Cationic lipids for use in lipid nanoparticles

Abstract

Compounds are provided having the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1, G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8 and X are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, nanoparticles comprising the compounds and methods for their use and preparation are also provided.

Description

(19) *EP004678164A2* (11) EP 4 678 164 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 14.01.2026 Bulletin 2026 / 03 (21) Application number: 25198053.8 (22) Date of filing: 16.07.2021 (51) International Patent Classification (IPC): A61K 9 / 00 (2006.01) (52) Cooperative Patent Classification (CPC): C07C 229 / 24; A61K 9 / 1272; A61K 9 / 5123; A61K 45 / 06; C07C 233 / 36; C07C 237 / 06 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME Designated Validation States: KH MA MD TN (30) Priority: 16.07.2020 US 202063052815 P 14.05.2021 US 202163188996 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 21751728.3 / 4 182 297 (71) Applicant: Acuitas Therapeutics Inc. Vancouver, British Columbia V6T 1W5 (CA) (72) Inventor: GATENYO, Julia Vancouver BC V6T 1Z3 (CA) (74) Representative: Grünecker Patent‑ und Rechtsanwälte PartG mbB Leopoldstraße 4 80802 München (DE) Remarks: •This application was filed on 26‑08‑2025 as a divisional application to the application mentioned under INID code 62. •Claims filed after the date of filing of the application (Rule 68(4) EPC). (54) CATIONIC LIPIDS FOR USE IN LIPID NANOPARTICLES (57) Compounds are provided having the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1, G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8 and X are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, nanoparticles comprising the compounds and methods for their use and preparation are also provided. EP 4 67 8 16 4 A 2 Processed by Luminess, 75001 PARIS (FR) Description BACKGROUND Technical Field

[0001] The present disclosure generally relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles to facilitate the intracellular delivery of therapeutic agents, such as nucleic acids (e.g., oligonucleotides, messenger RNA), both in vitro and in vivo. Description of the Related Art

[0002] There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system. Nucleic acid based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism in order to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products aswould be useful in the treatment of, for example, diseases related toadeficiencyof a proteinor enzyme.The therapeutic applicationsof translatable nucleotidedelivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether or not indigenous to the system. The expression products of the nucleic acid can augment existing levels of protein, replace missing or non- functional versions of a protein, or introduce new protein and associated functionality in a cell or organism.

[0003] Some nucleic acids, such asmiRNA inhibitors, can be used to effect expression of specific cellular products that are regulated by miRNA as would be useful in the treatment of, for example, diseases related to deficiency of protein or enzyme. The therapeutic applications ofmiRNA inhibition are extremely broad as constructs can be synthesized to inhibit oneormoremiRNA thatwould in turn regulate theexpressionofmRNAproducts.The inhibitionofendogenousmiRNAcan augment its downstream target endogenous protein expression and restore proper function in a cell or organism as a means to treat disease associated to a specific miRNA or a group of miRNA.

[0004] Other nucleic acids can down-regulate intracellular levels of specific mRNA and, as a result, down-regulate the synthesis of the correspondingproteins throughprocesses suchasRNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotide and RNAi are also extremely broad, since oligonucleotideconstructs canbesynthesizedwithanynucleotidesequencedirectedagainst a targetmRNA.Targetsmay include mRNAs from normal cells, mRNAs associated with disease-states, such as cancer, and mRNAs of infectious agents, suchas viruses. Todate, antisenseoligonucleotide constructshave shown theability to specifically down-regulate target proteins through degradation of the cognate mRNA in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, two problems currently face the use of oligonucleotides in therapeutic contexts. First, free RNAs are susceptible to nuclease digestion in plasma. Second, free RNAs have limited ability to gain access to the intracellular compartment where the relevant translation machinery resides. Lipid nanoparticles formed from cationic lipids with other lipid components, suchasneutral lipids, cholesterol,PEG,PEGylated lipids, andoligonucleotideshavebeenused toblock degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides.

[0006] There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles would provide optimal drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. In addition, these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages. BRIEF SUMMARY

[0007] In brief, the present disclosure provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including for example, all sterols) and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids such as antisense and / or messenger RNA. Methods for use of such lipid nanoparticles for treatment or prevention (e.g., vaccination) of various diseases or conditions, such as those caused by infectious entities and / or insufficiency of a protein, are also provided. 2 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0008] In one embodiment, compounds having the following structure (I) are provided: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1, G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8 and X are as defined herein.

[0009] Lipidnanoparticles (LNPs) comprisingoneormoreof thecompoundsof structure (I) anda therapeuticagent, and pharmaceutical compositions comprising the same, are also provided. In some embodiments, the nanoparticles further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids. Such LNPs are useful for delivery of the therapeutic agent, for example for treatment of a disease or vaccination against a viral pathogen.

[0010] These and other aspects of the disclosure will be apparent upon reference to the following detailed description. DETAILED DESCRIPTION

[0011] In the following description, certain specific details are set forth in order to provide a thorough understanding of variousembodiments of the disclosure.However, oneskilled in theartwill understand that thedisclosuremaybepracticed without these details.

[0012] The present disclosure is based, in part, upon the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of an active or therapeutic agent such as a nucleic acid into a cell of a mammal. In particular, embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic acid-lipid nanoparticle compositions previously described. In other embodiments, the disclosed lipids, and lipid nanoparticles comprising the same, have increased safety and / or tolerability when used for delivery of active agents, such as nucleic acids.

[0013] In particular embodiments, the present disclosure provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for expression of protein encoded by mRNA. In other embodiments, these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for down- regulating (e.g., silencing) the protein levels and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes. In yet other embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody.

[0014] The lipid nanoparticles and compositions of embodiments of the present disclosure may be used for a variety of purposes, including thedelivery of encapsulatedor associated (e.g., complexed) therapeutic agents suchasnucleic acids to cells, both in vitro and in vivo. Accordingly, embodiments of the present disclosure provide methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein.

[0015] As described herein, embodiments of the lipid nanoparticles of the present disclosure are particularly useful for the delivery of nucleic acids, including,e.g.,mRNA, antisense oligonucleotide, plasmidDNA,microRNA (miRNA),miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of certain 3 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments of the present disclosure may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit processes that terminate expression of mRNA (e.g., miRNA inhibitors). In certain embodiments, the protein expressed by the nucleic acid is an antigen, and the LNPs thus induce an immune response (e.g., vaccination). Alternatively, the lipid nanoparticles and compositions of embodiments of the present disclosure may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of embodiments of the present disclosuremayalso be used for co-delivery of different nucleic acids (e.g.,mRNAandplasmidDNA) separately or in combination, such as may be useful to provide an effect requiring co-localization of different nucleic acids (e.g., mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).

[0016] Nucleic acids for usewith embodiments of this disclosuremaybepreparedaccording to anyavailable technique. For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA. In vitro transcription describes a process of template-directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3 and SP6coliphage) linked toadownstreamsequenceencoding thegeneof interest. TemplateDNAcanbeprepared for in vitro transcription fromanumberof sourceswithappropriate techniqueswhicharewell known in theart including,but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription andRNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesesMethods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012).

[0017] Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNApolymeraseandadenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large ScaleRNAProductionSystem (Promega),MegaScript Transcription kits (LifeTechnologies), aswell aswith commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409‑46; Kamakaka, R. T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1‑11.6.17; Beckert, B. And Masquida, B.,(2010)Synthesis ofRNAby InVitroTranscription inRNA inMethods inMolecular Biology v. 703 (Neilson,H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101‑114; all of which are incorporated herein by reference).

[0018] The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolationof themRNA transcripts arewell known in theart.Well knownprocedures includephenol / chloroformextractionor precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-freeRNAoligonucleo- tides, RNA v.10, 889‑893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0019] Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full- length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double- stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templatesandself-complementary3’ extension. It hasbeendemonstrated that thesecontaminantswithdsRNAstructures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques to remove these dsRNA contaminants have been developed and are known in the art including but not limited to scaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves 4 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and CellMetabolismModulation inMethods inMolecularBiologyv.969 (Rabinovich,P.H.Ed), 2013).HPLCpurifiedmRNAhas been reported to be translated at much greater levels, particularly in primary cells and in vivo.

[0020] Asignificant variety ofmodificationshavebeendescribed in theartwhichareused toalter specificpropertiesof in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5’ and 3’ termini of themRNA.EndogenouseukaryoticmRNA typically contain a capstructureon the5’-endof amaturemoleculewhichplays an important role inmediating binding of themRNACapBinding Protein (CBP), which is in turn responsible for enhancing mRNAstability in the cell and efficiency ofmRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5’-cap contains a 5’‑5’-triphosphate linkage between the 5’-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide ismethylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5’-nucleotides on the 2’-hydroxyl group.

[0021] Multiple distinct cap structures can be used to generate the 5’-cap of in vitro transcribed synthetic mRNA. 5’- capping of synthetic mRNA can be performed co-transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, the Anti-Reverse Cap Analog (ARCA) cap contains a 5’‑5’-triphosphate guanine-guanine linkagewhere oneguanine contains anN7methyl group aswell as a 3’-O-methyl group.However, up to 20%of transcripts remain uncappedduring this co-transcriptional process and the synthetic cap analog is not identical to the 5’-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5’-cap structure that more closely mimics, either structurally or functionally, the endogenous 5’-cap which have enhanced binding of cap binding proteins, increased half-life and reduced susceptibility to 5’ endonucleases and / or reduced 5’ decapping. Numerous synthetic 5’-cap analogs have been developed and are known in the art to enhancemRNA stability and translatability (see,e.g., Grudzien-Nogalska, E., Kowalska, J., Su,W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0022] On the3’-terminus, a longchain of adeninenucleotides (poly-A tail) is normally added tomRNAmoleculesduring RNAprocessing. Immediately after transcription, the 3’ end of the transcript is cleaved to free a 3’ hydroxyl towhich poly-A polymerase adds a chain of adenine nucleotides to theRNA in a process called polyadenylation. The poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373‑377; Guhaniyogi, J. And Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11‑23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611‑613).

[0023] Poly (A) tailing of in vitro transcribedmRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase. The first caseallows in vitro transcriptionofmRNAwithpoly (A) tails of defined length, dependingon thesizeof thepoly (T) tract, but requires additionalmanipulation of the template. The latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3’termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length. 5’-capping and 3’-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.

[0024] In addition to 5’ cap and 3’ poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency of translation and stability. It is well known in the art that pathogenic DNAandRNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structure and nucleosidemodifications sincemost nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn can inhibit effectivemRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see, e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifica- tions suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523‑532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013; Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine IntomRNAYieldsSuperiorNonimmunogenic VectorWith Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833‑1840). The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleosidemodifications are available thatmaybe incorporated aloneor in combination 5 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 with other modified nucleosides to some extent into the in vitro transcribed mRNA (see, e.g., US 2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.

[0025] Other components of mRNA which can be modified to provide benefit in terms of translatability and stability include the 5’ and 3’ untranslated regions (UTR).Optimization of theUTRs (favorable 5’ and 3’UTRs canbeobtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribedmRNA (see, e.g.,Pardi, N.,Muramatsu, H.,Weissman,D., Kariko, K., In vitro transcription of longRNAcontainingmodified nucleosides in SyntheticMessenger RNAandCell MetabolismModulation inMethods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0026] In addition tomRNA, other nucleic acid payloadsmay be used for this disclosure. For oligonucleotides,methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).

[0027] For plasmidDNA, preparation for usewith embodiments of this disclosure commonly utilizes, but is not limited to, expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K. L. and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:II:1.7:1.7.1‑1.7.16; Rozkov, A., Larsson, B., Gillström, S., Björnestedt, R. and Schmidt, S. R., (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99: 557‑566; and US 6,197,553 B1). Plasmid isolationcanbeperformedusingavarietyof commercially availablekits including, but not limited toPlasmidPlus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits as well as with commercially available reagents.

[0028] Various exemplary embodiments of the cationic lipids of the present disclosure, lipid nanoparticles and compositions comprising the same, and their use to deliver active (e.g., therapeutic agents), such as nucleic acids, to modulate gene and protein expression, are described in further detail below.

[0029] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0030] Unless the context requires otherwise, throughout the present specification and claims, theword "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open and inclusive sense, that is, as "including, but not limited to."

[0031] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to which this disclosure belongs. As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0033] Thephrase "induceexpression of a desired protein" refers to theability of a nucleic acid to increaseexpression of thedesired protein. Toexamine theextent of protein expression, a test sample (e.g.,asampleof cells in culture expressing thedesiredprotein) or a testmammal (e.g., amammal suchasahumanorananimal)model suchasa rodent (e.g.,mouse) or a non-human primate (e.g., monkey)model is contactedwith a nucleic acid (e.g., nucleic acid in combinationwith a lipid of the present disclosure). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g., a sample of cells in culture expressing the desired protein) or a control mammal (e.g., amammal suchasahumanorananimal)model suchasa rodent (e.g.,mouse)ornon-humanprimate (e.g., monkey)model that is not contactedwith or administered the nucleic acid.When the desired protein is present in a control sample or a controlmammal, the expression of a desired protein in a control sample or a controlmammalmaybe assigned a value of 1.0. In particular embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or thecontrolmammal is greater than1, for example, about1.1, 1.5, 2.0. 5.0or 10.0.Whenadesiredprotein is not present ina control sampleoracontrolmammal, inducingexpressionofadesiredprotein isachievedwhenanymeasurable level of the desiredprotein in the test sample or the testmammal is detected.Oneof ordinary skill in the artwill understandappropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can 6 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 produce fluorescence or luminescence under appropriate conditions.

[0034] The phrase "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal) model such as a rodent (e.g.,mouse) or a non-humanprimate (e.g.,monkey)model is contactedwith a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., amammal suchasahumanorananimal)model suchasa rodent (e.g.,mouse)ornon-humanprimate (e.g., monkey) model that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of a target gene is achievedwhen the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In otherwords, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a testmammal relative to the level of target gene expression in a control sample or a controlmammal not contactedwith or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein ormRNA levels using techniques known to thoseof skill in the art, suchas,e.g.,dot blots, northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

[0035] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce thedesired effect,e.g.,an increaseor inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product that is not present in theabsenceof thenucleic acid. In thecasewhere theexpressionproduct is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such asmRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater. Inhibition of expression of a target gene or target sequence is achievedwhen the value obtainedwith a nucleic acid such as antisense oligonucleotide relative to the control is about 95%,90%,85%,80%, 75%,70%,65%,60%, 55%,50%,45%,40%, 35%,30%,25%,20%, 15%,10%,5%,or 0%. Suitable assays formeasuring expression of a target geneor target sequence include,e.g., examination of protein orRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence, or luminescence of suitable reporter proteins, aswell as phenotypic assays known to those of skill in the art.

[0036] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single‑ or double-stranded form and includesDNA, RNA, and hybrids thereof. DNAmay be in the formof antisensemolecules, plasmidDNA, cDNA,PCRproducts, or vectors.RNAmaybe in the formof small hairpinRNA (shRNA),messengerRNA (mRNA), antisenseRNA,miRNA,micRNA,multivalentRNA, dicer substrateRNAor viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphor- othioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605‑2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91‑98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0037] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0038] "Gene product," as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide. 7 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0039] The term "lipid" refers to agroupof organic compounds that include, but arenot limited to, estersof fatty acidsand aregenerally characterizedbybeingpoorly soluble inwater, but soluble inmanyorganic solvents. Theyareusually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0040] A "steroid" is a compound comprising the following carbon skeleton:

[0041] Non-limiting examples of steroids include cholesterol, and the like.

[0042] A "cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively chargedorneutral formdependingonpH.The ionizationof thecationic lipid affects the surfacechargeof the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance, and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3‑17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8:1188‑1196 (2001)) critical to the intracellular delivery of nucleic acids.

[0043] The term "polymer conjugated lipid" refers to amolecule comprisingboth a lipid portion andapolymer portion. An exampleof a polymer conjugated lipid is apegylated lipid. The term "pegylated lipid" refers toamolecule comprisingbotha 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.

[0044] The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic format aselectedpH.Atphysiological pH, such lipids include, but arenot limited to, phosphotidylcholines such as 1,2-Distearoyl-sn-glycero‑3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero‑3-phosphocholine (DPPC), 1,2-Di- myristoyl-sn-glycero‑3-phosphocholine (DMPC), 1-Palmitoyl‑2-oleoyl-sn-glycero‑3-phosphocholine (POPC), 1,2-dio- leoyl-sn-glycero‑3-phosphocholine (DOPC), phophatidylethanolamines such as 1,2-Dioleoyl-sn-glycero‑3-phos- phoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0045] The term "charged lipid" refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range, e.g., pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylammonium-propanes, (e.g., DO- TAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol).

[0046] The term "lipid nanoparticle" refers to particles having at least one dimension on the order of nanometers (e.g., 1‑1,000 nm) which include one or more of the compounds of structure (I) or other specified cationic lipids. In some embodiments, lipid nanoparticles comprising the disclosed cationic lipids (e.g., compounds of structure (I)) are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g.,cell, tissue, organ, tumor, and the like). In someembodiments, the lipidnanoparticlescomprisea compound of structure (I) and a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipient selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid,may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by themechanisms of the host organism or cells, e.g., an adverse immune response.

[0047] In variousembodiments, the lipid nanoparticleshaveameandiameter of fromabout 30nm toabout 150nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, fromabout 80 nm to about 90 nm, fromabout 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of 8 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 preparation are disclosed in, e.g., U.S. Patent Pub. Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0048] As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g.,mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0049] As used herein, the term "aqueous solution" refers to a composition comprising water.

[0050] "Serum-stable" in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.

[0051] "Systemic delivery," as used herein, refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0052] "Local delivery,"asusedherein, refers todeliveryof anactiveagentdirectly toa target sitewithinanorganism.For example, an agent can be locally delivered by direct injection into a disease site suchas a tumor, other target site suchas a site of inflammation, or a target organsuchas the liver, heart, pancreas, kidney, and the like. Local delivery canalso include topical applications or localized injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.

[0053] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated, and having, for example, from one to twenty-four carbon atoms (C1‑C24 alkyl), four to twenty carbon atoms (C4‑C20 alkyl), six to sixteen carbon atoms (C6‑C16 alkyl), six to nine carbon atoms (C6‑C9 alkyl), one to fifteen carbon atoms (C1‑C15 alkyl),one to twelve carbon atoms (C1‑C12 alkyl), one to eight carbon atoms (C1‑C8 alkyl) or one to six carbon atoms (C1‑C6 alkyl) andwhich is attached to the rest of themolecule by a single bond, e.g.,methyl, ethyl, n-propyl, 1-methylethyl (iso propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is substituted or unsubstituted.

[0054] "Alkylene" refers to a straight or branched divalent hydrocarbon chain linking the rest of themolecule to a radical group, consisting solely of carbon and hydrogen, which is saturated, and having, for example, from one to twenty-four carbon atoms (C1‑C24 alkylene), one to fifteen carbon atoms (C1‑C15 alkylene),one to twelve carbon atoms (C1‑C12 alkylene), one to eight carbon atoms (C1‑C8 alkylene), one to six carbon atoms (C1‑C6 alkylene), two to four carbon atoms (C2‑C4alkylene), one to twocarbonatoms (C1‑C2alkylene),e.g.,methylene,ethylene,propylene,n-butylene,and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. Thepoints of attachment of the alkylene chain to the rest of themolecule and to the radical group canbe throughone carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain is substituted or unsubstituted.

[0055] "Alkene" and "alkenylene" refer to an alkyl and alkylene, respectively, comprising at least one carbon-carbon double bond. Alkenes and alkenylenes include the same number of carbon atoms as alkyl and alkylene as defined above, except that alkenes and alkenylenes must include at least two carbons. Unless stated otherwise specifically in the specification, alkenes and alkenylenes are substituted or unsubstituted.

[0056] The term "substituted" used herein means any of the above groups (e.g., alkyl or alkylene) wherein at least one hydrogenatom is replacedbyabond toanon-hydrogenatomsuchas,butnot limited to: ahalogenatomsuchasF,Cl,Br, or I; oxo groups (=O); hydroxyl groups (-OH); carboxyl groups ‑(CO2H); C1‑C12 alkyl groups; ‑(C=O)OR’; ‑O(C=O)R’; ‑C(=O) R’; ‑OR’; ‑S(O)xR’; ‑S-SR’; ‑C(=O)SR’; ‑SC(=O)R’; ‑NR’R’; ‑NR’C(=O)R’; ‑C(=O)NR’R’; ‑NR’C(=O)NR’R’; ‑OC(=O)NR’R’; ‑NR’C(=O)OR’; ‑NR’S(O)xNR’R’; ‑NR’S(O)xR’; and ‑S(O)xNR’R’, wherein: R’ is, at each occurrence, independently H or C1‑C15 alkyl and x is 0, 1 or 2. In some embodiments the substituent is a C1‑C12 alkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, thesubstituent is ahydroxyl group. Inotherembodiments, the substituent is analkoxygroup (-OR’). In otherembodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR’R’).

[0057] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event of circum- stances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0058] The disclosure disclosed herein is alsomeant to encompass all pharmaceutically acceptable compounds of the compound of structure (I) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopesof hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, suchas 2H, 3H, 11C, 13C, 14C, 9 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I, respectively. These radiolabeled compounds could beuseful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds of structure (I), (IA) or (IB), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., 3H, and carbon‑14, i.e., 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0059] Substitution with heavier isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0060] Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds of structure (I) cangenerally bepreparedby conventional techniques known to those skilled in theart or by processes analogous to those described in thePreparations andExamples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0061] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0062] "Mammal" includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

[0063] "Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0064] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0065] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formedwith inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor‑10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane‑1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene‑1,5-disulfonic acid, naphtha- lene‑2-sulfonic acid, 1-hydroxy‑2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0066] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness andpropertiesof the freeacids,whicharenot biologically or otherwiseundesirable.Thesesalts areprepared fromaddition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylami- noethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, cho- line, betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic basesare isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0067] Often crystallizations produce a solvate of a compound of the disclosure (i.e., a compound of structure (I)). As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the disclosure with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. Solvates of compound of the disclosure may be true solvates, while in other cases the compound of the disclosuremaymerely retain adventitious water or be amixture of water plus someadventitious solvent.

[0068] A "pharmaceutical composition" refers to a formulation of a compound of the disclosure and amedium generally accepted in theart for thedelivery of thebiologically active compound tomammals,e.g., humans.Suchamedium includes all pharmaceutically acceptable carriers, diluents or excipients therefor. 10 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0069] "Effective amount" or "therapeutically effective amount" refers to that amount of a compound of the disclosure which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a lipid nanoparticle of the disclosurewhich constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, themanner of administration, and the ageof themammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0070] "Treating" or "treatment" as used herein covers the treatment of the disease or condition of interest in amammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing thediseaseor condition fromoccurring in amammal, in particular,whensuchmammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet beenworkedout) and it is therefore not yet recognizedasadiseasebut only as anundesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

[0071] The compounds of the disclosure, or their pharmaceutically acceptable salts may contain one or more stereocenters and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)‑ or (S)‑ or, as (D)‑ or (L)‑ for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (‑), (R)‑ and (S)‑, or (D)‑ and (L)‑ isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0072] A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers andmixtures thereof and includes "enantiomers," which refers to two stereoisomers whosemolecules are non-superimposable mirror images of one another.

[0073] A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds. Compounds

[0074] In an aspect, the disclosure provides novel lipid compounds which are capable of combining with other lipid components such as neutral lipids, charged lipids, steroids and / or polymer conjugated-lipids to form lipid nanoparticles with therapeutic agents, such as oligonucleotides. Without wishing to be bound by theory, it is thought that these lipid nanoparticles shield the therapeutic agent from degradation in the serum and provide for effective delivery of the therapeutic agent to cells in vitro and in vivo.

[0075] In one embodiment, the compounds have the following structure (I): 11 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: G1 and G2 are each independently C1‑C6 alkylene; L1 and L2 are each independently ‑O(C=O)‑ or ‑(C=O)O‑; R1aandR1bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R1a isHorC1‑C12alkyl, and R1b together with the carbon atom towhich it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond; R2aandR2bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R2a isHorC1‑C12alkyl, and R2b together with the carbon atom towhich it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R3aandR3bare, at eachoccurrence, independently either (a):HorC1‑C12alkyl; or (b)R3a isHorC1‑C12alkyl, and R3b together with the carbon atom towhich it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R4aandR4bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R4a isHorC1‑C12alkyl, and R4b together with the carbon atom towhich it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R5 and R6 are each independently H or methyl; R7 is ‑O(C=O)R10, ‑(C=O)OR10, ‑NR9(C=O)R10 or ‑(C=O)NR9R10; R8 is OH, ‑N(R11)(C=O)R12, ‑(C=O)NR11R12, ‑NR11R12, ‑(C=O)OR12 or ‑O(C=O)R12; R9 is H or C1‑C15 alkyl; R10 is C1‑C15 alkyl; R11 is H or C1‑C6 alkyl; R12 is C1‑C6 alkyl; X is ‑(C=O)‑ or a direct bond; and a, b, c and d are each independently an integer from 1 to 24; wherein each alkyl and alkylene and is independently optionally substituted.

[0076] In other embodiments, the compound has one of the following structures (IA) or (IB): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0077] In certain embodiments, G1 is C2‑C3 alkylene. In different embodiments, G1 is C4‑C6 alkylene. For example, in various embodiments, G1 is C2, C3, C4, C5 or C6 alkylene.

[0078] In other embodiments, G2 is C2‑C4 alkylene, for example C2‑C3 alkylene or C3‑C4 alkylene. In some embodi- ments, G2 is C2, C3 or C4 alkylene.

[0079] In various different embodiments, X is ‑(C=O)‑, while in different embodiments X is a direct bond.

[0080] In any of the foregoing embodiments, R7 is ‑O(C=O)R10 or ‑(C=O)OR10. In certain of these embodiments, R10 is linear C1‑C15 alkyl, such as linear C6‑C10 alkyl. In other such embodiments, R10 ismethyl or R10 is branchedC2‑C15 alkyl, such as branched C10‑C15 alkyl.

[0081] In still other of the foregoing embodiments, R7 is ‑NR9(C=O) or ‑(C=O)NR9R10. In some of these embodiments, R9 is H. In other of these embodiments, R9 and R10 are each independently C6‑C10 alkyl.

[0082] In other embodiments, at least one occurrence of R1a andR1b, R1a is H or C1‑C12 alkyl, andR1b together with the carbon atom to which it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond. 12 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0083] Inmore embodiments, for at least one occurrence of R4a and R4b, R4a is H or C1‑C12 alkyl, and R4b together with thecarbonatomtowhich it isbound is taken togetherwithanadjacentR4band thecarbonatom towhich it isbound to forma carbon-carbon double bond.

[0084] In still other embodiments, for at least one occurrence of R2a and R2b, R2a is H or C1‑C12 alkyl, and R2b together with the carbon atom towhich it is bound is taken together with an adjacent R2b and the carbon atom towhich it is bound to form a carbon-carbon double bond.

[0085] In other embodiments, for at least one occurrence of R3a and R3b, R3a is H or C1‑C12 alkyl, and R3b together with thecarbonatomtowhich it isbound is taken togetherwithanadjacentR3band thecarbonatom towhich it isbound to forma carbon-carbon double bond.

[0086] In various of the foregoing embodiments, R1a, R1b, R2a, R2b, R3a, R3b, R4a and R4b are, at each occurrence, independently H or C1‑C12 alkyl. In other embodiments, R2a, R2b, R3a andR3b are, at each occurrence, H. For example, in certain embodimentR1a andR4aare, at eachoccurrence,H. In different embodiments, at least oneofR1b andR4b isC1‑C8 alkyl. For example, in some embodiments C1‑C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- hexyl or n-octyl.

[0087] In other more specific embodiments, or both, independently has one of the following structures:

[0088] Inmore embodiments, a, b, c and d are each independently an integer from2 to 12. Inmore embodiments, a, b, c and d are each independently an integer from4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9 or 6 to 9. In still different embodiments, b and c are independently 5, 6, 7, 8, 9 or 10.

[0089] In some embodiments, one of R5 or R6 is methyl. In other embodiments, each of R5 and R6 is methyl.

[0090] In some embodiments, R8 is OH.

[0091] In other embodiments, R8 is ‑N(R11)(C=O)R12. In different embodiments, R8 is ‑(C=O)NR11R12. Inmore different embodiments, R8 is ‑NR11R12. In some of these foregoing embodiments, R11 and R12 are each independently H or C1‑C8 alkyl. In other of these embodiments, R11 and R12 are each independently H or C1‑C3 alkyl. For example, in some embodiments the C1‑C8 alkyl or C1‑C3 alkyl is unsubstituted or substituted with hydroxyl. In other different such embodiments, R11 and R12 are each methyl.

[0092] In other embodiments of the compound of structure (I), R8 is ‑(C=O)OR12, while in different embodiments R8 is ‑O(C=O)R12.

[0093] In specific embodiments of any of the foregoing compounds, R8 has one of the following structures:

[0094] -OH or 13 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0095] In various different embodiments, the disclosure provides a compound having one of the structures set forth in Table 1 below, or a pharmaceutically acceptable salt or tautomer thereof. Table 1. Representative Compounds No. Structure I‑1 I‑2 I‑3 I‑4 I‑5 14 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) No. Structure I‑6 I‑7 I‑8 I‑9 I‑10 15 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) No. Structure I‑11 I‑12 I‑13 I‑14 I‑15 16 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) No. Structure I‑16 I‑17 I‑18 I‑19 I‑20 17 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) No. Structure I‑21 I‑22 1‑23

[0096] It is understood that any embodiment of the compounds of structure (I), as set forth above, and any specific substituent and / or variable in the compound of structure (I), as set forth above,may be independently combinedwith other embodiments and / or substituents and / or variables of compounds of structure (I) to form embodiments of the disclosures not specifically set forth above. In addition, in the event that a list of substituents and / or variables is listed for any particular R group, G group, L group or variable a, b, c, d or n, in a particular embodiment and / or claim, it is understood that each individual substituent and / or variablemay be deleted from the particular embodiment and / or claim and that the remaining list of substituents and / or variables will be considered to be within the scope of the disclosure.

[0097] It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

[0098] In some embodiments, lipid nanoparticles comprising a compound of structure (I) are provided. The lipid nanoparticles optionally include excipients selected from a neutral lipid, a steroid and a polymer conjugated lipid.

[0099] In some embodiments, compositions comprising any one or more of the compounds of structure (I) and a therapeutic agent are provided. For example, in someembodiments, the compositions comprise any of the compounds of structure (I) and a therapeutic agent and one or more excipient selected from neutral lipids, steroids and polymer conjugated lipids.Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0100] In some embodiments, the neutral lipid is selected fromDSPC,DPPC,DMPC,DOPC, POPC,DOPEandSM. In someembodiments, the neutral lipid isDSPC. In various embodiments, themolar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

[0101] In various embodiments, the compositions further comprise a steroid or steroid analogue. In certain embodi- ments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1.

[0102] In various embodiments, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1‑(monomethoxy-polyethyleneglycol)‑2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O‑(2’,3’-di(tetradecanoyloxy)propyl‑1-O‑(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated cer- 18 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 amide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω-methoxy(polyethoxy)ethyl-N‑(2,3-di(tetradecanoxy) propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N‑(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100:1 to about 20:1.

[0103] In some embodiments, the composition comprises a pegylated lipid having the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R8andR9areeach independentlyastraight or branched, saturatedorunsaturatedalkyl chain containing from10 to30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0104] In some embodiments, R8 and R9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w ranges from about 42 to 55, for example about 49.

[0105] In some embodiments of the foregoing composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense and messenger RNA. In some of the foregoing embodiments, the composition comprises a lipid nanoparticle.

[0106] Some related embodiments provide a lipid nanoparticle comprising the compound of any one of the foregoing embodiments (e.g., a compound of structure (I)). In certain embodiments, the lipid nanoparticle further comprises a therapeutic agent (e.g., a nucleic acid such as antisense and messenger RNA).

[0107] In someembodiments, the lipid nanoparticle further comprisesoneormoreexcipient selected fromneutral lipids, steroids and polymer conjugated lipids. In some embodiments, the neutral lipids are selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In more specific embodiments, the neutral lipid is DSPC.

[0108] In somemore specific embodiments, themolar ratio of the compound to the neutral lipid ranges fromabout 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from 5:1 to 1: 1.

[0109] In certain embodiments, the polymer conjugated lipid is pegylated lipid. In certain more specific embodiments, the molar ratio of the compound to pegylated lipid ranges from about 100:1 to about 20:1.

[0110] In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyox- ypropylcarbamate. In other embodiments, the pegylated lipid has the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R8andR9areeach independentlyastraight or branched, saturatedorunsaturatedalkyl chain containing from10 to30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0111] In somemore specific embodiments of structure (II), R8 and R9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In more specific embodiments, the average w is about 49.

[0112] In other different embodiments, the disclosure is directed to a method for administering a therapeutic agent to a patient in need thereof, themethodcomprisingpreparingor providinganyof the foregoingcompositionsandadministering the composition to the patient

[0113] For the purposes of administration, embodiments of the compounds of the present disclosure (typically in the 19 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 form of lipid nanoparticles in combination with a therapeutic agent) may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of the present disclosure comprise a compound of structure (I) and one or more pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the compound of structure (I) is present in the composition in an amount which is effective to form a lipid nanoparticle and deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

[0114] Administration of the compositions of embodiments of the disclosure can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of embodiments of the disclosure may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The termparenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. Pharmaceutical compositions of embodiments of the disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient in someembodiments take the formof one ormore dosageunits, where for example, a tabletmaybeasingledosageunit, andacontainerof acompoundof anembodimentsof thedisclosure inaerosol formmay hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, seeRemington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure.

[0115] A pharmaceutical composition of embodiments of the disclosure may be in the form of a solid or liquid. In one aspect, thecarrier(s) areparticulate, so that thecompositionsare, for example, in tablet or powder form.Thecarrier(s)may be liquid, with the compositions being, for example, oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.

[0116] When intended for oral administration, the pharmaceutical composition of certain embodiments is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0117] As a solid composition for oral administration, the pharmaceutical composition of some embodiments may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one ormore inert diluents or edible carriers. In addition, one ormore of the followingmay bepresent: binders suchas carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

[0118] When the pharmaceutical composition of some embodiments is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0119] The pharmaceutical composition of some embodiments may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples.When intended for oral administration, preferredcomposition contain, in addition to acompoundof structure (I), one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0120] The liquid pharmaceutical compositions of embodiments of the disclosure, whether they be solutions, suspen- sions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphatesandagents for the adjustment of tonicity suchas sodiumchlorideor dextrose; agents toact as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0121] The pharmaceutical composition of embodiments of the disclosuremay be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as 20 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 water and alcohol, and emulsifiers and stabilizers. Thickening agentsmay be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0122] The pharmaceutical composition of embodiments of the disclosuremay include variousmaterials, whichmodify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. Thematerials that form the coating shell are typically inert, andmay be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.

[0123] The pharmaceutical composition of embodiments of the disclosure in solid or liquid form may include an agent that binds to the compound of the disclosure and thereby assists in the delivery of the LNP. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, or a protein.

[0124] The pharmaceutical composition of embodiments of the disclosure may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of LNPs of embodiments of the disclosure may be delivered in singlephase,bi-phasic, or tri-phasic systems inorder todeliver theactive ingredient(s).Deliveryof theaerosol includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit. One skilled in the art, without undue experimentation, may determine preferred aerosols.

[0125] The pharmaceutical compositions of embodiments of the disclosure may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administeredby injection can bepreparedby combining the lipid nanoparticles of the disclosurewith sterile, distilledwater or other carrier so as to forma solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interactwith thecompoundof thedisclosuresoas to facilitatedissolutionorhomogeneous suspension of the compound in the aqueous delivery system.

[0126] Thecompositionsof embodimentsof thedisclosure, or their pharmaceutically acceptable salts, areadministered ina therapeutically effectiveamount,whichwill varydependinguponavariety of factors including theactivity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, anddiet of thepatient; themodeand timeofadministration; the rateof excretion; thedrugcombination; the severity of the particular disorder or condition; and the subject undergoing therapy.

[0127] Compositions of embodiments of the disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of embodiments of the disclosure and one or more additional active agents, as well as administration of the composition of embodiments of the disclosure and each active agent in its own separate pharmaceutical dosage formulation. For example, a composition of embodiments of the disclosure and theother active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Where separate dosage formulations are used, the compounds of embodiments of the disclosure and one ormore additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.

[0128] Preparation methods for the above compounds and compositions are described herein below and / or known in the art.

[0129] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compoundsmayneed tobeprotectedby suitable protectinggroups. Such functional groups includehydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)‑R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd.,Wiley.Asoneof skill in theartwouldappreciate, theprotectinggroupmayalsobeapolymer resin suchasa Wang resin, Rink resin or 2-chlorotrityl-chloride resin.

[0130] It will also be appreciated by those skilled in the art, although such protected derivatives of compounds of this disclosure may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the disclosure which are pharmacologically active. Such derivatives may therefore be described as "prodrugs." All prodrugs of compounds of this disclosure are included within the scope of the disclosure.

[0131] Furthermore, compounds of embodiments of the disclosure which exist in free base or acid form can be 21 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 converted to their pharmaceutically acceptable salts by treatmentwith the appropriate inorganic or organic baseor acid by methods known to one skilled in the art. Salts of compounds of embodiments of the disclosure can be converted to their free base or acid form by standard techniques.

[0132] The followingGeneral ReactionScheme1 illustrates exemplarymethods tomake compounds of this disclosure, i.e., compounds of structure (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1 , G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b,R5, R6, R7, R8 andXare as defined herein. It is understood that one skilled in the artmay be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of structure (I) not specifically illustrated below by using the appropriate starting components andmodifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this disclosure.

[0133] General Reaction Scheme I provides an exemplary method for preparation of a compound of structure (I) (i.e., A5). a, b, c, d,G2, L1, L2, R1a,R1b,R2a,R2b,R3a, R3b,R4a,R4b,R5,R6,R8,R9 andR10 inGeneral reactionScheme1are as definedherein, andZ represents anactivatedanalogueofG1 sufficient for bond formationwith theNHgroupofA3 (e.g., an alkene or an alkylene terminating in an aldehyde, acid halide, acrylate, etc.). Intermediates and reagents (e.g., A1 andA2) 22 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 needed for preparation of the compounds according to General Reaction Scheme I can be purchased or prepared according to the examples below or methods known by one of ordinary skill in the art.

[0134] It should be noted that various alternative strategies for preparation of compounds of structure (I) are available to those of ordinary skill in the art. For example, other compounds of structure (I) wherein can be prepared according to analogousmethodsusing theappropriatestartingmaterial. Theuseofprotectinggroupsasneededandothermodification to the above General Reaction Scheme will be readily apparent to one of ordinary skill in the art.

[0135] The following examples are provided for purpose of illustration and not limitation. EXAMPLE 1 LUCIFERASE MRNA IN VIVO EVALUATION USING THE LIPID NANOPARTICLE COMPOSITIONS

[0136] Lipid nanoparticles were prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2015 / 199952 and WO 2017 / 004143, the full disclosures of which are incorporated herein by reference. Briefly, cationic lipid,DSPC, cholesterol andPEG-lipidwere solubilized in ethanol at amolar ratio of about 50:10:38.5:1.5 or about 47.5:10:40.8:1.7. Lipid nanoparticles (LNP) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. ThemRNA is diluted to 0.2mg / mL in 10 to 50mMcitrate or acetate buffer, pH4. Syringe pumpswere used tomix the ethanolic lipid solution with themRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) with total flow rates above 15 mL / min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 µm pore sterile filter. Lipid nanoparticle particle size was approximately 55‑95 nm diameter, and in some instances approximately 70‑90 nm diameter as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0137] Studies were performed in 6‑8 week old female C57BL / 6 mice (Charles River) or 8‑10 week old CD‑1 (Harlan) mice (Charles River) according to guidelines established by an institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle were systemically administered by tail vein injection and animals euthanized at a specific timepoint (e.g., 4 hours) post-administration. Liver and spleenwere collected in preweighed tubes, weights determined, immediately snap frozen in liquid nitrogen and stored at ‑80°C until processing for analysis.

[0138] For liver, approximately50mgwasdissected foranalyses ina2mLFastPrep tubes (MPBiomedicals,SolonOH). ¼" ceramic sphere (MPBiomedicals) is added to each tube and 500µL of Glo Lysis Buffer - GLB (Promega, MadisonWI) equilibrated to room temperature is added to liver tissue. Liver tissueswere homogenizedwith the FastPrep24 instrument (MPBiomedicals) at 2× 6.0m / s for 15 seconds. Homogenate was incubated at room temperature for 5minutes prior to a 1:4 dilution in GLB and assessed using SteadyGlo Luciferase assay system (Promega). Specifically, 50 µL of diluted tissuehomogenatewas reactedwith50µLofSteadyGlo substrate, shaken for10seconds followedby5minute incubation and then quantitated using a CentroXS3 LB 960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using the BCA protein assay kit (Pierce, Rockford IL). Relative luminescence units (RLU) were then normalized to total µg protein assayed. To convert RLU to ng luciferase a standard curve was generated with QuantiLum Recombinant Luciferase (Promega).

[0139] The FLuc mRNA (L‑6107 or L‑7202) from Trilink Biotechnologies will express a luciferase protein, originally isolated from the firefly, photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and poly- adenylated mRNA was fully substituted with respect to uridine and / or cytidine nucleosides. EXAMPLE 2 DETERMINATION OF PKA OF FORMULATED LIPIDS

[0140] As described elsewhere, the pKa of formulated cationic lipids is correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529‑8533; Semple et al, Nature Biotechnology 28, 172‑176 (2010)). The preferred range of pKa is ~5 to ~7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on fluorescence of 2‑(p-toluidino)‑6-napthalene sulfonic acid (TNS). Lipid nanoparticles comprising cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4mM total lipid were prepared using the in-line process as described in Example 1. TNSwas prepared asa100µMstock solution in distilledwater. Vesicleswerediluted to 24µMlipid in2mLof buffered solutionscontaining, 10 mMHEPES, 10mMMES, 10mMammoniumacetate, 130mMNaCl, where the pH ranged from2.5 to 11. An aliquot of the TNS solution was added to give a final concentration of 1 µM and following vortex mixing fluorescence intensity was measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis was applied to the fluorescence data and the 23 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 pKa was measured as the pH giving rise to half-maximal fluorescence intensity. EXAMPLE 3 DETERMINATION OF EFFICACY OF LIPID NANOPARTICLE FORMULATIONS CONTAINING VARIOUS CATIONIC LIPIDS USING AN IN VIVO LUCIFERASE MRNA EXPRESSION RODENT MODEL

[0141] Representative compounds of the disclosure shown in Table 2 were formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% Cholesterol / 1.5% PEG lipid 2‑[2‑(ω-methox- y(polyethyleneglycol2000)ethoxy]‑N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% Cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours following administration via tail vein injection as described in Example 1. The activity was compared at a dose of 1.0 or 0.5 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1. Compound numbers in Table 2 refer to the compound numbers of Table 1. Table 2. Novel Cationic Lipids and Associated Activity Cmp. No. pKa Liver Luc @ 1.0 mg / kg (ng luc / g liver) Structure I‑1 6.11 24031 ± 4777 I‑2 6.67 122 ± 18* * determined at 0.3 mg / kg 682 ± 147** ** deter- mined at 1.0 mg / kg I‑4 6.17 46932 ± 17399 I‑5 6.08 11720 ± 2439 24 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Cmp. No. pKa Liver Luc @ 1.0 mg / kg (ng luc / g liver) Structure I‑6 6.22 4093 ± 1036* * deter- mined at 0.3 mg / kg 11290 ± 6455** ** de- termined at 1.0 mg / kg I‑14 6.80 669±575 * * determined at 0.5 mg / kg I‑15 5.96 21357 ± 15325 I‑16 6.25 66763 ± 15823 I‑17 6.78 26691 ± 13186 I‑18 6.02 40650 ± 11479 25 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Cmp. No. pKa Liver Luc @ 1.0 mg / kg (ng luc / g liver) Structure I‑19 5.95 32706 ± 4621 I‑20 6.48 4140 ± 1117* * deter- mined at 0.3 mg / kg 17095 ± 8181** ** de- termined at 1.0 mg / kg I‑21 6.40 7723 ± 1714* * deter- mined at 0.3 mg / kg 20223 ± 5982** ** de- termined at 1.0 mg / kg I‑22 6.28 5291 ± 1348* * deter- mined at 0.3 mg / kg 17654 ± 8167** ** de- termined at 1.0 mg / kg I‑23 6.33 553 ± 153 * * deter- mined at 0.5 mg / kg EXAMPLE 4 SYNTHESIS OF BIS(2-BUTYLOCTYL) 7‑((3‑(DIMETHYLAMINO)PROPYL)(3‑(DIOCTYLAMINO)‑3-OXOPROPYL) AMINO)TRIDECANEDIOATE (COMPOUND 1‑7)

[0142] 26 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 Synthesis of acryloyl chloride

[0143] Acrylic acid (1.20 g, 16.65 mmol) was dissolved in 20mL anhydrous dichloromethane. Thionyl chloride (1.98 g, 16.65 mmol) was added dropwise while stirring under N2, and the reaction mixture was allowed to heat to reflux for 4 hr. After the completion of reaction, the crude product concentrated to yield a pale yellow liquid that used in the next step without further purification. Synthesis of N,N-Dioctylacrylamide (Intermediate A)

[0144] Acryloyl chloride (1.12 g, 12.37mmol) was added to a cooled solution (0 °C) of dioctylamine in dichloromethane with triethylamine (1 equiv) as a base. The reaction mixture was stirred at 0 °C for 1 h and additional 1 h at room temperature. The reactionmixturewasfilteredand the solutionobtainedwaswashedwithhydrochloric acid (1NHCl), then with sat NaHCO3 solution and brine. The solvent evaporated under reduced pressure to yield crude product (colorless liquid) which was used in the following step without further purification. Synthesis of bis(2-butyloctyl) 7-oxotridecanedioate

[0145] To a solution of 2-butyloctane‑1-ol (3.85 g, 20.66 mmol), 7-oxotridecanedioic acid (1.34 g, 5.17 mmol) and 4- dimethylaminopyridine (DMAP) (1.9 g, 15.55 mmol) in anhydrous DCM was added DCC (4.27 g, 20.69 mmol). The resulting mixture was allowed to stir overnight at room temperature. The solid (DCU) was then filtered and washed with DCM. The filtrate was concentrated. The residue (oil / solid) was purified by column chromatography on silica gel (0‑5% ethyl acetate in hexane). The desired product was obtained as a colorless oil (2.55 g, 42.86 mmol, 83%). 27 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 Synthesis of bis(2-butyloctyl) 7‑((3‑(dimethylamino)propyl)amino)tridecanedioate

[0146] A solution of 3‑(dimethylamino)‑1-propylamine (0.09 g, 0.88 mmol) and bis(2-butyloctyl) 7-oxotridecanedioate (0.37 g, 0.63 mmol) in DCE was treated with sodium triacetoxyborohydride (0.20 g, 0.94 mmol) and AcOH (55 µL, 0.98 mmol) overnight. Thesolutionwaswashedwith dilute aqueous sodiumhydroxide solution (1NNaOH). Theorganic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and the solvent removed. The residue was passed down a small pad of silica gel, washed with amixture of DCM / MeOH / Et3N (85:15:1). The filtrate was concentrated to give the desired product as a slightly yellow oil (240 mg, 0.35 mmol, 56%). Synthesis of I‑7

[0147] AnEtOH (10mL) solution of bis(2-butyloctyl) 7‑((3‑(dimethylamino)propyl)amino)tridecanedioate (210mg, 0.30 mmol) and N,N-Dioctylacrylamide (1.5 eq. 136 mg, 0.46 mmol) was stirred at room temperature overnight. The reaction mixture was heated to reflux for 7 days. Upon completion of the reaction, the solvent was removed. The residue was dissolved in a mixture of hexanes and EtOAc (19:1) and washed with saturated sodium bicarbonate solution, and brine. The extract was dried over sodium sulfate. The dried extract was filtered through a pad of silica gel. The pad was washed with a mixture of hexane / ethyl acetate / triethylamine (80:20:1). The washing was concentrated to give the crude desired product.

[0148] The crude product was further purified by flash dry column chromatography on silica gel (MeOH in chloroform, 0 to 5%). This gave the desired product as a colorless oil (30mg, 0.03mmol, 10%). 1HNMR (400MHz,CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.30‑3.16 (m, 4H), 2.74 (t, 7.2 Hz, 2H), 2.65‑2.24 (m, 17H), 1.80‑1.44 (m, 12H), 1.43‑1.15 (64H), 0.93‑0.82 (m, 18H). EXAMPLE 5 SYNTHESIS OF BIS(2-HEXYLDECYL) 7‑((4‑(DIHEXYLAMINO)‑4-OXOBUTYL)(2‑(DIMETHYLAMINO)ETHYL)AMI- NO)TRIDECANEDIOATE (COMPOUND 1‑19)

[0149] 28 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 Synthesis of N,N-dihexyl‑4-oxobutanamide (Intermediate B)

[0150] Butyrolactone (2.51 g, 29.15mmol) and dihexylamine (5.40 g, 29.13mmol) were heated for 4 days in a pressure flask at 61 °C. The reaction mixture was cooled to room temperature. The crude product was purified by column chromatography on silica gel (0% to 5% of MeOH in DCM) to yield N,N-dihexyl‑4-hydroxybutanamide as a slightly yellow oil (6.30 g, 79%).

[0151] N,N-dihexyl‑4-hydroxybutanamide (3.00 g, 11.05 mmol) was dissolved in DCM and treated with pyridinium chlorochromate (2.38 g, 11.05mmol) for two hours. Diethyl etherwas added and the supernatant filtered through silica gel bed. The solvent was removed from the filtrate and resultant oil dissolved in hexane. The suspension was filtered through silica gel bed and the solvent removed. The crude product (colorless liquid) was used in the following step without further purification. Synthesis of I‑19

[0152] A solution of N,N-dihexyl‑4-oxobutanamide (0.56 g, 1.97 mmol), and bis(2-hexyldecyl) 7‑((2‑(dimethylamino) ethyl)amino)tridecanedioate (0.44g, 0.56mmol, prepareaccording toproceduresofExample4) in 1,2-dichloroethane (10 mL) was stirred for 15 min, after which time sodium triacetoxyborohydride (0.41 g, 1.97 mmol) was added in one portion and stirred at room temperature for additional 16 hours. The mixture was concentrated. The residue was taken up in a mixture of hexane and ethyl acetate (96:4) and washed with saturated aqueous NaHCO3 solution and brine. The organic layer was separated, dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure to obtain colorlessoil. Thecrudeproductwaspurifiedbyflashcolumnchromatographyonsilicagel (MeOH inchloroform,0 to5%) to yield the desired product as a colorless oil (260 mg, 0.25 mmol, 45%). 1HNMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.7 Hz, 2H), 3.20 (t-like, 7.7 Hz, 2H), 2.56‑2.47 (m, 2H), 2.44 (t, 6.8 Hz, 2H), 2.39‑2.20 (m, 15H), 1.74‑1.45 (m, 12H), 1.42‑1.15 (72H), 0.93‑0.84 (m, 18H). EXAMPLE 6 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑((4‑(DIHEXYLAMINO)‑4-OXOBUTYL)(3‑(DIMETHYLAMINO)PROPYL) AMINO)NONADECANEDIOATE (COMPOUND I‑21)

[0153] Compound I‑21 was prepared according to the general procedures of Example 5 to yield 0.05 g of colorless oil, 0.03 mmol, 32%. 1HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.6 Hz, 2H), 3.20 (t-like, 7.6 Hz, 2H), 2.43‑2.23 (m, 13H), 2.20 (s, 6H), 1.75‑1.45 (m, 14H), 1.40‑1.12 (m, 68H), 0.93‑0.84 (m, 18H). EXAMPLE 7 SYNTHESIS OF BIS(2-BUTYLOCTYL) 7‑((4‑(DIHEXYLAMINO)‑4-OXOBUTYL)(3‑(DIMETHYLAMINO)PROPYL) AMINO)TRIDECANEDIOATE (COMPOUND 1‑20)

[0154] Compound I‑20 was prepared according to the general procedures of Example 5 to yield 0.06 g of colorless oil, 0.06 mmol, 41%. 1HNMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.27 (t-like, 7.6 Hz, 2H), 3.19 (t-like, 7.6 Hz, 2H), 2.62‑2.17 (m, 19H), 1.79‑1.43 (m, 14H), 1.42‑1.10 (m, 56H), 0.95‑0.81 (m, 18H). EXAMPLE 8 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑(N‑(3‑(DIMETHYLAMINO)PROPYL)‑6-METHOXY‑6-OXOHEXANAMIDO) NONADECANEDIOATE (COMPOUND I‑14)

[0155] 29 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55

[0156] Adipoyl chloride (0.12 g, 0.68 mmol) in anhydrous benzene (5 mL) was added via syringe to a solution of bis(2- butyloctyl) 10‑((3‑(dimethylamino)propyl)amino)nonadecanedioate (0.26 g, 0.34 mmol, prepared according to Example 4), triethylamine (0.3mL, 2.5mmol) andDMAP (5mg) in benzene (10mL) at RTover the period of 5min. Themixture was allowed to stir for 2 h and then Methanol (0.5 mL) was added to remove excess acyl chloride. The resulting mixture was stirred for another hour and then filtered throughapadof silica gel, washedwith amixture of hexane / EtOAc / Et3N (70:30:1) and concentrated. The residue was passed down a silica gel column (0‑4%MeOH in DCM gradient), yielding compound I‑14 as a colorless oil (0.28 g, 0.30 mmol, 89%). 1HNMR (400 MHz, CDCl3) δ: 4.52‑4.29 (br., estimated 0.3H, due to slow isomerization about amide bond), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H), 3.59 (quintet-like, 7.0 Hz, 0.7H), 3.14‑3.05 (m, 2H), 2.37‑2.24 (m, 10H), 2.23‑2.18 (m, 6H), 1.73‑1.54 (m, 12H), 1.48‑1.37 (m, 4H), 1.34‑1.14 (m, 52H), 0.93‑0.83 (m, 12H). EXAMPLE 9 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑(N‑(2‑(DIMETHYLAMINO)ETHYL)‑6-METHOXY‑6-OXOHEXANAMIDO) NONADECANEDIOATE (COMPOUND I‑15)

[0157] Compound I‑15 was prepared according to the general procedures of Example 8, to yield 0.18 g of colorless oil, 0.20mmol, 85%. 1HNMR (400MHz, CDCl3) δ: 4.53‑4.30 (br., 0.3H, due to slow isomerization about amide bond), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H), 3.58 (quintet-like, 7 Hz, 0.7H), 3.27‑3.15 (m, 2H), 2.46‑2.22 (m, 16H), 1.75‑1.54 (m, 10H), 1.50‑1.36 (m, 4H), 1.35‑1.09 (m, 52H), 0.94‑0.82 (m, 12H). EXAMPLE 10 SYNTHESIS OF BIS(2-HEXYLDECYL) 7‑(N‑(2‑(DIMETHYLAMINO)ETHYL)‑6-METHOXY‑6-OXOHEXANAMIDO) TRIDECANEDIOATE (COMPOUND I‑16)

[0158] Compound I‑16 was prepared according to the general procedures of Example 8 to yield 0.27 g of colorless oil, 0.29 mmol, 81%. 1HNMR (400 MHz, CDCl3) δ: 4.53‑4.30 (br., 0.3H, due to slow isomerization about amide bond), 3.99‑3.92 (m,4H),3.66 (s, 3H), 3.59 (quintet-like, 7.0Hz,0.7H),3.28‑3.14 (m,2H),2.46‑2.20 (m,16H),1.75‑1.53 (m,10H), 1.51‑1.36 (m, 4H), 1.35‑1.09 (m, 56H), 0.94‑0.81 (m, 12H). EXAMPLE 11 30 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 SYNTHESIS OF BIS(2-HEXYLDECYL) 7‑(N‑(3‑(DIMETHYLAMINO)PROPYL)‑8-METHOXY‑8-OXOOCTANAMIDO) TRIDECANEDIOATE (COMPOUND I‑17)

[0159] Compound I‑17 was prepared according to the general procedures of Example 8 to yield 0.08 g of colorless oil, 0.08 mmol, 75%. 1HNMR (400 MHz, CDCl3) δ: 4.53‑4.30 (br., 0.3H, due to slow isomerization about amide bond), 3.99‑3.91 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.15‑3.06 (m, 2H), 2.34‑2.23 (m, 10H), 2.22 (s, 6H), 1.75‑1.53 (m, 10H), 1.51‑1.38 (m, 4H), 1.37‑1.15 (m, 62H), 0.93‑0.82 (m, 12H). EXAMPLE 12 SYNTHESIS OF BIS(2-HEXYLDECYL) 7‑(N‑(2‑(DIMETHYLAMINO)ETHYL)‑8-METHOXY‑8-OXOOCTANAMIDO) TRIDECANEDIOATE (COMPOUND I‑18)

[0160] Compound I‑18 was prepared according to the general procedures of Example 8 to yield 0.15 g of colorless oil, 0.16 mmol, 79%. 1HNMR (400 MHz, CDCl3) δ: 4.53‑4.30 (br., 0.3H, due to slow isomerization about amide bond), 3.99‑3.92 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0Hz, 0.7H), 3.26‑3.14 (m, 2H), 2.47‑2.35 (m, 2H), 2.34‑2.20 (m, 14H), 1.73‑1.53 (m, 8H), 1.51‑1.39 (m, 4H), 1.38‑1.14 (m, 62H), 0.93‑0.82 (m, 12H). EXAMPLE 13 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑(N‑(3‑(DIMETHYLAMINO)PROPYL)‑6-METHOXY‑6-OXOHEXANAMIDO) NONADECANEDIOATE (COMPOUND I‑1)

[0161] Synthesis of Intermediate C

[0162] To a solution of acrylic acid (1.1 eq, 8.25mmol, 594mg), octanol (1 eq, 975mg, 7.5 mmol ) and DMAP (0.4 eq, 3 mmol, 366mg) inDCM(15mL)wasaddedDCC(1.4eq, 10.5mmol, 2.16g).The resultingmixturewasstirredatRTfor16h. The reactionmixturewasfilteredand thefiltrate concentrated.The residuewas takenup inhexanes (50mL)and loadedon 31 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 a column of silica gel. The column was washed with hexane (40 mL). The fractions were combined, re-loaded on the column and eluted with amixture of hexane and ethyl acetate (ca 99:1 or 98:2, 200mL). A colorless oil was obtained (986 mg, 71%). Synthesis of Compound I‑1

[0163] AnEtOH (10mL) solution of bis(2-butyloctyl) 10‑((4‑(dimethylamino)butyl)amino)nonadecanedioate (1 eq., 220 mg, 0.28mmol, prepared according to general procedures above), and intermediate C (2.75 eq. 0.77 mmol, 140mg) in a sealed pressure flask was stirred at RT for 4 days under Ar. The reaction mixture was concentrated. The residue was purified twice by flash dry column chromatography on silica gel (hexane-EtOAc-Et3N, 95:5:0 to 80:20:1 and MeOH in chloroform, 0 to 5%). The desired product was obtained as colorless oil (68 mg, 0.07 mmol, 25%) 1HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.9Hz, 2H), 3.97 (d, 5.8Hz, 4H), 2.69 (t, 7.2Hz, 2H), 2.38‑2.33 (m,4H), 2.33‑2.26 (m, 1H), 2.29 (t, 7.5Hz, 4H), 2.26‑2.22 (m, 2H), 2.21 (s, 6H), 1.61 (quintet-like, 7.0 Hz, 8H), 1.48‑1.08 (70H), 0.92‑0.86 (m, 15H). EXAMPLE 14 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑((5‑(DIMETHYLAMINO)PENTYL)(3‑(OCTYLOXY)‑3-OXOPROPYL)AMI- NO)NONADECANEDIOATE (COMPOUND 1‑2)

[0164] Compound I‑2 was prepared according to the general procedures of Example 13 to yield 0.05 g of colorless oil, 0.05mmol, 10%. 1HNMR (400MHz, CDCl3) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.2 Hz, 2H), 2.38‑2.21 (m, 17H), 1.61 (quintet-like, 7.0 Hz, 8H), 1.51‑1.10 (m, 72H), 0.93‑0.84 (m, 15H). EXAMPLE 15 SYNTHESIS OF BIS(2-BUTYLOCTYL) 7‑((4‑(DIMETHYLAMINO)BUTYL)(3‑(OCTYLOXY)‑3-OXOPROPYL)AMINO) TRIDECANEDIOATE (COMPOUND 1‑3)

[0165] Compound I‑3 was prepared according to the general procedures of Example 13 to yield 0.01 g of colorless oil, 0.01mmol, 11%. 1HNMR (400MHz, CDCl3) δ: 4.03 (t, 6.9 Hz, 2H), 3.96 (d, 5.6 Hz, 4H), 2.69 (t, 7.1 Hz, 2H), 2.38‑2.20 (m, 17H), 1.69‑1.56 (m, 10H), 1.48‑1.09 (m, 56H), 0.92‑0.84 (m, 15H). EXAMPLE 16 SYNTHESIS OF BIS(2-HEXYLDECYL) 7‑((4‑(DIMETHYLAMINO)BUTYL)(3‑(OCTYLOXY)‑3-OXOPROPYL)AMINO) TRIDECANEDIOATE (COMPOUND 1‑4)

[0166] Compound I‑4 was prepared according to the general procedures of Example 13 to yield 0.05 g of colorless oil, 0.05mmol, 13%. 1HNMR (400MHz, CDCl3) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.1 Hz, 2H), 2.39‑2.21 (m, 17H), 1.66‑1.09 (m, 82H), 0.88 (t, 7.0 Hz, 15H). EXAMPLE 17 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑((4‑(DIMETHYLAMINO)BUTYL)(OCTYL)AMINO)NONADECANEDIOATE (COMPOUND 1‑23)

[0167] 32 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 Synthesis of Compound I‑23

[0168] A solution of octanal (3.5 eq, 0.90 mmol, 115 mg, 0.141 mL) and bis(2-butyloctyl) 10‑((4‑(dimethylamino)butyl) amino)nonadecanedioate (200mg,0.26mmol, preparedaccording togeneral proceduresabove) in1,2-dichloroethane (5 mL) was stirred for 15 min, after which time sodium triacetoxyborohydride (3.5 eq, 0.9 mmol, 190 mg) was added in one portion. Stirring was continued at RT for 16 hours. The reactionmixture was concentrated. The residuewas purified twice by flash dry column chromatography on silica gel (hexane-EtOAc-Et3N, 95:5:0 to 80:20:1 and MeOH in chloroform, 0 to 5%). The desired product was obtained as colorless oil (203mg, 0.23mmol, 88%)). 1HNMR (400MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.40‑2.18 (m, 17H), 1.69‑1.56 (m, 6H), 1.52‑1.10 (m, 72H), 0.92‑0.86 (m, 15H). EXAMPLE 18 SYNTHESIS OF BIS(2-ETHYLHEXYL) 10‑((4‑(DIMETHYLAMINO)BUTYL)(6‑((2-HEXYLDECANOYL)OXY)HEXYL) AMINO)NONADECANEDIOATE (COMPOUND I‑22)

[0169] Compound I‑22 was prepared according to the general procedures of Example 17 to yield 0.19 g of colorless oil, 0.19 mmol, 80%. 1HNMR (400 MHz, CDCl3) δ: 4.06 (t, 6.7 Hz, 2H), 3.97 (d, 5.6 Hz, 4H), 2.39‑2.26 (m, 11H), 2.23 (s, 6H), 1.68‑1.10 (m, 83H), 0.94‑0.82 (m, 18H). EXAMPLE 19 SYNTHESIS OF BIS(2-BUTYLOCTYL) 10‑((4‑(DIMETHYLAMINO)BUTYL)(6‑((2-HEXYLDECANOYL)OXY)HEXYL) AMINO)NONADECANEDIOATE (COMPOUND I‑5)

[0170] Compound I‑5 was prepared according to the general procedures of Example 17 to yield 0.04 g of colorless oil, 0.04mmol, 73%. 1HNMR (400MHz, CDCl3) δ: 4.05 (t, 7.0 Hz, 2H), 3.96 (d, 5., 8 Hz, 4H), 2.38‑2.26 (m, 11H), 2.23 (s, 6H), 1.71‑1.09 (m, 99H), 0.95‑0.82 (m, 18H).

[0171] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification, including but not limited to U.S. Provisional Patent ApplicationNo. 63 / 052,815, filed July 16, 2020, and U.S. Provisional Patent Application No. 63 / 188,996, filed May 14, 2021, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other 33 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 changes can bemade to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments alongwith the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[0172] The application further encompasses the following embodiments: 1. A compound having a structure of Formula (I): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: G1 and G2 are each independently C1‑C6 alkylene; L1 and L2 are each independently ‑O(C=O)‑ or ‑(C=O)O‑; R1aandR1bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R1a isHorC1‑C12alkyl, and R1b together with the carbon atom towhich it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond; R2aandR2bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R2a isHorC1‑C12alkyl, and R2b together with the carbon atom towhich it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R3aandR3bare, at eachoccurrence, independently either (a):HorC1‑C12alkyl; or (b)R3a isHorC1‑C12alkyl, and R3b together with the carbon atom towhich it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R4aandR4bare, at eachoccurrence, independently either: (a)HorC1‑C12alkyl; or (b)R4a isHorC1‑C12alkyl, and R4b together with the carbon atom towhich it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R5 and R6 are each independently H or methyl; R7 is ‑O(C=O)R10, ‑(C=O)OR10, ‑NR9(C=O)R10 or ‑(C=O)NR9R10; R8 is OH, ‑N(R11)(C=O)R12, ‑(C=O)NR11R12, ‑NR11R12, ‑(C=O)OR12 or ‑O(C=O)R12; R9 is H or C1‑C15 alkyl; R10 is C1‑C15 alkyl; R11 is H or C1‑C6 alkyl; R12 is C1‑C6 alkyl; X is ‑(C=O)‑ or a direct bond; and a, b, c and d are each independently an integer from 1 to 24; wherein each alkyl and alkylene and is independently optionally substituted. 2. The compound of embodiment 1, having one of the following structures (IA) or (IB): 34 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 3. The compound of embodiment 1 or 2, wherein G1 is C2‑C3 alkylene. 4. The compound of embodiment 1 or 2, wherein G1 is C4‑C6 alkylene. 5. The compound of any one of embodiments 1‑4, wherein G2 is C2‑C4 alkylene. 6. The compound of embodiment 5, wherein G2 is C2‑C3 alkylene or C3‑C4 alkylene. 7. The compound of any one of embodiments 1‑6, wherein X is ‑(C=O)‑. 8. The compound of any one of embodiments 1‑6, wherein X is a direct bond. 9. The compound of any one of embodiments 1‑8, wherein R7 is ‑O(C=O)R10 or ‑(C=O)OR10. 10. The compound of embodiment 9, wherein R10 is linear C1‑C15 alkyl. 11. The compound of embodiment 10, wherein R10 is linear C6‑C10 alkyl. 12. The compound of embodiment 10, wherein R10 is methyl. 13. The compound of embodiment 10, wherein R10 is branched C2‑C15 alkyl. 14. The compound of embodiment 13, wherein R10 is branched C10‑C15 alkyl. 15. The compound of any one of embodiment 1‑8, wherein R7 is ‑NR9(C=O) or ‑(C=O)NR9R10. 16. The compound of embodiment 15, wherein R9 is H. 17. The compound of embodiment 15, wherein R9 and R10 are each independently C6‑C10 alkyl. 18. The compound of any one of embodiments 1‑17, wherein for at least one occurrence of R1a and R1b, R1a is H or C1‑C12 alkyl, andR1b together with the carbon atom towhich it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond. 19. The compound of any one of embodiments 1‑18, wherein for at least one occurrence of R4a and R4b, R4a is H or C1‑C12 alkyl, andR4b together with the carbon atom towhich it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond. 20. The compound of any one of embodiments 1‑19, wherein for at least one occurrence of R2a and R2b, R2a is H or C1‑C12 alkyl, andR2b together with the carbon atom towhich it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond. 35 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 21. The compound of any one of embodiments 1‑20, wherein for at least one occurrence of R3a and R3b, R3a is H or C1‑C12 alkyl, andR3b together with the carbon atom towhich it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond. 22. The compound of any one of embodiments 1‑17, wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a and R4b are, at each occurrence, independently H or C1‑C12 alkyl. 23. The compound of embodiment 22, wherein R2a, R2b, R3a and R3b are, at each occurrence, H. 24. The compound of embodiment 22 or 23, wherein R1a and R4a are, at each occurrence, H. 25. The compound of any one of embodiments 22‑24, wherein at least one of Rlb and R4b is C1‑C8 alkyl. 26. Thecompoundof embodiment 25,whereinC1‑C8alkyl ismethyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert- butyl, n-hexyl or n-octyl. 27. The compound of any one of embodiments 22‑26, wherein or or both, independently has one of the following structures: 28. The compound of any one of embodiments 1‑27, wherein a, b, c and d are each independently an integer from2 to 12 29. The compound of any one of embodiments 1‑27, wherein a, b, c and d are each independently an integer from4 to 36 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9 or 6 to 9. 30. The compound of any one of embodiments 1‑29, wherein one of R5 or R6 is methyl. 31. The compound of any one of embodiments 1‑30, wherein each of R5 and R6 is methyl. 32. The compound of any one of embodiments 1‑31, wherein R8 is OH. 33. The compound of any one of embodiments 1‑31, wherein R8 is - N(R11)(C=O)R12. 34. The compound of any one of embodiments 1‑31, wherein R8 is ‑(C=O)NR11R12. 35. The compound of any one of embodiments 1‑31, wherein R8 is ‑NR11R12. 36. The compound of any one of embodiments 33‑35, wherein R11 and R12 are each independently H or C1‑C8 alkyl. 37. The compound of any one of embodiments 33‑36, wherein R11 and R12 are each independently H or C1‑C3 alkyl. 38. The compound of embodiment 36 or 37, wherein C1‑C8 alkyl or C1‑C3 alkyl is unsubstituted or substituted with hydroxyl. 39. The compound of any one of embodiments 33‑36, wherein R11 and R12 are each methyl. 40. The compound of any one of embodiments 1‑31, wherein R8 is ‑(C=O)OR12. 41. The compound of embodiment 40, wherein R8 is ‑O(C=O)R12. 42. The compound of any one of embodiment 1‑31, wherein R8 has one of the following structures: -OH or 43. A compound selected from a compound in Table 1. 44. A lipid nanoparticle comprising the compound of any one of embodiments 1‑43 and a therapeutic agent. 45. The lipid nanoparticle of embodiment 44, further comprising one or more excipient selected from neutral lipids, steroids and polymer conjugated lipids. 46. The lipid nanoparticle of embodiment 45, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. 47. The lipid nanoparticle of embodiment 46, wherein the neutral lipid is DSPC. 48. The lipid nanoparticle of any one of embodiments 44‑47, wherein the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1. 49. The lipid nanoparticle of any one of embodiments 45‑48, wherein the steroid is cholesterol. 50.The lipid nanoparticleof embodiment 49,wherein themolar ratioof thecompound to cholesterol ranges from5:1 to 1:1. 51. The lipid nanoparticle of any one of embodiments 45‑50, wherein the polymer conjugated lipid is pegylated lipid. 52. The lipid nanoparticle of embodiment 51, wherein the molar ratio of the compound to pegylated lipid ranges from 37 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 about 100:1 to about 20:1. 53. The lipid nanoparticle of anyoneof embodiment 51or 52,wherein thepegylated lipid isPEG-DAG,PEG-PE,PEG- S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate. 54. The lipid nanoparticle of any one of embodiment 51 or 52, wherein the pegylated lipid has the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R8 and R9 are each independently a straight or branched, alkyl, alkenyl or alkynyl from 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. 55.The lipidnanoparticleof embodiment54,whereinR8andR9areeach independently straightalkyl chaincontaining from 12 to 16 carbon atoms. 56. The lipid nanoparticle of any one of embodiment 54 or 55, wherein the average w is about 49. 57. The lipid nanoparticle of any one of embodiments 44‑56, wherein the therapeutic agent comprises a nucleic acid. 58.The lipidnanoparticleof embodiment57,wherein thenucleicacid is selected fromantisenseandmessengerRNA. 59. A pharmaceutical composition comprising the lipid nanoparticle of any one of embodiments 44‑58 and a pharmaceutically acceptable diluent or excipient. 60. Amethod for treating or preventingadisease in a patient in need thereof, themethod comprisingadministering the lipid nanoparticle of any one of embodiments 44‑58, or the pharmaceutical composition of embodiment 59, to the patient. 61. Amethod for vaccinating a patient in need thereof against a viral pathogen, themethod comprising administering the lipid nanoparticle of any one of embodiments 44‑58, or the pharmaceutical composition of embodiment 59, to the patient, wherein the therapeutic agent is a viral antigen or a nucleic acid capable of transcribing a viral antigen. Claims 1. A lipid nanoparticle (LNP) comprising: i) a compound having a structure of Formula (I): 38 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: G1 and G2 are each independently C1‑C6 alkylene; L1 and L2 are each independently ‑O(C=O)‑ or ‑(C=O)O‑; R1a and R1b are, at each occurrence, independently either: (a) H or C1‑C12 alkyl; or (b) R1a is H or C1‑C12 alkyl, andR1b togetherwith the carbonatom towhich it is bound is taken togetherwithanadjacentR1band the carbon atom to which it is bound to form a carbon-carbon double bond; R2a and R2b are, at each occurrence, independently either: (a) H or C1‑C12 alkyl; or (b) R2a is H or C1‑C12 alkyl, andR2b togetherwith the carbonatom towhich it is bound is taken togetherwithanadjacentR2band the carbon atom to which it is bound to form a carbon-carbon double bond; R3a and R3b are, at each occurrence, independently either (a): H or C1‑C12 alkyl; or (b) R3a is H or C1‑C12 alkyl, andR3b togetherwith the carbonatom towhich it is bound is taken togetherwithanadjacentR3band the carbon atom to which it is bound to form a carbon-carbon double bond; R4a and R4b are, at each occurrence, independently either: (a) H or C1‑C12 alkyl; or (b) R4a is H or C1‑C12 alkyl, andR4b togetherwith the carbonatom towhich it is bound is taken togetherwithanadjacentR4band the carbon atom to which it is bound to form a carbon-carbon double bond; R5 and R6 are each independently H or methyl; R7 is ‑O(C=O)R10, ‑(C=O)OR10, ‑NR9(C=O)R10 or ‑(C=O)NR9R10; R8 is ‑N(R11)(C=O)R12, ‑(C=O)NR11R12, ‑NR11R12, ‑(C=O)OR12 or ‑O(C=O)R12; R9 is H or C1‑C15 alkyl; R10 is C1‑C15 alkyl; R11 is H or C1‑C6 alkyl; R12 is C1‑C6 alkyl; X is ‑(C=O)‑ or a direct bond; and a, b, c and d are each independently an integer from 1 to 24; wherein each alkyl and alkylene is independently optionally substituted with one or more halo, preferably fluoro, and ii) a therapeutic agent, preferably a nucleic acid, for use in a method of treating a disease caused by an infectious entity and / or insufficiency of a protein, said method including inducing expression of a protein in a subject. 2. The LNP for use according to claim 1, having one of the following structures (IA) or (IB): 39 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. 3. The LNP for use according to claim 1 or 2, wherein G1 is C2‑C3 alkylene or C4‑C6 alkylene. 4. The LNP for use according to any one of claims 1‑3, wherein G2 is C2‑C4 alkylene, C2‑C3 alkylene or C3‑C4 alkylene. 5. The LNP for use according to any one of claims 1‑4, wherein R7 is ‑O(C=O)R10 or ‑(C=O)OR10, preferably wherein: i) R10 is linear C1‑C15 alkyl; ii) R10 is linear C6‑C10 alkyl; iii) R10 is methyl; iv) R10 is branched C2‑C15 alkyl; or v) R10 is branched C10‑C15 alkyl. 6. The LNP for use according to any one of claims 1‑4, wherein R7 is ‑NR9(C=O) or ‑(C=O)NR9R10, preferably wherein: i) R9 is H; or ii) R9 and R10 are each independently C6‑C10 alkyl. 7. The LNP for use according to any one of claims 1‑6, wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a and R4b are, at each occurrence, independently H or C1‑C12 alkyl, preferably wherein: i) R2a, R2b, R3a and R3b are, at each occurrence, H; ii) R1a and R4a are, at each occurrence, H; iii) at least one of R1b and R4b is C1‑C8 alkyl; iv) C1‑C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl; or v) or both, independently has one of the following structures: 40 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 8. LNP for use according to any one of claims 1‑7, wherein a, b, c and d are each independently an integer from 2 to 12, preferably wherein a, b, c and d are each independently an integer from 4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9 or 6 to 9. 9. The LNP for use according to any one of claims 1‑8, wherein one of R5 or R6 is methyl, preferably wherein each of R5 and R6 is methyl. 10. The LNP for use according to any one of claims 1‑9, wherein R8 has the following structure: 11. The LNP for use according to claim 1, wherein the compound has one of the following structures: 41 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 42 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 43 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 44 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 or 12. The LNP for use according to any one of claims 1‑11, wherein the nucleic acid is messenger RNA. 13. The LNP for use according to claim 12, wherein the mRNA encodes an antigen or antibody, wherein the antigen or antibody protects against infection. 14. The LNP for use according to claim 12, wherein themessenger RNAencodes a genemodifying enzyme, wherein the lipid nanoparticle further comprises a DNA segment for incorporation into a host genome, or the lipid nanoparticle is used in combination with a DNA segment for incorporation into a host genome. 45 EP 4 678 164 A2 5 10 15 20 25 30 35 40 45 50 55 46 EP 4 678 164 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 20120251618 A

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[0140] (19) *EP004678164A3* (11) EP 4 678 164 A3 (12) EUROPEAN PATENT APPLICATION (88) Date of publication A3: 22.04.2026 Bulletin 2026 / 17 (43) Date of publication A2: 14.01.2026 Bulletin 2026 / 03 (21) Application number: 25198053.8 (22) Date of filing: 16.07.2021 (51) International Patent Classification (IPC): C07C 229 / 24 (2006.01) C07C 233 / 36 (2006.01) C07C 237 / 06 (2006.01) A61K 47 / 00 (2006.01) A61K 9 / 00 (2006.01) (52) Cooperative Patent Classification (CPC): C07C 229 / 24; A61K 9 / 1272; A61K 9 / 5123; A61K 45 / 06; A61P 31 / 12; C07C 219 / 06; C07C 233 / 36; C07C 237 / 06 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR Designated Extension States: BA ME Designated Validation States: KH MA MD TN (30) Priority: 16.07.2020 US 202063052815 P 14.05.2021 US 202163188996 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 21751728.3 / 4 182 297 (71) Applicant: Acuitas Therapeutics, Inc. Vancouver, British Columbia V6T 1Z3 (CA) (72) Inventor: GATENYO, Julia Vancouver BC V6T 1Z3 (CA) (74) Representative: Grünecker Patent‑ und Rechtsanwälte PartG mbB Leopoldstraße 4 80802 München (DE) (54) CATIONIC LIPIDS FOR USE IN LIPID NANOPARTICLES (57) Compounds are provided having the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1, G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8 and X are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, nanoparticles comprising the compounds and methods for their use and preparation are also provided. EP 4 67 8 16 4 A 3 Processed by Luminess, 75001 PARIS (FR) 2 EP 4 678 164 A3 5 10 15 20 25 30 35 40 45 50 55 3 EP 4 678 164 A3 5 10 15 20 25 30 35 40 45 50 55 4 EP 4 678 164 A3 5 10 15 20 25 30 35 40 45 50 55 要 摘 提供了具有以下结构的化合物： 或其药学上可接受的盐、互变异构体或立体 异构体，其中 a、b、c、d、G1、G2、L1、L2、R1a、R1b、R2a、R2b、R3a、R3b、 R4a、R4b、R5、R6、R7、R8 和 X 如本文所定义。还提供了所述化合物作为脂 质纳米颗粒制剂的组分用于递送治疗剂的用途、包含所述化合物的组合物及其 使用和制备方法。

Claims

1. A lipid nanoparticle (LNP) comprising: i) a compound having a structure of Formula (I): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: G1 and G2 are each independently C1-C6 alkylene; L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R1a and R1b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R1a is H or C1-C12 alkyl, and R1b together with the carbon atom to which it is bound is taken together with an adjacent R1b and the carbon atom to which it is bound to form a carbon-carbon double bond; R2a and R2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond; R3a and R3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R3a is H or C1-C12 alkyl, and R3b together with the carbon atom to which it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond; R4a and R4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R5 and R6 are each independently H or methyl; R7 is -O(C=O)R10, -(C=O)OR10, -NR9(C=O)R10 or -(C=O)NR9R10; R8 is -N(R11)(C=O)R12, -(C=O)NR11R12, -NR11R12, -(C=O)OR12 or -O(C=O)R12; R9 is H or C1-C15 alkyl; R10 is C1-C15 alkyl; R11 is H or C1-C6 alkyl; R12 is C1-C6 alkyl; X is -(C=O)- or a direct bond; and a, b, c and d are each independently an integer from 1 to 24; wherein each alkyl and alkylene is independently optionally substituted with one or more halo, preferably fluoro, and ii) a therapeutic agent, preferably a nucleic acid, for use in a method of treating a disease caused by an infectious entity and / or insufficiency of a protein, said method including inducing expression of a protein in a subject.

2. The LNP for use according to claim 1, having one of the following structures (IA) or (IB): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

3. The LNP for use according to claim 1 or 2, wherein G1 is C2-C3 alkylene or C4-C6 alkylene.

4. The LNP for use according to any one of claims 1-3, wherein G2 is C2-C4 alkylene, C2-C3 alkylene or C3-C4 alkylene.

5. The LNP for use according to any one of claims 1-4, wherein R7 is -O(C=O)R10 or -(C=O)OR10, preferably wherein: i) R10 is linear C1-C15 alkyl; ii) R10 is linear C6-C10 alkyl; iii) R10 is methyl; iv) R10 is branched C2-C15 alkyl; or v) R10 is branched C10-C15 alkyl.

6. The LNP for use according to any one of claims 1-4, wherein R7 is -NR9(C=O) or -(C=O)NR9R10, preferably wherein: i) R9 is H; or ii) R9 and R10 are each independently C6-C10 alkyl.

7. The LNP for use according to any one of claims 1-6, wherein R1a, R1b, R2a, R2b, R3a, R3b, R4a and R4b are, at each occurrence, independently H or C1-C12 alkyl, preferably wherein: i) R2a, R2b, R3a and R3b are, at each occurrence, H; ii) R1a and R4a are, at each occurrence, H; iii) at least one of R1b and R4b is C1-C8 alkyl; iv) C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl; or v) or both, independently has one of the following structures:

8. LNP for use according to any one of claims 1-7, wherein a, b, c and d are each independently an integer from 2 to 12, preferably wherein a, b, c and d are each independently an integer from 4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9 or 6 to 9.

9. The LNP for use according to any one of claims 1-8, wherein one of R5 or R6 is methyl, preferably wherein each of R5 and R6 is methyl.

10. The LNP for use according to any one of claims 1-9, wherein R8 has the following structure:

11. The LNP for use according to claim 1, wherein the compound has one of the following structures: or 12. The LNP for use according to any one of claims 1-11, wherein the nucleic acid is messenger RNA.

13. The LNP for use according to claim 12, wherein the mRNA encodes an antigen or antibody, wherein the antigen or antibody protects against infection.

14. The LNP for use according to claim 12, wherein the messenger RNA encodes a gene modifying enzyme, wherein the lipid nanoparticle further comprises a DNA segment for incorporation into a host genome, or the lipid nanoparticle is used in combination with a DNA segment for incorporation into a host genome.