Lipid nanoparticles having non-polar lipids for nucleic acid delivery to the liver

Abstract

The present disclosure provides a method for delivering a nucleic acid cargo to the liver of a subject, the method comprising administering to the subject a lipid nanoparticle encapsulating the nucleic acid and comprising a non-polar lipid. The lipid nanoparticle may exhibit a blebbed morphology, despite the absence or low levels of phospholipid. The disclosure further provides compositions for delivering nucleic acid cargo to the liver in order to treat various diseases, disorders and conditions in the subject.

Description

Technical Field

[0001] The present disclosure relates to lipid nanoparticle formulations for the delivery of cargo such as nucleic acid. Background

[0002] There is increasing interest in the use of lipid nanoparticle-targeted hepatic delivery to treat genetic disorders, cancer and infectious diseases of the liver, which account for millions of deaths world-wide (Witzigmann et al., 2020, Advanced Drug Delivery Reviews 159:344-363). Many of the current clinical interventions for inherited liver disorders and alcohol-related liver diseases have limited efficacy, with transplants being the only viable option in many cases. However, transplants may raise concerns of histocompatibility, create surgical risks, such as infections, and may require long-term immunosuppression (Zadory et al., 2022, Biomater. Sci., 10:6077-6115). Thus, lipid nanoparticle (LNP) therapy is an attractive option for avoiding or limiting the use of such invasive procedures.

[0003] An early example of a liver-tropic therapeutic formulation approved for clinical use is Onpattro™. The formulation is an LNP encapsulating short interfering RNA (siRNA) for the treatment of a liver-based rare genetic disorder, known as hereditary transthyretin amyloidosis, which causes amyloid fibril deposition in multiple organs. The Onpattro™ LNP formulation consists of four main lipid components, namely: ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a polyethylene glycol conjugated lipid (PEG-lipid) at respective molar amounts of 50 / 10 / 38.5 / 1.5. The success of this liver targeted mRNA-LNP delivery system paved the way for the clinical development of the leading LNP-based COVID-19 mRNA vaccines.

[0004] Of the four Onpattro™ lipid components, the ionizable cationic lipid is deemed most important for the in vitro and in vivo activity of the LNP system. Accordingly, most work in the field has focused primarily on improving this lipid component. The ionizable cationic lipid is positively charged at low pH, which facilitates association with the negatively charged nucleic acid but is neutral at physiological pH, making it more biocompatible in biological systems. Further, it has been suggested that after the lipid nanoparticles are taken up by a cell by endocytosis, the ability of these lipids to ionize at low pH enables endosomal escape. This in turn allows the nucleic acid to be released into the intracellular compartment.

[0005] As to the remaining lipid components, reports suggest that DSPC and cholesterol in empty Onpattro™-type LNP systems reside in outer lipid layers but that in systems loaded with siRNA, the DSPC and cholesterol is internalized together with siRNA in a hydrophobic core. It was found that the presence of both DSPC and cholesterol in LNPs is vital to the stable encapsulation of siRNA. (Kulkarni et al., 2019, Nanoscale, 11:21733-21739). It was further suggested that DSPC is required to stably retain cholesterol in the LNP formulations examined.

[0006] U.S. Patent No. 11,191,849 discloses liver-targeted mRNA-LNPs having 55 mol% ionizable cationic lipid, 41 mol% cholesterol and 3.3 mol% PEG2000-C-DMA. Inflammatory responses after administration of the LNP in mice were reduced compared to the base composition (PEG2000-C-DMA / cationic lipid / cholesterol / DSPC at 1.6 / 55 / 33 / 11 mol%) without impacting potency. The results observed were attributed in part to a high PEG-lipid conjugate content (>3 mol%).

[0007] Some recent studies have investigated improving in vivo delivery properties by introducing targeting lipid components into LNP formulations. One approach uses Selective Organ Targeting (SORT) LNPs (Wang et al., 2023, Nat. Protoc. 18:265-291), which are tuned for tissue-specific mRNA delivery by the introduction of a fifth targeting lipid component, such as a permanently charged anionic or cationic lipid. However, such LNPs are still reliant on maintaining the traditional four lipid components of the clinically approved Onpattro™ formulation. Moreover, the introduction of a fifth “SORT”, permanently charged cationic or anionic lipid may introduce toxicity in vivo.

[0008] Other investigators have examined the use of triacylglycerols, such as triolein, in LNPs in combination with palmitoylphosphatidycholine (POPC), although the focus of such studies was to improve the delivery of hydrophobic, small molecule drugs (e.g., doxorubicin) rather than nucleic acids (Zhigaltsev et al., 2012, 28:3633-3640). NMR and cryo-EM studies of triolein / POPC 60 / 40 (mol / mol) LNPs suggested that the particles formed by microfluidic mixing consist of POPC in outer monolayers surrounding a hydrophobic triglyceride core (Zhigaltsev et al., 2012, 28:36333640).

[0009] WO 2018 / 119514 describes triolein-containing LNPs for mRNA delivery to neurons. The studies suggested that cholesterol had limited impact on potency. In addition, the mRNA-LNPs examined in the studies included high levels of phospholipid (20-40 mol%), namely dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC). US2010 / 0297242 describes particles with LDL-like characteristics but similarly were prepared with phospholipid to impart stability. Further, Kulkarni et al., found that the incorporation of DOPE, dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (ESM) and cholesterol in lipid nanoparticles allowed effective encapsulation of siRNA, but the inclusion of triolein and trilinolein was reported to result in ineffective entrapment (Nanoscale, 2019, 11:21733-21739, supplemental Figure 2).

[0010] Despite the foregoing advances in the art, there is a continuing need for LNP formulations that have desirable properties for the delivery of nucleic acids to the liver. Summary

[0011] The present disclosure addresses one or more problems in the art and / or provides useful alternatives thereof.

[0012] The present disclosure is based in part on the finding that lipid nanoparticles (LNPs) prepared with non-polar lipids, such as non-polar glyceride lipids or analogues thereof and without phospholipid, or low levels thereof, have favourable nucleic acid delivery properties. In particular, the LNPs described herein have been found to exhibit increased nucleic acid delivery to the liver relative to an Onpattro™-type baseline LNP (also herein “Onpattro™-type formulation” or “baseline”), despite significant departures in lipid composition from this “gold standard” benchmark.

[0013] In one example of the disclosure, the LNPs having a combination of a non-polar lipid and lacking phospholipid or having low levels thereof, exhibit improved mRNA expression in the liver over the spleen. In some embodiments, such mRNA expression in the liver over the spleen is significantly improved relative to the Onpattro™-type baseline LNP.

[0014] The LNPs were also found to exhibit surprising morphologies as determined by cryogenic electron microscopy. As described herein, despite the lack of phospholipid, or low levels thereof, the particles surprisingly assumed a blebbed morphology.

[0015] According to one aspect of the disclosure, there is provided a lipid nanoparticle comprising: a nucleic acid cargo molecule; a sterol (including a derivative thereof) at a content of between 0 mol% and 50 mol%; an ionizable lipid present at a content of between 20 mol% and 70 mol%; a non-polar lipid present at a content of between 20 mol% and 80 mol%; a phospholipid (including a derivative thereof) at a content of 0 to 5 mol%; and a hydrophilic polymer-lipid conjugate at a content of between 0 mol% and 3 mol%, wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

[0016] According to a further aspect of the disclosure, there is provided a lipid nanoparticle comprising: encapsulated nucleic acid; an ionizable lipid; a homogeneous generally spherical hydrophobic core of a non-polar lipid and one or more peripheral bleb structures as observed by cryogenic electron microscopy (cryo-TEM); optionally a sterol; optionally a hydrophilic polymerlipid conjugate; and a phospholipid at a content of 0 to 5 mol%.

[0017] In one embodiment of either of the foregoing aspects of the disclosure, the non-polar lipid is a combination of two or more non-polar lipids.

[0018] In one example of either of the foregoing aspects of the disclosure or embodiments thereof, the non-polar lipid is a triglyceride.

[0019] In one embodiment, the triglyceride is selected from triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin and oleoyldipalmitin.

[0020] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the non-polar lipid is a diglyceride.

[0021] In one embodiment, the diglyceride is selected from glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate, glycerol dibehenate, glycerol dipalmitoleate, glycerl dioleate, glycerol dilinoleate, glycerol dilinolenate and glycerol di arachidonate.

[0022] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the non-polar lipid is a monoglyceride.

[0023] In one embodiment, the monoglyceride is selected from 1 auroyl-rac-glycerol, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopalmitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1-monomyristoyl-rac glycerol, 1-mono-palm itoyl-rac-glycerol, 2-monopalm itoylglycerol, 1-mono-palm itolenyl-rac-glycerol, 1-monostearoyl-rac-glycerol, 1-monoleoyl-rac-glycerol, 1-monolinoleoyl-rac-glycerol and 1-monolinolenoyl-rac-glycerol.

[0024] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the non-polar lipid is castor oil.

[0025] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the non-polar lipid is methyl ricinoleate.

[0026] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the non-polar lipid is present at a content between 22 mol% and 38 mol%, 25 mol% and 35 mol%, 25 mol% and 35 mol%, 27 mol% and 32.5 mol% or 28 mol% and 32 mol%.

[0027] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the ionizable lipid is an amino lipid.

[0028] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the ionizable lipid is present at a content between 23 mol% and 47 mol%, 25 mol% and 45 mol%, 27 mol% and 43 mol%, 28 mol% and 42 mol%, 29 mol% and 41.5 mol%, 29.5 mol% and 41 mol% or 30 mol% and 40 mol%.

[0029] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the phospholipid content is less than 4 mol%.

[0030] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the phospholipid content is less than 3 mol%.

[0031] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the phospholipid content is less than 2 mol%.

[0032] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the phospholipid content is less than 1 mol%.

[0033] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the phospholipid content is less than 0.5 mol%.

[0034] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the lipid nanoparticle has no detectable amount of phospholipid.

[0035] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the hydrophilic-polymer lipid is present at a content between 0.75 mol% and 2.5 mol%, at a content between 1 mol% and 2.25 mol%, at a content between 1.25 mol% and 2 mol% or at a content between 1.25 mol% and 1.75 mol%.

[0036] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the sterol is present and is a non-cationic lipid.

[0037] In one embodiment, the sterol is cholesterol.

[0038] In one embodiment, the sterol is present at a content between 22 mol% and 38 mol%, 25 mol% and 35 mol%, 27 mol% and 32.5 mol% or 28 mol% and 32 mol%.

[0039] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the LNP has an N / P charge ratio of the cationic charge (N) of the ionizable lipid to the anionic charge (P) of the nucleic acid cargo is between 1 and 15.

[0040] In one embodiment, the N / P charge ratio is between 2 and 6 or 3 and 6.

[0041] In one embodiment, the nucleic acid is selected from an siRNA, mRNA, a vector nucleic acid, an antisense oligonucleotide, a nucleic acid-protein complex and a nucleic acid-peptide complex.

[0042] In one embodiment, the nucleic acid is selected from siRNA, vector nucleic acid, and an antisense oligonucleotide. In one embodiment, the nucleic acid is mRNA.

[0043] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the lipid nanoparticle has a bleb structure as visualized by cryogenic electron microscopy (cryo-TEM).

[0044] In one embodiment, the lipid nanoparticle has a homogeneous core with one or more peripheral blebs as visualized by cryogenic electron microscopy (cryo-TEM).

[0045] In one example of either of the foregoing aspects of the disclosure or any embodiment thereof, the lipid nanoparticle has at least a 10% increase in expression of the protein or peptide at 4 hours, 24 hours, and / or 48 hours post-injection in the liver of a mammal as compared to baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol encapsulating the nucleic acid, but otherwise measured under identical conditions, wherein the expression is measured using luminescence.

[0046] In a further aspect of the disclosure, there is provided a method for delivery of mRNA or vector DNA for in vivo production of protein or peptide in the liver, the method comprising administering to a mammal a lipid nanoparticle as described in any one of the foregoing aspects or embodiments thereof, wherein the nucleic acid is mRNA or vector DNA and wherein the administering of the lipid nanoparticle results in an increased liver-specific expression of the protein or peptide encoded by the mRNA or vector DNA as compared to a baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol delivering the mRNA or vector DNA.

[0047] In one embodiment, the increased expression of the protein or peptide encoded by the mRNA in the liver over the spleen is at least 10% greater than the increased expression in a baseline LNP in the liver over the spleen measured under otherwise identical conditions.

[0048] In a further aspect of the disclosure, there is provided a method for delivery of siRNA or antisense oligonucleotide for in vivo silencing of a gene in the liver, the method comprising administering to a mammal a lipid nanoparticle of any one of the foregoing aspects or embodiments thereof, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased liver-specific silencing of the gene as compared to a baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol delivering the siRNA or antisense oligonucleotide.

[0049] In a further aspect of the disclosure, there is provided a method for delivering a nucleic acid to a hepatic cell to treat a disease, disorder or condition, the method comprising contacting the lipid nanoparticle of any one of the foregoing aspects or embodiments thereof with the hepatic cell in vivo or in vitro.

[0050] In a further embodiment of any one of the foregoing methods, the phospholipid content of the LNP is less than 4 mol%.

[0051] In a further embodiment of any one of the foregoing methods, the sterol is present at 25 to 45 mol% or 25 to 40 mol%.

[0052] In a further embodiment of any one of the foregoing methods, a hydrophilic polymer-lipid conjugate is present in the lipid nanoparticle at a content between 0 and 3 mol%. In a further embodiment of any one of the foregoing methods, the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticle at a content between 0.5 and 2.0 mol%. Brief Description of the Figures

[0053] Figure 1A shows ex vivo luminescence for TAG LNP and Onpattro™-type baseline LNP in the liver at 0.02 mg / kg luciferase at four hours post-administration to CD-I mice. The cargo was NTx firefly luciferase (fLuc) mRNA. Details of the TAG-LNP and Onpattro™-type baseline LNP lipid compositions are set forth in Table 1 of Example 1.

[0054] Figure IB shows ex vivo luminescence for TAG LNP and Onpattro™-type baseline LNP in the liver at 0.1 mg / kg luciferase at four hours post-administration to CD-I mice. The cargo was an internal NTx fLuc mRNA.

[0055] Figure IC shows ex vivo luminescence for TAG LNP and Onpattro™-type baseline LNP in the liver at 0.5 mg / kg luciferase at four hours post-administration to CD-I mice. The cargo was an internal NTx fLuc mRNA.

[0056] Figure ID shows representative whole-body images of the CD-I mice for the TAG LNP and Onpattro™-type baseline LNP at a dose of 0.5 mg / kg internal NTx fLuc mRNA. The images were taken at four hours post-administration. Intensity scales are the same for both images.

[0057] Figure 2A shows ex vivo luminescence for TAG LNP and Onpattro™-type baseline LNP in the liver and spleen at four hours post-administration to CD-I mice. The cargo was NTx fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0058] Figure 2B shows in vivo luminescence for Onpattro™-type baseline LNP and two different TAG LNPs having ionizable lipid / cholesterol / triolein / PEG2ooo-DMG but differing in the ionizable lipids (ionizable amino lipids 1 and 2 in Example 2). The results show luminescence of tissue homogenates from liver at a dose of 1 mg / kg at 24 hours post-administration to CD-I mice.

[0059] Figure 2C shows in vivo luminescence of spleen tissue homogenate at 24 hours post injection for the LNPs as set forth in Figure 2B.

[0060] Figure 2D shows in vivo luminescence for Onpattro™-type baseline LNP and two different TAG LNPs 1 and 3 with differing lipid composition. TAG LNP 1 is the previously described ionizable lipid / cholesterol / triolein / PEG2ooo-DMG formulation and TAG LNP 3 additionally comprises cholesteryl hemisuccinate (CHEMS). The results show luminescence of tissue homogenates from liver at a dose of 1 mg / kg at 24 hours post-administration to CD-I mice.

[0061] Figure 2E shows in vivo luminescence of spleen tissue homogenate at 24 hours post injection for the LNPs as set forth in Figure 2D.

[0062] Figure 3A shows a time course of ex vivo luminescence for TAG LNP (Table 1) and Onpattro™-type baseline LNP in the whole body and abdomen of CD-I mice at 4, 24 and 48 hours post-administration. The cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0063] Figure 3B shows in vivo luminescence for TAG LNP (Table 1) and Onpattro™-type baseline LNP in the whole body and abdomen of CD-I mice at 4-, 24- and 48-hours postadministration. The cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0064] Figure 4 shows ex vivo luminescence for TAG LNP (Table 1) and Onpattro™-type baseline LNP in the liver at 0.5 mg / kg luciferase at four hours post-administration to CD-I mice following 3 months storage at 4°C. The cargo was an internal NTx fLuc mRNA.

[0065] Figure 5 shows ex vivo luminescence for TAG LNP (Table 1) and Onpattro™-type baseline LNP in the liver at 0.5 mg / kg luciferase at four hours post-administration to CD-I mice following cryogenic storage at -80°C. The cargo was an internal NTx fLuc mRNA.

[0066] Figure 6 shows ex vivo luminescence for TAG LNP (Table 1), at N / P ratios of 3, 4, 5 and 6, in the liver at 0.5 mg / kg luciferase at four hours post-administration to CD-I mice. The cargo was an internal NTx fLuc mRNA.

[0067] Figure 7 shows ex vivo luminescence for a Comimaty™-type LNP comprising phospholipid (10 mol% DSPC) and lacking TAG (denoted +phospholipid (PL) -TAG), a Comimaty-type LNP with addition of TAG (+PL +TAG) and the inventive TAG LNP (-PL+TAG) in the liver dosed at 0.1 mg / kg luciferase and measured at four hours post-administration (i.v.) within CD-I mice. The formulations are set forth in Table 3. The cargo was MG fLuc mRNA and the dose was 0.1 mg / kg mRNA.

[0068] Figure 8A shows the total bilirubin levels (pmol / L) at 4, 24 and 48 hours postadministration of a TAG LNP and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total bilirubin levels (pmol / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA. The formulations are set forth in Table 4.

[0069] Figure 8B shows the total alanine transaminase (ALT) levels (U / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total alanine transaminase (ALT) levels (U / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0070] Figure 8C shows the total aspartate aminotransferase (AST) levels (U / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total aspartate aminotransferase (AST) levels (U / L) were measured at 4 hours postadministration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0071] Figure 8D shows the total alkaline phosphatase (AP) levels (U / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total alkaline phosphatase (AP) levels (U / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0072] Figure 8E shows the total albumin levels (g / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total albumin levels (g / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0073] Figure 8F shows the total amylase levels (U / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific time points. For Onpattro™-type baseline LNPs, total amylase levels (U / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0074] Figure 8G shows the total lactate dehydrogenase (LDH) levels (U / L) at 4, 24 and 48 hours post-administration of a TAG LNP (Table 4) and a PBS control injection measured from blood samples taken from CD-I mice at these specific timepoints. For Onpattro™-type baseline LNPs, total lactate dehydrogenase (LDH) levels (U / L) were measured at 4 hours post-administration only. In all cases, the cargo was MG fLuc mRNA and the dose was 0.5 mg / kg mRNA.

[0075] Figure 9A shows particle size (nm) for TAG LNP and Onpattro™-type baseline LNP formulations between various batches of the formulations. The lipid components of the LNPs are set forth in Table 4.

[0076] Figure 9B shows poly dispersity index (PDI) for TAG LNP and Onpattro™-type baseline LNP formulations between various batches of the formulations. The LNPs are set forth in Table 4.

[0077] Figure 9C shows mRNA encapsulation for TAG LNP and Onpattro™-type baseline LNP formulations between various batches of the formulations. The LNPs are set forth in Table 4. The cargo was fLuc mRNA obtained from a variety of sources.

[0078] Figure 9D shows fluorescence vs pH for TAG LNP and Onpattro™-type baseline LNP formulations between various batches of the formulations and the apparent pKa for each LNP. The LNPs are set forth in Table 4.

[0079] Figure 10A shows cryo-TEM images for TAG LNP formulations encapsulating fLuc mRNA. The arrows depict blebs. Details of the LNP formulation are set forth in Table 4.

[0080] Figure 10B shows cryo-TEM images for the Onpattro™-type baseline formulations encapsulating fLuc mRNA. Details of the LNP formulation are set forth in Table 4.

[0081] Figure 11 shows ex vivo luminescence for phospholipid-less LNPs (containing different non-polar glyceride lipids) in the liver at 0.1 mg / kg luciferase at four hours post-administration to CD-I mice. The cargo was an internal NTx fLuc mRNA. Details of the LNP formulation are set forth in Table 5. Detailed Description Non-polar lipid

[0082] The term "non-polar lipid" as used herein refers to a lipid that is neutral and uncharged at physiological pH, lacks a head group moiety (e.g., phosphate), and that comprises one or more lipophilic chains that are at least 10 carbon atoms in length that impart an overall hydrophobic character to the lipid. The non-polar lipid includes but is not limited to esterified fatty acids and glycerides described below.

[0083] In one example of the disclosure, the non-polar lipid has a structure of Formula I: R2 I b Ja Formula I or a tautomer or stereoisomer thereof, wherein RI, R2 and R3 are at each occurrence, independently, H or C1-C26 alkyl or a C1-C26 alkenyl having 1-6 double bonds, wherein at least one, at least two or all of Rl-X, R2-Y and R3-Z is the C1-C26 alkyl or alkenyl bonded to a heteroatom substituent represented by X, Y and Z, and wherein at least one of RI, R2 and R3 comprises at least 10 carbon atoms and terminates with a methyl; and wherein X, Y and Z are at each occurrence, a direct bond or are independently, the heteroatom substituent selected from -O-; —(C=O)O-; —O(C=O)-; — C(=O)-; —O(C=O)O-; —S(O)X-; — S—S-; — C(=O)S-; — SC(=O)-; —NR'—; —NR'C(=O) —; —C(=O)NR'—; — NR'C(=O)NR'—; —OC(=O)NR'—; —NR'C(=O)OR’—; —NR'S(O)XNR'—; —NR'S(O)XR’— ;and —S(O)XNR'—, wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2, and wherein a and b are independently 0 to 4, wherein the alkyl or alkenyl of RI, R2 and R3 is optionally substituted.

[0084] The term “optionally substituted” with reference to an RI, R2 or R3 alkyl or alkenyl means that at least one hydrogen atom of the alkyl or alkenyl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl is interrupted (i.e., a -(CH)2- group replaced) by a non-carbon atom or one or more substituents, provided the non-polar lipid retains sufficient hydrophobicity so as to form the hydrophobic core of the LNP. The substituent that replaces the hydrogen atom may include -OR’, -SR’ or -NR’. Substituents that may replace a methylene -(CH)2- group include but are not limited to -O-, -S- and -NR'-, wherein R' is as defined above.In one embodiment, X, Y and Z are independently selected from —(C=O)O-; —O(C=O)-; NR'C(=0) —; —C(=0)NR'—; —NR'C(=0)NR'—; —0C(=0)NR'—; and — NR'C(=0)0R’—In one embodiment, the X, Y and Z are independently selected from —(C=O)O-and —0(0=0)-. In one embodiment, the non-polar lipid, is a non-polar glyceride lipid or analogues thereof that includes triglycerides, diglycerides, monoglycerides or mixtures thereof.

[0085] In one embodiment, the non-polar glyceride lipid is a tri-acylglycerol (TAG), which refers to a glyceride having three fatty acid chains covalently bonded to a glycerol backbone through respective ester linkages. In one embodiment, all three fatty acids bonded to the glycerol backbone are the same. In alternative embodiments, the three fatty acids of the triglyceride are different, e.g., having different lengths and / or degrees of saturation.

[0086] In some embodiments, the TAG can be characterized by the degree of saturation of each fatty acid chain. In some embodiments, TAGs have SSS, SUS, UUU and USU- fatty acid chains and are therefore considered symmetrical triglycerides, where S represents a saturated fatty acid and U represents an unsaturated fatty acid. In other embodiments, asymmetrical TAGS are used in the LNP. In some embodiments, the sn-1 and sn-3 positions of the TAG contain different fatty acids, in which case the central carbon atom is a chiral carbon and the TAG is asymmetrical.

[0087] In embodiments of the disclosure, the triglyceride in the LNP is triolein, a symmetrical triglyceride derived from glycerol and three units of the unsaturated fatty acid oleic acid. The IUPAC name for triolein is 2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl (Z)-octadec- 9-enoate, and synonyms include glycerol trioleate, glycerol trioleyl, trielaidin, trioleoylglycerol, and trioleyl glycerol.

[0088] Non-limiting examples of the triglyceride include triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin, triricinolein, tribehenin, tripalmitolein, triarachidonin, glyceryl distearate oleate, glyceryl distearate linoleate, glyceryl palmitate oleate stearate, glyceryl palmitate dioleate, glyceryl stearate dioleate, glyceryl palmitate dilinoleate, glyceryl stearate oleate linoleate. In some embodiments, a mixture of two or more triglycerides is included in the LNP.

[0089] In some embodiments, the tri-acylglyceride is naturally derived. Examples of natural origin fatty acids used in some embodiments include soy oil, castor oil, sunflower oil, canola oil and palm oil, omega 3 fatty acid and omega 6 fatty acid. Further, examples of omega 3 fatty acids used in some embodiments include, but are not limited to, alpha- linolenic acid and docasahexaenoic acid. Examples of omega 6 fatty acids used in some embodiments include, but are not limited to, linoleic acid and gamma linolenic acid.

[0090] Additional commercially available triglycerides include Captex® and Sterotex®. In some embodiments, the TAG is selected from Captex™ 8000, Captex™ GTO, and Captex™ 1000.

[0091] In some embodiments, the number of carbon atoms on the aliphatic tails of the triglyceride can be used to classify the TAG as a medium chain triglyceride (MCT) having 6 to 12 carbon atoms (6, 7, 8, 9, 10, 11, 12). In some embodiments, the LNP contains one or more MCT, or mixtures thereof. In some embodiments, the medium chain triglyceride comprises a fatty acid selected from one or more of caproic acid, octanoic acid, capric acid, caprylic acid, and / or lauric acid. In some embodiments, the MCT is highly pure. In some embodiments, the MCT has a purity by weight % of equal to or greater than about: 90%, 95%, 97%, 98%, 99%, 100%, or ranges including and / or spanning the aforementioned values. In some embodiments, the MCT (or LCT) is present in the lipid-based particle composition at dry weight % of equal to or greater than about: 10%, 20%, 30%, 35%, 40%, 45%, 50%, or ranges including and / or spanning the aforementioned values.

[0092] In some embodiments, the triglyceride comprises a fatty acid greater than 12 carbons in length. In some embodiments, the lipid component comprises a long chain triglyceride (LCT) greater than or equal to 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length or ranges including and / or spanning the aforementioned values.

[0093] The term “diacylglycerol lipid” or “diacylglyceride” (DAG) herein refers to a glyceride having two fatty acid chains covalently bonded to a glycerol backbone through respective ester linkages. The DAG includes in some embodiments rac-1,3 or sn-1,2 lipids.

[0094] Exemplary diacylglycerides include, but are not limited to, glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate, glycerol dibehenate, glycerol dipalmitoleate, glycerol dioleate, glycerol dilinoleate, glycerol dilinolenate, glycerol di arachidonate, glycerol dicaprylate, l-stearoyl-3-oleyl-glycerol, l-stearoyl-2-oleyl-sn-glycerol or combinations thereof.

[0095] The term "monoacylglycerol lipid" or "monoacylglyceride" (MAG) as used herein refers to a glyceride having one fatty acid chain covalently bonded to a glycerol backbone through an ester linkage. In one embodiment, the monoacylglycerol lipid is a 1-monoacylglycerol or 2-monoacylglycerol, depending on the position of the ester bond on the glycerol backbone.

[0096] Exemplary monoacylglycerol lipids for incorporation in the LNP include, but are not limited to, glycerol monolaurate, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopalmitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1-monomyristoyl-rac glycerol, 1-mono-palm itoyl-rac-glycerol, 2-monopalmitoylglycerol, 1-mono-palmitolenyl-rac-glycerol, 1-monostearoyl-rac-glycerol, 1-monoleoyl-rac-glycerol, 1- monolinoleoyl-rac-glycerol, 1-monolinolenoyl-rac-glycerol or combinations thereof. Sterol The lipid nanoparticles in some non-limiting examples include a sterol.

[0097] The term “sterol” refers to steroids that are naturally-occurring or synthetic and includes sterol derivatives. The term includes phytosterols, zoosterols and derivatives thereof.

[0098] The term “sterol derivatives” refers to modified sterols or precursors thereof, including triterpenes.

[0099] The term “cholesterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl bonded to one of its rings, typically the A-ring and encompasses a cholesterol derivative.

[00100] The cholesterol derivative may be naturally-occurring or synthetic and includes but is not limited to a cholesterol molecule having a gonane structure and one or more additional functional groups, including derivatization of the terminal hydroxyl group.

[00101] In another embodiment, the LNP may comprise a triterpene. Non-limiting examples include squalene, achilleol A, polypodatetraene, malabaricane, lanostane, cucuribitacin, hopane, oleanane and urosolic acid.

[00102] In certain embodiments, the cholesterol derivative is a phytosterol. The phytosterol may be P-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol or a salt or ester thereof.

[00103] In certain embodiments, the cholesterol derivative is selected from P-sitosterol, P-sitosterol acetate, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3P[N-(N'N'-dimethylaminoethyl)carbamoyl cholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxy cholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, dehydroergosterol, 9, 11 -dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, daucosterol or a salt or ester thereof.

[00104] The sterol, cholesterol or derivative thereof may be conjugated to another moiety, such as an amino acid or an alkyl group.

[00105] In one embodiment, the sterol or derivative thereof is present at from 0 mol% to 50 mol%, 15 mol% to 50 mol%, 20 mol% to 45 mol% or 25 mol% to 40 mol%, based on the total lipid present in the lipid nanoparticle.

[00106] In one embodiment, the cholesterol or derivative thereof is present at from 0 mol% to 50 mol%, 15 mol% to 50 mol%, 20 mol% to 45 mol% or 25 mol% to 40 mol%, based on the total lipid present in the lipid nanoparticle.

[00107] In some embodiments, the LNP comprises a cholesteryl ester comprising a fatty acid conjugated via an ester group. For example, the cholesteryl ester content may be 0 to 10 mol%, 0 to 8 mol%, 0 to 5 mol% or 0 to 2 mol%.

[00108] In another embodiment, the LNP has low levels of cationic cholesterol lipids, such as 3P-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol). For example, the cationic cholesterol content may be less than 10 mol%, less than 8 mol%, less than 5 mol% or less than 2 mol%.

[00109] The LNP may further comprise a tocopherol as an additional component. The tocopherol includes an a-tocopherol, P-tocopherol, y-tocopherol, 6-tocopherol or a salt or ester thereof. The tocopherol may be present at 0.5 mol% to 20 mol%, 1 mol% to 15 mol% or 2 mol% to 10 mol%. Ionizable lipid

[00110] The LNP of the disclosure has an ionizable lipid, which includes one or a combination of two or more of such lipids. The ionizable lipid may be charged at low pH and bear substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. Without being limited by theory, after cellular uptake by endocytosis, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent membrane destabilization and release of the nucleic acid-based therapeutics into the cytoplasm to exert their effects.

[00111] In some embodiments, it is desirable to include less than 50 mol% ionizable lipid in the LNP. In certain embodiments, the ionizable lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.

[00112] In certain embodiments, the ionizable lipid content may be less than 48 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%. In certain embodiments, the lower limit of the ionizable lipid content may be greater than 5 mol%, greater than 8 mol%, greater than 10 mol%, greater than 12 mol%, greater than 14 mol%, greater than 15 mol%, greater than 16 mol%, greater than 18 mol% or greater than 20 mol%. Any one of the upper limits may be combined with any one of the lower limits to arrive at a suitable ionizable lipid content in the LNP.

[00113] In some embodiments, the ionizable lipid is an “ionizable, cationic lipid”, which refers to a lipid that at physiological pH, is in an electrostatically neutral form and that accepts protons, thereby becoming electrostatically positively charged. In some embodiments, the cationic lipid has a pKa that is between 6.0 and 8.5 or between 6.0 and 7.5.

[00114] In some embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group. Non-limiting examples of such lipids include, but are not limited to sulfur lipids, such as MF019 described herein and DODMA. Other lipids that may be used in the practice of the disclosure include MC3-and KC2-type lipids, which are well-known to those of skill in the art. In further embodiments, the ionizable lipid is selected from one or more lipids set forth in WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 24657; WO 2023 / 147657; WO2022 / 155728; WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; WO 2024 / 130421; WO 2024 / 065043; and US 2024 / 0294462, each incorporated herein by reference.

[00115] In one embodiment, the ionizable cationic lipid comprises an ionizable amino head group and at least two lipophilic groups, at least one of which comprises a heteroatom, such as an ester or one or more sulfur atoms. In some embodiments, at least one lipophilic group comprises distal branching and / or one or more cyclic groups. Examples of ionizable cationic lipids comprising an ionizable amino head group and two lipophilic chains, at least one chain comprising one or more sulfur atoms and / or ester groups are described in co-owned and co-pending WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; WO 2024 / 130421; and WO 2024 / 065043. Functional groups comprising one or more heteroatoms may be biodegradable in vivo.

[00116] In some embodiments, it is desirable to include less than 50 mol% cationic lipid in the LNP. That is, the ionizable lipid content may be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol% or less than 5 mol%.

[00117] In certain embodiments, the cationic lipid content is from 5 mol% to 50 mol% or 8 mol% to 47 mol% or 10 mol% to 50 mol% or 15 mol% to 45 mol% or 15 mol% to 35 mol% of total lipid present in the lipid nanoparticle.

[00118] The ionizable lipid component may include an ionizable anionic lipid as part of the ionizable lipid content. An example of such a lipid is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in co-pending and co-owned WO 2024 / 192528, which is incorporated herein by reference in its entirety. Hydrophilic polymer-lipid conjugate

[00119] In one embodiment, the lipid nanoparticle comprises a hydrophilic-polymer lipid conjugate capable of incorporation into the LNP. The conjugate includes a lipophilic moiety bonded to a polymer chain that is hydrophilic. The lipophilic moiety may be bonded to the polymer chain directly or via linkers known to those of ordinary skill in the art. Examples of hydrophilic polymers include polyethyleneglycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine and polyaspartamide. In one embodiment, the hydrophilic-polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate may also be a naturally-occurring or synthesized oligosaccharide-containing molecule, such as, for example, monosialoganglioside (Gmi). The ability of a given hydrophilic-polymer lipid conjugate to enhance the circulation longevity of the LNPs herein could be readily determined by those of skill in the art using known methodologies.

[00120] The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol%, or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid.

[00121] The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol%, or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.

[00122] In another embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate that is present in the nanoparticle at 0.5 mol% to 5 mol%, or at 0.5 mol% to 3 mol% or at 0.5 mol% to 2.5 mol% or at 0.5 mol% to 2.0 mol% or at 0.5 mol% to 1.8 mol% of total lipid.

[00123] In another embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate that is present in the nanoparticle at 0 mol% to 5 mol%, or at 0 mol% to 3 mol% or at 0 mol% to 2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid.

[00124] As discussed below, the hydrophilic polymer-lipid conjugate may be conjugated to a targeting ligand at its distal end. Low phospholipid content

[00125] The lipid nanoparticle has “substantially no phospholipid”, meaning that the lipid nanoparticle has less than 3 mol% of a phospholipid, such as a neutral phospholipid. In one example, the lipid nanoparticle has 3 mol% or less phospholipid content. Examples of neutral phospholipids include phosphatidylcholine or phosphatidylethanolamine such as distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolaine (DSPE), di oleoylphosphatidylethanolamine (DOPE), di oleoylphosphatidylcholine (DOPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC). In some embodiments, the phospholipid is a phospholipid-sterol conjugate, such as an SPC-cholesterol, OPC-cholesterol or PPC-cholesterol conjugate. Additional phospholipid-sterol conjugates are described in US 2011 / 0177156, which is incorporated herein by reference.

[00126] In one embodiment, the lipid nanoparticle has less than 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or 0 mol% phospholipid. Most advantageously, the LNP has no phospholipid, but small amounts may not impact the LNP properties described herein.

[00127] In another embodiment, the lipid nanoparticle has “substantially no neutral lipid” meaning that the lipid nanoparticle has 0 to 3 mol% of any polar, neutral lipid, excluding cholesterol or a cholesterol derivative and the non-polar glyceride or analogue thereof. The term “neutral lipid” refers to any of a number of polar lipid species, including vesicle-forming lipids, that exist either in an uncharged or neutral zwitterionic form at physiological pH. In another example, the lipid nanoparticle has less than 8, 6, 4 or 2 mol% neutral lipid, such as a phospholipid and / or phospholipid conjugate.

[00128] Examples of polar neutral lipids include sphingomyelin, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), di oleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC), diacylphosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), ceramide, cephalin, triglyceride and diacylglycerol. In some embodiments, the neutral lipid is a phospholipid-sterol conjugate, such as a SPC-cholesterol or PPC-cholesterol conjugate or those described in US2011 / 0177156.

[00129] In one embodiment, the lipid nanoparticle has less than 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or 0 mol% of any polar, neutral lipid. Most advantageously, the LNP has no polar neutral lipid, but small amounts may be included in the formulation without impacting the LNP properties described herein. Additional components

[00130] The LNP may comprise additional lipid components or modifications to the sterol (e.g., cholesterol), sterol derivative (e.g., cholesterol derivative) and / or hydrophilic polymer-lipid conjugate.

[00131] For example, the surface of the LNP may be grafted to comprise a targeting ligand. The targeting ligand may be conjugated to cholesterol, the cholesterol derivative and / or the hydrophilic polymer-lipid conjugate. The targeting ligand may be conjugated to the distal end of a hydrophilic polymer-lipid conjugate. In some embodiments, the targeting ligand may be used to target receptors on cells in vivo. In some embodiments, the targeting ligand may be conjugated to a small amount of a phospholipid that may be included in the LNP. In such embodiments, the phospholipid-targeting ligand conjugate is typically present at less than 3 mol%.

[00132] The ligand includes peptides, polypeptides or proteins and includes antibodies or fragments thereof. In one embodiment, the ligand may be a single-chain antibody fragment. Examples of ligands are described in WO 2024 / 119279, which is incorporated herein by reference. Nanoparticle preparation and morphology

[00133] Lipid nanoparticles can be prepared using any of a variety of suitable methods, such as a rapid mixing / ethanol dilution process. Examples of preparation methods are described in Jeffs, L.B., et al., Pharm Res, 2005, 22(3):362-72; and Leung, A.K., et al., The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[00134] For example, the method of preparing the lipid nanoparticles may comprise dissolving lipid components (e.g., ionizable lipid, a non-polar glyceride or analogue thereof, a sterol, and a hydrophilic polymer-lipid) at appropriate ratios in an organic solvent (e.g., ethanol). An aqueous buffer (e.g., sodium acetate or sodium citrate) is prepared separately at a suitable pH to ensure that the head group (e.g., amino group) of an ionizable lipid is protonated to facilitate electrostatic interaction with the negatively charged cargo, and the positively charged ionizable lipid. Such charge interaction improves encapsulation.

[00135] In some embodiments, an aqueous phase is subsequently combined with the organic solvent-lipid mixture. Combining the aqueous phase and the organic-solvent-lipid mixture may be carried out in a mixing device (e.g., in-line mixer), such as a T-junction mixer with specialized pumps (e.g., a T-tube mixer), a herringbone micromixer, a toroidal mixer, a multi-inlet vortex mixer or other suitable mixing devices known to those of skill in the art. In some embodiments, the mixing device refers to a device comprising two or more inlets meeting in a central mixing region and an outlet through which the mixture exits the device. The LNP formation may occur upon mixture of the aqueous phase and organic solvent-lipid mixture and / or subsequent to such mixing. (Kulkarni et al., 2019, Nanoscale, 11(18):9023-9031, which is incorporated herein by reference).

[00136] The aqueous phase typically comprises a buffer. Non-limiting examples of suitable buffers include MES, sodium acetate or phosphate buffered saline (PBS). Examples of suitable solvents to prepare the organic solvent-lipid mixture are organic solvents including ethanol, isopropanol, methanol and acetone.

[00137] The aqueous phase and organic-solvent lipid mixture may be introduced to the mixer as two separate respective streams via pumps. The volumetric flow rate of each stream may be the same or different and the respective flow rates of each stream may be adjusted to achieve optimal mixing and / or LNP formation.

[00138] In some embodiments, the LNPs are prepared by solvent injection. In one embodiment, such method comprises dissolving lipids in an organic solvent and subsequent step-wise dilution of the resultant solution with an aqueous solution (e.g., buffer). This controlled step-wise dilution is achieved by mixing the aqueous and lipid streams together in a container.

[00139] The lipid nanoparticles may have an average size of between 40 and 150 nm or between 40 and 140 nm or between 45 and 130 nm or any range therebetween. In another embodiment, the lipid nanoparticle has aPDI of less than 0.20 or less than 0.15 or less than 0.12, or less than 0.10.

[00140] The nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 12. In another embodiment, the nitrogen-to-phosphate ratio of the lipid nanoparticle may be between 1 and 6.

[00141] The LNP generally comprises a “core” region, which may be characterized as being electron dense and optionally has one or more peripheral blebs as visualized by cryo-TEM microscopy.

[00142] A “bleb” refers to a protrusion from the lipid nanoparticle that typically assumes a spherical or hemispherical shape.

[00143] The morphology is assessed visually by cryo-TEM microscopy as set forth in the Materials and Methods of the Examples herein.

[00144] An example of such a morphology is shown in Fig. 7A. The bleb or blebs may appear as spherical or hemispherical protrusions from the lipid nanoparticle (see Fig. 7A). In some embodiments, the core is hydrophobic and one or more bleb protrusions extend from the core. The bleb or blebs may have an aqueous compartment and comprise nucleic acid. In one embodiment, the non-polar glyceride or analogue thereof forms the hydrophobic core of the LNP. In one embodiment, the core formed by the non-polar glyceride lipid or analogue thereof is homogeneous as observed by cryo-TEM microscopy but has a periphery with the one or more blebs.

[00145] In some embodiments, the LNP is not a lipoplex. Lipoplexes are prepared by mixing preformed cationic liposomes with nucleic acid in an aqueous solution and may exhibit undesirable properties such as localization of the cargo on the particle surface. Lipoplexes lack the abovedescribed core of the LNP particle. Further, LNPs have a defined size, shape and morphology whereas lipoplexes lack such defined physical characteristics. (See Kubota et al., 2017, Int. J. Nanomedicine, 12:5121-5133 and Kulkarni et al., 2018, Nucleic Acid Therapeutics, 28(3):146-157, which are each incorporated herein by reference).

[00146] Thus, according to some embodiments, the LNPs disclosed herein have a defined mean particle size that ranges between 40 and 150 nm or between 40 and 140 nm or between 45 and 150 nm. In some embodiments, the LNPs herein have a PDI of less than 0.25, less than 0.20, less than 0.18, less than 0.16, less than 0.15 or less than 0.14.

[00147] As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid cargo within an LNP refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle. However, this excludes localization of the nucleic acid on the particle surface as in lipoplexes. In some examples of the disclosure, the nucleic acid is present in the bleb of the LNP. Nucleic acid cargo

[00148] In one embodiment, the cargo is a nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), micro RNA (miRNA), messenger RNA (mRNA) or DNA such as vector DNA or linear DNA. The nucleic acid length can vary and can include nucleic acid of 1-50,000 nucleotides in length. The nucleic acid can be in any form, including single stranded DNA or RNA, double stranded DNA or RNA, or hybrids thereof. Single stranded nucleic acid includes antisense oligonucleotides. The nucleic acid may be conjugated to another molecule, including a targeting moiety. An example of such a nucleic acid conjugate is an antibody-nucleic acid conjugate, or an oligosaccharide-nucleic acid conjugate, such as a GalNAc-nucleic acid conjugate.

[00149] In one embodiment, the cargo is an mRNA, which includes a polynucleotide that encodes at least one peptide, polypeptide or protein. The mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in co-pending WO 2023 / 184038, which is incorporated herein by reference.

[00150] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.

[00151] In those embodiments in which an mRNA is a chemically synthesized molecule, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[00152] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[00153] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[00154] The present disclosure may be used to encapsulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 515 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.

[00155] Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap is advantageous in that it may provide resistance to nucleases found in eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[00156] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 1 and 500 nucleotides in length or 50 and 500 nucleotides in length or longer.

[00157] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 1 and 500 nucleotides in length or 50 and 500 nucleotides in length or longer.

[00158] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5’ and 3’ ends and thus may be more stable in vivo due to its resistance to degradation by exonucleases. The circular mRNA may be prepared by any known method, including any one of the methods described in Deviatkin et al., 2023, “Cap-Independent Circular mRNA Translation Efficiency”, Vaccines, 11(2), 238, which is incorporated herein by reference. Translation of the circular mRNA is carried out by a cap-independent initiation mechanism.

[00159] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[00160] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA for in vitro studies or for in vivo studies in animal models to assess expression and biodistribution.

[00161] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[00162] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides for any therapeutic, prophylactic or diagnostic application may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. A wide variety of different siRNA molecules may be used to target a specific gene or transcript. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 1 to 30 nucleotides in length or 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-stranded and has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[00163] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing. The snRNA molecules may have therapeutic or diagnostic importance in diseases that are an outcome of splicing defects.

[00164] Examples of the nucleic acid cargo include but are not limited to antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozyme, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, pDNA or an aptamer.

[00165] In another embodiment, the cargo is a DNA vector. The encapsulated DNA vectors may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide. In another embodiment, the encapsulated DNA vectors may be administered to a subject for diagnosis of disease. The DNA vector may localize in target cells (e.g., rapidly dividing cells) and expression of encoded DNA may be used to provide a measurable signal. Accordingly, the nucleotide polymers can be nucleotide sequences including genomic DNA, cDNA, or RNA.

[00166] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain double-stranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and selfreplicating systems such as vector DNA.

[00167] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will most advantageously have some or all of the nucleotide linkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotri ester linkages.

[00168] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moieties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronically similar structures, including azasugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[00169] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A non-limiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11):1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[00170] The nucleic acids used in the present disclosure can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Known procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[00171] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs. Improvements in liver-specific expression

[00172] In some embodiments, the LNP exhibits “liver-specific expression” of the protein or peptide encoded by the cargo nucleic acid, meaning that the cargo nucleic acid has increased expression of the protein or peptide in the liver over the spleen by at least 1.5-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold or at least 60-fold. In some embodiments, the cargo nucleic acid is mRNA or vector DNA. In one example, the nucleic acid is mRNA. The improvements in liver-specific expression can be assessed relative to an “Onpattro™-type baseline formulation” or “baseline LNP”. The terms refer to an LNP having ionizable lipid / DSPC / chol / PEG2ooo-DMG lipid at 50 / 10 / 38.5 / 1.5 mol / mol, wherein the ionizable lipid is nor-MC3 (see composition in Table 1 herein).

[00173] In further embodiments, the inclusion of low levels of a hydrophilic-polymer lipid conjugate may improve liver-specific expression of LNPs having elevated levels of sterol or a sterol derivative relative to the Onpattro™-type baseline formulation. For example, the hydrophilic-polymer lipid conjugate content to achieve such improved liver targeting may be less than 2.5 mol%, less than 2.25 mol%, less than 2.0 mol%, less than 1.75 mol%, less than 1.5 mol%, less than 1.25 mol%, less than 1.00 mol%, less than 0.75 mol%, less than 0.50 mol%, less than 0.25 mol% or less than 0.20 mol%.

[00174] Alternatively or additionally, the increased expression of the protein or peptide encoded by the nucleic acid cargo (e.g., mRNA or vector DNA) in the liver over the spleen is at least 5% or 10% greater than the increased expression in an Onpattro™-type baseline LNP in the liver over the spleen measured under otherwise identical conditions and encapsulating the same cargo.

[00175] In one embodiment, the administration of a fresh sample of the lipid nanoparticle results (less than one month after preparation) in a five-fold increase in liver-specific expression of the protein or peptide encoded by the encapsulated mRNA or vector DNA as compared to a fresh sample of the Onpattro™-type baseline formulation.

[00176] In one embodiment, the administration of a fresh sample of the lipid nanoparticle results in a five-fold increase in liver-specific silencing of the gene as compared to a fresh sample of the Onpattro™-type baseline formulation.

[00177] In one embodiment, the administration of a frozen sample of the lipid nanoparticle results in a four-fold increase in liver-specific expression of the protein or peptide encoded by the encapsulated mRNA or vector DNA as compared to a frozen sample of the Onpattro™-type baseline formulation.

[00178] In one embodiment, the administration of a frozen sample of the lipid nanoparticle results in a four-fold increase in liver-specific silencing of the gene as compared to a frozen sample of the Onpattro™-type baseline formulation. Editing of genetic material of hepatic cells

[00179] The LNP may comprise a nucleic acid that encodes for a protein or peptide that forms part of a “genetic editor” or “editor”, which includes without limitation products or compositions that edit genetic material, e.g., nucleic acid is inserted, deleted, modified (e.g., epigenetic editing) or replaced in the genetic material of an organism at a site-specific location.

[00180] The editor may be used for ex vivo or in vivo genetic modification of a hepatic cell and includes post-translational modifications.

[00181] The genetic editor includes, without limitation, Cas-based (e.g., CRISPR or non-CRISPR), transcription activator-like effector nuclease (TALEN), megaTALs, zinc finger nuclease (ZFN), Adenosine Deaminase Acting on RNA (ADAR), prime editors, base editors, epigenetic, transposase, meganuclease, ARCUS gene editing systems or any variant or combination thereof. These editors are exemplary, however, and the disclosure includes any product or composition that can modify genetic material (including RNA transcripts and noncoding regions) of a hepatic cell to treat, prevent or ameliorate a disorder or disease. Without limitation, the editor may include those that are designed by a process referred to as Directed Nuclease Editor (DNE), which is known to those of skill in the art.

[00182] Cas-based editors comprise CRISPR and non-CRISPR gene editing systems. In addition, the editor includes those that cut DNA as well as epigenetic editing systems that modify nucleic acid markers, as discussed below.

[00183] The CRISPR nucleic acid editor most advantageously comprises nucleic acid (e.g., mRNA) encoding for one or more of a Class II Cas nuclease family of proteins and a guide RNA. The nucleases encoded by the nucleic acid are enzymes with DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by an appropriate guide RNA. The nuclease and guide RNA form a complex referred to as a ribonucleoprotein (RNP). In some embodiments, the nuclease is a Class II CRISPR enzyme, which is further subdivided into Types II, V and VI. According to one embodiment, the mRNA encodes for a Cas protein that is part of a Type II CRISPR / Cas system, such as a Cas9 protein or a Cpfl protein.

[00184] In another embodiment, the mRNA encodes for a Cas protein that is part of a Type V CRISPR / Cas system, such as Cas 12a. In another embodiment, the mRNA encodes for a Cas protein that is a Cas 13a, which is an RNA endonuclease and cleaves single-stranded RNA.

[00185] The guide RNA can direct the Cas nuclease to the target sequence on a target nucleic acid molecule, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or modulates the sequence. In some embodiments, the guide RNA binds to a class 2 nuclease, thereby providing specificity of cleavage.

[00186] Guide RNAs for the CRISPR / Cas9 nuclease system include CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a targeting sequence that is complementary to and hybridizes to a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to, and hybridize to, a portion of tracrRNA. In some embodiments, the crRNA can correspond to the structure of a naturally-occurring crRNA transcribed from a bacterial CRISPR locus, wherein the targeting sequence acts as a spacer for the CRISPR / Cas9 system. The flagpole corresponds to the part of the repetitive sequence adjacent to the spacer above the CRISPR locus.

[00187] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of a crRNA. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can comprise at least one mismatch.

[00188] The length of the targeting sequence may depend on the RNP system and components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequences are: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides in length can be included. In some embodiments, the targeting sequence can comprise a length of 18 to 24 nucleotides. In some embodiments, the targeting sequence can comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence can comprise a length of 20 nucleotides.

[00189] In some embodiments, the nucleic acid editor includes Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csblll, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[00190] As noted, non-CRISPR, Cas-based nucleic acid editors are encompassed by embodiments of the disclosure as well. A Cas-based editor may include a Cas enzyme fused to deaminase (Luo et al., 2020, Microbial Cell Factories, 19(93), incorporated herein by reference). An example is a cytosine base editor or an adenine base editor produced by fusing endonuclease Cas to cytosine deaminase pmCDAl or heterodimer adenine deaminase TadA-TadA. A further non-limiting example is Cas fused to reverse transcriptase (Mohr et al., 2018, Mol Cell., 72(4):700-714, incorporated herein by reference).

[00191] Fanzor is a eukaryotic RNA-guided endonuclease that could function as a nucleic acid editor. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference). In some embodiments, Fanzor proteins use RNA as a guide to target DNA precisely and can be modified to edit a hepatic cell using the LNPs described herein. In some examples, the compact Fanzor systems may have the ability to facilitate more improved delivery than CRISPR-Cas systems.

[00192] In those embodiments in which the editor is a TALEN, the LNP comprises a nucleic acid encoding a peptide having a Transcription Activator-Like (TAL) effector DNA binding domain, a fragment or a variant thereof. In an embodiment, the editor comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In an embodiment, the peptide having nuclease activity is a type-II restriction 1 -like endonuclease, e.g., a Fokl endonuclease.

[00193] In those embodiments in which the nucleic acid editor is a zinc finger nuclease (ZFN), the nucleic acid may encode a peptide having: a Zinc finger DNA binding domain, a fragment or a variant thereof; and / or nuclease activity, e.g., endonuclease activity. In an embodiment, the Zn finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or more Zinc fingers. In an embodiment, the peptide having nuclease activity is a type-II restriction 1-like endonuclease, e.g., a Fokl endonuclease.

[00194] Adenosine Deaminase Acting on RNA (ADAR) is another editor encompassed by embodiments of the disclosure that may be used for post-transcriptional modification of RNA. Examples include ADAR1 and ADAR2. AD ARI may catalyze posttranscriptional deamination of C6 of adenosines in dsRNA, converting them to inosines (see Song et al., 2022, PMC, 13(1):el665, incorporated herein by reference).

[00195] Meganucleases are enzymes in the endonuclease family that may induce homologous recombination, generate mutations and alter reading frames. The meganuclease includes homing endonucleases that are intron or intein endonucleases. In one embodiment, the meganuclease is from the LAGLID ADG family, a GIY-YIG endonuclease, an HNH endonuclease, a His-Cys box endonuclease or a PD-(DZE)XK endonuclease. Meganucleases may be combined with components of other editors. In one embodiment, a DNA binding domain from a transcription activator-like (TAL) effector is combined with a meganuclease to produce a “megaTAL”. In another embodiment, a meganuclease may be fused to a DNA end-processing enzyme to promote an error-prone non-homologous end joining.

[00196] ARCUS nuclease is an editor based on I-Crel, which is a kind of homing endonuclease that evolved in the algae Chlamydomonas reinhardtii. In some embodiments, the nuclease can deactivate itself after gene editing, thereby reducing off-targeting. ARCUS nucleases in some embodiments can generate a unique cleavage site that is a four-base-pair, 3’ overhang and may be able to carry out gene insertion, gene excision, gene repair or a combination thereof.

[00197] Epigenetic editing is also encompassed by examples of the disclosure. Such editing of genetic material does not cut nucleic acid but rather alters epigenomic marks “adorning” DNA. Changing the epigenic signature of a hepatic cell can serve to modify an epigenetic signature of the cell and change its transcriptional profile. In some embodiments, the epigenetic editing system may target and edit one or more methylation sites of a nucleic acid sequence. In some embodiments, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted to function as only delivery systems. In one embodiment, an epigenetic modifying enzyme or domain can be fused to the homing protein and local epigenetic modifications can be altered upon protein recruitment. A targeting protein that recognizes DNA sequences may be linked to an effector protein that alters epigenomic marks, such as methylation. Examples of targeting proteins include Transcription Activator-Like Effector (TALE), zinc finger proteins, and Cas systems, including but not limited to CRISPR-Cas. Nonlimiting examples of effector proteins include TET1, which induces demethylation of cytosine at CpG sites; LSD1, which induces demethylation of H3K4mel / 2, which also causes an indirect effect of deacetylation on H3K27; and CIB1 / CRY2, which is a cryptochrome / blue light activated complex allowing chromatin to be modified upon illumination.

[00198] Further examples of effector proteins include DNA methyltransferase, a fragment (e.g., a biologically active fragment) or variant thereof (e.g., DNMT1, DNMT2 DNMT3A, DNMT3B, DNMT3L, or CpG methyltransferase (M. Sssl)); or a poly comb repressive complex or a component thereof, e.g., PRC1 or PRC2, or PR-DUB, or a fragment (e.g., biologically active fragment) or a variant thereof.

[00199] In an embodiment, the epigenetic editor comprises a molecule that modifies chromatin architecture and / or modifies a histone. In an embodiment, the epigenetic modulator is a molecule that modifies chromatin architecture, e.g., a SWI / SNF remodeling complex or a component thereof. In an embodiment, the epigenetic modulator is a molecule that modifies a histone, e.g., methylates and / or acetylates a histone, e.g., a histone modifying enzyme or a fragment (e.g., biologically active fragment) or a variant thereof, e.g., HMT, HDM, HAT, or HD AC.

[00200] The examples below are intended to illustrate the preparation of specific lipid nanoparticle preparations and properties thereof but are in no way intended to limit the scope of the invention.

[00201] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated. Examples Materials and Methods LNP preparation

[00202] The LNPs were prepared by dissolving mRNA in 25 mM sodium acetate, pH 4.0, while the lipid components at the mole % specified were dissolved in absolute ethanol. The lipids in ethanol and the reporter (firefly luciferase) mRNA in buffer were combined in a 1:3 volume by volume ratio using a t-junction with dual-syringe. The solutions were pushed through the t-junction at a combined flow rate of 20 mL / min (5 mL / minute for the lipid-containing syringe, 15 mL / minute for the mRNA-containing syringe). The mixture was subsequently dialyzed overnight against -1000 volumes of lx phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cut-off 12 000-14 000 or 3 000-5 000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 10 000 or 100 000 MWCO (molecular weight cut-off), regenerated cellulose concentrator.

[00203] For cryogenic storage of LNPs, buffer excahnge to a low salt (e.g. 15 mM TRIS), pH 7 buffer containing 20% sucrose (w / v) was first performed. LNPs in sucrose buffer were then stored at -80°C. Upon thawing, LNPs were remved from the -80°C freezer and left to warm to room temperature on the bench.

[00204] Encapsulation efficiency was calculated by determining unencapsulated mRNA content by measuring the fluorescence upon the addition of RiboGreen™ to the mRNA-LNP (F) and comparing this value to the total mRNA content that is obtained upon lysis of the LNP by 2% Triton X-100 (Ft): % encapsulation = (Ft - F\) / F\ * 100.

[00205] The particle size and poly dispersity index (PDI) were characterized using a Zetasizer Ultra Red™ (Malvern Panalytical™). Measurement of luminescence in organs / tissues in vivo and ex vivo

[00206] For all in vivo studies, LNPs at an mRNA concentration of 0.02 to 0.5 mg / kg were injected intravenously (i.v.) in CD-I mice (6-8 weeks old, male and female) at a volume using the formula weight of the mouse (in grams) * 2 pL. The mRNA used in the studies was firefly luciferase (fLuc) that was either internally sourced (NanoVation Therapeutics™, NTx fLuc) or sourced from RNA Technologies and Therapeutics (https: / / www.matechnologies.com / ; MG fLuc). At 4 hours post-injection, luciferin substrate (15 mg / mL) was i.p. injected at a dose of 150 mg / kg. The mice were imaged in vivo using an in vivo imaging system (PhotonIMAGER Optima, BioSpaceLab™; https: / / biospacelab.com) within 10 minutes of the substrate administration. The organs were then harvested, placed in a petri dish and luminescence measured using the in vivo imaging system. For the liver, ex vivo bioluminescence was determined by summing the bioluminescence of the 5 individual liver lobes. Bioluminsecence reported was normalized to per mg tissue weight.

[00207] For the kinetics studies, the dose was 0.5 mg / kg MG fLuc and the substrate, luciferin, was i. p. administered at 4 hours, 24 hours and 48 hours post-injection, followed by in vivo imaging and tail vein bleed at each time point. Tissue homogenate assay

[00208] The LNPs at a luciferase mRNA concentration of 0.1 mg / mL were injected intravenously (i.v.) in mice at 1 mg / kg. Organs were harvested at 24 hours after the LNP injections.

[00209] Tissues were removed from the mice and placed in 2 mL tubes and snap frozen in liquid nitrogen. The tissues were subsequently stored at -80°C. An appropriate volume of GLO™ lysis buffer from Promega™ was added to each of the tubes, ensuring that the samples remained frozen before addition of the lysis buffer. Samples were placed in a FastPrep™ homogenizer and the homogenizer was operated at a speed of 6 m / s for 20 seconds and repeated 2 times for a total of three rounds. The homogenized samples were spun down for 10 minutes at 12,000 rpm at room temperature and subsequently 50 pL of homogenate in duplicate was added to a black plate. The plate was transferred to a plate reader and the fluorescence was read at 640 nm excitation / 720 nm emissions. Luminescence was determined by adding 50 pL of Steady Gio™ substrate into the homogenate sample and a luciferase signal was read. Apparent pKa

[00210] The apparent pKa was measured using a 6-( / ?-Toluidino)-2-naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; Jayaraman et al., 2012, Angew. Chern Int. Ed., 51:85298533, which are incorporated herein by reference for the purposes of determining apparent pKa). In the adapted method, a series of buffers are prepared spanning a pH range of 2.5-10.9 in varying pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.12 mM TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polystyrene 96-well plate, to yield a final concentration of 6.25 and 12 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using a BMG LabTech CLARIOstar PLUS™ microplate reader at lex=321 nm, kem=445 nm. The fluorescence is subsequently plotted against pH using a sigmoidal curve fit through Prism™, in which thepKa is determined to be the pH value with 50% of maximal fluorescent intensity. Cryo-TEM

[00211] LNPs were concentrated to between 5 and 10 mg / mL total lipid prior to cryo-TEM imaging. A defined volume, for example 2-4 pL, of the resulting LNP solution was applied to a copper grid, and plunge-frozen using an FEI Mark IV Vitrobot™ to generate vitreous ice. These grids were stored in liquid nitrogen until imaged by a Glacios TEM. The instrument was operated at 200 kV in low-dose conditions and the resulting images were obtained using a bottom-mount FEI Falcon™ direct electron detector camera at 47-88,000 X magnification with an under-focus of 0.5-2 pm in order to enhance contrast. Determination of N / P ratio of lipid nanoparticles

[00212] N / P values describe the ratio of anionic charge (P) to cationic charge (N) within an oligonucleotide-containing lipid nanoparticle. To calculate the total lipid weight required to obtain a specific N / P ratio, the average molecular weight per anionic charge (P) of a single nucleic acid base / phosphate / ribose monomer (-300 g / mol) versus the molecular weight per cationic charge of the ionizable lipid (N) was first calculated. From this molar ratio, required ionizable lipid weights were calculated by required N / P*calculated molar ratio*ionizable lipid molecular weight (g / mol)* required oligonucleotide weight (g). From this, total lipid weights (including non-charged lipid components) were calculated based on the desired lipid composition (i.e., mol% of individual lipids) and individual lipid molecular weights. Example 1: LNPs having a non-polar glyceride and lacking phospholipid (TAG LNP) exhibit improved liver delivery of cargo relative to gold-standard baseline lipid nanoparticles

[00213] This example demonstrates that a lipid nanoparticle comprising a triacylglycerol (triolein, C18:l), referred to herein as “TAG LNP” and lacking phospholipid surprisingly exhibits significantly improved delivery of nucleic acid to the liver relative to a gold standard Onpattro™-type baseline formulation. The Onpattro™-type baseline formulation was selected as a baseline as it is a known formulation designed specifically for liver-tropism and contains 10 mol% phospholipid (DSPC). The two formulations tested are set forth in Table 1 below: Table 1: Triacylglycerol (C18:l) mRNA-LNP formulations with or without phospholipid prepared to examine liver mRNA expression in vivo Sample LNP composition Molar ratios Phospholipid (molar) TAG LNP Ionizable lipid l / cholesterol / triolein / PEG2ooo- DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 0 Onpattro™-type baseline LNP nMC3 / cholesterol / DSPC / PEG2ooo-DMG / DiR 50 / 38.5 / 9.9 / 1.5 / 0.1 10

[00214] The ionizable lipid included in the Onpattro™-like baseline LNP was nor-MC3 (nMC3) as described in co-owned and co-pending WO 2022 / 246571 and the ionizable lipid 1 in the inventive TAG-LNP was compound 24 of co-owned U.S. Patent No. 12,121,591 (incorporated herein by reference).

[00215] The LNPs additionally contained 0.1 mol% of a fluorescent lipid probe, DiR (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide). DiR was added to the TAG-LNP composition at the expense of 0.05 mol% cholesterol and 0.05 mol% triacylglycerol. DiR was added to the Onpattro™-type baseline composition at the expense of 0.1 mol% DSPC.

[00216] The TAG-LNP and Onpattro™-type baseline LNP were administered i.v. at a dose of 0.02, 0.1 and 0.5 mg / kg luciferase mRNA to CD-I mice, followed by substrate administration (luciferin) four hours post-administration as described in the Materials and Methods.

[00217] The results for 0.02 - 0.5 mg / kg luciferase mRNA (NTx fLuc) are shown in Figure 1A-D. Surprisingly, it was observed that the TAG-LNP having no phospholipid exhibited 7.5, 11.3 and 4.4 times the luminescence in the liver relative to the Onpattro™-type baseline formulation containing 10 mol% DSPC, at 0.02, 0.1 and 0.5 mg / kg fLuc mRNA doses respectively. Representative live, whole body bioluminescence images (0.5 mg / kg NTx fLuc) are shown in Figure ID. Example 2: LNPs having a non-polar glyceride and lacking phospholipid (TAG LNP) exhibit improved liver selectivity over spleen relative to the Onpattro™-type baseline formulation

[00218] The results from Example 1 revealed a luminescence signal in the spleen. The results for a dose of 0.5 mg / kg fLuc mRNA were therefore assessed for liver and spleen luminescence ex vivo to determine the extent of liver selectivity relative to the spleen for the TAG-LNP and the Onpattro™-type baseline formulation.

[00219] The results in Figure 2A show that the TAG LNP (0.5 mg / kg fLuc mRNA; Table 1) had 50 times the luminescence in the liver relative to the spleen, while the Onpattro™-type baseline formulation (0.5 mg / kg fLuc mRNA) with 10 mol% phospholipid had only a 26 times luminescence over the spleen.

[00220] Thus, the results demonstrate that the TAG LNP has superior liver tropism relative to the spleen when compared to the Onpattro™-type baseline formulation.

[00221] In a separate study, the above TAG-LNP with a different ionizable amino lipid (lipid 2 in Table 2) or further comprising cholesteryl hemisuccinate (CHEMS) was also examined for liver tropism relative to the spleen. The additional formulations examined (TAG-LNP 2 and TAG-LNP 3) are set forth in Table 2 below. The in vivo luciferase expression in the liver and spleen was assessed by quantifying luminescence in tissue homogenates as set forth in the Material and Methods above. Table 2: Triacylglycerol (C18:l) mRNA-LNP formulations with variations in ionizable lipid and CHEM prepared to examine liver mRNA expression in vivo Sample LNP composition Ionizable lipid* nMC3 benchmark nMC3 / cholesterol / DSPC / PEG2ooo-DMG nMC3 TAG-LNP 1 Ionizable lipid l / cholesterol / triolein / PEG2ooo-DMG 1 TAG-LNP 2 Ionizable lipid 2 / cholesterol / triolein / PEG2ooo-DMG 2 TAG-LNP 3 Ionizable lipid l / cholesterol / CHEMS / triolein / PEG2ooo-DMG 1 *nMC3 is set forth in WO 2022 / 246571; ionizable lipid 1 is compound 24 in U.S. Patent No. 12,121,591 and ionizable lipid 2 is compound 27 in U.S. Patent No. 12, 121,591, each of which are incorporated herein by reference.

[00222] TAG-LNP 2 with ionizable lipid 2 also exhibited liver tropism over the spleen in the tissue homogenate study (Figures 2B and 2C). In addition, TAG-LNP 3 that contained the additional lipid component, CHEMS, exhibited increased liver expression over the spleen (Figures 2D and 2E). Example 3: Expression kinetics of LNP comprising the non-polar glyceride and lacking phospholipid

[00223] The expression pharmacokinetics of the TAG LNP formulation (Table 1) were next examined. CD-I mice were intravenously injected with TAG LNP formulations with MG fLuc as the cargo as per the Materials and Methods. The dose was 0.5 mg / kg MG fLuc and the substrate, luciferin, was i.p. administered at 4 hours, 24 hours and 48 hours post-injection, followed by in vivo imaging and tail vein bleed at each time point.

[00224] The in vivo luminescence results are shown in Figure 3 A and Figure 3B. The whole body and abdominal luminescence were similar, indicating the majority of the fLuc expression is from the liver (and spleen), and the results indicate that maximal luminescence signal was sustained for at least 24 h post LNP administration. Example 4: The impact of long-term storage at 4°C

[00225] The stability of TAG LNP versus Onpattro™-type baseline LNP formulations was next examined. Following 3-month LNP storage at +4°C, CD-I mice were intravenously injected with TAG LNP (Table 1) or Onpattro™-type baseline LNP with NTx fLuc as the cargo, followed by substrate administration (luciferin) four hours post-administration as described in the Materials and Methods.

[00226] The results in Figure 4 show that after prolonged storage at 4°C, TAG LNP showed 58x luciferase expression compared to Onpattro™-type baseline LNPs within the liver. This surprising result demonstrates significant improvement in long term stability of mRNA within TAG LNPs compared to Onpattro™-type baseline LNPs. Example 5: The impact of freeze-thaw on LNP activity

[00227] The impact of freeze / thawing LNP samples was next examined. Following LNP thawing, CD-I mice were intravenously injected with TAGLNP (Table 1) or Onpattro™-type baseline LNP with NTx fLuc as the cargo, followed by substrate administration (luciferin) four hours postadministration as described in the Materials and Methods.

[00228] The results in Figure 5 show that after freeze / thaw, TAG LNP maintained 4. lx superior luciferase expression compared to Onpattro™-type baseline LNPs within the liver. This result demonstrates cryogenic storage of TAG LNPs does not adversely affect in vivo potency in the liver compared to Onpattro™-type baseline LNPs. Example 6: The impact of varying N / P ratios in TAG-LNPs.

[00229] The impact of varying N / P ratios was next examined. CD-I mice were intravenously injected with TAG LNP (Table 1) having varying N / P ratios from 3 to 6, with NTx fLuc as the cargo, followed by substrate administration (luciferin) four hours post-administration as described in the Materials and Methods.

[00230] The results from Figure 6 show that TAG LNPs maintain similar levels of mRNA expression within the liver across all N / P ratios tested. Example 7: The impact of the inclusion of phospholipid on liver delivery for TAG LNPs

[00231] To determine the impact of phospholipid (PL) on liver delivery of triacylglycerol (TAG) LNPs, the following three LNP formulations were prepared. Table 3: Lipid formulations tested to examine the impact of phospholipid on liver delivery for TAG LNPs Sample Tradename DSPC (mol%) TAG LNP composition* Molar ratios +PL -TAG Comimaty™-type LNP 9.4 0 norMC 3 / chol esterol / D SPC / ALC-0159 / DiR 46.3 / 42.6 / 9.4 / 1.6 / 0.1 +PL +TAG Comimaty™-type LNP + TAG 9.4 21.3 norMC 3 / chol esterol / D SPC / triolein / ALC-0159 / DiR 46.3 / 21.3 / 9.4 / 21.3 / 1.6 / 0.1 -PL +TAG Inventive TAG LNP 0 31.7 norMC3 / cholesterol / tri olein / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1

[00232] Figure 7 shows that adding TAG to a baseline LNP containing 9.4 mol% DSPC (Comimaty™-type LNP) provided only a modest enhancement in liver delivery as measured by luminescence (compare +PL -TAG to +PL +TAG formulations, left and middle bars). However, the addition of TAG to a phospholipid-free LNP (-PL) increased the liver luminescence signal significantly (-PL +TAG, right bar).

[00233] The results at four hours post-administration show that the significant improvements in liver luminescence for TAG-containing LNPs are realized in the absence of phospholipid. Example 8: Liver toxicity studies

[00234] TAG LNP and Onpattro™-type baseline LNPs were prepared as set out in Table 4 to assess liver toxicity. The LNPs were dosed at 0.5 mg / kg fLuc mRNA to mice. Total bilirubin, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, amylase and lactate dehydrogenase (LDH) were measured. Table 4: Triacylglycerol (C18:l) mRNA-LNP formulations with or without phospholipid prepared to examine liver toxicity Sample LNP composition* Molar ratios Phospholipid (molar) Phosphate buffered saline (PBS) NA NA NA TAG-LNP Ionizable lipid 1 / cholesterol / triolein / PEG2ooo- DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 0 Onpattro™-type baseline LNP nMC3 / cholesterol / DSPC / PEG2ooo-DMG / DiR 50 / 38.5 / 9.9 / 1.5 / 0.1 10

[00235] The results presented in Figure 8A-G show that no acute liver toxicity was observed with the TAG-LNP. Example 9: Batch-to-batch consistency of TAG LNPs

[00236] To assess the variability of physiochemical properties of TAG LNPs between batches, the Onpattro™-type baseline and TAG LNPs were prepared as set out in Table 4 above. The studies varied the formulation volume (2-20 mL scale), initial total lipid concentration (5 - 20 mM), the fLuc mRNA source (internal NTx mRNA) or sourced from RNA Technologies and Therapeutics (MG mRNA), the device used for preparing the LNPs (T-mixer vs microfluidic chip) and the formulator.

[00237] The results show that the particle size (nm), poly dispersity index (PDI) and mRNA encapsulation % did not vary significantly between batches (Figures 9A-C).

[00238] The apparent pKa for the Onpattro™-type baseline LNPs were 6.38, while the TAG LNPs had a pKa of 6.58 (Figure 9D). Example 10: TAG LNP morphology differs from that of the Onpattro™-type baseline formulation

[00239] The morphology of the LNPs of Table 4 was assessed by Cryo-TEM. The Cryo-TEM images are shown in Figure 10A and 10B for the TAG LNPs and the Onpattro™-type baseline LNPs, respectively.

[00240] The images reveal that the LNPs contained small bleb-like structures as indicated by the arrows (Fig. 10A). By contrast, the Onpattro™-type baseline LNPs lacked blebs and were solid core, which is consistent with previous observations for Onpattro™-type baseline formulations. These results are surprising since the LNPs lacking a conventional bilayer forming lipid (e.g. DSPC phospholipid) would not be expected to form stable membranes surrounding an inner aqueous LNP compartment. Example 11: Formulations of triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), methyl ricinoleate (MR) and castor oil (CO) in phospholipid-less lipid nanoparticles

[00241] This example shows that a variety of different non-polar glycerides containing a single acyl chain (monoacylglycerols or MAGs), two acyl chains (diacylglycerols or DAGs), three acyl chains (triacylglycerols or TAGs), cholesteryl esters, esterified fatty acids can castor oil can be formulated in phospholipid-less lipid nanoparticles. Formulations tested are set forth in Table 5 below: Table 5: LNPs containing non-polar glycerides investigated to assess compatibility and potency of phospholipid-less LNPs Sample LNP composition* Molar ratios TAG-LNP Ionizable lipid 1 / cholesterol / triolein / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 DAG-LNP Ionizable lipid 1 / cholesterol / 1,3-di ol ein / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 MAG-LNP Ionizable lipid 1 / cholesterol / 1-Monooleyl-rac-glycerol / PEG2000-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 CE-LNP Ionizable lipid 1 / cholesterol / cholesteryl oleate / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 MR-LNP Ionizable lipid 1 / cholesterol / methyl ricinoleate / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1 CO-LNP Ionizable lipid 1 / cholesterol / castor oil / PEG2ooo-DMG / DiR 35 / 31.7 / 31.7 / 1.5 / 0.1

[00242] The results presented in Figure 11 show that phospholipid-less LNPs can be formulated with a variety of structurally distinct non-polar lipids, including glycerides and esterified fatty acids. Following intravenous administration in CD-I mice with NTx fLuc as the cargo, DAG-, CE-, MR- and CO-LNPs tested in this example demonstrated similar expression potency in the liver relative to the parent TAG-LNP.

Claims

1. A lipid nanoparticle comprising:(i) a nucleic acid cargo molecule;(ii) a sterol at a content of between 0 mol% and 50 mol%;(iii) an ionizable lipid present at a content of between 20 mol% and 70 mol%;(iv) a non-polar lipid present at a content of between 20 mol% and 80 mol%;(v) a phospholipid at a content of 0 to 5 mol%; and(vi) a hydrophilic polymer-lipid conjugate at a content of between 0 mol% and 3 mol%, wherein each mol% content is relative to total lipid present in the lipid nanoparticle.

2. A lipid nanoparticle comprising:(i)         encapsulated nucleic acid;(ii)         an ionizable lipid;(iii) a homogeneous generally spherical hydrophobic core of a non-polar lipid and one or more peripheral bleb structures as observed by cryogenic electron microscopy (cryo-TEM); (iv)        optionally a sterol;(iv)       optionally a hydrophilic polymer-lipid conjugate; and(v) a phospholipid at a content of 0 to 5 mol%.

3. The lipid nanoparticle of claim 1 or 2, wherein the non-polar lipid is a combination of two or more non-polar lipids.

4. The lipid nanoparticle of claims 1, 2 or 3, wherein the non-polar lipid is a triglyceride.

5. The lipid nanoparticle of claim 4, wherein the triglyceride is selected from triolein,tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, triarachidin and oleoyldipalmitin.

6. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is a diglyceride.

7. The lipid nanoparticle of claim 6, wherein the diglyceride is selected from glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol distearate, glycerol diarachidate,glycerol dibehenate, glycerol dipalmitoleate, glycerl dioleate, glycerol dilinoleate, glycerol dilinolenate and glycerol di arachidonate.

8. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is a monoglyceride.

9. The lipid nanoparticle of claim 8, wherein the monoglyceride is selected from lauroyl-rac-glycerol, glycerol monomyristate, glycerol monopalmitate, glycerol monostearate, glycerol monoarachidate, glycerol monobehenate, glycerol monopalmitoleate, glycerol monooleate, glycerol monolinoleate, glycerol monolinolenate, glycerol monoarachidonate, and glycerol monocaprylate, and / or for example 1-monomyristoyl-rac glycerol, 1-mono-palm itoyl-rac-glycerol, 2-monopalm itoylglycerol, 1-mono-palm itolenyl-rac-glycerol, 1-monostearoyl-rac-glycerol, 1-monoleoyl-rac-glycerol, 1- monolinoleoyl-rac-glycerol and 1-monolinolenoyl-rac-glycerol.

10. The lipid nanoparticle of any one of claims 1 to 4, wherein the non-polar lipid is castor oil.

11. The lipid nanoparticle of claim 1 or 2, wherein the non-polar lipid is methyl ricinoleate.

12. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 22 mol% and 38 mol%.

13. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 25 mol% and 35 mol%.

14. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar lipid is present at a content between 27 mol% and 32.5 mol%.

15. The lipid nanoparticle of claim 1, 2 or 3, wherein the non-polar glyceride or analogue thereof is present at a content between 28 mol% and 32 mol%.

16. The lipid nanoparticle of any one of claims 1 to 15, wherein the ionizable lipid is an amino lipid.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the ionizable lipid is present at a content between 23 mol% and 47 mol%.

18. The lipid nanoparticle of any one of claims 1 to 17, wherein the ionizable lipid is present at a content between 25 mol% and 45 mol%.

19. The lipid nanoparticle of any one of claims 1 to 18, wherein the ionizable lipid is present at a content between 27 mol% and 43 mol%.

20. The lipid nanoparticle of any one of claims 1 to 19, wherein the ionizable lipid is present at a content between 28 mol% and 42 mol%.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the ionizable lipid is present at a content between 29 mol% and 41.5 mol%.

22. The lipid nanoparticle of any one of claims 1 to 21, wherein the ionizable lipid is presentat a content between 29.5 mol% and 41 mol%.

23. The lipid nanoparticle of any one of claims 1 to 11, wherein the ionizable lipid is presentat a content between 30 mol% and 40 mol%.

24. The lipid nanoparticle of any one of claims 1 to 23, wherein the phospholipid content isless than 4 mol%.

25. The lipid nanoparticle of any one of claims 1 to 24, wherein the phospholipid content isless than 3 mol%.

26. The lipid nanoparticle of any one of claims 1 to 25, wherein the phospholipid content isless than 2 mol%.

27. The lipid nanoparticle of any one of claims 1 to 26, wherein the phospholipid content isless than 1 mol%.

28. The lipid nanoparticle of any one of claims 1 to 27, wherein the phospholipid content isless than 0.5 mol%.

29. The lipid nanoparticle of any one of claims 1 to 28, wherein the lipid nanoparticle has nodetectable amount of phospholipid.

30. The lipid nanoparticle of any one of claims 1 to 29, wherein the hydrophilic-polymer lipid is present at a content between 0.75 mol% and 2.5 mol%.

31. The lipid nanoparticle of any one of claims 1 to 30, wherein the hydrophilic-polymer lipid is present at a content between 1 mol% and 2.25 mol%.

32. The lipid nanoparticle of any one of claims 1 to 31, wherein the hydrophilic-polymer lipid is present at a content between 1.25 mol% and 2 mol%.

33. The lipid nanoparticle of any one of claims 1 to 32, wherein the hydrophilic-polymer lipid is present at a content between 1.25 mol% and 1.75 mol%.

34. The lipid nanoparticle of any one of claims 1 to 33, wherein the sterol is present and is a non-cationic lipid.

35. The lipid nanoparticle of any one of claims 1 to 34, wherein the sterol is cholesterol.

36. The lipid nanoparticle of any one of claims 1 to 35, wherein the sterol is present at a contentbetween 22 mol% and 38 mol%.

37. The lipid nanoparticle of any one of claims 1 to 36, wherein the sterol is present at a content between 25 mol% and 35 mol%.

38. The lipid nanoparticle of any one of claims 1 to 37, wherein the sterol is present at a content between 27 mol% and 32.5 mol%.

39. The lipid nanoparticle of any one of claims 1 to 38, wherein the sterol is present at a content between 28 mol% and 32 mol%.

40. The lipid nanoparticle of any one of claims 1 to 39, wherein an N / P charge ratio of the cationic charge (N) of the ionizable lipid to the anionic charge (P) of the nucleic acid cargo is between 1 and 15.

41. The N / P charge ratio of claim 40, wherein the N / P charge ratio is between 2 and 6.

42. The N / P charge ratio of claims 40 or 41, wherein the ratio is between 3 and 6.

43. The lipid nanoparticle of any one of claims 1 to 40, wherein the nucleic acid is selected from an siRNA, mRNA, a vector nucleic acid, an antisense oligonucleotide, a nucleic acid-protein complex, and a nucleic acid-peptide complex.

44. The lipid nanoparticle claim 43, wherein the nucleic acid is selected from siRNA, vector nucleic acid, and an antisense oligonucleotide.

45. The lipid nanoparticle of claim 43, wherein the nucleic acid is mRNA.

46. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has a bleb structure asvisualized by cryogenic electron microscopy (cryo-TEM).

47. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has a homogeneous core with one or more peripheral blebs as visualized by cryogenic electron microscopy (cryo-TEM).

48. The lipid nanoparticle of any one of claims 1 to 47, the lipid nanoparticle resulting in at least a 10% increase in expression of the protein or peptide at 4 hours, 24 hours, and / or 48 hours post-injection in the liver of a mammal as compared to baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol encapsulating the nucleic acid, but otherwise measured under identical conditions, wherein the expression is measured using luminescence.

49. A method for delivery of mRNA or vector DNA for in vivo production of protein or peptide in the liver, the method comprising administering to a mammal a lipid nanoparticle of any one of claims 1 to 48, wherein the nucleic acid is mRNA or vector DNA and wherein the administering of the lipid nanoparticle results in an increased liver-specific expression of the protein or peptide encoded by the mRNA or vector DNA as compared to a baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol delivering the mRNA or vector DNA.

50. A method for delivery of siRNA or antisense oligonucleotide for in vivo silencing of a gene in the liver, the method comprising administering to a mammal a lipid nanoparticle of any one of claims 1 to 48, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in an increased liverspecific silencing of the gene as compared to a baseline formulation of nor-MC3 ionizable lipid / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mol:mol delivering the siRNA or antisense oligonucleotide.

51. A method for delivering a nucleic acid to a hepatic cell to treat a disease, disorder or condition, the method comprising contacting the lipid nanoparticle of any one of claims 1 to 48 with the hepatic cell in vivo or in vitro.

52. The method of claim 49, wherein the increased expression of the protein or peptide encoded by the mRNA in the liver over the spleen is at least 10% greater than the increased expression in a baseline LNP in the liver over the spleen measured under otherwise identical conditions.

53. The method of any one of claims 49 to 52, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 5 mg / kg.

54. The method of any one of claims 49 to 53, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 4 mg / kg.

55. The method of any one of claims 49 to 54, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 3 mg / kg.

56. The method of any one of claims 49 to 55, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 2 mg / kg.

57. The method of any one of claims 49 to 56, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 1.5 mg / kg.

58. The method of any one of claims 49 to 57, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 1 mg / kg.

59. The method of any one of claims 49 to 58, comprising administration of a dose of the lipid nanoparticle between 0.075 mg / kg and 0.75 mg / kg.

60. The method of any one of claims 49 to 59, wherein the phospholipid content is less than 4 mol%.

61. The method of any one of claims 49 to 60, wherein the sterol is present at 25 to 45 mol%.

62. The method of claim 61, wherein the sterol is present at 25 to 40 mol%.

63. The method of any one of claims 49 to 62, wherein a hydrophilic polymer-lipid conjugateis present in the lipid nanoparticle at a content between 0 and 3 mol%.

64. The method of claim 63, wherein the hydrophilic polymer-lipid conjugate is present in the lipid nanoparticle at a content between 0.5 and 2.0 mol%.

Images