Advanced silicon-containing lipid nanoparticles for efficient human t cell transfection

Abstract

The invention relates to LipexSil® lipid-containing nanoparticle compositions, in which a silicon-containing ionizable lipid is combined with a certain ratio of other lipids consituting the lipid nanoparticles resulting in a remarkable T cell transfection. In certain embodiments, the formulation consists of four components (ionizable lipid, structural lipid, helper lipid, and shield lipid), while in other embodiments a fifth lipid component is included. This ionizable cationic lipid family is showing remarkably low in vitro toxicity on T cells. The invention describes the production and characterization of the lipid nanoparticles and in vitro experiments demonstrating that the corresponding formulations with the LipexSil® lipids [WO2024023174] are superior to the current approach, delivering their payload, or cargo (e.g. RNA, DNA, mRNA, microRNA, siRNA, pDNA, circular DNA, small biologically active molecules) into T cells.

Description

[0001] 120852P1420PC November 21, 2025 AldexChem Kft.

[0002] ADVANCED SILICON-CONTAINING LIPID NANOPARTICLES FOR EFFICIENT HUMAN T CELL TRANSFECTION

[0003] TECHNICAL FIELD

[0004] The present invention belongs to the field of biomedicine and drug delivery.

[0005] The invention relates to LipexSil® lipid-containing nanoparticle compositions, in which a silicon-containing ionizable lipid is combined with a certain ratio of other lipids consituting the lipid nanoparticles (LNPs) resulting in a remarkable T cell transfection. In certain embodiments, the formulation comprises four components (ionizable lipid, structural lipid, helper lipid, and shield lipid), while in other embodiments a fifth lipid component is included. This ionizable cationic lipid family is showing remarkably low in vitro toxicity on T cells. The invention describes the production and characterization of the lipid nanoparticles and in vitro experiments demonstrating that the corresponding formulations with the LipexSil® lipids [WO2024023174] are superior to the current approach, delivering their payload, or cargo (e.g. RNA, DNA, mRNA, microRNA, siRNA, pDNA, circular DNA, small biologically active molecules) into T cells.

[0006] BACKGROUND

[0007] In vitro T cell transfection has become an emerging field in immunotherapy, particularly for engineering Chimeric Antigen Receptor (CAR) T cells. This approach involves introducing genetic material into T cells to reprogram their functionality, enabling them to target specific antigens. The two primary methods for achieving this are viral vector-mediated transfection and non-viral approaches such as electroporation and LNPs. LNPs have emerged as a promising non-viral transfection tool, particularly for mRNA delivery. These nanoparticles encapsulate mRNA, protecting it from degradation and facilitating its entry into T cells. Once inside the cell, the mRNA is translated into the desired protein. This method is advantageous because it avoids genomic integration, providing transient expression of the therapeutic gene. Moreover, LNPs have been optimized for reduced cytotoxicity and improved delivery efficiency, making them a viable alternative to both viral vectors and electroporation [Front. Immunol. 13:867013, Biotech. Adv.

[0008] 2021, 49, 107760.] 120852P1420PC November 21, 2025 AldexChem Kft.

[0009] Recently, several research groups have developed an array of lipids, lipoids, lipid-polymer hybrid nanoparticles, systematically varying the lipid compositions, LNPs’ physico-chemical parameters etc., and showed enhanced T cell transfection [ACS Biomater. Sci. Eng. 2022, 8, 722−733; Adv. Mater.2024, 36, 2310168; Nano Lett. 2022, 22, 533-542; Biotechnol. Bioeng.2021, 118, 1674-1687; JCR, 2023, 353, 196-215; J. Biomed. Sci. 2024, 31, 5; Mol. Ther. Methods Clin. Dev. 2023, 31, 101110; J. Hematol. Oncol. 2024, 17, 45; Nat. Commun. 2024, 15, 590; JCR 2024, 373, 837-852, Adv. Funct. Mater. 2024, 34, 2404510].

[0010] Recent advancements in LNP formulations have significantly enhanced mRNA delivery and expression in T cells. Incorporating structural lipids such as l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or helper lipids such as P-sitosterol has been shown to increase fusogenicity, thereby improving endosomal escape and cytosolic translation. Additionally, optimizing the ionizable lipid content within LNPs has led to more efficient mRNA transfection, as demonstrated by the superior performance of certain ionizable lipids like C12-200 and cKK-E12 in delivering mRNA and inducing potent immune responses Biomater. Sci., 2023, 11, 964-974].

[0011] Recently, several patent applications have been published on T cell transfection with LNPs [CN 118221780 A, CN 118206664A, WO2024077232, W02021077067, W02021000041, W02020210901, CN117210479A, CN116731196A].

[0012] Liu et al. describe the transfection of Jurkat cells with LNP compositions based on lipids SM-102, ALC-0315 and DLin-MC3-DMA, including the use of compositions comprising sitosterol (Liu, H., Chen, M. Z., Payne, T., Porter, C. J. H., Pouton, C. W. and Johnston, A. P. R., Adv. Funct. Mater. 2024, 34, 2404510). As shown in Liu et al., the performance of different ionizable lipids varied strongly, so that the performance of other lipids in the transfection of T cells cannot be predicted.

[0013] Furthermore, the development of antigen-presenting cell mimetic LNPs has streamlined the ex vivo process by enabling simultaneous T cell activation and transfection (Adv. Mater. 2024, 36, 2313226). These activating LNPs mimic antigen-presenting cells, facilitating rapid, one-step activation and transfection of human T cells with CAR mRNA. This innovation has been validated in murine xenograft models, where the resulting mRNA CAR T cells effectively reduced tumour burden, highlighting the potential of LNPs as a promising platform for rapid mRNA CAR T cell production.

[0014] In vitro T cell transfection is a rapidly evolving field, with non-viral methods like LNPs offering safer and more scalable alternatives to traditional viral vectors. Continued research into 120852P1420PC November 21, 2025 AldexChem Kft.

[0015] optimizing these technologies is crucial to overcoming current limitations and expanding the clinical applicability of T cell-based therapies. Future efforts will likely focus on improving transfection efficiency, reducing costs, and developing robust protocols for large-scale production.

[0016] SUMMARY OF THE INVENTION

[0017] Surprisingly, the present inventors have found that certain silicon-containing ionizable lipids as described in W02024 / 023174 can be combined with a certain mixture of: a helper lipid, a structural lipid, a shield lipid and optionally a fifth lipid component resulting in highly efficient human T cells transfection. Liu et al. (Adv. Funct. Mater. 2024, 34, 2404510) describe the transfection of Jurkat T cells using LNP formulations containing well-known ionizable lipids such as SM-102, ALC-0315, and DLin-MC3-DMA, including compositions comprising P-sitosterol. Their study demonstrates that the transfection performance of these structurally different ionizable lipids varies substantially, even under otherwise identical formulation and dosing conditions. Therefore, the skilled person would recognize that the structure-activity relationship for ionizable lipids in T cell transfection is highly unpredictable, and performance cannot be extrapolated from one lipid scaffold to another. In addition to this general unpredictability, the present invention has disclosed that minor structural changes within a given lipid scaffold can lead to significant differences in T cell transfection efficiency.

[0018] This is further corroborated in the present application that shows that the specific arrangement of the silicon motif and ester linkages in the silicon-containing lipids is critical for efficient T cell transfection. Lipids bearing alternative Si-motifs or differently positioned ester linkages (Compounds 3, 10, 11, 12) exhibited substantially lower activity, confirming that the beneficial effect arises from a particular and previously unrecognized structural configuration.

[0019] Additionally, the present inventors have shown that compositions based on silicon-containing lipids with a different substitution pattern could be used for the targeting of spleen cells, but were not successful in T cell transfection (data not shown here).

[0020] Accordingly, the skilled person could not have reasonably predicted that such minor and specific modifications within the silicon and ester motifs would lead to an optimized class of ionizable lipids capable of enhanced T cell transfection.

[0021] Thus, in first aspect the present invention relates to lipid nanoparticle composition comprising: 120852P1420PC November 21, 2025 AldexChem Kft.

[0022] (a) an ionizable cationic lipid of formula (I):

[0023] T’-G’N I'D1

[0024] p

[0025] (I)

[0026] or a salt thereof,

[0027] wherein:

[0028] G1is unsubstituted C2-C9alkylene,

[0029] T* is

[0030] (a) b^Xa-R!

[0031]

[0032] or(b) R / ^Xa-b1

[0033] wherein

[0034] b1is a bond to G1,

[0035] Xi and X2are the same or different and each independently represents O or S,

[0036] R1is linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and X2;

[0037] R2is

[0038] linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and the carbon linking X1and X2;

[0039] R3 is

[0040] _ b2R / 1.six xsi>^2

[0041] R4 S. z \ Z \

[0042]

[0043] X3X3 or X3X3X3X3

[0044] wherein 120852P1420PC November 21, 2025 AldexChem Kft.

[0045] b2is a link to Xi,

[0046] Y i is -O- or -CH2- or -O-CH2-CH2- wherein -CH2- is attached to Si,

[0047] each X3is independently selected from the group consisting of C1-C4 alkyl or C1-C4 alkoxy,

[0048] R4is linear C2-C17alkyl, or non-linear C3-C17 alkyl, C3-C2o alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and Yi;

[0049] D1is selected from the group consisting of

[0050]

[0051] wherein

[0052] b4is a bond to nitrogen,

[0053] R9is selected from C1-C6alkyl, cyclopentyl, cyclohexyl, hydroxyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, 4-hydroxy benzyl,

[0054]

[0055] m is an integer selected from 1 and 6,

[0056] n is an integer selected from 0 and 6,

[0057] o is an integer selected from 0 and 6; provided that the sum of n+o is at least 1;

[0058] P is selected from

[0059] G2-T3

[0060] wherein 120852P1420PC November 21, 2025 AldexChem Kft.

[0061] G2is as defined above;

[0062] T3is

[0063]

[0064] b5X5-R13

[0065] wherein

[0066] b5is a bond to G2,

[0067] X4and X5can be the same or different and each independently is O, S.

[0068] R13 is linear C2-C17 alkyl, non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and X5;

[0069] (b) a structural lipid as second component selected from l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2dimyristoyl-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1- Pahnitoyl-2-Oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-dipahnitoyl-sn-glycero- 3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 - pahnitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);

[0070] (c) a helper lipid as third component selected from P-sitosterol, ergosterol, campesterol, stigmasterol, and pharmaceutically acceptable salts thereof, and mixtures thereof; (d) a shield lipid as fourth component selected from poly(ethylene glycol)-modified diacylglycerols (PEG-DAG), in particular a PEG-DAG selected from DSPE-PEG and DMG-PEG;

[0071] (e) and optionally a fifth lipid component selected from

[0072] (el) l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) (DOPG), dipahnitoylphosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3 - phosphoglycerol (DSPG), 1,2-dimyristoyl-sn-glycero-3 -phosphoglycerol (DMPG), 1 -pahnitoyl-2-oleoyl-sn-glycero-3 -phosphoglycerol (POPG), phosphatidylserine (PS; including dioleoyl PS (DOPS) and 18:0 PS), 1,2- dipahnitoyl-sn-glycero-3 -phosphate (DPP A), 1,2-dioleoyl-sn-glycero-3 - phosphate (DOPA), and pharmaceutically acceptable salts thereof; or 120852P1420PC November 21, 2025 AldexChem Kft.

[0073] (e2) Chrysin, Apigenin, Pinocembrin, Galangin and pharmaceutically acceptable salts thereof, and mixtures thereof;

[0074] wherein the composition comprises either four or five lipid components in total.

[0075] In a second aspect, the present invention relates to a lipid nanoparticle composition according to the invention for use in delivering one or more nucleic acids to human T cells.

[0076] In a third aspect, the present invention relates to a lipid nanoparticle composition according to the invention for use in delivering one or more nucleic acids to human T cells, wherein the lipid nanoparticle composition is selected from:

[0077] (i) a four-component system consisting of an ionizable cationic lipid of formula I as defined in (a), a structural lipid as defined in (b), a helper lipid as defined in (c), and a shield lipid as defined in (d); or

[0078] (ii) a five-component system further comprising a fifth lipid component as defined in (el) or (e2).

[0079] In a fourth aspect, the present invention relates to a lipid nanoparticle composition according to the invention for use in delivering one or more nucleic acids to human T cells, wherein the nucleic acid is selected from mRNA, cDNA, ceDNA, pDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof.

[0080] In a fifth aspect, the present invention relates to a lipid nanoparticle composition according to the invention for the treatment of a disease or condition selected from autoimmune diseases, cardiac fibrosis, B-cell malignancies, infectious diseases, transplant-related disorders involving human T cells.

[0081] In a sixth aspect, the present invention relates to a lipid nanoparticle composition according to the invention for the treatment of a disease or condition selected from autoimmune diseases, cardiac fibrosis, B-cell malignancies, infectious diseases, transplant-related disorders involving human T cells, wherein the nucleic acid is selected from mRNA, cDNA, ceDNA, pDNA, ceDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof.

[0082] In a seventh aspect, the present invention relates to a pharmaceutical composition comprising a lipid nanoparticle composition according to the present invention comprising one or 120852P1420PC November 21, 2025 AldexChem Kft.

[0083] more therapeutically active nucleic acids selected from mRNA, cDNA, ceDNA, pDNA, ceDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof and a pharmaceutically acceptable carrier.

[0084] DETAILED DESCRIPTION

[0085] The present invention is based on the surprising finding that novel LNP compositions based on a particular class of ionizable cationic lipids could be found that show high transfection efficiency on human T cells. The present invention provides novel LNP compositions, details of the formation of LNPs and their characterization as well as IC50 values of the ionizable cationic lipids and transfection experiments of loaded LNPs.

[0086] Thus, in first aspect the present invention relates to lipid nanoparticle composition comprising an ionizable cationic lipid of formula (I):

[0087] XD1

[0088] T1N

[0089] I

[0090] P

[0091]

[0092] (I)

[0093] or a salt thereof,

[0094] wherein:

[0095] G1is unsubstituted C2-C9alkylene,

[0096] T1is

[0097] R3R3

[0098] (a) b

[0099]

[0100] 1X2-RI or X2-b1

[0101] wherein

[0102] b1is a bond to G1,

[0103] Xi and X2are the same or different and each independently represents O or S, 120852P1420PC November 21, 2025 AldexChem Kft.

[0104] R1is linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and X2;

[0105] R2is

[0106] linear C1-C17 alkyl, non-linear C3-C17 alkyl, C3-C17 alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and the carbon linking X1and X2;

[0107] R3is

[0108] JK „b2

[0109] FV Si Si

[0110]

[0111] R4X X3X3or x3x3x3x3

[0112] wherein

[0113] b2is a link to Xi,

[0114] Y 1 is -O- or -CH2- or -O-CH2-CH2- wherein -CH2- is attached to Si,

[0115] each X3is independently selected from the group consisting of C1-C4 alkyl or C1-C4 alkoxy,

[0116] R4is linear C2-C17alkyl, or non-linear C3-C17 alkyl, C3-C2o alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and Yi;

[0117] D1is selected from the group consisting of

[0118]

[0119] wherein

[0120] b4is a bond to nitrogen, 120852P1420PC November 21, 2025 AldexChem Kft.

[0121] R9is selected from C1-C6alkyl, cyclopentyl, cyclohexyl, hydroxyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, 4-hydroxy benzyl,

[0122]

[0123] m is an integer selected from 1 and 6,

[0124] n is an integer selected from 0 and 6,

[0125] o is an integer selected from 0 and 6; provided that the sum of n+o is at least 1;

[0126] P is selected from

[0127] G2-T3

[0128] wherein

[0129] G2is as defined above;

[0130] T3isb5''X5-R13

[0131]

[0132] wherein

[0133] b5is a bond to G2,

[0134] X4and X5can be the same or different and each independently is O, S.

[0135] R13 is linear C2-C17 alkyl, non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and X5;

[0136] a structural lipid as second component, wherein said structural lipid is selected from l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 - phosphocholine (DOPC), l-Pahnitoyl-2-Oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2- 120852P1420PC November 21, 2025 AldexChem Kft.

[0137] dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero- phosphocholine (DUPC), 1 -pahnitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), a helper lipid as third component, wherein the helper lipid is selected from P-sitosterol, ergosterol, campesterol, stigmasterol, and pharmaceutically acceptable salts thereof, and mixtures thereof, a shield lipid as fourth component, wherein the shield lipid is selected from poly(ethylene glycol)-modified diacylglycerols (PEG-DAG), in particular a PEGDAG selected from DSPE-PEG and DMG-PEG, and optionally a fifth lipid component, wherein the fifth lipid component is selected from l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) (DOPG), dipahnitoylphosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero- 3 -phosphoglycerol (DSPG), l,2-dimyristoyl-sn-glycero-3 -phosphoglycerol (DMPG), 1- pahnitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), phosphatidylserine (PS; including dioleoyl PS (DOPS) and 18:0 PS), l,2-dipahnitoyl-sn-glycero-3 -phosphate (DPP A), l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA), and pharmaceutically acceptable salts thereof, or Chrysin, Apigenin, Pinocembrin, Galangin and pharmaceutically acceptable salts thereof, and mixtures thereof, wherein the composition comprises either four or five lipid components in total.

[0138] In the context of the present invention, by human T cells we mean CD4+ T cells, CD8+ T cells, y8 T cells, MAIT cells, NKT cells, Naive T cells, Effector T cells, Memory T cells, Treg cells.

[0139] As used herein, the term ‘lipid component’ refers to any molecule exhibiting lipophilic or amphiphilic character that contributes to the structural integrity, encapsulation efficiency, or functional performance of the lipid phase. This term encompasses classical lipids (e.g., phospholipids, ionizable lipids) as well as lipid-mimetic compounds, such as hydrophobic flavonoids (e.g. Chrysin, Apigenin, Pinocembrin, Galangin), sterols, or their derivatives, that can integrate into or interact with the lipid matrix.

[0140] By unsubstituted C2-C9 alkylene we mean ethylene (-(CH2)2-), propylene (-(CH2)3-), butylene (-(CH2)4-), pentylene (-(CH2)5-), hexylene (-(CH2)6-), heptylene (-(CH2)7-), octylene (-(CH2)8-), or nonylene (-(CH2)9-).

[0141] By linear C1-C17 alkyl we mean methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, or heptadecyl.

[0142] By non-linear C3-C17 alkyl we mean a saturated branched alkyl group containing 3 to 17 carbon atoms, e.g. isopropyl, isobutyl, 3 -methylpentyl, 2-propylpentyl, 2-methylhexyl, 2- 120852P1420PC November 21, 2025 AldexChem Kft.

[0143] ethylhexyl, 2-ethyldecyl, 2-propyldecyl, 2-butyldecyl, 2-pentyldecyl, 2-hexyldecyl, or 2-heptyldecyl.

[0144] By C1-C4 alkyl group we mean a linear or non-linear alkyl group containing 1 to 4 carbon atoms, preferably methyl.

[0145] By C3-C17 alkenyl containing one double bond or C3-C20 alkenyl containing one double bond we mean a linear or non-linear alkyl group as defined above containing 3 to 17 or 3 to 20 carbon atoms respectively, which contains one double bond in the chain, e.g. hex-3 -en-l-yl, oct-3 -en-l-yl, dec-3 -en-l-yl, dec-2-en-l-yl, undec-3-en-2-yl, preferably hex-3 -en-l-yl, oct-3 -en-l-yl.

[0146] By salts of the compounds of formula (I) we mean salts of the compounds of formula (I) with inorganic or organic acids. Preferred salts are those with pharmaceutically acceptable acids. The salts are e.g. chloride, sulfate, phosphate, formate, acetate, fumarate, maleate, oxalate, citrate or tartrate. The salts formed during purification or isolation are also subject of the invention.

[0147] By stereoisomers we mean optical and geometric isomers. The compounds of formula (I) may contain one or more asymmetric carbon atoms thus they can exist in the form of optical isomers, enantiomers or diastereomers. The compounds of formula (I) may contain double bounds and the groups attached to the double bond can have different (cis or trans) confirmations, e.g. cis or trans fatty acid moieties.

[0148] In a particular embodiment, the present invention relates to lipid nanoparticle composition of the present invention, which comprises an ionizable cationic lipid according to formula (I)

[0149] _. G1X^D1

[0150] T1N

[0151] I

[0152] P

[0153]

[0154] (I)

[0155] or a salt thereof,

[0156] wherein:

[0157] G1is unsubstituted C2-C9alkylene,

[0158] T1is 120852P1420PC November 21, 2025 AldexChem Kft.

[0159] , R3, R3

[0160] XI

[0161] (

[0162]

[0163] a) b1"^x'x2-R1 or(b) R2- X2-b1

[0164] wherein

[0165] b1is a bond to G1,

[0166] Xi and X2are the same or different and each independently represents O or S,

[0167] R1is linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and X2;

[0168] R2is

[0169] linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and the carbon linking X1and X2;

[0170] R3is

[0171] ^Yi. Ox„b2

[0172] R4 Si RZ Si S

[0173] / \ / 4 t i %

[0174]

[0175] X3X3or X3X3X3X3

[0176] wherein

[0177] b2is a link to Xi,

[0178] Y 1 is -O- or -CH2- or -O-CH2-CH2- wherein -CH2- is attached to Si,

[0179] each X3is independently selected from the group consisting of C1-C4 alkyl or C1-C4 alkoxy,

[0180] R4is linear C2-C17alkyl, or non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and Yi;

[0181] D1is selected from the group consisting of 120852P1420PC November 21, 2025 AldexChem Kft.

[0182]

[0183] wherein

[0184] b4is a bond to nitrogen,

[0185] R9is selected from C1-C6alkyl, cyclopentyl, cyclohexyl, hydroxyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, 4-hydroxy benzyl,

[0186]

[0187] m is an integer selected from 1 and 6,

[0188] n is an integer selected from 0 and 6,

[0189] o is an integer selected from 0 and 6; provided that the sum of n+o is at least 1;

[0190] P is selected from

[0191] G2-T3

[0192] wherein

[0193] G2is as defined above;

[0194] T3is

[0195] b s5t X5-R13

[0196] wherein

[0197] b5is a bond to G2,

[0198] X4and X5can be the same or different and each independently is O, S. 120852P1420PC November 21, 2025 AldexChem Kft.

[0199] R13 is linear C2-C17 alkyl, non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and X5.

[0200] In particular embodiments, each of Xi, X2, X4and X5is O.

[0201] In particular embodiments, G1is selected from C6-C9-alkenyl.

[0202] In particular embodiments, R3 is

[0203]

[0204] 3 3, wherein Y 1 is -CH2-, each of X3 is methyl, and R4 is selected from C5-C7-alkyl.

[0205] In particular embodiments, Ri, where present, or R2, where present, is selected from C7-C8-alkyl. In particular embodiments, where Ri is present, Ri is C7-alkyl. In particular embodiments, where R2 is present, R2 is Cg-alkyl.

[0206] In particular embodiments, G2is selected from C4-C6-alkenyl, an in particular is C5-alkenyl.

[0207] In particular embodiments, D1is

[0208]

[0209] ''m » wherein m is selected from 1 and 2.

[0210] In particular embodiments, each of Xi, X2, X4and X5is O, G1is selected from C6-C9-

[0211] alkenyl, R3 is

[0212]

[0213] 3 3, wherein Y 1 is -CH2-, each of X3 is methyl, and R4is selected from C5-C7-alkyl, R1, where present, is C7-alkyl, R2, where present, is Cg-alkyl, G2is Cs-alkenyl, and D1b4r7

[0214] i

[0215]

[0216] s ' / m ’ wherein m is selected from 1 and 2.

[0217] In certain embodiments the lipid nanoparticle composition is selected from:

[0218] (iii) a four-component system consisting of an ionizable cationic lipid of formula I as defined in (a), a structural lipid as defined in (b), a helper lipid as defined in (c), and a shield lipid as defined in (d); or

[0219] (iv) a five-component system further comprising a fifth lipid component as defined in (el) or (e2).

[0220] In some embodiment the fifth lipid component is selected from l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) (DOPG), dipahnitoylphosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3 -phosphoglycerol (DSPG), l,2-dimyristoyl-sn-glycero-3 -phosphoglycerol (DMPG), 1- 120852P1420PC November 21, 2025 AldexChem Kft.

[0221] pahnitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), phosphatidylserine (PS; including dioleoyl PS (DOPS) and 18:0 PS), l,2-dipahnitoyl-sn-glycero-3 -phosphate (DPP A), 1,2-dioleoyl-sn-glycero-3 -phosphate (DOPA), and pharmaceutically acceptable salts thereof.

[0222] In a particular embodiment, the fifth lipid component is l,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) and pharmaceutically acceptable salts.

[0223] In some embodiment the fifth lipid component is selected from Chrysin, Apigenin, Pinocembrin, Galangin and pharmaceutically acceptable salts thereof, and mixtures thereof.

[0224] In a particular embodiment, the fifth lipid component is Chrysin, and pharmaceutically acceptable salts thereof.

[0225] In a particular embodiment, the present invention relates to lipid nanoparticle composition, wherein said components are present at the following amounts, expressed as mole percent of total lipid: ionizable cationic lipid 20-70 mol %, structural lipid 2–40 mol %, helper lipid 5–40 mol %, shield lipid 0,5-10 mol %, wherein the total of components (ionizable cationic lipid, structural lipid, helper lipid shield lipid) equals 100 mol% (excluding solvent, buffer, counterions, and payload).

[0226] In a particular embodiment, the present invention relates to lipid nanoparticle composition, wherein said components are present at the following amounts, expressed as mole percent of total lipid: ionizable cationic lipid 20-70 mol %, structural lipid 2–40 mol %, helper lipid 5–40 mol %, shield lipid 0,5-10 mol %, fifth lipid component 1-25 mol %, wherein the total of components (ionizable cationic lipid, structural lipid, helper lipid shield lipid, fifth lipid component) equals 100 mol% (excluding solvent, buffer, counterions, and payload).

[0227] In certain embodiments, the present invention relates to lipid nanoparticle composition, wherein said helper lipid is P-sitosterol, ergosterol, campesterol, stigmasterol, and pharmaceutically acceptable salts thereof, and mixtures thereof.

[0228] In a particular embodiment, the present invention relates to lipid nanoparticle composition, wherein said helper lipid is P-sitosterol, and pharmaceutically acceptable salts thereof, and mixtures thereof.

[0229] In certain embodiments, the present invention relates to lipid nanoparticle composition, wherein said structural lipid is l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), l,2dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3- 120852P1420PC November 21, 2025 AldexChem Kft.

[0230] phosphocholine (DOPC), l-Palmitoyl-2-Oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-dipahnitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC)

[0231] In a particular embodiment, the present invention relates to lipid nanoparticle composition, wherein said structural lipid is DSPC, and pharmaceutically acceptable salts thereof, and mixtures thereof.

[0232] In certain embodiments, the said shield lipid is selected from PEG-DAG or a combination thereof, in particular PEG-DSPE or PEG-DMG.

[0233] In some embodiments the shield lipid is a PEG lipid comprising a PEG moiety from 1,000 to 20,000 daltons

[0234] In a particular embodiment the T’-G1moiety of said ionizable cationic lipid has one of the following structures:

[0235]

[0236] In another particular embodiments, the P moiety of said ionizable cationic lipid is a G2-T3moiety, with T3having one of the following structures:

[0237]

[0238] In certain embodiments, the lipid nanoparticle composition, wherein the P moiety of said ionizable cationic lipid is a G2-T3moiety with T3, wherein R13is linear C2-C17alkyl:

[0239]

[0240] In a particular embodiment, the lipid nanoparticle composition, wherein the P moiety of said ionizable cationic lipid is a T3-G2moiety, with T3having the following structures:

[0241] O

[0242]

[0243] 120852P1420PC November 21, 2025 AldexChem Kft.

[0244] In a particular embodiment, the lipid nanoparticle composition, wherein the P moiety of said ionizable cationic lipid is a T3-G2having the following structure:

[0245]

[0246] In some embodiments, the lipid nanoparticle composition, wherein the D1moiety of said ionizable cationic lipid has one of the following structures:

[0247]

[0248] ‘OH

[0249] In a particular embodiment, the lipid nanoparticle composition, wherein said ionizable cationic lipid has one of the following structures:

[0250]

[0251] In certain embodiments, the lipid nanoparticle composition comprises, based on the total lipid content, about 50 mol % of an ionizable cationic lipid, about 10 mol % DSPC, about 38.5 mol % P-sitosterol, and about 1.5 mol % DMG-PEG

[0252] In certain embodiments, the lipid nanoparticle composition comprises, based on the total lipid content, about 50 mol % of an ionizable cationic lipid, about 8 mol % DSPC, about 38.5 mol % P-sitosterol, and about 1.5 mol % DMG-PEG, 2 mol% DSPG.

[0253] In certain embodiments, the lipid nanoparticle composition comprises, based on the total lipid content, about 50 mol % of an ionizable cationic lipid, about 10 mol % DSPC, about 28.5 mol % P-sitosterol, and about 1.5 mol % DMG-PEG, 10 mol% Chrysin. 120852P1420PC November 21, 2025 AldexChem Kft.

[0254] In the context of the present application, the term “about” in the context of a numerical value refers to a range of plus / minus 5 % of the given value, e. g. when the numerical value is 50 mol %, the term “about 50 mol %” refers to a range from 47.5 mol % to 52.5 mol %.

[0255] In some embodiments, the LNP is loaded with at least one payload selected from the group consisting of mRNA, cDNA, ceDNA, pDNA, microRNA, siRNA, gRNA, CAS 9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, small biologically active molecule and any combination thereof.

[0256] In some embodiments, the lipid nanoparticle composition is used for delivering one or more nucleic acids to human T cells for the treatment of a disease or condition selected from autoimmune diseases, cardiac fibrosis, B-cell malignancies, infectious diseases, transplant-related disorders.

[0257] In some embodiments, the lipid nanoparticle composition is used for delivering one or more nucleic acids to human T cells, wherein the fifth lipid component DSPG or Chrysin.

[0258] In some embodiments, the lipid nanoparticle composition for use in delivering one or more nucleic acids to human T cells, wherein the nucleic acid is selected from mRNA, cDNA, ceDNA, pDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof.

[0259] Another aspect of the invention related to the lipid nanoparticle according to the present invention for use in the transfection of human T cells, wherein said lipid nanoparticle composition is loaded with at least one payload selected from the group consisting of mRNA, cDNA, ceDNA, pDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, small biologically active molecule and any combination thereof.

[0260] In another aspect, the present invention relates to a pharmaceutical composition comprising the lipid nanoparticle composition of the present invention and at least one payload selected from the group consisting of mRNA, cDNA, ceDNA, pDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, small biologically active molecule and any combination thereof.

[0261] In particular embodiments, the amount of payload being present is expressed by the ratio of the number of negative charges N of the payload being present to the number of positive charges P of the ionizable lipids being present. In particular embodiments, the N / P ratio is between about 6 and 12, and is in particular about 9. 120852P1420PC November 21, 2025 AldexChem Kft.

[0262] In particular embodiments, the payload is a mRNA or cDNA coding for a chimeric antigen receptor.

[0263] EXAMPLES

[0264] The various embodiments of this application can be more clearly understood through the following illustrative examples. These examples are provided for explanatory purposes and do not restrict the scope of the application.

[0265] LNP FORMULATION AND CHARACTERIZATION

[0266] Formulation:

[0267] All lipids were dissolved in 100% ethanol in a concentration ranging from 1-25 mM and stored at -20°C. Before formulating, lipids were heated to 37°C, vortexed for 2 minutes and cooled to room temperature. The mRNA was diluted to a final concentration of 0.1-0.065 pg / pL in 10 mM citrate (pH 4.0). Lipid and mRNA solutions where mixed in a 3:1 (mRNA aqueous: lipid in EtOH) ratio by manual mixing using pipettes, vortexed for 30 seconds and incubated at room temperature for 15 minutes. Next the samples were diluted lOx with PBS (pH 7.4). Formulations were placed onto Amicon® Ultra Centrifugal Filters (50 kDa MWCO). After buffer exchange 3 times with lOx volume of PBS (pH 7.4) of the original sample volume, samples were concentrated to their original volume.

[0268] Formulation A:

[0269] LNP composition (mol%)

[0270] Ionizable DSPC P- DMG-PEG N / P

[0271] lipid Sitosterol (2000) ratio

[0272] 50 10 38.5 1.5 9

[0273]

[0274] Formulation B:

[0275] LNP composition (mol%)

[0276] Ionizable DSPC P- DMG-PEG DSPG N / P

[0277] lipid Sitosterol (2000) ratio

[0278]

[0279] 120852P1420PC November 21, 2025 AldexChem Kft.

[0280] 50 8 38.5 1.5 2 9

[0281]

[0282] Formulation C:

[0283] LNP composition (mol%)

[0284] Ionizable DSPC P- DMG-PEG Chrysin N / P

[0285] lipid Sitosterol (2000) ratio

[0286] 50 10 28.5 1.5 10 9

[0287]

[0288] DLS (Size, PDI and Zeta potential):

[0289] Characterization of mRNA-LNPs was done with nanoparticle tracking analysis (NTA) (PMX-230 ZetaView TWIN Laser, Particle Metrix) to determine size, polydispersity and zeta potential of the LNP samples. All dialyzed sample were diluted 10000 times in 0.1x PBS buffer (pH 7.4) and size and zeta potential were measured.

[0290] Encapsulation assay:

[0291] Free and total mRNA concentrations were determined by the RiboGreen assay (Quant-it™ RiboGreen RNA Assay Kit, Invitrogen) according to the manufacturer’s protocol. In short, samples were 25x diluted in TE buffer pH 7.4. 25uL of all samples were mixed 1:1 with TE buffer, or with TE buffer containing 0.3% Triton X-100 buffer. Next samples were incubated in a 96 well plate at 37°C for 10 min. to lyse mRNA-LNP in the presence of Triton X-100. Meanwhile, the RiboGreen reagent was diluted 1:200 in TE buffer pH 7.5. After 10 minutes the plate with the samples was cooled to room temperature and 50 pL of RiboGreen solution was added to each well. The fluorescent signal in all wells was determined using a plate reader (excitation = 485, emission = 530). A standard curve (0.02-1.0 ng / pl RNA) was used to calculate the total and free concentration of mRNA in the LNP samples.

[0292] Results:

[0293] The physico-chemical properties (size, PDI, zeta potential, EE%) are summarized in Table 1 and demonstrate that the LNP compositions in accordance with the present invention have physicochemical properties that are similar to those of prior art composition that are frequently used for carrying and transporting cargo material. 120852P1420PC November 21, 2025 AldexChem Kft.

[0294] Table 1: Physico-chemical properties of the produced LNPs

[0295] Lipid Structure Formulation Size PDI zeta potential EE%

[0296] (nm) (mV)

[0297] 1 > > A 97.8 0.131 -4.97 74.7

[0298] 2 Xj A 107.3 0.134 -7.43 78.8

[0299] 3 A 118.4 0.133 -5.29 46.8

[0300] 4 A 113.3 0.116 -4.95 76.3

[0301] / z\z\z\z\z\XS^

[0302] 5 A 124.0 0.182 -3.27 71.2

[0303] 6 A 116.3 0.172 -6.21 65.0

[0304] 7 A 116.6 0.191 -3.32 59.0

[0305] 8 A 131.8 0.244 -7.34 75.9

[0306] 9 A 116.4 0.208 -3.82 59.9

[0307] 10 A 119.8 0.225 -5.20 40.7

[0308] 11 A 120.1 0.220 -4.35 80.3

[0309] 12 A 112.7 0.231 -4.21 68.1

[0310] 13 SM-102 ref. (CAS# 2089251- A 116.3 0.329 -5.38 73.0 47-6)

[0311] 14 ALC-0315 ref. (CAS# A 108.2 0.257 -4.68 48.9 2036272-55-4)

[0312]

[0313] 120852P1420PC November 21, 2025 AldexChem Kft.

[0314] 2 B 121.9 0.228 -11.18 59.7

[0315] 3 B 118.4 0.211 -13.16 51.5

[0316] 4 B 109.2 0.206 -11.69 74.8

[0317] 5 B 121.8 0.228 -12.20 79.7

[0318] 8 B 126.4 0.226 -12.00 76.6

[0319] 13 SM-102 ref. (CAS# 2089251- B 120.6 0.13 -8.9 91.3 47-6)

[0320] 14 ALC-0315 ref. (CAS# B 115.4 0.15 -12.6 56.2 2036272-55-4)

[0321] 2nH C 129.9 0.157 -6.27 78.3

[0322] 3 c 142.2 0.215 -8.64 73.4

[0323] .

[0324] 4 c 135.9 0.188 -7.17 74.8

[0325] 5 c 129.8 0.207 -8.27 77.0

[0326] 7 c 130.8 0.205 -7.86 76.4

[0327] 8 c 130.9 0.329 -7.58 75.4

[0328] 13 SM-102 ref. (CAS# 2089251- c 117.2 0.15 -7.3 87.5 47-6)

[0329] 14 ALC-0315 ref. (CAS# c 107.1 0.14 -11.5 44.6 2036272-55-4)

[0330]

[0331] 120852P1420PC November 21, 2025 AldexChem Kft.

[0332] HUMAN T CELL ISOLATION FROM PERIPHERAL BLOOD

[0333] Buffers and media:

[0334] Isolation Buffer: 2 mM EDTA, 2 % FBS in PBS

[0335] RPMI media:

[0336] for PBMC isolation: 10 % FBS, lx Glutamax

[0337] for T cell culturing: 10 % FBS, lx Glutamax, lx Penicillin- Streptomycin PBMC isolation was performed with standard protocol from human peripheral blood.

[0338] The yield of the isolation varies but approximately 16-20 ml blood yields 10 million T cells.

[0339] T cell isolation protocol

[0340] (Dynabeads™ Untouched™ Human T Cells Kit, standard protocol):

[0341] Preparation of cell mixture:

[0342] - 10 ml cell suspension is transferred to 15 ml tube and volume is increased to 15 ml - centrifuge at 360 g for 6 min

[0343] - media is discarded and Isolation Buffer is added proportional to cell number (e.g. 10 million cells 100 pl Isolation Buffer)

[0344] - to 100 pl cell suspension 20 pl FBS and 20 pl Antibody is added and the mixture is incubated at 4°C for 20 min

[0345] - add 4 ml Isolation Buffer, mix well with pipetting

[0346] - centrifuge at 360 g for 8 min

[0347] Washing of beads:

[0348] - use beads proportional to cell number (10 million cells - 100 pl beads)

[0349] - vortex tube with beads, take 100 pl into an Eppendorf tube

[0350] - add 1 ml Isolation Buffer and place it into the magnetic rack

[0351] - discard liquid from beads by pipetting

[0352] - add proportional amount of Isolation Buffer (100 pl for 1 million cells)

[0353] Isolation of T cells:

[0354] - remove supernatant from cell mixture and resuspend in proportional amount of Isolation Buffer (100 pl for 10 million cells)

[0355] - add washed beads

[0356] - incubate at RT for 15 minutes on an orbital shaker

[0357] - add proportional amount of Isolation Buffer (1.5 ml for 10 million cells but never less than 1 ml) 120852P1420PC November 21, 2025 AldexChem Kft.

[0358] - carefully resuspend with pipette

[0359] - put it on the magnetic rack for 2 minutes

[0360] - collect supernatant (containing T cells)

[0361] - to the magnetic beads add again 1.5 ml Isolation Buffer and resuspend carefully with a pipette

[0362] - put it on the magnetic rack for 2 minutes

[0363] - collect supernatant (containing T cells) and combine with the first isolate

[0364] - count cells

[0365] - centrifuge cells at 300 g for 5 min

[0366] - discard supernatant

[0367] - resuspend T cells in RPMI medium (with Pen / Strep) and culture in Petri dish or plate at the desired concentration in cell culture plates

[0368] IN VITRO TOXICITY STUDY ON HUMAN T CELL

[0369] Human T cells were isolated from human peripheral blood. Blood was drawn and PBMCs were isolated and incubated overnight in a 10 cm Petri dish in growth medium. On the next day T cells were isolated using the Dynabeads™ Untouched™ Human T Cells Kit as described above. T cells were maintained in RPMI medium supplemented with 10% FBS. Cell toxicity measurements were carried out in 384-well microtiter plates with 20,000 cells seeded in each well. Test measurements were carried out in triplicate, and control measurements in quadruplicate. The ethanolic lipid stock solutions were diluted with the medium to the target concentrations. After isolation, cells were treated with an increasing concentration of the lipid compounds (8.192, 20.48, 51.2, 128, 320, 800 pM). 48 h post-treatment, following visual inspection under an inverted microscope, cell viability was measured by resazurin assay. Viability of treated cells was calculated relative to that of ethanol vehicle controls. Average values and SD were calculated from repeated measurements. CRCs were obtained and corresponding IC50values were calculated where applicable. The determined IC50values are shown in Table 2.

[0370] Resazurin reagent:

[0371] Resazurin reagent (Sigma- Aldrich) was dissolved in PBS (pH 7.4) at 0.15 mg / ml concentration, 0.22 μm filtered and aliquoted at -20 °C. 10 pl resazurin stock solution was added to wells. After 18 hours incubation at 37 °C under 5 % CO2fluorescence (530 nm excitation / 580 nm emission) was recorded on a multimode microplate reader. 120852P1420PC November 21, 2025 AldexChem Kft.

[0372] Table 2: Determined IC50values of the lipids on human T cells

[0373] Lipid Structures IC50value (μM) 1 >800 YU

[0374] 2 >800 ^

[0375] 3 >800 U

[0376] ^

[0377] 4 >800

[0378] 5 >800

[0379] M M

[0380] 6 731 ^

[0381] 7 >800

[0382] 8 >800

[0383] 9 >800

[0384] 10 397

[0385] 11 >800

[0386] 12 W >800

[0387]

[0388] 120852P1420PC November 21, 2025 AldexChem Kft.

[0389] 13 SM-102 ref. (CAS# 2089251-47-6) 417 14 ALC-0315 ref. (CAS# 2036272-55-4) >800 15 87

[0390] 16 148

[0391] 17 75

[0392]

[0393] IN VIVO TRANSFECTION STUDY ON HUMAN T CELLS

[0394] Studies were carried out in 96 well round-bottom microtiter plates with 75,000 cells seeded in each well in 50 pl RPMI medium. After an overnight incubation at 37°C, cells were treated with 100 ng mRNA containing LNP formulations. LNP formulations were pretreated with 2x volume of 10% FBS (in PBS) for 15 min and diluted to their final concentration in cell culture media (2 ng / pl mRNA) before addition to cells (50 pl).

[0395] 24 h post-treatment, following visual inspection under an inverted microscope, 50 pl cell culture media was removed carefully not to discard the cells and cells were lysed with the addition of 12.5 pl 5x lysis buffer (Lysis Buffer, Promega) at 37°C for 10 min. After lysis 50 pl luciferin substrate (Bright-Glo™ Luciferase Assay System, Promega) was added and mixed with the lysates. The full volume of the mixture was transferred into white plates for luminescence measurement, and the results are shown in Table 3. 120852P1420PC November 21, 2025 AldexChem Kft.

[0396] Table 3: Luminescence signals (counts per second)

[0397] Luminescence signal (CPS) at 100 ng mRNA Lipid Structures Formulation Formulation Formulation A B C

[0398] 1 24400

[0399] 2 41454 22376 85163

[0400] 3 0000^ 16632 9990 21761

[0401] 4 27205 17480 49711

[0402] 5 21013 17446 22133

[0403] 6 24706

[0404] 7 'N27523 26842

[0405] 8 37551 26183 56988

[0406] 9 17281

[0407] 10 9844

[0408] 11 22369

[0409] 12 xxx,- ^“xxxxx, -x^™ 18224

[0410] 13 SM-102 ref. (CAS# 2089251-47- 26751 5236 8902

[0411] 6)

[0412] 14 ALC-0315 ref. (CAS# 2036272- 10634 5991 4776

[0413] 55-4)

[0414]

[0415] 120852P1420PC November 21, 2025 AldexChem Kft.

[0416] CONCLUSION

[0417] The present invention describes lipid nanoparticles that demonstrate significant human T cells transfection capabilities. The primary component of these LNPs is the proprietary LipexSil® ionizable lipid, which is combined with a certain ratio of other lipids constituting the LNPs resulting in a remarkable T cell transfection. In certain embodiments, the formulation consists of four components (ionizable lipid, structural lipid, helper lipid, and shield lipid), while in other embodiments a fifth lipid component is included. This has been validated through the transfection of Flue mRNA reporter. Furthermore, key feature of these ionizable lipids is the lower toxicity (based on IC50values) on human T cells relative to SM-102 as the reference lipid. ALC-0315 -based compositions on the other side, which appear to have a rather low toxicity on human T cells as well, do no result in efficient T cell transfection.

[0418] These lipids to be used in the LNP compositions in accordance with the present invention are relatively simple from synthetic point of view, and they can be produced from readily available raw materials. Based on these findings, these LNP formulations represent promising candidates for further clinical developments programs (e.g. CAR T cell therapies).

Claims

120852P1420PC November 21, 2025 AldexChem Kft.CLAIMS1. A lipid nanoparticle composition, comprising:(a) an ionizable cationic lipid of formula (I);Ip(I)or a salt thereof,wherein:G1is unsubstituted C2-C9alkylene,T1isR3R3(a) b1X2-RI X2-b1whereinb1is a bond to G1,Xi and X2are the same or different and each independently represents O or S,R1is linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and X2;R2islinear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and the carbon linking X1and X2;R3is_ / Yi.o.xb2zOx^b2sR4' Si SiR‘,:X3X3 or x3x3x3x3120852P1420PC November 21, 2025 AldexChem Kft.whereinb2is a link to Xi,Y i is -O- or -CH2- or -O-CH2-CH2- wherein -CH2- is attached to Si,each X3is independently selected from the group consisting of C1-C4 alkyl or C1-C4 alkoxy,R4is linear C2-C17alkyl, or non-linear C3-C17 alkyl, C3-C2o alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and Yj;D1is selected from the group consisting of. OHn, («)whereinb4is a bond to nitrogen,R9is selected from C1-C6alkyl, cyclopentyl, cyclohexyl, hydroxyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, 4-hydroxy benzyl,m is an integer selected from 1 and 6,n is an integer selected from 0 and 6,o is an integer selected from 0 and 6; provided that the sum of n+o is at least 1;P is selected from120852P1420PC November 21, 2025 AldexChem Kft.G2-T3whereinG2is as defined above;T3isb A5X5-R13whereinb5is a bond to G2,X4and X5can be the same or different and each independently is O, S.R13 is linear C2-C17 alkyl, non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and X5;(b) a structural lipid as second component selected from l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2dimyristoyl-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1- Pahnitoyl-2-Oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-dipahnitoyl-sn-glycero-3 - phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 - pahnitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);(c) a helper lipid as third component selected from P-sitosterol, ergosterol, campesterol, stigmasterol, and pharmaceutically acceptable salts thereof, and mixtures thereof;(d) a shield lipid as fourth component selected from poly(ethylene glycol)-modified diacylglycerols (PEG-DAG), in particular a PEG-DAG selected from DSPE-PEG and DMG-PEG;(e) and optionally a fifth lipid component selected from(el) l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) (DOPG), dipahnitoylphosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3 - phosphoglycerol (DSPG), 1,2-dimyristoyl-sn-glycero-3 -phosphoglycerol (DMPG), 1 -pahnitoyl-2-oleoyl-sn-glycero-3 -phosphoglycerol (POPG),120852P1420PC November 21, 2025 AldexChem Kft.phosphatidylserine (PS; including dioleoyl PS (DOPS) and 18:0 PS), 1,2- dipahnitoyl-sn-glycero-3 -phosphate (DPP A), 1,2-dioleoyl-sn-glycero-3 - phosphate (DOPA), and pharmaceutically acceptable salts thereof; or(e2) Chrysin, Apigenin, Pinocembrin, Galangin and pharmaceutically acceptable salts thereof, and mixtures thereof;wherein the composition comprises either four or five lipid components in total.

2. The lipid nanoparticle composition of claim 1, wherein the composition is selected from:(i) a four-component system consisting of an ionizable cationic lipid of formula I as defined in (a), a structural lipid as defined in (b), a helper lipid as defined in (c), and a shield lipid as defined in (d); or(ii) a five-component system further comprising a fifth lipid component as defined in (el) or (e2).

3. The lipid nanoparticle composition of any one of claims 1-2, which comprises an ionizable cationic lipid according to formula (I)., G1X XD1T1NIP(I)or a salt thereof,wherein:G1is unsubstituted C2-C9alkylene,T1isR3 x R3xr xr(a) b^Xj-R!or(b) R / ^Xa-b1whereinb1is a bond to G1,Xi and X2are the same or different and each independently represents O or S,120852P1420PC November 21, 2025 AldexChem Kft.R1is linear C1-C17alkyl, non-linear C3-C17alkyl, C3-C17alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and X2;R2islinear Cj-Cn alkyl, non-linear C3-C17 alkyl, C3-C17 alkenyl containing one double bond with the proviso that there is at least one -CH2- group between the double bond and the carbon linking Xi and X2;R3is_(X ^b.'Si Si Si ' / \ / xX3X3or X3X3X3Xwhereinb2is a link to Xi,Y i is -O- or -CH2- or -O-CH2-CH2- wherein -CH2- is attached to Si,each X3is independently selected from the group consisting of C1-C4 alkyl or C1-C4 alkoxy,R4is linear C2-C17alkyl, or non-linear C3-C17 alkyl, C3-C2o alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and Y;;D1is selected from the group consisting ofwhereinb4is a bond to nitrogen,R9is selected from C1-C6alkyl, cyclopentyl, cyclohexyl, hydroxyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, 4-hydroxy benzyl,120852P1420PC November 21, 2025 AldexChem Kft.m is an integer selected from 1 and 6,n is an integer selected from 0 and 6,o is an integer selected from 0 and 6; provided that the sum of n+o is at least 1; P is selected fromG2-T3whereinG2is as defined above;TJisbs5£^XS-R,3whereinb5is a bond to G2,X4and X5can be the same or different and each independently is O, S.R13 is linear C2-C17 alkyl, non-linear C3-C17 alkyl, C3-C20 alkenyl containing one double bond with the proviso there is at least one -CH2- group between the double bond and X5.

4. The lipid nanoparticle composition of any one of claims 1 to 3, wherein said fifth lipid component is selected from l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) (DOPG), dipahnitoylphosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3 -phosphoglycerol (DSPG), l,2-dimyristoyl-sn-glycero-3 -phosphoglycerol (DMPG), l-pahnitoyl-2-oleoyl-sn- glycero-3 -phosphoglycerol (POPG), phosphatidylserine (PS; including dioleoyl PS (DOPS) and 18:0 PS), l,2-dipahnitoyl-sn-glycero-3 -phosphate (DPP A), l,2-dioleoyl-sn-glycero-3- phosphate (DOPA), and pharmaceutically acceptable salts thereof5. The lipid nanoparticle composition of any one of claims 1 to 3, wherein said fifth lipid component is l,2-distearoyl-sn-glycero-3 -phosphoglycerol (DSPG) or a pharmaceutically acceptable salt thereof.120852P1420PC November 21, 2025 AldexChem Kft.

6. The lipid nanoparticle composition of any one of claims 1 to 3, wherein said fifth lipid component is selected from Chrysin, Apigenin, Pinocembrin, Galangin and pharmaceutically acceptable salts thereof, and mixtures thereof.

7. The lipid nanoparticle composition of any one of claims 1 to 3, wherein said fifth lipid component is Chrysin, or a pharmaceutically acceptable salt thereof.

8. The lipid nanoparticle composition of any one of claims 1, 2, 3, wherein said components are present at the following amounts, expressed as mole percent of total lipid: ionizable cationic lipid 20-70 mol %, structural lipid 2^0 mol %, helper lipid 5^10 mol %, shield lipid 0.5-10 mol %, wherein the total of components (ionizable cationic lipid, structural lipid, helper lipid shield lipid) equals 100 mol% (excluding solvent, buffer, counterions, and payload).

9. The lipid nanoparticle composition of any one of claims 1-7, wherein said components are present at the following amounts, expressed as mole percent of total lipid: ionizable cationic lipid 20-70 mol %, structural lipid 2–40 mol %, helper lipid 5^0 mol %, shield lipid 0.5- 10 mol %, and fifth lipid component 1-25 mol %; wherein the total of components (ionizable cationic lipid, structural lipid, helper lipid shield lipid, fifth lipid component) equals 100 mol% (excluding solvent, buffer, counterions, and payload).

10. The lipid nanoparticle composition of any one of claims 1 to 9, wherein said helper lipid is selected from P-sitosterol, ergosterol, campesterol, stigmasterol, and pharmaceutically acceptable salts thereof, and mixtures thereof.

11. The lipid nanoparticle composition of claim 10, wherein said helper lipid is P-sitosterol.

12. The lipid nanoparticle composition of any one of claims 1 to 11, wherein said structural lipid is DSPC.

13. The lipid nanoparticle composition of any one of claims 1 to 12, wherein said shield lipid is a PEG lipid comprising a PEG moiety from 1,000 to 20,000 daltons.

14. The lipid nanoparticle composition of any one of claims 1 to 13, wherein said shield lipid is poly(ethylene glycol)-modified diacylglycerols (PEG-DAG), in particular a PEG-DAG selected from DSPE-PEG and DMG-PEG.

15. The lipid nanoparticle composition of claim 14, wherein said shield lipid is DMG-PEG.

16. The lipid nanoparticle composition of any one of claims 1 to 15, wherein the T’-G1moiety of said ionizable cationic lipid has one of the following structures:120852P1420PC November 21, 2025 AldexChem Kft.

17. The lipid nanoparticle composition of any one of claims 1 to 16, wherein the P moiety of said ionizable cationic lipid is a G2-T3moiety, with T3having one of the following structures:ObAo-R1318. The lipid nanoparticle composition of any one of claims 1 to 17, wherein the P moiety of said ionizable cationic lipid is a G2-T3moiety with T3, wherein R!3is linear C2-C17 alkyl:ObAo-R1319. The lipid nanoparticle composition of any one of claims 1-18, wherein the P moiety of said ionizable cationic lipid is a G2-T3moiety, with T3having one of the following structures:o20. The lipid nanoparticle composition of any one of claims 1 to 19, wherein the P moiety of said ionizable cationic lipid is a G2-T3moiety having the following structure:o21. The lipid nanoparticle composition of any one of claims 1 to 20, wherein the D1moiety of said ionizable cationic lipid has one of the following structures:OH22. The lipid nanoparticle of any one of claims 1 to 15, wherein said ionizable cationic lipid has one of the following structures:120852P1420PC November 21, 2025 AldexChem Kft.

23. The lipid nanoparticle composition according to claim 22, wherein the following components are present in the following amounts expressed as mole percent of total lipid: ionizable cationic lipid 50 mol %, DSPC 10 mol %, B-sitosterol 38,5 mol %, DMG-PEG 1.5 mol %.

24. The lipid nanoparticle composition according to claim 22, wherein the following components are present in the following amounts expressed as mole percent of total lipid: ionizable cationic lipid 50 mol %, DSPC 8 mol %, B-sitosterol 38,5 mol %, PEG-DMG 1.5 mol %, DSPG 2 mol %.

25. The lipid nanoparticle composition according to claim 22, wherein the following components are present in the following amounts expressed as mole percent of total lipid: ionizable cationic lipid 50 mol %, DSPC 10 mol %, B-sitosterol 28.5 mol %, PEG-DMG 1.5 mol %, Chrysin 10 mol %.

26. The lipid nanoparticle composition of any one of claims 1 to 25 for use in delivering one or more nucleic acids to human T cells, wherein the nucleic acid is selected from mRNA, cDNA, ceDNA, pDNA, ceDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof.

27. The lipid nanoparticle composition any one of claims 1 to 26 for use in delivering one or more nucleic acids to human T cells for the treatment of a disease or condition selected from autoimmune diseases, cardiac fibrosis, B-cell malignancies, infectious diseases, transplant- related disorders.

28. The lipid nanoparticle composition any one of claims 1 to 26 for use in delivering one or more nucleic acids to human T cells for the treatment of a disease or condition selected from autoimmune diseases, cardiac fibrosis, B-cell malignancies, infectious diseases, transplant- related disorders involving human T cells, wherein the nucleic acid is selected from mRNA, cDNA, ceDNA, pDNA, ceDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof.

29. A pharmaceutical composition comprising a lipid nanoparticle composition of any one of claims 1 to 28 comprising one or more therapeutically active nucleic acids selected from mRNA, cDNA, ceDNA, pDNA, ceDNA, microRNA, siRNA, gRNA, CAS9 mRNA, saRNA, circRNA, modified RNA, antisense oligonucleotide, targeted nucleic acid, or a combination thereof and a pharmaceutically acceptable carrier.

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