of any of items 1 to 35, 81 to 87 and 90 for use in therapy.

[00204] 92. A composition of any of items 1 to 35, 81 to 87 and 90 for use in inducing an immune response in an individual.

[00205] Other aspects of this disclosure are disclosed here. BRIEF DESCRIPTION OF THE FIGURES

[00206] Figure 1 is an in vivo assay scheme for the BNT162b1 material.

[00207] Figure 2 shows RNA integrity determined by capillary electrophoresis. RNA LNPs were prepared using the aqueous-ethanol mixture protocol with 20 mM Tris added to the organic phase. LNPs were generated in Tris:acetate pH 4, pH 5.5, or pH 6.8, and the resulting primary LNPs were split: one portion was dialyzed against PBS (A); the other portion was dialyzed against Tris:acetate pH 7.4 (B). For comparison, the organic phase did not receive Tris, the LNPs were generated in Na-acetate buffer pH 5.5, and the material was dialyzed against Tris:acetate pH 7.4. All samples were stored for 50 h at room temperature.

[00208] Figure 3 shows the morphology of the selected LNP RNA compositions. Vitrified samples were analyzed by cryo-electron microscopy. For sample d028, a 2.5x magnification was used.

[00209] Figure 4 shows the immunogenicity of Petition 870260076800, dated 07 / 31 / 2026, page 111 / 442 99 / 377 mice were injected with RNA LNP compositions. 1 pg of RNA LNP compositions D028 (LNP A), D029 (LNP B), and D030 (LNP C) were injected intramuscularly into mice; a reference composition (ATM) and saline solution were used as controls. S1 protein expression (left panels) and S1 IgG generation (right panels) were monitored for 28 days. All RNA LNP compositions have comparable bioactivity to each other and to the reference composition.

[00210] Figure 5 shows the stability of the RNA composition of LNP D028 (A) and the RNA compositions of LNP D029 (B) and D030 (C). Squares: room temperature, diamonds: 5°C, triangles: -20°C, circles -70°C. Solid lines: particle size, RNA integrity or RNA content; dashed lines: PDI, LMS (indicates stable folded RNA) or RNA encapsulation.

[00211] Figure 6 shows the compositions of colloidal RNA LNPs with a buffer strength of 10 mM or 50 mM. Squares: room temperature, diamonds: 5°C, triangles: -20°C. Solid lines represent particle size and dashed lines represent PDI.

[00212] Figure 7 shows the stability of RNA in relation to the strength of the Tris buffer. The results represent the percentage of RNA present in the samples after specific times and conditions. RNA denotes complete RNA; LMS denotes the highly stable folded form of RNA; and Frag denotes RNA fragments in the sample.

[00213] Description of the sequences

[00214] The following table provides a listing of certain sequences referenced herein. Petition 870260076800, dated 07 / 31 / 2026, page 112 / 442 100 / 377 Table 1: Description of sequences SEQ ID NO: Description SEQUENCE Antigenic sequences of protein S 1 Protein S (aa) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDL FLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT TLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDL PQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYL QPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTE SIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGG VSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRRARSVASQSIIAYTMSLGAE NSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQY GSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPS KPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE NQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLSSNFGA ISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAA TKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNE VAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCS CLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT Petition 870260076800, dated 07 / 31 / 2026, page 113 / 442 101 / 377 2 Protein S(CDS) auguuguguuucuugugcugcugcuugugucuucucagugugugaauuug acaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggagug uauuauccugauaaaguguuugaucuucugugcugcacagcacacacacagaggaccug uuucugccaauuuuuagcaaugugacaugguuucaugcaauucauguguga acaaauggaacaaaaaagauuugauaauccugugcugccuuuuaaaugauggagug uauuuugcuucaacagaaaagucaauauauauaugaggauuuuggaaca acacuggaucuaaaacagucucugcuuugugaauauauagcaacaaagauggug gugauuaaagugugugaauuuucaguuuguaaugauccuuuucugggaguguau uaucacaaaaauaauaaauauaucuuggauggaauuccuu gcaaauaauuguacauuuugaauauuguugaauuagaguguauuccu gcaaauaauuguacauuuugaauauuguugaauuugaguguauuccu gcaaauaauuguacauuuugaauauuguugaauuugaguguauuccu ggauauuuuuaaaauuuuauucuaaacacacaccaauuaauuuagugagaucug ccucagggauuuuucugcucuggaaccucugguggauucugccaauuggcauuaau auuacaagauuucagacacugcuggcucugcacagaucuuaucugacaccug gaucucucuucuggauggaccagccggagcugcagcuuauauugugggcuaucug cagccaagaacauuucugcugaauauaaugaaaauggaacaauuacagaugcu guggauugugcugcuugucugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuuaauuuuagagugcagccaacagaa ucuauugugaguuuccaauauauacaaaucuguguccauuuggaaguguuu aaugcaacaagauuugcaucuguuguauagcauggaauagaaaaagaauuucuaau uguguggcugauuaauucuguugcuguguauaauagugcuuuuuuccacauuuaa uguuauggagugucuccaacaaaauuuaugauuuuauguuuucaaauguguau cgugauucuuuuugugaucagaggugaugagugagacagauugccccccggacag acaggaaaauugcugauuacaauuacaaacugccugaugaugauuuuacaggaugu gugauugcuuggaauucuaauauauauauaaucuaaagugggagaauuaa uaucuguacacacuguuuagaaaaucaaaacugugaaaccuuuugaaagagauauuu ucaacagaauuuaucaggcuggaucaacaccuuguuaauggagugaaggauuu aauuguuauuuuccauuucagagcuaugggauacca uaucagccauaugagugguggugcugucuuuugaacugcugcaugcaccugca acaguguguggaccuaaaaaucuacaaauuuagugaaaaauaaugugugaau uuuaauuuuaaugggauuaacaggaacaggagugcugacaaucuaauaaaaaa uuucugccuuuucagcaguuuggcagagauauugcagauaccacagaugcagug agaauccucagacauuuagaaauucugggauauuacaccuuguuuggu gugucugugauuacaccuggaacaaauacaucuaaucauggcugugcuguau caggauguguauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaauguguuucagacaagagcagga ugucugauuggagcagaacaugugaauauauucuaugaaugugauauuccaauu ggagcaggcauuuugugcaucuuaucagacacagacaaauuccccaaggagagca aguacuguggcaucucagucuauuauugcauacacaugucucugggagcagaa aauucuguggcauauucuaauauauucuaugcuauuccaacaacaaaauuuuuaccaau ucugugacaacagaaauuuuaccugugucuaugacaaaaacauucuggauugu accauguacauuugggagaucuacagaauguucuaauucugcugcugcugaguau ggaucuuuuuguacacagcugaauagagcuuuuaacaggaauugcuggugaacaag gauaaaaauacaggaagguuugcucaggugaaacaa ccaauuaaagauuuggaggauuuaauuugcaaccaa ccaauuaaagauuuggaggauuuaauuugcaagccagauucugccugauccuu aaaccuucuaaaagaucuuuuuauugaagaucugcuguuuuaauaaagugacacug gcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugca agagaucugauuugugcucagaaauuuaauggacugacagugcuccucuc cugacagaugaaauguugcucaguacacaucugcuuuacuggcuggaacaauu acaagcggauggacauuuggagcuggagcucugcagauuccuuuugcaaug cagauggcuuacagauuuaauggaauuggagugacacaauguuguuauaugaa aaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggau ucucugucuucucacagcuucugcugggaaaacugcagaguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggca auuucuucugugcugaaugauauucugucuagacuggauaaaguggaagcugaa gugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugug Petition 870260076800, dated 07 / 31 / 2026, page 114 / 442 102 / 377 acacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcu acaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuugugga aaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuu uuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcaccagca auuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaau ggaacacauugguuugugacacagagaaauuuuuaugaaccucagauuauuaca acagauaauacauuugugucaggaaauugugauguggugauuggaauugugaau aauacaguguaugauccacugcagccagaacuggauucuuuuaaagaagaacug gauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucu ggaaucaaugcaucuguggugaauauucagaaagaaauugauagacugaaugaa guggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauau gaacaguacauuaaauggccuugguacauuuggcuuggauuuauugcaggauua auugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucu uguuuaaaaggauguuguucuuguggaagcuguuguaaauuugaugaagaugau ucugaaccuguguuaaaaggagugaaauugcauuacaca 3 Proteína SRBD (amnoácido )(V05) MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD YSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCG PK 4 Protein SRBD (CDS) (V05) auguuuguguuucuugugcugcugcucuugugucuucucaguguguggugaga uuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaaga ucuccaaaaauuaaaugauuuauguuuacaaauguguaugcugauucuuuuu gugaucagagugaugaagugagauugcccccggacagagaaaaauu cuguuuagaaaaucaaaucugaaaccuuuugaaagagauauucaacagaaau uaucaggcuggaucaacacuuguaauggaguggaaggauuuaauuguuauuuu ccauuacagcuuggauuucagccaaccaauggugugggaucaucaucaugcauau 5 Protein SRBD / (amnoacid Fibritin) (V05) MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD YSVLYNSASFSTFKYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCG PKGSPGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG 6 Proteína SRBD / Fibritina (CDS) (V05) auguuuguguuucuugugcugcugccucuugugucuucucaguguguggugaga uuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaaga uuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugau uauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagug ucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuu gugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauu gcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuugg aauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacaga cuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauu uaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuu ccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauau agagugguggugcugucuuuugaacugcugcaugcaccugcaacagugugugga ccuaaaggcucccccggcuccggcuccggaucugguuauauuccugaagcucca agagaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggagggugcuguuccagggccccggc Petition 870260076800, dated 07 / 31 / 2026, page 115 / 442 103 / 377 7 Proteína SPP (amnoácido ) (V08 / V09) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDL FLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDL PQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYL QPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTE SIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGG VSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAE NSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQY GSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPS KPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPL LTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGA ISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAA TKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNE VAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCS CLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT Petition 870260076800, dated 07 / 31 / 2026, page 116 / 442 104 / 377 8 SPP Protein (CDS) (V08) auguuuguguuucuugugcugcugccucugugucuucucagugugugaauuug acaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggagug uauuauccugauaaaguguuuugacuucugugcugcacagcacacacacacagaggaccug uuucugccaauuuuuagcaaugugacaugguuucaugcaauucauguguga acaaauggaacaaaaagauuugauaauccugugcugccuuuuaaaugauggaggug uauuuugcuucaacagaaaagucaauauauauaugaggauuuuggaaca acacuggaucuaaaacagucucugcuauguugaauauauagcaacaaauauggu gugauuaaagugugugaauuucaguuuguaaugauccuuuucugggaguguau uaucacaaaaauaauaaauauauaucuuggauggaauuccu gcaaauaauuguacauuuugaauauugugcaauuugaucugga ggaaaacagggcaauuuuuaaaaucugagagaaauuuguguuuaaauauaugau ggauauuuuuaaaauuuuauucuaaacacacaccaauuaauuuagugagagaucug ccucagggauuuuucugcucuggaaccucuggugaucugccaauuggcauuaau auuacaagauuucagacacugcuggcucugcacagaucuuaucugacaccug gaucucucuucuggauggauggacagccggagcugcagcuuauauuguggcuaucug cagccaagaacauuucugcugaauauaugaaaauggaacaauuacagaugcu guggauugugcugcucugucugcugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuuaauuuuagagugcagccaacagaa ucuauugugaguuuccaauauauacaaaucuguguccauuuggaaguguuu aaugcaacaagauuugcaucuguuguauagcauggaauagaaaaagaauuucuaau uguguggcugauuaauucuguugcuguguauaauagugcuuuuuuccacauuuaa uguuauggagugucuccaacaaaauuuaugauuuuauguuuucaaauguguau cgugauucuuuuugugaucagaggugaugagugagacagauugccccccggacag acaggaaaauugcugauuacaauuacaaacugccugaugaugauuuuacaggaugu gugauugcuuggaauucuaauauauauauaaucuaaagugggagaauuaa uaucuguacacacuguuuagaaaaucaaaacugugaaaccuuuugaaagagauauuu ucaacagaauuuaucaggcuggaucaacaccuuguuaauggagugaaggauuu aauuguuauuuuccauuucagagcuaugggauacca uaucagccauaugagugguggugcugucuuuugaacugcugcaugcaccugca acaguguguggaccuaaaaaucuacaaauuuagugaaaaauaaugugugaau uuuaauuuuaaugggauuaacaggaacaggagugcugacaaucuaauaaaaaa uuucugccuuuucagcaguuuggcagagauauugcagauaccacagaugcagug agaauccucagacauuuagaaauucugggauauuacaccuuguuuggu gugucugugauuacaccuggaacaaauacaucuaaucauggcugugcuguau caggauguguauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaauguguuucagacaagagcagga ugucugauuggagcagaacaugugaauauauucuaugaaugugauauuccaauu ggagcaggcauuuugugcaucuuaucagacacagacaaauuccccaaggagagca aguacuguggcaucucagucuauuauugcauacacaugucucugggagcagaa aauucuguggcauauucuaauauauucuaugcuauuccaacaacaaaauuuuuaccaau ucugugacaacagaaauuuuaccugugucuaugacaaaaacauucuggauugu accauguacauuugggagaucuacagaauguucuaauucugcugcugcugaguau ggaucuuuuuguacacagcugaauagagcuuuuaacaggaauugcuggugaacaag gauaaaaauacaggaagguuugcucaggugaaacaa ccaauuaaagauuuggaggauuuaauuugcaaccaa ccaauuaaagauuuggaggauuuaauuugcaagccagauucugccugauccuu aaaccuucuaaaagaucuuuuuauugaagaucugcuguuuuaauaaagugacacug gcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugca agagaucugauuugugcucagaaauuuaauggacugacagugcuccucuc cugacagaugaaauguugcucaguacacaucugcuuuacuggcuggaacaauu acaagcggauggacauuuggagcuggagcucugcagauuccuuuugcaaug cagauggcuuacagauuuaauggaauuggagugacacaauguuguuauaugaa aaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggau ucucugucuucucacagcuucugcugggaaaacugcagaguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggca auuucuucugugcugaaugauauucugucuagacuggauccuccugaagcugaa gugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugug Petition 870260076800, dated 07 / 31 / 2026, page 117 / 442 105 / 377 acacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcu acaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuugugga aaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuu uuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcaccagca auuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaau ggaacacauugguuugugacacagagaaauuuuuaugaaccucagauuauuaca acagauaauacauuugugucaggaaauugugauguggugauuggaauugugaau aauacaguguaugauccacugcagccagaacuggauucuuuuaaagaagaacug gauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucu ggaaucaaugcaucuguggugaauauucagaaagaaauugauagacugaaugaa guggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauau gaacaguacauuaaauggccuugguacauuuggcuuggauuuauugcaggauua auugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucu uguuuaaaaggauguuguucuuguggaagcuguuguaaauuugaugaagaugau ucugaaccuguguuaaaaggagugaaauugcauuacaca Petition 870260076800, dated 07 / 31 / 2026, page 118 / 442 106 / 377 9 Proteína SPP (CDS) (V09) auguucguguuccuggugcugcugccucugguguccagccagugugugaaccug accaccagaacacagcugccuccagccuacaccaacagcuuuaccagaggcgug uacuaccccgacaagguguucagauccagcgugcugcacucuacccaggaccug uuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggc accaauggcaccaagagauucgacaaccccgugcugcccuucaacgacggggug uacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcacc acacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacgug gucaucaaagugugcgaguuccaguucugcaacgaccccuuccugggcgucuac uaccacaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagc gccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaa ggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgac ggcuacuucaagaucuacagcaagcacaccccuaucaaccucgugcgggaucug ccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaac aucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggc gauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccug cagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgcc guggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaa uccaucgugcgguuccccaauacaccaaucugugcccccuucggcgagguguuc aaugccaccagauucgccucuguguacgccuggugaaccggaagcggaucagcaau ugcguggccgacuacuccgugcuguaacaacuccgccagcuucagcaccuucaag ugcuacgggugucccuaccaagcugaacgaccugugcuucacaaacguguac gccgacagcuucgugauccgggaugaagugcggcagauugccccuggagaugaagugcggcagauugccccuggagacag acaggcaagaucgccgacuacaacuacaagcugcccgacgacuucaccggugu gugauugccuggagaacagcaacaaccuggacuccaaagugcggcagacuucaccggugu gugauugccuggagaacaagcaacaaccuggacuccaaagucggggcacacuacaau uaccuguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaacucaau uccaccgagaucuaucagauccaugcggcagcacccccuuguaacgggugaaggcuac aacugcuacuucccacugcaguccuacggcuucagcccacaaauggcguggc uaucagcccuacagaguggugcugagcuucgaacugcugcaugccccugcc acagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaac uucaacuucaacggccugaccggcaccgggugcugacagagagcaacaagaag uuccugcauuccagcaguuuggccgggauaucgccgauaccacagagcgccguu agaaucccagacacuggaaauccuggacaucacccuugcagcuucggcga gugucugugaucaccccuggcaccaacaccagcaaucacaccagcaaucaggcagucuguac caggacgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggc ugucugaucggagccgagcacgugaacaauagcuacgagugcgacauccccauc ggcgcuggaaucugcgccagcuaccagacacagacaaacagcccucggagagcc agaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgag aacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccauc agcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugc accauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguac ggcagcuucugcacccagcugaauagagcccugacagggaucgccguggaacag gacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccu ccuaucaaggacuucggcggcuucaauuucagccagauucugcccgauccuagc aagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacug gccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgcc agggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucug cugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaauc acaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaug cagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgag aaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggac agccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccagaaugcccaggcacugaacacccuggucaagcagcuguccuccaacuucggcgcc aucagcucugugcugaacgauauccugagcagacggacccuccugaggccgag gugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgug Petition 870260076800, dated 07 / 31 / 2026, page 119 / 442 107 / 377 acccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgcc accaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggc aagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuu cugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagcc aucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaac ggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucacc accgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaac aauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacug gacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagc ggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgag guggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguac gagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacug auugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagc ugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgau ucugagcccgugcugaagggcgugaaacugcacuacaca Foldon 10 Foldon (aa) GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG 11 Foldon (CDS) ggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaa gauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggaggugcuguuccaggggccccggc 5′-UTR (hAg-Kozak) 12 |5′-utr |acuaguauucucuguccccacagacucagagagaacccgccacc 3′-UTR (FI Element) 13 3′-UTR AGUCCCCCCACCUCGGGUCCAGGUAUGCCCACCUCCACCUGCCCCACUC ACCACCUCUGCUACCACCUCCCAAGCACGCAGCAAUGCAGCAAAAA GCCACACC A30L70 14 A30L70 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DETAILED DESCRIPTION OF INVENTION

[00215] Although the present disclosure is described in more detail below, it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used in this descriptive report is only for the purpose of describing particular embodiments and is not intended to limit the scope of this disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used in this report Petition 870260076800, dated 07 / 31 / 2026, page 120 / 442 108 / 377 descriptive terms have the same meanings commonly understood by a technician versed in the subject.

[00216] The elements of this disclosure will be described in more detail below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any way, and in any number, to create additional embodiments. The various examples and preferred embodiments described should not be interpreted as limiting this disclosure only to the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all the elements described in this application should be considered disclosed by the description of this application, unless the context indicates otherwise.For example, if in one preferred embodiment the composition (or formulation) comprises a cryoprotectant and in another preferred embodiment the cationically ionizable lipid has the structure I-3, then in yet another preferred embodiment the composition (or formulation) comprises a cryoprotectant and the cationically ionizable lipid with the structure I-3.

[00217] Preferably, the terms used herein are defined as described in A Multilingual Glossary of Biotechnological Terms: (IUPAC Recommendations), HGW Leuenberger, B. Nagel and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). Petition 870260076800, dated 07 / 31 / 2026, page 121 / 442 109 / 377

[00218] The practice of this disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques which are explained in the literature in the field (cf., for example, Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, Organische Chemie, VCH, 1990; Beyer / Walter, Lehrbuch der Organischen Chemie, S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, Organische Chemie, VCH, 1995; March, Advanced Organic Chemistry, John Wiley & Sons, 1985; Rompp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[00219] Throughout this descriptive report and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" shall be understood as implying the inclusion of a stated member, whole or step or group of members, whole numbers or steps, but not the exclusion of any other member, whole number or step or group of members, whole numbers or steps. The term "essentially consisting of" means the exclusion of other members, whole numbers or steps of any essential meaning. The term "comprising" encompasses the term "essentially consisting of" which, in turn, encompasses the term "consisting of". Thus, in each occurrence in the present application, the term "comprising" may be replaced by the term "essentially consisting of" or Petition 870260076800, dated 07 / 31 / 2026, page 122 / 442 110 / 377 consisting of. Similarly, in each occurrence in this application, the term essentially consisting of may be replaced by the term consisting of.

[00220] The terms "a," "an," and similar references used in the context of the description of this disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The citation of value ranges in this descriptive report is intended only as a shorthand method for referring individually to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated into the descriptive report as if it were individually cited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by the context.The use of any and all examples or exemplary language (e.g., such as) provided in this descriptive report is intended only to better illustrate this disclosure and does not represent a limitation on the scope of this disclosure otherwise claimed. No language in the descriptive report should be interpreted as indicating any unclaimed element essential to the practice of this disclosure.

[00221] Where used herein, and / or shall be considered as specific disclosure of each of the two specified features or components with or without the other. For example, X and / or Y shall be considered as specific disclosure. Petition 870260076800, dated 07 / 31 / 2026, pp. 123 / 442 111 / 377 of each of (i) X, (ii) Y and (iii) X and Y, as if each were presented individually in this descriptive report.

[00222] In the context of this disclosure, the term "approximately" denotes a range of precision that one skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the stated numerical value of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and, for example, ±0.01%. As will be appreciated by one skilled in the art, the specific deviation from a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than a man-made or engineered technical effect.

[00223] The citation of value ranges in this descriptive report is intended only as a shorthand method for individually referring to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated into the descriptive report as if it were individually cited herein.

[00224] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention does not have the right to precede such disclosure by virtue of the Petition 870260076800, dated 07 / 31 / 2026, page 124 / 442 112 / 377 prior invention. Definitions

[00225] Definitions that apply to all aspects of this disclosure will be provided below. The following terms have the following meanings, unless otherwise indicated. Any undefined terms have their meanings recognized in the prior art.

[00226] Terms such as reduce or inhibit, as used in this descriptive report, mean the ability to cause an overall decrease, for example, of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more, in level. The term inhibit or similar phrases include complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[00227] Terms such as increase or improve in an embodiment refer to an increase or improvement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[00228] Physiological pH as used here refers to a pH of approximately 7.5.

[00229] Physiological conditions as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human being. Preferably, physiological conditions mean a physiological pH and / or a temperature of approximately 37°C.

[00230] As used in this disclosure, Petition 870260076800, dated 07 / 31 / 2026, p. 125 / 442 113 / 377 % (w / v) (or % w / v) refers to weight per volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (ml).

[00231] As used in this disclosure, % by weight or % (w / w) (or % w / w) refers to the percentage by weight, which is a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the composition in grams (g).

[00232] Regarding the presence of divalent inorganic ions, in particular divalent inorganic cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in a concretization, sufficiently low to prevent RNA degradation. In a concretization, the concentration or effective concentration of divalent inorganic ions is below the catalytic level for the hydrolysis of phosphodiester bonds between RNA nucleotides. In a concretization, the concentration of free divalent inorganic ions is 20 μM or less. In a concretization, there are no or practically no free divalent inorganic ions.

[00233] Osmolarity refers to the concentration of a given solute expressed as the number of osmoles of solute per kilogram of solvent.

[00234] The term freezing refers to the solidification of a liquid, usually with the removal of heat.

[00235] The term aqueous phase as used in this descriptive report in relation to a composition / formulation Petition 870260076800, dated 07 / 31 / 2026, page 126 / 442 114 / 377 comprising particles, in particular LNPs, means the mobile or liquid phase, that is, the continuous aqueous phase including all components dissolved therein, but (formally) excluding the particles. Thus, if particles, such as LNPs, are dispersed in an aqueous phase and the aqueous phase is substantially free of compound X, the aqueous phase is free of X in whatever way is practically and realistically feasible, for example, the concentration of compound X in the aqueous composition is less than 1% by weight. However, it is possible that, at the same time, the particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

[00236] The term protonated form as used herein in relation to a base (e.g., an organic primary amine such as Tris) means the conjugate acid of the base, wherein the conjugate acid contains a proton that is removable by deprotonation resulting in the base. For example, the protonated form of Tris has the formula [H3N(CH2CH2OH)3]+. A buffer substance as used herein refers to a mixture of the base and its protonated form (e.g., a mixture of Tris and [H3N(CH2CH2OH)3]+). Consequently, the amount of a buffer substance contained in a composition is the sum of the amounts of both the base and the conjugate acid in the composition.

[00237] The term recombinant in the context of this disclosure means made through genetic engineering. In one embodiment, a recombinant object in the context of this disclosure is not occurring naturally. Petition 870260076800, dated 07 / 31 / 2026, page 127 / 442 115 / 377

[00238] The term naturally occurring, as used herein, refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and that has not been intentionally modified by humans in a laboratory is naturally occurring. The term found in nature means present in nature and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but which may be discovered and / or isolated in the future from a natural source.

[00239] As used in this descriptive report, the terms ambient temperature and ambient temperature are used interchangeably in this descriptive report and refer to temperatures of at least about 15°C, preferably from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[00240] The term alkyl refers to a monoradical of a linear or branched saturated hydrocarbon. Preferably, the alkyl group comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, abbreviated as C1-12 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, abbreviated as C1-10 alkyl), more preferably, 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl (also called 2-propyl or 1-methylethyl), Petition 870260076800, dated 07 / 31 / 2026, pp. 128 / 442 116 / 377 butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, secpentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decile, n-undecyl, n-dodecyl and the like. A substituted alkyl group means that one or more (such as the maximum number of hydrogen atoms bonded to an alkyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 and 2) hydrogen atoms of the alkylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent, as specified in this descriptive report. Examples of a substituted alkyl group include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[00241] The term alkylene refers to a diradical of a linear or branched saturated hydrocarbon. Preferably, the alkylene comprises from 1 to 12 (as, 1 to 10) carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Examples of 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 isomers of butylene (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), Petition 870260076800, dated 07 / 31 / 2026, pp. 129 / 442 117 / 377 1,4-butylene, 1,1-iso-butylene, 1,2-iso-butylene and 1,3-isobutylene), 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. example, 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. Linear alkylene moieties with at least 3 carbon atoms and a free valence at each end may also be designated as a methylene multiple (e.g., 1,4-butylene may also be called tetramethylene).Generally, instead of using the ylene ending for alkylene moieties as specified above, one can also use the diyl ending (for example, 1,2-butylene can also be called butan-1,2-diyl). A substituted alkylene means that one or more (such as 1 to the maximum number of hydrogen atoms bonded to an alkylene group, for example, 1, 2, 3, 4, 5, 6, ...) are substituted. 7, 8, 9, or even 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is Petition 870260076800, dated 07 / 31 / 2026, pp. 130 / 442 118 / 377 a first-level substitute, as specified in this descriptive report.

[00242] The term alkenyl refers to a monoradical of a linear or branched unsaturated hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in the alkenyl group can be equal to the integer that 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 unequal, rounding the result of the division to the next integer. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (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 12 (as well as 2 to 10) carbon atoms, that is, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (as well as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, as well as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 12, abbreviated as C2-12 alkenyl, (for example, 2 to 10) carbon atoms and 1, 2, 3, 4, 5 or 6 (for example, 1, 2, 4 or 5) carbon-carbon double bonds, more preferably comprising 2 to 8 carbon atoms and 1, 2, 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 10 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the... Petition 870260076800, dated 07 / 31 / 2026, pp. 131 / 442 119 / 377 cis (Z) or trans (E) configuration. Examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl. 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9dodecenyl, 10-dodecenyl, 11-dodecenyl and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.A substituted alkenyl group means that one or more (such as 1 to the maximum number of hydrogen atoms bonded to an alkenyl group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 and 2) hydrogen atoms of the alkenyl group are replaced by a non-hydrogen substituent (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent as specified herein.

[00243] The term alkenylene refers to a Petition 870260076800, dated 07 / 31 / 2026, pp. 132 / 442 120 / 377 diradical of a linear or branched unsaturated hydrocarbon having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in the alkenylene group can be equal to the integer that is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is unequal, rounding the result of the division to the next integer. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (as well as 2 to 10) carbon atoms, that is, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises 2 to 12 (such as 2 to 10 carbon atoms) and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably comprising 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the cis (Z) or trans (E) configuration. Examples of alkenylene groups include ethen-1,2-di-yl, vinylidene (also called ethenylidene), 1-propen-1,2-di-yl, 1-propen-1,3-di-yl, and 1-propen-1,3-di-yl. (Petition 870260076800, dated 07 / 31 / 2026, p. 133 / 442) 121 / 377 di-yl, 1-propen-2,3-di-yl, allylidene, 1-buten-1,2-diyl, 1buten-1,3-diyl, 1-buten-1,4-diyl, 1-buten-2,3-diyl, 1buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1,2-diyl, 2-buten-1,3-diyl, 2-buten-1,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl and the like. If an alkenylene group is bonded to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A substituted alkenylene means that one or more (such as 1 to the maximum number of hydrogen atoms bonded to an alkenylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent as specified herein.

[00244] The term cycloalkylene represents non-aromatic cyclic versions of alkylene and is a geminal, vicinal, or isolated diradical. In certain embodiments, the cycloalkylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and / or (ii) has 3 to 14 members (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment, cycloalkylene is a mono-, bi-, or tricyclic cycloalkylene with 3 to 14 members (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14 members, such as 3- to 12-membered or 3- to 10-membered). Generally, instead of using the ylene ending for cycloalkylene fractions as specified Petition 870260076800, dated 07 / 31 / 2026, pp. 134 / 442 122 / 377 above, the diyl ending can also be used (for example, 1,2-cyclopropylene can also be called cyclopropan-1,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene and adamantanylene (for example, tricyclo[3.3.1.13,7]decan-2,2-di-yl). A substituted cycloalkylene means that one or more (such as 1 to the maximum number of hydrogen atoms bonded to a cycloalkylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent as specified here.

[00245] The term cycloalkenylene represents non-aromatic cyclic versions of alkenylene and is a geminal, vicinal, or isolated diradical. Generally, the maximum number of carbon-carbon double bonds in the cycloalkenylene group can be equal to the integer number calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2 and, if the number of carbon atoms in the cycloalkenylene group is unequal, rounding the result of the division to the next integer. For example, for a cycloalkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (as 1 to 4), i.e., 1, 2, 3, Petition 870260076800, dated 07 / 31 / 2026, pp. 135 / 442 123 / 377 4, 5, or 6 carbon-carbon double bonds. In certain embodiments, cycloalkenylene (i) is monocyclic or polycyclic (as bi- or tricyclic) and / or (ii) has 3 to 14 members (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13- or 3- to 12-membered or 3- to 10-membered). In one embodiment, cycloalkenylene is a mono-, bi-, or tricyclic compound with 3 to 14 members (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13- or 14-membered groups, such as 3 to 12-membered or 3 to 10-membered groups) cycloalkenylene. Examples of cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. A substituted cycloalkenylene means that one or more (such as 1 to the maximum number of hydrogen atoms bonded to a cycloalkenylene group, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 and 5, 1 and 4, or 1 and 3, or 1 and 2) hydrogen atoms of the cycloalkenylene group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a first-level substituent as specified here.

[00246] The term aromatic used in the context of hydrocarbons means that every molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of this disclosure. Similarly, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated, the structure Petition 870260076800, dated 07 / 31 / 2026, pp. 136 / 442 The resulting hydrogenated bi- or polycyclic compound (such as 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of this disclosure (even if one ring, as in 1,2-dihydronaphthyl, is still aromatic).

[00247] Typical first-level substituents are preferably selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2z(CH3)z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl, or isopropyl. Particularly preferred first-level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3.

[00248] The expression after thawing the frozen composition, as used here in the context of a frozen composition, means that the frozen composition must be thawed before characteristics (such as RNA integrity and / or size (Z-mean) and / or size distribution and / or the PDI of the LNPs contained in the composition) can be measured.

[00249] A monovalent compound refers to a compound with only one functional group of interest. For example, a monovalent anion refers to a compound with only one negatively charged group, preferably under physiological conditions.

[00250] A divalent or dibasic compound refers to a compound with two functional groups of interest. For example, a dibasic organic acid has two acidic groups. Petition 870260076800, dated 07 / 31 / 2026, pp. 137 / 442 125 / 377

[00251] A polyvalent or polybasic compound refers to a compound with three or more functional groups of interest. For example, a polybasic organic acid has three or more acidic groups.

[00252] The term RNA integrity means the percentage of complete (i.e., unfragmented) RNA to the total amount of RNA (i.e., unfragmented RNA plus fragmented RNA) contained in a sample. RNA integrity can be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of ​​the main RNA peak (i.e., the peak area of ​​the complete (i.e., unfragmented) RNA), determining the peak area of ​​the total RNA, and dividing the peak area of ​​the main RNA peak by the peak area of ​​the total RNA.

[00253] The term cryoprotectant refers to a substance that is added to a preparation (e.g., formulation or composition) to protect the active ingredients of the preparation during the freezing steps.

[00254] According to the present disclosure, the term peptide includes oligo- and polypeptides and refers to substances comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about fifty, about one hundred or one hundred or about fifty consecutive amino acids linked together by peptide bonds. The term protein refers to large peptides, in particular peptides with at least about 151 amino acids, but the terms peptide and protein are commonly used herein as synonyms. Petition 870260076800, dated 07 / 31 / 2026, pp. 138 / 442 126 / 377

[00255] A therapeutic protein has a positive or beneficial effect on a subject's disease condition or state when provided to the subject in a therapeutically effective amount. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to improve, alleviate, relieve, reverse, delay the onset, or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such a disease or pathological condition. The term therapeutic protein includes whole proteins or peptides, and may also refer to their therapeutically active fragments. It may also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

[00256] According to the present disclosure, it is preferable that a nucleic acid such as RNA (e.g., mRNA) encoding a peptide or protein, once absorbed or introduced, i.e., transfected or transduced, into a cell which may be present in vitro or in an individual, results in the expression of said peptide or protein. The cell may express the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), may secrete the encoded peptide or protein, or may express it on its surface.

[00257] According to the present disclosure, terms Petition 870260076800, dated 07 / 31 / 2026, pp. 139 / 442 127 / 377 as nucleic acid expression and nucleic acid coding or similar terms are used interchangeably in this descriptive report and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce said peptide or polypeptide.

[00258] The term portion refers to a fraction. With respect to a particular structure, such as an amino acid sequence or protein, the term portion thereof may designate a continuous or discontinuous fraction of said structure.

[00259] The terms part and fragment are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of that structure. When used in the context of a composition, the term part means a portion of the composition. For example, a part of a composition can be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90% or 99%) of that composition.

[00260] Fragment, with reference to an amino acid sequence (peptide or protein), refers to a part of an amino acid sequence, that is, a sequence that represents the amino acid sequence shortened at the N-terminal and / or C-terminal. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating an open reading frame. Petition 870260076800, dated 07 / 31 / 2026, pp. 140 / 442 A truncated 128 / 377 fragment that does not have the 3' end of the open reading frame. A shortened N-terminal fragment (C-terminal fragment) is obtained, for example, by translating a truncated open reading frame that does not have the 5' end of the open reading frame, provided that the truncated open reading frame includes a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of an amino acid sequence.

[00261] According to this disclosure, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it was derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective nucleic acid binding. For example, a pharmacologically active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the fragment was derived. A part or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular, at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 Petition 870260076800, dated 07 / 31 / 2026, pp. 141 / 442 129 / 377 or at least 50 consecutive amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular, up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the peptide or protein.

[00262] By variant here we mean an amino acid sequence that differs from a parental amino acid sequence by virtue of at least one amino acid modification. The parental amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or it may be a modified version of a wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parental amino acid sequence, for example, from 1 to about 20 amino acid modifications and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

[00263] Wild type or WT or native type here means an amino acid sequence found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

[00264] For the purposes of this disclosure, variants of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or variants of Petition 870260076800, dated 07 / 31 / 2026, pages 142 / 442 130 / 377 amino acid substitution. The term variant includes all mutants, splicing variants, post-translational modified variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those that occur naturally. The term variant includes, in particular, fragments of an amino acid sequence.

[00265] Amino acid insertion variants comprise insertions of one or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with an insertion, one or more amino acid residues are inserted at a particular location in an amino acid sequence, although random insertion with appropriate sorting of the resulting product is also possible. Amino acid addition variants comprise amino and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be at any position in the protein.Amino acid deletion variants that involve deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to modifications in amino acid sequence positions that are not conserved between homologous proteins or peptides and / or to the... Petition 870260076800, dated 07 / 31 / 2026, pp. 143 / 442 131 / 377 Substitution of amino acids for others with similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, that is, substitutions of amino acids with or without similar charge. A conservative amino acid change involves the substitution of one from a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions in the following groups: - glycine, alanine; - valine, isoleucine, leucine; - aspartic acid, glutamic acid; - asparagine, glutamine; - serine, threonine; - lysine, arginine; and - phenylalanine, tyrosine.

[00266] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said amino acid sequence will be at least about Petition 870260076800, dated 07 / 31 / 2026, pages 144 / 442 132 / 377 of 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given by at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids.In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be done with tools known in the art, preferably using best sequence alignment, for example, using Align, using default settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[00267] Sequence similarity indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. Sequence identity between two sequences of Petition 870260076800, dated 07 / 31 / 2026, pp. 145 / 442 133 / 377 amino acids indicates the percentage of amino acids that are identical between the sequences. Sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[00268] The terms % identical and % identity or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids that are identical in an optimal alignment between the sequences to be compared. The said percentage is purely statistical and the differences between the two sequences may be, but not necessarily, randomly distributed along the entire length of the sequences to be compared. Comparisons of two sequences are generally performed by comparing the sequences, after optimal alignment, with respect to a comparison segment or window, in order to identify local regions of matching sequences. Optimal alignment for a comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Mathematics. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol.48, 443, with the help of the Pearson and Lipman similarity search algorithm, 1988, Proc. Natl Acad. Science USA 88, 2444, or with the help of computer programs using the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the National Genetics Information Center website. Petition 870260076800, dated 07 / 31 / 2026, pp. 146 / 442 134 / 377 US National Centre for Biotechnology Information (NCBI) (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgiPAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) Expect (i) Threshold set to 10; (ii) Word size set to 28; (iii) Maximum matches in a query range set to 0; (iv) Match / mismatch scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low-complexity regions in use. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Maximum matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extent: 1; and (vii) adjustment of the conditional compositional scoring matrix.

[00269] Percent identity is obtained by determining the number of identical positions in which the sequences to be compared correspond, dividing that number by the number of positions compared (for example, the number of positions in the reference sequence) and multiplying that result by 100.

[00270] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the sequence of Petition 870260076800, dated 07 / 31 / 2026, pp. 147 / 442 135 / 377 reference. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides in some embodiments, continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[00271] Homologous amino acid sequences exhibit, according to the disclosure, at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98%, or at least 99% identity of amino acid residues.

[00272] The amino acid sequence variants described here can be easily prepared by the specialist, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for the preparation of peptides or proteins with substitutions, additions, insertions or deletions is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described here can be easily prepared with the aid of known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[00273] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a functional fragment or functional variant. The term functional fragment or Petition 870260076800, dated 07 / 31 / 2026, pp. 148 / 442 136 / 377 A functional variant of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term functional fragment or functional variant, as used herein, refers in particular to a variant molecule or sequence comprising an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, for example, inducing an immune response.In one embodiment, modifications to the amino acid sequence of the molecule or parental sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present; for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the molecule or parental sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be increased compared to the molecule or parental sequence.

[00274] A sequence of amino acids (peptide, Petition 870260076800, dated 07 / 31 / 2026, pp. 149 / 442 137 / 377 protein or polypeptide) derived from a designated amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence that is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a specific amino acid sequence may be variants of that specific sequence or a fragment thereof. For example, it will be understood by a person skilled in the art that antigens suitable for use herein may be altered so that they vary in sequence from the natural or native sequences from which they were derived, while maintaining the desirable activity of the native sequences.

[00275] Isolated means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as, for example, a host cell. In a preferred embodiment, the RNA (such as mRNA) used in the present disclosure is in substantially purified form. In one embodiment, a solution (preferably an aqueous solution) of RNA (such as mRNA) in substantially purified form contains a first buffer system. Petition 870260076800, dated 07 / 31 / 2026, pp. 150 / 442 138 / 377

[00276] The term genetic modification or simply modification includes the transfection of cells with nucleic acid. The term transfection refers to the introduction of nucleic acids, in particular RNA, into a cell. For the purposes of this disclosure, the term transfection also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, wherein the cell may be present in a subject, for example, a patient. Thus, according to this disclosure, a cell for transfection of a nucleic acid described herein may be present in vitro or in vivo, i.e., the cell may be part of an organ, tissue and / or organism of a patient. According to the disclosure, transfection may be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is expressed only transiently. RNA may be transfected into cells to transiently express its encoded protein.Since the nucleic acid introduced in the transfection process is generally not integrated into the nuclear genome, the foreign nucleic acid will be diluted by mitosis or degraded. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, a stable transfection must occur. Such a stable transfection can be achieved using virus-based systems or transposon-based systems for transfection. Generally, the antigen encoding the nucleic acid is transiently transfected into the cells. Petition 870260076800, dated 07 / 31 / 2026, pp. 151 / 442 139 / 377 RNA can be transfected into cells to transiently express its encoded protein.

[00277] According to this disclosure, an analog of a peptide or protein is a modified form of said peptide or protein from which it is derived and has at least one functional property of said peptide or protein. For example, a pharmacologically active analog of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the analog was derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and / or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and / or proteins or peptides.In one embodiment, protein or peptide analogs include modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective / blocking groups, proteolytic cleavage, or binding to an antibody or other cellular ligand. The term analog also extends to all functional chemical equivalents of said proteins and peptides.

[00278] Activation or stimulation, as used in this descriptive report, refers to the state of an immune effector cell, such as a T cell, that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term immune effector cells Petition 870260076800, dated 07 / 31 / 2026, page 152 / 442 140 / 377 activated refers, among other things, to immune effector cells that are undergoing cell division.

[00279] The term priming refers to a process in which an immune effector cell, such as a T cell, has its first contact with its specific antigen and causes differentiation into effector cells, such as effector T cells.

[00280] The term clonal expansion or expansion refers to a process in which a specific entity is multiplied. In the context of this disclosure, the term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell that recognizes that antigen is amplified. Preferably, clonal expansion leads to the differentiation of immune effector cells.

[00281] An antigen, according to this disclosure, encompasses any substance that will induce an immune response and / or any substance against which an immune response or an immune mechanism, such as a cellular response, is directed. This also includes situations where the antigen is processed into antigenic peptides and an immune response or an immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, an antigen refers to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T lymphocytes (T cells). According to this disclosure, the term antigen includes any molecule comprising at least one Petition 870260076800, dated 07 / 31 / 2026, page 153 / 442 141 / 377 epitope, such as a T-cell epitope. Preferably, an antigen in the context of this disclosure is a molecule that, optionally after processing, induces an immune reaction that is preferably specific for the antigen (including cells expressing the antigen). In one embodiment, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[00282] According to the present disclosure, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response can be either a humoral or cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably by an antigen-presenting cell, in the context of MHC molecules, resulting in an immune response against the antigen. An antigen is preferably a product that corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or an antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, for example, a viral protein or a part thereof.

[00283] The term disease-associated antigen is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains epitopes that will stimulate the Petition 870260076800, dated 07 / 31 / 2026, pp. 154 / 442 142 / 377 a host's immune system to produce a specific cellular antigen immune response and / or a humoral antibody response against disease. Disease-associated antigens include pathogen-associated antigens, that is, antigens that are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens) or cancer-associated antigens, typically tumors, such as tumor antigens.

[00284] In a preferred embodiment, the antigen is a tumor antigen, that is, a part of a tumor cell, in particular those that occur mainly intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, that is, an antigen derived from a pathogen, for example, from a virus, bacterium, unicellular organism or parasite, for example, a viral antigen, such as viral ribonucleoprotein or coat protein. In particular, the antigen must be presented by MHC molecules that result in modulation, namely activation of immune system cells, preferably CD4+ and CD8+ lymphocytes, notably through modulation of the activity of a T cell receptor.

[00285] The term tumor antigen refers to a constituent of cancer cells that may be derived from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to antigens that are produced intracellularly or as surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, αΐ-fetoprotein, and isoferritin. Petition 870260076800, dated 07 / 31 / 2026, page 155 / 442 143 / 377 and fetal sulfoglycoprotein, α2-H-ferroprotein and γ-fetoprotein, as well as various viral tumor antigens. According to this disclosure, a tumor antigen preferably comprises any antigen that is characteristic of tumors or cancers, as well as of tumor or cancer cells with respect to type and / or level of expression.

[00286] The term viral antigen refers to any viral component with antigenic properties, that is, capable of provoking an immune response in an individual. The viral antigen can be a viral ribonucleoprotein or an envelope protein.

[00287] The term bacterial antigen refers to any bacterial component with antigenic properties, that is, capable of provoking an immune response in an individual. The bacterial antigen can be derived from the cell wall or the cytoplasmic membrane of the bacterium.

[00288] The term epitope refers to an antigenic determinant in a molecule, such as an antigen, that is, a part or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies, T cells or B cells, in particular, when presented in the context of MHC molecules. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein, and is preferably between about 5 and about 100, preferably between about 5 and about 50, more preferably between about 8 and about 0, most preferably between about 10 and about 25 amino acids in length, for example, the Petition 870260076800, dated 07 / 31 / 2026, pp. 156 / 442 144 / 377 epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of this disclosure be a T-cell epitope.

[00289] Terms such as epitope, antigen fragment, immunogenic peptide, and antigen peptide are used interchangeably in this descriptive report and preferably refer to an incomplete representation of an antigen that is preferentially capable of eliciting an immune response against the antigen or a cell that expresses or understands and preferentially presents the antigen. Preferably, the terms refer to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. The term epitope refers to a part or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells, or antibodies.An antigen epitope may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, or between about 5 and about 50, more preferably between about 8 and about 30, or even more preferably between about 8 and about 25 amino acids in length, for example, the epitope may preferably be 9, 10, 11, 12, 13, 14,. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in Petition 870260076800, dated 07 / 31 / 2026, pp. 157 / 442 145 / 377 length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term epitope includes T-cell epitopes.

[00290] The term T-cell epitope refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term major histocompatibility complex and the abbreviation MHC include MHC class I and MHC class II molecules and refer to a complex of genes that is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune reactions, where MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and display self-antigens (peptide fragments of the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell.In the case of MHC / class I peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long, although longer or shorter peptides can be effective. In the case of MHC / class II peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long, and in particular are about 13 to about 18 amino acids long, while longer and shorter peptides can be effective.

[00291] The peptide and protein antigen can be of Petition 870260076800, dated 07 / 31 / 2026, pp. 158 / 442 146 / 377 to 100 amino acids, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide may have more than 50 amino acids. In some embodiments, the peptide may have more than 100 amino acids.

[00292] The peptide or protein antigen can be any peptide or protein that can induce or enhance the immune system's ability to develop antibodies and T-cell responses to the peptide or protein.

[00293] In one embodiment, the vaccine antigen, that is, an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. Preferably, the vaccine antigen, if recognized by an immune effector cell, is capable of inducing, in the presence of appropriate co-stimulatory signals, stimulation, initiation and / or expansion of the immune effector cell bearing an antigen receptor that recognizes the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or is present on the surface of a cell, preferably an antigen-presenting cell. In one embodiment, an antigen is presented by a diseased cell (such as a tumor cell or an infected cell). In one embodiment, an antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of the MHC.In one embodiment, the binding of a TCR, when expressed by T cells and / or present on T cells, to an antigen. Petition 870260076800, dated 07 / 31 / 2026, pp. 159 / 442 147 / 377 presented by cells, such as antigen-presenting cells, results in stimulation, initiation and / or expansion of said T cells. In one embodiment, the binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferentially release cytotoxic factors, for example, perforins and granzymes.

[00294] In one embodiment, an antigen receptor is an antibody or B cell receptor that binds to an epitope on an antigen. In another embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[00295] The term expressed on the cell surface or associated with the cell surface means that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from outside said cell, for example, by antibodies located outside the cell. In this context, a part preferably has at least 4, preferably at least 8, preferably at least 12, most preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that may be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated Petition 870260076800, dated 07 / 31 / 2026, page 160 / 442 148 / 377 on the surface of a cell can be a transmembrane protein with an extracellular portion or it can be a protein associated with the surface of a cell by interaction with another protein that is a transmembrane protein.

[00296] Cell surface or surface of a cell is used according to its normal meaning in the art and therefore includes the outer part of the cell that is accessible to binding by proteins and other molecules. An antigen is expressed on the cell surface if it is located on the surface of said cells and is accessible to binding by, for example, antigen-specific antibodies added to the cells.

[00297] The term extracellular portion or exodomain in the context of this disclosure refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside that cell, for example, by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

[00298] The terms T cell and T lymphocyte are used interchangeably here and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells. The term antigen-specific T cell or similar terms refers to a T cell that recognizes the antigen to which the T cell is directed, particularly when presented on the surface of antigen-presenting cells or diseased cells, such as cancer cells in the context of molecules Petition 870260076800, dated 07 / 31 / 2026, page 161 / 442 149 / 377 of MHC and preferentially performs effector functions of T cells. T cells are considered antigen-specific if the cells kill target cells that express an antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, lymphokine synthesis (such as interferon-γ) can be measured. In certain embodiments of the present disclosure, RNA (in particular mRNA) encodes at least one epitope.

[00299] The term target will mean an agent such as a cell or tissue that is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, that is, a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses an antigen and preferentially presents said antigen with MHC class I.

[00300] Antigen processing refers to the degradation of an antigen into processing products that are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T cells.

[00301] By antigen-responsive CTL is meant a CD8+ T cell that responds to an antigen or to a peptide derived from that antigen, which is presented with MHC of Petition 870260076800, dated 07 / 31 / 2026, page 162 / 442 150 / 377 class I on the surface of antigen-presenting cells.

[00302] According to the disclosure, CTL responsiveness may include sustained calcium flux, cell division, cytokine production such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and target-specific cytolytic killing of tumor antigen expression cells. CTL responsiveness can also be determined using an artificial reporter that accurately indicates CTL responsiveness.

[00303] The terms immune response and immune reaction are used interchangeably in this descriptive report in their conventional meaning and refer to an integrated bodily response to an antigen and preferably refer to a cellular immune response, a humoral immune response, or both. According to the disclosure, the term immune response to or immune response against an agent, such as an antigen, cell, or tissue, refers to an immune response, such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of the development of antibodies against one or more antigens and expansion of antigen-specific T lymphocytes, preferably CD4+ and CD8+ T lymphocytes, more preferably CD8+ T lymphocytes, which can be detected in various in vitro proliferation or cytokine production assays.

[00304] The terms induce an immune response and induce an immune response and similar terms in the context Petition 870260076800, dated 07 / 31 / 2026, pp. 163 / 442 Paragraphs 151 / 377 of this disclosure refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventive / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen from a virus (such as influenza virus (A, B, or C), CMV, or RSV)). Inducing in this context may mean that there was no immune response against a specific antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a specific antigen or pathogen before induction, and after induction, said immune response is enhanced. Thus, inducing the immune response in this context also includes enhancing the immune response.Ideally, after inducing an immune response in an individual, that individual is protected against developing a disease, such as an infectious disease or a cancerous disease, or the disease condition is improved by inducing an immune response.

[00305] The terms cellular immune response, cell response, cell-mediated immunity, or similar terms are intended to encompass a cell-directed response characterized by the expression of an antigen and / or presentation of a class I or class II CHM antigen. The cellular response refers to cells called T cells or T lymphocytes, which act as helper or killer cells. Helper T cells (also called CD4+ T cells) Petition 870260076800, dated 07 / 31 / 2026, page 164 / 442 152 / 377 play a central role in regulating the immune response, and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill cells such as diseased cells.

[00306] The term humoral immune response refers to a process in living organisms in which antibodies are produced in response to agents and organisms, which they ultimately neutralize and / or eliminate. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to antigens of a single specificity. After binding to an appropriate antigen and receiving several other activation signals, B lymphocytes divide, producing memory B cells as well as clones of antibody-secreting plasma cells, each producing antibodies that recognize the antigenic epitope identical to that recognized by their antigen receptor. Memory B lymphocytes remain dormant until subsequently activated by their specific antigen. These lymphocytes provide the cellular basis for memory and the resulting increase in antibody response when re-exposed to a specific antigen.

[00307] The term antibody, as used herein, refers to an immunoglobulin molecule that is capable of specifically binding to an epitope on an antigen. In particular, the term antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bridges. The term antibody includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, Petition 870260076800, dated 07 / 31 / 2026, pp. 165 / 442 153 / 377 chimeric antibodies and combinations of any of the above. Each heavy chain is composed of a variable heavy chain region (VH) and a constant heavy chain region (CH). Each light chain is composed of a variable light chain region (VL) and a constant light chain region (CL). The variable regions and constant regions are also referred to here as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called structural regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are designated HCDR1, HCDR2, and HCDR3, while the CDRs of a VL are designated LCDR1, LCDR2, and LCDR3.The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), where CH can be further subdivided into a CH1 constant domain, a hinge region, and CH2 and CH3 constant domains (arranged from amino-terminal to carboxy-terminal in the following order: CH1, CH2, CH3). The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Petition 870260076800, dated 07 / 31 / 2026, page 166 / 442 154 / 377 Antibodies can be intact immunoglobulins derived from natural or recombinant sources and can be immunoactive moieties of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[00308] The term immunoglobulin refers to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or the B cell receptor (BCR). Immunoglobulins are characterized by a structural domain, namely the immunoglobulin domain, possessing a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains that are linked by disulfide bridges.These chains are primarily composed of immunoglobulin domains, such as the VL domain (variable light chain), CL domain (constant light chain), VH domain (variable heavy chain), and the CH domains (constant heavy chain) CH1, CH2, CH3, and CH4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, ... Petition 870260076800, dated 07 / 31 / 2026, page 167 / 442 155 / 377 γ, which represent the different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. Unlike the heavy chains of soluble immunoglobulins, membrane heavy chains or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminal. In mammals, there are two types of light chains, namely lambda and kappa. Immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different immunoglobulin isotypes, while the variable part is highly diversified and responsible for antigen recognition.

[00309] The terms vaccination and immunization describe the process of treating an individual for therapeutic or prophylactic reasons and refer to the procedure of administering one or more immunogen(s) or antigen(s) or their derivatives, in particular in the form of RNA (especially mRNA) that encodes it, as described herein, to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by the presentation of said one or more immunogen(s) or antigen(s).

[00310] By cell characterized by presenting an antigen or cell presenting an antigen or MHC molecules presenting an antigen on the surface of a cell presenting an antigen or similar expressions means a cell such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen-presenting cell presenting the antigen. Petition 870260076800, dated 07 / 31 / 2026, page 168 / 442 156 / 377 or an antigenic peptide, directly or after processing, in the context of MHC molecules, preferably MHC class I and / or MHC class II molecules, more preferably MHC class I molecules.

[00311] In the context of this disclosure, the term transcription refers to a process in which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) can be translated into peptide or protein.

[00312] With regard to RNA, the term expression or translation refers to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of an amino acid sequence to form a peptide or protein.

[00313] The term optional or optionally as used herein means that the event, circumstance or condition described below may or may not occur, and that the description includes instances where said event, circumstance or condition occurs and instances where it does not occur.

[00314] The prodrugs of a specific compound described herein are those compounds which, after administration to an individual, undergo chemical conversion under physiological conditions to provide the specific compound. Additionally, prodrugs can be converted into the particular compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted into the particular compound when, for example, placed in a transdermal pad reservoir with a suitable enzyme or chemical reagent. Examples of prodrugs Petition 870260076800, dated 07 / 31 / 2026, page 169 / 442 157 / 377 drugs are esters (using an alcohol or carboxy group contained in the particular compound) or amides (using an amino or carboxy group contained in the particular compound) that are hydrolyzable in vivo. Specifically, any amino group contained in the particular compound that possesses at least one hydrogen atom can be converted into a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[00315] Isomers are compounds with the same molecular formula but differ in structure (structural isomers) or in the geometric (spatial) positioning of functional groups and / or atoms (stereoisomers). Enantiomers are a pair of stereoisomers that are non-superimposable mirror images of each other. A racemic mixture or racemate contains a pair of enantiomers in equal quantities and is indicated by the prefix (±). Diastereomers are stereoisomers that are not superimposable and are not mirror images of each other. Tautomers are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; that is, tautomers are in dynamic chemical equilibrium with each other. An example of tautomers are the keto-enol-tautomerism isomers.Conformers are stereoisomers that can be interconverted only by rotations about formally simple bonds and include - in particular - those that lead to different three-dimensional shapes of (hetero)cyclic rings, such as chair, half-chair, etc. Petition 870260076800, dated 07 / 31 / 2026, page 170 / 442 158 / 377 boat and cyclohexane boat twist.

[00316] The term average diameter refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, average diameter, diameter or size for particles is used as a synonym for this Z-average value.

[00317] The polydispersity index is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of the average diameter. Under certain prerequisites, it can be taken as a measure of the size distribution of a set of nanoparticles.

[00318] The radius of gyration (here abbreviated as Rg) of a particle about an axis of rotation is the radial distance from a point on the axis of rotation at which, if all the mass of the particle is considered concentrated, its moment of inertia about the given axis would be the same as with its actual mass distribution. Mathematically, Rg is the square root of the average distance of the particle's components from its center of mass or from a given axis. For example, for a macromolecule composed of n mass elements, of masses mi (i = 1, 2, 3, ..., n), located at fixed distances si from the center of mass, Rg is the square root of the average mass of si² over all the mass elements and can be calculated as follows: Petition 870260076800, dated 07 / 31 / 2026, page 171 / 442 159 / 377 / η / n 11 / 2 Mς^·2 / ;H�

[00319] The radius of gyration can be determined or calculated experimentally, for example, using light scattering. In particular, for small scattering vectors qa the structure function S is defined as follows: S(q) « N · (1 - ?-^) where N is the number of components (Guinier's law).

[00320] The D10 value, in particular, with respect to a quantitative distribution of particle sizes, is the diameter at which 10% of the particles have a diameter smaller than that value. The D10 value is a means of describing the proportion of the smallest particles within a particle population (such as within a particle peak obtained from a field flux fractionation).

[00321] The D50 value, in particular, with respect to a quantitative particle size distribution, is the diameter at which 50% of the particles have a diameter smaller than this value. The D50 value is a means of describing the average particle size of a particle population (such as within a particle peak obtained from a field flux fractionation).

[00322] The D90 value, in particular, with respect to a quantitative distribution of particle sizes, is the diameter at which 90% of the particles have a diameter smaller than this value. The D95, D99, and D100 values ​​have corresponding meanings. The D90, D95, D99, and D100 values ​​are means of describing the proportion of Petition 870260076800, dated 07 / 31 / 2026, page 172 / 442 160 / 377 larger particles within a particle population (such as within a particle peak obtained from a field flux fractionation).

[00323] The hydrodynamic radius (sometimes called the Stokes radius or Stokes-Einstein radius) of a particle is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only its size but also the effects of the solvent. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags along a greater number of water molecules as it moves through the solution. Since the actual dimensions of the particle in a solvent are not directly measurable, the hydrodynamic radius can be defined by the Stokes-Einstein equation: kB· T / 4 ——-6 · π · η · D where kB is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, for example, using dynamic light scattering (DLS). Thus, a procedure for determining the hydrodynamic radius of a particle or a population of particles (such as the hydrodynamic radius of particles like LNPs contained in a formulation or composition as disclosed in this descriptive report or the hydrodynamic radius of a particle peak obtained by subjecting such a formulation or Petition 870260076800, dated 07 / 31 / 2026, page 173 / 442 161 / 377 composition for field flow fractionation) is to measure the DLS signal of said particle or particle population (such as the DLS signal of particles like LNPs contained in a formulation or composition as disclosed in this descriptive report or the DLS signal of a particle peak obtained from subjecting such formulation or composition to field flow fractionation).

[00324] The term aggregate, as used in this descriptive report, refers to a cluster of particles, in which the particles are identical or very similar and adhere to each other in a non-covalent manner (e.g., through ionic interactions, hydrogen-bonding interactions, dipole interactions and / or van der Waals interactions).

[00325] The term light scattering, as used here, refers to the physical process in which light is forced to deviate from a straight path along one or more paths due to localized non-uniformities in the medium through which the light passes.

[00326] The term UV stands for ultraviolet and designates a range of the electromagnetic spectrum with wavelengths from 10 nm to 400 nm, that is, shorter than visible light but longer than X-rays.

[00327] The term multi-angle light scattering or MALS as used here refers to a technique for measuring light scattered by a sample at a plurality of angles. Multi-angle means, in this respect, that the scattered light can be detected at different discrete angles measured, for example, by a single detector moved in a strip. Petition 870260076800, dated 07 / 31 / 2026, p. 174 / 442 162 / 377 including the selected specific angles or an array of detectors fixed at specific angular locations. In a preferred embodiment, the light source used in MALS is a laser source (MALLS: multi-angle laser light dispersing). Based on the MALS signal of a composition comprising particles and using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), it is possible to determine the radius of gyration (Rg) and therefore the size of said particles. Preferably, the Zimm plot is a graphical representation using the following equation: -^- = MwP(0)-2á2cM3P2(0) Λ C where c is the mass concentration of particles in the solvent (g / mL); A2 is the second virial coefficient (mol^mL / g2); P(θ) is a shape factor related to the dependence of the intensity of scattered light on the angle; Rθ is the excess Rayleigh ratio (cm-1); and K* is an optical constant that is equal to 4π2ηα (dn / dc )2À0-4Na-1, where ηo is the refractive index of the solvent at the wavelength of the incident radiation (vacuum), λ0 is the wavelength of the incident radiation (vacuum) (nm), NA is Avogadro's number (mol-1) and dn / dc is the increment of the differential refractive index (mL / g) (cf., for example, Buchholz et al. (Electrophoresis 22 (2001)). 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the graph of Berry is calculated using the following term: Re K*c Petition 870260076800, dated 07 / 31 / 2026, page 175 / 442 163 / 377 where c, Hθ and K* are as defined above. Preferably, the Debye plot is calculated in the following term: K*c ^h where c, Hθ and K* are as defined above.

[00328] The term dynamic light scattering or DLS as used here refers to a technique for determining the size and size distribution profile of particles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is fired through a polarizer and onto a sample. The scattered light then passes through a second polarizer where it is detected and the resulting image is projected onto a screen. The particles in the solution are struck by the light and diffract the light in all directions. The light diffracted from the particles can interfere constructively (bright regions) or destructively (dark regions). This process is repeated at short time intervals and the resulting set of spot patterns is analyzed by an autocorrelator that compares the light intensity at each point over time.

[00329] The term static light scattering or SLS as used in this descriptive report refers to a technique for determining the size and size distribution profile of particles, in particular with respect to the radius of spin of the particles and / or the molar mass of the particles. High-intensity monochromatic light, usually a laser, is shone into a solution containing the particles. One or more detectors are used to measure the scattering intensity at one or more angles. Petition 870260076800, dated 07 / 31 / 2026, page 176 / 442 164 / 377 angular dependence is required to obtain accurate molar mass and size measurements for all radius macromolecules. Therefore, simultaneous measurements at multiple angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[00330] The term nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), their combinations and their modified forms. The term includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single- or double-stranded molecule and linearly or covalently circularly closed. A nucleic acid may be isolated. The term isolated nucleic acid means, according to this disclosure, that the nucleic acid (i) has been amplified in vitro, for example, via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, for example, an RNA polymerase) for RNA, (ii) has been recombinantly produced by cloning, (iii) has been purified, for example, by cleavage and separation by gel electrophoresis, or (iv) has been synthesized, for example, by chemical synthesis.

[00331] The term nucleoside (here abbreviated as N) refers to compounds that can be considered 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. Petition 870260076800, dated 07 / 31 / 2026, page 177 / 442 165 / 377 and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[00332] The five standard nucleosides that generally make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated to their one-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly written as dT (d stands for deoxy), as it contains a 2'-deoxyribofuranose moiety instead of the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). On the other hand, uridine is found in RNA and not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they would be represented as A, C, and G, while in DNA they would be represented as dA, dC, and dG.

[00333] A modified purine (A or G) or pyrimidine (C, T or U) base fraction is preferably modified by one or more alkyl groups, more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base fractions include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil and N(1)-C1-4 alkyl-uracil, preferably N7-methylguanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyluracil and N(1)-methyl-uracil.

[00334] Here, the term DNA refers to a molecule Petition 870260076800, dated 07 / 31 / 2026, p. 178 / 442 166 / 377 nucleic acid that includes deoxyribonucleotide residues. In preferred embodiments, DNA contains all or most deoxyribonucleotide residues. As used herein, deoxyribonucleotide refers to a nucleotide lacking a hydroxyl group at the 2' position of a β-Dribofuranosyl group. DNA includes, without limitation, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinant DNA, as well as modified DNA that differs from natural DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to the internal nucleotides of the DNA or to the end(s) of the DNA. It is also considered here that the nucleotides in DNA can be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides.For the purposes of this disclosure, these altered DNAs are considered analogs of naturally occurring DNA. A molecule contains a majority of deoxyribonucleotide residues if the content of deoxyribonucleotide residues in the molecule is greater 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 (regardless of whether the nucleotide residues are standard nucleotide residues (i.e., Petition 870260076800, dated 07 / 31 / 2026, page 179 / 442 167 / 377 of natural occurrence) or their analogues).

[00335] DNA can be recombinant DNA and can be obtained by cloning a nucleic acid, in particular cDNA. cDNA can be obtained by reverse transcription of RNA. RNA

[00336] According to the present disclosure, the term RNA means a nucleic acid molecule that includes ribonucleotide residues. In preferred embodiments, RNA contains all or most ribonucleotide residues. As used herein, ribonucleotide refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, without limitation, double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinant 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 the addition of non-nucleotide material to the internal nucleotides of the RNA or to the RNA end(s).It is also contemplated here that the nucleotides in RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) may be referred to as analogs of naturally occurring RNAs. Petition 870260076800, dated 07 / 31 / 2026, page 180 / 442 168 / 377 natural. A molecule contains the majority of ribonucleotide residues if the ribonucleotide residue content in the molecule is greater 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 (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogs).

[00337] RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (sRNAA), 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).

[00338] In a preferred embodiment, RNA comprises an open reading frame (ORF) that encodes a peptide or protein.

[00339] The term in vitro transcription or IVT as used here means that transcription (i.e., RNA generation) is conducted in a cell-free manner. That is, IVT does not use living / cultured cells, but rather the transcription machinery extracted from cells (e.g., cell lysates or their isolated components, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). Petition 870260076800, dated 07 / 31 / 2026, page 181 / 442 169 / 377

[00340] According to the present disclosure, the term mRNA means messenger RNA and refers to a transcript that can be generated using a DNA template and can encode a peptide or protein. Typically, an mRNA comprises a 5'-UTR, a peptide / protein coding region, and a 3'-UTR. In the context of the present disclosure, mRNA is preferably generated by in vitro transcription (IVT) from a DNA template. As stated above, the in vitro transcription methodology is known to those skilled in the art, and a variety of in vitro transcription kits are commercially available.

[00341] mRNA is single-stranded, but it can contain self-complementary sequences that allow parts of the mRNA to fold and pair up to form double helices.

[00342] According to this disclosure, dsRNA stands for double-stranded RNA and is RNA with two partially or completely complementary strands.

[00343] In preferred embodiments of the present disclosure, mRNA refers to an RNA transcript that codes for a peptide or protein.

[00344] In one embodiment, RNA that preferentially codes for a peptide 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, Petition 870260076800, dated 07 / 31 / 2026, page 182 / 442 170 / 377 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.

[00345] In one embodiment, RNA (such as mRNA) contains a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The methodology for in vitro transcription is known to those skilled in the art; cf., for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, a variety of in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (such as the TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as the HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as systems RiboMAX™, HeLaScribe®, Riboprobe®), Jena Bioscience (as SP6 or T7 transcription kits) and Epicenter (as AmpliScribe™). To provide 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 made and / or added to the mRNA. Petition 870260076800, dated 07 / 31 / 2026, page 183 / 442 171 / 377 after transcription.

[00346] In one embodiment, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from an appropriate DNA template. The promoter to control transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[00347] In certain embodiments of the present disclosure, the RNA (such as mRNA) is RNA replicon (such as mRNA replicon) or simply a replicon, in particular self-replicating RNA (such as self-replicating mRNA) or self-amplifying RNA (or self-amplifying mRNA). In a particularly preferred embodiment, the replicon or self-replicating RNA (such as self-replicating mRNA) is derived from or comprises elements derived from an ssRNA virus, in particular a positive-strand ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA Petition 870260076800, dated 07 / 31 / 2026, pp. 184 / 442 172 / 377 typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in transcription, modification, and replication of viral RNA and in protein modification) and structural proteins (forming the viral particle). There are usually two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF starting near the 5' end of the genome, while the alphavirus structural proteins are encoded together by a second ORF that is found downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1.In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule resembling eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly as a messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Petition 870260076800, dated 07 / 31 / 2026, page 185 / 442 173 / 377 Alphavirus-based transreplication systems depend on alphavirus nucleotide sequence elements in two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Transreplication requires the presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain elements of the alphaviral sequence to allow recognition and RNA synthesis by the alphaviral replicase.

[00348] In one embodiment of the present disclosure, RNA (such as mRNA) contains one or more modifications, for example, in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, to increase the expression of RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence that codes for the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein. Such modifications are described, for example, in documents WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5' and / or 3' untranslated regions (UTRs) such as the introduction of a UTR that is not related to the coding region of said RNA; the substitution of one or more naturally occurring nucleotides with synthetic nucleotides; and optimization Petition 870260076800, dated 07 / 31 / 2026, pp. 186 / 442 174 / 377 of codons (for example, to alter, preferably increase, the GC content of the RNA). The term modification in the context of modified mRNA according to this disclosure refers preferably to any modification of an mRNA that is not naturally present in said RNA (such as mRNA).

[00349] In some embodiments, RNA (such as mRNA) comprises a 5'-cap structure. In one embodiment, the mRNA does not have uncapped 5'-triphosphates. In another embodiment, RNA (such as mRNA) may comprise a conventional 5'-cap and / or a 5'-cap analog. The term conventional 5'-cap refers to a cap structure found at the 5' end of an mRNA molecule and generally consists of a guanosine 5'-triphosphate (Gppp) that is connected via its triphosphate portion to the 5' end of the next mRNA nucleotide (i.e., the guanosine is connected via a 5'-to-5' triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at the N7 position (resulting in the m7Gppp cap structure).The term 5'-cap analog refers to a 5'-cap based on a conventional 5'-cap, but which has been modified at the 2' or 3' position of the m7guanosine structure to avoid integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those with one or more substitutions on the bridging oxygen and no bridging on the phosphate bridge, such as phosphorothioate-modified 5'-cap analogs on the β-phosphate (such as m27,2'OG(5')ppSp(5')G (referred to as betaS-ARCA or β-S-ARCA)), as described in... Petition 870260076800, dated 07 / 31 / 2026, page 187 / 442 175 / 377 PCT / EP2019 / 056502. The provision of an RNA (such as mRNA) with a 5'-cap structure as described in this descriptive report can be achieved by in vitro transcription of a DNA template in the presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is transcriptionally co-incorporated into the generated RNA chain (such as mRNA), or the RNA (such as mRNA) can be generated, for example, by in vitro transcription, and the 5'-cap structure can be attached to the mRNA post-transcriptionally using capping enzymes, for example, vaccinia virus capping enzymes.

[00350] In some embodiments, RNA (as mRNA) comprises a 5'-cap structure selected from the group consisting of m27.2'OG(5')ppSp(5')G (in particular, its diastereomer D1), m27.3'OG(5')ppp(5')G, and m27.3'-OGppp(m12'-O)ApG.

[00351] In some embodiments, RNA (such as mRNA) comprises a cap0, cap1, or cap2, preferably cap1 or cap2. According to the present disclosure, the term cap0 means the structure m7GpppN, where N is any nucleoside containing an OH moiety at the 2' position. According to the present disclosure, the term cap1 means the structure m7GpppNm, where Nm is any nucleoside containing an OCH3 moiety at the 2' position. According to the present disclosure, the term cap2 means the structure m7GpppNmNm, where each Nm is independently any nucleoside containing an OCH3 moiety at the 2' position.

[00352] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure: Petition 870260076800, dated 07 / 31 / 2026, pp. 188 / 442 176 / 377

[00353] The D1 diastereomer of beta-S-ARCA or betaS-ARCA(D1) is the beta-S-ARCA diastereomer that elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and therefore exhibits a shorter retention time. HPLC is preferably an analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably in the format: 5 μm, 4.6 x 250 mm, is used for separation, where a flow rate of 1.3 ml / min can be applied. In one embodiment, a methanol-ammonium acetate gradient is used, for example, a linear gradient of 0-25% methanol in 0.05 M ammonium acetate, pH = 5.9, over 15 min. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[00354] The analog 5'-cap m27.3'-OGppp(m12'-O)ApG (also referred to as m27.3'OG(5')ppp(5')m2'-OApG), which is a building block of a cap1, has the following structure: Petition 870260076800, dated 07 / 31 / 2026, pp. 189 / 442 177 / 377 cap0 mRNA comprising

[00355] An example of β-SARCA and mRNA has the following structure: mRNA

[00356] An exemplary cap0 mRNA comprising the following structure: m27,3'OG(5')ppp(5')G and mRNA has the mRNA

[00357] An exemplary cap1 mRNA comprising Petition 870260076800, dated 07 / 31 / 2026, pp. 190 / 442 178 / 377 m27'3'-OGppp(mi2'-O)ApG and mRNA have the H2N HO CH3 THE CH3 —OP—O—PR—O—P—O |Y |β |α following structure: THE mRNA

[00358] As used herein, the term polyA tail or poly-A sequence refers to an uninterrupted or interrupted sequence of adenylate residues that is typically located at the 3' end of an RNA molecule (such as mRNA). Poly-A tails or poly-A sequences are known to those skilled in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. The RNAs (as mRNAs) disclosed in this descriptive report may have a poly-A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a DNA-encoded poly-A tail transcribed by a template-dependent RNA polymerase.

[00359] It has been demonstrated that a poly-A tail of approximately 120 A nucleotides has a beneficial influence on mRNA levels in transfected eukaryotic cells, as well as on the levels of protein that is translated from a frame of Petition 870260076800, dated 07 / 31 / 2026, pp. 191 / 442 179 / 377 open reading that is present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[00360] The poly-A tail can be of any length. In some embodiments, a poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and in particular, about 120 A nucleotides. In this context, essentially consists of means that the majority of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly-A tail are A nucleotides, but allows the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, it means that all nucleotides in the poly-A tail, i.e., 100% of the nucleotides in the poly-A tail, are A nucleotides. The term A nucleotide or A refers to adenylate.

[00361] In some embodiments, a poly-A tail is attached during RNA transcription, for example, during the preparation of transcribed RNA in vitro, based on a DNA template comprising repeated dT (deoxythymidylate) nucleotides on the strand complementary to the coding strand. The DNA sequence encoding a polyA tail (coding strand) is referred to as a poly(A) cassette. Petition 870260076800, dated 07 / 31 / 2026, pp. 192 / 442 180 / 377

[00362] In some embodiments, the poly(A) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). This random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides long. Such a cassette is disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 may be used in this disclosure. A poly(A) cassette consisting essentially of dA nucleotides, but interrupted by a random sequence with an equal distribution of the four nucleotides (dA, dC, dG, dT) and with a length of, for example, 5 to 50 nucleotides, shows, at the DNA level, that the constant propagation of plasmid DNA in E. coli is encompassed and is also associated, at the RNA level, with beneficial properties regarding the support of RNA stability and translation efficiency.Consequently, in some embodiments, the poly-A tail contained in an mRNA molecule described here consists essentially of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). This random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides long.

[00363] In some embodiments, no nucleotide other than A nucleotides flanks a poly-A tail at its 3' end, that is, the poly-A tail is not masked or followed at its 3' end by a nucleotide other than A.

[00364] In one embodiment, the poly-A sequence comprises at least 100 nucleotides. In a Petition 870260076800, dated 07 / 31 / 2026, page 193 / 442 181 / 377 embodiment, the poly-A sequence comprises or consists of the nucleotide sequence with SEQ ID NO: 14. In one embodiment, the poly-A sequence has a nucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 14.

[00365] In some embodiments, the RNA (such as mRNA) used in this disclosure comprises a 5'-UTR and / or a 3'-UTR. The term untranslated region or UTR refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) may be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5' end, upstream of the start codon of a protein-coding region. A 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of a protein-coding region, but the term 3'-UTR preferably does not include the poly-A sequence.Thus, the 3'-UTR is upstream of the poly-A sequence (if present), for example, directly adjacent to the poly-A sequence. The incorporation of a 3'-UTR into the 3' untranslated region of an RNA molecule (preferably mRNA) can result in increased translation efficiency. A synergistic effect can be obtained by incorporating two or more of these 3'-UTRs (which are preferentially arranged). Petition 870260076800, dated 07 / 31 / 2026, pp. 194 / 442 182 / 377 in a head-to-tail orientation; cf., for example, Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs can be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced. In one particular embodiment, the 3'-UTR is derived from a globin gene or mRNA, such as an alpha2-globin, alpha1-globin, or beta-globin gene or mRNA, preferably beta-globin, most preferably human beta-globin. For example, the RNA (preferably mRNA) can be modified by replacing the existing 3'-UTR or by inserting one or more, preferably two, copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, most preferably human beta-globin.

[00366] In some embodiments, the RNA (such as mRNA) used in the present disclosure comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 12.

[00367] In some embodiments, the RNA (such as mRNA) used in the present disclosure comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 13.

[00368] RNA (such as mRNA) can have ribonucleotides modified to increase its stability and / or decrease its immunogenicity and / or decrease its immunogenicity. Petition 870260076800, dated 07 / 31 / 2026, pp. 195 / 442 183 / 377 cytotoxicity. For example, in one embodiment, the uridine in the RNA (such as mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[00369] In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (ψ), Nl-methyl-pseudouridine (η1ψ), 5-methyl-uridine (m5U) and combinations thereof.

[00370] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in RNA (such as mRNA) may be any one or more of 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U),5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl Petition 870260076800, dated 07 / 31 / 2026, page 196 / 442, 184 / 377 uridina (cmnm5U), 5-carboximetilaminometil-2-thio-uridina (cmnm5s2U), 5 -propinyl-uridina, 1-propinyl-pseudouridina, 5-taurinometil-uridina (Tm5U), 1-taurinometilpseudouridina, 5-taurinometil-2-thio-uridina (Tm5s2U), 1taurinometil-4-thio-pseudouridina), 5-metil-2-thio-uridina (m5s2U), 1-metil-4-thio-pseudouridina (m1s44), 4-thio-1metil-pseudouridina, 3-metil-pseudouridina (m34), 2-thio-1 metil-pseudouridina, 1-metil-1-deaza-pseudouridina, 2-thio1-metil-1-deaza-pseudouridina, di-hidrouridina (D), dihidropseudouridina, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3carboxypropyl)pseudouridine (acp3 ψ), 5(isopentenylaminomethyl)uridine (inm5U), 5(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thiouridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (4m), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any another modified uridine known in the state of the art. Petition 870260076800, dated 07 / 31 / 2026, page 197 / 442 185 / 377

[00371] An RNA (preferably mRNA) that is modified by pseudouridine (partially or completely replacing, preferably completely, uridine) is referred to herein as Ψ-modified, while the term η1Ψ-modified means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely replacing, preferably completely, uridine). Furthermore, the term m5U-modified means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely replacing, preferably completely, uridine). These Ψ or η1Ψ or m5U modified RNAs generally exhibit decreased immunogenicity compared to their unmodified forms and are therefore preferred in applications where the induction of an immune response must be avoided or minimized.

[00372] The RNA (preferably mRNA) codons used in this disclosure may be further optimized, for example, to increase the GC content of the RNA and / or replace codons that are rare in the cell (or subject) in which the peptide or protein of interest is to be expressed with codons that are frequent synonymous codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA used in this disclosure is encoded by a coding sequence that is codon-optimized and / or whose G / C content is increased compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or increased in G / C content compared to the Petition 870260076800, dated 07 / 31 / 2026, pp. 198 / 442 186 / 377 corresponding sequence regions of the wild-type coding sequence. In one embodiment, codon optimization and / or increasing the G / C ratio preferentially does not alter the sequence of the encoded amino acid sequence.

[00373] The term codon-optimized refers to altering codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferentially altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, coding regions are preferentially codon-optimized for optimal expression in a subject to be treated using the RNA (preferably mRNA) described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequency of tRNA occurrence in cells. Thus, the RNA sequence (preferably mRNA) can be modified so that codons for which frequently occurring tRNAs are available are inserted instead of rare codons.

[00374] In some embodiments, the guanosine / cytosine (G / C) ratio of the RNA (preferably mRNA) coding region described herein is increased compared to the G / C ratio of the corresponding wild-type RNA coding sequence, wherein the amino acid sequence encoded by the RNA (preferably mRNA) is not preferentially modified compared to the amino acid sequence encoded by the wild-type RNA. This RNA sequence modification is based on Petition 870260076800, dated 07 / 31 / 2026, page 199 / 442 187 / 377 The fact that the sequence of any RNA region to be translated is important for the efficient translation of that RNA (preferably mRNA). Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Regarding the fact that several codons code for one and the same amino acid (the so-called degeneracy of the genetic code), the codons most favorable for stability can be determined (the so-called alternative use of codons). Depending on the amino acid to be encoded by the RNA (preferably mRNA), there are several possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that code for the same amino acids but do not contain them. A and / or U nucleotides, or contain a lower content of A and / or U nucleotides.

[00375] In various embodiments, the G / C content of the mRNA coding region described here is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% or even more compared to the G / C content of the wild-type RNA coding region.

[00376] A combination of the modifications described above, namely, incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with nucleotides Petition 870260076800, dated 07 / 31 / 2026, pages 200 / 442 188 / 377 synthetics (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (η1Ψ) or 5-methyluridine (m5U) for uridine) and codon optimization have a synergistic influence on RNA stability (preferably mRNA) and increased translation efficiency.Thus, in a preferred embodiment, the RNA (preferably mRNA) used in the present disclosure, in particular an RNA (preferably mRNA) encoding an antigen or epitope to induce an immune response disclosed in this descriptive report, contains a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, namely, (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a polyA sequence; (iii) alteration of 5'- and / or 3'-UTRs (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mW) or 5-methyluridine (m5U) for uridine), and (v) codon optimization. In one embodiment, the RNA comprises a cap1 or cap2 structure, preferably a cap1. In one embodiment, the poly-A sequence comprises at least 100 nucleotides. In one embodiment, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14. In one embodiment, the 5' UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence. Petition 870260076800, dated 07 / 31 / 2026, page 201 / 442 189 / 377 of SEQ ID NO: 12. In one embodiment, the 3' UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with the nucleotide sequence of SEQ ID NO: 13.

[00377] Some aspects of dissemination involve the targeted delivery of the RNA (preferably mRNA) disseminated herein to certain cells or tissues. In one embodiment, dissemination involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically the spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically the spleen, is particularly preferred if the RNA (preferably mRNA) delivered is RNA (preferably mRNA) encoding an antigen or epitope to induce an immune response. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.The lymphatic system is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or central lymphoid organs... Petition 870260076800, dated 07 / 31 / 2026, page 202 / 442 190 / 377 peripheral lymph nodes, which include the lymph nodes and spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

[00378] Lipid-based RNA delivery systems (such as mRNA) have an inherent preference for the liver. Hepatic accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates). In one embodiment, the target organ is the liver and the target tissue is hepatic tissue. Delivery to such target tissue is preferred, in particular, if the presence of mRNA or the encoded peptide or protein in this organ or tissue is desired and / or if it is desired to express large quantities of the encoded peptide or protein and / or if the systemic presence of the encoded peptide or protein, particularly in significant quantities, is desired or required.

[00379] In one embodiment, following administration of the RNA LNP compositions described herein, at least one portion of the RNA is delivered to a target cell or target organ. In one embodiment, at least one portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is RNA (preferably mRNA) encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein. In one embodiment, the target cell is a liver cell. In one embodiment, the target cell is a muscle cell. In one embodiment, the target cell is an endothelial cell. In one embodiment, the target cell is a tumor cell or a cell in the tumor microenvironment. In one embodiment, the target cell is a blood cell. In one Petition 870260076800, dated 07 / 31 / 2026, page 203 / 442 In one embodiment, the target cell is a cell in the lymph nodes. In another embodiment, the target cell is a cell in the lung. In another embodiment, the target cell is a blood cell. In another embodiment, the target cell is a skin cell. In another embodiment, the target cell is a spleen cell. In another embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In another embodiment, the target cell is a dendritic cell in the spleen. In one concretization, the target cell is a T cell. In another concretization, the target cell is a B cell. In another concretization, the target cell is an NK cell. In another concretization, the target cell is a monocyte. Thus, the The RNA LNP compositions described herein can be used to deliver RNA (preferably mRNA) to such a target cell. Consequently, the present disclosure also refers to a method for delivering RNA (preferably mRNA) to a target cell in a subject comprising administering the RNA LNP compositions described herein to the subject. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is RNA (preferably mRNA) encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein.

[00380] Coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or RNA (preferably mRNA), to serve as a template for the synthesis of other polymers and macromolecules in biological processes having a defined nucleotide sequence. Petition 870260076800, dated 07 / 31 / 2026, pp. 204 / 442 192 / 377 (i.e., rRNA, t...

Claims

CLAIMS 1. Composition characterized in that it comprises lipid nanoparticles (LNPs) dispersed in an aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the aqueous phase comprises a buffer system comprising a buffer substance and a monovalent anion, the buffer substance being selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris) and its protonated form, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris-methane) and its protonated form, and triethanolamine (TEA) and its protonated form, the monovalent anion being selected from the group consisting of morpholinoethanesulfonic acid (MES) anion, 3-(N-morpholino)propanesulfonic acid (MOPS) anion and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) anion; The concentration of the buffer substance in the composition is at most about 25 mM;and the aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions, and substantially free of ethylenediaminetetraacetic acid (EDTA) anions, wherein the concentration of the monovalent anion in the composition is at least equal to the concentration of the buffer substance in the composition.

2. Composition according to claim 1, characterized in that: (i) the buffer substance is Tris and its protonated form; (ii) the concentration of the buffer substance in the composition is at most about 20 mM, or at most about 15 mM, or at most about 10 mM, or about 10 mM; (iii) the aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or anions of Petition 870260076800, dated 07 / 31 / 2026, p.391 / 442 2 / 18 dibasic organic acid and / or polybasic organic acid anions, or the aqueous phase is substantially free of inorganic anions sulfate anions, carbonate anions, dibasic organic acid anions and polybasic organic acid anions; (iv) the concentration of the monovalent anion in the composition is greater than the concentration of the buffer substance in the composition; (v) the pH of the composition is between about 6.5 and about 8.0, between about 6.9 and about 7.9, or between about 7.0 and about 7.8; (vi) water is the major component in the composition and / or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v / v); (vii) the osmolality of the composition is at most about 400 x 10-3 osmol / kg; (viii) the RNA concentration in the composition is about 5 mg / l about 150 mg / l, about 10 mg / l about 130 mg / l, or about 30 mg / l about 120 mg / l; (ix) the composition comprises: a.a. a cryoprotectant, at a concentration of at least about 1% w / v; b. a cryoprotectant, wherein the cryoprotectant comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols, or is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof; c. a cryoprotectant, wherein the cryoprotectant comprises sucrose and / or glycerol; d. a cryoprotectant, at a concentration of at least about 1% w / v, wherein the cryoprotectant comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols, or is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof; e.a cryoprotectant, at a concentration of at least about 1% w / v, wherein the cryoprotectant comprises sucrose and / or glycerol; or the composition is substantially free of a cryoprotectant; and / or (x) the aqueous phase does not comprise a chelating agent.

3. Composition according to any one of claims 1 to 2, characterized in that the cationically ionizable lipid: (i) has the structure of Formula (I): R3 ^G3 Λ1. / N. XL2 R1 Ό1 Ό2 R2 (I) or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-,S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-,C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, OC(=O)NRa- or -NRaC(=O)O- or a direct linkage; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C2-C12 alkenylene; G3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; Petition 870260076800, dated 07 / 31 / 2026, p.393 / 442 4 / 18 R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; ex is 0, 1 or 2; (ii) is selected from the following structures B to F: or; (iii) is a lipid with the structure Petition 870260076800, dated 07 / 31 / 2026, page 394 / 442 5 / 18.

4. Composition according to claim 3, characterized in that the cationically ionizable lipid has the structure of Formula (I), wherein (i) R1 and R2 each independently have the following structure: R7a wherein: R7a and R7b are, in each occurrence independently, C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b and a are each selected such that R1 and R2 each independently comprise from 6 to 20 carbon atoms; or (ii) R1 and R2 both independently have one of the following structures: Petition 870260076800, dated 07 / 31 / 2026, p. 395 / 442 6 / 18 5. Composition, according to any one of claims 1 to 4, characterized in that: (i) the LNPs further comprise a lipid conjugated with a polymer, a neutral lipid and a steroid.

6. Composition according to claim 5, characterized in that the lipid conjugated with polymer comprises: (i) a pegylated lipid; (ii) a pegylated lipid, wherein the pegylated lipid has the following structure: R13 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R12 and R13 are each independently a linear or branched alkyl chain, saturated or unsaturated, containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages; ew has an average value ranging from 30 to 60; (iii) a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material; or Petition 870260076800, dated 07 / 31 / 2026, p.396 / 442 7 / 18 (iv) a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material, wherein the polysarcosine-lipid conjugate or conjugate of polysarcosine and a lipid-like material is a selected member of the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate and a mixture thereof.

7. Composition according to any one of claims 5 or 6, characterized in that the neutral lipid is: (i) a phospholipid; (ii) a phospholipid selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins;(iii) a phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibeenoylphosphatidylcholine (DBPC), ditricosanoylphos phatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyoleoyl-phosphatidylcholine (POPC), 1,2-di-Ooctadecenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylPetition 870260076800, of 07 / 31 / 2026, p. 397 / 442 8 / 18 phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE) and diphytanoylphosphatidylethanolamine (DPyPE).; 8. Composition, according to any one of claims 5 to 7, characterized in that the steroid comprises a sterol such as cholesterol.

9. Composition, according to any one of claims 1 to 8, characterized in that: (i) the LNPs comprise at least about 75%, at least about 80% of the RNA comprised in the composition; (ii) the RNA is encapsulated within or associated with the LNPs; (iii) the RNA comprises a modified nucleoside in place of uridine, or the RNA comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methylpseudouridine (η1ψ) and 5-methyl-uridine (m5U); (iv) the RNA comprises at least one or all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence; (v) the RNA comprises at least one or all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence, where: a. the poly-A sequence comprises at least 100 A nucleotides; b.the poly-A sequence comprises at least 100 A nucleotides and the poly-A sequence is an interrupted sequence of A nucleotides; and / or c. the 5' cap is a cap1 or cap2 structure; and / or (vi) the RNA: Petition 870260076800, dated 07 / 31 / 2026, page 398 / 442 9 / 18 a. encodes one or more polypeptides; b. encodes one or more polypeptides, wherein one or more polypeptides comprise an epitope to induce an immune response against an antigen in a subject; c. comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant; d. comprises an ORF encoding a full-length SARS-CoV-2 S protein variant with proline residue substitutions at positions 986 and 987 of SEQ ID NO: 1; and / or e.It comprises an ORF encoding a full-length SARS-CoV-2 S protein variant, wherein the SARS-CoV-2 S protein variant has at least 80% identity with SEQ ID NO:

7.

10. Composition, according to any one of claims 1 to 9, characterized in that: (i) the composition is in frozen form; (ii) the composition is in frozen form, wherein the RNA integrity after thawing the frozen composition is at least 50% compared to the RNA integrity before the composition was frozen; and / or (iii) the composition is in frozen form, wherein the size (Z-mean) and / or size distribution and / or polydispersity index (PDI) of the LNPs after thawing the frozen composition is equal to the size (Z-mean) and / or size distribution and / or PDI of the LNPs before the composition was frozen. Petition 870260076800, dated 07 / 31 / 2026, pp. 399 / 442 10 / 18 11. Composition, according to any one of claims 1 to 9, characterized in that the composition is in liquid form.

12. Method for preparing a composition comprising LNPs dispersed in a final aqueous phase, characterized in that it comprises: (I) preparing a formulation comprising LNPs dispersed in the final aqueous phase, wherein the LNPs comprise cationically ionizable lipid and RNA; and (II) optionally freezing the formulation at about -10°C or below, thereby obtaining the composition, wherein step (I) comprises: (a) preparing an RNA solution containing water and a first buffer system; (b) preparing an ethanolic solution comprising cationically ionizable lipid and, if present, one or more additional lipids; (c) mixing the RNA solution prepared in (a) with the ethanolic solution prepared in (b), thereby preparing a first intermediate formulation comprising the LNPs dispersed in a first aqueous phase comprising the first buffer system;(d) filtering the first intermediate formulation prepared in (c) using a final aqueous buffer solution comprising the final buffer system, thereby preparing the formulation comprising the LNPs dispersed in the final aqueous phase; wherein the LNPs comprise a cationically ionizable lipid and RNA; the final aqueous phase comprises a final buffer system comprising a final buffer substance and a final monovalent anion, the final buffer substance being selected from the group consisting of Tris and its protonated form, Bis-Tris-methane and its protonated form, and TEA and its protonated form and the final monovalent anion being selected from the group consisting of MES anion, MOPS anion and HEPES anion; the concentration of the final buffer substance in the composition is at most about 25 mM;and the final aqueous phase is substantially free of inorganic phosphate anions, substantially free of citrate anions, and substantially free of EDTA anions, where the concentration of the final monovalent anion in the composition is at least equal to the concentration of the final buffer substance in the composition.

13. Method according to claim 12, characterized in that: (i) step (I) further comprises one or more selected dilution and filtration steps; (ii) step (I) comprises: (a') providing an aqueous RNA solution; (b') providing a first aqueous buffer solution comprising a first buffer system; (c') mixing the aqueous RNA solution provided in (a') with the first aqueous buffer solution provided in (b'), thereby preparing an RNA solution containing water and the first buffer system; (d') preparing an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids; (e') mixing the RNA solution prepared in (c') with the ethanolic solution prepared in (d'), thereby preparing a first intermediate formulation. Petition 870260076800, dated 07 / 31 / 2026, p. 401 / 442 12 / 18 comprising LNPs dispersed in a first aqueous phase comprising the first buffer system;(f') optionally filtering the first intermediate formulation prepared in (e') using an additional aqueous buffer solution comprising an additional buffer system, thereby preparing an additional intermediate formulation comprising the LNPs dispersed in an additional aqueous phase comprising the additional buffer system, wherein the other aqueous buffer solution may be identical to or different from the first aqueous buffer solution; (g') optionally repeating step (f') one or two or more times, wherein the additional intermediate formulation comprising the LNPs dispersed in the additional aqueous phase comprising the additional buffer system obtained after step (f') of one cycle is used as the first intermediate formulation of the next cycle, wherein in each cycle the additional aqueous buffer solution may be identical to or different from the first aqueous buffer solution;(h') filter the first intermediate formulation obtained in step (e'), if step (f') is absent, or the additional intermediate formulation obtained in step (f'), if step (f') is present and step (g') is not present, or the additional intermediate formulation obtained after step (g'), if steps (f') and (g') are present, using a final aqueous buffer solution comprising the final buffer system and having a pH of at least 6.0; and (i') optionally dilute the formulation obtained in step (h') with a dilution solution; Petition 870260076800, dated 07 / 31 / 2026, page 402 / 442 13 / 18 thus preparing the formulation comprising the LNPs dispersed in the final aqueous phase; (iii) the filtration is tangential flow filtration or diafiltration; and / or (iv) the method comprises (II) freezing the formulation at about 10°C or less.; 14. Method according to any one of claims 12 to 13, characterized in that: (i) the final buffer substance is Tris and its protonated form; (ii) the concentration of the final buffer substance in the composition is at most about 20 mM, at most about 15 mM, at most about 10 mM, or about 10 mM; and / or (iii) the final aqueous phase is substantially free of inorganic sulfate anions and / or carbonate anions and / or dibasic organic acid anions and / or polybasic organic acid anions, or substantially free of inorganic sulfate anions, carbonate anions, dibasic organic acid anions and polybasic organic acid anions.

15. Method, according to any one of claims 12 to 14, characterized in that (i) the RNA solution prepared in step (a) further comprises one or more di- and / or polybasic organic acid anions and step (d) is conducted under conditions that remove one or more di- and / or polybasic organic acid anions resulting in the formulation comprising the LNPs dispersed in the final aqueous phase with the final aqueous phase being substantially free of one or more di- and / or polybasic organic acid anions present in the RNA solution prepared in step (a); or (ii) the first aqueous buffer solution and the first aqueous phase comprise one or more di- and / or polybasic organic acid anions. Petition 870260076800, dated 07 / 31 / 2026, p.403 / 442 14 / 18 polybasic and at least one of the steps (f') to (h') is conducted under conditions that remove one or more anions of di- and / or polybasic organic acids from the first intermediate formulation and / or from the additional intermediate formulation; and / or where (i) the RNA solution obtained in step (a) or (ii) the first aqueous buffer solution has a pH less than 6.0, or at most about 5.0, or at most about 4.

5.

16. Method according to claim 15, characterized in that one or more di- and / or polybasic organic acid anions comprise citrate anions and / or EDTA anions. 17.A method according to any one of claims 12 to 14, characterized in that: (i) the first buffer system used in step (a) comprises the final buffer substance and the final monovalent anion used in step (d); or (ii) the first buffer system used in step (a) comprises the final buffer substance and the final monovalent anion used in step (d), wherein the buffer system and pH of the first buffer system used in step (a) are identical to the buffer system and pH of the final aqueous buffer solution used in step (d); or (iii) each of the first buffer systems and each additional buffer system used in steps (b'), (f') and (g') comprises the final buffer substance and the final monovalent anion used in step (h'); or (iv) each of the first buffer system and each additional buffer system used in steps (b'), (f') and (g') comprises the final buffer substance and the final monovalent anion used in step (h'), wherein the buffer system and the pH of Petition 870260076800, dated 07 / 31 / 2026, page.404 / 442 15 / 18 each of the first aqueous buffer solution and of each additional aqueous buffer solution used in steps (b'), (f') and (g') are identical to the buffer system and pH of the final aqueous buffer solution.

18. Method, according to any one of claims 12 to 17, characterized in that: (i) the concentration of the final monovalent anion in the composition is greater than the concentration of the final buffer substance in the composition; (ii) the pH of the composition is between about 6.5 and about 8.0, between about 6.9 and about 7.9, or between about 7.0 and about 7.8; (iii) water is the main component in the formulation and / or composition and / or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v / v); (iv) the osmolality of the composition is at most about 400 x 10-3 osmol / kg; (v) the RNA concentration in the composition is about 5 mg / l about 150 mg / l, about 10 mg / l about 130 mg / l or about 30 mg / l about 120 mg / l; and / or (vi) (i) step (I) further comprises diluting the formulation prepared in (d) with a dilution solution, or step (i') is present, wherein the dilution solution comprises a cryoprotectant;and / or (ii) the formulation obtained in step (I) and the composition comprise: a. a cryoprotectant at a concentration of at least about 1% w / v; b. a cryoprotectant, wherein the cryoprotectant comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols, or is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof; c. a cryoprotectant, wherein the cryoprotectant comprises sucrose and / or glycerol; d. a cryoprotectant, at a concentration of at least about 1% w / v, wherein the cryoprotectant comprises one or more compounds selected from the group consisting of carbohydrates and sugar alcohols, or is selected from the group consisting of sucrose, glucose, glycerol, sorbitol and a combination thereof; and e. a cryoprotectant, at a concentration of at least about 1% w / v, wherein the cryoprotectant comprises sucrose and / or glycerol;or the formulation obtained in step (I) and the composition are substantially free of a cryoprotectant.; 19. Method according to any one of claims 12 to 18, characterized in that the cationically ionizable lipid is as defined in claim 3 or 4.

20. Method according to any one of claims 12 to 19, characterized in that: (i) the ethanolic solution prepared in step (b) or (d') further comprises a polymer-conjugated lipid, a neutral lipid and a steroid, and the LNPs further comprise a polymer-conjugated lipid, a neutral lipid and a steroid; or (ii) the ethanolic solution prepared in step (b) or (d') further comprises a polymer-conjugated lipid, a neutral lipid and a steroid, and the LNPs further comprise a polymer-conjugated lipid, a neutral lipid and a steroid, wherein the additional lipids are as defined in claims 5 to 8. Petition 870260076800, dated 07 / 31 / 2026, pp. 406 / 442 17 / 18 21. Method according to any one of claims 12 to 20, characterized in that: (i) the cationically ionizable lipid, the polymer-conjugated lipid, the neutral lipid and the steroid are present in the ethanolic solution in a molar ratio of 20% to 60% of the cationically ionizable lipid, 0.5% to 15% of the polymer-conjugated lipid, 5% to 25% of the neutral lipid and 25% to 55% of the steroid, or in a molar ratio of 45% to 55% of the cationically ionizable lipid, 1.0% to 5% of the polymer-conjugated lipid, 8% to 12% of the neutral lipid and 35% to 45% of the steroid; (ii) the final aqueous phase does not comprise a chelating agent; (iii) the LNPs comprise at least about 75%, or at least about 80% of the RNA comprised in the composition; (iv) the RNA is encapsulated within or associated with the LNPs; and / or (v) the RNA is as defined in claim 9.

22. Method, according to any one of claims 12 to 21, characterized in that it does not comprise step (II).

23. Composition characterized in that it is preparable by the method as defined by any one of claims 12 to 22, wherein the composition is in frozen form or in liquid form.

24. Ready-to-use pharmaceutical composition characterized in that it is preparable by a method in which: (i) the method comprises the steps of providing a frozen composition prepared by the method as defined in any one of claims 12 to 21 and thawing the frozen composition, thereby obtaining the ready-to-use pharmaceutical composition; or (ii) the method comprises the step of providing a liquid composition prepared by the method as defined in any one of claims 12, 13(i) to (iii) and 14 to 22, thereby obtaining the ready-to-use pharmaceutical composition.

25. Composition according to any one of claims 1 to 11, 23 and 24, characterized in that it is for use in therapy or for use in inducing an immune response in a subject.

26. Use of a composition as defined in any one of claims 1 to 11, 23 and 24, characterized in that it is for the manufacture of a medicament for use in therapy.

27. Use of a composition as defined in any one of claims 1 to 11, 23 and 24, characterized in that it is for the manufacture of a medicament to induce an immune response in a subject.

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