Helper lipids in nucleic acid delivery

Abstract

The present invention relates to helper lipids and their use in lipid nanoparticles, lipid nanoparticle formulations comprising these helper lipids, alone or in combination with other lipids. The lipid nanoparticles formulations may be formulated with nucleic acids for their delivery to target tissues after local or systemic administration.

Description

[0001] Foreignfiling text P24-220

[0002] - 1 -

[0003] Helper lipids in nucleic acid delivery

[0004] Technical Field

[0005] 5 The present invention relates to helper lipids and their use in lipid nanoparticles, lipid nanoparticle formulations comprising these helper lipids, alone or in combination with other lipids. The lipid nanoparticles formulations may be formulated with nucleic acids for their delivery to target tissues after local or systemic administration.

[0006] Background

[0007] Nucleic acids represent a new and promising class of therapeutics. Rather than treating conditions by dosing small molecules or proteins, DNA or RNA is administered to patients, whose cells then produce the desired pharmaceutically

[0008] 15 relevant protein from these “blueprints.” As nucleic acids are much more stable and easier to produce and modify than fully translated proteins, there is enormous potential for these drugs to prevent, treat or cure disease, with applications ranging from cancer to chronic conditions like cystic fibrosis and even neurological disorders. Recently, nucleic acid therapeutics have come into the public eye in the

[0009] 20 form of mRNA vaccines against the SARS-CoV-2 virus.

[0010] Among the diverse types of delivery vehicles, lipid-based systems are the industryleading standard to deliver such payloads, as witnessed by several marketed products. Lipid nanoparticles (LNP) are multi-component lipid-based delivery systems, comprising one or more ionizable and / or permanently cationic lipids, one

[0011] 25 or more polymer-conjugated lipids and one or more helper lipids - all having a strategic role to the particle's formation, performance, and stability. Different compositions of RNA-LNP drug products are presently either marketed or in clinical phase III trials (Schoenmaker et al., 2021).

[0012] The number of patent literature relating to use of RNA-LNP as a vaccine or

[0013] 30 therapeutic has increased dramatically over the last five years. Leaving aside the patents focusing on the nucleic acid drug substance, significant importance has been given to the patent literature directed to LNP delivery system itself, especially regarding its composition and molar ratios of the individual components. Early work Foreignfiling text P24-220

[0014] - 2 - on LNPs was carried out by Pieter Cullis and co-workers (UBC), and LNP technology was further developed by several companies, e.g. WO 2009 / 082817 or WO 2011 / 140627.

[0015] In recent years, significant efforts were invested to find alternative solutions and / or

[0016] 5 enhance delivery efficiency. In some approaches, a fifth or sixth lipidic component has been added to the LNP, to control the tissue tropism and / or stability of the LNP formulations, moving, thus, away from the conventional 4-lipidic component LNP systems, e.g. W02022 / 204043 or WO2022 / 180213 A1. As an alternative to this approach, others have focused on increasing the level of specific lipid component in the LNP to allow an extrahepatic delivery, e.g. WO2020 / 219941 , WO2021 / 250263 or WO2023 / 184038.

[0017] Currently, much effort has been put on identifying novel lipid-based delivery systems' compositions that can enhance or improve delivery efficiency. This invention focuses to improve further the standard LNP composition by optimizing

[0018] 15 the helper lipid component of the multicomponent LNP systems encapsulating nucleic acids.

[0019] While much effort has been put on identifying lipid nanoparticle-based vehicles containing different lipid compositions, there is still a significant need for improvements in these delivery systems with enhanced delivery and stability of

[0020] 20 payloads, in particular nucleic acid payloads.

[0021] Of the different lipid components included in the multicomponent LNP systems, the ionizable lipid is considered the most important for the nucleic acid encapsulation, in vitro and in vivo performance of the LNP. Accordingly, most of the scientific efforts have been focused on optimizing the ionizable lipid structure to ensure better and

[0022] 25 safer RNA delivery. With respect to the remaining lipidic components, less attention has been devoted on their contribution to the LNP stability and biological performance. From these, the polymer-conjugated lipid is required to provide circulation longevity, while the helper lipids, namely cholesterol and a phospholipid (e.g., DSPC) have a structural and stability-related role for the LNP formulation.

[0023] 30

[0024] Recent publications have highlighted the importance of the helper lipids type and content on the morphology, circulation time and extrahepatic biodistribution of the resulting LNP (Barbieri et al., 2024). These studies have revealed that by increasing Foreignfiling text P24-220

[0025] - 3 - the level of the helper lipids in the classical LNP composition (e.g., composition applied for the Onpattro benchmark LNP), this can lead to prolonged circulation times, leading to extrahepatic biodistribution (e.g., to immune cells in the bone marrow and / or the spleen) with signigifcant application in diseases like cancer or

[0026] 5 vaccines for infectious diseases.

[0027] This highlights the importance of these components, not only for the structure and stability of the LNP formulations, but also on their biological performance. Thus, there is a need in the art to modify lipid particle compositons to further improve physicochemical properties, stability as well as in vitro and in vivo behaviour including in vivo extra hepatic delivery of the resulting lipid particles (e.g. LNPs).

[0028] Summary of the invention

[0029] The present invention addresses one or more of the foregoing needs in the prior art and / or provides useful alternatives to known compositions for the delivery of

[0030] 15 pharmaceutical agents.

[0031] The present invention is based on the finding that phosphonolipids can be beneficially used as helper lipids in lipid particle for drug delivery.

[0032] In one aspect, the invention relates to a lipid particle comprising at least one pharmaceutical agent and at least one phosphonolipid.

[0033] 20 In one embodiment of the invention, the lipid particle comprises at least one pharmaceutical agent, at least one cationic lipid, at least one phosphonolipid and at least one conjugated lipid.

[0034] In another aspect, the present invention relates to novel phosphonolipids that can be used in lipid particle for drug delivery.

[0035] 25 In yet another aspect, the present invention relates to a pharmaceutical composition comprising the lipid particle comprising at least one pharmaceutical agent and at least one phosphonolipid as defined in the present invention.

[0036] Detailed description of the invention

[0037] 30

[0038] It has been found that phosphonolipids can be used in lipid particles (e.g. LNPs) for drug delivery. The resulting compositions can exhibit improved stability and / or delivery of the payload. Foreignfiling text P24-220

[0039] - 4 -

[0040] In a first aspect the present invention relates to a lipid particle comprising at least one pharmaceutical agent and at least one phosphonolipid.

[0041] As used herein, the terms "a", "an" and "the" and similar references used in the

[0042] 5 context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0043] As used herein, the term "lipid particle" refers to particles having a certain dimension which include one or more specified lipids. In some embodiments, the lipid particle is included in a formulation that can be used to deliver a pharmaceutical agent, to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).

[0044] As used herein, the term "lipid" refers to a group of organic compounds that include,

[0045] 15 but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents.

[0046] For non-viral nucleic acid delivery vehicles, particle encapsulation of nucleic acids physically protects nucleic acids from degradation and, depending on the specific

[0047] 20 chemistry, can aid in cellular uptake and endosomal escape.

[0048] Lipid particles (e.g. LNPs) according to the invention include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations. LPX described herein is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid and a phase comprising a dispersion of lipids. In some embodiments, the lipid

[0049] 25 phase comprises liposomes. In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous

[0050] 30 environment.

[0051] In one embodiment of the invention, the lipid particle is a lipid nanoparticle. Foreignfiling text P24-220

[0052] - 5 -

[0053] The term “lipid nanoparticle" relates to a nano-sized lipid particle comprising a pharmaceutical agent, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least 1 nm and below 1000 nm. Preferably, the size of a particle is its diameter. Preferably, the pharmaceutical agent is a nucleic acid, more

[0054] 5 preferably a RNA or a mRNA, as described herein and at least one phosphonolipid In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of a pharmaceutical agent, preferably a nucleic acid, in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0055] In some embodiments, LNPs comprise at least four components: a pharmaceutical agent, preferably a nucleic acid, a cationic lipid, a phosphonolipid, and a conjugated lipid. In some embodiments, LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid dissolved in an aqueous buffer.

[0056] 15

[0057] The term "pharmaceutical agent" refers to a biologically active component with a therapeutic and / or diagnostic effect including treating, preventing or curing diseases, or aiding in the diagnosis of medical conditions. The pharmaceutical agent can be a organic molecule, inorganic molecule, nucleic acid, protein, peptide,

[0058] 20 nucleic acid, targeting agent, an isotopically labelled chemical compound, vaccine, an immunological agent, or an agent useful in bioprocessing.

[0059] In a another embodiment of the invention, the pharmaceutical agent is a nucleic acid. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic

[0060] 25 acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a nucleic acid is DNA. In some embodiments, a nucleic acid is RNA. In some embodiments, a nucleic acid is a mixture of DNA and RNA. A nucleic acid may be present as a single-stranded or double-stranded and

[0061] 30 linear or covalently circularly closed molecule.

[0062] In some embodiments, nucleic acid particles (especially mRNA particles) comprise more than one type of nucleic acid molecules, where the molecular parameters of Foreignfiling text P24-220

[0063] - 6 - the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[0064] 5 In a another embodiment of the invention, the pharmaceutical agent is ribonucleic acid (RNA). In a particular embodiment the nucleic acid is a messenger RNA (mRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), single guide RNA (sgRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, all nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small

[0065] 15 nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, viral satellite RNA, selfamplifying mRNA, or Trans-amplifying mRNA.aaln preferred embodiment the agent is a ribonucleic acid (RNA), a small interfering RNA (siRNA), a micro RNA (miRNA), an antisense oligo nucleotides, a messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA or an immune stimulating nucleic acid. In a further preferred

[0066] 20 embodiment the agent is a messenger RNA (mRNA).

[0067] In one embodiment, the lipid particle (e.g. LNP) comprises a phosphonolipid and a nucleic acid, preferably a RNA, more preferably an mRNA.

[0068] In another embodiment the nucleic acid is DNA. In a particular embodiment the

[0069] 25 nucleic acid is pDNA.

[0070] In the lipid particles (e.g. LNPs) of the invention, the nucleic acid may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from enzymatic degradation. In preferred embodiments, a LNP comprising a

[0071] 30 nucleic acid, such as an RNA (e.g., mRNA or siRNA), is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the LNP is not substantially degraded after exposure of the particle to a nuclease at 37 degrees centigrade for Foreignfiling text P24-220

[0072] - 7 - at least 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the LNP is not substantially degraded after incubation of the particle in serum at 37 degrees centigrade for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments,

[0073] 5 the nucleic acid (e.g., nucleic acid, such as siRNA or mRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulations of the present invention is that the lipid particle (e.g. LNP) compositions are substantially non toxic to mammals such as humans. The term "fully encapsulated" indicates that the nucleic acid in the lipid particle is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than 25 percent of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100 percent of free nucleic acid, more preferably less than 10 percent, and most preferably less than 5 percent of the nucleic acid in the particle is degraded. In the

[0074] 15 context of nucleic acid therapeutic agents, full encapsulation may be determined by an Oligreen(R) assay. Oligreen(R) is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single- stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, CA). "Fully encapsulated" also indicates that the lipid particles (e.g. LNPs) are serum-stable, that is, that they do

[0075] 20 not rapidly decompose into their component parts upon in vivo administration.

[0076] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles (e.g. LNPs). In preferred embodiments, the nucleic acid (e.g., RNA, mRNA or siRNA) is fully encapsulated

[0077] 25 within the lipid portion of the lipid particles (e.g., LNP), such that from 30 percent to 100 percent, from 40 percent to 100 percent, from 50 percent to 100 percent, from 60 percent to 100 percent, from 70 percent to 100 percent, from 80 percent to 100 percent, from 90 percent to 100 percent, from 30 percent to 95 percent, from 40 percent to 95 percent, from 50 percent to 95 percent, from 60 percent to 95 percent,

[0078] 30 percent, from 70 percent to 95 percent, from 80 percent to 95 percent, from 85 percent to 95 percent, from 90 percent to 95 percent, from 30 percent to 90 percent, from 40 percent to 90 percent, from 50 percent to 90 percent, from 60 percent to 90 percent, from 70 percent to 90 percent, from 80 percent to 90 percent, or at least 30 Foreignfiling text P24-220

[0079] - 8 - percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent (or any fraction thereof or range therein) of the lipid particles (e.g., LNPs)

[0080] 5 have the nucleic acid encapsulated therein.

[0081] Typically, the lipid particles (e.g., LNPs) of the invention have a lipid:pharmaceutial agent ratio (mass / mass ratio) of from 1 to 100. In some instances, the lipid:pharmaceutial agent ratio (mass / mass ratio) ranges from 1 to 50, from 2 to 25, from 3 to 20, from 4 to 15, or from 5 to 10.

[0082] Typically, the lipid particles (e.g., LNPs) of the invention have a mean diameter of from 40 nm to 500 nm. In preferred embodiments, the lipid particles (e.g., LNPs) of the invention have a mean diameter of from 40 nm to 200 nm, from 40 nm to 180

[0083] 15 nm, from 40 nm to 160 nm, from 40 nm to 150 nm, from 40 nm to 130 nm, from 40 nm to 120 nm, from 40 nm to 100 nm, from 50 nm to 120 nm, from 50 nm to 100 nm, from 60 nm to 120 nm, from 60 nm to 110 nm, from 60 nm to 100 nm, from 60 nm to 90 nm, from 60 nm to 80 nm, from 70 nm to 120 nm, from 70 nm to 110 nm, from 70 nm to 100 run, from 70 nm to 90 nm, from 70 nm to 80 nm, or less than 120

[0084] 20 nm, 110 nm, 100 nm, 90 nm, or 80 nm (or any fraction thereof or range therein). The particle size may be determined by dynamic light scattering (DLS).

[0085] The present invention also provides a pharmaceutical composition comprising a lipid particle (e.g., LNP) described herein and a pharmaceutically acceptable carrier.

[0086] 25

[0087] In a further aspect, the present invention provides a method for introducing one or more active agents or therapeutic agents (e.g., nucleic acid) into a cell, comprising contacting the cell with a lipid particle (e.g., LNP) described herein. In one embodiment, the cell is in a mammal and the mammal is a human. In another

[0088] 30 embodiment, the present invention provides a method for the in vivo delivery of one or more active agents or therapeutic agents (e.g., nucleic acid), comprising administering to a mammalian subject a lipid particle (e.g., LNP) described herein. In a preferred embodiment, the mode of administration includes, but is not limited Foreignfiling text P24-220

[0089] - 9 - to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is a human.

[0090] 5 In another embodiment, the polydispersity index (PDI) of the plurality of lipid particles (e.g. LNPs) in a composition is less than 0.3, 0.25, 0.2, 0.15, 0.12 or 0.10. The PDI may be determined by dynamic light scattering (DLS).

[0091] The lipid particles (e.g. LNPs) herein may exhibit a particularly high encapsulation efficiency (EE) of the pharmaceutical agent (e.g. RNA or mRNA). As used herein, the term "encapsulation," with reference to incorporating the nucleic acid within a lipid nanoparticle refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle, including a lipophilic or the aqueous portion. Thus, in one embodiment, the encapsulation efficiency is at least

[0092] 15 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 92 percent. The EE may be determined by Quant-iT RiboGreen RNA assay.

[0093] The present disclosure also provides lipid particles (e.g. LNPs) described according to the molar ratio between the positively charged amine groups of the amine lipid

[0094] 20 (N) and the negatively charged phosphate groups (P) of the oligonucleotide to be encapsulated. This may be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is between 4 and 15 or between 4.5 and 12 or between 5 and 10 or between 5.5 and 8. In one embodiment, the N / P ratio of the lipid particle (e.g. LNP) is at least 1 , 2, 3, 4, 4.25, 4.50, 4.75, 5.0, 5.25,

[0095] 25 5.5, 5.75, 6.0 or 6.25. The upper limit may be 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9 or 8. The disclosure also encompasses a combination of any two of the upper and lower limits.

[0096] As used herein, the term "phosphonolipid" refers to a class of lipids that contain a

[0097] 30 phosphonate group instead of the typical phosphate group found in phospholipids. In one embodiment, a phosphonolipid is characterized by a glycerol backbone, two fatty acid tails, and a phosphonate group, R-PO(OR)2 group, wherein R is an optionally substituted organic group, e.g. alkyl or aryl. Foreignfiling text P24-220

[0098] - 10 -

[0099] The synthesis and properties of certain phosphonolipids (e.g., DEPN-8) are described in the literature, e.g. in Turcotte et al., 1977 or Skita et al. 1995. So far, their application has been published in the development of more stable lung surfactant preparations, e.g. in Notter et al., 2016.

[0100] 5

[0101] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (I) wherein

[0102] X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2- C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, and a

[0103] 15 pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0104] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (I)

[0105] 20 wherein

[0106] X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2-

[0107] 25 C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0108] In another embodiment, the lipid particle comprises a phosphonolipid according to

[0109] 30 the formula (I) Foreignfiling text P24-220

[0110] - 11 - wherein

[0111] 5 X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2- C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, with the proviso that X contains at least one heteroatom, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0112] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (II) , wherein

[0113] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce-

[0114] 20 C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0115] A and B independently of one another are absent, -O-, -OC(=O)- or -NRaC(=O)-, wherein Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and Z is optionally substituted C1-C12 alkyl, C2-C12 alkenyl, cycloalkyl or

[0116] 25 heterocycloalkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0117] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (II)

[0118] 30 Foreignfiling text P24-220

[0119] - 12 -

[0120] 5 , wherein

[0121] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0122] A and B are -OC(=O)-, and

[0123] Z is optionally substituted cyclohexyl or alkoxy, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0124] In another embodiment, the lipid particle comprises a phosphonolipid according to

[0125] 15 the formula (III) wherein

[0126] 20

[0127] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0128] A and B independently of one another are absent, -O-, -OC(=O)-, -C(=O)O-,-S-, - C(=O)S-, -SC(=O)-, -S(=O)-, -S(=O)2-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -

[0129] 25 OC(=O)NRa-, -NRaC(=O)O

[0130] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and

[0131] D is absent, hydrogen, hydroxy, -N+RaRbRc-, or optionally substituted cycloalkyl, heterocycloalkyl or C1-C12 heteroalkyl wherein

[0132] 30

[0133] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling text P24-220

[0134] - 13 -

[0135] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (III) wherein

[0136] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0137] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0138] B is -OC(=O)-,

[0139] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and,

[0140] D is -N+RaRbRc-, wherein

[0141] 15

[0142] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is C3-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0143] 20

[0144] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (III) wherein

[0145] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24

[0146] 30 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0147] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0148] B is -OC(=O)-,

[0149] L is optionally substituted C3 alkyl, Foreignfiling text P24-220

[0150] - 14 -

[0151] D is -N+RaRbRc-, wherein

[0152] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0153] 5

[0154] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (IV) wherein

[0155] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce-

[0156] 15 C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0157] L is optionally substituted C3 alkyl, and

[0158] D is -N+RaRbRc-, wherein

[0159] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally

[0160] 20 substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0161] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (V) wherein

[0162] 30

[0163] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, Foreignfiling text P24-220

[0164] - 15 -

[0165] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and

[0166] D is hydrogen or -N+RaRbRc-, wherein

[0167] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally

[0168] 5 substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0169] In one embodiment, the phosphonolipid is a compound according to formula (V) wherein

[0170] 15 R1and R2independently of one another are optionally substituted C6-C24 alkenyl or C6-C24 heteroalkenyl,

[0171] L is optionally substituted Ci-Ce alkyl, and

[0172] D is hydrogen or -N+RaRbRc-, wherein

[0173] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally

[0174] 20 substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0175] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (la) wherein

[0176] X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2-

[0177] 30 C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling text P24-220

[0178] - 16 -

[0179] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (la) wherein

[0180] X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2- C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0181] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (la)

[0182] 15 wherein

[0183] X, Y and Z independently of one another are optionally substituted C1-C24 alkyl, C2-

[0184] 20 C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl, C2-C24 heteroalkynyl, with the proviso that X contains at least one heteroatom, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0185] 25

[0186] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (la) wherein Foreignfiling text P24-220

[0187] - 17 -

[0188] X and Y independently of one another are hydroxy or optionally substituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl or C2- C24 heteroalkynyl; whereas, only one of X or Y can be hydroxy; and

[0189] 5 Z is optionally substituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl or C2-C24 heteroalkynyl; and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0190] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Ila) wherein

[0191] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0192] 20 A and B independently of one another are absent, -O-, -OC(=O)- or -NRaC(=O)-, wherein Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and Z is optionally substituted C1-C12 alkyl, C2-C12 alkenyl, cycloalkyl or heterocycloalkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0193] 25

[0194] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Ila) wherein Foreignfiling text P24-220

[0195] - 18 -

[0196] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0197] A and B are -OC(=O)-, and

[0198] 5 Z is optionally substituted cyclohexyl or alkoxy, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0199] In another embodiment, the lipid particle comprises a phosphonolipid according to (Illa), wherein

[0200] 15

[0201] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0202] A and B independently of one another are absent, -O-, -OC(=O)-, -C(=O)O-,-S-, - C(=O)S-, -SC(=O)-, -S(=O)-, -S(=O)2-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -

[0203] 20 OC(=O)NRa-, -NRaC(=O)O

[0204] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and

[0205] D is absent, hydrogen, hydroxy, -N+RaRbRc-, or optionally substituted cycloalkyl, heterocycloalkyl or C1-C12 heteroalkyl wherein

[0206] 25

[0207] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0208] In another embodiment, the lipid particle comprises a phosphonolipid according to

[0209] 30 the formula (Illa) Foreignfiling text P24-220

[0210] - 19 - (Illa), wherein

[0211] 5

[0212] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24 heteroalkynyl,

[0213] A and B independently of one another are absent, -O-, -OC(=O)-, -C(=O)O-, -S-, -C(=O)S-, -SC(=O)-, -S(=O)-, -S(=O)2-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, -NRaC(=O)O-, -C(=O)NRaC(=O)- or -O-P(=O)(O-)O-, wherein

[0214] Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenylyl, cycloalkylyl or heterocycloalkylyl, and

[0215] 15

[0216] D is absent, hydrogen, hydroxy, -N+RaRbRc-, or optionally substituted cycloalkyl, heterocycloalkyl or C1-C12 heteroalkyl, wherein

[0217] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer

[0218] 20 and isotopically labeled derivative thereof.

[0219] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Illa)

[0220] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce-

[0221] 25 C24 alkenyl, C6-C24 heteroalkyl or C6-C24 heteroalkenyl,

[0222] A is -OC(=O)-, -O-, or -NRaC(=O)-,

[0223] B is -OC(=O)- or -NRaC(=O)-, wherein

[0224] Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6

[0225] 30 heteroalkenylyl, cycloalkylyl or heterocycloalkylyl, and

[0226] D is -N+RaRbRc-, wherein

[0227] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, Foreignfiling text P24-220

[0228] - 20 - with the proviso that if A and B are both -OC(=O)-, L is optionally substituted C2-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0229] 5

[0230] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Illa) (Illa), wherein

[0231] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0232] 15

[0233] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0234] B is -OC(=O)-,

[0235] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and,

[0236] D is -N+RaRbRc-, wherein

[0237] 20

[0238] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is C3-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0239] 25

[0240] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Illa) (Illa), wherein Foreignfiling text P24-220

[0241] - 21 -

[0242] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0243] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0244] 5 B is -OC(=O)-,

[0245] L is optionally substituted C2 alkyl,

[0246] D is -N+RaRbRc-, wherein

[0247] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0248] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (Iva) , wherein

[0249] 20 R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0250] L is optionally substituted C3 alkyl, and

[0251] D is -N+RaRbRc-, wherein

[0252] 25 Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0253] In another embodiment, the lipid particle comprises a phosphonolipid according to

[0254] 30 the formula (Va) Foreignfiling text P24-220

[0255] - 22 - wherein

[0256] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0257] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and

[0258] D is hydrogen or -N+RaRbRc-, wherein

[0259] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt,

[0260] 15 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0261] In one embodiment, the phosphonolipid is a compound according to formula (Va) wherein

[0262] R1and R2independently of one another are optionally substituted C6-C24 alkenyl or C6-C24 heteroalkenyl,

[0263] 25

[0264] L is optionally substituted Ci-Ce alkyl, and

[0265] D is hydrogen or -N+RaRbRc-, wherein

[0266] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0267] 30

[0268] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (VI) Foreignfiling text P24-220

[0269] - 23 - wherein

[0270] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0271] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0272] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0273] 20

[0274] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 heteroalkyl or C6-C24 heteroalkenyl, and

[0275] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0276] 25

[0277] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein Foreignfiling text P24-220

[0278] - 24 -

[0279] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and

[0280] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt,

[0281] 5 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0282] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0283] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0284] 15 C6-C24 alkenyl, and

[0285] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted Ci-Ce alkyl or C2-C6 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0286] 20 In one embodiment, the phosphonolipid is a compound according to formula (VI)

[0287] 25 wherein

[0288] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0289] C6-C24 alkenyl, and

[0290] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C3 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate,

[0291] 30 tautomer, stereoisomer and isotopically labeled derivative thereof.

[0292] In one embodiment, the phosphonolipid is a compound according to formula (VI) Foreignfiling text P24-220 wherein

[0293] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and

[0294] R3, R4and R5independently of one another are a free bond, hydrogen or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0295] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0296] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0297] 20 C6-C24 alkenyl, and

[0298] R3, R4and R5independently of one another are hydrogen or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0299] 25

[0300] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0301] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and Foreignfiling text P24-220

[0302] - 26 -

[0303] R3, R4and R5are methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0304] In another embodiment, the lipid particle comprises a phosphonolipid according to

[0305] 5 the formula (VII), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0306] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (VIII),

[0307] 15

[0308] (VIII), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer,

[0309] 20 stereoisomer and isotopically labeled derivative thereof.

[0310] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (IX),

[0311] 25

[0312] (IX), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer,

[0313] 30 stereoisomer and isotopically labeled derivative thereof.

[0314] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (X), Foreignfiling text P24-220

[0315] - 27 -

[0316] 5 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0317] In another embodiment, the lipid particle comprises a phosphonolipid according to the formula (XI), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0318] 15

[0319] In another embodiment, the lipid particle comprises a phosphonolipid selected from the list consisting of formula (VII), (VIII), (IX), (X) and (XI). In another embodiment, the lipid particle comprises a phosphonolipid selected from the list consisting of formula (VII), (VIII), (IX) and (X).

[0320] 20

[0321] In another embodiment of the invention, the lipid particle comprises at least one pharmaceutical agent (e.g. RNA or mRNA), at least one cationic lipid, at least one helper lipid and at least one conjugated lipid, wherein at least one helper lipid is a phosphonolipid as defined above.

[0322] 25

[0323] In another embodiment of the invention, the lipid particle comprises at least one pharmaceutical agent (e.g. RNA or mRNA), at least one cationic lipid, at least one phosphonolipid and at least one conjugated lipid.

[0324] In another embodiment of the invention, the lipid particle consisting of at least one pharmaceutical agent (e.g. RNA or mRNA), at least one cationic lipid, at least one

[0325] 30 phosphonolipid and at least one conjugated lipid.

[0326] The term "cationic lipid" refers to a lipid capable of being positively charged. This primary component is responsible for electrostatic complexation of negatively Foreignfiling text P24-220

[0327] - 28 - charged nucleic acids during formulation. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable lipids that can exist in a positively charged or neutral form depending on pH. In some embodiments, a cationic lipid has a net positive charge only at certain

[0328] 5 pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution as well as the ability to form non- bilayer structures critical to the intracellular delivery of nucleic acids. For purposes of the present disclosure, cationically ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.

[0329] In one embodiment, the lipid particle (e.g. LNP) comprises at least one cationic lipid

[0330] 15 comprising from 5 mol percent to 85 mol percent, 20 mol percent to 80 mol percent, 30 mol percent to 70 mol percent, 20 mol percent to 50 mol percent, of the total lipid present in the lipid particle (e.g. LNP).

[0331] The term "conjugated lipid" refers to a molecule comprising both a lipid portion and

[0332] 20 a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like. The term "pegylated lipid" is used interchangeably with "PEGylated lipid."

[0333] 25

[0334] In one embodiment, the lipid particle (e.g. LNP) comprises at least one conjugated lipid comprising from 0.1 mol percent to 10 mol percent, 0.5 mol percent to 5 mol percent, 1 mol percent to 3 mol percent, 1 mol percent to 2 mol percent or 1.5 mol percent, of the total lipid present in the lipid particle (e.g. LNP).

[0335] 30

[0336] The term "helper lipid" refers to any amphipathic lipid or any other neutral lipid or anionic lipid that do not primarily mediate electrostatic complexation of nucleic acids, but contribute to bilayer structure, particle formation and stability. Foreignfiling text P24-220

[0337] - 29 -

[0338] The term "amphipathic lipid" refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics

[0339] 5 derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phosphonolipids, phospholipids, aminolipids, and sphingolipids.

[0340] Representative examples of phospholipids include, but are not limited to,

[0341] 15 phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, di stearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds

[0342] 20 lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and beta -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

[0343] 25 The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterol, cholesterol, cerebrosides, and diacylglycerols.

[0344] 30

[0345] In a particular embodiment, the additional at least one helper lipid is a sterol. In another particular embodiment, the lipid particle additionally comprises a sterol. Foreignfiling text P24-220

[0346] - 30 -

[0347] As used therein, the term "sterol" refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring. Examples of sterols include cholesterol, or a cholesterol derivative, the latter referring to a cholesterol molecule having a gonane

[0348] 5 structure and one or more additional functional groups. Cholesterol derivatives include but are not limited to P-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2 '-hydroxy ethyl ether, cholesteryl-4'- hydroxybutyl ether, 3P[N-(N'N'-dimethylaminoethyl)carbamoyl cholesterol (DC-Chol), 24(S)- hydroxycholesterol, 25 -hydroxy cholesterol, 25(R)- 27-hydroxycholesterol, 22-oxacholesterol, 23- oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7- hydroxycholesterol, 19-hydroxy cholesterol, 22-hydroxycholesterol, 25 -hydroxy cholesterol, 7- dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, dehydroergosterol,

[0349] 15 dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22- dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol or a salt or ester thereof.

[0350] 20

[0351] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-

[0352] 25 glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0353] The lipid particles (e.g. LNPs) according to the invention comprise at least one or

[0354] 30 more helper lipids as defined above, wherein at least one helper lipid is a phosphonolipid. Foreignfiling text P24-220

[0355] - 31 -

[0356] In one embodiment, the lipid particle (e.g. LNP) comprises only one helper lipid which is a phosphonolipid.

[0357] In case the lipid particle (e.g. LNP) comprises only one helper lipid which is a

[0358] 5 phosphonolipid, the phosphonolipid is present at least 15 mole percent; at least 20 mole percent, at least 30 mole percent, at least 40 mole percent, at least 45 mole percent, at least 50 mole percent, at least 55 mole percent, at least 55 mole percent, at least 60 mole percent, at least 65 mole percent, at least 70 mole percent, at least 75 mole percent, at least 80 mole percent or at least 85 mole percent based on the total lipid present in the lipid particle (e.g. LNP).

[0359] In one embodiment, the lipid particle (e.g. LNP) comprises two or more helper lipids, wherein all helper lipids are phosphonolipid. In this embodiment, the lipid particle comprises two or more phosphonolipids.

[0360] 15

[0361] In one embodiment, the lipid particle (e.g. LNP) comprises two helper lipids, wherein one helper lipid is a phosphonolipid and the other helper lipid is not a phosphonolipid.

[0362] 20 In one embodiment, the at least one helper lipid is present at least 50 mole percent; at least 55 mole percent, at least 60 mole percent, at least 65 mole percent, at least 70 mole percent, at least 75 mole percent, at least 80 mole percent or at least 85 mole percent based on the total lipid present in the lipid particle (e.g. LNP).

[0363] 25 In one embodiment, the lipid particle (e.g. LNP) comprises two helper lipids, wherein one helper lipid is a phosphonolipid and the other helper lipid is a sterol.

[0364] In one embodiment, the lipid particle (e.g. LNP) comprises two helper lipids, wherein one helper lipid is a phosphonolipid and the other helper lipid is a sterol and wherein

[0365] 30 the phosphonolipid comprising from 5 mole percent to 15 mol percent of the total lipid present in the lipid particle (e.g. LNP) and the steroid comprising from 30 mole percent to 50 mol percent of the total lipid present in the lipid particle (e.g. LNP). Foreignfiling text P24-220

[0366] - 32 -

[0367] In one embodiment, the combined (i) sterol content (e.g., cholesterol or cholesterol derivative thereof); and (ii) phosphonolipid content is at least 50 mole percent; at least 55 mole percent, at least 60 mole percent, at least 65 mole percent, at least 70 mole percent, at least 75 mole percent, at least 80 mole percent or at least 85

[0368] 5 mole percent based on the total lipid present in the lipid particle (e.g. LNP).

[0369] In one embodiment, the lipid particle (e.g. LNP) according to claim, comprises at least one pharmaceutical agent (e.g. RNA or mRNA), at least one cationic lipid comprising from 20 mol percent to 85 mol percent of the total lipid present in the particle, at least one helper lipid comprising from 5 mole percent to 80 mol percent of the total lipid present in the particle, and at least one conjugated lipid comprising from 0.5 mol percent to 10 mol percent of the total lipid present in the particle, wherein at least one helper lipid is a phosphonolipid.

[0370] In one embodiment, the lipid particle (e.g. LNP) according to claim, comprises at

[0371] 15 least one pharmaceutical agent (e.g. RNA or mRNA), at least one cationic lipid comprising from 20 mol percent to 85 mol percent of the total lipid present in the particle, at least one phosphonolipid comprising from 5 mole percent to 80 mol percent of the total lipid present in the particle, and at least one conjugated lipid comprising from 0.5 mol percent to 10 mol percent of the total lipid present in the

[0372] 20 particle.

[0373] In one embodiment, the phosphonolipid is a compound according to formula (I), (II), (III), (IV), (V) or (VI) as defined in the present application.

[0374] 25 In one embodiment, the phosphonolipid is a compound selected from the list consisting of DEPN-8, IND-1233, IND-1234, IND-1235 and IND-1236. In another embodiment, the phosphonolipid is selected from the list consisting of IND-1233, IND-1234, IND-1235, IND-1236.

[0375] 30 In one embodiment, the phosphonolipid is selected from the list consisting of DEPN- 8, IND-1233, IND-1234, IND-1235, IND-1236. In another embodiment, the phosphonolipid is selected from the list consisting of I ND- 1233, I ND- 1234, IND- 1235, IND-1236. Foreignfiling text P24-220

[0376] - 33 -

[0377] Lipid nanoparticles (LNPs) formulated with at least one phosphonolipid as a helper lipid exhibit advantageous physicochemical properties, including optimal particle size, a favorable polydispersity index (PDI), high encapsulation efficiency, and / or

[0378] 5 elevated total RNA concentration.

[0379] Furthermore, these LNPs demonstrate enhanced stability, particularly in terms of storage stability across varying temperatures. Notably, the mean particle size of LNPs containing at least one phosphonolipid as a helper lipid remains stable during storage after repeated freeze-thaw cycles, whereas the size of LNPs formulated with at least one phospholipid as a helper lipid tend to increase.

[0380] Furthermore, in the in vitro performance, lipid nanoparticles (LNPs) formulated with at least one phosphonolipid as a helper lipid exhibit enhanced RNA delivery. As

[0381] 15 example, a higher luciferase activity was measured after treatment of human liver (HepG2) and murine muscle (C2C12) and human lung (A549) with LNP formulated with DEPN-8 as a helper lipid (instead of DSPC or DPPC).

[0382] Furthermore, in the in vivo performance, lipid nanoparticles (LNPs) formulated with

[0383] 20 at least one phosphonolipid as a helper lipid exhibit enhanced biological activity. As example, Lipid nanoparticles (LNPs) formulated with DEPN-8 as a helper lipid (instead of DSPC) exhibit 2-fold higher luciferase expression (in vivo BLI- biodistribution) at 6h hours post dose and 3-fold higher liver tissue localization (ex vivo BLI luciferase activity) at 24h hours post dose, when injected intravenously in

[0384] 25 mouse animal model.

[0385] LNP formulated with the novel phosphonolipids as a helper lipid exhibit advantageous physicochemical properties, including optimal particle size, a favorable polydispersity index (PDI), high encapsulation efficiency, and / or elevated

[0386] 30 total RNA concentration.

[0387] Furthermore, depending on the cell type, differences in the in vitro performance for the different novel phosphonolipids which were incooperated into LNP were found. Foreignfiling text P24-220

[0388] - 34 -

[0389] As example, a high luciferase activity was measured after treatment of human liver (HepG2) and human lung (A549) with LNP containing I ND- 1234 or I ND- 1233 (instead of DSPC). This indicates that a specific cell interaction can be mediated by an lipid particle (e.g. LNP) formulation containing the different novel

[0390] 5 phosphonolipids.

[0391] Further advantages of the lipid particles and phosphonolipids according to the invention can be found by e.g. (i) analyzing the in vivo tolerability by determining the concentrations of cytokines and chemokines (e.g. IL-6 and MCP-1) in animal blood at different time points post dosing to evaluate the in vivo tolerability of the LNP formulations containing the novel phosphonolipids, and / or (ii) by analyzing the enzymatic degradation & chemical stability by investigating the enzymatic degradation and chemical stability of novel phosphonlipids using chromatography and / or mass spectrometry based methods, and / or (iii) by analyzing the interaction

[0392] 15 of blood proteins with LNP containing phosphonolipids by measuring the adsorption of blood proteins onto the surface to understand the in vitro and in vivo performance of the LNP containing phosphonolipids, and / or (iv) analyzing the stabilization of LNP containing phosphonolipids to increase formulation stability at > 0°C using e.g. freeze-drying or other methods to enable the storage of the LNP formulation at >

[0393] 20 0°C, and / or (v) optimization of LNP containg phosphonolipids to reach extrahepatic tissue in vivo by in vivo testing of different formulations, and / or (vi) functionalization of LNP containing phosphonolipids with targeting ligands (small molecules, peptides or proteins) to mediate a specific cell interaction.

[0394] 25 Based on the structural properties of the phosphonolipid, LNP with phosphonolipids can have enhanced stability compared to LNP with classic phospholipids.

[0395] DSPC (Phospholipid) features a phospho-choline headgroup and contains ester bonds that are enzymatically cleavable, allowing for rapid degradation. The

[0396] 30 degradation process involves enzymes such as esterase and phospholipase, contributing to its quick clearance in vivo. In contrast, IND-1234 (Phosphonolipid) also has a phosphono-choline headgroup and contains ester bonds; however, the Foreignfiling text P24-220

[0397] - 35 -

[0398] P-C bond is moderately stable. The degradation of IND-1234 primarily relies on esterase, resulting in a moderate clearance rate in vivo.

[0399] The term "optional" or "optionally" as used herein means that the subsequently

[0400] 5 described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

[0401] In the present invention, a structural formula of a compound may represent an amorphous or kristalline form of the compound. Additionally, the structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present disclosure includes all isomers such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures

[0402] 15 and is not limited to the description of the formula. Furthermore, in the present specification, a structural formula of a compound may represent a specific salt and / or solvate of said compound. It is to be understood, however, that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g. , hydrates) and is not limited to the description of the specific salt and / or solvate.

[0403] 20

[0404] Isomers are compounds having the same molecular formula but differ in structure (structural isomers) or in the geometrical (spatial) positioning of the functional groups and / or atoms (stereoisomers). Enantiomers are a pair of stereoisomers which are non- superimposable mirror-images of each other. A racemic mixture or

[0405] 25 racemate contains a pair of enantiomers in equal amounts and is denoted by the prefix (plus or minus). Diastereomers are stereoisomers which are non- superimposable and which 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

[0406] 30 individual atoms or groups of atoms; i.e. , the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto- enol-tautomerism. "Conformers" are stereoisomers that can be interconverted just by rotations formally single bonds, and include - in particular - those leading to Foreignfiling text P24-220

[0407] - 36 - different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0408] The term "solvate" as used herein refers to an addition complex of a dissolved

[0409] 5 material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n- propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A hydrate is a solvate wherein the solvent is water.

[0410] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0411] 15

[0412] Pharmaceutically acceptable salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Salts which are not themselves suitable for pharmaceutical uses but can be used, for example, for isolation, purification or storage of the compounds according to the

[0413] 20 invention are also included.

[0414] In isotopically labeled derivative of a compound of the invention, one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced

[0415] 25 by a deuterium or tritium atom. Exemplary isotopes which can be used in the present disclosure include deuterium, tritium,11C,13C,14C,15N,18F,32P,32S,35S,36CI, and125l.

[0416] Alkyl in the context of the invention represents an optionally substituted straight¬

[0417] 30 chain or branched alkyl radical having 1 to 50 carbon atoms.

[0418] C1-C3 alkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical having 1 to 3 carbon atoms. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n- Foreignfiling text P24-220

[0419] - 37 - propyl; and isopropyl. Ci-Ce alkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical having 1 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl;

[0420] 5 n-pentyl; isopentyl; and n-hexyl. C3-C6 alkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical having 3 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; and isopentyl. C1-C12 alkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical having 1 to 12 carbon atoms. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; isopentyl; n-hexyl; 2-methylpentyl; n-heptyl; 2-methylhexyl; and n-octyl. C1-C24 alkyl in the context of the invention represents an optionally substituted straight-chain or

[0421] 15 branched alkyl radical having 1 to 24 carbon atoms. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; isopentyl; n-hexyl; 2- methylpentyl; n-heptyl; 2-methylhexyl; n-octyl; 2-ethylheptyl; n-nonyl; 3-methyloctyl; n-decyl; 2-propylnonyl; n-undecyl; 3-methyldecyl; n-dodecyl; 2-ethyldecyl; n-

[0422] 20 tridecyl; 3-methylundecyl; n-tetradecyl; 2-propyltridecyl; n-pentadecyl; 3- methyltetradecyl; n-hexadecyl; n-heptadecyl; n-octadecyl; n-nonadecyl; and n- icosyl. C6-C24 alkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical having 6 to 24 carbon atoms. The following may be mentioned by way of example and by way of preference: n-hexyl; 2-

[0423] 25 methylpentyl; n-heptyl; 2-methylhexyl; n-octyl; 2-ethylheptyl; n-nonyl; 3-methyloctyl; n-decyl; 2-propylnonyl; n-undecyl; 3-methyldecyl; n-dodecyl; 2-ethyldecyl; n- tridecyl; 3-methylundecyl; n-tetradecyl; 2-propyltridecyl; n-pentadecyl; n-hexadecyl; n-heptadecyl; n-octadecyl; n-nonadecyl; n-icosyl; n-docosyl; n-tricosyl; n-tetracosyl; n-pentacosyl; n-hexacosyl; n-heptacosyl; n-octacosyl; n-nonacosyl; n-triacontyl; n-

[0424] 30 hentriacontyl; n-dotriacontyl; n-tritriacontyl; and n-tetratriacontyl.

[0425] A radical in the context of the invention represents an atom or group of atoms bearing one or more unpaired electrons, which can act as a substituent within a Foreignfiling text P24-220

[0426] - 38 - chemical structure. Depending on its position within the compound, the radical may be a monoradical or a diradical. Specifically, a monoradical (e.g., substituent Z) is typically a radical substituent located at a terminal position of the molecule. A diradical (e.g., substituent L) typically serves as a bridging substituent connecting

[0427] 5 two parts of the compound, except in cases where one of the connected parts is absent, in which case it may behave as a monoradical.

[0428] Heteroalkyl in the context of the invention represents an optionally substituted straight-chain or branched alkyl radical as defined above, wherein at least one carbon atom, preferably one to five carbon atoms, are independently replaced by a heteroatom or heteroatom functionality, selected from -NH-, -N=, -O-, -S-S-, -S-, - S(=O)- or -S(=O)2-.

[0429] Alkenyl in the context of the invention represents an optionally substituted straight¬

[0430] 15 chain or branched alkenyl radical having one to five double bonds and 2 to 50 carbon atoms. C2-C3alkenyl in the context of the invention represents an optionally substituted straight-chain or branched alkenyl radical having one or two double bonds and 2 to 3 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; and allyl. C2-C6alkenyl in the context of the

[0431] 20 invention represents an optionally substituted straight-chain or branched alkenyl radical having one or two double bonds and 2 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop-1-en- 1-yl; iso-propenyl; n-but-1-en-1-yl; n-but-2-en-1-yl; n-but-3-en-1-yl; 2-methylprop-1- en-1-yl; 2-methylprop-2-en-1-yl; n-pent-1-en-1-yl; and 2-methylbut-2-en-1-yl. C3-C6

[0432] 25 alkenyl in the context of the invention represents an optionally substituted straightchain or branched alkenyl radical having one or two double bonds and 3 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: n-prop-1-en-1-yl; iso-propenyl; n-but-1-en-1-yl; n-but-2-en-1-yl; n-but-3- en-1-yl; 2-methylprop-1-en-1-yl; and 2-methylprop-2-en-1-yl. C2-Ci2alkenyl in the

[0433] 30 context of the invention represents an optionally substituted straight-chain or branched alkenyl radical having one to five double bonds and 2 to 12 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop-1-en-1-yl; iso-propenyl; n-but-1-en-1-yl; n-but-2-en-1-yl; n-but-3-en-1- Foreignfiling text P24-220

[0434] - 39 - yl; 2-methylprop-1-en-1-yl; 2-methylprop-2-en-1-yl; n-pent-1-en-1-yl; 2-methylbut-2- en-1-yl; n-hex-1-en-1-yl; 3-methylpent-1-en-1-yl; n-hept-1-en-1-yl; 2-methylhex-2- en-1-yl; n-oct-1-en-1-yl; and 3-methylhept-1-en-1-yl. C2-C24alkenyl in the context of the invention represents an optionally substituted straight-chain or branched alkenyl

[0435] 5 radical having one to five double bonds and 2 to 24 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop- 1-en-1-yl; iso-propenyl; n-but-1-en-1-yl; n-but-2-en-1-yl; n-but-3-en-1-yl; 2- methylprop-1-en-1-yl; 2-methylprop-2-en-1-yl; n-pent-1-en-1-yl; 2-methylbut-2-en- 1-yl; n-hex-1-en-1-yl; 3-methylpent-1-en-1-yl; n-hept-1-en-1-yl; 2-methylhex-2-en-1- yl; n-oct-1-en-1-yl; 3-methylhept-1-en-1-yl; n-non-1-en-1-yl; 2-methyloct-2-en-1-yl; n-dec-1-en-1-yl; 3-methylnon-1-en-1-yl; n-undec-1-en-1-yl; 2-methyldec-2-en-1-yl; n-dodec-1-en-1-yl; 3-methylundec-1-en-1-yl; n-tridec-1-en-1-yl; 2-methyldodec-2- en-1-yl; n-tetradec-1-en-1-yl; 3-methyltridec-1-en-1-yl; n-pentadec-1-en-1-yl; 2- methyltetradec-2-en-1-yl; n-hexadec-1-en-1-yl; 3-methylpentadec-1-en-1-yl; n-

[0436] 15 heptadec-1-en-1-yl; 2-methylhexadec-2-en-1-yl; n-octadec-1-en-1-yl; 3- methylheptadec-1-en-1-yl; n-nonadec-1-en-1-yl; 2-methyloctadec-2-en-1-yl; n-icos- 1-en-1-yl; and 3-methylnonadec-1-en-1-yl. C6-C24alkenyl in the context of the invention represents an optionally substituted straight-chain or branched alkenyl radical having one to five double bonds and 6 to 24 carbon atoms. The following

[0437] 20 may be mentioned by way of example and by way of preference: n-hex-1-en-1-yl; 3-methylpent-1-en-1-yl; n-hept-1-en-1-yl; 2-methylhex-2-en-1-yl; n-oct-1-en-1-yl; 3- methylhept-1-en-1-yl; n-non-1-en-1-yl; 2-methyloct-2-en-1-yl; n-dec-1-en-1-yl; 3- methylnon-1-en-1-yl; n-undec-1-en-1-yl; 2-methyldec-2-en-1-yl; n-dodec-1-en-1-yl; 3-methylundec-1-en-1-yl; n-tridec-1-en-1-yl; 2-methyldodec-2-en-1-yl; n-tetradec-1-

[0438] 25 en-1-yl; 3-methyltridec-1-en-1-yl; n-pentadec-1-en-1-yl; 2-methyltetradec-2-en-1-yl; n-hexadec-1-en-1-yl; 3-methylpentadec-1-en-1-yl; n-heptadec-1-en-1-yl; 2- methylhexadec-2-en-1-yl; n-octadec-1-en-1-yl; 3-methylheptadec-1-en-1-yl; n- nonadec-1-en-1-yl; 2-methyloctadec-2-en-1-yl; n-icos-1-en-1-yl; n-docos-1-en-1-yl; n-tricos-1-en-1-yl; n-tetracos-1-en-1-yl; n-pentacos-1-en-1-yl; n-hexacos-1-en-1-yl;

[0439] 30 n-heptacos-1-en-1-yl; n-octacos-1-en-1-yl; n-nonacos-1-en-1-yl; n-triacont-1-en-1- yl; and n-hentriacont-1-en-1-yl. Foreignfiling text P24-220

[0440] - 40 -

[0441] Heteroalkenyl in the context of the invention represents an optionally substituted straight-chain or branched alkenyl radical as defined above, wherein at least one carbon atom, preferably one to five carbon atoms, are independently replaced by a heteroatom or heteroatom functionality, selected from -NH-, -N=, -O-, -S-S-, -S-, -

[0442] 5 S(=O)- or -S(=O)2.

[0443] Alkynyl in the context of the invention represents an optionally substituted straightchain or branched alkynyl radical having one to five triple bonds and 2 to 50 carbon atoms. C2-C3alkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical having one or two triple bonds and 2 to 3 carbon atoms. The following may be mentioned by way of example and by way of preference: ethynyl; and prop-2-yn-1-yl. C2-C6alkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical having one or two triple bonds and 2 to 6 carbon atoms. The following may

[0444] 15 be mentioned by way of example and by way of preference: ethynyl; prop-1-yn-1-yl; prop-2-yn-1-yl; but-1-yn-1-yl; but-2-yn-1-yl; but-3-yn-1-yl; 2-methylprop-1-yn-1-yl; 2- methylprop-2-yn-1-yl; pent-1-yn-1-yl; and 2-methylbut-2-yn-1-yl. C3-C6alkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical having one or two triple bonds and 3 to 6 carbon atoms.

[0445] 20 The following may be mentioned by way of example and by way of preference: prop- 1-yn-1-yl; prop-2-yn-1-yl; but-1-yn-1-yl; but-2-yn-1-yl; but-3-yn-1-yl; 2-methylprop-1- yn-1-yl; and 2-methylprop-2-yn-1-yl. C2-Ci2alkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical having one to five triple bonds and 2 to 12 carbon atoms. The following may be

[0446] 25 mentioned by way of example and by way of preference: ethynyl; prop-1-yn-1-yl; prop-2-yn-1-yl; but-1-yn-1-yl; but-2-yn-1-yl; but-3-yn-1-yl; 2-methylprop-1-yn-1-yl; 2- methylprop-2-yn-1-yl; pent-1-yn-1-yl; 2-methylbut-2-yn-1-yl; hex-1-yn-1-yl; 3- methylpent-1-yn-1-yl; hept-1-yn-1-yl; 2-methylhex-2-yn-1-yl; oct-1-yn-1-yl; and 3- methylhept-1-yn-1-yl. C2-C24alkynyl in the context of the invention represents an

[0447] 30 optionally substituted straight-chain or branched alkynyl radical having one to five triple bonds and 2 to 24 carbon atoms. The following may be mentioned by way of example and by way of preference: ethynyl; prop-1-yn-1-yl; prop-2-yn-1-yl; but-1- yn-1-yl; but-2-yn-1-yl; but-3-yn-1-yl; 2-methylprop-1-yn-1-yl; 2-methylprop-2-yn-1-yl; Foreignfiling text P24-220

[0448] - 41 - pent-1-yn-1-yl; 2-methylbut-2-yn-1-yl; hex-1-yn-1-yl; 3-methylpent-1-yn-1-yl; hept-1- yn-1-yl; 2-methylhex-2-yn-1-yl; oct-1-yn-1-yl; 3-methylhept-1-yn-1-yl; non-1-yn-1-yl;

[0449] 2-methyloct-2-yn-1-yl; dec-1-yn-1-yl; 3-methylnon-1-yn-1-yl; undec-1-yn-1-yl; 2- methyldec-2-yn-1-yl; dodec-1-yn-1-yl; 3-methylundec-1-yn-1-yl; tridec- 1-yn-1-yl; 2-

[0450] 5 methyldodec-2-yn-1-yl; tetradec- 1-yn-1-y I; 3-methyltridec-1-yn-1-yl; pentadec-1-yn- 1-yl; 2-methyltetradec-2-yn-1-yl; hexadec- 1-yn-1-yl; 3-methylpentadec-1-yn-1-yl; heptadec- 1-yn-1-yl; 2-methylhexadec-2-yn-1-yl; octadec- 1-yn-1-yl; 3- methylheptadec-1-yn-1-yl; nonadec-1-yn-1-yl; 2-methyloctadec-2-yn-1-yl; icos-1- yn-1-yl; docos-1-yn-1-yl; tricos- 1-yn-1-yl; tetracos- 1-yn-1-y I; pentacos- 1-yn-1-yl; hexacos- 1-yn-1-y I; heptacos- 1-yn-1-y I; octacos-1-yn-1-yl; nonacos-1-yn-1-yl; triacont-1-yn-1-yl; and hentriacont-1-yn-1-yl. C6-C24alkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical having one to five triple bonds and 6 to 24 carbon atoms. The following may be mentioned by way of example and by way of preference: n-hex-1-yn-1-yl; 3-

[0451] 15 methylpent-1-yn-1-yl; n-hept-1-yn-1-yl; 2-methylhex-2-yn-1-yl; n-oct-1-yn-1-yl; 3- methylhept-1-yn-1-yl; n-non-1-yn-1-yl; 2-methyloct-2-yn-1-yl; n-dec-1-yn-1-yl; 3- methylnon-1-yn-1-yl; n-undec-1-yn-1-yl; 2-methyldec-2-yn-1-yl; n-dodec-1-yn-1-yl;

[0452] 3-methylundec-1-yn-1-yl; n-tridec-1-yn-1-yl; 2-methyldodec-2-yn-1-yl; n-tetradec-1- yn-1-yl; 3-methyltridec-1-yn-1-yl; n-pentadec-1-yn-1-yl; 2-methyltetradec-2-yn-1-yl;

[0453] 20 n-hexadec-1-yn-1-yl; 3-methylpentadec-1-yn-1-yl; n-heptadec-1-yn-1-yl; 2- methylhexadec-2-yn-1-yl; n-octadec-1-yn-1-yl; 3-methylheptadec-1-yn-1-yl; n- nonadec-1-yn-1-yl; 2-methyloctadec-2-yn-1-yl; n-icos-1-yn-1-yl; n-docos-1-yn-1-yl; n-tricos-1-yn-1-yl; n-tetracos-1-yn-1-yl; n-pentacos-1-yn-1-yl; n-hexacos-1-yn-1-yl; n-heptacos-1-yn-1-yl; n-octacos-1-yn-1-yl; n-nonacos-1-yn-1-yl; n-triacont-1-yn-1-

[0454] 25 yl; and n-hentriacont-1-yn-1-yl.

[0455] Heteroalkynyl in the context of the invention represents an optionally substituted straight-chain or branched alkynyl radical as defined above, wherein at least one carbon atom, preferably one to five carbon atoms, are independently replaced by a

[0456] 30 heteroatom or heteroatom functionality, selected from -NH-, -N=, -O-, -S-S-, -S-, - S(=O)- or -S(=O)2. Foreignfiling text P24-220

[0457] - 42 -

[0458] Aryl in the context of the invention represents an optionally substituted aromatic cycle (aromatic radical) which can be either monocyclic or polycyclic, having 3 to 8 atoms per ring. The following may be mentioned by way of example and by way of preference: phenyl; naphtyl; fluorenyl; anthraceneyl; and phenanthrenyl.

[0459] 5

[0460] Heteroaryl in the context of the invention represents an optionally substituted aromatic heterocycle (heteroaromatic radical) which can be either monocyclic or polycyclic, having 3 to 8 atoms per ring, wherein at least one ring contains at least one heteroatom or heteroatom functionality, preferably one to five heteroatoms or heteroatom functionalities, independently selected from -NH-, -N=, -O-, -S-S-, -S-, - S(=O)- or -S(=O)2- and is attached via a ring carbon atom or a heteroatom wherein the one or more rings can have 4 to 8 ring atoms. The following may be mentioned by way of example and by way of preference: pyridyl; furanyl; thienyl; quinolinyl; isoquinolinyl; indolyl; benzofuranyl; and benzothiazolyl.

[0461] 15

[0462] Cycloalkyl in the context of the invention represents an optionally substituted saturated or partially saturated cycloalkyl group, which can be either monocyclic or polycyclic, having 3 to 8 atoms per ring, whereby the polycyclic cycloalkyl group can be fused ring structures, spirocycles, bridged rings or a cycloalkyl group fused with

[0463] 20 one or more aromatic rings. The following may be mentioned by way of example and by way of preference: cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cycloheptyl; cyclohexenyl; cyclopentenyl; cyclobutenyl; cycloheptenyl; cyclooctenyl; bicyclo[1.1.0]butyl; bicyclo[2.2.0]hexyl; bicyclo[3.3.0]octyl; bicyclo[3.2.0]heptyl; bicyclo[2.2.0]hexenyl; bicyclo[3.3.0]octenyl; decalyl; norbornyl; cubyl;

[0464] 25 bicyclo[4.2.0]octan-7-ylidene; decahydronaphthalen-2-ylidene; bicyclo[2.2.0]hexan- 2-ylidene; bicyclo[4.2.0]octa- 1 (6) ;2;4-trien-7-ylidene; 2;3-dihydro- 1 H-inden- 1 - ylidene; 2;3-dihydro-1 H-inden-2-ylidene; spiro[3.3]heptan-2-ylidene; spiro[3.5]nonan-7-ylidene; spiro[3.5]nonan-2-ylidene; spiro[5.5]undecan-3-ylidene; adamantan-2-ylidene; bicyclo[3.1.1]heptan-3-ylidene; bicyclo[2.2.2]octan-2-

[0465] 30 ylidene; bicyclo[2.1.1]hexan-2-ylidene; bicyclo[2.2.1]heptan-2-ylidene; cholesteryl; cholesteryl-derivatives and adamantyl. Foreignfiling text P24-220

[0466] - 43 -

[0467] Heterocycloalkyl in the context of the invention represents an optionally substituted saturated or partially saturated heterocyclic group, which can be either monocyclic or polycyclic, having 3 to 8 atoms per ring, wherein at least one ring contains at least one heteroatom or heteroatom functionality, preferably one to five heteroatoms or

[0468] 5 heteroatom functionalities, independently selected from -NH-, -N=, -O-, -S-S-, -S-, - S(=O)- or -S(=O)2- and is attached via a ring carbon atom or a heteroatom; whereby the polycyclic heterocycloalkyl group can be fused ring structures; spirocycles; bridged rings or a heterocycloalkyl group fused with one or more aromatic rings. The following may be mentioned by way of example and by way of preference: pyrrolidinyl; tetrahydrofuranyl; oxetan-3-ylidene; oxolan-3-ylidene; oxan-4-ylidene; oxepan-4-ylidene; morpholinyl; piperidinyl; furanyl; thienyl; thiazolyl; indolinyl; quinolinyl; tetrahydroquinolinyl; isoquinolinyl; benzothiazolyl; benzofuranyl; bicyclo[2.2.1]heptyl; 2-oxo-2,3-dihydro-1 H-indol-3-ylidene; 4,5,6,7-tetrahydro-1- benzothiophen-4-ylidene; 1 H,4H,5H,6H-cyclopenta[b]pyrrol-4-ylidene;

[0469] 15 decahydroquinolin-4-ylidene; octahydro-2H- 1 -benzopyran-4-ylidene; hexahydro- 1 H-cyclopenta[c]furan-5-ylidene; 2,2-dioxo-2lambda6-thiaspiro[3.3]heptan-6- ylidene; 2-thiaspiro[3.3]heptan-6-ylidene; 2-oxaspiro[3.3]heptan-6-ylidene; 2- azabicyclo[2.2.2]octan-5-ylidene and 2-oxabicyclo[2.2.2]octan-5-ylidene.

[0470] 20 Alkoxy in the context of the invention represents an optionally substituted straightchain or branched alkoxy radical. A straight-chain or branched alkoxy radical having 1 to 6 carbon atoms is preferred. The following radicals may be mentioned by way of example and by way of preference: methoxy; ethoxy; n-propoxy; isopropoxy; n- butoxy; tert-butoxy; n-pentoxy and n-hexoxy.

[0471] 25

[0472] Hydroxyalkyl in the context of the invention represents an alkyl group as defined above that contains one or more hydroxyl (-OH) functional groups.

[0473] Optionally substituted in the context of the invention means that the referenced

[0474] 30 chemical moiety may be unsubstituted or substituted with one or more chemical groups. Substituted with a chemical group in the context of the invention means that one or more hydrogen atoms are replaced by a different chemical group. Foreignfiling text P24-220

[0475] - 44 -

[0476] In one embodiment of the invention, substituted means substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, hydroxy, oxo, thioxo, alkoxy, halogen, -CN, -NO2, -N3, -ORa, -NRaRb, -N+RaRbRc, -

[0477] 5 NRaC(=O)Rb, -SRa, -S-SRa, -C(=O)Ra, -CH(ORa)(ORb), -C(=O)ORa, -C(=O)SRa-, - C(=O)Ra, -OC(=O)Ra, -SC(=O)Ra, -C(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, - NRaC(=NRb)NRcRd, -NRaC(=NRb)Rc, -C(=NRa)NRbRc, -NRaC(=O)NRbRc, -S(=O)Ra, -SO2Ra, -NRaSORb, -NRaSO2Rb, -S(=O)NRaRb, -C(=S)NRaRb, -NRaC(=S)Rb, - SO2NRaRb, and -OSOsR3, -OPC>3RaRb, -N=NRb, -OSiRaRbRc; wherein Ra, Rb, Rcand Rdindependently of one another are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and alkoxy, and wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, Ra, Rb, Rcand Rdare optionally substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl,

[0478] 15 cycloalkyl, heterocycloalkyl, hydroxy, oxo, thioxo, alkoxy, halogen, -CN, -NO2, -N3, wherein Ra’, Rb’, Rc’ and Rd’ independently of one another are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and alkoxy.

[0479] 25

[0480] In another aspect, the present invention relates to novel phosphonolipids that can be used in lipid particle (e.g. LNP) for drug delivery. Those novel phosphonolipids can also be used in lipid particles according to the invention.

[0481] 30 In one embodiment, the phosphonolipid is a compound according to formula (III) Foreignfiling text P24-220

[0482] - 45 - wherein

[0483] 5

[0484] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0485] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0486] B is -OC(=O)-,

[0487] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, cycloalkyl or heterocycloalkyl, and,

[0488] D is hydrogen or -N+RaRbRc-, wherein

[0489] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,

[0490] 15 with the proviso that if A and B are both -OC(=O)-, L is C3-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0491] In one embodiment, the phosphonolipid is a compound according to formula (III)

[0492] 20 wherein

[0493] 25 R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0494] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0495] B is -OC(=O)-,

[0496] 30 L is optionally substituted C3 alkyl,

[0497] D is hydrogen or -N+RaRbRc-, wherein Foreignfiling text P24-220

[0498] - 46 -

[0499] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0500] 5 In one embodiment, the phosphonolipid is a compound according to formula (IV) wherein

[0501] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0502] 15 L is optionally substituted C3 alkyl, and D is hydrogen or -N+RaRbRc-, wherein

[0503] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0504] 20

[0505] In one embodiment, the phosphonolipid is a compound according to formula (V) wherein

[0506] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0507] 30

[0508] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, cycloalkyl or heterocycloalkyl, and

[0509] D is hydrogen or -N+RaRbRc-, wherein Foreignfiling text P24-220

[0510] - 47 -

[0511] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0512] 5 In one embodiment, the phosphonolipid is a compound according to formula (V) wherein

[0513] R1and R2independently of one another are optionally substituted C6-C24 alkenyl or C6-C24 heteroalkenyl,

[0514] L is optionally substituted Ci-Ce alkyl, and

[0515] 15 D is hydrogen or -N+RaRbRc-, wherein

[0516] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0517] 20 In one embodiment, the phosphonolipids is a compound according to formula (Illa) (Illa), wherein

[0518] 25

[0519] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0520] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0521] B is -OC(=O)-,

[0522] 30

[0523] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, cycloalkyl or heterocycloalkyl, and,

[0524] D is hydrogen or -N+RaRbRc-, wherein Foreignfiling text P24-220

[0525] - 48 -

[0526] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is C3-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and

[0527] 5 isotopically labeled derivative thereof.

[0528] In one embodiment, the phosphonolipids is a compound according to formula (Illa), wherein

[0529] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24 heteroalkynyl,

[0530] A is -OC(=O)- or -NRaC(=O)-,

[0531] B is -OC(=O)- or -NRaC(=O)-, wherein

[0532] Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,

[0533] 15 L is optionally substituted Ci-Ce alkyl, Ci-Ce heteroalkyl C2-C6 alkenyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and,

[0534] D is hydrogen or -N+RaRbRc-, wherein

[0535] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,

[0536] 20 with the proviso that if A and B are both -OC(=O)-, L is C2-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0537] In one embodiment, the phosphonolipids is a compound according to formula (Illa),

[0538] 25 wherein

[0539] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 heteroalkyl or C6-C24 heteroalkenyl,

[0540] A is-OC(=O)-, -O-, or -NRaC(=O)-,

[0541] B is -OC(=O)- or -NRaC(=O)-,

[0542] 30 L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenylyl, cycloalkylyl or heterocycloalkylyl, and

[0543] D is -N+RaRbRc-, wherein Foreignfiling text P24-220

[0544] - 49 -

[0545] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is optionally substituted C2-C6 alkyl,

[0546] 5 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0547] In one embodiment, the phosphonolipid is a compound according to formula (Illa), wherein

[0548] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0549] A is -OC(=O)-, -O- or -NRaC(=O)-,

[0550] B is -OC(=O)-,

[0551] 15 L is optionally substituted C2 alkyl,

[0552] D is hydrogen or -N+RaRbRc-, wherein

[0553] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0554] 20

[0555] In one embodiment, the phosphonolipid is a compound according to formula (Illa) wherein

[0556] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24

[0557] 25 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0558] A is -OC(=O)- or -NRaC(=O)-,

[0559] B is -OC(=O)- or -NRaC(=O)-,

[0560] L is optionally substituted Ci-Ce alkyl, Ci-Ce heteroalkyl C2-C6 alkenyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl, and,

[0561] 30 D is hydrogen or -N+RaRbRc-, wherein

[0562] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is C3-C6 alkyl, Foreignfiling text P24-220

[0563] - 50 - and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0564] In one embodiment, the phosphonolipid is a compound according to formula (Illa)

[0565] 5 wherein

[0566] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0567] L is optionally substituted C2-C6 alkyl, and

[0568] D is hydrogen or -N+RaRbRc-, wherein

[0569] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0570] 15

[0571] In one embodiment, the phosphonolipid is a compound according to formula (IVa) , wherein

[0572] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24

[0573] 25 heteroalkynyl,

[0574] L is optionally substituted C2-C6 alkyl, and

[0575] D is hydrogen or -N+RaRbRc-, wherein

[0576] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,

[0577] 30 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0578] In one embodiment, the phosphonolipid is a compound according to formula (IVa) wherein Foreignfiling text P24-220

[0579] - 51 -

[0580] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0581] L is optionally substituted C2 alkyl, and

[0582] 5 D is hydrogen or -N+RaRbRc-, wherein

[0583] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0584] In one embodiment, the phosphonolipid is a compound according to formula (IVa) wherein

[0585] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0586] 15 L is optionally substituted C2 alkyl, and

[0587] D is -N+RaRbRc-, wherein

[0588] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0589] 20

[0590] In one embodiment, the phosphonolipid is a compound according to formula (Illa) or (IVa), wherein

[0591] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl,

[0592] 25 L is optionally substituted C2 alkyl, and

[0593] D is -N+RaRbRc-, wherein

[0594] Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted Ci-Ce alkyl or C2-C6 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0595] 30

[0596] In one embodiment, the phosphonolipid is a compound according to formula (IVa) wherein Foreignfiling text P24-220

[0597] - 52 -

[0598] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl,

[0599] L is optionally substituted C2 alkyl, and

[0600] D is -N+RaRbRc-, wherein

[0601] 5 Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted Ci-Ce alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0602] In one embodiment, the phosphonolipid is a compound according to formula (IVa) wherein

[0603] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl,

[0604] L is optionally substituted C2 alkyl, and

[0605] D is -N+RaRbRc-, wherein

[0606] 15 Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0607] In one embodiment, the phosphonolipid is a compound according to formula (IVa)

[0608] 20 wherein

[0609] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl,

[0610] L is optionally substituted C2 alkyl, and

[0611] D is -N+RaRbRc-, wherein

[0612] 25 Ra, Rb, and Rcare hydrogen, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0613] In one embodiment, the phosphonolipid is a compound according to formula (IVa) wherein

[0614] 30 R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl,

[0615] L is optionally substituted C2 alkyl, and

[0616] D is -N+RaRbRc-, wherein Foreignfiling text P24-220

[0617] - 53 -

[0618] Ra, Rb, and Rcare methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0619] In one embodiment, the phosphonolipid is a compound according to formula (Va)

[0620] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl,

[0621] L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, cycloalkyl or heterocycloalkyl,

[0622] 15 and

[0623] D is hydrogen or -N+RaRbRc-, wherein

[0624] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0625] 20

[0626] In one embodiment, the phosphonolipid is a compound according to formula (Va) wherein

[0627] R1and R2independently of one another are optionally substituted C6-C24 alkenyl or C6-C24 heteroalkenyl,

[0628] 25 L is optionally substituted Ci-Ce alkyl, and

[0629] D is hydrogen or -N+RaRbRc-, wherein

[0630] Ra, Rb, and Rcindependently of one another are a bond, hydrogen, optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0631] 30

[0632] In one embodiment, the phosphonolipid is a compound according to formula (VI) Foreignfiling text P24-220

[0633] - 54 - wherein

[0634] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl, C6-C24 heteroalkynyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl, and

[0635] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0636] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0637] 20

[0638] R1and R2independently of one another are optionally substituted C6-C24 alkyl, Ce- C24 alkenyl, C6-C24 heteroalkyl or C6-C24 heteroalkenyl, and

[0639] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0640] 25

[0641] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein Foreignfiling text P24-220

[0642] - 55 -

[0643] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and

[0644] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt,

[0645] 5 solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0646] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0647] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0648] 15 C6-C24 alkenyl, and

[0649] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted Ci-Ce alkyl or C2-C6 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0650] 20 In one embodiment, the phosphonolipid is a compound according to formula (VI)

[0651] 25 wherein

[0652] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0653] C6-C24 alkenyl, and

[0654] R3, R4and R5independently of one another are a free bond, hydrogen or optionally substituted C1-C3 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate,

[0655] 30 tautomer, stereoisomer and isotopically labeled derivative thereof.

[0656] In one embodiment, the phosphonolipid is a compound according to formula (VI) Foreignfiling text P24-220

[0657] - 56 - wherein

[0658] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and

[0659] R3, R4and R5independently of one another are a free bond, hydrogen or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0660] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0661] R1and R2independently of one another are optionally substituted C6-C24 alkyl or

[0662] 20 C6-C24 alkenyl, and

[0663] R3, R4and R5independently of one another are hydrogen or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0664] 25

[0665] In one embodiment, the phosphonolipid is a compound according to formula (VI) wherein

[0666] R1and R2independently of one another are optionally substituted C6-C24 alkyl or C6-C24 alkenyl, and Foreignfiling text P24-220

[0667] - 57 -

[0668] R3, R4and R5are methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0669] In one embodiment, the phosphonolipid is a compound according to formula (VII),

[0670] 5 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0671] Compound of formula (VII) is also referenced as I ND- 1233 in the present disclosure.

[0672] In one embodiment, the phosphonolipid is a compound according to formula (VIII),

[0673] 15

[0674] (VIII), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer,

[0675] 20 stereoisomer and isotopically labeled derivative thereof.

[0676] Compound of formula (VIII) is also referenced as IND-1234 in the present disclosure.

[0677] 25

[0678] In one embodiment, the phosphonolipid is a compound according to formula (IX),

[0679] 30

[0680] (IX), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof. Foreignfiling text P24-220

[0681] - 58 -

[0682] Compound of formula (IX) is also referenced as IND-1235 in the present disclosure.

[0683] In one embodiment, the phosphonolipid is a compound according to formula (X), and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

[0684] Compound of formula (X) is also referenced as IND-1236 in the present disclosure.

[0685] In another embodiment, the phosphonolipid is a compound selected from the list consisting of formula (VII), (VIII), (IX) and (X).

[0686] In another aspect, the present invention relates to a pharmaceutical composition

[0687] 15 comprising the lipid particle (e.g. LNP) for drug delivery comprising at least one pharmaceutical agent (e.g. RNA or mRNA) and at least one phosphonolipid as defined above.

[0688] In another aspect, the present invention relates to a pharmaceutical composition

[0689] 20 comprising at least one phosphonolipid as defined above.

[0690] In one embodiment, the pharmaceutical composition comprises a lipid particle (e.g. LNP), preferably a lipid nanoparticle, comprising one pharmaceutical agent, preferably a nucleic acid, more preferably a RNA, at least one cationic lipid, at least

[0691] 25 one helper lipid, and at least one conjugated lipid, wherein at least one helper lipid is a phosphonolipid.

[0692] All embodiments as defined above for the aspect of the lipid particle (e.g. LNP) for drug delivery and the aspect of the phosphonolipid according to the invention, also

[0693] 30 apply for the aspect of the pharmaceutical composition.

[0694] In another embodiment, the pharmaceutical composition additionally comprises at least one pharmaceutically acceptable excipient. Foreignfiling text P24-220

[0695] - 59 -

[0696] In some embodiments, the lipid particle (e.g. LNP) is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic

[0697] 5 benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0698] The lipid particles (e.g. LNPs) described herein may be used to treat and / or prevent any disease, disorder or condition in a mammalian subject. This includes a disease, disorder or condition, such as cancer, infectious diseases such as bacterial, viral, fungal or parasitic infections, inflammatory and / or autoimmune disorders, including treatments that induce immune tolerance and cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis. Examples of cancers include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine

[0699] 15 cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting

[0700] 20 examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of some other non-liver cancer cell type), and hepatoblastoma.

[0701] Non-limiting examples of other diseases, disorders or conditions that may be treated

[0702] 25 by the lipid particles (e.g. LNPs) described herein and that may be attributed at least in part to an immunological disorder include colitis, Crohn's disease, allergic encephalitis, allograft transplant / graft vs. host disease (GVHD), diabetes and multiple sclerosis.

[0703] 30 The lipid particles (e.g. LNPs) described herein may also be used in other applications besides the treatment and / or prevention of a disease or disorder. The lipid particles (e.g. LNPs) may be used to treat conditions such as aging, Foreignfiling text P24-220

[0704] - 60 - preventative medicine and / or as part of a personalized medicine regime. In further embodiments, the lipid particle (e.g. LNP) is used in a diagnostic application.

[0705] In one embodiment, the lipid particle (e.g. LNP) is part of a pharmaceutical

[0706] 5 composition administered parenterally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral administration. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0707] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0708] In another aspect, the present invention relates to a method for in vivo delivery of a

[0709] 15 pharmaceutical agent (e.g. RNA or mRNA) to a mammalian subject, the method comprising: administering to the mammalian subject a lipid particle (e.g. LNP) as described above.

[0710] All embodiments as defined above for the aspect of the lipid particle (e.g. LNP) for drug delivery and the aspect of the phosphonolipid according to the invention, also

[0711] 20 apply for the aspect of the pharmaceutical composition.

[0712] In another aspect, the present invention relates to a use of the lipid particle (e.g. LNP) as described above in vivo or in vitro delivery and expression of the pharmaceutical agent (e.g. RNA or mRNA) to mammalian cells.

[0713] 25

[0714] In another aspect, the present invention relates to a use of the lipid particle (e.g. LNP) as described above for the manufacture of a medicament for in vivo or in vitro delivery of the pharmaceutical agent (e.g. RNA or mRNA) to mammalian cells.

[0715] Examples:

[0716] 30

[0717] Example 1 : Phosphonolipid synthesis and characterization

[0718] 1. 1 Synthesis procedure and characterization of IND 1233 Foreignfiling text P24-220

[0719] - 61 -

[0720] Synthesis procedure

[0721] In a 500 mL round bottom flask equipped with a magnetic stirrer bar, palmitic acid (25.0 g, 97.49 mmol, 2.6 eq.) was dissolved in DCM (300 mL). Subsequently, 3- (benzyloxy)propane-1 ,2-diol (6.83 g, 37.50 mmol, 1.0 eq.), EDC.HCI (28.75 g, 150

[0722] 5 mmol, 4 eq.) and DMAP (1.83 g, 15 mmol, 0.4 eq.) and the reaction mixture was stirred at 25°C for 15 hrs. After completion of the reaction (checked by TLC), the organic layer was washed with an aqueous solution of NaHCOs (50 mL) followed by brine solution. The organic layer was dried over Na2SC>4, filtered, and concentrated under reduced pressure. The final product 3-(benzyloxy) propane- 1 ,2-diyldipalm itate was purified by column chromatography (SiC>2, 0-5% EtOAc in Hexane) and obtained as a white solid (21.30 g, 86% yield).

[0723] A solution of 3-(benzyloxy)propane-1 ,2-diyldipalmitate 11.0 g (15.38 mmol) in 140 ml of THF was hydrogenated (50 psi) over 700 mg of 20% Pd(OH)2 / C for 20 min at room temperature using parr shaker. The catalyst was removed by filtration and the

[0724] 15 filtrate was concentrated and washed with hexane to afford 3-hydroxypropane-1 ,2- diyldipalmitate as a crude white solid (8.10 g, 85%). The filtered Pd(OH)2 / C and celite were then transferred to a 20% HCI solution and stored in a palladium waste container.

[0725] In a flame-dried 250 mL round bottom flask equipped with a magnetic bar, diethyl

[0726] 20 (3-bromopropyl)phosphonate (4.80 g, 18.50 mmol, 1.0 eq.) was dissolved in dry DCM (50 mL). The solution was cooled to -40°C and TMSBr (6.23 g, 40.70 mmol, 2.2 eq.) was added drop by drop over 10 minutes. This was stirred for 20 minutes at -40°C, before warming to room temperature, at which point it was stirred for 3 hours. The solvent was removed under a high vacuum in inert condition at 50 °C for

[0727] 25 1.5 h. The residue was dissolved in ethanol-free chloroform (50 mL) and PCIs (9.24 g, 44.4 mmol, 2.4 eq.) was added in one portion at room temperature. The solution was heated to 50°C for 1.5 hours, then after the solvent was again removed under high vacuum in inert condition at 50 °C for 1.5 h obtained (3-bromopropyl) phosphonic dichloride. The residue of (3-bromopropyl) phosphonic dichloride was

[0728] 30 once again dissolved in ethanol-free chloroform (10 mL / g) and cooled to 0°C. A mixture of Et3N (2.59 mL, 18.50 mmol, 1.0 eq.) and 3-hydroxypropane-1 ,2- diyldipalmitate (5.27 g. 9.26 mmol, 0.5 eq.) in ethanol-free chloroform (10 ml / g) was added dropwise. After the addition, the solution was warmed to room temperature, Foreignfiling text P24-220

[0729] - 62 - allowing it to stir for 48 hours. Water (2 mL / g of alcohol) was added to the flask and stirred for 1 hour before removing the solvent under vacuum (60°C water bath). The residue was dissolved in (10:10:1) CHCI3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1.5 hours. The solution

[0730] 5 was filtered and washed with the same solvent, and the filtrate was concentrated by rotary evaporation. The residue was then taken up in DCM (20 mL / g), gently washed with water (10 mL / g) and brine (10 mL / g), dried over MgSCL, filtered and concentrated under vacuum and the crude of 2,3-bis(palmitoyloxy)propyl (3- bromopropyl)phosphonate was used further. The crude of 2,3- bis(palmitoyloxy)propyl (3-bromopropyl)phosphonate was redissolved in (10:17:17) CHCI3:iPrOH:MeCN (20 mL / g) and 40% aq. Me3N (19.0 mL, 280 mmol, 15.1 eq.) and allowed to stir for 48 hours at 60°C. After cooling and concentrating the solution under vacuum, it was once again dissolved in (10:10:1) CHCI3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1 hour.

[0731] 15 After filtering and rinsing with the same solvent, the filtrate was concentrated under a vacuum, taken up in DCM (20 mL / g), gently washed with water (10 mL / g) and brine (10 mL / g) and dried over MgSCL, filtered, and concentrated by rotary evaporation. The compound was precipitated using CHCI3and acetone as off white solid (4.20 g). The residue was purified by normal column chromatography using

[0732] 20 neutral alumina as the stationary phase and Hexane / CHCI3 / MeOH as the mobile phase. First, we used 0-100% CHCI3 / hexane then 0-20% Methanol / CHCI3. The final compound IND1233 was eluted at 10-20% methanol in CHCI3as an off-white solid (3.5 g, 26%).

[0733] 25 Column chromatography and TLC

[0734] Reagents and solvents for column chromatography and TLC (MeOH, Chloroform, DCM, Ethyl acetate and Hexane) were of reagent grade and acquired from Sigma- Aldrich. They were used without further purification.

[0735] For column chromatography, silica gel (230-400 mesh) and neutral alumina was

[0736] 30 used. Thin layer chromatography was carried out with ready-to-use aluminum plates coated with silica gel from Sigma-Aldrich). The detection was done by application of staining reagent phosphomolybdic acid (PMA). The staining reagent PMA was prepared by dissolving 10 g PMA in 100 mL EtOH. Foreignfiling text P24-220

[0737] - 63 -

[0738] NMR

[0739] NMR spectra were recorded using Bruker 400 MHz NMR spectrometer. Calibration of the spectra was done using solvent residual peaks. Coupling constants J were

[0740] 5 given in Hz. The following abbreviations were used to describe the signals in the NMR spectra: s - singlet, d - doublet, t - triplet, q - quartet, quint - quintet, and m - complex multiplet. Abbreviation “br” is used to describe broad signals: e.g. “br s” stands for “broad singlet”.

[0741] LC-MS

[0742] LC MS were recorded using Shimadzu LCMS 8045.

[0743] 7.2 Synthesis procedure and characterization of IND 1234

[0744] In a 500 mL round bottom flask equipped with a magnetic stirrer bar, stearic acid

[0745] 15 (25 g, 87.88 mmol, 2.6 eq.) was dissolved in DCM (300 mL). Subsequently, 3- (benzyloxy)propane-1 ,2-diol (6.52 g, 33.80 mmol, 1.0 eq.), EDC.HCI (25.92 g, 135.20 mmol, 4 eq.) and DMAP (1.65 g, 13.52 mol, 0.4 eq.) and the reaction mixture was stirred at 25 °C for 15 hrs. After completion of the reaction (checked by TLC), the organic layer was washed with an aqueous solution of NaHCCh (50 mL) followed

[0746] 20 by brine solution and the organic layer was dried over Na2SC>4, filtered, and concentrated under reduced pressure. The final product was purified by column chromatography (SiC>2, 0-5% EtOAc in Hexane) and obtained 3- (benzyloxy)propane-1 ,2-diyldistearate as a white solid (21.10 g, 87% yield).

[0747] A solution of 11.0 g (15.38 mmol) of the 3-(benzyloxy)propane-1 ,2-diyldistearate in

[0748] 25 140 ml of THF was hydrogenated (50 psi) over 770 mg (7% w / w) of 20% Pd(OH)2 / C for 20 min at room temperature using parr shaker. The catalyst was removed by filtration and the filtrate was concentrated and washed with hexane to afford 3- hydroxypropane-1 ,2-diyldistearate as a crude white solid (8.41 g, 87.5 %). This was used further without purification. The filtered Pd(OH)2 / C and celite were then

[0749] 30 transferred to a 20% HCI solution and stored in a palladium waste container. In a flame-dried 250 mL round bottom flask equipped with a magnetic bar, diethyl (3- bromopropyl)phosphonate (4.80 g, 18.50 mmol, 1.0 eq.) was dissolved in dry DCM (50 mL). The solution was cooled to -40°C and TMSBr (6.23 g, 40.70 mmol, 2.2 eq.) Foreignfiling text P24-220

[0750] - 64 - was added drop by drop over 10 minutes. This was stirred for 20 minutes at -40°C before warming to room temperature, at which point it was stirred for 3 hours. The solvent was removed under a high vacuum in inert condition at 50 °C for 1.5 h. The residue was dissolved in ethanol-free chloroform (50 mL) and PCIs (9.24 g, 44.4

[0751] 5 mmol, 2.4 eq.) was added in one portion at room temperature. The solution was heated to 50°C for 1.5 hours then after the solvent was again removed under high vacuum in inert condition at 50°C for 1.5 h to obtain (3-bromopropyl) phosphonic dichloride. The residue of (3-bromopropyl) phosphonic dichloride was once again dissolved in ethanol-free chloroform (10 mL / g) and cooled to 0°C. A mixture of Et3N (2.59 mL, 18.50 mmol, 1.0 eq.) and 3-hydroxypropane-1 ,2-diyldistearate (5.79 g. 9.26 mmol, 2.0 eq.) in ethanol-free chloroform (10 mL / g) was added dropwise. After the addition, the solution was warmed to room temperature, allowing it to stir for 48 hours. Water (2 mL / g of alcohol) was added to the flask and stirred for 1 hour before removing the solvent under vacuum (60°C water bath). The residue was dissolved

[0752] 15 in (10:10:1) CHCl3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1.5 hours. The solution was filtered and washed with the same solvent, and the filtrate was concentrated by rotary evaporation. The residue was then taken up in DCM (20 mL / g), gently washed with water (10 mL / g) and brine (10 mL / g), dried over MgSC>4, filtered and concentrated under vacuum

[0753] 20 and the crude of 2,3-bis(stearoyloxy)propyl (3-bromopropyl)phosphonate was used further. The crude of 2,3-bis(stearoyloxy)propyl (3-bromopropyl)phosphonate was re-dissolved in (10:17:17) CHCI3:iPrOH:MeCN (20 mL / g) and 40% aq. Me3N (18.60 mL, 139.8 mmol, 15.1 eq.) and allowed to stir for 48 hours at 60 °C. After cooling and concentrating the solution under vacuum, it was once again dissolved in

[0754] 25 (10:10:1) CHCI3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1 hour. After filtering and rinsing with the same solvent, the filtrate was concentrated under a vacuum, taken up in DCM (20 mL / g), gently washed with water (10 mL / g) and brine (10 mL / g) and dried over MgSCL, filtered and concentrated by rotary evaporation. The compound was precipitated

[0755] 30 using CHCI3and acetone as off white solid (4.40 g). The residue was purified by normal column chromatography using neutral alumina as the stationary phase and Hexane / CHCI3 / MeOH as the mobile phase. First, we used 0-100% CHCI3 / Hexane Foreignfiling text P24-220

[0756] - 65 - then 0-20% Methanol / CHCh. The final compound IND1234 was eluted at 10-20% Methanol / CHCh as an off-white solid (3.7 g, 25%).

[0757] Column chromatography and TLC

[0758] 5 Reagents and solvents for column chromatography and TLC (MeOH, Chloroform, DCM, Ethyl acetate and Hexane) were of reagent grade and acquired from Sigma- Aldrich. They were used without further purification.

[0759] For column chromatography, silica gel (230-400 mesh) and neutral alumina was used. Thin layer chromatography was carried out with ready-to-use aluminum plates coated with silica gel from Sigma-Aldrich). The detection was done by application of staining reagent phosphomolybdic acid (PMA). The staining reagent PMA was prepared by dissolving 10 g PMA in 100 mL EtOH.

[0760] NMR

[0761] 15 spectra were recorded using Bruker 400 MHz NMR spectrometer. Calibration of the spectra was done using solvent residual peaks. Coupling constants J were given in Hz. The following abbreviations were used to describe the signals in the NMR spectra: s - singlet, d - doublet, t - triplet, q - quartet, quint - quintet, and m - complex multiplet. Abbreviation “br” is used to describe broad signals: e.g. “br s” stands for

[0762] 20 “broad singlet”

[0763] LC-MS

[0764] LC MS were recorded using Shimadzu LCMS 8045.

[0765] 1.3 Synthesis procedure and characterization of I ND 1235

[0766] 25

[0767] To a solution of glycerol (183g, 2 mol, 20 eq.), and imidazole (20g, 0.59 mol, 3 eq.) in DCM (300 mL) and DMF (120 mL), tert-butyldimethylsilyl chloride (15g, 0.099 mol, 1 equiv.) in DCM (20 mL) was added dropwise at -10°C. After complete addition, the reaction was stirred for 1 h. The resulting mixture was extracted with

[0768] 30 DCM. The organic layer was washed with water and brine twice and dried over anhydrous sodium sulfate. Solvent was evaporated under reduced pressure and purified by column chromatography with 0-40% EtOAc in hexane and obtained desired product 3-((tert-butyldimethylsilyl)oxy)propane-1 ,2-diol 18g (90%). Foreignfiling text P24-220

[0769] - 66 -

[0770] In a 500 mL round bottom flask equipped with a magnetic stir bar, oleic acid (10 g, 35.40 mmol, 2.5 eq.) was dissolved in DCM (100 mL). To this solution was added EDC.HCI (10.85 g, 56.64 mmol, 4 eq.) and DMAP (0.691 g, 5.66 mmol, 0.4 eq.) and the reaction mixture was stirred for 10 min followed by addition of 3-((tert-

[0771] 5 butyldimethylsilyl)oxy)propane-1 ,2-diol (2.87g, 14.16 mmol, 1 eq.) at rt and the reaction was stirred overnight. After completion of the reaction (checked by TLC), the organic layer was washed with an aqueous solution of NaHCCh (50 mL) followed by brine solution and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The final product was purified by column chromatography (SiC>2, 0-5% EtOAc in Hexane) and obtained 3-((tert- butyldimethylsilyl)oxy)propane-1 ,2-diyl dioleate as a liquid (9 g, 90% yield).

[0772] To a 250 mL round bottom flask equipped with magnetic stir bar was added 3-((tert- butyldimethylsilyl)oxy)propane-1 ,2-diyl dioleate (7g, 9.52 mmol, 1 eq.) in dry THF (50 mL) and maintained at -10°C. To this was added pyridine (2.30 mL, 28.58 mmol,

[0773] 15 3 eq.) followed by dropwise addition of HF-Pyridine (70%) (3.341 mL, 28.58 mmol, 3 eq.). The reaction was then stirred for 4h at room temperature. After completion, the reaction was extracted with water followed by aq. NaHCOs wash. The organic layer was dried over Na2SC>4, filtered, and concentrated under reduced pressure for 4-5 h to remove volatile impurities and obtained desired compound 3-

[0774] 20 hydroxypropane-1 ,2-diyl dioleate as liquid (5.6 g, 92% yield).

[0775] In a flame-dried three neck 250 mL round bottom flask equipped with a magnetic bar, diethyl(3-bromopropyl)phosphonate (4.80 g, 18.50 mmol, 1.0 eq.) was dissolved in dry DCM (50 mL). The solution was cooled to -40°C and TMSBr (6.23 g, 40.70 mmol, 2.2 eq.) was added dropwise over 10 minutes. This was stirred for

[0776] 25 20 minutes at -40 °C before warming to room temperature, the reaction was stirred for 3 h. After 3 h, the solvent was removed under high vacuum in inert condition at 50 °C for 1.5 h. The residue obtained was further dissolved in ethanol-free chloroform (50 mL) and PCIs (9.24 g, 44.4 mmol. 2.4 eq.) was added in one portion at room temperature. The reaction mixture was then heated to 50 °C for 1.5 h, then

[0777] 30 again solvent was removed under high vacuum in inert condition at 50 °C for 1.5 h to obtain (3-bromopropyl) phosphonic dichloride. The residue of (3-bromopropyl) phosphonic dichloride was once again dissolved in ethanol-free chloroform 50 mL and cooled to 0°C. A mixture of Et3N (2.59 mL, 18.50 mmol, 1.0 equiv.) and 3- Foreignfiling text P24-220

[0778] - 67 - hydroxypropane- 1 ,2-diyl dioleate (5.6 g. 9.3 mmol, 0.5 eq.) in ethanol-free chloroform (20 mL) was added dropwise. After the addition, the solution was warmed to room temperature and stirred for 48 h. After 48 h, water (2 mL / g) was added to the flask and stirred for 1 h before removing the solvent under vacuum

[0779] 5 (60°C in water bath). The residue was dissolved in 10:10:1 CHCI3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1 .5 h. The solution was filtered and washed with the same solvent, and the filtrate was concentrated by rotary evaporation. The residue was then taken up in DCM (100 mL) and gently washed with water (50 mL) and brine (50 mL), dried over MgSCL, filtered and concentrated under vacuum and re-dissolved in 10:17:17 CHCI3:PrOH:MeCN (100 mL / g) and 40% aq.Me3N (19 mL, 286 mmol, 15.1 eq.) and allowed to stir for 48 hours at 60°C. After cooling and concentrating the solution under vacuum, it was once again dissolved in 10:10: 1 CHCI3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1 hour.

[0780] 15 After filtering and rinsing with the same solvent, the filtrate was concentrated under vacuum, taken up in DCM (100 mL) and gently washed with water (50 mL) and brine (50 mL) and dried over MgSC>4, filtered and concentrated by rotary evaporation. The compound was precipitated using CHCI3and acetone as waxy compound (3.8 g). The precipitated residue was purified by normal column chromatography using

[0781] 20 neutral alumina as the stationary phase and Hexane / CHCI3 / MeOH as the mobile phase. We used 0-100% of CHCI3 / Hexane followed by 0-20% Methanol / CHCI3. The final compound IND1235 was eluted using 10-20% Methanol / CHCI3as a waxy compound (2.5 g, 27% yield).

[0782] 25 Column chromatography and TLC

[0783] Reagents and solvents for column chromatography and TLC (MeOH, Chloroform, DCM, Ethyl acetate and Hexane) were of reagent grade and acquired from Sigma- Aldrich. They were used without further purification.

[0784] For column chromatography, silica gel (230-400 mesh) and neutral alumina was

[0785] 30 used. Thin layer chromatography was carried out with ready-to-use aluminum plates coated with silica gel from Sigma-Aldrich). The detection was done by application of staining reagent phosphomolybdic acid (PMA). The staining reagent PMA was prepared by dissolving 10 g PMA in 100 mL EtOH. Foreignfiling text P24-220

[0786] - 68 -

[0787] NMR

[0788] NMR spectra were recorded using Bruker 400 MHz NMR spectrometer. Calibration of the spectra was done using solvent residual peaks. Coupling constants J were

[0789] 5 given in Hz. The following abbreviations were used to describe the signals in the NMR spectra: s - singlet, d - doublet, t - triplet, q - quartet, quint - quintet, and m - complex multiplet. Abbreviation “br” is used to describe broad signals: e.g. “br s” stands for “broad singlet”.

[0790] LC-MS

[0791] LC MS were recorded using Shimadzu LCMS 8045.

[0792] 7.4 Synthesis procedure and characterization of IND 1236

[0793] To a solution of glycerol (368 g, 4 mol, 20 eq.), and imidazole (41 g, 0.6 mol, 3 eq.)

[0794] 15 in DCM (600 mL) and DMF (240 mL), tert-butyldimethylsilyl chloride (30 g, 0.2 mol, 1 eq.) in DCM (40 mL) was added dropwise at -10°C. After complete addition, the reaction was stirred for 1 h. The resulting mixture was extracted with DCM and the organic layer was washed with water and brine twice and dried over anhydrous sodium sulfate. Solvent was evaporated under reduced pressure and purified by

[0795] 20 column chromatography with 0-40% EtOAc in Hexane to obtain 37 g of desired compound 3-((tert-butyldimethylsilyl)oxy)propane-1 ,2-diol as colorless liquid in 90% yield.

[0796] In a 500 mL round bottom flask equipped with a magnetic stir bar, oleic acid (10 g, 35.40 mmol, 2.5 eq.) was dissolved in DCM (100 mL). To this solution was added

[0797] 25 EDC.HCI (10.85 g, 56.64 mmol, 4 eq.) and DMAP (0.691 g, 5.66 mmol, 0.4 eq.) and the reaction mixture was stirred for 10 min followed by addition of 3-((tert- butyldimethylsilyl)oxy)propane-1 ,2-diol (2.87 g, 14.16 mmol, 1 eq.) at room temperature (rt) and the reaction was stirred for overnight at rt. After completion of the reaction (checked by TLC), the organic layer was washed with an aqueous

[0798] 30 solution of NaHCOs (50 mL) followed by brine solution. The organic layer was dried over Na2SC>4. filtered, and concentrated under reduced pressure. The final product was purified by column chromatography (SiC>2, 0-5% EtOAc in Hexane) and Foreignfiling text P24-220

[0799] - 69 - obtained 3-((tert-butyldimethylsilyl)oxy)propane-1 ,2-diyl dioleate as a liquid (9 g, 90% yield).

[0800] To a 250 mL RBF equipped with magnetic stir bar was added 3-((tert- butyldimethylsilyl)oxy)propane-1 ,2-diyl dioleate (7 g, 9.52 mmol, 1 equiv.) in dry

[0801] 5 THF (50 mL) and maintained at -10°C. To this solution pyridine (2.30 mL, 28.58 mmol, 3 equiv.) was added followed by dropwise addition of HF-Pyridine (70%) (3.341 mL, 28.58 mmol, 3 eq.). The reaction was then stirred for 4h at room temperature. After completion, the reaction was extracted with water followed by aq. NaHCOs wash. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure for 4-5 h to remove volatile impurities and obtained desired compound 3-hydroxypropane-1 ,2-diyl dioleate as liquid (5.6 g, 92% yield).

[0802] In a flame-dried three neck 250 mL round bottom flask equipped with a magnetic bar, diethyl(3-bromopropyl)phosphonate (4.80 g, 18.50 mmol, 1.0 eq.) was dissolved in dry DCM (50 mL). The solution was cooled to -40 °C and TMSBr (6.23

[0803] 15 g, 40.70 mmol, 2.2 eq.) was added dropwise over 10 minutes. This was stirred for 20 minutes at -40°C before warming to room temperature, the reaction was stirred for 3 h. After 3 h, the solvent was removed under high vacuum in inert condition at 50 °C for 1.5 h. The residue was dissolved in ethanol-free chloroform (50 mL) and PCIs (9.24 g, 44.4 mmol. 2.4 eq.) was added in one portion at room temperature.

[0804] 20 The reaction mixture was then heated to 50 °C for 1.5 h, then again solvent was removed under high vacuum in inert condition at 50 °C for 1.5 h obtained (3- bromopropyl) phosphonic dichloride. The residue of 3 (3-bromopropyl) phosphonic dichloride was once again dissolved in ethanol-free chloroform 50 mL and cooled to 0°C. A mixture of Et3N (2.59 mL, 18.50 mmol, 1.0 eq.) and 3-hydroxypropane-1 ,2-

[0805] 25 diyl dioleate (5.6 g. 9.3 mmol, 0.5 eq.) in ethanol-free chloroform (20 mL) was added dropwise. After the addition, the solution was warmed to room temperature and stirred for 48 h. After 48 h, water (2 mL / g) was added to the flask and stirred for 1 h before removing the solvent under vacuum (60°C in water bath). The residue was dissolved in 10:10:1 CHCl3:MeOH:H2O (10 mL / g) and stirred with Amberlite™ ion

[0806] 30 exchange resin (IRC-120 H) (20 mL / g) for 1.5 h. The solution was filtered and washed with the same solvent, and the filtrate was concentrated by rotary evaporation. The residue was then taken up in DCM (100 mL) and gently washed with water (50 mL) and brine (50 mL), dried over MgSCL, filtered and concentrated Foreignfiling text P24-220

[0807] - 70 - under vacuum and re-dissolved in 10:17:17 CHCh rOKMeCN (20 mL / g) and 25% aq.NHs (15 mL, 0.78 mol, 41 eq.) was added dropwise at 0 °C and allowed to stir for 48 h at room temperature. After that, the solution was concentrated under vacuum, it was once again dissolved in 10:10:1 CHCl3:MeOH:H2O (10 mL / g) and

[0808] 5 stirred with Amberlite™ ion exchange resin (IRC-120 H) (20 mL / g) for 1 hour. After filtering and rinsing with the same solvent, the filtrate was concentrated under a vacuum, taken up in DCM (100 mL), gently washed with water (50 mL) and brine (50 mL) and dried over MgSC>4, filtered and concentrated by rotary evaporation. The final compound IND1236 was precipitated using CHCh and acetone as waxy compound and the product formation was confirmed by 1 HNMR and direct mass.

[0809] Column chromatography and TLC

[0810] Reagents and solvents for column chromatography and TLC (MeOH, Chloroform, DCM, Ethyl acetate and Hexane) were of reagent grade and acquired from Sigma-

[0811] 15 Aldrich. They were used without further purification.

[0812] Thin layer chromatography was carried out with ready-to-use aluminum plates coated with silica gel from Sigma-Aldrich). The detection was done by application of staining reagent phosphomolybdic acid (PMA). The staining reagent PMA was prepared by dissolving 10 g PMA in 100 mL EtOH.

[0813] 20

[0814] NMR

[0815] NMR spectra were recorded using Bruker 400 MHz NMR spectrometer. Calibration of the spectra was done using solvent residual peaks. Coupling constants J were given in Hz. The following abbreviations were used to describe the signals in the

[0816] 25 NMR spectra: s - singlet, d - doublet, t - triplet, q - quartet, quint - quintet, and m - complex multiplet. Abbreviation “br” is used to describe broad signals: e.g. “br s” stands for “broad singlet”.

[0817] LC-MS

[0818] 30 LC MS were recorded using Shimadzu LCMS 8045

[0819] Compounds used in the examples can be found in table 1. Foreignfiling text P24-220

[0820] - 71 -

[0821] Table 1

[0822] 5

[0823] 15

[0824] 20

[0825] 25

[0826] 30 Foreignfiling text P24-220

[0827] -72 -

[0828] 5

[0829] 15

[0830] 20

[0831] 25

[0832] 30 Foreignfiling text P24-220

[0833] -73-

[0834] 5

[0835] 15

[0836] 20

[0837] 25

[0838] 30 Foreignfiling text P24-220

[0839] - 74 -

[0840] 5

[0841] 15

[0842] Example 2: Lipid nanoparticle preparation

[0843] 20

[0844] 2. 1 Preparation of lipid nanoparticle composition via microfluidic mixing

[0845] LNP with the lipid components at the molar ratio (mol %) specified in table 2 were prepared by mixing an ethanolic lipid solution with FLuc mRNA aqueous solution in 50 mM citrate buffer at pH 4, using a commercial microfluidic-based mixing (NanoAssemblr® Ignite®; Precision NanoSystems, Vancouver, Canada) at an

[0846] 25 mRNA:lipid phase flow rate ratio = 3:1 and total flow rate = 12 mL / min. The formulations were dialyzed overnight (at 2-8 °C) against 100-fold volume of 1x dPBS or the appropriate storage matrix buffer, using Slide-A-Lyzer G3 dialysis cassettes (MWCO 10 kDa). In case of long-term storage, the formulations were up- concentrated, after dialysis, to the desired concentration using Amicon Ultra-15-30K

[0847] 30 centrifugal units, introduced to the appropriate storage matrix by dilution and sterile filtered through Millex®-GV syringe filters (pore size 0.22 pm). Foreignfiling text P24-220

[0848] LNP compositions can be found in table 2 (l-lipid =lonizable lipid; P-lipid = Phospho- / Phosphonolipid; Molar ratio = Molar ratio of Ionizable lipid:Sterol:Phospho- / Phosphonolipid:PEG-lipid in %; RNA = RNA concentration in mg / mL; N / P = N / P ratio).

[0849] 5

[0850] Table 2

[0851] 15

[0852] 20

[0853] 25

[0854] 30 Foreignfiling_text P24-220

[0855] -76- Foreignfiling_text P24-220

[0856] -77 - Foreignfilingjext P24-220

[0857] -78- Foreignfiling text P24-220

[0858] - 79 -

[0859] Example 3: LNP physicochemical characterization

[0860] 3. 1 Measurement of particle size and polydispersity index (PDI)

[0861] The particle size and polydispersity (PDI) of the lipid nanoparticles were measured by dynamic light scattering (DLS) using DynaPro plate reader III instrument from

[0862] 5 Wyatt, USA.

[0863] 3.2 Measurement of encapsulation efficiency and total RNA concentration The RNA encapsulation efficiency and total RNA concentration was quantified by Quant-iT RiboGreen RNA assay (Invitrogen, USA), following manufacturer’s instructions. A RNA standard curve and comparing fluorescence in the presence and absence of Triton X- 100, using a Tecan Infinite M200 Pro Multi Mode Microplate Reader.

[0864] Briefly, the encapsulation efficiency was determined using the RNA binding dye RiboGreen by comparing fluorescence between samples in the presence and

[0865] 15 absence of 1% Triton X-100. In the absence of detergent, fluorescence can be measured from accessible free RNA only, whereas in the presence of detergent, fluorescence is measured from the total RNA amount. The fluorescence of samples in the presence of the detergent Triton X-100 was also used to calculate the total RNA concentration based on a calibration curve. Lipid nanoparticle samples or PBS

[0866] 20 (negative control) were diluted with 1xTE buffer (Thermo Fisher Scientist) down to a RNA concentration of -1200 ng / mL. Aliquots of each diluted sample was further diluted 1 :1 in 1xTE buffer (measuring accessible RNA) or 1 :1 in 1xTE buffer containing 2% Triton-X100 (measuring total mRNA, both accessible within the particle and free RNA). Samples were prepared in triplicate. Samples were

[0867] 25 incubated 10 min at 37°C to ensure sufficient lipid dissociation. Quant-iT RiboGreen RNA reagent (1 :100 dilutions from the stock solution in TE buffer) was then added 1 :1 to each sample and the fluorescence of the dye was measured at an excitation wavelength of 485 nm and emission 535 nm (Tecan Infinite M200 Pro Multimode Plate Reader).

[0868] 30

[0869] The RNA accessibility was determined as follows: the total RNA concentration was determined using an RNA calibration curve in 1xTE buffer with 1% Triton X-100.

[0870] Physicochemical characterization of LNP compositions can be found in table 3 Foreignfiling text P24-220

[0871] Table 3

[0872] 5

[0873] 15

[0874] 20

[0875] 25

[0876] 30 Foreignfiling text P24-220

[0877] 5

[0878] 15

[0879] 20

[0880] 25

[0881] 30 Foreignfiling text P24-220

[0882] - 82 -

[0883] 5

[0884] A panel of Luc mRNA LNP compositions were prepared according to Table 1 with varying amounts of phospho- / phosphonolipid (DEPN-8, DSPC or DOPE)(comp. no. 1-42) . The ionizable lipid and PEG-lipid mol-% was kept constant. The phospho- / phosphonolipids were added from 5 mol-% to 15 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid content to 100 mol-%. For all variations tested, the DEPN-8 LNP compositions showed comparable physicochemical properties as observed for the phospholipid-type LNPs.

[0885] A panel of Luc mRNA LNP compositions were prepared according to Table 1 with

[0886] 15 increasing amount of DEPN-8 (comp, no 17, 46-49). The ionizable lipid MC3 was selected for the testing. The ionizable lipid and PEG-lipid mol-% was kept constant. DEPN-8 was added from 10 mol-% to 48.5 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid content to 100 mol-%.

[0887] With increasing mol% DEPN-8 and decreasing mol% cholesterol a trend towards

[0888] 20 larger diameter and PDI was observed.

[0889] The phosphonolipid content of the LNP was increased up to 70 mol% at the expense of both ionizable lipid and cholesterol according to Table 1 (comp. no. 17, 50-55). The ionizable lipid was MC3.

[0890] 25 A trend towards larger diameter and PDI as well as lower encapsulation efficiency was observed with increasing mol% of DEPN-8 and decreasing mol% of cholesterol and MC3.

[0891] To understand the effect of N / P ratio on the properties & performance of LNP the

[0892] 30 nitrogen-to-phosphate ratio (N / P) ratio was varied from 3:1 to 12:1 (comp, no 56- 59). A higher N / P ratio typically results in more positive charged LNP, improving interaction with negatively charged cell membranes and transfection efficiency.

[0893] The composition of LNP was 30:18.5:50:1.5 mol%. Foreignfiling text P24-220

[0894] - 83 -

[0895] A trend towards larger diameter and PDI as well as lower encapsulation efficiency was observed with a decreasing N / P ratio.

[0896] A panel of Luc mRNA LNP compositions were prepared according to Table 1 with

[0897] 5 varying type of phosphonolipid (IND-1233, IND-1234, IND-1235) (comp. no. 60-65). The ionizable lipid and PEG-lipid mol-% was kept constant. The phosphonolipid was added at 10 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid content to 100 mol-%. For all variations tested, the phosphonolipid LNP compositions showed comparable physicochemical properties as observed for the phospholipid-type LNPs.

[0898] Example 3: Lipid nanoparticle storage stability

[0899] The LNP long-term storage stability was examined upon storing MC3-based LNP, loaded with 0.5 mg / ml mRNA encoding for FLuc (LNP composition No. 20, 46, 49)

[0900] 15 either at room temperature (25 °C), in the refrigerator (2-8 °C) or in the freezer (-20 °C, -80 °C) over a period of 12 months.

[0901] Composition of LNPs was 10% mol, 50% mol MC3 ionizable lipid, 38.5% mol Cholesterol / Synthechol, 1.5% mol DMG-PEG.

[0902] The physicochemical properties were tested after 0, 2 weeks, 1 month, 3 months, 6

[0903] 20 months and 12 months.

[0904] The mRNA integrity is presented as a percentage relative mRNA integrity at time point 0.

[0905] As shown in table 4, the DEPN-8 LNP compositions revealed comparable

[0906] 25 physicochemical properties as observed for the phospholipid-type LNPs.

[0907] Table 4

[0908] 30 Foreignfiling text P24-220

[0909] -84-

[0910] 5

[0911] 15

[0912] 20

[0913] 25

[0914] 30 Foreignfiling text P24-220

[0915] - 85 -

[0916] 5

[0917] The freeze-thaw stability was examined in Luc mRNA LNP compositions using MC3 ionizable lipid. The Composition of LNPs was 10% mol phospho- / phosphonolipid, 50% mol MC3 ionizable lipid, 38.5% mol Cholesterol / Synthechol, 1.5% mol DMG- PEG, and 0.5 mg / ml Luc mRNA according to Table 1.

[0918] Prepared LNP were frozen at -80 °C. On completion of freezing, LNP were thawed at room temperature. The freezing and simultaneous thawing made one cycle of freeze and thaw (F / T). The size distribution of LNP was analyzed under repeated F / T cycles (1 to 3 cycles).

[0919] 15 As shown in table 5, the mean particle size of the DEPN-8 LNP composition remained stable after repeated freeze and thaw.

[0920] Table 5

[0921] 20

[0922] 25

[0923] Example 4: In vitro experiments

[0924] The effect of the phosphonolipid in the LNP composition on mRNA delivery and

[0925] 30 protein expression was tested in vitro.

[0926] HepG2 cells were maintained at 37 °C in a 5% (vol / vol) CO2 atmosphere in Eagle’s Minimium Essential Medium with 10% (vol / vol) fetal bovine serum (FBS), 1x non- essential amino acid (ThermoFisher), 200 mM glutamine and 1x PenStrep (all from Foreignfiling text P24-220

[0927] - 86 -

[0928] Sigma-Aldrich, Germany). C2C12 cells were maintained at 37 °C in a 5% (vol / vol) CO2 atmosphere in Dulbecco's Modified Eagle Medium (high glucose) with 10% (vol / vol) FBS and 1x PenStrep. A549 cells were maintained at 37 °C in a 5% (vol / vol) CO2 atmosphere in Dulbecco's Modified Eagle Medium (low glucose) with 10%

[0929] 5 (vol / vol) FBS and 1x PenStrep. Jurkat cells were maintained at 37 °C in a 5% (vol / vol) CO2 atmosphere in RPMI-1640 Medium containing 2 mM L-glutamine with 10% (vol / vol) fetal bovine serum (FBS), 1 mM Pyruvate and 1x PenStrep (all from Sigma-Aldrich, Germany). RAW264.7 cells were maintained at 37 °C in a 5% (vol / vol) CO2 atmosphere in DM EM (Dulbecco's Modified Eagle Medium) with 1 g / L D- / L-Glucose and pyruvate (Gibco) with 10% (vol / vol) fetal bovine serum (FBS), 200 mM glutamine and 1x PenStrep (all from Sigma-Aldrich, Germany). All cells were kept in culture for a maximum of 20 passages and were passaged 2-3 times per week.

[0930] The in vitro performance of LNP containing the phospholipid DEPN-8 was evaluated

[0931] 15 in terms of luciferase activity upon transfecting cell lines derived from human liver (HepG2) and murine muscle (C2C12), human lung (A549), human spleen (Jurkat) and murine macrophage (RAW264.7) with LNP based on different ionizable lipids (MC3, ALC-315 or TOT3) according to table 6 phospho- / phosphonolipid molar ratio of 10%. Cell lines were exposed to LNP compositions at 25 ng mRNA / well for 24

[0932] 20 hours and were evaluated for luciferase activity and viability as shown in table 6 (P- lipid = Phospho- / Phosphonolipid).

[0933] Table 6

[0934] 25

[0935] 30 Foreignfiling text P24-220

[0936] - 87 -

[0937] 5

[0938] Understanding the effect of the phospho- / phosphonolipid molar ratio on the properties & performance of LNP containing phosphonolipid instead of phospholipid.

[0939] A panel of Luc mRNA LNP compositions were prepared according to Table 1 with varying amounts of phospho- / phosphonolipid. The ionizable lipid and PEG-lipid mol- % was kept constant. The phospho- / phosphonolipids were added from 5 mol-% to 15 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid content to 100 mol-%.

[0940] Cell lines HepG2 and C2C12 were exposed to LNP compositions at 25 ng mRNA / well for 24 hours and were evaluated for luciferase activity and viability.

[0941] 15

[0942] FIG. 2A-C shows transfection efficiency results from luciferase / cell viability assay of (FIG. 2A-C) HepG2 cells and (FIG. 2C-F) C2C12 cells upon treatment with LNP compositions containing phospho- / phosphonolipids DEPN-8, DSPC, DOPE and DPPC. LNP composition containing DEPN-8 showed improved or comparable in vitro performance compared to phospholipid-type formulations in both tested cell

[0943] 20 lines.

[0944] Cell lines HepG2 (Table 7) and C2C12 (Table 8) were exposed to LNP compositions for 24 hours and were evaluated for luciferase activity and viability. 10mol% DEPN- 8 showed highest luciferase activity and 50mol% DEPN-8 (N / P 3-12) showed a

[0945] 25 significant lower luficerase activity (could be related to bigger particle).

[0946] Table 7

[0947] 30 Foreignfiling text P24-220

[0948] - 88 -

[0949] 5

[0950] Table 8

[0951] 15

[0952] 20

[0953] 25

[0954] A panel of Luc mRNA LNP compositions were prepared according to Table 1 with varying mol% of DEPN-8 (10 - 48.5%) as phosphonolipid.

[0955] 30 Cell lines HepG2 and C2C12 were exposed to LNP compositions at 25 ng mRNA / well for 24 hours and were evaluated for luciferase activity and viability.

[0956] FIG. 3 shows transfection efficiency results from luciferase / cell viability assay of (FIG. 3A) HepG2 cells and (FIG. 3B) C2C12 cells upon treatment with LNP Foreignfiling text P24-220

[0957] - 89 - compositions. For both cell types, cell treated with LNP containing 20 mol% of DEPN-8 show the highest luciferase activity.

[0958] Evaluating the in vitro performance of LNP containing phosphonolipid.

[0959] 5 A panel of Luc mRNA LNP compositions were prepared according to Table 1 containing three different novel phosphonolipids. Cell lines A549 cells (Table 9), HepG2 (Table 10) and C2C12 (Table 11) and were exposed to LNP compositions at 10-100 ng mRNA / well for 24 hours and were evaluated for luciferase activity and viability. mRNA was successfully delivered via LNP containing the three novel phosphonolipids into all three cell lines (Table 8-10).

[0960] A549 (Table 8) and HepG2 cells (Table 9) treated with LNP containing the novel phosphonolipids IND-1235 exhibit a significant higher luciferase activity compared to LNP containing IND-1233 or IND-1234. For C2C12 cells (Table 10) treated with

[0961] 15 LNP containing the novel phosphonolipids, no significant difference between LNP containing IND1233-1235 was determined.

[0962] Table 9

[0963] 20

[0964] 25

[0965] 30

[0966] Tabel 10 Foreignfiling text P24-220

[0967] - 90 -

[0968] 5

[0969] Table 11

[0970] 15

[0971] 20

[0972] 25

[0973] Example 5: In vivo experiments

[0974] For in vivo evaluation, the MC3-based LNP formulations, composed either of DSPC

[0975] 30 (LNP composition No.20) or DEPN-8 instead (LNP composition No.17), were administered intravenously to BALB / c mice to compare the levels of luciferase expression (in vivo BLI-biodistribution, FIG. 1A) and tissue localization (ex vivo BLI, FIG. 1 B). The control was phosphate buffered saline (PBS). Foreignfiling text P24-220

[0976] - 91 -

[0977] BALB / c mice were treated with 0.25 mg / kg mRNA delivered through IV injection of LNP compositions. Bioluminescent signals were measured at 2, 6 and 24 h postinjection in living mice and the main organs from sacrificed animals were quantitatively analyzed.

[0978] 5 The DEPN-8 LNP composition behaved similar to the phospholipid-type LNP. Luc mRNA accumulated predominantly in the liver, followed by the spleen, lung and kidneys.

[0979] Table 12 (in vivo)

[0980] 15

[0981] Table 13 (ex vivo)

[0982] 20

[0983] The DEPN-8 LNP composition behaved similar to the phospholipid-type LNP. Luc

[0984] 25 mRNA accumulated predominantly in the liver, followed by the spleen, lung and kidneys.

[0985] Further for in vivo evaluation, LNP formulations containing a novel ionizable lipid, composed either of DSPC, DEPN-8, IND-1233, IND-1234, IND-1235 or IND-1236

[0986] 30 were administered intravenously to BALB / c mice to compare the levels of luciferase expression (Table 14) and tissue localization (Table 15). Foreignfiling text P24-220

[0987] - 92 -

[0988] BALB / c mice were treated with 0.25 mg / kg mRNA delivered through IV injection of LNP compositions. Bioluminescent signals were measured at 6 and 24 h postinjection in living mice and the main organs from sacrificed animals were quantitatively analyzed.

[0989] 5

[0990] The DSPC-LNP composition behaved similar to the LNP with IND-1234 (phosphonolipid as DSPC analogon). LNP with IND-1233 showed an overall reduced luciferase activity. LNP with IND-1235 and IND-1236 showed an overall increased luciferase activity after 6 h compared to DSPC-LNP.

[0991] Table 14 (in vivo) Values are given in (ph / s)

[0992] 15 The DSPC-LNP composition and all phosphonolipid-type LNP accumulated predominantly in the liver, followed by the spleen, lung and kidneys.

[0993] Table 15 (ex vivo) Values are given as %

[0994] 20

[0995] 25

[0996] 30 Foreignfiling text P24-220

[0997] - 93 -

[0998] References

[0999] • Schoenmaker, L., Witzigmann, D., Kulkarni, J. A., Verbeke, R., Kersten, G.F.A., Jiskoot, W. & Crommelin, D.J.A. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 601, 120586 (2021). DOI:

[1000] 5 10.1016 / j.ijpharm.2021.120586.

[1001] • Wang, Z., Schwan, A.L., Lairson, L.L., O'Donnell, J.S., Byrne, G.F., Foye, A., Holm, B.A. & Notter, R.H. Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine. Am. J. Physiol. 285, L550-L559 (2003). DOI: 10.1152 / ajplung.00346.2002.

[1002] • Notter, R.H., et al. Synthetic lung surfactants containing SP-B and SP-C peptides

[1003] 15 plus novel phospholipase-resistant lipids or glycerophospholipids. PeerJ 4, e2635 (2016). DOI: 10.7717 / peerj.2635.

[1004] • Turcotte, S., et al. BBA Lipids Lipid Metabol 488, 235-248 (2000). DOI: 10.1016 / S1388-1981 (00)00024-400024-4).

[1005] 20

[1006] • Skita, V., et al. Bilayer characteristics of a diether phosphonolipid analog of the major lung surfactant glycerophospholipid dipalmitoyl phosphatidylcholine. J. Lipid Res. 36, 1116-1127 (1995). DOI: 10.1016 / S0022-2275(20)30703-330703- 3).

[1007] 25

[1008] • Barbieri, S., et al. Controlled release of therapeutic agents from lipid-based formulations. J. Controlled Release 374, 280-292 (2024). DOI: 10.1016 / j.jconrel.2024.08.016.

[1009] 30

Claims

Foreignfiling text P24-220- 94 -Claims1. A lipid particle, comprising a) at least one pharmaceutical agent, b) at least one cationic lipid,5 c) at least one phosphonolipid, and d) at least one conjugated lipid.

2. A lipid particle according to claim 1 , comprising a) at least one pharmaceutical agent, b) at least one cationic lipid comprising from 20 mol percent to 85 mol percent of the total lipid present in the particle, c) at least one phosphonolipid comprising from 5 mole percent to 85 mol percent of the total lipid present in the particle, and d) at least one conjugated lipid comprising from 0.5 mol percent to 10 mol15 percent of the total lipid present in the particle.

3. A lipid particle according to claim 1 or 2, wherein the at least one phosphonolipid comprising from 5 mole percent to 20 mol percent of the total lipid present in the particle.

204. A lipid particle according to any of claims 1 to 3, wherein the lipid particle is a lipid nanoparticle.

5. A lipid particle according to any of claims 1 to 4, wherein the pharmaceutical25 agent is a nucleic acid.

6. A lipid particle according to any of claims 1 to 5, additionally comprising a sterol.

7. A lipid particle according to any of claims 1 to 6, wherein the phosphonolipid is30 a compound according to the formula (la)Foreignfiling text P24-220- 95 -wherein5 X and Y independently of one another are hydroxy or optionally substituted Ci- 024 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl or C2-C24 heteroalkynyl, whereas, only one of X or Y can be hydroxy,Z is optionally substituted C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 heteroalkyl, C2-C24 heteroalkenyl or C2-C24 heteroalkynyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

8. A lipid particle according to any of claims 1 to 7, wherein the phosphonolipid is15 a compound according to formula (Illa)20 (Illa), whereinR1and R2independently of one another are optionally substituted C6-C24 alkyl, C6-C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24 heteroalkynyl,A and B independently of one another are absent, -O-, -OC(=O)-, -C(=O)O-,25 -S-, -C(=O)S-, -SC(=O)-, -S(=O)-, -S(=O)2-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, -NRaC(=O)O-, -C(=O)NRaC(=O)- or -O-P(=O)(O-)O-, whereinRais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,30 L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenylyl, cycloalkylyl or heterocycloalkylyl,D is absent, hydrogen, hydroxy, -N+RaRbRc-, or optionally substituted cycloalkyl, heterocycloalkyl or C1-C12 heteroalkyl, whereinForeignfiling text P24-220- 96 -Ra, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

59. A lipid particle according to any of claims 1 to 8, wherein the phosphonolipid is a compound according to formula (Illa)(Illa), whereinR1and R2independently of one another are optionally substituted C6-C24 alkyl, C6-C24 alkenyl, C6-C24 heteroalkyl or C6-C24 heteroalkenyl,A is -OC(=O)-, -O-, or -NRaC(=O)-,15B is -OC(=O)- or -NRaC(=O)-, whereinRais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,L is optionally substituted Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenylyl, cycloalkylyl or heterocycloalkylyl, and20D is -N+RaRbRc-, whereinRa, Rb, and Rcindependently of one another are a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is optionally substituted C2-C6 alkyl,25 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

10. A lipid particle according to any of claims 1 to 9, wherein the phosphonolipid is selected from the list consisting of formulaForeignfiling text P24-220- 97 -20 and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

11. A compound of formula (Illa)25(Illa), whereinR1and R2independently of one another are optionally substituted C6-C24 alkyl,30 C6-C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24 heteroalkynyl,A is -OC(=O)- or -NRaC(=O)-,B is -OC(=O)- or -NRaC(=O)-, whereinForeignfiling text P24-220Rais a free bond, hydrogen or optionally substituted C1-C12 alkyl or C2-C12 alkenyl,L is optionally substituted Ci-Ce alkyl, Ci-Ce heteroalkyl C2-C6 alkenyl, C2-C6 heteroalkenyl, cycloalkyl or heterocycloalkyl,5 D is hydrogen or -N+RaRbRc-, whereinRa, Rb, and Rcindependently of one another are a free bond, hydrogen, or optionally substituted C1-C12 alkyl or C2-C12 alkenyl, with the proviso that if A and B are both -OC(=O)-, L is C2-C6 alkyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

12. A compound according to claim 11 of formula (IVa)whereinR1and R2independently of one another are optionally substituted C6-C24 alkyl,20 C6-C24 alkenyl, C6-C24 alkynyl, C6-C24 heteroalkyl, C6-C24 heteroalkenyl or C6-C24 heteroalkynyl,L is optionally substituted C2-C6 alkyl,D is hydrogen or -N+RaRbRc-, whereinRa, Rb, and Rcindependently of one another are a free bond, hydrogen, or25 optionally substituted C1-C12 alkyl or C2-C12 alkenyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

13. A compound according to claim 11 or 12, wherein,R1and R2independently of one another are optionally substituted C6-C24 alkyl30 or C6-C24 alkenyl,L is optionally substituted C2 alkyl,D is -N+RaRbRc-, whereinForeignfiling text P24-220- 99 -Ra, Rb, and Rcindependently of one another are a free bond, hydrogen, or methyl, and a pharmaceutically acceptable salt, solvate, hydrate, tautomer, stereoisomer and isotopically labeled derivative thereof.

514. A compound according to any of claims 11 or 13, wherein the compund is selected from the list consisting of formulaand a pharmaceutically acceptable salt, solvate, hydrate, tautomer,25 stereoisomer and isotopically labeled derivative thereof.

15. A pharmaceutical composition comprising a lipid particle according to any of claims 1 to 10 or a compound according to any of claims 11 to 14.30

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