Vaccine delivery method and composition for use thereof

Abstract

Provided is a method for inducing an immune response in a subject to treat or prevent a disease or disorder, the method comprising administering a vaccine comprising a lipid nanoparticle encapsulating nucleic acid encoding an antigenic protein, peptide or fragment thereof, a neutral lipid content of from 30 mol% to 70 mol%, a sterol or derivative thereof and an ionizable cationic amino lipid at between 5 and 50 mol%, and a hydrophilic polymer-lipid conjugate that is present at a lipid content of 0 mol% to 5 mol%, wherein the administering of the lipid nanoparticle results in the immune response against the antigenic protein, peptide or fragment thereof expressed by the mRNA in the subject, wherein each mol% is measured relative to a total lipid content of the lipid nanoparticle. The disclosure further provides vaccine compositions and use of such compositions to induce an immune response in a subject.

Description

VACCINE DELIVERY METHOD AND COMPOSITION FOR USE THEREOFTechnical Field

[0001] The present disclosure relates to methods of vaccine delivery and compositions for use thereof.Background

[0002] Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™. Onpattro™ is a lipid nanoparticle-based short interfering RNA (siRNA) drug formulation for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. The Onpattro™ LNP formulation consists of four main lipid components, namely: ionizable amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and polyethylene glycol conjugated lipids (PEG-lipids) at respective molar amounts of 50 / 10 / 38.5 / 1.5. Onpattro™ is still considered the gold standard for comparison in studies of LNP -mediated efficacy and current approaches to LNP design make few deviations from the four-component system.

[0003] Of these four components, the ionizable lipid makes up the bulk of the Onpattro™ formulation (50 mol%) and is considered important for the in vitro and in vivo activity of the LNP. Accordingly, most work in the field has focused on improving this lipid component. The ionizable lipid, which is typically an amino lipid, has been carefully designed so that it is charged at low pH and near-neutral at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid during initial formulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. 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.

[0004] With respect to the remaining three lipid components, hydrophilic polymer-lipid conjugates, such as the PEG-lipid, are well known for improving circulation longevity of the LNP. Furthermore, hydrophilic polymer-lipid conjugates are thought to play a key role in extending the storage stability of LNPs by reducing aggregation of the particles over time. Cholesterol functionsto stabilize the particle by broadening the phase transition temperature. Generally, however, comparatively less attention has been devoted to studying DSPC beyond its role as a structural lipid.

[0005] The success of the Onpattro™ LNP delivery system paved the way for the clinical development of the leading LNP -based COVID-19 mRNA vaccines, which rely on a similar formulation of lipids. It is widely believed that the efficacy of an mRNA vaccine will increase as protein expression is increased and prolonged (Schlake et al., 2012, RNA Biology, 9(11): 1319- 1330). Thus, previous and on-going efforts in vaccine development are aimed at increasing mRNA expression from the cargo. For instance, the LNP -based COVID-19 mRNA vaccines were designed with mRNA having an RNA CAP, polyA tail and untranslated regions that impact mRNA stability, thereby improving mRNA antigenic expression. Codon optimization and sequence modifications of the open reading frame can also improve protein expression. (See Kim et al., 2022, Mol Cell Toxiol., 18(1): 1-8). However, engineering mRNA structural elements to improve expression is time consuming and could suffer from scale-up challenges.Summary

[0006] The present disclosure addresses one or more of the foregoing problems in the prior art and / or provides useful alternatives to known vaccine compositions.

[0007] The present disclosure is based on the finding that an LNP formulation (herein “IcLNP™”) that includes elevated levels of a neutral lipid relative to an Onpattro™ benchmark LNP (herein “Onpattro™-type formulation” or “baseline LNP formulation”) can provide a therapeutically effective immune response in a subject against an antigenic protein, peptide or fragment thereof expressed by an encapsulated nucleic acid. In some examples of the disclosure, and without being bound by theory, it has been found that an equivalent immune response is mounted by the nucleic acid-containing-lcLNP™ relative to Onpattro™-type formulation, despite a significantly lower expression level from the IcLNP™ after local injection.

[0008] The inventors further discovered that the IcLNP™ formulations lacking a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid) resulted in an increased immune response in an otherwise identical IcLNP™ formulation that lacked the hydrophilic polymer-lipid conjugate.

[0009] According to one aspect of the disclosure, there is provided a method for inducing an immune response in a subject to treat, prevent or ameliorate a disease, condition or disorder, the method comprising administering to the subject a vaccine comprising a lipid nanoparticle encapsulating an nucleic acid encoding an antigenic protein, peptide or fragment thereof, at least 25 mol% of a neutral lipid, a sterol or derivative thereof at a content of at least 10 mol%, and an ionizable cationic amino lipid at between 10 and 40 mol%, optionally the ionizable cationic amino lipid having one or more lipophilic chains with a biodegradable group, wherein the administering of the lipid nanoparticle results in the immune response against the antigenic protein, peptide or fragment thereof expressed by the mRNA in the subject, and wherein each mol% is measured relative to a total lipid content of the lipid nanoparticle.

[0010] In one embodiment of the foregoing method, the neutral lipid has a phosphocholine head group and is optionally a phosphatidylcholine lipid or a sphingolipid.

[0011] In one embodiment of the foregoing method, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

[0012] In one embodiment of the foregoing method, the sphingolipid is sphingomyelin.

[0013] In one embodiment of the foregoing method, the neutral lipid is present at a content of at least 30 mol%.

[0014] In one embodiment of the foregoing method, the neutral lipid is present at a content of at least 35 mol%.

[0015] In one embodiment of the foregoing method, the neutral lipid is present at a content of at least 40 mol%.

[0016] In one embodiment of the foregoing method, the neutral lipid is present at 45 mol%.

[0017] In one embodiment of the foregoing method, the neutral lipid is present at 65 mol% or less.

[0018] In one embodiment of the foregoing method, the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

[0019] In one embodiment of the foregoing method, the hydrophilic polymer-lipid conjugate is a polyethylene-glycol lipid conjugate, a polysaccharide-lipid conjugate or a polysarcosine-lipid conjugate.

[0020] In one embodiment of the foregoing method, the lipid nanoparticle comprises or consists essentially of the neutral lipid, the sterol or derivative thereof, and the ionizable cationic amino lipid and wherein the lipid nanoparticle has 0-1.0 mol% or 0-0.5 mol% of the hydrophilic polymerlipid conjugate.

[0021] In one embodiment of the foregoing method, the lipid nanoparticle has 0-0.2 mol% of the hydrophilic polymer-lipid conjugate.

[0022] In one embodiment of the foregoing method, the lipid nanoparticle has 0-0.1 mol% of the hydrophilic polymer-lipid conjugate.

[0023] In one embodiment of the foregoing method, the lipid nanoparticle is used to treat or prevent a disease or disorder that is an infectious disease, cancer or an autoimmune disorder.

[0024] In one embodiment of the foregoing method, the lipid nanoparticle is used to treat or prevent a disease or disorder that is an infectious disease.

[0025] In one embodiment of the foregoing method, the lipid nanoparticle is used to treat a disease or disorder that is an infectious disease and wherein the infectious disease is a viral disease.

[0026] In one embodiment of the foregoing method, the lipid nanoparticle is used to treat a disease or disorder that is cancer.

[0027] In one embodiment of the foregoing method, an antibody response is raised against the antigenic protein, peptide or fragment thereof expressed by the nucleic acid in the subject.

[0028] In one embodiment of the foregoing method, the antibody response comprises IgG against the antigenic protein, peptide or fragment thereof expressed by the nucleic acid in the subject.

[0029] In one embodiment of the foregoing method, the lipid nanoparticle is administered at a dosage of from 0.01 mg to 20 mg.

[0030] In one embodiment of the foregoing method, the lipid nanoparticle is administered at a dosage of from 0.5 mg to 15 mg.

[0031] In one embodiment of the foregoing method, the lipid nanoparticle is administered at a dosage from 0.5 mg to 10 mg.

[0032] In one embodiment of the foregoing method, the lipid nanoparticle is administered at a dosage from 0.5 mg to 5 mg.

[0033] In one embodiment of the foregoing method, the lipid nanoparticle is administered at a dosage from 0.5 mg to 1 mg.

[0034] In one embodiment of the foregoing method, further comprising administering a booster dose of the vaccine.

[0035] According to another aspect of the disclosure, there is provided a vaccine comprising a lipid nanoparticle, the lipid nanoparticle comprising:(i) a nucleic acid encoding an antigenic protein, peptide or fragment thereof;(ii) a neutral lipid content of from 30 mol% to 70 mol%;(iii) an ionizable cationic lipid content of from 5 mol% to 50 mol%;(iv) a sterol selected from cholesterol or a derivative thereof; and(v) a hydrophilic polymer-lipid conjugate that is present at a lipid content of 0 mol% to 5 mol%, wherein each lipid content is relative to a total lipid content of the lipid nanoparticle.

[0036] According to another aspect of the disclosure, there is provided a vaccine comprising a lipid nanoparticle comprising encapsulated nucleic acid encoding an antigenic protein, peptide or fragment thereof and 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, an ionizable cationic lipid and a sterol, the lipid nanoparticle inducing at least an equivalent immune response in an animal as measured by IgG levels against the antigenic protein, peptide or a fragment thereof at 21 days and / or 35 days post-injection as compared to a baseline LNP formulation of ALC-0315 / 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 IgG level is measured using electrochemiluminescence.

[0037] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

[0038] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid is distearoylphosphatidylcholine (DSPC) or dipalmitoyl-phosphatidylcholine (DPPC).

[0039] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid is a mixture of two phosphatidylcholine lipids.

[0040] In one example of any one the foregoing aspects or embodiments thereof, the mixture comprises distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

[0041] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid content is between 40 mol% and 60 mol%.

[0042] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid content is between 42 mol% and 58 mol%.

[0043] In one example of any one the foregoing aspects or embodiments thereof, the neutral lipid content is between 45 mol% and 55 mol%.

[0044] In one example of any one the foregoing aspects or embodiments thereof, the ionizable cationic lipid is an amino lipid having at least one lipophilic chain and wherein the at least one lipophilic chain comprises a biodegradable group.

[0045] In one example of any one the foregoing aspects or embodiments thereof, wherein the ionizable cationic lipid is present at less than 40 mol%.

[0046] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

[0047] In one example of any one the foregoing aspects or embodiments thereof, the hydrophilic polymer-lipid conjugate is a polyethyleneglycol-lipid conjugate a polysaccharide-lipid conjugate or a polysarcosine-lipid conjugate.

[0048] In one example of any one the foregoing aspects or embodiments thereof, the hydrophilic polymer lipid conjugate is present at 0 to 1.0 mol%.

[0049] In one example of any one the foregoing aspects or embodiments thereof, the hydrophilic polymer lipid conjugate is present at 0 to 0.8 mol%.

[0050] In one example of any one the foregoing aspects or embodiments thereof, the hydrophilic polymer lipid conjugate is present at 0 to 0.5 mol%.

[0051] In one example of any one the foregoing aspects or embodiments thereof, the sterol is present at from 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0052] In one example of any one the foregoing aspects or embodiments thereof, the sterol is present at from 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

[0053] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle exhibits an IgG response in vivo at 1 pg that is equivalent or greater than an IgG response resulting from a lipid nanoparticle having the baseline LNP formulation and that is otherwise identical.

[0054] According to a further aspect of the disclosure, there is provided a lipid nanoparticle lacking a stabilizer to induce an immune response against the antigenic protein, peptide or fragment thereof expressed by an mRNA, wherein the lipid nanoparticle comprises an ionizable cationic amino lipid with one or more biodegradable groups in one or more of its lipophilic moieties or chains.

[0055] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle is present at a dosage from 0.01 mg to 20 mg.

[0056] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle is present at a dosage from 0.5 mg to 15 mg.

[0057] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle is present at a dosage from 0.5 mg to 10 mg.

[0058] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle is present at a dosage from 0.5 mg to 5 mg.

[0059] In one example of any one the foregoing aspects or embodiments thereof, the lipid nanoparticle is present at a dosage from 0.5 mg to 1 mg.Brief description of the drawings

[0060] Figure 1A shows the luciferase expression level in mice as indicated by bioluminescence imaging using IVIS® Lumina II In Vivo Imaging System at 4 hours post-injection with 10 mg ofluciferase mRNA encapsulating LNPs having 10 mol% DSPC (ALC-0315:DSPC:Chol:PEG2ooo- DMG (50:10:38.5: 1.5, mol:mol) (“Onpattro-type formulation”)); or 50 mol% DSPC (ALC- 0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mokmol)) ("IcLNP™”); and an untreated mouse used as a negative control.

[0061] Figure IB shows the luciferase expression level in mice as indicated by bioluminescence imaging using IVIS® Lumina II In Vivo Imaging System at 24 hours post-injection of a mouse for the formulations described in Figure 1A.

[0062] Figure 2 shows the electrochemiluminescence signals of the induced IgG levels against the receptor binding domain (RBD) of the spike protein in mice serum as indicated at 35 days postinjection of a boost dose of mRNA vaccine LNPs having 10 mol% DSPC (ALC- 0315:DSPC:Chol:PEG2ooo-DMG (50:10:38.5: 1.5, mokmol) (“Onpattro™-type formulation”)); or 50 mol% DSPC (ALC-0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mokmol) (“ALC IcLNP”).

[0063] Figure 3 shows the electrochemiluminescence signals of the induced IgG levels against the spike protein in mice serum as indicated at Day 21 and Day 35 post-injection of a prime dose at Day 0 and a boost dose at Day 21, respectively, of the following formulations: negative control phosphate buffered saline (PBS); mRNA vaccine LNPs having 10 mol% DSPC (ALC- 0315:DSPC:Chol:PEG2ooo-DMG (50: 10:38.5: 1.5, mokmol) (“Onpattro™-type formulation”)); ALC IcLNP™ formulation having 50 mol% DSPC (ALC-0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mokmol)); a negative control luc IcLNP™ formulation having 50 mol% DSPC (ALC-0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mokmol)); and IcLNP™ formulations with various ionizable lipids as indicated having 50 mol% DSPC (ionizable lipid:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1: 1.5, mokmol). Apart from the negative control, each formulation comprises mRNA encoding for the Sars-Cov2 spike protein.

[0064] Figure 4A shows the electrochemiluminescence signals of the induced IgG levels against the spike protein in mice serum as indicated at Day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21, respectively, of the following formulations: ionizable lipid 3:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mokmol) or ionizable lipid 3:DSPC:Chol (27.4:50:22.6 mokmol). Each formulation comprises mRNA encoding for the Sars-Cov2 spike protein.

[0065] Figure 4B shows the electrochemiluminescence signals of the induced IgG levels against the spike protein in mice serum as indicated at Day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21, respectively, of the following formulations: Nor MC3:DSPC:Chol (27.4:50:22.6, mol:mol encapsulating luciferase; ALC-0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5, mol:mol) encapsulating mRNA for Sars-Cov2 spike protein; and Ionizable lipid 1 :DSPC:Chol (27.4:50:22.6, mokmol) encapsulating mRNA for Sars-Cov2 spike protein (see also Table 5).

[0066] Figure 4C shows the electrochemiluminescence signals of the induced IgG levels against receptor binding protein (RBC) in mice serum as indicated at Day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21, respectively, of the formulations set out in Figure 4B above (see also Table 5).

[0067] Figure 5A shows the luciferase expression level in mice as indicated by bioluminescence imaging using Lumina II In Vivo Imaging System (IVIS®) at 4 hours post-injection with 1 mg of LNPs encapsulating luciferase mRNA having PEG (Ionizable lipid 3:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5 mokmol)); lacking PEG (Ionizable lipid 3:DSPC:Chol (27.4:50:22.6)) ("PEGless IcLNP™”); and an untreated mouse used as a negative control. The formulations and ionizable lipid are described in Table 4.

[0068] Figure 5B shows the luciferase expression level in mice as indicated by bioluminescence imaging using the IVIS® imaging system at 24 hours post-injection of mice for the PEG- containing, PEGless formulations described in Figure 5A. An untreated mouse was used as a negative control.

[0069] Figure 6A shows the interleukin-6 (IL-6) pg / mL in mice measured ex vivo as indicated at 6 hours post-injection after a prime dose at Day Oof the following two PEG-containing baselines and PEGless formulation: ALC-0315:DSPC:Chol:PEG2ooo-DMG (50:10:38.5: 1.5 mokmol) containing mRNA encoding luciferase (ALC 50 / 10 luc); ALC-0315:DSPC:Chol:PEG2ooo-DMG (27.4:50:21.1 : 1.5 mokmol) containing mRNA coding for SARS-CoV2 Spike Protein (ALC 50 / 10); and ionizable lipid l :DSPC:Chol (Ionizable lipid l :DSPC:Chol (27.4:50:22.6 mokmol) containing mRNA coding for SARS-CoV2 Spike protein. A mouse treated with phosphate buffered saline (PBS) was used as a negative control. The formulations and ionizable lipid are described in described in Table 6.

[0070] Figure 6B shows the interferon gamma (IFN-y) pg / mL in mice measured ex vivo at 6 hours after the prime doseof the same formulations and negative control as described in Figure 6A (see also Table 6) encapsulating mRNA encoding luciferase or SARS-CoV2.

[0071] Figure 6C shows the interferon gamma (TNF-a) pg / mL in mice measured ex vivo at 6 hours after the primedoseof the same formulations and negative control as described in Figure 6A (see also Table 6) encapsulating mRNA encoding luciferase or SARS-CoV2.

[0072] Figure 7A shows the interferon gamma (IFN-y) activity / 300,000 splenocytes in mice measured ex vivo as indicated at day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21 of the of the same formulations and negative control as described in Figure 6A (see also Table 6) encapsulating mRNA encoding luciferase or SARS-CoV2.

[0073] Figure 7B shows the interleukin-4 (IL-4) activity / 300, 000 splenocytes in mice measured ex vivo as indicated at day 35 post-injection after the prime dose and booster of the same formulations and negative control as described in Figure 6A encapsulating mRNA encoding luciferase or SARS-CoV2 RBD (see also Table 6).

[0074] Figure 8A shows the percentage of activated CD4+cells positive for various T-helper type 1 (TH-type l)cytokines of mice measured ex vivo, namely interferon gamma (IFN-y+), tumour necrosis factor alpha (TNF-a+), IFN-y+ and TNF-a+, interleukin-2 (11-2+) and minute levels of T helper type 2 (TH-type 2) cytokine interleukin-4 (IL-4+)) at Day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21. The same formulations were injected as in Figure 6 A (see also Table 6) with mRNA encoding luciferase or SARS-CoV2 Spike protein. The negative control was PBS treated mice.

[0075] Figure 8B shows the percentage of activated CD8+cells positive for various type 1 cytokines in mice measured ex vivo as indicated (IFN-y+, TNF-a+, IFN-y+ and TNF-a+, 11-2+) and type 2 cytokine IL-4+ at Day 35 post-injection after a prime dose at Day 0 and a boost dose at Day 21. The same formulations and PBS (negative control) were injected as in Figure 6A (see also Table 6) encapsulating mRNA encoding luciferase or SARS-CoV2 Spike protein.

[0076] Figure 9A shows the percentage inhibition of functional antibody ex vivo that inhibits ACE2 binding to SARS CoV-2 RBD (at 1 / 300 dilution) in the serum of mice after a prime dose at Day 0. The same formulations were injected as in Figure 6A (see also Table 6) encapsulatingmRNA encoding luciferase or SARS-CoV2 Spike protein. The negative control was PBS treated mice.

[0077] Figure 9B shows the percentage inhibition of functional antibody ex vivo that inhibits ACE2 binding to SARS CoV-2 RBD (at 1 / 1000 dilution) in the serum of mice after a boost dose at Day 21. The same formulations and PBS (negative control) were injected as in Figure 6A (see also Table 6) encapsulating mRNA encoding luciferase or SARS-CoV2 Spike protein.

[0078] Figure 10 shows the ECL signal (IgG levels against SARS-CoV2 spike protein) for the control and PEGless LNPs set forth in Table 7. The negative control was PBS treated mice.Detailed Description

[0079] The lipid nanoparticle described herein encapsulates nucleic acid, such as an RNA (e.g., mRNA) or DNA, encoding an antigen or fragment thereof that provides an immune response in a subject against such antigen or fragment thereof. In certain advantageous embodiments, the LNP comprises ionizable cationic lipid, a neutral lipid, a sterol and optionally a hydrophilic polymerlipid conjugate, and in which the neutral lipid is present at a mol% of at least 20 mol% or at least 30 mol% and in which the ionizable lipid is present at less than 40 mol%. At set forth herein, elevated neutral lipid mol% beyond what is used in conventional formulations for nucleic acid delivery may provide an immune response against an expressed antigen or fragment thereof that is comparable to the benchmark Onpattro™-type formulations, which are similar to those used in current Covid-19 vaccines.

[0080] The nucleic acid includes, without limitation, RNA, including small activating RNA (saRNA), self-replicating RNA (srRNA), transamplifying RNA (taRNA), circular RNA (circRNA), transfer RNA (tRNA), and peptide nucleic acid (PNA); and DNA such as vector DNA and linear DNA, or hybrids thereof. 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.

[0081] A “nanoparticle” or “lipid nanoparticle” refers to a lipid-containing particle having a particle size (diameter) below 1 pm as measured by dynamic light scattering (DLS) (see Materials and Methods of the Example section). In some embodiments, the average diameter of nanoparticles in a nanoparticle preparation is 1 pm or smaller (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or smaller). In some embodiments, the lipid nanoparticle has a diameter of between 50 nm and 300 nm or between 60 nm and 250 nm.

[0082] In some embodiments, the nucleic acid does not encode for a reporter protein or peptide such as eGFP. In some embodiments, the LNP is part of a pharmaceutical composition that has a nucleic acid cargo and has a therapeutic, diagnostic or prophylactic effect on a subject.

[0083] In some embodiments, the lipid nanoparticle described herein comprises a cargo that is messenger RNA. As used herein, the term “messenger RNA” or “mRNA”, refers to a polynucleotide that encodes and expresses at least one peptide, polypeptide or protein. The term is meant to include, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA).

[0084] As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid within a lipid nanoparticle refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle. In one example of the disclosure, the nucleic acid is present in the core of the LNP.

[0085] The concentration of mRNA in the LNP may be between 0.01 and 20 mg / mL or between 0.01 and 10 mg / mL or between 0.05 and 5 mg / mL or between 0. 075 and 4 mg / mL.

[0086] 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.

[0087] In those embodiments in which an mRNA is chemically synthesized, 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, pyrrol o-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, C 5 -methyl cytidine, 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).

[0088] 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.

[0089] 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.

[0090] The present disclosure may be used to formulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to formulate and encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 2-20 kb, about 2-15 kb, about 2-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about8-20 kb, or about 8-15 kb in length.

[0091] 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 provides resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0092] 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 50 and 500 nucleotides in length.

[0093] 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 50 and 500 nucleotides in length or longer.

[0094] 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.

[0095] 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 are incorporated into mRNA for in vivo studies in animal models to assess biodistribution.

[0096] The lipid nanoparticle described herein may comprise encapsulated DNA vector. As used herein, the term “DNA vector” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein and that is either circular or has been linearized.

[0097] The DNA vector may replicate autonomously, or it may replicate by being inserted into the genome of the host cell, by methods well known to those of skill in the art. Vectors that replicate autonomously will have an origin of replication or autonomous replicating sequence (ARS) that is functional in a host cell. The DNA vector may be included in more than one host cell, e.g., in E. coli for cloning and construction, and in a mammalian cell for expression.

[0098] 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.

[0099] 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 preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0100] The DNA vector may include nucleic acid 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.

[0101] 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.

[0102] The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrientsensitive 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.

[0103] The nucleic acids used in the present method 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. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[0104] 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.

[0105] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.Neutral Lipid

[0106] The term “neutral lipid” refers to a lipid that exists either in an uncharged or neutral zwitterionic form at physiological pH and has one or more tail moieties. In some embodiments, the neutral lipid includes an amphipathic lipid that allows for the formation of particles. In alternative embodiments, the lipid nanoparticle comprises elevated levels (>20 mol%) of a structural lipid that is a non-cationic lipid. In one embodiment, the neutral lipid is amphipathic and has at least two tails and a polar region, such as a head group. The term includes lipids with choline head groups (e.g., phosphatidylcholine), meaning an amphipathic lipid that has a choline head group and that bears no or substantially no net charge at physiological pH. The neutral lipid also includes phospholipids conjugated to sterols.

[0107] The neutral lipid has a head group. As used herein “head group”, means a moiety that imparts polarity to the lipid and comprises one or more electronegative atoms, such as nitrogen, oxygen and / or phosphorus. Generally, the electronegative atom or atoms are part of one or more functional groups. In some embodiments, the head group is zwitterionic. In some embodiments, the head group comprises a phosphate group and a nitrogen atom. In some embodiments, the head group is zwitterionic and has no net charge at physiological pH.

[0108] In some embodiments, the neutral lipid is selected so that the lipid nanoparticle has “substantially no net charge” at physiological pH, which means a net surface charge of about zero, or near neutral at physiological pH, such as without limitation about -2.5 mV to about 2.5 mV.

[0109] The term “consists essentially of’ or “consisting essentially” with reference to one or more lipid components in a lipid nanoparticle, means that the lipid nanoparticles contains no more than 5 mol% of additional lipid components besides those listed.

[0110] Neutral lipids include, but are not limited to, phosphotidylcholines such as 1,2-Distearoyl- sn-glycero-3 -phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 ,2-Dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1 -Palmitoyl-2-oleoyl-sn-gly cero-3 - phosphocholine (POPC), 1,2-dioleoyl-sn-gly cero-3 -phosphocholine (DOPC), phophatidylethanolamines such as 1,2-Dioleoyl-sn-gly cero-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0111] The neutral lipid content may include mixtures of two or more types of different structural lipids. In one embodiment, the phosphatidylcholine lipid content is a mixture of DSPC and DOPC and POPC. In such embodiments, the mixture may have a DSPC content of at least 20 or 30 mol%.

[0112] The neutral lipid content in some embodiments is greater than 20 mol%, greater than 25 mol%, greater than 30 mol%, greater than 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, greater than 46 mol%, greater than 48 mol% or greater than 50 mol%. In some embodiments, the upper limit of helper lipid content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol% or 45 mol%. The disclosure also encompasses sub-ranges of any combination of the foregoing numerical upper and lower limits.

[0113] For example, in certain embodiments, the phosphatidylcholine lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0114] In one embodiment, the neutral lipid is DSPC. For example, in certain embodiments, the DSPC lipid content is from 20 mol% to 80 mol% or 25 mol% to 60 mol% or 30 mol% to 60 mol% or 35 mol% to 60 mol% or 40 mol% to 60 mol% or 42 mol% to 58 mol%, or 43 mol% to 57 mol% or 44 mol% to 56 mol% or 45 mol% to 55 mol% of total lipid present in the lipid nanoparticle.

[0115] The LNP may comprise additional structural lipids besides a phosphatidylcholine lipid. For example, the LNP may comprise structural lipids that have a net positive or negative charge at physiological pH. Generally, such lipids will be present at less than 10 mol% or less than 5 mol%.

[0116] The structural lipid content is determined based on the total amount of lipid in the lipid nanoparticle, including the sterol.Ionizable lipid

[0117] The LNP of the disclosure has an ionizable lipid. The ionizable lipid may be charged at low pH and have substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initialformulation. Since the ionizable lipid is near neutral at physiological pH, toxicity and renal clearance is reduced. 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.

[0118] In some embodiments, it is desirable to include less than 50 mol% ionizable lipid. 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%.

[0119] 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.

[0120] As used herein, the term "cationic lipid" refers to a lipid that, at a given pH, such as physiological pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically positively charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol (i.e., a cLogP) greater than 8. In some embodiments, the cationic lipid has an apparent pKa that is between 5.0 and 8.0 when formulated in an LNP.

[0121] In some embodiments, the LNP has an apparent pKa of between 5.0 and 7.5, between 6.5 and 7.5 or between 6.8 and 7.3. The apparent pKa is measured using a 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; and Jayaraman et al., 2012, Angew. Chem Int. Ed., 51 :8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). According to the method, a series of buffers are prepared spanning a pH range of 2-11 in 0.5 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 0.06 mM of TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polysterene 96-well plate, to yield a final concentration of 6.25 and 6 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using an SpectraMax™ M5 microplate reader at lex=321 nm, lem=445 nm. The fluorescence isthen plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0122] In some embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, C 16 to Cl 8 alkyl chains, optionally ether linkages between its head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, but are not limited to sulfur lipids, such as MF019 described in WO 2022 / 155728A1 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 / 246571; WO 2023 / 147657; WO2022 / 155728; WO 2023 / 215989; WO 2024 / 065041; WO 2024 / 065042; PCT / CA2023 / 051727 filed on December 21, 2023; WO 2024 / 065043; and U.S. patent application No. 18 / 442,431 filed on February 15, 2024, each incorporated herein by reference.

[0123] 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 or a moiety comprising one or more heteroatoms, 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; PCT / CA2023 / 051727 filed on December 21, 2023; and WO 2024 / 065043. Functional groups comprising one or more heteroatoms may be biodegradable in vivo.

[0124] In one embodiment, the ionizable cationic lipid has a protonatable amino head group; at least two lipophilic moieties, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic moieties are directly bonded; each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the lipid has (i) an apparent pKaof between 6 and 7.5 (measured as per the TNS assay); and (ii) a ClogP of at least 11.

[0125] Optionally, at least one of the lipophilic moieties bonded to the head group has a biodegradable group, including but not limited to an ester group or a moiety comprising an ester (in any orientation). In one embodiment, at least one of the lipophilic moieties comprises a sulfuratom. In another embodiment, at least one of the lipophilic moieties comprises an ester or a moiety comprising an ester, one or more sulfur atoms or any combination thereof.

[0126] In one embodiment, the lipid nanoparticle comprises a “sulfur-containing amino lipid”, which is an ionizable lipid having at least one ionizable nitrogen (tertiary nitrogen) and one or more sulfur atoms in at least one of its lipophilic chains. In one embodiment, the sulfur-containing amino lipid further comprises a biodegradable group such as an ester moiety in one or more of its lipophilic chains. Non-limiting examples of sulfur-containing lipids are provided in Table 3 and in the foregoing co-owned PCT applications. The incorporation of the biodegradable group(s) into the lipid increases metabolism post-administration and improves clearance of the lipid from the body following delivery of the active agent to a target area. Generally, such lipids have decreased toxicity when compared to similar lipids without the biodegradable groups.

[0127] Sulfur-containing ionizable lipids and / or ionizable lipids with one or more biodegradable groups (e.g., esters, groups comprising an ester or a disulfide) in one or more of their lipophilic chains have been shown to be particularly efficacious when formulated in LNPs, including unshielded LNPs, as described in the Example section herein.

[0128] Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) in the lipophilic chains of the ionizable cationic lipid include cycloalkyl groups (mono or polycyclic); — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — 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.

[0129] By the term “biodegradable group”, with reference to a group in a lipophilic chain of an ionizable lipid, includes a functional group with one or more electronegative atoms (e.g., O, N, S or P) that is metabolized in vivo by an enzyme, thereby increasing its metabolism relative to an otherwise identical ionizable lipid that does not contain such group. Non-limiting examples include— O-; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — 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.

[0130] Non-limiting examples of atoms or substituents that may replace a hydrogen atom in the ionizable cationic lipid include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0131] In one embodiment, the ionizable amino lipid is a “non-furan ring-containing ionizable lipid”, which is an ionizable lipid that lacks a furan ring, such as an oxolane in the head group moiety. In one embodiment, the ionizable lipid is a “non-MC3 type lipid”, which is an ionizable lipid that lacks in its head group a terminal ionizable amino group that is a methyl 4- (dimethylamino)butanoate. In another embodiment, the ionizable lipid is a non-furan ringcontaining ionizable lipid that is also a non-MC3 type lipid. In one embodiment, the ionizable amino lipid is a “non-cyclopentane ring-containing ionizable lipid”, which is an ionizable lipid that lacks a cyclopentane moiety in the head group moiety.

[0132] In one non-limiting example, at least one of the lipophilic groups or moieties of the ionizable lipid has the formula:or is an amino acid or a sarcosine group. The ester (which may be denoted as “E”) is present in either orientation (i.e., the carbonyl (C=O) is bonded to R1or the -O- of the ester is bonded to R1).

[0133] In one embodiment, R1and R2are, independently, linear, cyclic and / or branched optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation; and n is 4 to 8. In an alternative embodiment, R2is C2-C12 alkyl (including alkenyl).

[0134] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipid in the LNP. That is, the ionizable cationic lipid content may be less than 50 mol%, less than 45mol%, 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%.

[0135] In certain embodiments, the ionizable 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.

[0136] 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 U.S. PCT / CA2024 / 050347 filed on March 22, 2024, which is incorporated herein by reference in its entirety.Sterol

[0137] The LNP further includes a sterol in some embodiments. The term “sterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring.

[0138] Examples of sterols include cholesterol, or a cholesterol derivative, the latter referring to a cholesterol molecule having a gonane structure and one or more additional functional groups.

[0139] The cholesterol derivative includes P-sitosterol, 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 -hydroxy cholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23- oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7- hydroxy cholesterol, 19-hydroxy cholesterol, 22-hydroxy cholesterol, 25-hydroxy cholesterol, 7- dehydrocholesterol, 5a-cholest-7-en-3P-ol, 3,6,9-trioxaoctan-l-ol-cholesteryl-3e-ol, 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 or a salt or ester thereof. 1

[0140] In one embodiment, the sterol is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0141] In another embodiment, the sterol is cholesterol and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0142] In another embodiment, the sterol is a cholesterol derivative and is present at from 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol% or 30 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

[0143] In one embodiment, the combined (i) sterol content (e.g., cholesterol or cholesterol derivative thereof); and (ii) neutral lipid content is at least 50 mol%; at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol% or at least 85 mol% based on the total lipid present in the lipid nanoparticle.Hydrophilic polymer-lipid conjugate

[0144] In one embodiment, the lipid nanoparticle comprises a hydrophilic-polymer lipid conjugate capable of incorporation into the LNP. In one embodiment, the conjugate includes a vesicle- forming lipid having a polar head group, and covalently attached to the head group, a polymer chain that is hydrophilic. In another embodiment, the conjugate includes a lipophilic moiety covalently attached to the hydrophilic polymer directly or via a linker region. 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 monosialoganglioside (GMI).

[0145] In some embodiments, LNPs without hydrophilic polymer lipid conjugate or low amounts thereof are advantageous as such formulations may allow a greater IgG response after a booster dose.

[0146] 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. In certain embodiments, 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 or at 0 mol% to 1.7 mol% or at 0 mol% to 1.6 mol% or at 0 mol% to 1.5 mol% or at 0 mol% to 1.4 mol% or at 0 mol% to 1.3 mol% or at 0 mol% to 1.2 mol% or at 0 mol% to 1.1 mol% or at 0 mol% to1.0 mol% or at 0 mol% to 0.9 mol% or at 0 mol% to 0.8 mol% or at 0 mol% to 0.7 mol% or at 0 mol% to 0.6 mol% or at 0 mol% to 0.5 mol% or at 0 mol% to 0.4 mol% or at 0 mol% to 0.3 mol% or at 0 mol% to 0.2 mol% or at 0 mol% to 0.1 mol%.

[0147] In another embodiment, the PEG-lipid conjugate 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. In certain embodiments, the PEG-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% to2.5 mol% or at 0 mol% to 2.0 mol% or at 0 mol% to 1.8 mol% of total lipid or at 0 mol% to 1.7 mol% or at 0 mol% to 1.6 mol% or at 0 mol% to 1.5 mol% or at 0 mol% to 1.4 mol% or at 0 mol% to 1.3 mol% or at 0 mol% to 1.2 mol% or at 0 mol% to 1.1 mol% or at 0 mol% to 1.0 mol% or at 0 mol% to 0.9 mol% or at 0 mol% to 0.8 mol% or at 0 mol% to 0.7 mol% or at 0 mol% to 0.6 mol% or at 0 mol% to 0.5 mol% or at 0 mol% to 0.4 mol% or at 0 mol% to 0.3 mol% or at 0 mol% to 0.2 mol% or at 0 mol% to 0.1 mol%.

[0148] The lipid nanoparticle in some examples has “substantially no hydrophilic polymer-lipid conjugate” or is “non-sterically stabilized” or “uncoated”, meaning the lipid nanoparticle has less than 0.8 mol% total hydrophilic-polymer lipid conjugate or other surface stabilizer content as measured based on the total lipid content of the nanoparticle.

[0149] As used herein, the term “surface stabilizer” is a macromolecule, including a protein, polysaccharide or polymer, including a block copolymer, that is used to stabilize a lipid nanoparticle, and in which at least a portion (e.g., hydrophilic) is present on the surface of the lipid nanoparticle. Such molecules are employed by those of skill in the art to prevent aggregation, improve shelf life and / or improve the stability of the particle after administration, such as the circulation lifetime of the lipid nanoparticle. The term includes surface stabilizers that are knownto control the size of lipid nanoparticles, such as amphiphilic polymers (e.g., block co-polymer). As would be appreciated by those of skill in the art, a hydrophobic portion of the surface stabilizer may partition in a lipophilic portion of the lipid nanoparticle.

[0150] Examples of such macromolecules include the hydrophilic polymer-lipid conjugates described above or the amphiphilic polymer, such as an amphiphilic block copolymer (e.g., a poloxamer, such as polyethylene-propylene glycol copolymer or polyoxvethylene- polyoxypropylene copolymer) as described in US2021 / 0046192, which is incorporated herein by reference. Such stabilizer(s) may be present in the nanoparticle at 0 mol% to 1.5 mol% or at 0 mol% to 1.4 mol% or at 0 mol% to 1.3 mol% or at 0 mol% to 1.2 mol% or at 0 mol% to 1.1 mol% or at 0 mol% to 1.0 mol% or at 0 mol% to 0.9 mol% or at 0 mol% to 0.8 mol% or at 0 mol% to 0.7 mol% or at 0 mol% to 0.6 mol% or at 0 mol% to 0.5 mol% or at 0 mol% to 0.4 mol% or at 0 mol% to 0.3 mol% or at 0 mol% to 0.2 mol% or at 0 mol% to 0.1 mol%.

[0151] In one embodiment, the amphiphilic polymer, such as an amphiphilic block copolymer (e.g., a poloxamer) may be present in the nanoparticle at low levels, such as at 0 mol% to 1.5 mol% or at 0 mol% to 1.4 mol% or at 0 mol% to 1.3 mol% or at 0 mol% to 1.2 mol% or at 0 mol% to 1.1 mol% or at 0 mol% to 1.0 mol% or at 0 mol% to 0.9 mol% or at 0 mol% to 0.8 mol% or at 0 mol% to 0.7 mol% or at 0 mol% to 0.6 mol% or at 0 mol% to 0.5 mol% or at 0 mol% to 0.4 mol% or at 0 mol% to 0.3 mol% or at 0 mol% to 0.2 mol% or at 0 mol% to 0.1 mol%.

[0152] In further embodiments, the LNP lacks a surface stabilizer that is a protein, referred to as a protein stabilizer. This includes an apolipoprotein stabilizer, derivative or mimetic thereof (see e.g., WO 2023 / 233042, which is incorporated herein by reference). Such apolipoprotein may be selected from one or a combination of apo Al, apo Al - Milano, apo A2, apo A4, apo A5, apo B48, apo Bl 00, apo C-l, apo C-l I, apo C-lll, apo C-IV, apo D, apo E, apo F, apo H, apo L and apo M. In some embodiments, the protein stabilizer content is less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In some embodiments, the protein stabilizer content is less than 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15 or 0.10 mol% as measured based on the total lipid content of the nanoparticle. In further embodiments, the protein stabilizer content mol% content is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%,0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%. Since lipid nanoparticles can adsorb proteins after administration, the protein content is measured in vitro prior to administration.

[0153] Alternatively, in some embodiments a lipid nanoparticle preparation lacks or has low levels thereof of one or more stabilizing agents, which includes a surface stabilizer as described above and a cryoprotectant that functions as a stabilizer. In some embodiments, the lipid nanoparticle preparation has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, a lipid nanoparticle preparation having a plurality of LNPs has low levels or lacks glycerol and / or propylene glycol as a cryoprotectant, such as at concentration levels less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v in the preparation. In some embodiments, the lipid nanoparticle preparation consists essentially of buffer, meaning that a lipid nanoparticle preparation comprises buffer and has less than 2 w / v, 1.75 w / v, 1.50 w / v, 1.25 w / v, 1.00 w / v, 0.75 w / v, 0.50 w / v, 0.25, 0.10 or 0.05 w / v of one or more cryoprotectants in the preparation. In some embodiments, the lipid nanoparticle preparation comprises no cryoprotectant, such as for example, glycerol and / or propylene glycol.

[0154] In another embodiment, the lipid nanoparticle is “PEG-less”, meaning that the lipid nanoparticle has no detectable amounts of polyethylene-glycol lipid conjugate. In further embodiments, the lipid nanoparticle is “PEGless”, meaning that the LNP lacks a surface stabilizer comprising a hydrophilic polymer with a polyethylene oxide repeating unit or includes low levels thereof, such as less than 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mol% as measured based on the total lipid content of the lipid nanoparticle. In further embodiments, surface stabilizer comprising a hydrophilic polymer has a mol% content that is between 0 and 0.75 mol%, 0 and 0.70 mol%, 0 and 0.65 mol%, 0 and 0.60 mol%, 0 and 0.55 mol%, 0 and 0.50 mol%, 0 and 0.45 mol%, 0 and 0.40 mol%, 0 and 0.35 mol%, 0 and 0.30 mol%, 0 and 0.25 mol%, 0 and 0.20 mol%, 0 and 0.15 mol% or 0 and 0.10 mol%.Nanoparticle preparation and morphology

[0155] Delivery vehicles incorporating the nucleic acid (e.g., mRNA or DNA) can be prepared using 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.

[0156] Without being bound by theory, the mechanism whereby a lipid nanoparticle comprising encapsulated nucleic acid can be formed using the rapid mixing / ethanol dilution process can be hypothesized as beginning with formation of a dense region of hydrophobic mRNA-ionizable lipid core at low pH (e.g., pH 4) surrounded by a monolayer of helper lipid / cholesterol that fuses with smaller empty vesicles as the pH is raised due to the conversion of the ionizable cationic lipid to the neutral form. As the proportion of bilayer helper lipid increases, the bilayer lipid progressively forms blebs and the ionizable lipid migrates to the interior hydrophobic core. At high enough helper lipid contents, the exterior bilayer preferring helper lipid can form a complete lipid layer, such as a continuous or discontinuous bilayer, around the interior trapped volume.

[0157] The LNP may comprise a “core” region. Unexpectedly, it has been observed that LNPs with elevated levels of neutral lipid have a core that is non-homogeneous in that it includes both an electron dense region and an aqueous portion or compartment as visualized by cryo-EM microscopy. In some embodiments, the core may be characterized as non-solid. Without being limiting, the electron dense region within the core may be partially surrounded by the aqueous portion or compartment within the enclosed space as observed by cryo-TEM. The aqueous portion may form a distinct aqueous region or compartment within the lipid nanoparticle. In other words, it is believed that the aqueous portion or compartment is not merely a hydration layer. Such nonsolid core particles are described in co-owned and co-pending WO 2022 / 251959, the contents of which are incorporated herein by reference.

[0158] In one embodiment, at least one about fifth of the core (trapped volume) contains the aqueous portion or compartment, and in which the electron dense region within the core is partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about quarter of the core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In a further embodiment, at least one about one third of the core contains the aqueous portion or compartment, and in which the electron dense region is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM. In another embodiment, at least one about one half ofthe core contains the aqueous portion or compartment, and in which the electron dense core is either partially contiguous with the lipid layer comprising the bilayer, as determined qualitatively by cryo-EM.

[0159] In another embodiment, the electron dense region of the LNP unexpectedly appears to be completely surrounded by the aqueous portion of the core as visualized by cryo-TEM microscopy. This morphology is observed in a single plane and a portion of the electron dense region as observed is contiguous with the lipid layer (e.g., bilayer) but cannot be seen since this portion is not within the plane that can be visualized.

[0160] In one embodiment, the electron dense region is generally spherical in shape. In another embodiment, the electron dense region is hydrophobic.

[0161] The lipid nanoparticles herein may exhibit particularly high trapping efficiencies of mRNA. Thus, in one embodiment, the trapping efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85 or 90%.

[0162] In a further embodiment, the helper lipid is present in the lipid layer comprising the bilayer.

[0163] The lipid nanoparticle may comprise a single bilayer or may be a combination of a bilayer and a monolayer in some embodiments. In one embodiment, the lipid layer is a continuous bilayer that surrounds the core.

[0164] In certain embodiments the electron dense region of the core is separated from the lipid layer comprising the bilayer by the aqueous portion or compartment. For example, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which at least 20%, 30%, 40%, 50%, 60% or 70% of the particles as determined by cryo-EM microscopy have a core with an electron dense region and an aqueous portion or compartment and in which the aqueous portion or compartment is partially surrounded by the lipid layer comprising the bilayer as visualized by cryo-EM microscopy.

[0165] In another embodiment, and without being limiting, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have an elongate shape (e.g., generally oval-shaped) as determined qualitatively by cryo-EM microscopy. In this latter embodiment, theelectron dense region of the core may be partially surrounded the aqueous space as visualized by cryo-EM microscopy.

[0166] In one embodiment, the lipid nanoparticle is part of a preparation of lipid nanoparticles, and wherein the electron dense region of at least 20% of the lipid nanoparticles are either (i) enveloped by the aqueous portion, or (ii) is partially surrounded by the aqueous portion and wherein a portion of a periphery of the electron dense region is contiguous with the lipid layer, as visualized by cryo-EM microscopy in a single plane.

[0167] In certain embodiments, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles as determined by cryo-EM microscopy have a core with an electron dense region that is contiguous with the lipid layer comprising the bilayer as visualized by cryo-EM microscopy.

[0168] In another embodiment, and without being limiting, the disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles in which generally at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core comprising an electron dense region that appears to be surrounded or enveloped by a continuous aqueous space disposed between the lipid layer (e.g., bilayer) and the electron dense region, as visualized in one plane by cryo-EM microscopy.

[0169] LNPs are visualized by cryo-TEM as described in Kulkami et al., 2019, Nanoscale, 11 :21733, which is incorporated herein by reference.

[0170] In another embodiment, the poly dispersity index (PDI) of the LNP preparation is less than 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.12 or 0.10.

[0171] In another embodiment, the particle size distribution is such that at least 90% of the particles in the LNP preparation of the disclosure have a diameter of between 40 nm and 200 nm, between 45 and 175 nm or between 50 and 150 nm.

[0172] According to some embodiments, the LNPs disclosed herein have a defined mean particle size that ranges between 40 and 250 nm or between 40 and 200 nm or between 45 and 150 nm or between 50 and 150 nm or between 50 and 120 nm or between 50 and 140 nm. In some embodiments, at least 90% of the particles have a size within any one of the foregoing ranges usingdynamic light scattering as set forth in the Materials and Methods herein. In some embodiments, the particle size distribution is + / - 40, 30 or 20 nm, meaning the lipid nanoparticles in a lipid nanoparticle preparation do not vary significantly in size. 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.

[0173] The lipid nanoparticles herein may exhibit particularly high encapsulation efficiencies of nucleic acid. As used herein, the term “encapsulation,” with reference to incorporating the nucleic acid within a lipid nanoparticle refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle, including a lipophilic or the aqueous portion. In one embodiment, the nucleic acid is present at least in the core of the LNP.

[0174] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%. The encapsulation efficiency of the nucleic acid is determined as set forth in the Materials and Methods section in the Examples herein.

[0175] Embodiments of the present disclosure also provide lipid nanoparticles described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the oligonucleotide to be encapsulated. This may be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is between 3 and 15, 4 and 15 or between 4.5 and 10 or between 5 and 10 or between 5.5 and 8.

[0176] In one embodiment, the N / P ratio of the lipid nanoparticle is at least 3, 3.25, 3.50, 3.75, 4, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0 or 6.25. The upper limit may be 15, 14, 13, 12, 11, 10, 9 or 8. The disclosure also encompasses a combination of any two of the upper and lower limits.

[0177] In one embodiment, the lipid nanoparticle has a weight nucleic acid / micromole of total lipid that is 0.05: 1 to 1 : 1. In one embodiment, the lower limit is 0.06: 1, 0.08:1, 0.10: 1, 0.12: 1, 0.14: 1, 0.16: 1, 0.18: 1, 0.20: 1, 0.22: 1, 0.24: 1, 0.26: 1, 0.28:1, 0.30: 1, 0.32: 1, 0.34: 1, 0.36: 1, 0.38:1 or 0.40: 1 weight nucleic acid / micromole of total lipid. In another embodiment, the upper limit is 0.80: 1, 0.82: 1, 0.84: 1, 0.86: 1, 0.88: 1, 0.90: 1, 0.92: 1, 0.94: 1, 0.96:1 or 0.98: 1 weight nucleic acid / micromole of total lipid. The disclosure also encompasses a combination of any two of the upper and lower limits.

[0178] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8.

[0179] In one embodiment, the LNP dosage is 0.5 pg to 1 pg. In another embodiment, the LNP dosage is 0.5 pg to 1.5 pg. In yet another embodiment, the LNP dosage is 0.5 pg to 1.25 pg. In yet another embodiment, the LNP dosage is 0.5 pg to 2 pg.

[0180] The terms “dosage” or “dose” as used herein denotes any forms of the active ingredient formulation that contain an amount sufficient to produce a therapeutic effect with one and / or more dose administrations.Immune response

[0181] As used herein, “expression” of a nucleic acid refers to expression of an encoded peptide (e.g., an antigen), polypeptide, or protein and also can include, as indicated by context, the post- translational modification of the peptide, polypeptide or fully assembled protein.

[0182] As used herein, “inducing” an immune response refers to causing an adaptive immune response, which in some embodiments includes production of a measurable level of IgG against the peptide (e.g., an antigen), polypepide, or protein encoded by the mRNA encapsulated in the vaccine LNPs.

[0183] By “equivalent immune response” to Onpattro™-type formulation with reference to a vaccine comprising the LNP of the disclosure, it is meant that the LNP vaccine results in an immune response at an applicable time point post-administration of a booster dose that does not vary by more than + / - 20%, + / - 15%, + / - 10%, + / -5%, or + / - 2% relative to the gold standard Onpattro™-type vaccine formulation.

[0184] In one embodiment, the vaccine comprising the lipid nanoparticle has at least an equivalent immune response relative to the Onpattro™-type formulation after being administered as a booster dose via intramuscular injection. In yet another embodiment, the vaccine comprising the lipid nanoparticle shows a comparable immune response after being administered to the patient as a booster dose via intramuscular injection.

[0185] As described in Example 2, the vaccine comprising the lipid nanoparticle of the disclosure may elicit or induce at least an equivalent IgG level relative to Onpattro™-type against the antigenic protein, peptide or a fragment in the subject at 21 days and / or 35 days post-injection. Whether or not the vaccine of the disclosure induces such enhanced IgG response against theantigenic protein, peptide or a fragment can be determined by electrochemiluminescence. In such embodiments, IgG levels are measured in the blood sample of the subject. In particular, according to such embodiments, a vaccine comprising the lipid nanoparticle is prepared encapsulating mRNA coding for the antigen and IgG levels in a blood sample are evaluated using an assay, such as those commercially available from Mesoscale Discovery (MSD) to detect electrochemiluminescence.

[0186] In some embodiments, to assess whether a given vaccine comprising the lipid nanoparticle induces an equivalent or increased immune response in a subject at 21 days and / or 35 days post-injection, the vaccine of the disclosure is compared to the Onpattro™-type vaccine formulation of Example 2. The two vaccines being compared are subjected to the same experimental methods and materials as set forth in Example 2. The two formulations being compared will comprise identical ionizable cationic lipids and cargo but vary in the amount of neutral lipid.

[0187] In one embodiment, the vaccine comprising the lipid nanoparticle exhibits at least an equivalent or an increased IgG response against the receptor-binding domain of the spike protein encoded by the encapsulated mRNA as measured in vivo in serum samples at 21 days postinjection as compared to an Onpattro™-type lipid nanoparticle vaccine formulation of ALC- 0315 / DSPC / cholesterol / PEG-lipid at 50 / 10 / 38.5 / 1.5, mokmol, wherein the immune response is measured in a mouse model by detection of the IgG levels in the serum sample. The IgG level is determined by electrochemiluminescence as set forth in Example 2. The increase in immune response is determined by collecting blood samples from saphenous bleeds on Day 21 and Day 35 and comparing IgG levels against receptor binding domain (RBD) of the spike protein to that of IgG levels of the Onpattro™-type vaccine formulation. The observed ECL signal was plotted for each sample to compare immune response.

[0188] In one embodiment, the immune response in the serum sample is assessed after 21 days following intramuscular injection. In another embodiment the immune response in the serum sample is checked following 35 days after intramuscular injection. In one embodiment, a booster dose of the vaccine is further administered to the subject at day 21 after an initial prime dose at day 0. The blood sample from the subject is collected by saphenous bleeds at day 21, and by cardiac puncture at day 35. In one embodiment, the IgG levels quantified to assess the immuneresponse induced by the vaccines is measured in the serum extracted from the blood sample. Electrochemiluminescence (ECL) signals are used to measure the IgG response, and the ECL signals are measured by an assay commercially available from Mesoscale Discovery (MSD).

[0189] In one embodiment, the IgG targets the receptor binding domain of the spike protein. In one embodiment the IgG levels are measured at Day 21 following an initial prime dose of the vaccine. In another embodiment IgG levels were measured at Day 21 and Day 35 following a prime dose and a booster dose respectively.Clinical and non-clinical uses of the LNP herein

[0190] In some embodiments, the vaccine lipid nanoparticle comprising nucleic acid is part of a pharmaceutical composition and is administered to treat, ameliorate, prevent and / or delay any disease, disorder or condition in a mammalian subject. The pharmaceutical composition may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0191] The pharmaceutical composition comprising the LNP may be used to treat, ameliorate, prevent and / or delay any disease, disorder or condition that is an infectious disease, such as viral, bacterial, fungal or parasitic infections, cancer and other cell proliferative disorders, inflammatory and / or autoimmune disorders, including treatments that induce immune tolerance and cardiovascular diseases such as hypertension, cardiac arrhythmia and restenosis.

[0192] The term “prevent”, as used herein, includes prevention of a disease or disorder, such as risk factor reduction or reducing progress of the disease or disorder.

[0193] As used herein, the term “ameliorate” means to reduce, prevent, alleviate, and / or lessen the impact of a disease, symptom or condition, to bring about an improvement in the disease, symptom or condition, or at least a partial relief of one or more symptoms associated with the disease or condition. The term, as used herein, may include a detectable or measurable improvement in a subject’s condition, disease or symptom thereof. A detectable or measurable improvement includes a decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the condition or disease, or an improvement in a symptom or an underlying cause of the disease, including a reversal of the disease. A successful treatment outcome can lead to a “therapeutic effect,” or “benefit” ofameliorating, decreasing, reducing, inhibiting, suppressing, limiting, controlling, or preventing the occurrence, frequency, severity, progression, or duration of a disease or condition, or consequences of the disease or condition in a subject.

[0194] The vaccine LNP described herein may provide active or acquired immunity against an infectious disease or a causative agent. In one embodiment, the vaccine comprising the LNP contains at least one RNA (e.g., mRNA) molecule encoding a foreign antigen, protein or a peptide fragment from the disease-causing agent against which an adaptive immune response is desired. The peptide encoded by the encapsulated mRNA can be expressed in the form of a weakened antigen, protein or binding domain.

[0195] In some embodiments, the type of disease to be treated or prevented is an infectious disease caused by a pathogen such as a virus, bacterium, protozoan, parasite (e.g., helminth), or other pathogen, e.g., including those that enter intracellularly and are attacked, e.g., by cytotoxic T lymphocytes. In one embodiment, the disease is a chronic infectious disease. In another embodiment, the disease is an acute infectious disease. In another embodiment, the disease is a viral infectious disease.

[0196] In some embodiments, the infection to be treated is caused by a virus, such as COVID, influenza, severe acute respiratory syndrome (SARS), hepatitis (e.g., hepatitis A, B, C, D or E), , infections caused by human T-lymphotrophic virus (HTLV), infections caused by herpes virus, Epstein-Barr virus or a human papilloma virus, infections caused by human immunodeficiency virus (HIV), including, for example, HIV resulting in mycobacterial infection, AIDS-related cachexia, AIDS-related cytomegalovirus infection, HIV-associated nephropathy, AIDS-related cryptococcal meningitis, AIDS-related neutropenia, Pneumocysitis jiroveci (Pneumocystis carinii) infections, AIDS-related toxoplasmosis, herpes, herpes zoster (chicken pox also known as varicella), measles, such as German measles (rubella virus), diseases caused by flavi viruses, such as yellow fever, dengue fever, or diseases caused by Marburg or Ebola viruses, e.g., hemorrhagic infectious diseases), genital herpes, orofacial herpes, rabies, rotaviral gastroenteritis, viral encephalitis (e.g., Japanese encephalitis, western equine encephalitis or tick-borne encephalitis), viral meningitis, Argentine haemorrhagic fever, Ross river virus infection, West Nile virus (WNV) disease, human papillomavirus (HPV) infections, viral respiratory infections such as adenovirus infections, avian (H5N1) influenza, respiratory syncytial virus (RSV) infections, viralsinusitis, Coccidioidomycosis and swine (H1N1) influenza, viral skin diseases such as B19 parvovirus infections, warts, genital herpes, orofacial herpes, shingles, viral inner ear infections, fetal cytomegalovirus syndrome, lassa fever, Marburg hemorrhagic fever, Nipah virus disease, hantavirus, smallpox, rubella, mumps and polio.

[0197] In some embodiments, the LNP herein is a vaccine that elicits an immune response against Coronavirus spp., whose immunogens include, but are not limited to, those derived from a SARS CoV-1, SARS-CoV-2(12); human influenza virus, and Neisseria meningitidis for which useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein.

[0198] Examples of bacterial infectious diseases include Legionnaires' disease (e.g. caused by Legionella), gastric ulcer (e.g., caused by Helicobacter), cholera (e.g., caused by Vibrio), E. coli infections, staphylococcal infections, Salmonella infections, streptococcal infections, tetanus (e.g., caused by Clostridium tetani), leprosy, diseases caused by Bordetella pertussis (whooping cough), diphtheria, tetanus, tuberculosis, typhoid, Clostridium difficile-associated diarrhea (CDAD), bacterial encephalitis, meningococcal infections, meningitis caused by Haemophilus influenza type b (Hib), pneumococcal infection, sexually transmitted diseases such as chlamydial diseases, chancroid, syphilis, non-septic bacterial infections such as cellulitis, lyme disease, methicillin-resistant Staphylococcus aureus (MRSA) infection, pseudomonas infections, boutonneuse fever, leptospirosis, rheumatic fever, botulism, rickettsial disease, mastoiditis, legionellosis, sepsis such as bacteremia, sepsis / septic shock, sepsis in premature infants, gonococcal infections, urinary tract infection, vaginal infections (bacterial), foodborne illnesses such as brucellosis (e.g., caused by Brucella species), or those caused by Clostridium perfringens (Epsilon toxin) or E. coli 0157 :H7 (Escherichia coli), Salmonellosis (caused by Salmonella species), shigellosis, vibriosis, listeriosis, bubonic plague, anthrax, glanders (e.g., caused by Burkholderia mallei), melioidosis (e.g, caused by Burkholderia pseudomallei), psittacosis (e.g., caused by Chlamydia psittaci), Q fever (e.g., caused by Coxiella burnetii) and tularemia (e.g., caused by Fancisella tularensis).

[0199] Examples of parasitic infections (including single cell or multicellular parasites) include protozoan infectious diseases such as malaria, sleeping sickness, leishmaniasis, toxoplasmosis, e.g., infections caused by plasmodium, trypanosomes, leishmania and toxoplasma), cysticercosis,diarrheal infections including amoebiasis, , cryptosporidiosis, giardiasis, cyclosporiasis, .nematode / roundworm infections, trichomoniasis, echinococcosis, trematode, fluke infections, trichinellosis, babesiosis, hypoderma myiasis, diphyllobothriasis, and trypanosomiasis.

[0200] Examples of fungal diseases include skin diseases such as candidiasis, onychomycosis, tinea captis, i.e., scalp ringworm, tinea corporis e.g., body ringworm, tinea cruris, e.g., “jock itch”, tinea pedis, e.g., athlete's foot and sporotrichosis, aspergillosis infections and fungal vaginal infections.

[0201] The vaccine composition may be administered to treat, ameliorate, prevent and / or delay cancer and other cell proliferative disorders.

[0202] In some embodiments, the cancer vaccine may activate cytotoxic T cells in the subject, and the activated T cells recognize and act against specific types of cancer cells and / or induce the production of antibodies in the body that bind to cancer antigens present on the surface of cancerous cells.

[0203] Examples of cancers that may be treated, prevented or ameliorated by the vaccine lipid nanoparticles include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, bladder cancer, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of some other non-liver cancer cell type), and hepatoblastoma.

[0204] In some embodiments, the vaccine LNP may be a tolerogenic vaccine that is administered to treat, ameliorate, prevent and / or delay autoimmune and / or inflammatory disorders by inducing immune tolerance. By introducing modified mRNA encoding a self-antigen and delivering it using a vaccine lipid nanoparticle, the immune response can be at least partially suppressed. (See Wardell and Levings, 2021, Nature Biotechnology, 39:419-421, which is incorporated herein by reference).

[0205] The term “immune tolerance” refers to a state of unresponsiveness or partial responsiveness of the immune system to substances or tissue that have the capacity to elicit animmune response. Immune tolerance provided by tolerogenic vaccines is induced by prior exposure to specific antigen and / or antigens. In some embodiments the tolerogenic lipid nanoparticles are designed for the treatment, amelioration, prevention and / or prophylaxis of an allergic disease / susceptibility or an autoimmune disorder.

[0206] Non-limiting examples of autoimmune disorders that may be treated, prevented or ameliorated by the vaccine lipid nanoparticles include, but are not limited to, food intolerance, such a gluten intolerance or lactose intolerance. In some embodiments, the nanoparticle for inducing immune tolerance comprises mRNA that encodes for gluten / gliadin or tolerogenic epitope thereof, or lactose or a tolerogenic epitope thereof. In some embodiments the nanoparticle comprises mRNA that encodes for an antigen associated with an autoimmune disorder (e.g., rheumatoid arthritis, type 1 diabetes, multiple sclerosis, Crohn's disease, systemic lupus erythematosus, scleroderma, alopecia areata, antiphospholipid antibody syndrome, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, ulcerative colitis, inflammatory myopathies, polymyositis, myasthenia gravis, primary biliary cirrhosis, psoriasis, Sjogren's syndrome, vitiligo, gout, atopic dermatitis, auto-immune bullous skin disease (e.g., pemphigus, pemphigoid), autoimmune hepatitis, autoimmune pancreatitis, and the like). Exemplary autoimmune diseases and / or inflammatory conditions targeted by the lipid nanoparticles include, but are not limited to, systemic and organ specific autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue- dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease, Dego's disease, dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, grave's disease, guillain-barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis,polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff- man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosi, and other organ- specific inflammatory disorders.

[0207] Non-limiting examples of inflammatory disorders include asthma (either allergic or non- allergic), allergic conditions (such as, for example, food allergies (peanut, egg, etc.), venom allergy, allergy to animals, drug allergy, allergy caused by pollen, birch, dust mite feces, and cat dandruff, anaphylaxis, hyper IgE syndrome, allergic rhinitis, allergic conjunctivitis and allergic enterogastritis), allergy to medication, adverse immune effects caused by Factor VIII and therapeutic proteins, life threatening allergic drug allergies, immune response to adenovirus gene therapy vectors, atopic disorders (such as, for example, atopic dermatitis, urticaria (including chronic idiopathic urticaria and chronic spontaneous urticaria), eczema), bullous pemphigoid, respiratory disorders (such as allergic and nonallergic asthma, chronic obstructive pulmonary disease (COPD)), nasal polyposis and other conditions involving airway inflammation (such as, for example, eosinophilia, fibrosis and excess mucus production including cystic fibrosis and pulmonary fibrosis, systemic sclerosis (SSc)); gastrointestinal disorders or conditions (such as, for example, inflammatory bowel diseases (IBD) and eosinophilic esophagitis (EE), and eosinophilic- mediated gastrointestinal disease, ulcerative colitis and Crohn's disease); systemic lupus erythematosus, liver disorders or conditions (such as, for example, cirrhosis, and hepatocellular carcinoma), scleroderma; fibrotic diseases or disorders (such as, for example, fibrosis of the liver (such as, for example, fibrosis caused by a hepatitis B and / or C virus)), scleroderma; solid tumors or cancers such as leukemia (such as, for example, B cell chronic lymphocytic leukaemia), glioblastoma, lymphoma (such as, for example, Hodgkin's lymphoma) and mastocytosis.

[0208] Vaccines to treat, prevent and / or ameliorate a cardiovascular disease or disorder comprise a lipid nanoparticle with an encapsulated mRNA encoding for an antigenic protein, peptide or fragment thereof. By way of example, the vaccine lipid nanoparticle may be induced to form antibodies directed to angiopoietin-like 3 (ANGPTL3) in vivo. Such vaccine compositions can be used for the prevention and / or treatment of ANGPTL3 -related cardiovascular disorders or diseases.

[0209] Examples of cardiovascular diseases or disorders that may be treated, prevented or ameliorated by the vaccine lipid nanoparticles include, but are not limited to, coronary heart disease, arteriosclerosis, apoplexy and hypertension.

[0210] In some embodiments at least two mRNAs encoding different peptides are encapsulated. In another embodiment three or more mRNAs encoding different peptides are encapsulated within the nanoparticle.Vaccine compositions

[0211] In one embodiment, the vaccine comprising the lipid nanoparticle is part of a pharmaceutical composition.

[0212] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients. Examples of pharmaceutically acceptable salts and / or excipients can be found in Remingtons, The Science and Practice of Pharmacy, October 31, 2011, 21stEdition, Walters Kluwer, which is incorporated herein by reference. The pharmaceutically acceptable salt may be any suitable inorganic or organic salt known to those of ordinary skill in the art.

[0213] The terms “subject,” “individual,” and “patient” may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like). In various embodiments, the subject can be a human under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care of a physician or other health worker. The term patient as used herein includes a human or a non-human subject.

[0214] An “antigen” or a fragment thereof refers to any protein, peptide or fragment thereof that can stimulate a desired immune response in the body of a subject and includes without limitation the production of one or more antibodies, sensitized T lymphocytes, or both.

[0215] “Administering” and similar terms used herein (grammatical variations) refer to the introduction of a vaccine comprising the lipid nanoparticles to the body of a subject, using any route of administration known to those skilled in the art. Exemplary routes of administration include an intramuscular, buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, intravenous, mucosal, oral, rectal,subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0216] In some embodiments, the vaccine comprising the lipid nanoparticle is administered orally, for instance, in a pharmaceutical composition suitable to deliver a protein, peptide or fragments thereof. In some embodiments, the compositions are administered through parenteral injection, intranasally, or via an intramuscular injection.

[0217] In some aspects, the vaccine is administered to the subject one or more times, such as at times including but not limited to 1 to 7 days, 1 to 3 weeks, or about 1, 2, 3, 4, or more months (e.g., up to about 6 months) before the subject is exposed to an agent causing an infectious disease, including a viral disease (e.g., caused by a coronavirus). In some embodiments, the compositions are administered to the subject one or more times after exposure to an agent causing an infectious disease, e.g., at 1 hour, 6 hours, 12 hours, 1 day, 2 days, 4 days or more (e.g., up to about 2 weeks) after exposure.

[0218] The term, “treating”, and similar terms (grammatical variants), refer to clinical interventions that aim to alter the natural course of the individual being treated and may be performed either for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, lipid nanoparticles of the disclosure are used to prevent or delay the development of a disease or to slow the progression of a disease.

[0219] In some embodiments, the vaccine comprises an adjuvant. The adjuvant may include a pharmacological and / or immunological agent that modifies, e.g., enhances, the effect of the antigen. In some embodiments, the adjuvant increases the immunogenicity of the antigen. For example, an adjuvant may be recognized by the innate immune system and may elicit an innate immune response. In some embodiments, an adjuvant is included in the formulation to modify the immune response by boosting it such as to provide increased levels of antibodies and / or a longer lasting immune response. In some embodiments, this may reduce the amount of antigen required.In some embodiments, the adjuvant may enhance the efficacy of the vaccine by activating particular cell types of the immune system, for example by activating the T cells instead of antibody-secreting B cells. Such adjuvants include, but are not limited to, Freund’s, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.

[0220] The examples below are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and properties thereof but are in no way intended to limit the scope of the invention.EXAMPLESMaterials and MethodsVaccine LNP preparation

[0221] 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 mRNA encoding SarsCov2 spike protein, obtained from RNA Technologies and Therapeutics™ Inc. in buffer were combined in a 1 :3 volume by volume ratio using a t-junction with dual-syringe pumps. 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 at least -100 volumes of 1 x phosphate buffered saline, pH 7.4 using Spectro / Por dialysis membranes (molecular weight cut-off 12000-14000 Da). The LNPs were concentrated as required with an Amicon Ultra™ 10000 MWCO (molecular weight cut-off), regenerated cellulose concentrator.

[0222] 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 - Fi) / Ftx 100.

[0223] The particle size and poly dispersity index (PDI) were characterized using a Zetasizer Nano ZS™.Modified LNP preparation method

[0224] The above LNP preparation method was modified to formulate LNPs without PEG-lipid (see Example 4 below). After T-mixing, 25 mM sodium acetate was added at a 1:3 volume ratio before dialysis and gently mixed, followed by transfer to dialysis in 1 X PBS followed by processing as usual.In vivo imaging

[0225] LNPs encapsulating mRNA encoding luciferase were injected at time 0 to three mice. Shortly prior to imaging, D-luciferin (as listed in Cayman™ chemicals catalog# 14681) was administered to the mice at a dose of 150 mg / kg intraperitoneally. A 15 mg / mL stock of D-luciferin was prepared using IX Dulbecco’s Phosphate Buffered Saline (containing no calcium or magnesium) and 10 pL / gram of body weight was administered intraperitoneally to each of the mice.

[0226] At 4 and 24 hours after LNP injection, the mice were anesthetized using 2.5-3% isoflurane in a chamber and were subsequently placed on the imaging platform maintained under 2% isoflurane through a nose cone.

[0227] All mice were imaged 10 mins post D-luciferin injection at an exposure time of 15 seconds. Bioluminescence imaging was performed using IVIS® Lumina II In Vivo Imaging System (IVIS). An untreated mouse was used as a negative control for background luminescence.Immune response studies

[0228] Mice (C57BL6) that were 6-8 weeks old were injected intramuscularly with the indicated doses of LNPs. Mice were injected intramuscularly (IM) at Day 0 (prime dose) and at Day 21 (boost dose). Blood samples were collected by saphenous bleeds at 6 hours post IM injections for innate immune responses and at 21 days (prime IgG levels) post IM injections for prime serum IgG levels. At 35 days post IM injection (terminal end point), blood samples were collected by cardiac puncture to measure boost serum IgG levels. Prime and boost serum IgG levels were measured using Mesoscale Discovery (MSD). Neutralizing antibodies that inhibit the binding of angiotensin converting enzyme 2 (ACE2) to receptor binding domain (RBD) of spike protein were measured using MSD. Serum samples taken at 6 hours were used to measure levels of cytokines for innate immune responses. At D35 post IMs, spleens were extracted andsplenocytes wereisolated to determine T cell responses. T cell responses were measured by stimulating splenocytes ex vivo with a spike peptide pool. IFN-g / IL-4 enzyme-linked immunospot (ELISpot) and intracellular cytokine staining were used to measure T cell responses.Example 1: Luciferase expression levels in an Onpattro™-type formulation is significantly higher compared to IcLNP™ formulation at 4 hours after intramuscular injection

[0229] Onpattro™-type LNP and IcLNP that encapsulate mRNA encoding luciferase were prepared. The two formulations are set forth in Table 1 below:Table 1: Formulations examined in vivo for luciferase mRNA expression

[0230] ALC-0315 in the baseline LNP is an ionizable cationic lipid described in WO 2018 / 081480, which is incorporated herein by reference. The structure of ALC-0315 is provided below:

[0231] Mice were injected intramuscularly with 10 pg of ALC-0315 Onpattro™-type formulation, 10 pg of ALC-0315 IcLNP formulations or uninfected (untreated or negative control). Both LNP formulations encapsulated luciferase mRNA. Luciferase expression was assessed at 4and 24 hours after LNP injection and luciferin injection using the in vivo imaging procedure set forth in the Materials and Methods.

[0232] The results are shown in Figure 1. The Onpattro™-type gold standard formulation showed significantly higher mRNA expression levels compared to IcLNP formulation at both 4 hours (Figure 1A) and 24 hours (Figure IB). No luminescence was observed in the untreated mouse.Example 2: An equivalent immune response is raised against mRNA expressed antigen in IcLNP™ formulation as compared to gold standard Onpattro™-type formulation

[0233] Onpattro™-type LNP and IcLNP were prepared that encapsulate mRNA encoding Sars- Cov2 spike protein. The two formulations are set forth in Table 2 below:Table 2: Formulations examined in vivo for IgG immune response

[0234] The physiochemical properties of the lipid nanoparticles were measured as described in the Materials and Methods. The particle size (nm), encapsulation % and polydispersity index (PDI) were all within acceptable ranges.

[0235] The mice in the study were injected intramuscularly with 1 pg of IcLNP™ formulations and Onpattro™-type formulations encapsulating SARS CoV-2 spike mRNA at Day 0 (prime dose) and at Day 21 (boost dose).

[0236] As set out in the Materials and Methods, blood samples were collected by saphenous bleeds on day 21 (prime IgG levels) and by cardiac puncture on Day 35 (boost IgG levels). Serum was extracted from blood samples and was stored at -80 degrees Celsius until analysis. Meso Scale Discovery (MSD) was used to measure IgG levels against SARS CoV-2 spike protein and RBD.

[0237] The observed Electrochemiluminescence (ECL) signal was plotted as the levels of IgG antibodies detected in serum samples for each group.

[0238] The results are shown in Figure 2. Unexpectedly, at day 35, the IcLNP™ exhibited comparable IgG levels against SARS-CoV2 RBD as the Onpattro™-type gold standard formulation. This is despite findings described in Example 1 showing that significantly lower expression levels of mRNA are observed after local injection when formulated in the IcLNP™.Example 3: A range of vaccine IcLNP™ formulations result in improved immune response against mRNA expressed antigen

[0239] In view of the unexpected results set forth in Example 2, additional vaccine candidates were screened for an IgG response against Sars-Cov2 spike protein as shown in Table 3 below. Phosphate Buffered Saline (PBS) and ALC luc IcLNP™ were used as negative controls. The two formulations from Example 2 (Table 2) were used as positive controls.Table 3: Formulations screened in vivo for IgG immune response

[0240] A total of 32 mice were selected. All subjects selected for the study were 6- to 8-week old female C57BL / 6 mice. The mice were pre-dosed with subcutaneous Metacam (5 mg / kg) before they were subjected to prime dose of the formulation at Day 0, and a boost dose at Day 21. Blood samples were collected and Meso Scale Discovery (MSD) was used to measure IgG levels against SARS CoV-2 spike protein as described in the Materials and Methods.

[0241] The observed electrochemiluminescence (ECL) signal was plotted as the levels of IgG antibodies detected in serum samples for each group.

[0242] The results are shown in Figure 3. Unexpectedly, at day 35, the IcLNP™ formulations of Table 3 exhibited comparable IgG levels against SARS-CoV2 spike protein as the Onpattro™- type gold standard formulation.Example 4: Formulations without PEG-lipid (PEG-less LNP) exhibit increased IgG levels against spike protein relative to IcLNP™ PEG-containing formulations

[0243] LNPs having elevated levels of neutral lipid (e.g., a phosphatidylcholine lipid) and lacking a PEG-lipid were next tested to determine IgG responses in mice. The LNPs lacking PEG- lipid were prepared using the modified LNP method described above. Two separate studies were conducted using different ionizable lipids.

[0244] Unexpectedly, the PEG-less LNPs exhibited a significantly higher IgG response after a boost dose than a PEG-containing counterpart formulation.

[0245] The formulations examined in the first study are set forth in Table 4 below. The ionizable lipid was ionizable lipid 3 (compound 24 of PCT / CA2023 / 051727 filed on December 21, 2023).Table 4: PEG-less formulations and baseline formulations screened in vivo for IgG immune response in study 1

[0246] The physiochemical properties of the lipid nanoparticles were measured as described in the Materials and Methods. The particle size (nm), encapsulation % and polydispersity index (PDI) were all within acceptable ranges.

[0247] Mice were injected intramuscularly at Day 0 (prime dose) and at Day 21 (boost dose with 1 pg of IcLNP™ formulations and PEG-less formulations encapsulating SARS CoV-2 spike mRNA.

[0248] Blood samples were collected and IgG levels were assayed as set forth in the Materials and Methods.

[0249] The observed Electrochemiluminescence (ECL) signal was plotted as the levels of IgG antibodies detected in serum samples for each group.

[0250] The results of the first study (conducted in two separate experiments) are shown in Figure 4 A. Unexpectedly, at day 35 after the prime dose, the PEG-less IcLNP™ exhibited higher IgG levels against SARS-CoV2 RBD than the IcLNP™ formulation with 1.5 mol% PEG-DMG.

[0251] The second study examined the IgG response to PEGless IcLNP formulated with ionizable lipid 1, namely compound 16 of W02024 / 065042.

[0252] The formulations tested are set out in Table 5 below.Table 5: PEG-less formulations and baseline formulations screened in vivo for IgG immune response in study 2

[0253] Similar to the first study, at day 35 after the prime dose, the PEG-less IcLNP™ with ionizable lipid 1 unexpectedly exhibited higher IgG levels against SARS-CoV2 RBD than the IcLNP formulation with 1.5 mol% PEG-DMG (Figures 4B and 4C).Example 5: Formulations without PEG-lipid (PEG-less IcLNP™) exhibit improved local delivery relative to baseline formulations containing PEG-lipid after intramuscular injection

[0254] Given the unexpected IgG immune response results above in Example 4, LNPs lacking a PEG-lipid were next tested to determine tissue distribution in mice and compared to a baseline LNP containing PEG-lipid. The LNPs lacking PEG-lipid were prepared using the modified LNP method described above and encapsulated mRNA coding for luciferase.

[0255] The baseline LNP containing PEG and PEGless IcLNP compositions were the same as those set forth in Table 4 of Example 4 above (study 1). Mice were injected intramuscularly with the LNPs at a dose of 1 pg and at 4 and 24 hours, d-Luciferin was subsequently injectedintraperitoneally and images were taken using IVIS imaging as described in the procedure set forth in the Materials and Methods.

[0256] The IVIS images at 4 and 24 hours are shown in Figure 5 A and Figure 5B. As shown in Figure 5A, at 4 hours, the mRNA was present in both the liver (see arrow) and at the site of injection for the benchmark LNP containing PEG. However, the PEGless IcLNP™ only exhibited mRNA expression at the site of injection. Similar results were observed at 24 hours. These results show that the PEGless IcLNP™ exhibits improved localized delivery relative to the benchmark.Example 6: Formulations without PEG-lipid (PEG-less IcLNP™) generate robust innate immune responses

[0257] LNPs lacking a PEG-lipid were next tested to determine innate immune responses in mice by ex vivo measurement of interleukin 6 (IL-6), interferon gamma (IFN-y) and tumour necrosis factor alpha (TNF-a) after LNP injections. The LNPs encapsulated mRNA encoding for SARS CoV-2 spike mRNA. The benchmark LNP contained ALC-0315:DSPC:Chol:PEG2ooo-DMG (50:10:38.5: 1.5 mokmol) with mRNA luciferase (ALC 50 / 10 luc) or with mRNA encoding SARS CoV-2 spike protein (ALC 50 / 10 Onpattro™) and the PEGless formulation contained ionizable lipid l :DSPC:Chol (27.4:50:22.6). The baseline formulations were prepared using the standard LNP preparation method and the LNPs lacking PEG-lipid were prepared using the modified LNP method described above in the Materials and Methods. The formulations that were tested are set out in Table 6 below. Phosphate buffered saline (PBS) was a negative control.Table 6: PEG-less formulations and baseline formulations screened ex vivo for immune responses

[0258] Mice were injected with the formulations at day 0 and with a booster at day 21 at a dose of 1 pg and MSD was used to measure the IL-6, IFN-y and TNF-a levels ex vivo as per the Materials and Methods.

[0259] As can be seen in Figure 6A, unexpectedly, the PEGless IcLNP formulation (PEGless IcLNP™) exhibited increased IL-6 relative to the benchmarks (ALC 50 / 10 luc and ALC 50 / 10). The IFN-y and TNF-a levels were robust as well for the PEGless IcLNP as the respective levels were increased or comparable to the PEG-containing benchmarks (Figures 6B and 6C).Example 7: Formulations without PEG-lipid (PEG-less IcLNP™) generate robust T cell responses

[0260] PEGless LNPs were next tested to determine interferon gamma (IF y) and interleukin-4 (IL-4) responses in mice relative to the PEG-containing benchmark LNPs. The LNPs encapsulated mRNA coding for SARS CoV-2 spike mRNA and the formulations tested are set forth in Table 5 above.

[0261] Mice were injected with the formulations at day 0 and with a booster at day 21 at a dose of 1 pg and Elispot was used to measure IFN y and IL-4 responses from stimulated splenocytes. Splenocytes were extracted from the mice at day 35 and stimulated ex vivo with the spike peptide pool to examine the T cell response ex vivo as described in the Materials and Methods.

[0262] As can be seen in Figure 7A, unexpectedly, the PEGless IcLNP formulation (PEGless IcLNP™) exhibited increased IFN y relative to the PEG-containing benchmarks (ALC 50 / 10 luc and ALC 50 / 10). The IL-4 level of the PEGless formulation was robust as well (comparable to the benchmark; Figure 7B).Example 8: Formulations without PEG-lipid (PEG-less IcLNP™) generate robust cellular responses

[0263] LNPs lacking a PEG-lipid were next tested to determine CD4+cellular immune responses in mice. The LNPs tested included the benchmark and PEGless IcLNP™ set forth in Table 6above. Mice were injected with 1 jug of the formulations at day 0 and day 21 and Intracellular Cytokine Staining (flow experiment) was used to measure the percentage of activated CD4+T cells with cytokine levels ex vivo as indicated in the Materials and Methods.

[0264] The percentage of activated CD4+or CD8+T cells positive for interferon gamma (IFNg+), tumour necrosis factor a (TNFa+), IFN y and TNF-a cytokines (IFNg+ / TNFa+cytokines), interleukin-2 (IL-2+) and interleukin-4 (IL-4+) levels were measured ex vivo.

[0265] The results presented in Figure 8A show that the PEGless LNP formulation caused a robust T-helper Type 1 (TH-type 1) IFNg+, IFNg+ / +TNFa+and IL-2+cellular response in CD4+T cells. Likewise, in CD8+T cells, the results in Figure 8B show that the PEGless LNP formulation caused a robust type 1 IFNg+ / +TNFa+, IL-2+immune response.Example 9: LNPs without PEG-lipid (PEG-less IcLNP™) generate functional antibodies responses

[0266] LNPs lacking a PEG-lipid were next tested to determine the functional antibody mounted against ACE2 binding to SARS CoV-2 receptor binding domain (RBD) in mice. The LNPs tested included PEGless LNP and the benchmark and PEGless IcLNP™ set forth in Table 6 of Example 6 above. Mice were injected with the formulations at a dose of 1 pg with prime and boost doses as per the procedure set forth in the Materials and Methods. The percentage inhibition of ACE2 binding to SARS CoV-2 RBD due to antibody was measured at dilutions of 1 / 300 and 1 / 1000.

[0267] The results in Figure 9A and 9B show that the PEGless IcLNP™ generate equivalent functional antibodies against the RBD as the benchmark LNP as measured by percentage inhibition.Example 10: LNPs without PEG-lipid generate robust IgG response

[0268] LNPs lacking a PEG-lipid were next tested to determine the IgG levels mounted against SARS CoV-2 spike protein in mice and compared with an ALC-0315 benchmark containing 1.5 mol% PEG and a norMC3 PEGless formulation.

[0269] C57BL6 mice were injected intramuscularly with 1 pg of LNP formulations encapsulating SARS CoV-2 spike mRNA. PBS and uLNP encapsulating luciferase mRNA were used as negative controls and ALC-0315 LNP was used as a positive control (benchmark). Mice were injected intramuscularly on Day 0 (prime dose) and at Day 21 (boost dose). Blood sampleswere collected by cardiac puncture at Day 35 (boost IgG levels). Serum was extracted from blood samples and was stored at -80 degrees until analysis. MSD (Mesoscale discovery) was used to measure IgG levels against SARS CoV-2 spike protein. The observed ECL signal was plotted as the levels of IgG antibodies detected in serum samples for each group.Table 7: PEG-less formulations and baseline formulations assessed for IgG response

[0270] As shown in Figure 10, the ECL signals, which are indicative of IgG levels against SARS CoV-2 spike protein, were comparable for the ALC-0315 benchmark (containing 1.5 mol% PEG2000-DMG) and samples without PEG-lipid (Ionizable lipid 4 and 5:DSPC:Chol (27.4:50:22.6)). This is unexpected due to the absence of PEG-DMG.

[0271] The examples are intended to illustrate the preparation of specific lipid nanoparticle mRNA preparations and properties thereof but are in no way intended to limit the scope of the invention.

[0272] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.

Claims

CLAIMS:

1. A method for inducing an immune response in a subject to treat, prevent or ameliorate a disease, condition or disorder, the method comprising administering to the subject a vaccine comprising a lipid nanoparticle encapsulating an nucleic acid encoding an antigenic protein, peptide or fragment thereof, at least 25 mol% of a neutral lipid, a sterol or derivative thereof at a content of at least 10 mol%, and an ionizable cationic amino lipid at between 10 and 40 mol%, optionally the ionizable cationic amino lipid having one or more lipophilic chains with a biodegradable group, wherein the administering of the lipid nanoparticle results in the immune response against the antigenic protein, peptide or fragment thereof expressed by the mRNA in the subject, and wherein each mol% is measured relative to a total lipid content of the lipid nanoparticle.

2. The method of claim 1, wherein the neutral lipid has a phosphocholine head group and is optionally a phosphatidylcholine lipid or a sphingolipid.

3. The method of claim 2, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

4. The method of claim 2, wherein the sphingolipid is sphingomyelin.

5. The method of any one of claims 1 to 4, wherein the neutral lipid is present at a content of at least 30 mol%.

6. The method of claim 5, wherein the neutral lipid is present at a content of at least 35 mol%.

7. The method of claim 5, wherein the neutral lipid is present at a content of at least 40 mol%.

8. The method of claim 5, wherein the neutral lipid is present at 45 mol%.

9. The method any one of claims 1 to 8, wherein the neutral lipid is present at 65 mol% or less.

10. The method of any one of claims 1 to 9, wherein the lipid nanoparticle further comprises a hydrophilic polymer-lipid conjugate.

11. The method of claim 10, wherein the hydrophilic polymer-lipid conjugate is a polyethylene-glycol lipid conjugate, a polysaccharide-lipid conjugate or a polysarcosine-lipid conjugate.

12. The method of any one of claims 1 to 9, wherein the lipid nanoparticle comprises or consists essentially of the neutral lipid, the sterol or derivative thereof, and the ionizable cationic amino lipid and wherein the lipid nanoparticle has 0-1.0 mol% or 0-0.5 mol% of the hydrophilic polymer-lipid conjugate.

13. The method of any one of claims 1 to 9, wherein the lipid nanoparticle has 0-0.2 mol% of the hydrophilic polymer-lipid conjugate.

14. The method of any one of claims 1 to 9, wherein the lipid nanoparticle has 0-0.1 mol% of the hydrophilic polymer-lipid conjugate.

15. The method of any one of claims 1 to 14, wherein the lipid nanoparticle is used to treat or prevent a disease or disorder that is an infectious disease, cancer or an autoimmune disorder.

16. The method of any one of claims 1 to 14, wherein the lipid nanoparticle is used to treat a disease or disorder that is an infectious disease and wherein the infectious disease is a viral disease.

17. The method of any one of claims 1 to 14, wherein the lipid nanoparticle is used to treat a disease or disorder that is cancer.

18. The method of any one of claims 1 to 17, wherein an antibody response is raised against the antigenic protein, peptide or fragment thereof expressed by the nucleic acid in the subject.

19. The method of claim 18, wherein the antibody response comprises IgG against the antigenic protein, peptide or fragment thereof expressed by the nucleic acid in the subject.

20. The method of any one of claims 1 to 19, wherein the lipid nanoparticle is administered at a dosage of from 0.01 mg to 20 mg.

21. The method of claim 20, wherein the lipid nanoparticle is administered at a dosage of from 0.5 mg to 15 mg.

22. The method of claims 20 or 21, wherein the lipid nanoparticle is administered at a dosage from 0.5 mg to 10 mg.

23. The method of any one of claims 20 to 22, wherein the lipid nanoparticle is administered at a dosage from 0.5 mg to 5 mg.

24. The method of any one of claims 20 to 23, wherein the lipid nanoparticle is administered at a dosage from 0.5 mg to 1 mg.

25. The method of any one of claims 1 to 24, further comprising administering a booster dose of the vaccine.

26. A vaccine comprising a lipid nanoparticle, the lipid nanoparticle comprising:(i) a nucleic acid encoding an antigenic protein, peptide or fragment thereof;(ii) a neutral lipid content of from 30 mol% to 70 mol%;(iii) an ionizable cationic lipid content of from 5 mol% to 50 mol%;(iv) a sterol selected from cholesterol or a derivative thereof; and(v) a hydrophilic polymer-lipid conjugate that is present at a lipid content of 0 mol% to 5 mol%, wherein each lipid content is relative to a total lipid content of the lipid nanoparticle.

27. A vaccine comprising a lipid nanoparticle comprising encapsulated nucleic acid encoding an antigenic protein, peptide or fragment thereof and 20 to 70 mol% of a neutral lipid relative to total lipid present in the lipid nanoparticle, an ionizable cationic lipid and a sterol, the lipid nanoparticle inducing at least an equivalent immune response in an animal as measured by IgG levels against the antigenic protein, peptide or a fragment thereof at 21 days and / or 35 days postinjection as compared to a baseline LNP formulation of ALC-0315 / 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 IgG level is measured using electrochemiluminescence.

28. The vaccine of claim 26 or 27, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), di oleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1- palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and / or dipalmitoyl-phosphatidylcholine (DPPC).

29. The vaccine of any one of claims 26 to 28, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC) or dipalmitoyl-phosphatidylcholine (DPPC).

30. The vaccine of any one of claims 26 to 28, wherein the neutral lipid is a mixture of two phosphatidylcholine lipids.

31. The vaccine of claim 30, wherein the mixture comprises distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC).

32. The vaccine of any one of claims 26 to 31, wherein the neutral lipid content is between 40 mol% and 60 mol%.

33. The vaccine of claim 32, wherein the neutral lipid content is between 42 mol% and 58 mol%.

34. The vaccine of claim 32, wherein the neutral lipid content is between 45 mol% and 55 mol%.

35. The vaccine of claim 26 or 27, wherein the ionizable cationic lipid is an amino lipid having at least one lipophilic chain and wherein the at least one lipophilic chain comprises a biodegradable group.

36. The vaccine of any one of claims 26 to 35, wherein the ionizable cationic lipid is present at less than 40 mol%.

37. The vaccine of claim 26 or 27, further comprising a hydrophilic polymer-lipid conjugate.

38. The vaccine of claim 37, wherein the hydrophilic polymer-lipid conjugate is a polyethyleneglycol-lipid conjugate a polysaccharide-lipid conjugate or a polysarcosine-lipid conjugate.

39. The vaccine of any one of claims 1 to 26 or 37, wherein the hydrophilic polymer lipid conjugate is present at 0 to 1.0 mol%.

40. The vaccine of claim 39, wherein the hydrophilic polymer lipid conjugate is present at 0 to 0.8 mol%.

41. The method of claim 40, wherein the hydrophilic polymer lipid conjugate is present at 0 to 0.5 mol%.

42. The vaccine of claim 26 or 27, wherein the sterol is present at from 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

43. The vaccine of claim 26 or 27, wherein the sterol is present at from 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

44. The vaccine of claim 27, wherein the lipid nanoparticle exhibits an IgG response in vivo at 1 pg that is equivalent or greater than an IgG response resulting from a lipid nanoparticle having the baseline LNP formulation and that is otherwise identical.

45. Use of the vaccine of any one of claims 26 to 44 to induce an immune response against the antigenic protein, peptide or fragment thereof expressed by the mRNA.

46. The vaccine of any one of claims 26 to 44, wherein the lipid nanoparticle is present at a dosage from 0.01 mg to 20 mg.

47. The vaccine of claim 46, wherein the lipid nanoparticle is present at a dosage from 0.5 mg to 15 mg.

48. The vaccine of claim 47, wherein the lipid nanoparticle is present at a dosage from 0.5 mg to 10 mg.

49. The vaccine of claim 48, wherein the lipid nanoparticle is present at a dosage from 0.5 mg to 5 mg.

50. The vaccine of claim 49, wherein the lipid nanoparticle is present at a dosage from 0.5 mg to 1 mg.

Images