Lipid nanoparticles comprising elevated native lipids and targeting moieties for targeted delivery of nucleic acids

Abstract

Provided herein are lipid nanoparticles encapsulating nucleic acids having a neutral lipid in a molar ratio of at least 30%, a sterol or a derivative thereof, and a targeting moiety anchored in its lipid layer by a lipophilic moiety. Methods of using the lipid nanoparticles for targeted delivery in vivo are also provided. Such lipid nanoparticles may have significantly improved delivery and targeting to extrahepatic tissues and / or organs.

Description

Technical Field

[0001] The present disclosure relates to lipid nanoparticle formulations for the delivery of nucleic acids.

[0002] background

[0003] Lipid nanoparticle (LNP) formulations represent a revolution in nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro, developed by Alnylam. TM Onpattro TM The success of this LNP delivery system paves the way for the clinical development of a major LNP-based mRNA vaccine for COVID-19.

[0004] Onpattro TM The LNP formulation consists of four major lipid components: an ionizable amino lipid (DLin-MC3-DMA or "MC3" (dilinoleyl-methyl-4-dimethylaminobutyrate)), distearoylphosphatidylcholine (DSPC), cholesterol, and a polyethylene glycol-conjugated lipid (PEG-lipid), each in a molar ratio of 50 / 10 / 38.5 / 1.5. TM It is still considered the gold standard for comparison in LNP-mediated efficacy studies, and the design methods of LNPs currently used in the clinic show little deviation compared with the four-component system.

[0005] Among these four components, ionizable lipids constitute Onpattro TM The main body of the preparation and is present at a 50% molar ratio. Ionizable lipids are considered to be crucial for the in vitro and in vivo activity of LNP systems, and therefore most of the work in this field has focused on improving this lipid component. Ionizable lipids, which are usually amino lipids, have been carefully designed so that they are charged at low pH and near-neutral physiological pH. This allows electrostatic interactions between lipids and negatively charged nucleic acids during initial formulation. Because ionizable lipids are close to neutral at physiological pH, toxicity and renal clearance are reduced. After endocytosis, the acidic environment of the endosome causes the net positive charge of the ionizable amino lipid to increase, which promotes fusion with the anionic lipids of the endosomal membrane and subsequent destabilization of the membrane and the release of nucleic acid-based therapeutics into the cytoplasm to exert their effects.

[0006] Regarding the remaining three lipid components, it is well known that PEG-lipids are used to improve the circulation lifetime of LNPs and cholesterol is used to stabilize the particles. However, relatively little attention has generally been devoted to studying neutral lipids beyond their structural roles.

[0007] The liver is a major organ in which Onpattro TM Four-component LNP accumulation. While delivery to the liver holds therapeutic potential, the ability of LNPs to accumulate in organs and tissues beyond the liver would greatly expand the clinical applications of these delivery systems. Extrahepatic delivery could improve the treatment and / or prevention of cancer, cardiovascular disease, infectious diseases, and other conditions.

[0008] However, efforts to target tissues (extrahepatic organs and tissues) beyond the intravenous approach to the liver have been less successful. In order to improve the delivery of LNP to extrahepatic tissues, particles should have an enhanced circulation life. As mentioned above, the conventional method realizing this purpose comprises designing LNPs with long-acting PEG coatings, commonly referred to as "stealth" liposomes. Nevertheless, comprising PEG-lipid in LNP often results in a low transfection efficacy. Alternatively, mixing multiple permanent positively charged lipids can enhance the transfection of multiple extrahepatic tissues after intravenous administration. However, this lipid is toxic, has potentially limited clinical application.

[0009] Studies have found that DSPC and cholesterol help to stably encapsulate siRNA in LNPs (Kulkarni et al., 2019, Nanoscale, 11: 21733-21739). Despite these findings, subsequent in vivo studies by another group failed to show any significant benefit due to the regulation of DSPC levels in LNPs to improve extrahepatic delivery of siRNA. These studies used Onpattro® LNPs with 10 and 40% molar ratios of DSPC. TM The results showed that 10% molar ratio of DSPC / PEG-DMP was effective in the in vitro silencing of siRNA gene in vitro (Ordobadi, 2019, “Lipid Nanoparticles for Delivery of Bioactive Molecules”, a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The University of British Columbia). TM The formulation had similar liver accumulation and blood circulation lifespan to the 40% molar ratio DSPC formulation (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5% molar ratio). In addition, LNPs containing 40% molar ratio DSPC siRNA (siRNA-LNPs) performed comparable to the 10% molar ratio DSPC formulation in bone marrow gene silencing.

[0010] The conjugation of various targeting moieties to the siRNA itself or their incorporation into the formulation as part of surface modification has been investigated by various groups as an alternative to passive targeting. TM Four component LNP preparations are accumulated in a large number in liver (liver) tissue, and most of ligand targeting strategies focus on improving the delivery to hepatocyte.For example, Chen et al., (2014, Journal of Controlled Release, 196:106-112) describe that the LNP with hepatocyte specific targeting ligand GalNAc-PEG is used for improving liver gene silencing by increasing the uptake of functionalized LNP in liver.WO 2010 / 144740 describes four component liposome preparations with ionizable lipid (MC3) / DSPC / chol / PEG-lipid, wherein improves FVII siRNA silencing in liver by adding GalNAc to liposome surface.The LNP containing mannose has also been used for targeting HepG2 hepatocyte.

[0011] Despite these previous efforts, there remains a significant need in the art to improve the targeted delivery of nucleic acids to the liver and / or extrahepatic tissues or organs using LNPs.

[0012] Overview

[0013] The inventors have discovered that by using lipid nanoparticles (LNPs) with elevated levels of neutral lipids in combination with moieties on the surface of the LNPs for binding to a subset of target cells, surprising improvements in nucleic acid expression in certain tissues and / or organs can be achieved. The inventive LNPs described herein are therefore capable of employing two levels of targeting, namely targeting the desired tissue or organ by including elevated levels of neutral lipids and actively targeting the cell type of interest having a surface receptor that binds to the targeting moiety on the LNP.

[0014] In some advantageous examples herein, present disclosure provides lipid nanoparticles (LNPs) comprising three or four lipid components for delivering nucleic acids. The three or four lipid components include ionizable lipids, such as neutral lipids of phospholipids, sterols, and optional hydrophilic polymer-lipid conjugates. In particular, neutral lipids are present in a content higher than that of conventional LNPs, such as at least 20% mol ratio, at least 30% mol ratio, at least 36% mol ratio, or at least 40% mol ratio (relative to the total lipid content of LNP).

[0015] According to one aspect of the present disclosure, a lipid nanoparticle is provided, comprising: (i) a nucleic acid; (ii) a neutral lipid content greater than 35 mol%; (iii) an ionizable lipid content of 5 mol% to 50 mol%; (iv) a sterol or a derivative thereof; and (v) a targeting moiety attached to a lipophilic moiety present in a lipid layer of the nanoparticle, the targeting moiety optionally being attached to the lipophilic moiety via a linker, wherein each % molar ratio is relative to the total lipid content of the lipid nanoparticle, and wherein the lipid nanoparticle comprises a core, the core optionally comprising an electron-dense region and an aqueous portion, and wherein the core is at least partially surrounded by the lipid layer as seen by cryo-electron microscopy (cryo-EM).

[0016] According to another aspect of the present disclosure, there is provided a nucleic acid-encapsulating lipid nanoparticle having at least 38% by molar ratio of a neutral lipid, a sterol, or a derivative thereof and a targeting moiety anchored in a lipid bilayer or a monolayer thereof by a lipophilic moiety, wherein the targeting moiety is present in an amount of less than 2.5% by molar ratio, and wherein a linker is optionally present between the lipophilic moiety and the targeting moiety.

[0017] According to one embodiment, the linker is a hydrophilic polymer conjugated to a lipophilic moiety at one end and to a targeting moiety at the other end.

[0018] According to one embodiment, lipid nanoparticles are prepared by ethanol injection, which includes the step of lowering the pH of the solution outside the nanoparticles after the nanoparticles are formed, thereby preparing a core comprising an electron-dense region and an aqueous portion.

[0019] In another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoyl-phosphatidylcholine (DMPC) or dipalmitoyl-phosphatidylcholine (DPPC).

[0020] According to another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC).

[0021] In another embodiment, the phosphatidylcholine content is 38 mol% to 60 mol%.

[0022] In another embodiment, the phosphatidylcholine content is 40% to 60% mol%, 42% to 60% mol%, 45% to 60% mol%, 46% to 60% mol%, or 48% to 60% mol%.

[0023] In another embodiment, the cationic lipid is an amino lipid.

[0024] According to another embodiment, the ionizable cationic lipid is present in a molar ratio of less than 20%.

[0025] In another embodiment, the lipid nanoparticle comprises a hydrophilic polymer-lipid conjugate present at a lipid content of 0% mol to 5% mol or 0.5% mol to 5% mol.

[0026] In another embodiment, the sterol is present in a molar ratio of 15% to 45% based on the total lipid present in the lipid nanoparticle.

[0027] According to another embodiment, the sterol is present in a molar ratio of 18% to 40% based on the total lipids present in the lipid nanoparticles.

[0028] According to another embodiment, the lipid nanoparticles exhibit at least a 10% increase in biodistribution in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without a targeting moiety and / or an Onpattro-type formulation that encapsulates the nucleic acid but is additionally measured under the same set of conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0029] In another embodiment, the lipid nanoparticles exhibit at least a 10% increase in mRNA expression in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without a targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0030] According to another embodiment, the lipid nanoparticles exhibit at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without a targeting moiety and / or an encapsulated nucleic acid but additionally measured under the same set of conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0031] In another embodiment, the lipid nanoparticles exhibit at least a 10% increase in mRNA expression in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without a targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0032] According to another embodiment, the targeting moiety is present in less than 2% molar ratio.

[0033] In another embodiment, the targeting moiety is present at less than 1.8% molar ratio.

[0034] According to another embodiment, the targeting moiety is present in less than 1.5% molar ratio.

[0035] In another embodiment, the targeting moiety is present at less than 1.2% molar ratio.

[0036] According to another aspect, there is provided a method for delivering a nucleic acid to a cell for treating a disease, disorder or condition, the method comprising contacting the lipid nanoparticle of any one of the preceding aspects or embodiments with the cell in vivo or in vitro.

[0037] According to one embodiment, the nucleic acid accumulates in the spleen, bone marrow, heart, lungs and / or kidneys of the individual for at least one day after administration.

[0038] In one embodiment, the disease, disorder or condition is an autoimmune disorder.

[0039] In another embodiment, the disease, disorder or condition is an infectious disease.

[0040] In one embodiment, the disease, disorder or condition is cancer.

[0041] In another embodiment, the cells are stem cells.

[0042] In one embodiment, the stem cells are hematopoietic stem or progenitor cells.

[0043] In another embodiment, the cell is a T cell.

[0044] In another aspect, there is provided a use of the lipid nanoparticles of any of the above aspects or embodiments for delivering nucleic acids to mammalian cells in vivo or in vitro.

[0045] In another aspect, there is provided a use of the lipid nanoparticles according to any of the above aspects or embodiments in the preparation of a medicament for delivering a nucleic acid to a mammalian cell in vivo or in vitro.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Particle size, encapsulation efficiency, and polydispersity index (PDI) of exemplary lipid nanoparticles (LNPs) of the present disclosure surface modified with varying amounts of an arginine-glycine-aspartic acid (RGD) peptide targeting moiety attached to a lipid via PEG (LNPs B and C) are shown compared to a non-targeted control (LNP A). The LNPs A to C tested are described in Table 1 of Example 1 and encapsulated mRNA encoding firefly luciferase (Fluc).

[0048] Figure 2 Shown are luminescence intensities per μg protein for exemplary lipid nanoparticles (LNPs) of the present disclosure surface-modified with an arginine-glycine-aspartic acid (RGD) peptide targeting moiety attached to a lipid via PEG (LNP E; squares) compared to a non-targeting control (LNP D; circles) at various doses. LNPs were added to the A7 astrocyte cell line at the indicated mRNA doses. The LNPs D and E tested are described in Table 2 of Example 2.

[0049] Figure 3A Luminescence intensity in the liver of exemplary lipid nanoparticles (LNPs) of the present disclosure surface-modified with varying amounts of an arginine-glycine-aspartic acid (RGD) peptide targeting moiety attached to a lipid by PEG is shown compared to a non-targeting control (LNP A). The LNPs AC tested are described in Table 1 of Example 1 and encapsulate mRNA encoding firefly luciferase (Fluc).

[0050] Figure 3B Luminescence intensity in bone marrow of exemplary lipid nanoparticles (LNPs) of the present disclosure surface-modified with varying amounts of an arginine-glycine-aspartic acid (RGD) peptide targeting moiety attached to the lipid via PEG (LNPs B and C) is shown. The LNPs B and C tested are described in Table 1 of Example 1 and encapsulate mRNA encoding firefly luciferase (Fluc).

[0051] Figure 4A The results show that mice were injected with phosphate buffered saline (PBS), Onpattro TM Type preparation (B), lcLNP without ligand TM (C) lcLNP with aCD117 ligand TM (D) and lcLNP with aCD5 ligand TM (E) Afterwards, lineages in the bone marrow - c-Kit + (LK) and pedigree - c-Kit + Sca1 +Enhanced green fluorescent protein (GFP) in (LSK) cell populations. The recipe is listed in Table 3 and the gating scheme is listed in Table 4.

[0052] Figure 4B The results show that mice were injected with phosphate buffered saline (PBS), Onpattro TM Type preparation (B), lcLNP without ligand TM (C) 1cLNP with aCD117 ligand TM (D) and 1cLNP with aCD5 ligand TM (E) Afterwards, multipotent progenitor cell lineages - ckit + Sca1 + CD34 + (MPP), short-term HSC lineage - ckit + Sca1 + CD34 - CD135 + (ST-HSC) and lineage - ckit + Sca1 + CD34 - CD135 - Enhanced green fluorescent protein (GFP) in the (LT-HSC) cell population was added. The formulation is listed in Table 3 and the gating scheme is listed in Table 4.

[0053] Figure 4C The results show that mice were injected with phosphate buffered saline (PBS), Onpattro TM Type preparation (B), lcLNP without ligand TM (C) lcLNP with aCD117 ligand TM (D) and lcLNP with aCD5 ligand TM (E) After that, the lineage - ckit + Sca1 + CD34 - CD135 - CD48 - CD150 + Enhanced green fluorescent protein (GFP) in cell populations. The recipe is listed in Table 3 and the gating scheme is listed in Table 4.

[0054] Figure 5Cryo-electron microscopy images of lipid nanoparticles consisting of 50% molar ratio DSPC, i.e., MF019 / DSPC / Chol / PEG-DMG (27.4 / 50 / 21.1 / 1.5 mol:mol), encapsulating mRNA encoding luciferase. MF019 is an ionizable cationic lipid disclosed in WO2022 / 155728A1, which is incorporated herein by reference.

[0055] Details

[0056] The lipid nanoparticles described herein comprise a targeting moiety and an ionizable lipid, an elevated level of a neutral lipid such as a phosphatidylcholine lipid (e.g., DSPC) or a sphingolipid (e.g., sphingomyelin lipid), and a sterol. In certain embodiments, the neutral lipid is a phosphatidylcholine lipid present in a % molar ratio of at least 35%, at least 40%, or at least 42%, and wherein the ionizable lipid is present in a 45% or 40% molar ratio. As set forth herein, in certain non-limiting examples of the present disclosure, relative to Onpattro TM The LNPs, or the same LNPs without a targeting moiety, comprising a neutral lipid in combination with a targeting ligand at a higher % molar ratio than that used in conventional formulations for nucleic acid delivery provide improved selective delivery of nucleic acids to the liver and / or extrahepatic tissues.

[0057] neutral lipids

[0058] Neutral lipids are amphipathic lipids that allow particle formation and that are substantially free of net charge at physiological pH. The term includes zwitterionic lipids, such as, but not limited to, phospholipids. In alternative embodiments, the lipid nanoparticles comprise the structural lipids of non-cationic lipids. In certain embodiments, LNPs are substantially free of net charge.

[0059] As used herein, "substantially no net charge" with respect to LNPs refers to a net surface charge of about zero or nearly neutral at physiological pH, for example, but not limited to, about -2.5 mV to about 2.5 mV.

[0060] In certain embodiments, neutral lipid is phosphatidylcholine lipid.Phosphatidylcholine lipid can be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC).In another embodiment, phosphatidylcholine lipid can be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and their mixture.The phosphatidylcholine lipid component can comprise the mixture of the different neutral lipids of two or more types.

[0061] In certain embodiments, the phosphatidylcholine content is greater than 20% mol ratio, greater than 25% mol ratio, greater than 30% mol ratio, greater than 32% mol ratio, greater than 34% mol ratio, greater than 36% mol ratio, greater than 38% mol ratio, greater than 40% mol ratio, greater than 42% mol ratio, greater than 44% mol ratio, greater than 46% mol ratio, greater than 48% mol ratio or greater than 50% mol ratio. In certain embodiments, the upper limit of the neutral lipid content is 70% mol ratio, 65% mol ratio, 60% mol ratio, 55% mol ratio, 50% mol ratio or 45% mol ratio. The present disclosure also encompasses sub-ranges of any combination of the aforementioned upper and lower numerical limits.

[0062] For example, in some embodiments, the phosphatidylcholine lipid content is from 20% to 80% mol%, or from 25% to 60% mol%, or from 30% to 60% mol%, or from 35% to 60% mol%, or from 40% to 60% mol%, or from 42% to 58% mol%, or from 43% to 57% mol%, or from 44% to 56% mol%, or from 45% to 55% mol%, of the total lipid present in the lipid nanoparticle.

[0063] In certain embodiments, the lipid nanoparticles comprise 35 to 60% or 35 to 55% mol of one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC). In certain embodiments, the lipid nanoparticles comprise 40 to 60% or 40 to 55% mol of one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC).

[0064] In one embodiment, most advantageously, the neutral lipid is DSPC. In certain examples of the present disclosure, relative to Onpattro TM In the presence of LNPs, the presence of DSPC lipids at elevated levels improved the biodistribution of LNPs relative to other neutral phospholipids.

[0065] The term "Onpattro" with respect to LNPs compared to the LNPs of the present disclosure TM Type "is an ionizable lipid with 50 / 10 / 38.5 / 1.5 mol / mol / DSPC / chol / PEG 2000 -DMG lipid LNPs where the ionizable lipid is the same as that of the LNP being evaluated.

[0066] In certain embodiments, the DSPC lipid content is from 20% to 80% molar ratio, or from 25% to 60% molar ratio, or from 30% to 60% molar ratio, or from 35% to 60% molar ratio, or from 38% to 60% molar ratio, or from 40% to 60% molar ratio, or from 42% to 60% molar ratio, or from 43% to 60% molar ratio, or from 44% to 60% molar ratio, or from 45% to 60% molar ratio, or from 46% to 60% molar ratio, or from 48% to 60% molar ratio, of the total lipid present in the lipid nanoparticle. In certain embodiments, the DSPC lipid content is from 30% to 55% molar%, or from 35% to 55% molar%, or from 38% to 55% molar%, or from 40% to 55% molar%, or from 42% to 55% molar%, or from 43% to 55% molar%, or from 44% to 55% molar%, or from 45% to 55% molar%, of the total lipid present in the lipid nanoparticle.

[0067] In one embodiment, the neutral lipid is DOPC. In some examples of the present disclosure, DOPC lipids improve the biodistribution of LNPs relative to other neutral phospholipids when increased. In certain embodiments, the DOPC lipid content is 20% to 80% mol ratio, or 25% to 60% mol ratio, or 30% to 60% mol ratio, or 35% to 60% mol ratio, or 40% to 60% mol ratio, or 42% to 60% mol ratio, or 43% to 60% mol ratio, or 44% to 60% mol ratio, or 45% to 60% mol ratio, or 46% to 60% mol ratio, or 48% to 60% mol ratio of the total lipid present in the lipid nanoparticles.

[0068] In one embodiment, neutral lipid is DPPC. In some examples of the present disclosure, DPPC lipid improves the biodistribution of LNP relative to other neutral phospholipids when increasing. In certain embodiments, DPPC lipid content is 20% mol ratio to 80% mol ratio, or 25% mol ratio to 60% mol ratio, or 30% mol ratio to 60% mol ratio, or 35% mol ratio to 60% mol ratio, or 40% mol ratio to 60% mol ratio, or 42% mol ratio to 60% mol ratio, or 43% mol ratio to 60% mol ratio, or 44% mol ratio to 60% mol ratio, or 45% mol ratio to 60% mol ratio, or 46% mol ratio to 60% mol ratio, or 48% mol ratio to 60% mol ratio of the total lipid present in the lipid nanoparticle.

[0069] Neutral lipids can also include sphingolipids, such as ceramides, sphingomyelins, cerebrosides, gangliosides, or derivatives, such as, but not limited to, reduced analogs thereof, which lack double bonds in the sphingosine unit. In certain embodiments, the sphingolipids are present in a range of 20% to 80% mol ratio, or 25% to 60% mol ratio, or 30% to 60% mol ratio, or 35% to 60% mol ratio, or 40% to 60% mol ratio, or 42% to 58% mol ratio, or 43% to 57% mol ratio, or 44% to 56% mol ratio, or 45% to 55% mol ratio of the total lipids present in the lipid nanoparticles. In certain embodiments, sphingomyelin is present at 20% to 80% molar%, or 25% to 60% molar%, or 30% to 60% molar%, or 35% to 60% molar%, or 40% to 60% molar%, or 42% to 58% molar%, or 43% to 57% molar%, or 44% to 56% molar%, or 45% to 55% molar% of the total lipid present in the lipid nanoparticle.

[0070] In certain embodiments, the sphingomyelin content of the lipid nanoparticles is less than 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "sphingomyelin-free," meaning that there is no detectable sphingomyelin in the LNP (less than 0.5% mol%) or the LNPs are substantially sphingomyelin-free, meaning that there is less than 5% sphingomyelin in the LNP.

[0071] LNP can comprise other neutral lipids except phosphatidylcholine lipid.For example, LNP can be included in other lipids with net positive charge or negative charge under physiological pH.In another example, LNP can also comprise one or more fusion lipids (relative to phosphatidylcholine lipid) of lesser amount, such as DOPE, and they are cone-shaped, thereby promote fusion with cell membrane.Usually, relative to the total lipid present in LNP, this non-phosphatidylcholine lipid is present in LNP with less than 10% mol ratio, less than 9% mol ratio, less than 8% mol ratio, less than 7% mol ratio, less than 6% mol ratio or less than 5% mol ratio.

[0072] Fusogenic lipids, such as dioleoylphosphatidylethanolamine (DOPE) inclusion is thought to promote the delivery of nucleic acids in vitro or in vivo. However, the present disclosure generally does not approve of including such lipids. Therefore, in certain instances, the fusogenic lipid content of the lipid nanoparticles is less than 5% mol ratio, less than 4% mol ratio, less than 3% mol ratio, less than 2% mol ratio, less than 1% mol ratio, less than 0.75% mol ratio, or less than 0.5% mol ratio. In certain embodiments, the LNP is "fusogenic lipid-free", meaning that there is no detectable amount of fusogenic lipid (less than 0.5% mol ratio) in the LNP or that the LNP is substantially fusogenic lipid-free, meaning that the fusogenic lipid content measured relative to the total lipid content in the LNP is less than 5% mol ratio.

[0073] In certain instances, the DOPE content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "DOPE-free," meaning that there is no detectable DOPE (less than 0.5% mol%) in the LNPs or the LNPs are substantially DOPE-free, meaning that DOPE is less than 5% mol% measured relative to the total lipid content in the LNPs.

[0074] In other embodiments, it may be advantageous to include a mixture of different phosphatidylcholine lipids in the LNP. However, in certain instances, the phosphatidylcholine lipid content includes less than 5, 4, or 3 different phosphatidylcholine lipids.

[0075] In certain embodiments, the lipid nanoparticles have an egg phosphatidylcholine (EPC) content of less than 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "EPC-free," meaning that there is no detectable EPC (less than 0.5% mol) in the LNPs, or the LNPs are substantially EPC-free, meaning that there is less than 5% mol of EPC in the lipid nanoparticles as measured relative to the total lipid content in the LNPs.

[0076] In another embodiment, the structural, neutral, zwitterionic or non-cationic lipid component of the lipid nanoparticles consists of less than 20, 10 or 5% molar ratio of non-phosphatidylcholine lipids, such as POPC (measured relative to the total phosphatidylcholine, structural lipid or neutral lipid content).

[0077] In certain embodiments, the transition temperature of the structured, neutral, zwitterionic or non-cationic lipid, such as a phospholipid with a choline head group, is at least 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C or 38°C. A hydrophilic polymer-lipid conjugate is typically included in the LNP to avoid fusion and aggregation of the particles. Without wishing to be bound by any particular theory, it is believed that during particle formation using the mixing method described in co-owned and co-pending U.S. Provisional Patent Application No. 63 / 588,167, filed on October 5, 2023, fusion and aggregation of lipid nanoparticles that do not have a hydrophilic polymer lipid conjugate (or a low level thereof) can be avoided by selecting a structured, neutral, zwitterionic or non-cationic lipid that is in a gel phase rather than a disordered liquid crystal phase at and above room temperature. Inclusion of such structured, neutral, zwitterionic or noncationic lipids in lipid nanoparticles may also improve blood stability after injection.

[0078] In one embodiment, the neutral lipid or mixture thereof has a phase transition temperature of at least 38, 39 or 40°C when incorporated into lipid nanoparticles.

[0079] Neutral lipid content was determined based on the total amount of lipids (mol:mol) in the lipid nanoparticles including sterols.

[0080] Ionizable lipids

[0081] The LNPs of the present disclosure comprise ionizable lipids. Ionizable lipids can be charged at low pH and have essentially no net charge at physiological pH. This allows for electrostatic interactions between lipids and negatively charged nucleic acid cargo during initial formulation. Since ionizable lipids are close to neutral at physiological pH, toxicity and renal clearance are reduced. Without being limited by theory, after endocytosis, the acidic environment of the endosome causes an increase in the net positive charge of the ionizable amino lipids, which promotes fusion with the anionic lipids of the endosome membrane and subsequent membrane destabilization and release of nucleic acid-based therapeutic agents into the cytoplasm to exert their effects.

[0082] In certain embodiments, the LNPs have an apparent pKa of 5.0 to 8.5, 5.0 to 8.0, 5.0 to 7.5, 6.5 to 7.5, or 6.8 to 7.3. Apparent pKa was measured using a 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay modified from previous studies by 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 purpose of determining apparent pKa). According to this method, a series of buffers spanning the pH range of 2 to 11 were prepared 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 to 0.2 mM LNPs. 0.06 mM TNS solution was then mixed with 175 μL of LNPs at each buffer pH in triplicate in a black polystearyl 96-well plate to produce final concentrations of 6.25 and 6 μM lipids and TNS, respectively, in each well. SpectraMax TM M5 microplate reader in λ ex =321nm, λ em =445nm. Then use Prism TM A sigmoidal curve was fitted to plot fluorescence versus pH, where the pKa was determined as the pH value with 50% of the maximum fluorescence intensity.

[0083] In certain embodiments, it is desirable to include less than 50% ionizable lipid in the LNP. That is, the ionizable lipid content can be less than 50% ionizable lipid, less than 45% ionizable lipid, less than 40% ionizable lipid, less than 35% ionizable lipid, less than 30% ionizable lipid, less than 25% ionizable lipid, less than 20% ionizable lipid, less than 15% ionizable lipid, less than 10% ionizable lipid, or less than 5% ionizable lipid, as measured based on the total lipid content of the LNP.

[0084] In certain embodiments, the ionizable lipid content is from 5% to 50% by mole, or from 8% to 47% by mole, or from 10% to 50% by mole, or from 15% to 45% by mole, or from 15% to 35% by mole of the total lipid present in the lipid nanoparticle.

[0085] Ionizable lipids may be referred to as "cationic lipids". As used herein, the term "cationic lipid" refers to a lipid that is electrostatically neutral at a given pH, such as physiological pH, and accepts protons at a lower pH, thereby becoming positively charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) greater than 8. In some embodiments, the cationic lipid has a pKa between 5.0 and 7.0.

[0086] In certain embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid comprises a protonable tertiary amine (e.g., pH titratable) head group and two C16 to C18 alkyl chains with 0 to 3 double bonds. Such lipids include, but are not limited to, thiolipids, such as MF019 and DODMA described herein. Other lipids that can be used to implement the present disclosure include MC3-type and KC2-type lipids, which are well known to those skilled in the art. In other embodiments, the ionizable lipid is selected from WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 246571; WO 2023 / 147657; WO 2022 / 155728; PCT / CA2023 / 050644, filed on May 11, 2023; PCT / CA2023 / 051272, filed on September 27, 2023; PCT / CA2023 / 051273, filed on September 27, 2023; U.S. Provisional Patent Application No. 63 / 434,506, filed on December 22, 2022; PCT / CA2023 / 051274, filed on September 27, 2023; and one or more lipids described in U.S. Provisional Patent Application No. 63 / 445,854, filed on February 15, 2023, each of which is incorporated herein by reference.

[0087] 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 or carbon atom, each of the two lipophilic moieties being directly bonded to the central nitrogen or carbon atom; each lipophilic chain having a total of 15 to 40 carbon atoms; and wherein the lipid has (i) a pKa of 6 to 8.0 (e.g., when formulated); and (ii) a logP of at least 11.

[0088] Optionally, at least one lipophilic moiety bonded to the head group has a biodegradable group. In a non-limiting example, at least one of the lipophilic moieties has an ester group and a sulfur atom in any orientation (e.g., see U.S. Provisional Patent Application No. 63 / 434,506, filed December 22, 2022, which is incorporated herein by reference). In one embodiment, the ionizable cationic lipid has at least one lipophilic moiety of the formula:

[0089]

[0090] In one embodiment, R 1 and R 2 are independently linear, cyclic or branched optionally substituted C3-C 20 alkyl, and optionally having varying degrees of unsaturation; and n is 2 to 8 or 4 to 8.

[0091] In certain embodiments, it is desirable to include less than 50% ionizable cationic lipids in the LNP. That is, the ionizable cationic lipid content can be less than 50% ionizable lipid, less than 45% ionizable lipid, less than 40% ionizable lipid, less than 35% ionizable lipid, less than 30% ionizable lipid, less than 25% ionizable lipid, less than 20% ionizable lipid, less than 15% ionizable lipid, less than 10% ionizable lipid, or less than 5% ionizable lipid.

[0092] In certain embodiments, the ionizable cationic lipid content is from 5% to 50% by mole, or from 8% to 47% by mole, or from 10% to 50% by mole, or from 15% to 45% by mole, or from 15% to 35% by mole of the total lipid present in the lipid nanoparticle.

[0093] It is believed that the inclusion of permanently positively charged lipids in LNPs, such as dimethyldioctadecyl ammonium bromide (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium (DOSPA) and cholesterol-imidazole (CHIM), promotes nucleic acid transfection in vitro or in vivo. Such permanently charged lipids have non-ionizable quaternary amines and are therefore permanently charged. However, the present disclosure generally does not favor the inclusion of such permanently charged lipids. Thus, in certain instances, the lipid nanoparticles have a permanently positively charged lipid content of less than 5 mol%, less than 4 mol%, less than 3 mol%, less than 2 mol%, less than 1 mol%, less than 0.75 mol%, or less than 0.5% molar ratio. In certain embodiments, the LNP is "free of permanently charged cationic lipids," meaning that there is no detectable amount of permanently charged cationic lipids (less than 0.5% molar ratio) in the LNP or the LNP is substantially free of permanently charged cationic lipids, meaning that there is less than 5% molar ratio or less than 3% molar ratio of permanently charged cationic lipids measured relative to the total lipid content in the LNP.

[0094] In certain instances, the DDAB content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "DDAB-free," meaning there is no detectable DDAB (less than 0.5% mol%) in the LNPs, or the LNPs are substantially DDAB-free, meaning less than 5% mol% DDAB measured relative to the total lipid content in the LNPs.

[0095] In some instances, the DOTMA content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In some embodiments, the LNPs are "DOTMA-free," meaning that there is no detectable DOTMA (less than 0.5% mol%) in the LNPs or the LNPs are substantially DOTMA-free, meaning that less than 5% mol% DOTMA is measured relative to the total lipid content in the LNPs.

[0096] In certain instances, the DOTAP content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "DOTAP-free," meaning that there is no detectable DOTAP in the LNPs (less than 0.5% mol%) or the LNPs are substantially DOTAP-free, meaning that DOTAP is less than 5% mol% measured relative to the total lipid content in the LNPs.

[0097] In certain instances, the DOSPA content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, less than 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In certain embodiments, the LNPs are "DOTAP-free," meaning that there is no detectable DOSPA (less than 0.5% mol%) in the LNPs or the LNPs are substantially free of DOSPA, meaning that DOSPA is less than 5% mol% measured relative to the total lipid content in the LNPs.

[0098] In some instances, the CHIM content of the lipid nanoparticles is less than 10% mol%, less than 8% mol%, 5% mol%, less than 4% mol%, less than 3% mol%, less than 2% mol%, less than 1% mol%, less than 0.75% mol%, or less than 0.5% mol%. In some embodiments, the LNPs are "CHIM-free," meaning there is no detectable CHIM in the LNP (less than 0.5% mol%) or the LNPs are substantially CHIM-free, meaning less than 5% mol% CHIM measured relative to the total lipid content in the LNP.

[0099] The ionizable lipid component can include an ionizable anionic lipid as a part of an ionizable lipid component. The example of such a lipid is cholesterol hemisuccinate (CHEMS). Other examples of ionizable anionic lipids are described in co-pending and co-owned U.S. Provisional Patent Application No. 63 / 453,766, filed March 22, 2023, entitled "Ionizable Anionic Lipids," which is incorporated herein by reference in its entirety.

[0100] In certain embodiments, the ionizable cationic lipid is not a lipid structure, including but not limited to C12-200 (see Khare et al., 2022, AAPS Journal, 24:8, incorporated herein by reference) and related structures known to those skilled in the art.

[0101] sterols

[0102] The term "sterol" refers to naturally occurring or synthetic steroids. The term includes cholesterol, plant sterols, zoosterols, and their derivatives.

[0103] The term "sterol derivative" refers to a modified sterol or its precursors, including triterpenes.

[0104] The term "cholesterol" refers to a naturally occurring or synthetic compound having a sterane backbone and having a hydroxyl group bonded to one of its rings, usually the A ring.

[0105] The LNPs may alternatively or additionally comprise a “cholesterol derivative.” Cholesterol derivatives may be naturally occurring or synthetic and include, but are not limited to, cholesterol molecules having a sterane structure and one or more other functional groups including derivatization of a terminal hydroxyl group.

[0106] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fuccasterol, or stigmasterol, dihydrocholesterol, α-cholesterol, epi-cholesterol, streptosterol, cholestanol, cholestanone, cholestanone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N,N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxapropanol, 23-oxapropanol, 24-oxapropanol, cycloasterol, 22-ketosterol, 20- Hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesterol-3β-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanosterol, lupenosterol, sitolcalciferol, calcipotriol, coproposterol, cholecalciferol, lupenosterol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fuccasterol, coproposterol or its salts or esters.

[0107] In one embodiment, the sterol is present at 15% to 50% mol%, 18% to 45% mol%, 20% to 45% mol%, 25% to 45% mol%, or 30% to 45% mol% based on the total lipid present in the lipid nanoparticle.

[0108] In another embodiment, the sterol is cholesterol and is present at 15% to 50% mol%, 18% to 45% mol%, 20% to 45% mol%, 25% to 45% mol%, or 30% to 45% mol% based on the total lipids present in the lipid nanoparticle.

[0109] In another embodiment, the sterol is a cholesterol derivative and is present at 15% to 50% mol%, 18% to 45% mol%, 20% to 45% mol%, 25% to 45% mol%, or 30% to 45% mol% based on the total lipids present in the lipid nanoparticles.

[0110] In one embodiment, the combined (i) sterol content (e.g., cholesterol or a cholesterol derivative thereof); and (ii) phosphatidylcholine lipid content is at least 50% molar, at least 55% molar, at least 60% molar, at least 65% molar, at least 70% molar, at least 75% molar, at least 80% molar, or at least 85% molar, based on the total lipid present in the lipid nanoparticle.

[0111] In one embodiment, the molar ratio of sterol to ionizable lipid is from 0.70 to 1.30 or any range therebetween.

[0112] Hydrophilic polymer-lipid conjugates

[0113] In non-limiting examples, lipid nanoparticles include the hydrophilic polymer lipid conjugates that can be incorporated into the LNP.Conjugates include lipophilic moiety (such as lipid moiety) and hydrophilic polymer chains, optionally with a connector (such as succinate) between lipophilic moiety and polymer chain. The example of hydrophilic polymers includes polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyl oxazoline, polyethyl oxazoline, polyhydroxyethyl oxazoline, polyhydroxypropyl oxazoline, polysarcosine and polyasparagine. In one embodiment, the hydrophilic polymer lipid conjugates are PEG-lipid conjugates. The hydrophilic polymer lipid conjugates can also be naturally occurring or synthetic molecules containing oligosaccharides, such as monosialoganglioside (G M1 The ability of a given hydrophilic-polymer lipid conjugate to enhance the circulation life of the LNPs herein can be readily determined by one skilled in the art using known methods.

[0114] The hydrophilic polymer lipid conjugate may be present in the nanoparticle at 0.5% to 5% mol%, or 0.5% to 3% mol%, or 0.5% to 2.5% mol%, or 0.5% to 2.0% mol%, or 0.5% to 1.8% mol% of the total lipid.

[0115] In certain embodiments, the hydrophilic polymer lipid conjugate may be absent or present in the nanoparticle. For example, the hydrophilic polymer-lipid conjugate may be present at 0% to 5% by mole, or 0% to 3% by mole, or 0% to 2.5% by mole, or 0% to 2.0% by mole, or 0% to 1.8% by mole of the total lipids.

[0116] In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0.5% to 5% mol ratio, or 0.5% to 3% mol ratio, or 0.5% to 2.5% mol ratio, or 0.5% to 2.0% mol ratio, or 0.5% to 1.8% mol ratio of the total lipid. In certain embodiments, the PEG-lipid conjugate can be present in the nanoparticle at 0% to 5% mol ratio, or 0% to 3% mol ratio, or 0% to 2.5% mol ratio, or 0% to 2.0% mol ratio, or 0% to 1.8% mol ratio of the total lipid.

[0117] In one embodiment, the hydrophilic polymer-lipid is selected based on its exchangeability with the lipid nanoparticle.Since the hydrophilic polymer-lipid conjugate is at least partially lost from the LNP upon reaching the target site in vivo, this property can promote in vivo efficacy.

[0118] In such embodiments, the lipid portion of the hydrophilic polymer-lipid conjugate typically has an acyl chain length of less than 18 and has 0 to 2 double bonds in one or both of the acyl chains. In certain embodiments, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate selected from dimyristoylphosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), dioleoylphosphatidylethanolamine-PEG (DOPE-PEG), dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), dimyristoyl diglycerol-PEG (DMG-PEG), or cholesterol-PEG (Chol-PEG).

[0119] In one embodiment, the hydrophilic polymer lipid conjugate is not DSPE-PEG. In other embodiments, the DSPE-PEG content is less than 0.5% mol%, 0.45% mol%, 0.40% mol%, 0.35% mol%, 0.30% mol%, 0.25% mol%, 0.20% mol%, or 0.15% mol%.

[0120] In another embodiment, in a hydrophilic polymer lipid conjugate, a cleavable linker is present between the lipid moiety and the hydrophilic polymer. This linker can be cleaved by being exposed to low pH, reducing agents or proteases present in vivo. The example of a cleavable linker includes esters, ethers, phosphoramidates, hydrazones, β-thiopropionates, disulfide groups and peptides (Romberg et al., 2008, Pharmaceutical Research, 25: 55-71, incorporated herein by reference).

[0121] Additional lipid components

[0122] LNP can comprise other lipid components except those mentioned above (neutral lipid, cholesterol, ionizable cationic lipid and optional hydrophilic polymer-lipid conjugate). Without limitation, such other lipid components can be present with less than 10% mol ratio, 9% mol ratio, 8% mol ratio, 7% mol ratio, 6% mol ratio, 5% mol ratio, 4% mol ratio, 3% mol ratio, 2% mol ratio, 1% mol ratio or 0.5% mol ratio (relative to the total lipid in LNP). Such other lipids include lipids comprising targeting moieties, charged lipids (charged cations or anionic lipids at physiological pH) or other lipid components such as vitamins (e.g., tocopherols). In certain embodiments, LNP is essentially composed of neutral lipid, cholesterol, ionizable cationic lipid and optional hydrophilic polymer-lipid conjugate, which means that any other lipid is present in an amount less than 5% mol ratio relative to the total lipid in LNP.

[0123] In one embodiment, the LNP lacks a ligand-lipid conjugate for targeting stem or progenitor cells. In such an embodiment, a ligand-lipid conjugate is undesirable because it can induce an immune response. Conversely, targeting can be achieved by lcLNP due to the elevated phosphatidylcholine content. TM Thus, in certain embodiments, the ligand-lipid conjugate is present at less than 1% molar ratio, less than 0.5% molar ratio, or 0% molar ratio.

[0124] Additional components may include anionic phospholipids such as phosphatidylserine, and / or ionizable anionic lipids. An example of such a lipid is cholesterol hemisuccinate (CHEMS). Other examples of ionizable anionic lipids are described in a co-pending and co-owned U.S. provisional patent filed on March 23, 2023, entitled "Ionizable Anionic Lipids," which is incorporated herein by reference.

[0125] Alternatively or additionally, the additional lipid component can include a permanently charged cationic lipid, including a lipid with a quaternary ammonium cation (e.g., DOTMA, DOSPA, DDAB, CHIM, and DOTAP) or a permanently charged anionic lipid, such as phosphatidylserine. In certain instances, such a permanently charged lipid is most advantageously present at less than 10% molar ratio, 9% molar ratio, 8% molar ratio, 7% molar ratio, 6% molar ratio, 5% molar ratio, 4% molar ratio, 3% molar ratio, 2% molar ratio, 1% molar ratio, 0.5% molar ratio, or 0.25% molar ratio relative to the total lipid content.

[0126] Nanoparticle preparation and morphology

[0127] Lipid nanoparticles incorporating oligonucleotides can be prepared using a variety of suitable methods, such as the rapid mixing / ethanol dilution method. Examples of preparation methods are described in Jeffs, LB, et al., Pharm Res, 2005, 22(3):362-72; and Leung, AK, 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.

[0128] Without being bound by theory, the mechanism that can use rapid mixing / ethanol dilution method to form the lipid nanoparticles comprising the oligonucleotide of encapsulation can be assumed to be that under low pH (for example, pH 4), the compact area of ​​hydrophobic RNA-ionizable lipid core is formed, which is surrounded by the monolayer of neutral lipid / cholesterol, and as pH raises, due to the conversion of ionizable cationic lipid to neutral form, the monolayer of neutral lipid / cholesterol merges with smaller vesicles. Along with the increase of double-layer neutral lipid ratio, double-layer lipid forms bubble gradually, and ionizable lipid migrates to inner hydrophobic core. Under sufficiently high neutral lipid content, the outer double layer of preferred neutral lipid can form complete double layer around the inner trapped volume.

[0129] The LNP can include a "core" region. The core can be considered heterogeneous in that it includes an electron dense region and an aqueous portion that optionally at least partially surrounds the electron dense region. The electron dense region was observed by cryo-electron microscopy using the methods described in the Materials and Methods section of this article. Without limitation, the electron dense region within the core can be partially surrounded by the aqueous portion within the enclosed space, as observed by cryo-electron microscopy. The aqueous portion forms different aqueous compartments within the lipid nanoparticle. In other words, in certain embodiments, the aqueous portion is not just a hydration layer. In addition, such particles are described in co-owned and co-pending WO 2023 / 184038, the contents of which are incorporated herein by reference. Figure 5 is a reproduction of the LNP with electron dense region and aqueous portion from co-owned and co-pending WO 2023 / 184038 of FIG. 16 .

[0130] In one embodiment, at least about one-fifth of the core (entrapped volume) comprises an aqueous portion, and wherein the electron-dense region is partially adjacent to the lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy. In another embodiment, at least about one-quarter of the core comprises an aqueous portion, and wherein the electron-dense core is partially adjacent to the lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy. In another embodiment, at least about one-third of the core comprises an aqueous portion, and wherein the electron-dense region is partially adjacent to the lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy. In another embodiment, at least about half of the core comprises an aqueous portion, and wherein the electron-dense core is partially adjacent to the lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy.

[0131] In one embodiment, the electron dense region is generally spherical. In another embodiment, the electron dense region is hydrophobic.

[0132] In another embodiment, the electron-dense region of the LNP unexpectedly appears to be completely surrounded by the aqueous portion, as seen by cryo-electron microscopy. This morphology is observed in a single plane, and a portion of the electron-dense region is adjacent to the bilayer, but is not visible because it is not in the visible plane.

[0133] Lipid nanoparticles can comprise single double layer or comprise multiple lipid layers (i.e. multilayer).Comprising one or more double-layer lipid layers can form a continuous layer around the core, or can be discontinuous.In certain embodiments, the lipid layer can be a combination of double layer and single layer.In a non-limiting example, the lipid layer is a continuous double layer around the core.

[0134] Thus, in certain embodiments, the electron-dense region of the core is separated from the lipid layer comprising the bilayer by an aqueous portion or compartment. For example, the present disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles have a core having an electron-dense region partially surrounded by an aqueous portion as determined by cryo-electron microscopy, and wherein the aqueous portion is partially surrounded by the lipid layer comprising the bilayer as observed by cryo-electron microscopy.

[0135] In another embodiment, and not by way of limitation, the present disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have an elongated shape (e.g., typically elliptical) as determined qualitatively by cryo-electron microscopy. In the latter embodiment, the electron-dense region of the core may be partially surrounded by an aqueous space as observed by cryo-electron microscopy.

[0136] In one embodiment, the lipid nanoparticle is part of a lipid nanoparticle formulation, and wherein at least 20% of the electron dense region of the lipid nanoparticle is either (i) encapsulated by the aqueous portion, or (ii) partially surrounded by the aqueous portion, and wherein a portion of the periphery of the electron dense region is adjacent to the lipid layer as observed by cryo-electron microscopy.

[0137] In certain embodiments, the present disclosure provides lipid nanoparticle formulations comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core as determined by cryo-electron microscopy, the core having an electron-dense region adjacent to the lipid layer comprising the bilayer as observed by cryo-electron microscopy.

[0138] In another embodiment, and not by way of limitation, the present disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60% or 70% of the particles have a core comprising an electron-dense region surrounded or wrapped by a continuous aqueous space disposed between a lipid layer (e.g., a bilayer) and the electron-dense region as observed by cryo-electron microscopy.

[0139] LNPs can be visualized by cryo-electron microscopy as described in the Examples section below.

[0140] In another embodiment, the polydispersity index (PDI) of the LNP formulation is less than 0.3, 0.25, 0.2, 0.15, 0.12, or 0.10.

[0141] In another embodiment, the particle size distribution is such that 90% of the particles in the LNP formulations of the present disclosure have a diameter between 40 nm and 200 nm, 40 nm and 150 nm, 40 nm and 140 nm, 45 to 150 nm, 50 nm and 120 nm, or 50 to 140 nm.

[0142] The lipid nanoparticles herein can exhibit particularly high nucleic acid encapsulation efficiencies. As used herein, the term "encapsulation" with respect to the incorporation of nucleic acids into lipid nanoparticles refers to any association of the nucleic acid with any lipid component or compartment of the lipid nanoparticle, including the lipophilic or aqueous portion. In one embodiment, the nucleic acid is present at least in the core of the LNP.

[0143] In one embodiment, the encapsulation efficiency is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 92%.The encapsulation efficiency of nucleic acids is determined as described in the Materials and Methods section of the Examples herein.

[0144] Embodiments of the present disclosure also provide lipid nanoparticles described according to the molar ratio between the positively charged amine groups (N) of the amine lipid and the negatively charged phosphate groups (P) of the oligonucleotide to be encapsulated. This can be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticle is 4 to 15, or 4.5 to 10, or 5 to 10, or 5.5 to 8.

[0145] 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 can be 15, 14, 13, 12, 11, 10, 9 or 8. The present disclosure also includes any two combinations of the upper and lower limits.

[0146] In one embodiment, the lipid nanoparticles have a total lipid weight nucleic acid / micromole of 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 total lipid weight nucleic acid / micromole. 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 total lipid weight nucleic acid / micromole. The present disclosure also includes combinations of any two of the upper and lower limits.

[0147] In one embodiment, the mRNA copy number / LNP is 1 to 10 or 4 to 8.

[0148] Targeting moiety

[0149] Lipid nanoparticles contain a targeting moiety associated therewith that promotes the binding of LNP to target cells and enters target cells by endocytosis. The targeting moiety is any molecule or fragment thereof that binds to target cells on the LNP surface, such as by being present in a cell surface receptor or epitope on the target cell. In some instances, the targeting moiety is selected to identify certain subsets of cells, such as pathological cells, such as malignant cells or infectious agents. In certain embodiments, the targeting moiety is referred to as a ligand.

[0150] The binding affinity of a targeting moiety to a target cell can be detected by any method known in the art, for example, by any standard in vitro assay, such as ELISA, flow cytometry, immunocytochemistry, surface plasmon resonance, etc. Fragments of a targeting moiety are considered targeting moieties as used herein and can be used in certain embodiments of the present disclosure (provided that the fragment can bind to an appropriate cell surface epitope).

[0151] Examples of targeting moieties include antibodies, nanobodies, proteins including but not limited to DARPins and antibodies or fragments thereof, peptides, carbohydrates (e.g., monosaccharides and polysaccharides), aptamers, small molecules, etc. Non-limiting examples of targeting moieties are described in Friedl et al., 2021, Adv. Funct. Mater. 31: 2103347, which is incorporated herein by reference.

[0152] Non-limiting binding pairs are antibody-antigen, nanobody-antigen, DARPin-receptor, hormone-receptor, enzyme-substrate, nutrient (eg vitamin)-transporter, growth factor-growth factor receptor, and carbohydrate-lectin.

[0153] In one embodiment, the targeting moiety is a protein or peptide comprising an immunoglobulin antigen-binding sequence, such as an antibody or fragment thereof. In another embodiment, the targeting moiety is an antigen-binding antibody fragment lacking an Fc sequence. Such a targeting moiety is an immunoglobulin Fc sequence. ab Fragments include immunoglobulin F(ab)2 fragments, Fv antibody fragments, or single-chain Fv antibody fragments (scFv). These fragments can be enzymatically derived or recombinantly produced.

[0154] In another embodiment, the targeting moiety may be a Nanobody, which is a heavy chain antibody with a VHH. Nanobodies may be desirable in certain examples of the present disclosure because they may be easier to scale up than polyclonal antibodies and / or may have improved stability.

[0155] In one embodiment, the targeting moiety is to form binding pairs with tyrosine kinase growth factor receptors, which are overexpressed on the cell surface in many tumors. Exemplary tyrosine kinase growth factors are VEGF receptors, FGF receptors, PDGF receptors, IGF receptors, EGF receptors, TGF-α receptors, TGF-β receptors, HB-EGF receptors, ErbB2 receptors, ErbB3 receptors and ErbB4 receptors. EGF receptor vIII and ErbB2 (HER2) receptors are particularly preferred for cancer treatment using the lipid nanoparticles of the present invention because these receptors are specific to cancer cells such as malignant cells. Alternatively, the targeting moiety is selected to identify cells that need genetic correction or genetic alteration by introducing beneficial genes, for example: endocrine cells, in vitro embryonic cells, germ cells, stem cells or germ cells in epithelial cells, genetically defective organisms.

[0156] A non-limiting example of a surface-modified LNP for cancer treatment is an anti-HER ScFv targeting HER2 expressed on breast cancer cells. Binding to the HER2 extracellular domain can cause inhibition of its activity. In another embodiment, HER2 activity in breast cancer cells can be inhibited by using ankyrin repeat proteins (DARPin). According to such an embodiment, LNP can be modified with a bispecific anti-tumor DARPin (bipDARPins) having two binding moieties that recognize the two extracellular domains of HER2. Both targeting moieties can play a role in capturing and stabilizing the inactive conformation of HER2. This is particularly effective for promoting apoptosis in HER2-dependent tumor cells. (See Stüber et al., 2021, Communications Biology 4 (762); incorporated by reference).

[0157] In another example, LNP can be surface modified to bind T cells, thereby introducing nucleic acid cargo. In certain instances, the targeted T cells include CD5+ or CD4+ T cells. A non-limiting example is the delivery of nucleic acids encoding chimeric antigen receptors to T cells. This treatment produces chimeric antigen receptor T cells (i.e., CAR T cells), which have been genetically engineered to produce artificial T cell receptors specific for the desired target antigen. The resulting CAR T cells can be used to target antigens present on the surface of a subset of cell types. After binding to the surface antigen, the CAR T cells are activated and exert the desired therapeutic and / or preventive effects on the target cells in vivo. This can include cell proliferation, cytotoxicity, and / or increased secretion of factors that can affect other cells, including but not limited to cytokines, interleukins, and / or growth factors, to destroy cells. This CART therapy can be used to treat or prevent a variety of disease indications, including cancer, immune disorders, or cardiovascular disorders. For example, this method can be used to treat heart damage by delivering mRNA encoding anti-fibrosis CAR to T lymphocytes in vivo. The mRNA is formulated in an LNP modified with a targeting portion of CD5. This approach can be used to generate anti-fibrotic CAR T cells in vivo. (Rurik et al., 2022, Science, 7:375(6576):91-96, incorporated herein by reference). Targeted LNPs with improved biodistribution in liver or extrahepatic tissues / organs can be used to deliver mRNA encoding chimeric antigen receptors to a variety of target cells to treat a variety of diseases or disease states.

[0158] Another non-limiting example of using LNPs to target cell subsets includes conjugating CD4 antibodies to LNPs to specifically target CD4 including T cells. + This type of LNP targeting can be used to introduce nucleic acids into T cells in vivo and can be used for immunotherapy, such as treating HIV or other diseases. (Tombicz et al., 2021, Mol Ther, 29(11):3293-3304, incorporated herein by reference).

[0159] Antibody-conjugated LNPs can target receptors present on stem and progenitor cells, such as HSPCs. Examples are CD117, CD49d, CD44, and IL-6R receptors expressed on HSPCs. Thus, LNPs can include anti-CD49d, CD44, and IL-6R antibodies.

[0160] In certain embodiments, the lipid nanoparticle comprises two or more different targeting moieties.

[0161] The part can be connected to the LNP by any suitable method available in the art. Connection can be covalent or non-covalent, for example, by adsorption or complex formation. The connection preferably includes a lipophilic molecule portion that can be put together with the part by forming a covalent bond or a non-covalent bond. The lipophilic molecule portion can be referred to as an "anchor". The anchor is distributed in a lipophilic environment such as a bilayer, thereby connecting the part to the LNP. The method for connecting the part by the lipophilic portion is known in the art.

[0162] A particularly suitable mode of attachment of the ligand to the LNP is by using a ligand conjugated to a lipophilic anchor via an intermediate polymer linker, such as, but not limited to, a hydrophilic polymer. Targeting moieties conjugated to a lipophilic anchor, such as a lipid, via a hydrophilic polymer intermediate linker advantageously become stably associated with the LNP of the present disclosure.

[0163] The size of the linker can vary depending on the ligand. In one embodiment, the linker size varies between 0.50 kDa and 20 kDa, 1 to 10 kDa, or 1.5 to 8 kDa. End-functionalized polymers that selectively react with functional groups on the ligand can be used. Typically, the linker is polyethylene glycol (PEG), although other polymer linkers of varying lengths known to those skilled in the art can also be used.

[0164] The targeting moiety can also be conjugated directly to the lipophilic moiety. For example, the targeting moiety can be a sugar group that is part of a synthetic or naturally occurring glycolipid. Thus, the term "conjugated" or "conjugate" used to refer to a molecule comprising a targeting moiety and a lipophilic anchor includes targeting moiety-lipid conjugates prepared by synthetic conjugation methods or naturally occurring moieties having a lipophilic region.

[0165] The targeting moiety conjugated to the lipophilic anchor (directly or through a linker) can be incorporated into the lipid nanoparticles by including the conjugated lipid in the lipid mixture used to prepare the lipid nanoparticles. For example, using the ethanol rapid mixing technique, a lipid (e.g., glycolipid) directly conjugated to the targeting moiety or a lipid (e.g., polymer, such as PEG) conjugated through a linker can be added to an ethanol lipid solution (comprising lipid components including ionizable lipids, phosphatidylcholines, sterols, and hydrophilic polymer-lipid conjugates), which is mixed with a buffered solution of nucleic acids to form LNP particles in a three-way adapter mixer, and then treated to increase the pH of the external solution to above the pKa of the ionizable lipid.

[0166] In another example, after preparation, use the rear technology of insertion to modify LNP with the targeting moiety that is conjugated to lipophilic anchor.If the targeting moiety is sensitive to conditions (for example peptide or protein, as antibody) used during preparation, for example the high ethanol concentration that uses in the ethanol rapid mixing technology, then this method can be used.In an exemplary embodiment, lipid-PEG-maleimide conjugates can be used with LNP particle and protein or peptide, for example antibody is conjugated.This lipid-PEG-maleimide conjugates can be connected with the antibody functionalized with N-succinimide S-acetylthioacetate through the sulfhydryl group on the antibody.In practice of the present invention, can use other rear technology of insertion that targeting moiety is introduced the surface of LNP to the responsive LNP of preparation condition.

[0167] In another example, the targeting moiety (directly or indirectly linked to the lipophilic moiety via a linker) is present at less than 3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1.0% molar ratio relative to the total lipid content of the LNP.

[0168] In another example, the targeting moiety (linked directly or indirectly through a linker to the lipophilic moiety) is present at 0.25 to 3% molar ratio, 0.30 to 1.5% molar ratio, or 0.35 to 1.25% molar ratio.

[0169] Nucleic acid cargo

[0170] The lipid nanoparticles contain a nucleic acid cargo. As used herein, the term "encapsulation" with respect to the incorporation of a nucleic acid into a nanoparticle refers to any association of the nucleic acid with any component or compartment of the lipid nanoparticle. In one embodiment, the nucleic acid is incorporated into the core of the lipid nanoparticle (visible by cryo-electron microscopy). In another embodiment, the nucleic acid is bound between two closely juxtaposed lipid layers.

[0171] Nucleic acids include, but are not limited to, oligonucleotides, vector DNA, or mRNA.

[0172] Oligonucleotide cargo

[0173] Oligonucleotide cargo includes interfering RNA and antisense oligonucleotides described in more detail below. "Oligonucleotide" or "oligonucleotide cargo" is a single-stranded or double-stranded RNA or DNA molecule and has a length of 5 to 500 nucleotides. The term includes single-stranded antisense oligonucleotides (ASOs), and generally 30 to 500 nucleotides or shorter, double-stranded silencing RNA molecules (siRNAs) with a length of 3 to 40 nucleotides.

[0174] short interfering RNA

[0175] In one embodiment, the oligonucleotide is a "short interfering RNA" or "siRNA," which is an RNA molecule that is capable of reducing or inhibiting the expression of a target gene or nucleic acid sequence in a cell. In one embodiment, the short interfering RNA can mediate the degradation of a target mRNA, as measured in vitro or in vivo. In such an embodiment, the siRNA can act by base pairing with the complementary sequence of the target mRNA (when single-stranded) and induce mRNA cleavage.

[0176] siRNA is double-stranded and can have a variety of lengths, but is typically less than 35 nucleotides in length. In certain embodiments, the siRNA has a length such as 1 to 35 nucleotides, or 15 to 30 nucleotides, or 20 to 25 nucleotides.

[0177] In those embodiments where the siRNA reduces expression of a target gene or sequence by complementary base pairing and mRNA degradation, the siRNA can have substantial or complete identity to the gene encoding the target sequence, or can contain mismatch regions (i.e., mismatch motifs). The sequence of the siRNA can correspond to the full-length target sequence or a subsequence thereof.

[0178] The double-stranded siRNA encapsulated in the LNP can comprise double-stranded RNA, for example double-stranded small interfering RNA, asymmetric interfering RNA (aiRNA) or pre-miRNA or the heterozygous molecule that comprises RNA and DNA.In one embodiment, double-stranded RNA is self-complementary.In such embodiment, siRNA can form stem-loop or hairpin structure at one end.

[0179] The siRNA encompassed by the embodiments of the present disclosure can be used to suppress the expression of a wide range of target polynucleotides. The siRNA molecules directed to the specific polynucleotides used for any treatment, prevention, or diagnosis can be easily prepared according to methods known in the art. The siRNA target site can be selected and chemically synthesized, produced by in vitro transcription, or expressed from a vector or PCR product to produce the corresponding interfering RNA.

[0180] As described above, the siRNAs described herein may contain a "mismatch motif" or "mismatch region," which refers to a portion of an siRNA sequence that does not have 100% complementarity with its target sequence. The siRNA may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. A mismatch motif or region may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.

[0181] The nucleotides of the siRNA may or may not be chemically modified. Examples of optional modifications include, but are not limited to, 2'-O-alkyl modifications such as 2'-O-Me or 2'-O-methoxyethyl modifications and 2'-halogen modifications such as 2'-fluoro modifications. In other embodiments, the siRNA comprises one, two, three, four or more 2'-deoxynucleotides, for example, in the sense strand and / or antisense strand of the double-stranded region. Alternatively or additionally, in certain embodiments, the siRNA comprises a phosphate backbone modification.

[0182] In siRNA, the antisense strand and the sense strand can be designed such that when they form a duplex due to complementary base pairing, they can anneal without overhangs at both ends of the duplex, thereby forming blunt ends, or have overhangs at one or more of the 3' end of the sense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and the 5' end of the antisense strand. In certain embodiments, there is no 5' overhang and no 3' antisense overhang, but there is a 3' sense overhang. In other aspects, there is no 5' overhang, but there are a 3' antisense overhang and a 3' sense overhang. The overhangs may contain T or U nucleotides.

[0183] In certain embodiments, siRNA is covalently bound to one or more other moieties to form a conjugate. In certain embodiments, the conjugate is selected based on their ability to promote siRNA delivery to an organism or cell. siRNA can be combined with a moiety at, for example, the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or at a position not at the 3' end or 5' end of any chain.

[0184] Examples of conjugates include, but are not limited to, one or more antibodies or fragments thereof, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell penetrating peptides, polymers, and sugar groups, including sugar monomers, oligosaccharides, and modifications thereof. In a non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc).

[0185] Antisense oligonucleotides (ASOs)

[0186] In one embodiment, the nucleic acid cargo is an "antisense oligonucleotide" or "ASO," which is a single-stranded nucleic acid (e.g., RNA or DNA) that binds to a target nucleic acid sequence through base pairing. The ASO can have substantial or complete identity to the gene encoding the target sequence, or can contain mismatch regions (i.e., mismatch motifs). The sequence of the ASO can correspond to the full-length target sequence or a subsequence thereof.

[0187] ASOs can reduce or inhibit the expression of a target gene or nucleic acid sequence in a cell through a variety of mechanisms, some of which are described below. In one embodiment, an ASO forms part of a gene editing complex and is used to direct a nuclease to a target site for site-specific cleavage of DNA.

[0188] In those embodiments in which ASO reduces the expression of a target gene or sequence by complementary base pairing and mRNA degradation, ASO exerts its effect and induces mRNA cutting by base pairing with the complementary sequence of the target mRNA. ASO can prevent or reduce the translation of the complementary RNA chain by binding to RNA. ASO can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, the target sequence can be degraded by the enzyme RNase H present in the nucleus and / or cytoplasm. In one embodiment, ASO is a "gapmer" sequence, which comprises 2 to 5 chemically modified nucleotides on each end of the central gap region (e.g., 8 to 10 bases "gaps") of DNA. Chemically modified nucleotides reduce the degradation of nucleases and increase the affinity of ASO to the target sequence. The gap allows the formation of a hybridization sequence that can be cut by RNase H. In addition, it is possible to use known methods to chemically modify oligonucleotides to raise RNase H.

[0189] In another embodiment, the ASO can bind to the target mRNA and block gene expression. ASOs that act by blocking gene expression are called "steric blockers" and block the binding of ribosomes, thereby preventing or reducing translation of the target nucleotide sequence.

[0190] In another embodiment, ASOs can modulate the splicing of pre-mRNA sequences. ASOs can be designed to target sequences within pre-mRNA to affect splicing and increase the production of desired isoforms. ASOs can be used, for example, to remove mutant exons, thereby restoring the proper reading frame and producing a more functional protein product.

[0191] In one embodiment, the ASO comprises from about 15 to about 500 nucleotides, or from about 20 to about 300 nucleotides, or from about 25 to about 200 nucleotides, or from about 30 to about 150 nucleotides.

[0192] Generally, an ASO may contain a "mismatch motif" or "mismatch region," which refers to a portion of the ASO sequence that does not have 100% complementarity with its target sequence. An ASO may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be contiguous or separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. Mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.

[0193] The nucleotides of ASO may or may not be chemically modified. Modification can improve the stability of ASO, for example, in addition to the protection provided by LNP, also increase nuclease resistance. In addition, in some examples of the present disclosure, chemical modification improves effectiveness and / or selectivity by increasing the binding affinity of ASO to its complementary sequence. The example of optional modification includes but is not limited to 2'-O-alkyl modifications such as 2'-O-Me or 2'-O-methoxyethyl modifications and 2'-halogen modifications such as 2'-fluoro modifications. In other embodiments, ASO comprises one, two, three, four or more 2'-deoxynucleotides, for example, in the sense strand and / or antisense strand of the double-stranded region. Alternatively or additionally, in certain embodiments, ASO comprises a phosphate backbone modification, such as a thiophosphate backbone modification. Additional main chain modifications include main chain analogs, such as locked nucleic acids (LNA). Non-limiting examples are structures containing a methylene bridge between the 2' and 4' positions of ribose, which "locks" the ribose ring in a conformation that is conducive to binding to a complementary nucleic acid sequence. The relevant bridge modification is a bridge nucleic acid (BNA). Other examples include ASOs with a peptide backbone (PNA), CpG oligomers, and other oligomers known to those skilled in the art.

[0194] The ASOs encapsulated in LNPs are typically single-stranded. However, in certain embodiments of the present disclosure, the ASOs have self-complementary sequences. In such embodiments, the ASOs may form one or more stem-loop or hairpin structures within the strand.

[0195] In certain embodiments, an ASO is covalently bound to one or more other moieties to form a conjugate. In certain embodiments, conjugates are selected based on their ability to facilitate delivery of the ASO into an organism or cell. The ASO can be bound to a moiety, for example, at the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or at a position that is not at the 3' or 5' end of either strand.

[0196] Examples of conjugates include, but are not limited to, one or more antibodies or fragments thereof, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell penetrating peptides, polymers such as hydrophilic polymers such as polyethylene glycol, and sugar groups, including sugar monomers, oligosaccharides, and modifications thereof. In one non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc).

[0197] Methods for designing antisense oligonucleotides are known in the art and can be readily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence can be based on analysis of the selected target sequence and the secondary structure, T m, binding energy and relative stability. ASO sequences can be designed computationally or obtained experimentally.

[0198] vector DNA

[0199] The lipid nanoparticles described herein may comprise an encapsulated DNA vector. The term "DNA vector" as used herein refers to a polynucleotide encoding at least one peptide, polypeptide or protein, which is circular or linearized.

[0200] DNA vectors can replicate autonomously by methods well known in the art, or can replicate by insertion into the genome of a host cell. Autonomously replicating vectors will have an origin of replication or autonomously replicating sequence (ARS) that functions in the host cell. The DNA vector can be used in more than one host cell, for example, Escherichia coli (E. coli) for cloning and construction, and mammalian cells for expression.

[0201] In order to repair, enhance or block or reduce the expression of a cellular protein or peptide, a DNA vector may be administered to an individual.Thus, the nucleotide polymer may be a nucleotide sequence comprising genomic DNA, cDNA or RNA.

[0202] As will be appreciated by those skilled in the art, a vector can encode a promoter region, an operator region, or a structural region. A DNA vector can contain double-stranded DNA or can be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes comprising operator control and terminator regions, and self-replicating systems such as vector DNA.

[0203] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triplex-forming oligonucleotides. In order to have extended activity, single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted by stable, non-phosphodiester linkages, such as phosphorothioate, phosphorodithioate, phosphoselenate, or O-alkylphosphotriester linkages.

[0204] DNA vectors can include nucleic acids modified in one or more sugar moieties and / or one or more pyrimidine or purine bases. Such sugar modifications can include using halogen, alkyl, amine, azido or functionalized to ether or ester to replace one or more hydroxyls. In another embodiment, the entire sugar can be substituted by spatial and electronic similar structures, including aza-sugar and carbocyclic sugar analogs. The modification of the purine or pyrimidine base moiety includes, for example, alkylated purine and pyrimidine, acylated purine or pyrimidine or other heterocycle substitutes well known by persons skilled in the art.

[0205] In certain embodiments, a modified molecule such as a peptide, protein, steroid or sugar can be used to partially modify the DNA vector. Using this molecule to modify the DNA vector can facilitate delivery to the target site of interest. In certain embodiments, this modification causes the DNA vector to shift across the nucleus of the target cell. As an example, the modifier can bind to a specific portion of the DNA vector (usually not encoding the target gene), but also has a nuclear homing effect, such as a peptide or other modifier of a nuclear localization signal. A non-limiting example of a regulator is the steroid-peptide nucleic acid conjugate described in Rebuffat et al., 2002, Faseb J. 16 (11): 1426-8, which is incorporated herein by reference.

[0206] DNA vectors can contain sequences encoding different proteins or peptides. Promoters, enhancers, stress or chemically regulated promoters, antibiotic sensitive regions or nutrient sensitive regions, and sequences encoding therapeutic proteins can be included as needed. Non-coding sequences can also be present in DNA vectors.

[0207] The nucleic acid that is used for this method can be separated from natural origin, obtain from the source such as ATCC or GenBank library or prepare by synthetic method.Synthetic nucleic acid can be prepared by various solutions or solid phase methods.Usually, solid phase synthesis is preferred.Extensively available detailed description of the method for solid phase synthesis nucleic acid by phosphite-triester, phosphotriester and H-phosphonate chemistry.

[0208] 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.

[0209] In another embodiment, the DNA vector is a nanoparticle or a minicircle.

[0210] The DNA vector can be part of a CRISPR / Cas9 or zinc finger nuclease gene editing system. In another embodiment, the DNA vector is used in diagnostic applications.

[0211] mRNA

[0212] The lipid nanoparticles described herein may include 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 includes, but is not limited to, circular or linear mRNA, as well as small activating RNA (saRNA) and trans-amplifying RNA (taRNA).

[0213] The concentration of mRNA in the LNP can be 0.01 to 20 mg / mL or 0.01 to 10 mg / mL or 0.05 to 5 mg / mL or 0.075 to 4 mg / mL.

[0214] mRNA used herein includes modified and unmodified mRNA. In one embodiment, mRNA comprises one or more coding regions and non-coding regions. mRNA can be purified from natural sources, produced using a recombinant expression system and optionally purified, or can be chemically synthesized.

[0215] In those embodiments in which the mRNA is chemically synthesized, the mRNA can include nucleoside analogs, e.g., analogs with chemically modified bases or sugars, and / or backbone modifications. In certain embodiments, the mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiopyrimidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcyt ... -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); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0216] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, in certain embodiments, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can 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.

[0217] In certain embodiments, in vitro synthesized mRNA can be purified prior to encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0218] The present disclosure can be used to prepare and encapsulate mRNA of various lengths. In certain embodiments, the present disclosure can be used to prepare and encapsulate in vitro synthesized mRNA with a length range of about 1 to 20 kb, about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 2 to 20 kb, about 2 to 15 kb, about 2 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0219] In those embodiments where the mRNA is linear, synthesis includes the addition of a "cap" at the 5' end and a "tail" at the 3' end. The presence of the cap provides resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0220] In certain embodiments, mRNA includes 5' and / or 3' untranslated regions. In certain embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron response element. In certain embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides.

[0221] In certain embodiments, the 3' untranslated region includes one or more polyadenylation signals, binding sites for proteins that affect the stability of mRNA localization in the cell, or one or more binding sites for miRNA. In certain embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or longer.

[0222] In another embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5' and 3' ends and is therefore more stable in vivo due to its resistance to exonuclease degradation. Circular mRNA can be prepared by any known method, including any of the methods described in Deviatkin et al., 2023, "Cap-Independent Circular mRNA Translation Efficiency", Vaccines, 11 (2), 238, which is incorporated herein by reference. Translation of circular mRNA is carried out by a cap-independent initiation mechanism.

[0223] While mRNA provided by an in vitro transcription reaction may be desirable in certain embodiments, other sources of mRNA are also contemplated, such as mRNA produced by bacteria, fungi, plants, and / or animals.

[0224] The mRNA sequence may contain a reporter gene sequence, although its inclusion in pharmaceutical formulations for administration is optional and is typically omitted. Such a sequence is incorporated into mRNA for in vivo studies in animal models to assess biodistribution.

[0225] Edit Goods

[0226] In one embodiment, the LNP-encapsulated cargo edits cells to produce desired modifications to treat, prevent, or ameliorate a disease or condition.

[0227] As used herein, the term "editing cargo" includes protein- and / or nucleic acid-based cargo that causes modification of cells at specific loci to produce desired modifications to treat, prevent, or ameliorate a disease or condition.

[0228] As used herein, the term "nucleic acid editor" includes protein- and / or nucleic acid-based systems that cause modification of any nucleic acid at a specific locus in a cell to produce a desired modification to treat, prevent, or ameliorate a disease or disease state.

[0229] The cargo may comprise a nucleic acid encoding a protein or peptide that forms part of a nucleic acid editing complex. A "nucleic acid editing complex" includes, but is not limited to, protein- and / or nucleic acid-based systems in which nucleic acids are inserted, deleted, modified (e.g., epigenetic editing), or replaced at site-specific locations in the genetic material of an organism.

[0230] Nucleic acid editing complexes can be used for genetic modification of cells, including post-translational modifications.

[0231] Alternatively or additionally, the cargo comprises a peptide or protein that is part of an editor or forms an editing complex.

[0232] Nucleic acid editing complexes include, but are not limited to, Cas-based (e.g., CRISPR or non-CRISPR), transcription activator-like effector nucleases (TALENs), megaTALs, zinc finger nucleases (ZFNs), adenosine deaminases (ADARs) acting on RNA, lead editors, base editors, epigenetic, transposases, giant nucleases, ARCUS gene editing goods, or any variants or combinations thereof. These nucleic acid editing goods are exemplary and include any goods capable of modifying the genetic material of a cell (including RNA transcripts and non-coding regions) to treat, prevent, or improve a condition or disease. Without limitation, gene editing goods may include those designed by methods known to those skilled in the art as directed nuclease editors (DNEs).

[0233] CAS-based editing products include CRISPR and non-CRISPR gene editing products. In addition, editing products include those that cut DNA and epigenetic editing products that modify nucleic acid tags, as described below.

[0234] CRISPR gene editing cargo most advantageously includes nucleic acids (e.g., mRNA) encoding one or more class II Cas nuclease family proteins and guide RNAs. The nuclease encoded by the nucleic acid is an enzyme with DNA endonuclease activity and can be guided by an appropriate guide RNA to cut the desired nucleic acid target. The nuclease and the guide RNA form a complex called a ribonucleoprotein (RNP). In certain embodiments, the nuclease is a class II CRISPR enzyme, which is further subdivided into type II, type V, and type VI. According to one embodiment, the mRNA encodes a Cas protein, which is part of a type II CRISPR / Cas system, such as a Cas9 protein or a Cpf1 protein.

[0235] In another embodiment, the mRNA encodes a Cas protein that is part of a type V CRISPR / Cas system, such as Cas12a. In another embodiment, the mRNA encodes a Cas protein that is Cas 13a, which is an RNA endonuclease and cuts single-stranded RNA.

[0236] The guide RNA is capable of guiding the Cas nuclease to a target sequence on a target nucleic acid molecule, wherein the guide RNA hybridizes to the target sequence and the Cas nuclease cuts or regulates the sequence. In certain embodiments, the guide RNA is combined with Class 2 nucleases, thereby providing specificity for cutting.

[0237] The guide RNA of the CRISPR / Cas9 nuclease system includes CRISPR RNA (crRNA) or tracr RNA (tracr). In certain embodiments, crRNA can include a targeting sequence that is complementary to and hybridizes with a target sequence on a target nucleic acid molecule. CrRNA can also include a flagpole that is complementary to and hybridizes with a portion of tracrRNA. In certain embodiments, crRNA can correspond to the structure of a naturally occurring crRNA transcribed from a bacterial CRISPR locus, wherein the targeting sequence serves as a spacer sequence of the CRISPR / Cas9 system. The flagpole corresponds to the portion of the repetitive sequence adjacent to the spacer sequence above the CRISPR locus.

[0238] The guide RNA of the RNP can target any sequence of interest through the targeting sequence of the crRNA. In certain embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can be 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain at least one mismatch.

[0239] The length of the targeting sequence can depend on the RNP system and the components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Therefore, a targeting sequence having a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides can be included. In certain embodiments, the targeting sequence can comprise a length of 18 to 24 nucleotides. In certain embodiments, the targeting sequence can comprise a length of 19 to 21 nucleotides. In certain embodiments, the targeting sequence can comprise a length of 20 nucleotides.

[0240] In certain embodiments, the editing system comprises Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl11, Csb2, Csb3, Csx17, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof.

[0241] As described above, non-CRISPR, Cas-based gene editing products are also included in the embodiments of the present disclosure. Cas-based editing systems can include Cas enzymes fused to deaminases (Luo et al, 2020, Microbial Cell Factories, 19 (93), incorporated herein by reference). An example is a cytosine base editor or adenine base editor produced by fusing the endonuclease Cas to the cytosine deaminase pmCDA1 or the heterodimeric adenine deaminase Tada-Tada. Another non-limiting example is Cas fused to a reverse transcriptase (Mohr et al., 2018, Mol Cell., 72 (4): 700-714, incorporated herein by reference).

[0242] Fanzor is a eukaryotic RNA-guided endonuclease that can function as a gene editor in certain embodiments herein. (See Saito et al., 2023, Nature 620: 660-668, which is incorporated herein by reference). In certain embodiments, the Fanzor protein uses RNA as a guide to precise target DNA and can be modified to edit cells using LNPS described herein. In certain instances, compact Fanzor cargo can have the ability to be more easily improved than CRISPR-Cas cargo.

[0243] In those embodiments where the cargo is a TALEN, the cargo comprises a nucleic acid encoding a peptide having a transcription activator-like (TAL) effector DNA binding domain, a fragment or variant thereof. In embodiments, the system comprises a nucleic acid encoding a peptide having nuclease activity, such as endonuclease activity. In embodiments, the peptide having nuclease activity is a type II restriction endonuclease, such as a Fok1 endonuclease.

[0244] In those embodiments where the cargo is a ZFN, the nucleic acid can encode a peptide having: a zinc finger DNA binding domain, a fragment or variant thereof; and / or nuclease activity, such as endonuclease activity. In embodiments, the Zn finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or more zinc fingers. In embodiments, the peptide having nuclease activity is a type II restriction endonuclease, such as a Fok1 endonuclease.

[0245] Adenosine deaminases (ADARs) acting on RNA are another editing cargo encompassed by embodiments of the present disclosure, which can be used for post-transcriptional modification of RNA. Examples include ADAR1 and ADAR2. ADAR1 can catalyze the post-transcriptional deamination of C6 of adenosine in dsRNA, converting them to inosine (see Song et al, 2022, PMC, 13(1):e1665, incorporated herein by reference).

[0246] Large nucleases are enzymes in the endonuclease family that can induce homologous recombination, produce mutations, and change reading frames. Large nucleases include homing endonucleases of introns or protein intron endonucleases. In one embodiment, the large nuclease is from the LAGLIDADG family, GIY-YIG endonuclease, HNH endonuclease, His-Cys box endonuclease, or PD-(D / E)XK endonuclease. Large nucleases can be combined with components of other gene editing systems. In one embodiment, the DNA binding domain from a transcription activator-like (TAL) effector is combined with a large nuclease to produce "megaTAL". In another embodiment, the large nuclease can be fused to a DNA end processing enzyme to promote error-prone non-homologous end joining.

[0247] ARCUS nucleases are a gene editing system based on I-CreI, a homing endonuclease that evolved in Chlamydomonas reinhardtii. In certain embodiments, the nuclease is capable of inactivating itself after gene editing, thereby reducing off-target effects. In certain embodiments, ARCUS nucleases are capable of generating a unique cleavage site that is four base pairs with a 3' overhang and can perform gene insertion, gene excision, gene repair, or a combination thereof.

[0248] Epigenetic editing is also included in the examples disclosed herein. This editing of genetic material does not cut nucleic acids, but changes epigenetic markers to "modify" DNA. Changing the epigenetic characteristics of a cell can be used to modify the epigenetic characteristics of a cell and change its transcriptional profile. In certain embodiments, the epigenetic editing system can target and edit one or more methylation sites of a nucleic acid sequence. In certain embodiments, a genomic homing protein with an engineered or naturally occurring nuclease function for gene editing can be mutated and adapted to function only as a delivery system. In one embodiment, an epigenetic modification enzyme or domain can be fused to a homing protein, and local epigenetic modifications can change when the protein is recruited. Targeted proteins that recognize DNA sequences can be connected to effector proteins that change epigenetic markers, such as methylated proteins. Examples of targeted proteins include transcription activator-like effectors (TALEs), zinc finger proteins, and Cas systems including but not limited to CRISPR-Cas. Non-limiting examples of effector proteins include TET1, which induces cytosine demethylation at CpG sites; LSD1, which induces demethylation of H3K4me1 / 2, which also causes the indirect effect of deacetylation of H3K27; and CIB1 / CRY2, which is a cryptand / blue light-activated complex that allows chromatin to be modified upon illumination.

[0249] Other examples of effector proteins include DNA methyltransferases, fragments (e.g., biologically active fragments) or variants thereof (e.g., DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L or CpG methyltransferase (M.SSSL)); or a polycomb repressive complex or a component thereof, such as PRC1 or PRC2, or a PR-DUB, or a fragment (e.g., biologically active fragment) or variant thereof.

[0250] In one embodiment, the epigenetic editor comprises a molecule that modifies chromatin structure and / or modifies histones. In one embodiment, the epigenetic regulator is a molecule that modifies chromatin structure, such as a SWI / SNF remodeling complex or a component thereof. In one embodiment, the epigenetic regulator is a molecule that modifies histones such as methylated histones and / or acetylated histones, such as a histone modifying enzyme or a fragment thereof (e.g., a biologically active fragment) or a variant thereof, such as an HMT, HDM, HAT, or HD AC.

[0251] Improved targeted expression and biodistribution of ligand-LNPs with increased neutral lipid content

[0252] As described in the Examples section, the targeting moiety-LNPs of the present disclosure with elevated neutral lipid content can provide a more effective and efficacious alternative to Onpattro® as described in the Examples section herein. TM In another embodiment, the baseline formulation can be (a) an otherwise identical LNP having a 10% molar lower level of the same neutral lipid; (b) an otherwise identical LNP having an N / P of 1 or 3 when the LNP has an N / P of equal to or greater than 4; and / or (c) an otherwise identical LNP having a total lipid weight nucleic acid / micromole of 0.20:1 when the lipid nanoparticle has a total lipid weight nucleic acid / micromole of 0.05:1 to 1:1, which is less than the total lipid weight nucleic acid / micromole of the lipid nanoparticles of the present disclosure.

[0253] In one embodiment, LNP of the present disclosure shows biodistribution that is increased than relevant baseline in liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin and / or ear in a specified mouse model. In another embodiment, this includes biodistribution of increase to extrahepatic tissue selected from spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin and / or ear relative to relevant baseline. In another embodiment, this includes biodistribution of increase to extrahepatic tissue selected from spleen or bone marrow relative to relevant baseline. Whether LNP encapsulated oligonucleotide shows this enhanced biodistribution to one or more given tissues or organs relative to baseline oligo-LNP formulation is determined by biodistribution studies in the in vivo mouse model as described in detail in the Examples section. Fluorescent lipid markers (DiD, as described in the Materials and Methods section) are used to assess biodistribution of oligo-LNP in given tissues or organs relative to baseline.

[0254] In one embodiment, the biodistribution of the lipid nanoparticles in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin and / or ear of a mouse is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% relative to any one of the above-mentioned relevant baselines, wherein biodistribution is measured in a mouse model by detecting lipid markers at 1, 4, 10 and / or 24 hours after administration. As described in the Examples section, tissue homogenates of one or more of the aforementioned tissues or organs are measured.

[0255] The percent increase in fluorescence relative to the relevant baseline is determined by comparing the fluorescence signal of the LNP assessed in the relevant tissue and / or organ / mg of tissue homogenate to the tissue homogenate fluorescence signal resulting from baseline LNP administration.

[0256] The oligo-LNPs being compared were prepared using the same materials and methods. In other words, the two formulations being compared had the same ionizable lipids, PEG-lipids, and sterols and were prepared using rapid ethanol injection as described in the Materials and Methods section.

[0257] Biodistribution was assessed at the same time points (1, 4, 10 and / or 24 hours) after administration to the same mice, and marker lipids were measured using the same analytical techniques (see Materials and Methods section).

[0258] In those embodiments where the baseline formulation has a 10% lower molar ratio of neutral lipids (e.g., DSPC or sphingomyelin) than the LNPs of the present disclosure, the neutral lipids can be reduced in the baseline at the expense of an equal ratio of cholesterol and ionizable lipids, but keeping the ionizable lipid:cholesterol (mol:mol) constant between the baseline and the LNPs of the present disclosure.

[0259] Clinical and nonclinical uses of LNPs in this article

[0260] In certain embodiments, the LNPs encapsulating nucleic acids are part of a pharmaceutical composition and are administered to treat and / or prevent a disease state. Treatment can provide prophylaxis (prevention), improvement, or therapeutic benefit. The pharmaceutical composition will be administered in any suitable dosage.

[0261] The nucleic acid-LNPs described herein can be used to treat and / or prevent any disease, disorder, or condition in a mammalian subject. This includes diseases, conditions, or conditions such as cancer, infectious diseases such as bacterial, viral, fungal, or parasitic infections, inflammatory and / or autoimmune disorders, including therapies that induce immune tolerance, and cardiovascular diseases such as hypertension, arrhythmias, and restenosis.

[0262] Examples of cancer include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach cancer (gastric cancer), esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), central nervous system cancer, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteogenic sarcoma, and blood cancer. 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.

[0263] Non-limiting examples of other diseases, disorders, or conditions that can be treated by the oligo-LNPs herein and that can be attributable, at least in part, to an immune disorder include colitis, Crohn's disease, allergic encephalitis, allograft transplantation / graft-versus-host disease (GVHD), diabetes, and multiple sclerosis.

[0264] The LNPs herein can also be used in applications other than treating and / or preventing diseases or conditions. LNPs can be used to treat disease states such as aging, preventative medicine, and / or as part of a personalized medicine regimen. In other embodiments, LNPs are used in diagnostic applications.

[0265] In one embodiment, the LNP is part of a pharmaceutical composition that is administered parenterally, i.e., intra-arterially, intravenously, subcutaneously, or intramuscularly. In another embodiment, the pharmaceutical composition is administered intratumorally. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinally, intrathecally, or by other topical routes. In certain embodiments, the oligo-LNP is applied or administered to the skin.

[0266] The pharmaceutical composition comprises a pharmaceutically acceptable salt and / or excipient. As used herein, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts derived from various organic and inorganic counterions well known in the art, and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and, when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, methanesulfonate, acetate, maleate, and oxalate. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

[0267] As used herein, the term "excipient" refers to a substance used to formulate an active pharmaceutical ingredient (API) into a pharmaceutical preparation. Non-limiting examples include mannitol, Starch, magnesium stearate, sodium saccharin, talc, cellulose, cross-linked carboxymethyl cellulose sodium, glucose, gelatin, sucrose, magnesium carbonate, etc. Acceptable excipients are non-toxic and can be any solid, liquid, or semi-solid excipient commonly available to those skilled in the art.

[0268] The compositions described herein can be administered to an individual. As used herein, the term individual includes human or non-human individuals, including mammals. Oligo-LNPs can be administered as part of a prophylactic treatment, and thus the individual is not limited to a patient.

[0269] The examples are intended to illustrate the preparation of specific lipid nanoparticle oligomer formulations and their properties, but are in no way intended to limit the scope of the invention.

[0270] Unless otherwise stated, the articles "a" or "an" as used herein are intended to include both the singular and the plural of the term or phrase referred to herein. Example

[0271] Materials and methods

[0272] Preparation of surface-modified lipid nanoparticles containing nucleic acids

[0273] Unless otherwise stated, LNPs were prepared by dissolving mRNA or plasmid DNA (pDNA) in 25 mM sodium acetate, pH 4.0, while lipid components at the specified % molar ratio were dissolved in anhydrous ethanol. The ethanolic lipid solution and the buffered nucleic acid cargo were combined in a 1:3 volume ratio using a three-way connector with a dual syringe. The solution was pushed through the three-way connector at a combined flow rate of 20 mL / min (5 mL / min for the syringe containing lipids and 15 mL / min for the syringe containing mRNA). The Spectro / Port® was then used to analyze the lipids. TM The mixture was dialyzed overnight against at least about 100 volumes of 1× phosphate buffered saline (PBS) (pH 7.4) using a dialysis membrane (molecular weight cut-off 12,000-14,000 Da). TM The LNPs were concentrated using a 10,000 MWCO (molecular weight cutoff) regenerated cellulose concentrator. The targeting moiety, the lipophilic moiety, was a DSPE-PEG lipid conjugated to an arginine-glycine-aspartic acid (RGD) peptide. The ionizable lipid was nor-MC3 (described in WO2022 / 246571, incorporated herein by reference). The remaining lipids included 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), distearoylphosphatidylcholine (DSPC), and cholesterol (Chol).

[0274] By adding RiboGreen to mRNA-LNP(Fi) TM The encapsulation efficiency was calculated by measuring the fluorescence at 4 ℃ and comparing this value with the total mRNA content obtained by lysing the LNPs with 2% Triton X-100 (Ft) to determine the unencapsulated mRNA content: % encapsulation = (Ft) t -F i ) / F t ×100.

[0275] Using the ZetasizerNano ZS TM Characterize particle size and polydispersity index (PDI).

[0276] In vitro analysis of A7 astrocytes

[0277] A7 astrocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For cell treatment, 10,000 cells were added to each well of a 96-well plate. After 24 hours, the culture medium was aspirated and replaced with culture medium containing diluted LNPs at a relevant concentration within the range of 0.03 to 10 μg / mL mRNA. Expression analysis was performed 24 hours later, and luciferase levels were measured using the Steady-Glo luciferase kit (Promega). Cells were lysed using Glo lysis buffer (Promega).

[0278] Tissue homogenate assay

[0279] Tissues were removed from mice and placed in 2 mL tubes and snap frozen in liquid nitrogen. Tissues were then frozen at -80°C. An appropriate volume of Promega TM GLO TM Lysis buffer is added to each tube, ensuring that the samples remain frozen before adding lysis buffer. TM In homogenizer, operate homogenizer and repeat 2 times, 3 rounds in total.The homogenized sample is centrifuged at room temperature, and the homogenized product is subsequently added to the black board.The plate is transferred to a plate reader and read under 640nm excitation / 720nm emission.

[0280] Cryo-electron microscopy

[0281] Prior to cryo-EM imaging, LNPs were concentrated to an estimated total lipid content of 15 to 25 mg / mL. A defined volume, e.g., 2 to 4 μL, of the resulting LNP solution was added to a glow-discharged copper grid and dip-frozen using a FEI Mark IV Vitrobot to produce vitreous ice. These grids were stored in liquid nitrogen until imaging using a FEI Titan Krios or FEI GlaciosTEM. The instrument was operated at 200 kV under low-dose conditions, and images were acquired using a bottom-mounted FEI Falcon direct electron detector camera at 47 to 88,000× magnification with an underfocus of 0.5 to 2 μm to enhance contrast.

[0282] Example 1: Particle properties of LNPs with elevated neutral lipids and modified with targeting moieties

[0283] The encapsulation efficiency, PDI and particle size of the following lipid nanoparticles A to E with different amounts of RGD-PEG-lipid and different types of sterols (cholesterol and β-sitosterol) were measured (see Materials and Methods section). All formulations tested contained elevated levels of neutral lipids (40% mol DSPC). Formulations A to D contained cholesterol as the sterol and included different % mol ratios of the targeting moiety lipid conjugate (RGD modified). Formulation E contained β-sitosterol as the sterol and 1.5% mol % of the targeting moiety lipid conjugate (RGD modified). The ionizable lipid was norMC3 (described in WO 2022 / 246571).

[0284] Table 1: Physicochemical particle properties of LNPs modified with targeting moieties having elevated neutral lipids

[0285]

[0286]

[0287] The results are shown in Figure 1 and show that, in non-limiting embodiments of the present disclosure, less than 2% molar ratio of targeting moiety to lipophilic moiety contained in the particles achieves suitable PDI, particle size and encapsulation efficiency.

[0288] Example 2: Dose-dependent in vitro activity of LNPs with elevated neutral lipids and modified with targeting moieties

[0289] The dose-dependent activities of the following lipid nanoparticles G, H, and I in Table 2 below in A7 astrocytes were analyzed according to the Materials and Methods section.

[0290] Table 2: In vitro activity of LNPs modified with targeting moieties having elevated neutral lipids

[0291]

[0292] The results are shown in Figure 2 and showed that, in non-limiting embodiments of the present disclosure, targeting moiety-modified LNPs exhibited enhanced transfection when measured in astrocytes.

[0293] Example 3: Tissue expression of mRNA-LNPs with targeting moieties and elevated neutral lipid content

[0294] The in vivo expression of lipid nanoparticles B and C of Table 1 (see Example 1) with different amounts of RGD-PEG-lipid (see Materials and Methods section) in mice was determined.

[0295] All formulations tested contained elevated levels of neutral lipids (40% DSPC by mole) and 1% and 2% RGD-PEG-lipids. Formulations contained nMC337.9%:40% DSPC:Chol 21.1%:DSPE-PEG2k-RGD1% (LNP B) and nMC336.9%:40% DSPC:Chol 21.1%:DSPE-PEG2k-RGD 2%.

[0296] Liver and bone marrow homogenates were analyzed, and the results are shown in Figure 3A and 3B middle.

[0297] Notably, Formulations B and C showed increased mRNA expression in the bone marrow relative to the liver. These results indicate that LNPs with elevated neutral lipids and 1% and 2% molar ratios of targeting moieties exhibit enhanced extrahepatic expression as measured in vivo.

[0298] Example 4: Expression of mRNA-LNPs with Elevated Neutral Lipid Content and Targeting Moieties to Cell Surface Markers of Hematopoietic Stem and Progenitor Myeloid Cells

[0299] The effect of LNPs modified with anti-CD117 antibodies with 50% molar ratio of DSPC on the expression of cargo in bone marrow hematopoietic stem cells and / or progenitor cells (HSPC) in vivo was evaluated. As a negative control, the same LNPs were prepared using antibodies against the cell surface marker (anti-CD5) of T cells. The model used was C57B1 / 6 mice.

[0300] Specifically, the following eGFP mRNA formulations containing nMC3 ionizable lipids (WO2022 / 246571), DSPC, cholesterol, PEG-lipids, and antibody-conjugated lipids were compared in this example (reported as % molar ratios).

[0301] Table 3: Formulations containing eGFP mRNA for in vivo testing

[0302]

[0303] The gating scheme is as follows:

[0304] Table 4: Gating scheme for viable hematopoietic stem and progenitor cells (HSPCs)

[0305]

[0306] for Figure 4A and 4B , with LNP C(nMC31cLNP TM) were compared with the control group and statistically analyzed using two-way anova. Figure 4C , with LNP C(nMC31cLNP TM ) compared with the control group, and statistically analyzed using one-way anova.

[0307] The results showed that in hematopoietic cells, formulation D (aCD117-1cLNP) with antibodies against the CD117 cell surface marker TM ) Compared to Onpattro, 1cLNP without antibody and aCD5 targeting T cell marker showed higher eGFP expression ( Figure 4A to C).

[0308] This description is intended to illustrate embodiments and examples of the present invention, but is in no way intended to limit the scope of the invention, which is defined by the appended claims.

[0309] Unless otherwise stated, the articles "a" or "an" as used herein are intended to include both the singular and the plural of the term or phrase referred to herein.

Claims

1. A lipid nanoparticle comprising: (i) nucleic acids; (ii) a neutral lipid content greater than 35% by mole; (iii) an ionizable cationic lipid content of 5 mol% to 50 mol%; (iv) sterol or its derivative; and (v) a targeting moiety attached to a lipophilic moiety present in the lipid layer of the nanoparticle, the targeting moiety optionally being attached to the lipophilic moiety via a linker, Wherein each % molar ratio is relative to the total lipid content of the lipid nanoparticles, And optionally, wherein the lipid nanoparticle comprises a core comprising an electron dense region and an aqueous portion, and wherein the core is at least partially surrounded by the lipid layer as seen by cryo-electron microscopy (cryo-EM).

2. The lipid nanoparticle according to claim 1, wherein The linker is a hydrophilic polymer conjugated to the lipophilic moiety at one end and to the targeting moiety at the other end.

3. The lipid nanoparticle of claim 1 or 2, wherein the lipid nanoparticle is prepared by ethanol injection, the ethanol injection comprising a step of lowering the pH of the solution outside the nanoparticle after the nanoparticle is formed, thereby preparing a core comprising the electron-dense region and the aqueous portion.

4. The lipid nanoparticle of claim 1, 2 or 3, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoyl-phosphatidylcholine (DMPC) or dipalmitoyl-phosphatidylcholine (DPPC).

5. The lipid nanoparticle of claim 4, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC). 6 . The lipid nanoparticle according to claim 1 , wherein the phosphatidylcholine content is 38 to 60 mol %.

7. The lipid nanoparticles according to claim 6, wherein the phosphatidylcholine content is 40 mol% to 60 mol%.

8. The lipid nanoparticles according to claim 7, wherein the phosphatidylcholine content is 42 mol% to 60 mol%.

9. The lipid nanoparticles according to claim 8, wherein the phosphatidylcholine content is 45 mol% to 60 mol%.

10. The lipid nanoparticles according to claim 9, wherein the phosphatidylcholine content is 46 mol% to 60 mol%. The lipid nanoparticles according to claim 10 , wherein the phosphatidylcholine content is 48 to 60 mol %.

12. The lipid nanoparticle of any one of claims 1 to 11, wherein the cationic lipid is an amino lipid.

13. The lipid nanoparticle of any one of claims 1 to 12, wherein the ionizable cationic lipid is present in a molar ratio of less than 20%.

14. The lipid nanoparticle of any one of claims 1 to 13, further comprising a hydrophilic polymer-lipid conjugate present at a lipid content of 0.5 mol% to 5 mol%.

15. The lipid nanoparticle of any one of claims 1 to 14, wherein the sterol is present in a molar ratio of 15% to 45% based on the total lipid present in the lipid nanoparticle.

16. The lipid nanoparticle of any one of claims 1 to 15, wherein the sterol is present in a molar ratio of 18% to 40% based on the total lipid present in the lipid nanoparticle.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the lipid nanoparticle exhibits at least a 10% increase in biodistribution in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without the targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid but additionally measured under the same set of conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

18. The lipid nanoparticle of any one of claims 1 to 16, wherein the lipid nanoparticle exhibits at least a 10% increase in mRNA expression in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without the targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

19. The lipid nanoparticle of any one of claims 1 to 16, wherein the lipid nanoparticle exhibits at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without the targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid but additionally measured under the same set of conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

20. The lipid nanoparticle of any one of claims 1 to 16, wherein the lipid nanoparticle exhibits at least a 10% increase in mRNA expression in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to an otherwise identical baseline formulation without a targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid, and wherein biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present in a molar ratio of less than 2%.

22. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present in a molar ratio of less than 1.8%.

23. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present in a molar ratio of less than 1.5%.

24. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present in a molar ratio of less than 1.2%.

25. A method of delivering a nucleic acid to a cell for treating a disease, disorder or condition, the method comprising contacting the lipid nanoparticle of any one of claims 1 to 24 with the cell in vivo or in vitro.

26. The method of claim 25, wherein the nucleic acid accumulates in the spleen, bone marrow, heart, lungs, and / or kidneys of the individual for at least one day after administration.

27. The method of claim 25 or 26, wherein the disease, disorder or condition is an autoimmune disorder.

28. The method of claim 25 or 26, wherein the disease, disorder or condition is an infectious disease.

29. The method of claim 25 or 26, wherein the disease, disorder or condition is cancer.

30. The method of claim 25 or 26, wherein the cells are stem cells.

31. The method of claim 30, wherein the stem cell is a hematopoietic stem or progenitor cell.

32. The method of any one of claims 25 to 29, wherein the cell is a T cell.

33. Use of the lipid nanoparticle of any one of claims 1 to 24 for delivering nucleic acids to mammalian cells in vivo or in vitro.

34. Use of the lipid nanoparticle of any one of claims 1 to 24 in the preparation of a medicament for delivering nucleic acids to mammalian cells in vivo or in vitro.

35. A lipid nanoparticle encapsulating a nucleic acid and having at least 38% molar ratio of neutral lipids, sterols or derivatives thereof and a targeting portion anchored to the lipid nanoparticle via a lipophilic portion, wherein a linker is optionally present between the lipophilic portion and the targeting portion, wherein the lipid nanoparticle comprises a core as viewed by cryo-electron microscopy (cryo-EM), the core comprising an electron-dense region and an aqueous portion, and wherein the core is at least partially surrounded by the lipid layer.

Images