Lipid nanoparticle

Abstract

The present invention relates to a lipid nanoparticle comprising: (a) an ionizable lipid; (b) a helper lipid; (c) a sterol; and (d) a PEGylated lipid, wherein: (i) the PEGylated lipid comprises a hydrocarbon chain comprising more than 14 C atoms; and / or (ii) the PEGylated lipid is at a concentration of at least 3 %mol.

Description

[0001] LIPID NANOPARTICLE

[0002] Cross reference to related applications

[0003] This application claims priority from GB2502437.3 filed on 19 February 2025 and GB2519601.5 filed on 19 November 2025, the contents of which are herein incorporated by reference.

[0004] Field of invention

[0005] The present invention relates to a lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain comprising more than 14 C atoms. The lipid nanoparticle may further comprise at least one targeting peptide. The lipid nanoparticle may be used in the treatment of a disease.

[0006] Background of the invention

[0007] Lipid nanoparticles (LNPs) are non-viral vectors which can be used for the delivery of payloads such as nucleic acids. LNPs are passively targeted to the liver and spleen, where they are taken up by cells for clearance from the body. LNPs have been successfully used to deliver mRNAs to liver cells, for example as part of a gene therapy. However, clinical efficacy for LNPs has been limited to hepatic diseases and vaccination due to liver accumulation. Developing LNPs which target non-liver cells and tissues remains a challenge.

[0008] Summary of the invention

[0009] The present invention relates to lipid nanoparticles comprising a PEGylated lipid comprising a hydrocarbon chain comprising more than 14 C atoms. The present inventors have surprisingly found that the inclusion of longer hydrocarbon lipid chain reduces shedding of the PEGylated lipid, causing it to remain within the lipid nanoparticle for an increased duration. This in turn decreases uptake of the lipid nanoparticle by the liver. Additionally, by using a higher concentration of PEGylated lipid in the nanoparticle, the lipid nanoparticle remains in circulation for longer. By combining the longer PEGylated lipids with targeting peptides specific to a particular cell, tissue or organ, a synergistic increase in the uptake by the target cell, tissue or organ is achieved. The lipid nanoparticles of the invention can thereforebe used to deliver a therapeutic payload to target cells with increased efficiency and reduced off target effects.

[0010] Accordingly, in one aspect, the present invention provides a lipid nanoparticle comprising:

[0011] (a) an ionizable lipid;

[0012] (b) a helper lipid;

[0013] (c) a sterol; and

[0014] (d) a PEGylated lipid,

[0015] wherein:

[0016] (i) the PEGylated lipid comprises a hydrocarbon chain comprising more than 14 C atoms; and / or

[0017] (ii) the PEGylated lipid is at a concentration of at least 3 %mol.

[0018] In a second aspect, the present invention provides a lipid nanoparticle comprising:

[0019] (a) an ionizable lipid;

[0020] (b) a helper lipid;

[0021] (c) a sterol;

[0022] (d) a PEGylated lipid; and

[0023] (e) a second PEGylated lipid,

[0024] wherein the PEGylated lipid is DSG-PEG comprising a PEG chain of 750 Da or 1 kDa, the PEGylated lipid is at a concentration about 10.29 %mol, the second PEGylated lipid is DSPE-PEG comprising a PEG chain of 3.4 kDa, and the second PEGylated lipid is at a concentration of about 0.09 %mol.

[0025] In a third aspect, the present invention provides a lipid nanoparticle comprising:

[0026] (a) an ionizable lipid;

[0027] (b) a helper lipid;

[0028] (c) a sterol;

[0029] (d) a PEGylated lipid; and

[0030] (e) a second PEGylated lipid,

[0031] wherein

[0032] (i) the ionizable lipid is SM102 at a concentration of about 39.98 %mol;(ii) the helper lipid is DSPC at a concentration of about 11.49 %mol;

[0033] (iii) the sterol is cholesterol at a concentration of about 38.15 %mol;

[0034] (iv) the PEGylated lipid is DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and

[0035] (v) the second PEGylated lipid is DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

[0036] In a fourth aspect, the present invention provides a lipid nanoparticle of the invention for use in a method of medical treatment, wherein the treatment comprises administering the lipid nanoparticle to a subject.

[0037] In a fifth aspect, the present invention provides a method of producing a polypeptide in cell, the method comprising contacting the cell with a lipid nanoparticle of the invention, wherein the lipid nanoparticle comprises a nucleic acid encoding the polypeptide.

[0038] In a sixth aspect, the present invention provides a method of delivering a payload to a cell, the method comprising contacting the cell with a lipid nanoparticle of the invention, wherein the lipid nanoparticle comprises the payload.

[0039] The present invention also provides a method of medical treatment, wherein the treatment comprises administering the lipid nanoparticle to a subject.

[0040] Description of the figures

[0041] Figure 1 Scheme illustrating in vivo barcoded LNP delivery experiments in tumour bearing mice. LNPs were generated, each comprising a different combination of targeting peptides and a unique DNA barcode. Following injection of the LNPs into the tail vein of mice, tissues were harvested for analysis. DNA sequencing was carried out to identify the barcodes present in each tissue. Results show the selectivity of the different LNPs for each tissue.

[0042] Figure 2 Dynamic Light Scattering (DLS) data of LNP samplesFigure 2 shows DLS data (intensity and volume distributions) of LNP samples. Samples: (A): formulation 1; (B): formulation 2; (C): formulation 3; (D): formulation 4; (E): formulation 5; (F): formulation 6; (G): formulation 7; (H): formulation control; (I): control.

[0043] Figure 3 DLS data of LNP samples with increasing concentrations of PEG-Lipid-DBCO Figure 3 shows DLS data (intensity and volume distributions) of LNP samples with increasing concentrations of PEG-Lipid-DBCO. Samples: (A): sample ID 1; (B): sample ID 2; (C): sample ID 3; (D): sample ID 4; (E): sample ID 5; (F): sample ID 6.

[0044] Figure 4 Dynamic Light Scattering (DLS) data of LNP samples. DLS data (intensity and volume distributions) of LNP samples. From left to right: Bare LNP formulation 1; Onpattro LNP.

[0045] Figure 5 In vivo data from barcoded LNP delivery experiments in tumour bearing mice.

[0046] Violin plot of the normalized reads per bDNA of the tumour divided by the normalized reads per bDNA in the liver. Mean and standard deviation are represented in white. Tumour / Liver data for each sample is represented as points. Data for mice sacrificed after injection of pooled LNPs.

[0047] Figure 6 Dynamic Light Scattering (DLS) data of LNP samples. DLS data (intensity and volume distributions) of LNP samples. From top to bottom: Bare LNP formulation 1; LNP samples functionalized with peptide combinations: NG006, NG005, NG008.

[0048] Figure 7 In vivo data from barcoded LNP delivery experiments in tumour bearing mice.

[0049] Violin plot of the normalized reads per bDNA of the tumour divided by the normalized reads per bDNA in the liver. Mean and standard deviation are represented in white. Tumour / Liver data for each sample is represented as points. Data for mice sacrificed after injection of pooled LNPs.

[0050] Detailed Description

[0051] General definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art to which this invention belongs.In general, the term “comprising" is intended to mean including but not limited to. For example, the phrase “a lipid nanoparticle comprising a PEGylated lipid" should be interpreted to mean that the lipid nanoparticle has a PEGylated lipid, but the lipid nanoparticle may comprise further elements. In some embodiments of the invention, the word “comprising" may be replaced with the phrase “ consisting of" . The term “consisting of" is intended to be limiting. For example, the phrase “a lipid nanoparticle consisting of an ionizable lipid, a helper lipid, a sterol and a PEGylated lipid" should be interpreted to mean that the lipid nanoparticle has an ionizable lipid, a helper lipid, a sterol and a PEGylated lipid and contains no further components.

[0053] In some embodiments of the invention, the word “comprising” may be replaced with the phrase “consisting essentially of’ . The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

[0054] The singular forms “a”, “an"", and “the"" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an amino acid" includes one or more instances or versions of such amino acids.

[0055] The term “about"" as applied to one or more values of interest refers to values that are similar to the stated value of interest. The term “about"" refers to a range of values which fall within + / - 10 % of the recited value. For example, when a PEGylated lipid is at a concentration of about 2 %mol, the PEGylated lipid may be at a concentration of between 1.8 and 2.2 %mol.

[0056] The terms “protein"" and “polypeptide"" are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0057] The terms “nucleic acid molecule"", “polynucleotide"" and “nucleotide sequence"" are intended to refer to a polymeric chain of any length of nucleotides, including deoxyribonucleotides, ribonucleotides, or analogues thereof. For example, the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The nucleic acid molecule, polynucleotide or nucleotide sequence may consist of DNA. Thenucleic acid molecule, polynucleotide or nucleotide sequence may be mRNA. Since the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0058] The unit “%moF refers to the amount of a particular component in a composition as measured in moles divided by the total amount of all components in a composition as measured in moles, i.e. the molar concentration of the component expressed as a percentage. For example, if a lipid nanoparticle comprises a PEGylated lipid at a concentration of 3 %mol, then 3% of the molecules making up the solution used to prepare the lipid nanoparticle are the PEGylated lipid, while the remaining 97% of the molecules making up the solution used to prepare the lipid nanoparticle are other components. When calculating the concentration of the components of a lipid nanoparticle in %mol, only the lipid components (i.e. the ionizable lipid, PEGylated lipid(s), helper lipid and sterol) should be considered. The payload, e.g. nucleic acid, should not be taken into account. For example, when calculating the concentration of ionizable lipid in a lipid nanoparticle of the invention, the following equation can be used:

[0059] [ionizable lipid]%mol

[0060] = 100

[0061]

[0062] wherein n(x) = the amount of molecule x in moles in the solution used to prepare the lipid nanoparticles.

[0063] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0064] Lipid nanoparticle

[0065] Lipid nanoparticles are spherical vesicles comprising a phospholipid layer comprised of ionizable lipids. Lipid nanoparticles are capable of carrying a payload for delivery to target cells. The ionizable lipids are positively charged at low pH and neutral at physiological pH. Lipid nanoparticles can be taken up by cells via endocytosis. The positive charge of theionizable lipids at low pH then allows for endosomal escape and release of the payload into the cytoplasm. In addition to the ionizable lipid, lipid nanoparticles typically comprise a helper lipid and a sterol to improve stability, and one or more PEGylated lipids to reduce opsonisation. The ratio of these different components can significantly affect the properties of the lipid nanoparticle.

[0066] In some embodiments, the nanoparticle composition includes one or more other components, including, but not limited to, one or more pharmaceutically acceptable excipients, small hydrophobic molecules, therapeutic agents, carbohydrates, polymers, permeability enhancing molecules, and surface altering agents.

[0067] The present invention provides a lipid nanoparticle comprising:

[0068] (a) an ionizable lipid;

[0069] (b) a helper lipid;

[0070] (c) a sterol; and

[0071] (d) a PEGylated lipid,

[0072] wherein:

[0073] (i) the PEGylated lipid comprises a hydrocarbon chain comprising more than 14 C atoms; and / or

[0074] (ii) the PEGylated lipid is at a concentration of at least 3 %mol.

[0075] The hydrodynamic radius of the lipid nanoparticle may be measured by dynamic light scattering (DLS). In some embodiments, the hydrodynamic radius of the lipid nanoparticle is about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm. In some embodiments, the hydrodynamic radius of the lipid nanoparticle is between 50 and 100 nm, between 55 and 100 nm, between 60 and 100 nm, between 65 and 100 nm, between 70 and 100 nm, between 75 and 100 nm, between 80 and 100 nm, between 85 and 100 nm, between 90 and 100 nm, or between 95 and 100 nm. In some embodiments, the hydrodynamic radius of the lipid nanoparticle is between 50 and 90 nm, between 60 and 90 nm, between 55 and 80 nm, between 55 and 75 nm, between 55 and 70 nm, or between 60 and 70 nm. In someembodiments, the hydrodynamic radius of the lipid nanoparticle is between 50 nm and 100 nm, preferably wherein the hydrodynamic radius is about 60 nm or about 70 nm.

[0076] The lipid nanoparticles of the invention may be highly homogenous. The poly dispersity of the lipid nanoparticle, as measured by DLS, can be used as a measured of homogeneity. For example, the poly dispersity of the lipid nanoparticles of the invention may be low. For example, the poly dispersity may be less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1, or less than 0.05. In some embodiments, the poly dispersity of the lipid nanoparticles of the invention is less than 0.2. In some embodiments, the poly dispersity of the lipid nanoparticles is between 0.01 and 0.2.

[0077] The zeta potential of the lipid nanoparticle is a measure of the electrostatic potential of the surface of the lipid nanoparticle. Zeta potential may be measured by laser droplet electrophoresis. At neutral pH, it is desirable for the zeta potential to be close to 0 (i.e. the surface charge of the lipid nanoparticle is close to neutral at neutral pH). Strongly anionic lipid nanoparticles may be repelled from negatively charged cell membranes, thereby reducing uptake. On the other hand, strongly cationic lipid nanoparticles may interact non-specifically with negatively charged cell membranes, resulting in disruption of the cell membranes and / or non-specific delivery of payload, resulting in cytotoxicity.

[0078] In some embodiments, the zeta potential of the lipid nanoparticle at neutral pH is between -50 and 30 mV, between -40 and 30 mV, between -40 and 20 mV, between -40 and 10 mV, between -30 and 20 mV, between -30 and 10 mV, between -20 and 10 mV, between -10 and 10 mV, between -5 and 10 mV, or between -5 and 5 mV. In some embodiments, the zeta potential of the lipid nanoparticle at neutral pH is between -10 and 10 mV. In some embodiments, the zeta potential of the lipid nanoparticle is about 0 mV, e.g. between -5 and 5 mV.

[0079] The hydrodynamic radius, poly dispersity, and zeta potential of the lipid nanoparticle may be measured as described in the Examples.

[0080] PEGylated lipidThe lipid (hydrocarbon) chain of the PEGylated lipid is buried within the phospholipid layer of the lipid nanoparticle, while the PEG molecule is exposed on the surface of the nanoparticle. The PEG forms a hydrophilic corona about the lipid nanoparticle, which presents a steric barrier. The presence of the steric barrier reduces the uptake of lipid nanoparticle from circulation by the liver and spleen. The PEGylated lipid is shed from the lipid nanoparticle over time, thus allowing the lipid nanoparticle to be cleared from circulation by the liver and spleen. A PEGylated lipid comprises a hydrocarbon chain and a PEG chain. The term “ hydrocarbon chain" refers to the lipid chain to which the PEG chain is attached, but does not comprise the PEG chain. Without wishing to be bound by theory, it is believed that by using a longer or branched hydrocarbon chain, the PEGylated lipid will take longer to be shed from the lipid nanoparticle, for example due to increased Van der Waals interactions with the lipid layer. The reduced shedding of the PEGylated lipid increases the time spent by the lipid nanoparticle in circulation and thereby increases the opportunity for uptake by target cells and organs other than the liver and spleen.

[0081] A PEGylated lipid may be a phospholipid according to Formula (I):

[0082]

[0083] in which Rp represents a PEG moiety and R1 and R2 represent hydrocarbon chains with or without saturation which may be the same or different. The hydrocarbon chains (e.g. R1 and R2) may be palmitic acid, lauric acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. The PEGylated lipid may be DSPE-PEG.

[0084] A PEGylated lipid may be a glycerol -based lipid according to Formula (II):

[0085]

[0086] in which Rp represents a PEG moiety and R1 and R2 represent hydrocarbon chains with or without saturation which may be the same or different. The hydrocarbon chains (e.g. R1 and R2) may be palmitic acid, lauric acid, myristic acid, myristoleic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. The PEGylated lipid may be DSG-PEG.

[0087] In some embodiments, the hydrocarbon chain is a branched or linear alkyl, alkenyl, or alkynyl chain. In some embodiments, the hydrocarbon chain comprises between 16 and 25 C atoms. In some embodiments, the hydrocarbon chain comprises between 16 and 20 C atoms. In some embodiments, the hydrocarbon chain comprises at least 16 C atoms. In some embodiments, the hydrocarbon chain comprises at least 18 C atoms. In some embodiments, the hydrocarbon chain comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 C atoms. In some embodiments, the hydrocarbon chain is 16 or 18 C atoms in length. In some embodiments, the PEGylated lipid is at a concentration of 4-15 %mol, 5-14 %mol, 6-13 %mol, 7-12 %mol, 8-12 %mol,9-ll %mol, 10-11 %mol, or 10-10.5 %mol. In some embodiments, the PEGylated lipid is at a concentration of 10-10.5 %mol. In some embodiments, the PEGylated lipid is at a concentration of about 10 %mol. In some embodiments, the PEGylated lipid is at a concentration of about 10.29 %mol. In some embodiments, the PEGylated lipid is at a concentration of about 10.38 %mol. In some embodiments, the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. In some embodiments, the PEGylated lipid is palmitic acid-PEG, l,2-dioleoyl-sn-glycero-3-PEG, DLG-PEG, DMG-PEG, DPG-PEG, DSG-PEG, DOG-PEG, DLG-PEG, DPPE-PEG, DOPE-PEG, DMPE-PEG, DSPE-PEG, or DOTA-PEG. In some embodiments, the PEGylated lipid is DSG-PEG or DSPE-PEG. Insome embodiments, the PEG chain is between 0.5 kDa and 2 kDa. In some embodiments, the PEG chain is about 1 kDa. In some embodiments, the PEG chain is about 750 Da.

[0088] In some embodiments, the lipid nanoparticle comprises a second PEGylated lipid comprising a second hydrocarbon chain and a second PEG chain. In some embodiments, the second PEGylated lipid differs from the first PEGylated lipid only in the PEG chain, i.e. the hydrocarbon chain of the second PEGylated lipid is the same, but the PEG chain is different. In some embodiments, the second PEGylated lipid comprises both a different hydrocarbon chain and a different PEG chain.

[0089] In some embodiments, the second hydrocarbon chain is a branched or linear alkyl, alkenyl, or alkynyl chain. In some embodiments, the second hydrocarbon chain comprises between 16 and 25 C atoms. In some embodiments, the second hydrocarbon chain comprises between 16 and 20 C atoms. In some embodiments, the second hydrocarbon chain comprises at least 16 C atoms. In some embodiments, the second hydrocarbon chain comprises at least 18 C atoms. In some embodiments, the second hydrocarbon chain comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 C atoms. In some embodiments, the second hydrocarbon chain is 16 or 18 C atoms in length. In some embodiments, the second PEGylated lipid is at a concentration of 0-10.38 %mol. In some embodiments, the second PEGylated lipid is at a concentration of 0-15 %mol, 0-12 %mol, 0-10 %mol, 0-9 %mol, 0-8 %mol, 0-7 %mol, 0-6 %mol, 0-5 %mol, 0-4 %mol, 0-3 %mol, 0-2 %mol, 0-1 %mol, 0-0.5 %mol or 0-0.15 %mol. In some embodiments, the second PEGylated lipid is at a concentration of 0.05-0.15 %mol, optionally about 0.09 %mol. In some embodiments, the second PEGylated lipid is DSG-PEG or DSPE-PEG. In some embodiments, the second PEG chain is between 1 and 5 kDa. In some embodiments, the second PEG chain is between 2 and 5 kDa. In some embodiments, the second PEG chain is between 3 and 4 kDa. In some embodiments, the second PEG chain is about 3.4 kDa.

[0090] In some embodiments, where the lipid nanoparticle comprises both a first and second PEGylated lipid, the total concentration of the PEGylated lipids (i.e. the concentration of the first PEGylated lipid plus the concentration of the second PEGylated lipid) is 4-15 %mol, 5-14 %mol, 6-13 %mol, 7-12 %mol, 8-12 %mol, 9-11 %mol, 10-11 %mol, or 10-10.5 %mol. In some embodiments, the total concentration of the PEGylated lipids is 10-10.5 %mol. Insome embodiments, the total concentration of PEGylated lipids is about 10 %mol. In some embodiments, the total concentration of PEGylated lipids is about 10.38 %mol.

[0091] In some embodiments, the present invention provides a lipid nanoparticle comprising:

[0092] (a) an ionizable lipid;

[0093] (b) a helper lipid;

[0094] (c) a sterol;

[0095] (d) a PEGylated lipid; and

[0096] (e) a second PEGylated lipid,

[0097] wherein the PEGylated lipid is DSG-PEG comprising a PEG chain of 750 Da or 1 kDa, the PEGylated lipid is at a concentration about 10.29 %mol, the second PEGylated lipid is DSPE-PEG comprising a PEG chain of 3.4 kDa, and the second PEGylated lipid is at a concentration of about 0.09 %mol.

[0098] In some embodiments, the present invention provides a lipid nanoparticle comprising:

[0099] (a) SM102 at a concentration of about 39.98 %mol;

[0100] (b) DSPC at a concentration of about 11.49 %mol;

[0101] (c) cholesterol at a concentration of about 38.15 %mol;

[0102] (d) DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and

[0103] (e) DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

[0104] Ionizable lipid

[0105] As described above, the ionizable lipid is the major component of the lipid nanoparticle. Ionizable lipids are positively charged at pH conditions below the acid-dissociation constant (pKa) of the ionizable lipid allowing the formation of lipid nanoparticles in combination with the other lipid components. Ionizable lipids enable nucleic acid encapsulation through electrostatic interactions with their negatively charged phosphate backbone. At pH conditions around the pKa (e.g. physiological conditions), the majority of the ionizable lipid is uncharged, preventing disruption of cell membranes and / or off-target delivery of the payload, thereby preventing toxicity. Upon uptake by the endosome, the pH conditions below the pKacauses the ionizable lipid to become positively charged. The ionizable lipid then interacts with the anionic lipids of the endosomal bilayer, disrupting the bilayer and lipid nanoparticle triggering the release of the lipid nanoparticle payload into the cell.

[0106] Ionizable lipids accordingly comprise a chemical moiety which is protonated in pH conditions below the pKa and deprotonated in pH conditions around the pKa. For example, the ionizable lipid typically comprises a tertiary amine.

[0107] In some embodiments, the ionizable lipid is at a concentration of 35-51 %mol, 38-48 %mol, 44-51 %mol or 35-45 %mol. In some embodiments, the ionizable lipid is at a concentration of about 40 %mol. In some embodiments, the ionizable lipid is at a concentration of about 39.98 %mol. In some embodiments, the ionizable lipid comprises a tertiary amine. In some embodiments, the ionizable lipid is selected from the group consisting of KL10, KL25, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), 1, 2-di oleyloxy -N,N-dimethylaminopropane (DODMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl -3 - [(9Z, 12Z)-octadeca-9, - 12-di en- 1 -yloxy ]propan- 1 -amine (Octyl-CLinDM A), (2R)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien- l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(313)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,-12-dien-l -yloxy]propan-l -amine (Octyl-CLinDMA (2S)). In some embodiments, the ionizable lipid is Dlin-MC3-DMA, ALC-0315, or SM-102. In some embodiments, the ionizable lipid is Dlin-MC3-DMA or SM-102.

[0108] Helper lipid

[0109] As described above, helper lipids improve the stability of the lipid nanoparticle during storage and circulation. Helper lipids may also aid in the uptake of the lipid nanoparticle into the cell. Helper lipids are typically non-cationic. In some embodiments, the helper lipid may be a lipid of Formula (III):

[0110]

[0111] in which Rp represents a phospholipid moiety and R1 and R2 represent hydrocarbons chains that may be the same or different. In some embodiments, the phospholipid moiety is selected from phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. In some embodiments, the hydrocarbon chain is selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0112] In some embodiments, the helper lipid is at a concentration of 8-12 %mol, 9-11 %mol, or 10-11 %mol. In some embodiments, the helper lipid is at a concentration of about 11.5 %mol. In some embodiments, the helper lipid is at a concentration of about 11.49 %mol. In some embodiments, the helper lipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 , 2-dipalmitoyl-sn-gly cero-3 -phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2-dilinolenoyl-sn-gly cero-3 -phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-gly cero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-g lycero-3-phosphoethanolam ine, 1 ,2-dilinoleoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2-dioleoyl-sn-gly cero-3 -phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, the helper lipid is l,2-Distearoyl-sn-glycerol-3-PC (DSPC).

[0113] Sterol

[0114] As described above, sterols improve the stability of the lipid nanoparticle during storage and circulation, by regulating the fluidity and thickness of the lipid bilayer. Sterols also improve the rigidity of the nanoparticle membrane, reducing leakage of the payload from the lipid nanoparticle. In some embodiments, the sterol is at a concentration of 35-45 %mol, 35-40%mol, or 40-45 %mol. In some embodiments, the sterol is at a concentration of about 38 %mol. In some embodiments, the sterol is at a concentration of about 38.15 %mol. In some embodiments, the sterol is selected from cholesterol, tomatidine, tomatine, ursolic acid, alphatocopherol, fecosterol, sitosterol, campesterol, brassicasterol, stigmasterol, and ergosterol. In some embodiments, the sterol is cholesterol.

[0115] Targeting peptides

[0116] The lipid nanoparticles of the invention may comprise at least one targeting peptide, at least two targeting peptides, or at least three targeting peptides. The targeting peptides of the invention are capable of binding to target molecules presented on the surface of target cells, thereby promoting endocytosis of the lipid nanoparticle by the target cells. For example, the targeting peptides may bind to receptors presented on the cell surface of target cells.

[0117] The targeting peptide is typically attached to either the second PEGylated lipid or to the sterol via a PEG chain. The PEG chain to which the targeting peptide is attached is typically longer than the PEG chain of the first PEGylated lipid. This means that the targeting peptides are presented on the surface of the lipid nanoparticle and can bind to target molecules despite the steric hindrance provided by the PEG corona. By varying the length of the PEG chain to which the targeting peptides are attached the affinity and selectivity of the lipid nanoparticle for the target cell, tissue or organ can be modified.

[0118] The combination of the PEGylated lipid comprising a hydrocarbon chain comprising more than 14 C atoms and the targeting peptides work synergistically to increase the uptake of the lipid nanoparticle of the invention.Thus, in some embodiments, each of the targeting peptides can specifically bind to a target molecule, wherein the target molecule is present on the cell surface of a target cell. In some embodiments, the targeting peptides can bind specifically to a target cell type.

[0119] In some embodiments, the targeting peptides are conjugated (a) to the second PEGylated lipid via the PEG chain or (b) to the sterol via a PEG chain. In this way, the targeting peptides are presented on the surface of the lipid nanoparticle. In some embodiments, the targeting peptides are conjugated via a linker. The length of the linker may be optimised in order to improve binding to the target molecule. In some embodiments, the second PEGylated lipid is conjugated to the targeting peptides via a click chemistry linker. Click chemistry linkers are known in the art and described in, for example, Lang and Chin, Bioorthogonal Reactions for Labeling Proteins, ACS Chemical Biology 9(1), 16-20, 2014 and Chen and Wu, Selectiv chemical labelling of proteins, Org. Biomol. Chem., 14, 5417-5439, 2016. Any suitable click chemistry reaction may be used, for example Diels-Alder cycloadditions (e.g. bicyxlononyne(BCN)-Azide, tetrazine-BCN) and copper-free strain-promoted azide - alkyne click chemistry (SPAAC) such as Azide-Dibenzocyclooctyne (DBCO). In some embodiments, other protein labelling techniques can be used to conjugate targeting peptides to the second PEGylated lipid, for example streptavidin / avidin, EDC / NHS, and maleimide / cysteine conjugation. In some embodiments, the PEGylated lipid is conjugated to the targeting peptides via azide-DBCO click chemistry or maleimide-cysteine linkage.

[0120] In some embodiments, the second PEGylated lipid comprises a DBCO moiety. In some embodiments, the DBCO moiety is conjugated to the second PEGylated lipid via a PEG chain.

[0121] In some embodiments, the targeting peptides comprise fewer than 50 amino acids, fewer than 45 amino acids, fewer than 40 amino acids, fewer than 35 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, or fewer than 20 amino acids. In some embodiments, the at least one targeting peptide comprises between 5 and 50 amino acids, between 5 and 45 amino acids, between 5 and 40 amino acids, between 5 and 35 amino acids, between 5 and 30 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, or between 5 and 15amino acids. In some embodiments, the targeting peptides comprise between 5 and 20 amino acids. In some embodiments, the targeting peptides comprise fewer than 20 amino acids. In some embodiments, the targeting peptide comprise about 10 amino acids.

[0122] In some embodiments, the targeting peptides are not antibodies or antibody fragments. It can be advantageous for the targeting peptides to have a low affinity (i.e. lower than a typical antibody) in order to allow for superselectivity. In some embodiments, the targeting peptides have an affinity (Kd) greater than 1 nM, greater than 10 nM, greater than 100 nM, or greater than 1 pM. Affinity may be measured using any suitable technique known in the art, for example surface plasmon resonance (SPR).

[0123] In some embodiments, the lipid nanoparticle comprises at least two targeting peptides, wherein the at least two targeting peptides specifically bind to different target molecules on the same cell or to different epitopes on the same target molecule. The use of at least two targeting peptides may increase the selectivity of the lipid nanoparticle for a target cell, tissue, or organ.

[0124] In some embodiments, the lipid nanoparticle comprises at least three targeting peptides, wherein the at least three targeting peptides specifically bind to different target molecules on the same cell or to different epitopes on the same target molecule. The use of at least three targeting peptides may increase the selectivity of the lipid nanoparticle for a target cell, tissue, or organ.

[0125] The molecular ratio of the targeting peptides refers to the molecular ratio of the peptides in the composition used in the formulation of the lipid nanoparticle. The molecular ratio correlates to the ratio of targeting peptides presented on the surface of the lipid nanoparticle. In some embodiments, the molecular ratio of the at least two or at least three targeting peptides increases the selectivity of the lipid nanoparticle for the target cell type.

[0126] The properties of the lipid nanoparticle may be tweaked in order to increase the selectivity of the lipid nanoparticle for a given cell type. For example, the hydrodynamic radius of the lipid nanoparticle, molecular ratio of the targeting peptides, number of the targeting peptides,binding affinity of the targeting peptides for their targets and PEG and / or linker length can all be varied to tune the selectivity of the lipid nanoparticle. Xiaohe Tian et al., On the design of precision nanomedicines. Sci. Adv.6, eaat0919(2020).DOI:10.1126 / sciadv.aat0919 describes how computer modelling can be used to predict super selectivity of a multivalent binding construct for a given target cell. For example, the use of at least two or at least three low affinity targeting peptides can provide super selectivity, wherein low affinity corresponds to a low binding energy. For example, the targeting peptide may have an affinity for their respective targets of between -8 and -100 kaT, between -10 and -50 kaT, or preferably between -10 and -20 U '. In some embodiments of the present invention, the lipid nanoparticle is super selective for a target cell, tissue or organ.

[0127] Reduced uptake by liver cells

[0128] As described above, the lipid nanoparticles of the invention have reduced uptake by the liver and spleen due to the presence of a PEGylated lipid comprising a hydrocarbon chain comprising more than 14 C atoms. In some embodiments, the lipid nanoparticle has reduced uptake by liver and / or spleen cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length. An “equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length” is a lipid nanoparticle that is the same as the test lipid nanoparticle, but for the length of the hydrocarbon chain.

[0129] In some embodiments, the lipid nanoparticle has at least 2 times, at least 3 times, at least 4 times, or at least 5 times reduced uptake by liver and / or spleen cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length.

[0130] Uptake by target cells may be measured by administering the lipid nanoparticle comprising FLuc mRNA to a mouse via tail vein injection and measuring luciferase expression post injection. Uptake by target cells may be measured as described in the Examples.

[0131] Increased uptake by target cells, tissues or organsAs described above, the lipid nanoparticles of the invention remain in circulation longer due to the presence of a PEGylated lipid comprising a hydrocarbon chain comprising more than 14 C atoms. This results in an increase in uptake of the lipid nanoparticle by target cells, tissues and / or organs of interest. For example, the target organ may be the heart, lungs, kidneys, large intestine, small intestine, brain, pancreas, skin, bladder, bones, ovaries, testes, glands, lymph nodes, spinal cord, or eyes. The target tissues may be muscle tissue, heart tissue, bone marrow, skin tissue, epithelial tissue, kidney tissue, brain tissue, endothelial tissue, bladder tissue, ovarian tissue, pancreatic tissue or blood. The target cells may be lung cells, stem cells, mesenchymal cells, muscle cells, nerve cells, stem cells, bone cells, hematopoietic cells, fat cells, skin cells, epithelial cells, B cells, T cells, leukocytes, or sex cells. The target cells may be cancer cells.

[0132] In order to determine whether a lipid nanoparticle is specific for a given target cell type, tissue or organ, the skilled person can inject the lipid nanoparticle comprising a DNA barcode into a mouse, harvest cell, tissue, or organ samples 4, 8 or 24 hours post administration, and measure the levels of the DNA barcode in the samples by quantitative sequencing. If the DNA barcode appears in the target cell, tissue, or organ at a higher level than other non-target cells, tissue, or organs then the lipid nanoparticle is specific for the target cell, tissue, or organ. The method may be carried out as described in the Examples.

[0133] In some embodiments, the lipid nanoparticle has an increased uptake by target cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length.

[0134] In some embodiments, uptake by target cells is at least 2 times, at least 3 times, at least 4 times, at least 5 times higher than uptake of an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length. In some embodiments, uptake by target cells is at least 2 times higher an uptake of an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length.In some embodiments, the lipid nanoparticle has at least 70%, at least 80%, at least 90%, or at least 100% uptake by target cells compared to a lipid nanoparticle comprising:

[0135] (a) SM102 at a concentration of about 39.98 %mol;

[0136] (b) DSPC at a concentration of about 11.49 %mol;

[0137] (c) cholesterol at a concentration of about 38.15 %mol;

[0138] (d) DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and

[0139] (e) DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

[0140] In some embodiments, the lipid nanoparticle has an increased uptake by target cells compared to an equivalent lipid nanoparticle lacking the targeting peptides.

[0141] In some embodiments, the uptake by target cells is at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher than uptake of an equivalent lipid nanoparticle lacking the targeting peptides.

[0142] Uptake by target cells can be measured in vitro by mRNA encoding reporter proteins such as luciferase, green fluorescent protein (GFP), and mCherry. For LNPs comprising siRNA cargos reporter protein knockdown assays are commonly performed in cells which have been engineered to constitutively express the reporter protein. In vivo luminescent signals from luciferase expression can be assessed by an imaging system to inform on biodistribution. To evaluate both mRNA transfection efficiency and LNP accumulation, reporter-loaded LNPs can be labelled with near-infrared dyes. For cellular-level analysis, flow cytometry can be performed on tissues after delivering fluorescent protein mRNAs or NIR dye-stained LNPs.

[0143] Uptake by target cells may be measured by administering the lipid nanoparticle comprising FLuc mRNA to a mouse via tail vein injection and measuring luciferase expression post injection. Uptake by target cells may be measured by in vivo bioluminescence imaging (BLI) or as described in the Examples.

[0144] PayloadThe lipid nanoparticles of the invention can be used to deliver a payload to target cells, tissues or organs. The payload may be any suitable payload. For example, the lipid nanoparticle may be used as a gene therapy vector for delivery of a therapeutic transgene. Alternatively, the lipid nanoparticle may be used to deliver cancer treatments to cancer cells, such as cytotoxic molecules or radioligands. The lipid nanoparticles may be used to deliver diagnostic labels. The lipid nanoparticle may be used to deliver therapeutic molecules. The lipid nanoparticles may be used to deliver a gene editing system, for example a CRISPR-Cas gene editing system comprising a Cas nuclease, a guide RNA and optionally a DNA molecule for insertion into the genome.

[0145] In some embodiments, the lipid nanoparticle further comprises a payload. In some embodiments, the payload is a nucleic acid, such as RNA, siRNA, mRNA, antisense oligonucleotides or DNA. In some embodiments, the payload comprises an RNA molecule, a DNA molecule, a polypeptide, a cytotoxic molecule, a radioligand, an antibiotic, an antiviral, an immunogen or a therapeutic transgene. In some embodiments, the payload comprises a component of a gene editing system, optionally wherein the component comprises a gRNA, a DNA molecule, a Cas nuclease, a Cas nickase, a deaminase, a cytosine base editor, and / or an adenine base editor.

[0146] RNA molecules which can be used as payloads include mRNA, miRNA, aiRNA and siRNA.

[0147] Methods of preparing a lipid nanoparticle

[0148] Lipid nanoparticles of the invention may be produced according to standard methods known in the art. Lipid nanoparticles may be produced via ethanol injection or microfluidic mixing. For example, Lipid-Based Nanoparticles for Drug / Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development Meenu Mehta, Thuy Anh Bui, Xinpu Yang, Yagiz Aksoy, Ewa M. Goldys, and Wei Deng ACS Materials Au 2023 3 (6), 600-619 DOI: 10.1021 / acsmaterialsau.3c00032 describes methods of preparing lipid nanoparticles. Lipid nanoparticles of the invention may be prepared according to the methods described in the Examples.Targeting peptides may be conjugated to the lipid nanoparticles of the invention after they have been prepared. For example, targeting peptides may be conjugated to the second PEGylated lipid via azide-DBCO click-chemistry or maleimide linkage. For example, azide and dibenzocyclooctyne (DBCO) groups can be present on the second PEG chain and targeting peptide, respectively, or vice versa. Upon addition of the targeting peptide to the lipid nanoparticle comprising the second PEG chain, the azide and DBCO will selectively react, thereby stably conjugating the targeting peptide to the second PEG chain.

[0149] Alternatively, the second PEG chain may comprise a maleimide group and the targeting peptide may comprise a cysteine residue. Upon addition of the targeting peptide to the lipid nanoparticle comprising the second PEG chain, the maleimide and cysteine residue will selectively react, thereby stably conjugating the targeting peptide to the second PEG chain.

[0150] Pharmaceutical composition

[0151] In some embodiments, there is provided a composition comprising the lipid nanoparticle of the invention and a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable" means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered.

[0152] Any suitable pharmaceutically acceptable carrier, diluent or excipient can be used in the preparation of a pharmaceutical composition (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).

[0153] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g. saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluids), microemulsions, liposomes, or other ordered structure suitable to accommodate a high product concentration (e.g. microparticles or nanoparticles). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol or sorbitol, or salts such as sodium chloride in the composition.

[0154] Methods of medical treatment / medical uses

[0155] The terms “treatment” , “treating" and the like refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptoms thereof from appearing or worsening and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease.

[0156] The lipid nanoparticles of the invention may be used for delivering a therapeutic payload to a target cell, tissue or organ. The skilled person will therefore understand that the lipid nanoparticles may be useful in the treatment of a large number of diseases. For example, the lipid nanoparticles may be used as a gene therapy vector, drug vector, vaccine or cancer treatment.

[0157] In some embodiments, the present invention provides a method of medical treatment comprising administering the lipid nanoparticle of the invention to a subject. In some embodiments, the method of medical treatment is a method of gene therapy. In some embodiments, the method is a method of treating cancer.

[0158] The invention provides methods involving administering lipid nanoparticles or pharmaceutical compositions including the same. Compositions of the invention, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition and / or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of the administration; the particular composition; the mode of administration; and the like. Compositions in accordance with the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of a composition of the present disclosure will be decided by an attending physician within the scope of soundmedical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the payloads employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.

[0159] A lipid nanoparticle or composition may be administered by any route. In some embodiments, compositions of the invention, including prophylactic, diagnostic, or imaging compositions including one or more nanoparticle compositions of the invention, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, trans- or intra-dermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, a composition may be administered intravenously, intramuscularly, intradermally, or subcutaneously.ASPECTS OF THE INVENTION

[0160] 1. A lipid nanoparticle comprising:

[0161] (a) an ionizable lipid;

[0162] (b) a helper lipid;

[0163] (c) a sterol; and

[0164] (d) a PEGylated lipid,

[0165] wherein:

[0166] (i) the PEGylated lipid comprises a hydrocarbon chain comprising more than 14 C atoms; and / or

[0167] (ii) the PEGylated lipid is at a concentration of at least 3 %mol.

[0168] 2. The lipid nanoparticle of aspect 1, wherein the hydrocarbon chain is a branched or linear alkyl, alkenyl, or alkynyl chain.

[0169] 3. The lipid nanoparticle of aspect 1 or aspect 2, wherein the hydrocarbon chain comprises at least 16 C atoms.

[0170] 4. The lipid nanoparticle of any one of the preceding aspects, wherein the hydrocarbon chain comprises at least 18 C atoms.

[0171] 5. The lipid nanoparticle of any one of the preceding aspects, wherein the hydrocarbon chain is 16 or 18 C atoms in length.

[0172] 6. The lipid nanoparticle of any one of the preceding aspects, wherein the ionizable lipid is at a concentration of 35-51 %mol, optionally about 40 %mol.

[0173] 7. The lipid nanoparticle of any one of the preceding aspects, wherein the ionizable lipid is at a concentration of about 39.98 %mol.8. The lipid nanoparticle of any one of the preceding aspects, wherein the ionizable lipid comprises or is Dlin-MC3-DMA, ALC-0315, or SM-102, optionally wherein the ionizable lipid comprises or is SM-102.

[0174] 9. The lipid nanoparticle of any one of the preceding aspects, wherein the helper lipid is at a concentration of 8-12 %mol, optionally about 11 %mol.

[0175] 10. The lipid nanoparticle of any one of the preceding aspects, wherein the helper lipid is at a concentration of about 11.49 %mol.

[0176] 11. The lipid nanoparticle of any one of the preceding aspects, wherein the helper lipid comprises oris l,2-Distearoyl-sn-glycerol-3-PC (DSPC).

[0177] 12. The lipid nanoparticle of any one of the preceding aspects, wherein the sterol is at a concentration of 35-45 %mol, optionally about 38 %mol.

[0178] 13. The lipid nanoparticle of any one of the preceding aspects, wherein the sterol is at a concentration of about 38.15 %mol.

[0179] 14. The lipid nanoparticle of any one of the preceding aspects, wherein the sterol comprises or is cholesterol.

[0180] 15. The lipid nanoparticle of any one of the preceding aspects, wherein the PEGylated lipid is at a concentration of 5-15 %mol, optionally about 10 %mol.

[0181] 16. The lipid nanoparticle of any one of the preceding aspects, wherein the PEGylated lipid is at a concentration of about 10.29 %mol or about 10.38 %mol.

[0182] 17. The lipid nanoparticle of any one of the preceding aspects, wherein the PEGylated lipid comprises or is distearoyl-glycerol-PEG (DSG-PEG) or distearoyl-glycero-3-phosphoethanolamine-PEG (D SPE-PEG) .18. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle has reduced uptake by liver and / or spleen cells compared to an equivalent lipid nanoparticle (i) comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length and / or (ii) comprising a PEGylated lipid at a concentration of less than 3 %mol.

[0183] 19. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle has at least 2 times, at least 3 times, at least 4 times, or at least 5 times reduced uptake by liver and / or spleen cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid (i) comprising a hydrocarbon chain that is 14 C atoms in length and / or (ii) comprising a PEGylated lipid at a concentration of less than 3 %mol.

[0184] 20. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle has an increased uptake by target cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a lipid chain that is 14 C atoms in length.

[0185] 21. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle has at least 70%, at least 80%, at least 90%, or at least 100% uptake by target cells compared to a lipid nanoparticle comprising:

[0186] (a) SM102 at a concentration of about 39.98 %mol;

[0187] (b) DSPC at a concentration of about 11.49 %mol;

[0188] (c) cholesterol at a concentration of about 38.15 %mol;

[0189] (d) DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and

[0190] (e) DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

[0191] 22. The lipid nanoparticle of aspect 20 or aspect 21, wherein the target cells are lung cells, stem cells, mesenchymal cells, muscle cells, nerve cells, stem cells, bone cells, hematopoietic cells, fat cells, skin cells, epithelial cells, B cells, T cells, leukocytes, or sex cells.

[0192] 23. The lipid nanoparticle of any one of aspects 20-22, wherein the target cells are cancer cells.24. The lipid nanoparticle of any one of aspects 18 to 23, wherein uptake by cells is measured by administering the lipid nanoparticle comprising FLuc mRNA to a mouse via tail vein injection and measuring luciferase expression post injection in the cells.

[0193] 25. The lipid nanoparticle of any one of aspects 18 to 24, wherein uptake by target cells is at least 2 times, at least 3 times, at least 4 times, at least 5 times higher than uptake of an equivalent lipid nanoparticle (i) comprising a PEGylated lipid comprising a lipid chain that is 14 C atoms in length and / or (ii) comprising a PEGylated lipid at a concentration of less than 3 %mol.

[0194] 26. The lipid nanoparticle of any one of the preceding aspects, wherein the PEGylated lipid comprises a PEG chain of between 0.5 and 2 kDa, optionally about 750 Da or about 1 kDa.

[0195] 27. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle comprises a second PEGylated lipid comprising a second hydrocarbon chain, wherein the hydrocarbon chain comprises at least 14 C atoms.

[0196] 28. The lipid nanoparticle of aspect 27, wherein the second hydrocarbon chain is a branched or linear alkyl, alkenyl, or alkynyl chain.

[0197] 29. The lipid nanoparticle of aspect 27 or aspect 28, wherein the second hydrocarbon chain comprises at least 16 C atoms.

[0198] 30. The lipid nanoparticle of any one of the aspects 27 to 29, wherein the second hydrocarbon chain comprises at least 18 C atoms.

[0199] 31. The lipid nanoparticle of any one of aspects 27 to 30, wherein the second hydrocarbon chain is 16 or 18 C atoms in length.

[0200] 32. The lipid nanoparticle of any one of aspects 27 to 31, wherein the second PEGylated lipid comprises a PEG chain that is between 1 kDa and 5 kDa, optionally about 3.4 kDa.33. The lipid nanoparticle of any one of aspects 27 to 32, wherein the second PEGylated lipid comprises or is DSG-PEG or DSPE-PEG.

[0201] 34. The lipid nanoparticle of any one of aspects 27 to 33, wherein the second PEGylated lipid is at a concentration of:

[0202] (a) 0-15 %mol, 0-12 %mol, 0-10 %mol, 0-9 %mol, 0-8 %mol, 0-7 %mol, 0-6 %mol, 0- 5 %mol, 0-4 %mol, 0-3 %mol, 0-2 %mol, 0-1 %mol, 0-0.5 %mol or 0-0.15 %mol; (b) 0.05-0.15 %mol; or

[0203] (c) 0.09 %mol.

[0204] 35. The lipid nanoparticle of any one of aspects 27 to 34, wherein the total concentration of the PEGylated lipids is 5-15 %mol, optionally about 10 %mol.

[0205] 36. The lipid nanoparticle of any one of aspects 27 to 35, wherein the total concentration of the PEGylated lipids is about 10.38 %mol.

[0206] 37. The lipid nanoparticle of any one of aspects 27 to 36, wherein the second PEGylated lipid comprise a DBCO moiety.

[0207] 38. The lipid nanoparticle of aspect 37, wherein the DBCO moiety is conjugated to the second PEGylated lipid via a PEG chain.

[0208] 39. A lipid nanoparticle comprising:

[0209] (a) an ionizable lipid;

[0210] (b) a helper lipid;

[0211] (c) a sterol;

[0212] (d) a PEGylated lipid; and

[0213] (e) a second PEGylated lipid,

[0214] wherein the PEGylated lipid is DSG-PEG comprising a PEG chain of 750 Da or 1 kDa, the PEGylated lipid is at a concentration about 10.29 %mol, the second PEGylated lipid is DSPE-PEG comprising a PEG chain of 3.4 kDa, and the second PEGylated lipid is at a concentration of about 0.09 %mol.

[0215] 40. A lipid nanoparticle comprising:

[0216] (a) an ionizable lipid;

[0217] (b) a helper lipid;

[0218] (c) a sterol;

[0219] (d) a PEGylated lipid; and

[0220] (e) a second PEGylated lipid,

[0221] wherein

[0222] (i) the ionizable lipid is SM102 at a concentration of about 39.98 %mol;

[0223] (ii) the helper lipid is DSPC at a concentration of about 11.49 %mol;

[0224] (iii) the sterol is cholesterol at a concentration of about 38.15 %mol;

[0225] (iv) the PEGylated lipid is DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and

[0226] (v) the second PEGylated lipid is DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

[0227] 41. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle comprises at least one targeting peptide, at least two targeting peptides, or at least three targeting peptides.

[0228] 42. The lipid nanoparticle of aspect 41, wherein the targeting peptides are conjugated (a) to the second PEGylated lipid via a PEG chain or (b) to the sterol via a PEG chain.

[0229] 43. The lipid nanoparticle of aspect 41 or aspect 42, wherein the second PEGylated lipid is conjugated to the targeting peptides via azide-DBCO click-chemistry or maleimide-cysteine linkage.

[0230] 44. The lipid nanoparticle of any one of aspects 41 to 43, wherein each of the targeting peptides can specifically bind to a target molecule, wherein the target molecule is present on the cell surface of a target cell.45. The lipid nanoparticle of any one of aspects 41 to 44, wherein the targeting peptides can bind specifically to a target cell, tissue or organ, optionally wherein the target cell or target tissue is not a liver or spleen cell or liver or spleen tissue and / or the organ is not the liver or spleen.

[0231] 46. The lipid nanoparticle of any one of aspects 41 to 45, wherein the targeting peptides comprise fewer than 20 amino acids.

[0232] 47. The lipid nanoparticle of any one of aspects 41 to 46, wherein the targeting peptides comprise between 5 and 20 amino acids.

[0233] 48. The lipid nanoparticle of any one of aspects 41 to 47, wherein the targeting peptides are not antibodies or antibody fragments.

[0234] 49. The lipid nanoparticle of any one of aspects 41 to 48, wherein the targeting peptides have a Kd greater than 1 nM, greater than 10 nM, greater than 100 nM, or greater than 1 pM.

[0235] 50. The lipid nanoparticle of any one of aspects 41 to 49, wherein the lipid nanoparticle has an increased uptake by target cells compared to an equivalent lipid nanoparticle lacking the targeting peptides.

[0236] 51. The lipid nanoparticle of aspect 50, wherein the uptake is measured by administering the lipid nanoparticle comprising FLuc mRNA to a mouse via tail vein injection and measuring luciferase expression post injection in the target cells.

[0237] 52. The lipid nanoparticle of aspect 50 or aspect 51, wherein uptake by target cells is at least 2 times higher, at least 3 times higher, at least 4 times higher or at least 5 times higher than uptake of an equivalent lipid nanoparticle lacking the targeting peptides.

[0238] 53. The lipid nanoparticle of any one of aspects 50 to 52, wherein the lipid nanoparticle comprises at least two or at least three targeting peptides, wherein the at least two or at leastthree targeting peptides specifically bind to different target molecules or to different epitopes on the same target molecule.

[0239] 54. The lipid nanoparticle of aspect 53, wherein the molecular ratio of the at least two or at least three targeting peptides increases the selectivity of the lipid nanoparticle for the target cell type.

[0240] 55. The lipid nanoparticle of any one of the preceding aspects, wherein the lipid nanoparticle further comprises a payload.

[0241] 56. The lipid nanoparticle of aspect 55, wherein the payload comprises a nucleic acid, optionally an RNA, an siRNA, an mRNA, an antisense oligonucleotide, a gRNA or a DNA.

[0242] 57. The lipid nanoparticle of aspect 55 or aspect 56, wherein the payload comprises an RNA molecule, a DNA molecule, a polypeptide, a cytotoxic molecule, a radioligand, an antibiotic, an antiviral, an immunogen, or a therapeutic transgene.

[0243] 58. The lipid nanoparticle of any one of aspects 55 to 57, wherein the payload comprises a component of a gene editing system, optionally wherein the component comprises a gRNA, a Cas nuclease, a Cas nickase, a deaminase, a cytosine base editor, and / or an adenine base editor.

[0244] 59. The lipid nanoparticle of any one of aspects 55 to 58, wherein the payload has a therapeutic effect in a target cell, optionally wherein the target cell is not a liver cell or spleen cell.

[0245] 60. The lipid nanoparticle of any one of the preceding aspects, wherein the hydrodynamic radius of the lipid nanoparticle is between 50 nm and 100 nm, preferably wherein the hydrodynamic radius is about 50nm, about 60 nm or about 70 nm.

[0246] 61. The lipid nanoparticle of any one of the preceding aspects for use in a method of medical treatment, wherein the treatment comprises administering the lipid nanoparticle to a subject.62. The lipid nanoparticle for use according to aspect 61, wherein the method of medical treatment is a method of gene therapy, optionally wherein the lipid nanoparticle comprises a therapeutic transgene.

[0247] 63. The lipid nanoparticle for use according to aspect 61, wherein the method of medical treatment is a method of treating cancer.

[0248] 64. A method of producing a polypeptide in a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of aspects 1 to 60, wherein the lipid nanoparticle comprises a nucleic acid encoding the polypeptide.

[0249] 65. A method of delivering a payload to a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of aspects 1 to 60, wherein the lipid nanoparticle comprises the payload.EXAMPLES

[0250] Example 1

[0251] Lipid nanoparticle formulation

[0252] LNPs encapsulating DNA barcodes (b-DNA) were prepared as follows. An ethanol phase containing SM-102 lipid, DSPC, cholesterol, and PEGylated lipid, with molar ratios as set out in Tables 1 to 4 below. The aqueous phase was composed of b-DNAs dissolved in 10 mM sodium acetate buffer at pH 4.5, with a lipid to nucleic acid ratio of 39.8 (w / w). LNPs were formulated by ethanol injection of the lipid solution into the nucleic acid-containing acetate buffer at a volume ratio of 1 :4 (v / v). The resulting LNPs were buffer swapped to PBS 3 times using an Amicon Ultra-15 centrifulgal filter 100 kDa MWCO by spinning at 4 000 xg for 20 min, each time.

[0253]

[0254] Table 1: Details of LNP formulation

[0255]

[0256] Table 2: LNP formulations with variable amounts of PEGylated lipids

[0257]

[0258] Table 3: LNP formulations with shorter hydrocarbon chain and variable amounts of PEGylated lipid

[0259]

[0260]

[0261] Table 4: LNP formulations with variable amounts of DBCO PEGylated lipid

[0262] Example 2

[0263] Functionalisation of LNPs with targeting peptides

[0264] LNPs of formulations 1-7 as well as controls 1 and 2 as set out in Table 5 were prepared according to the method set out in Example 1. Each of formulations 1 to 7 were reacted with a different set of targeting peptides. LNPs in PBS were reacted overnight at 4°C with equimolar mixtures of HER2 targeting peptides modified with azidopentanoic acid with a 3-fold molar excess. Unreacted peptides were removed using Amicon Ultra- 15 centrifulgal filter 100 kDa MWCO by spinning at 4000 xg for 20 min. The sample was further concentrated and stored in PBS. The final sample is filtered through a 0.22 pm filter.

[0265] Example 3

[0266] Measuring Hydrodynamic radius of LNPs

[0267] LNPs of formulations 1-7 as well as controls 1 and 2 as set out in Table 5 were prepared according to the method set out in Example 1. Each of formulations 1 to 7 were functionalised according to the method set out in Example 2. A Zetasizer Nano is used to measure the Z-average hydrodynamic radius and poly dispersity index (PDI). The hydrodynamic radius of the LNPs is given in Table 6 and Figure 2. The results show that all formulations tested were able to form LNPs of a suitable size, acceptable PDI and low inter-batch variability.

[0268]

[0269] Table 5: LNP formulations

[0270]

[0271]

[0272] Table 6: Size and poly dispersity results as measured by dynamic light scattering (DLS)

[0273] Example 4

[0274] LNPs with variable PEGylated lipid-DBCO concentration

[0275] LNPs with compositions as set out in Table 7 were prepared according to the method set out in Example 1. A Zetasizer Nano is used to measure the Z-average hydrodynamic radius and poly dispersity index (PDI). The hydrodynamic radius of the LNPs is given in Table 8 and Figure 3. The results show that all formulations tested were able to form LNPs.

[0276] Table 7: LNP formulations

[0277]

[0278]

[0279]

[0280] Table 8: Size and poly dispersity results as measured by dynamic light scattering (DLS)

[0281] Example 5

[0282] Measuring nucleic acid encapsulation efficiency

[0283] Encapsulation efficiency and total encapsulated barcoded DNA (bDNA) in LNP formulations, including formulations 1-7 and controls 1 and 2, were determined by a modified RediPlate RiboGreen (ThermoFisher) assay where the calibration curve with standards of known concentration was performed with samples of pooled and quantified bDNA, instead of manufacturer supplied RNA standards. Samples and standards were prepared following the manufacturer’s instructions, i.e. 180 pL of the RediPlate (ThermoFisher) Tris-EDTA buffer is added to the plate, followed by 20 pL of standard or LNP sample.. Samples and standards were then incubated for 5 to 20 minutes and fluorescence read with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. 50 pL of 10% Triton X-100 in Tris-EDTA buffer was then added to both the standards and samples, disrupting the LNPs, and fluorescence immediately read again with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Both samples and standards were tested in triplicate. Encapsulation efficiency (EE) was calculated as follows:

[0284]

[0285] where [Cl] is the bDNA concentration without Triton X-100, and [C2] is the bDNA concentration with Triton X-100, as interpolated from the calibration curve of bDNA standards performed at the same time.

[0286]

[0287] Table 9: Encapsulation efficiency and concentration of encapsulated nucleic acid, as determined by the modified RediPlate Ribogreen (ThermoFisher) assay. Fluorescence results measured by the GloMax Discover plate reader before and after LNP disruption with Triton X-100. Mean fluorescence was interpolated from calibration curves with known concentrations of the same nucleic acid. Results recorded after LNP production.

[0288] Example 6

[0289] In vivo barcoded LNP delivery

[0290] Mice are inoculated with HER2-positive tumours prior to administration of LNPs of formulations 1-7 and controls 1 and 2 as described in Examples 1 to 3 and 5. Mice are dosed with P-Oestradiol in drinking water beginning 7 days prior to inoculation. Mice are randomised into three groups once tumours reach a mean volume of 140-200 mm3(roughly 10 days after inoculation). A sample of each LNP formulation is administered to each of the three groups of mice following randomisation, and each group is sacrificed at a different timepoint.

[0291] A pool of different b-DNA LNPs is prepared right before injection by mixing together equal parts of the previously prepared b-DNA LNPs. The LNP pool is administered systemically to mice via tail vein injection at a dosage of 0.4 mg kg '. Free DNA is used as a further control. In-life blood samples are collected every 30 min to determine b-DNA LNP circulation times. Tissues samples (liver, spleen and tumour) are harvested 4, 8 and 24 hours post-administration,snap-frozen in liquid nitrogen, disrupted into powder and stored at -20 °C until further analysed.

[0292] Example 7

[0293] NGS library pool preparation

[0294] To prepare samples, approximately 30 pg of dry homogenized sample is suspended in a DNA-stabilizing lysis buffer and proteinase K, to remove protein and RNA contaminants. Following this step, the barcoded DNA (b-DNA) is then amplified by PCR. Libraries are combined in equimolar amounts to produce a library pool for next-generation sequencing (NGS), which is stored at -20 °C until sequencing.

[0295] Example 8

[0296] Counter screening of FLuc mRNA delivery in vivo

[0297] After analysing in vivo b-DNA delivery, 4 LNPs, comprising LNPs with C18 / 14-PEG lipids, with and without targeting peptides, are selected for counter-screening by delivering FLuc mRNA. Mice were administered FLuc mRNA-LNP via tail vein injection. Luciferase expression is evaluated using an IVIS Spectrum imaging system post-injection. Mice are then injected with D-luciferin at a dose of 150 mg / kg by intraperitoneal (i.p.) injection. Bioluminescence is quantified by measuring total flux in the region of interest. Ex vivo imaging is performed on heart, liver, spleen, and tumour after resection.

[0298] Example 9

[0299] Lipid nanoparticle formulation

[0300] LNPs encapsulating DNA barcodes were prepared as described in Example 1 and Table 1, and compared to the known LNP used in Onpattro™ (makeup shown in Table 10). The hydrodynamic radius of these LNPs was measured as described in Example 3 (Table 11 and Figure 4). Encapsulation efficiency was measured according to the method described in Example 5 (Table 12).

[0301]

[0302]

[0303] Table 10: Details of Onpattro™ LNP formulation

[0304]

[0305] Table 11: Size and polydispersity results as measured by dynamic light scattering (DLS) o: samples in Figure 1 and 2.

[0306]

[0307] Table 12: Encapsulation efficiency and concentration of encapsulated nucleic acid, as determined by the modified RediPlate Ribogreen (ThermoFisher) assay. Fluorescence results measured by the GloMax Discover plate reader before and after LNP disruption with Triton X-100. Mean fluorescence was interpolated from calibration curves with known concentrations of the same nucleic acid.

[0308] Example 10

[0309] In vivo barcoded LNP deliveryMice were inoculated with HER2 -positive tumours prior to administration of Bare and Onpattro™ as described in Example 9 and Tables 1 and 10, respectively. Mice were dosed with P-Oestradiol in drinking water beginning 7 days prior to inoculation. Mice were randomised once tumours reach a mean volume of 140-200 mm3 (roughly 10 days after inoculation). A sample of each LNP was administered to the mice following randomisation, and each group is sacrificed.

[0310] A pool of different b-DNA LNPs was prepared right before injection by mixing together equal parts of the previously prepared b-DNA LNPs. The LNP pool was administered systemically to mice via tail vein injection at a dosage of 0.4 mg kg-1. Tissues samples (liver, spleen and tumour) were harvested post-administration, snap-frozen in liquid nitrogen, disrupted into powder and stored at -20 °C until further analysed. Results show an increased ratio of DNA barcodes delivered to the tumour compared to the liver for the Bare formulation of Table 1 compared to the Onpattro™ formulation (Figure 5).

[0311] Example 11

[0312] Functionalisation of LNPs with targeting peptides

[0313] LNPs of formulations NG005, NG006, and NG008 as well benchmark LNP as set out in Table 13 were prepared according to the method set out in Example 1. Each of formulations were reacted with a different set of targeting peptides, as per Table 14. LNPs in PBS were reacted overnight at 4°C with equimolar mixtures of HER2 targeting peptides modified with azidopentanoic acid with a 3 -fold molar excess. Unreacted peptides were removed using Amicon Ultra-15 centrifugal filter 100 kDa MWCO by spinning at 4000 xg for 20 min. The sample was further concentrated and stored in PBS. The final sample is filtered through a 0.22 pm filter.

[0314] Example 12

[0315] Measuring Hydrodynamic radius of LNPs

[0316] LNPs and controls as set out in Table 13 were prepared according to the method set out in Example 1. Each of formulation was functionalised according to the method set out in Example 11. A Zetasizer Nano is used to measure the Z-average hydrodynamic radius and poly dispersity index (PDI). The hydrodynamic radius of the LNPs is given in Table 15 andFigure 6. The results show that all formulations tested were able to form LNPs of a suitable size, acceptable PDI and low inter-batch variability.

[0317]

[0318] Table 13: LNP formulations

[0319]

[0320] Table 14: Peptides functionalised in each LNP

[0321]

[0322]

[0323]

[0324] Table 15: Size and poly dispersity results as measured by dynamic light scattering (DLS)

[0325] Example 13

[0326] Measuring nucleic acid encapsulation efficiency

[0327] Encapsulation efficiency and total encapsulated barcoded DNA (bDNA) in LNP formulations was determined by a modified RediPlate RiboGreen (ThermoFisher) assay where the calibration curve with standards of known concentration was performed with samples of pooled and quantified bDNA, instead of manufacturer supplied RNA standards. Samples and standards were prepared following the manufacturer’s instructions, i.e. 180 pL of the RediPlate (ThermoFisher) Tris-EDTA buffer is added to the plate, followed by 20 pL of standard or LNP sample. Samples and standards were then incubated for 5 to 20 minutes and fluorescence read with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. 50 pL of 10% Triton X-100 in Tris-EDTA buffer was then added to both the standards and samples, disrupting the LNPs, and fluorescence immediately read again with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Both samples and standards were tested in triplicate. Encapsulation efficiency (EE) was calculated as follows:

[0328] 100

[0329]

[0330] where [Cl] is the bDNA concentration without Triton X-100, and [C2] is the bDNA concentration with Triton X-100, as interpolated from the calibration curve of bDNA standards performed at the same time.

[0331]

[0332] Table 16: Encapsulation efficiency and concentration of encapsulated nucleic acid, as determined by the modified RediPlate Ribogreen (ThermoFisher) assay. Fluorescence results measured by the GloMax Discover plate reader before and after LNP disruption with Triton X-100. Mean fluorescence was interpolated from calibration curves with known concentrations of the same nucleic acid. Results recorded after LNP production.

[0333] Example 14

[0334] In vivo barcoded LNP delivery

[0335] Mice were inoculated with HER2 -positive tumours prior to administration of pooled LNPs as described in Examples 11 to 13. Mice were dosed with P-Oestradiol in drinking water beginning 7 days prior to inoculation. Mice were randomised into three groups once tumours reach a mean volume of 140-200 mm3(roughly 10 days after inoculation). A sample of each LNP formulation was administered to each of the three groups of mice following randomisation, and each group is sacrificed.

[0336] A pool of different b-DNA LNPs was prepared right before injection by mixing together equal parts of the previously prepared b-DNA LNPs. The LNP pool was administered systemically to mice via tail vein injection at a dosage of 0.4 mg kg1. In-life blood samples were collected every 30 min to determine b-DNA LNP circulation times. Tissues samples (liver, spleen and tumour) were harvested 24 hours post-administration, snap-frozen in liquid nitrogen, disrupted into powder and stored at -20 °C until further analysed. Results of the tumour / liver ratio at 24h can be seen in Figure 7.

Claims

CLAIMS1. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) a helper lipid;(c) a sterol; and(d) a PEGylated lipid,wherein:(i) the PEGylated lipid comprises a hydrocarbon chain comprising more than 14 C atoms; and / or(ii) the PEGylated lipid is at a concentration of at least 3 %mol.

2. The lipid nanoparticle of claim 1, wherein the hydrocarbon chain is at least 16 or at least 18 C atoms in length.

3. The lipid nanoparticle of claim 1 or claim 2, wherein the ionizable lipid is at a concentration of 35-51 %mol, optionally about 40 %mol.

4. The lipid nanoparticle of any one of the preceding claims, wherein the ionizable lipid comprises or is Dlin-MC3-DMA, ALC-0315, or SM-102, optionally wherein the ionizable lipid comprises or is SM-102.

5. The lipid nanoparticle of any one of the preceding claims, wherein the helper lipid is at a concentration of 8-12 %mol, optionally about 11 %mol.

6. The lipid nanoparticle of any one of the preceding claims, wherein the helper lipid comprises oris l,2-Distearoyl-sn-glycerol-3-PC (DSPC).

7. The lipid nanoparticle of any one of the preceding claims, wherein the sterol is at a concentration of 35-45 %mol, optionally about 38 %mol.

478. The lipid nanoparticle of any one of the preceding claims, wherein the sterol comprises or is cholesterol.

9. The lipid nanoparticle of any one of the preceding claims, wherein the PEGylated lipid is at a concentration of 5-15 %mol, optionally about 10 %mol.

10. The lipid nanoparticle of any one of the preceding claims, wherein the PEGylated lipid comprises or is distearoyl-glycerol-PEG (DSG-PEG) or distearoyl-glycero-3-phosphoethanolamine-PEG (D SPE-PEG) .

11. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle:(a) has reduced uptake by liver and / or spleen cells compared to an equivalent lipid nanoparticle (i) comprising a PEGylated lipid comprising a hydrocarbon chain that is 14 C atoms in length and / or (ii) comprising a PEGylated lipid at a concentration of less than 3 %mol and / or(b) has an increased uptake by target cells compared to an equivalent lipid nanoparticle comprising a PEGylated lipid comprising a lipid chain that is 14 C atoms in length.

12. The lipid nanoparticle of any one of the preceding claims, wherein the PEGylated lipid comprises a PEG chain of between 0.5 and 2 kDa, optionally about 750 Da or about 1 kDa.

13. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle comprises a second PEGylated lipid comprising a second hydrocarbon chain, wherein the hydrocarbon chain comprises at least 14 C atoms.

14. The lipid nanoparticle of claim 13, wherein the second hydrocarbon chain comprises at least 16 or at least 18 C atoms.

15. The lipid nanoparticle of claim 13 or claim 14, wherein the second PEGylated lipid comprises a PEG chain that is between 1 kDa and 5 kDa, optionally about 3.4 kDa.

16. The lipid nanoparticle of any one of claims 13 to 15, wherein the second PEGylated lipid comprises or is DSG-PEG or DSPE-PEG.

17. The lipid nanoparticle of any one of claims 13 to 16, wherein the second PEGylated lipid is at a concentration of:(a) 0-15 %mol, 0-12%mol, 0-10 %mol, 0-9 %mol, 0-8 %mol, 0-7 %mol, 0-6 %mol, 0-5 %mol, 0-4 %mol, 0-3 %mol, 0-2 %mol, 0-1 %mol, 0-0.5 %mol or 0-0.15 %mol;(b) 0.05-0.15 %mol; or(c) 0.09 %mol.

18. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) a helper lipid;(c) a sterol;(d) a PEGylated lipid; and(e) a second PEGylated lipid,wherein:(i) the PEGylated lipid is DSG-PEG comprising a PEG chain of 750 Da or 1 kDa, the PEGylated lipid is at a concentration about 10.29 %mol, the second PEGylated lipid is DSPE-PEG comprising a PEG chain of 3.4 kDa, and the second PEGylated lipid is at a concentration of about 0.09 %mol; or(ii)(A) the ionizable lipid is SM102 at a concentration of about 39.98 %mol;(B) the helper lipid is DSPC at a concentration of about 11.49 %mol;(C) the sterol is cholesterol at a concentration of about 38.15 %mol;(D) the PEGylated lipid is DSG-PEG comprising a PEG chain of about 750 Da or about 1 kDa at a concentration of about 10.29 %mol; and(E) the second PEGylated lipid is DSPE-PEG-DBCO comprising a PEG chain of about 3.4 kDa at a concentration of about 0.09 %mol.

19. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle comprises at least one targeting peptide, at least two targeting peptides, or at least three targeting peptides.

20. The lipid nanoparticle of claim 19, wherein the targeting peptides are conjugated (a) to the second PEGylated lipid via a PEG chain or (b) to the sterol via a PEG chain.

21. The lipid nanoparticle of claim 19 or claim 20, wherein each of the targeting peptides can: (i) specifically bind to a target molecule, wherein the target molecule is present on the cell surface of a target cell and / or (ii) bind specifically to a target cell, tissue or organ, optionally wherein the target cell or target tissue is not a liver or spleen cell or liver or spleen tissue and / or the organ is not the liver or spleen.

22. The lipid nanoparticle of any one of claims 19 to 21, wherein the lipid nanoparticle has an increased uptake by target cells compared to an equivalent lipid nanoparticle lacking the targeting peptides.

23. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle further comprises a payload.

24. The lipid nanoparticle of claim 23, wherein the payload comprises:(a) a nucleic acid, optionally an RNA, an siRNA, an mRNA, an antisense oligonucleotide, a gRNA or a DNA;(b) an RNA molecule, a DNA molecule, a polypeptide, a cytotoxic molecule, a radioligand, an antibiotic, an antiviral, an immunogen, or a therapeutic transgene; and / or(c) a component of a gene editing system, optionally wherein the component comprises a gRNA, a Cas nuclease, a Cas nickase, a deaminase, a cytosine base editor, and / or an adenine base editor.

25. The lipid nanoparticle of any one of the preceding claims for use in a method of medical treatment, wherein the treatment comprises administering the lipid nanoparticle to a subject.50

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