Lipid nanoparticles containing increased neutral lipids and targeting moieties for targeted delivery of nucleic acids - Patent Application 20070122999

Abstract

The present invention provides lipid nanoparticles that encapsulate nucleic acid and have at least 30mol% of neutral lipid, sterol or its derivative, and targeting moiety fixed to lipid layer via lipophilic moiety.Furthermore, the method of using lipid nanoparticles for in vivo targeted delivery is also provided.Such lipid nanoparticles can show significantly improved delivery and targeting to extrahepatic tissues and / or organs.

Description

[Technical Field]

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

[0002] Lipid nanoparticle (LNP) formulations represent a major advancement in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™, developed by Alnylam. Onpattro™ is a lipid nanoparticle-based short interfering RNA (siRNA) drug intended for the treatment of polyneuropathy caused by hereditary transthyretin amyloidosis. The success of this LNP delivery system has paved the way for the clinical development of a leading LNP-based COVID-19 mRNA vaccine.

[0003] The Onpattro™ LNP formulation consists of four main lipid components: a so-called ionized amino lipid (DLin-MC3-DMA or "MC3" (dilinoleylmethyl-4-dimethylaminobutyrate)), distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-conjugated lipid (PEG lipid) in molar amounts of 50 / 10 / 38.5 / 1.5, respectively. Onpattro™ is still considered the gold standard for comparison in LNP-mediated efficacy studies, and current approaches to the design of LNPs for clinical use have rarely deviated from the four-component system.

[0004] Of these four components, ionizable lipids constitute the majority of the Onpattro™ formulation, present at 50 mol%. Ionizable lipids are believed to be critical for the in vitro and in vivo activity of LNP systems, and therefore most research in this area has focused primarily on improving the efficacy of this lipid component. Ionizable lipids, typically amino lipids, are carefully designed to be charged at low pH and nearly neutral at physiological pH. This allows electrostatic interactions between the lipid and the negatively charged nucleic acid during initial formulation. Because ionizable lipids are nearly neutral at physiological pH, toxicity and renal clearance are reduced. After endocytosis, the acidic environment of the endosome increases the net positive charge of the ionizable amino lipid, which promotes fusion with the anionic lipid in the endosomal membrane, subsequently destabilizing the membrane and releasing the nucleic acid-based therapeutic into the cytoplasm where it exerts its effects.

[0005] Regarding the remaining three lipid components, it is well known that PEG-lipids improve the circulation lifetime of LNPs and cholesterol has a particle stabilizing function, but in general, relatively little attention has been paid to studying the neutral lipid components in LNPs beyond their structural role.

[0006] The liver is the primary organ where Onpattro™ 4-component LNPs accumulate following intravenous administration. While delivery to the liver has therapeutic potential, the ability of LNPs to accumulate in organs and tissues other than the liver greatly expands the clinical utility of these delivery systems. Extrahepatic delivery may improve the treatment and / or prevention of diseases such as cancer, cardiovascular disease, infectious diseases, and other conditions.

[0007] However, efforts to target tissues other than the liver (extrahepatic organs and tissues) using the intravenous route have been less successful. To improve LNP delivery to extrahepatic tissues, the particle's circulatory lifetime must be extended. As mentioned above, traditional approaches to achieve this involve designing LNPs with a long-lasting PEG coating, often referred to as "stealth" liposomes. Nevertheless, the inclusion of PEG-lipids in LNPs often results in low transfection efficacy. Alternatively, the incorporation of various permanently positively charged lipids can enhance transfection in many extrahepatic tissues after intravenous administration. However, such lipids can be toxic, potentially limiting their clinical application.

[0008] Studies have shown that DSPC and cholesterol contribute to the stable encapsulation of siRNA in LNPs (Kulkarni et al., 2019, Nanoscale, 11:21733-21739). Despite these findings, subsequent in vivo studies by another group failed to demonstrate any clear benefit from adjusting the DSPC level in LNPs to improve extrahepatic delivery of siRNA. These studies investigated extrahepatic siRNA gene silencing in vivo using Onpattro™-type LNPs (MC3 / Chol / DSPC / PEG-DMP) incorporating DSPC at 10 and 40 mol% (Ordobadi, 2019, "Lipid Nanoparticles for Delivery of Bioactive Molecules," a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of British Columbia). The 10 mol% DSPC Onpattro™ formulation was shown to have similar liver accumulation and blood circulation lifetime as a 40 mol% DSPC formulation (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5 mol%). Furthermore, 40 mol% DSPC siRNA-containing LNPs (siRNA-LNPs) performed comparable to the 10 mol% DSPC formulation in bone marrow gene silencing.

[0009] Conjugating various targeting moieties to the siRNA itself or including them as part of the formulation as a surface modification has been investigated by various groups as an alternative to passive targeting. However, because the Onpattro™ four-component LNP formulation primarily accumulates in liver tissue, most ligand targeting strategies have focused on improving delivery to hepatocytes. For example, Chen et al. (2014, Journal of Controlled Release, 196:106-112) described the use of LNPs in combination with a hepatocyte-specific targeting ligand, GalNAc-PEG, to increase the uptake of functionalized LNPs in the liver and improve hepatic gene silencing. WO 2010 / 144740 describes a four-component liposomal formulation containing ionizable lipid (MC3) / DSPC / chol / PEG lipids, and the addition of GalNAc to the liposome surface improved FVII siRNA silencing in the liver. Mannose-containing LNPs have also been used to target HepG2 hepatocytes.

[0010] Despite these previous efforts, there remains a need in the art for improved targeted delivery of nucleic acids to the liver and / or extrahepatic tissues or organs using LNPs. (overview)

[0011] The present inventors have discovered that by using lipid nanoparticles (LNPs) with elevated levels of neutral lipids in combination with moieties on the LNP surface that bind to subsets of target cells, surprising improvements in nucleic acid expression in specific tissues and / or organs can be achieved. Thus, the LNPs of the invention described herein can employ two levels of targeting: targeting to a desired tissue or organ by including high levels of neutral lipids, and active targeting to cell types of interest that have surface receptors that bind to targeting moieties on the LNP.

[0012] In a particular advantageous embodiment of the present disclosure, the present invention provides a lipid nanoparticle (LNP) for nucleic acid delivery, which comprises three or four lipid components.The three or four lipid components include an ionizable lipid, a neutral lipid such as a phospholipid, a sterol, and optionally a hydrophilic polymer-lipid conjugate.In particular, the neutral lipid is present at a higher content than in conventional LNPs, for example, at least 20 mol%, at least 30 mol%, at least 36 mol%, or at least 40 mol% (relative to the total lipid content of the LNP).

[0013] According to one aspect of the present disclosure, there is provided a lipid nanoparticle comprising: (i) a nucleic acid; (ii) a neutral lipid at a content greater than 35 mol%; (iii) an ionizable lipid at a content of 5 mol% to 50 mol%; (iv) a sterol or a derivative thereof; and (v) a targeting moiety bound to a lipophilic moiety present in a lipid layer of the nanoparticle, the targeting moiety optionally being bound to the lipophilic moiety via a linker, wherein each mol% is relative to the total lipid content of the lipid nanoparticle, and the lipid nanoparticle optionally comprises a core comprising an electron-dense region and an aqueous portion, the core being at least partially surrounded by the lipid layer as visualized by cryo-electron microscopy.

[0014] According to another aspect of the present disclosure, there is provided a lipid nanoparticle encapsulating a nucleic acid and having at least 38 mol % of a neutral lipid, a sterol or derivative thereof, and a targeting moiety immobilized in the lipid bilayer or lipid monolayer via a lipophilic moiety, wherein the targeting moiety is present at less than 2.5 mol %, and a linker is optionally present between the lipophilic moiety and the targeting moiety.

[0015] According to one embodiment, the linker is a hydrophilic polymer conjugated at one end to a lipophilic moiety and at its other end to a targeting moiety.

[0016] According to one embodiment, lipid nanoparticles are prepared by ethanol injection, which includes a step of lowering the pH of the solution outside the nanoparticles after nanoparticle formation, thereby producing a core containing an electron-dense region and an aqueous portion.

[0017] In another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoyl-phosphatidylcholine (DMPC), or dipalmitoyl-phosphatidylcholine (DPPC).

[0018] According to another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC).

[0019] In a further embodiment, the phosphatidylcholine content is 38 mol % to 60 mol %.

[0020] In another embodiment, the phosphatidylcholine content is 40 mol% to 60 mol%, 42 mol% to 60 mol%, 45 mol% to 60 mol%, 46 mol% to 60 mol%, or 48 mol% to 60 mol%.

[0021] In another embodiment, the cationic lipid is an amino lipid.

[0022] According to a further embodiment, the ionizable cationic lipid is present at less than 20 mol %.

[0023] In another embodiment, the lipid nanoparticles comprise a hydrophilic polymer-lipid conjugate present at a lipid content of 0 mol% to 5 mol% or 0.5 mol% to 5 mol%.

[0024] In a further embodiment, the sterol is present at 15 mol % to 45 mol % based on the total lipid present in the lipid nanoparticle.

[0025] According to a further embodiment, the sterol is present at 18 mol % to 40 mol % based on the total lipids present in the lipid nanoparticles.

[0026] According to further embodiments, the lipid nanoparticles exhibit at least a 10% increase in biodistribution in the liver, spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin and / or ears compared to an otherwise identical baseline formulation not containing a targeting moiety and / or an Onpattro-type formulation encapsulating a nucleic acid but measured under an otherwise identical set of conditions, and biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0027] In another embodiment, the lipid nanoparticles demonstrate 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 formulation encapsulating a nucleic acid, and biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0028] According to further embodiments, the lipid nanoparticles exhibit at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin and / or ears compared to an otherwise identical baseline formulation not containing a targeting moiety and / or an Onpattro-type formulation encapsulating a nucleic acid but measured under an otherwise identical set of conditions, and biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0029] In another embodiment, the lipid nanoparticles demonstrate 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 formulation encapsulating a nucleic acid, and biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

[0030] According to another embodiment, the targeting moiety is present at less than 2 mol %.

[0031] In a further embodiment, the targeting moiety is present at less than 1.8 mol %.

[0032] According to a further embodiment, the targeting moiety is present at less than 1.5 mol %.

[0033] In a further embodiment, the targeting moiety is present at less than 1.2 mol %.

[0034] According to a further aspect, there is provided a method of delivering a nucleic acid to a cell to treat a disease, disorder, or condition, the method comprising contacting a lipid nanoparticle according to any one of the above aspects or embodiments with a cell in vivo or in vitro.

[0035] According to one embodiment, the nucleic acid accumulates in the spleen, bone marrow, heart, lungs and / or kidneys of the subject at least one day after administration.

[0036] In one embodiment, the disease, disorder or condition is an autoimmune disease.

[0037] In another embodiment, the disease, disorder or condition is an infectious disease.

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

[0039] In a further embodiment, the cell is a stem cell.

[0040] In one embodiment, the stem cells are hematopoietic stem cells or hematopoietic progenitor cells.

[0041] In a further embodiment, the cell is a T cell.

[0042] In another aspect, there is provided a use of a lipid nanoparticle according to any of the above aspects or embodiments for in vivo or in vitro delivery of a nucleic acid to a mammalian cell.

[0043] In another aspect, there is provided a use of a lipid nanoparticle according to any preceding aspect or embodiment for the manufacture of a medicament for in vivo or in vitro delivery of a nucleic acid to a mammalian cell. [Brief explanation of the drawings]

[0044] [Figure 1] Figure 1 shows the particle size, encapsulation efficiency, and polydispersity index (PDI) of example lipid nanoparticles (LNPs) of the present disclosure modified with varying amounts of arginine-glycine-aspartic acid (RGD) peptide targeting moieties attached to the lipid via PEG (LNPs B and C) and a non-targeting control (LNP A). The LNPs investigated, A-C, are listed in Table 1 in Example 1 and encapsulate mRNA encoding firefly luciferase (Fluc).

[0045] [Figure 2] Figure 1 shows the luminescence intensity per μg protein at various doses for example lipid nanoparticles (LNPs) of the present disclosure modified with an arginine-glycine-aspartic acid (RGD) peptide targeting moiety linked to the lipid via PEG (LNP E; squares) and a non-targeted control (LNP D; circles). LNPs were added to the A7 astrocyte cell line at the indicated mRNA doses. The LNPs D and E investigated are listed in Table 2 of Example 2.

[0046] [Figure 3A]The luminescence intensity in the liver of example lipid nanoparticles (LNPs) of the present disclosure modified with varying amounts of arginine-glycine-aspartic acid (RGD) peptide targeting moieties conjugated to lipids via PEG (LNP B and C) is shown compared to a non-targeted control (LNP A). The LNPs investigated, A-C, are listed in Table 1 of Example 1 and encapsulate mRNA encoding firefly luciferase (Fluc).

[0047] [Figure 3B] Figure 1 shows the luminescence intensity in bone marrow of example lipid nanoparticles (LNPs) of the present disclosure modified with varying amounts of arginine-glycine-aspartic acid (RGD) peptide targeting moieties attached to lipids via PEG (LNPs B and C). The LNPs investigated, B and C, are listed in Table 1 of Example 1 and encapsulate mRNA encoding firefly luciferase (Fluc).

[0048] [Figure 4A] Enhanced green fluorescent protein (GFP) expression in Lineage-c-Kit+ (LK) and Lineage-c-Kit+Sca1+ (LSK) cell populations in bone marrow after injection of mice with phosphate-buffered saline (PBS), Onpattro™ formulations (B), lcLNP™ without a ligand (C), lcLNP™ with aCD117 ligand (D), and lcLNP™ with aCD5 ligand (E). The formulations are shown in Table 3, and the gating scheme is shown in Table 4.

[0049] [Figure 4B]Enhanced green fluorescent protein (GFP) expression in multipotent progenitor cell Lineage- ckit+ Sca1+ CD34+ (MPP), short-term HSC Lineage- ckit+ Sca1+ CD34- CD135+ (ST-HSC), and Lineage- ckit+ Sca1+ CD34- CD135- (LT-HSC) cell populations after injection of mice with phosphate-buffered saline (PBS), Onpattro™-type formulations (B), lcLNP™ without ligands (C), lcLNP™ with aCD117 ligands (D), and lcLNP™ with aCD5 ligands (E). The formulations are shown in Table 3, and the gating scheme is shown in Table 4.

[0050] [Figure 4C] Enhanced green fluorescent protein (GFP) expression in the Lineage- ckit+ Sca1+ CD34- CD135- CD48- CD150+ cell population after injection of mice with phosphate-buffered saline (PBS), Onpattro™-type formulations (B), lcLNP™ without ligands (C), lcLNP™ with aCD117 ligands (D), and lcLNP™ with aCD5 ligands (E). The formulations are shown in Table 3, and the gating scheme is shown in Table 4.

[0051] [Figure 5] Cryo-TEM image of lipid nanoparticles composed of 50 mol% 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 WO 2022 / 155728 A1, incorporated herein by reference. DETAILED DESCRIPTION OF THE INVENTION

[0052] (Detailed explanation) The lipid nanoparticles described herein comprise a targeting moiety, such as an ionizable lipid, a high level of a neutral lipid, such as a phosphatidylcholine lipid (e.g., DSPC) or a sphingolipid (e.g., a sphingomyelin lipid), and a sterol. In certain embodiments, the neutral lipid is a phosphatidylcholine lipid present at a mol% of at least 35 mol%, at least 40 mol%, or at least 42 mol%, and the ionizable lipid is present at less than 45 mol% or less than 40 mol%. As described herein, in certain non-limiting examples of the present disclosure, the inclusion of a neutral lipid at a mol% higher than that used in conventional formulations for nucleic acid delivery in combination with a targeting ligand improves the selective delivery of nucleic acids to the liver and / or extrahepatic tissues compared to Onpattro™-type LNPs or identical LNPs without a targeting moiety. neutral lipid

[0053] Neutral lipids are amphipathic lipids that allow particle formation and have essentially no net charge at physiological pH.The term includes, but is not limited to, zwitterionic lipids, such as phospholipids.In alternative embodiments, lipid nanoparticles comprise structured lipids that are non-cationic lipids.In certain embodiments, LNPs have essentially no net charge.

[0054] As used herein, "substantially no net charge" with respect to LNPs means a net surface charge of about zero or near neutral at physiological pH, e.g., but not limited to, about -2.5 mV to about 2.5 mV.

[0055] In some embodiments, the neutral lipid is a phosphatidylcholine lipid. The phosphatidylcholine lipid can be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). In another embodiment, the phosphatidylcholine lipid can be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and mixtures thereof. The phosphatidylcholine lipid component can comprise a mixture of two or more different neutral lipids.

[0056] In some embodiments, the phosphatidylcholine content is greater than 20 mol%, greater than 25 mol%, greater than 30 mol%, greater than 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, greater than 46 mol%, greater than 48 mol%, or greater than 50 mol%. In some embodiments, the upper limit of the neutral lipid content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, or 45 mol%. The present disclosure also includes subranges of any combination of the upper and lower limits of the aforementioned numerical values.

[0057] For example, in certain embodiments, the phosphatidylcholine lipid content is between 20 mol% and 80 mol%, or between 25 mol% and 60 mol%, or between 30 mol% and 60 mol%, or between 35 mol% and 60 mol%, or between 40 mol% and 60 mol%, or between 42 mol% and 58 mol%, or between 43 mol% and 57 mol%, or between 44 mol% and 56 mol%, or between 45 mol% and 55 mol% of the total lipid present in the lipid nanoparticle.

[0058] In some embodiments, the lipid nanoparticles comprise 35-60 mol% or 35-55 mol% of any one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the lipid nanoparticles comprise 40-60 mol% or 40-55 mol% of any one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC).

[0059] In one embodiment, the neutral lipid is most advantageously DSPC. In some examples of the present disclosure, the DSPC lipid, present at higher levels compared to Onpattro™-type LNPs, improves the biodistribution of the LNPs compared to other neutral phospholipids.

[0060] The term "Onpattro™ type" refers to LNPs compared to LNPs of the present disclosure that have an ionizable lipid / DSPC / chol / PEG2000-DMG lipid ratio of 50 / 10 / 38.5 / 1.5 mol / mol, where the ionizable lipid is the same as that of the LNP being evaluated.

[0061] In certain embodiments, the DSPC lipid content is between 20 mol% and 80 mol%, or between 25 mol% and 60 mol%, or between 30 mol% and 60 mol%, or between 35 mol% and 60 mol%, or between 38 mol% and 60 mol%, or between 40 mol% and 60 mol%, or between 42 mol% and 60 mol%, or between 43 mol% and 60 mol%, or between 44 mol% and 60 mol%, or between 45 mol% and 60 mol%, or between 46 mol% and 60 mol%, or between 48 mol% and 60 mol% of the total lipid present in the lipid nanoparticle. In certain embodiments, the DSPC lipid content is between 30 mol% and 55 mol%, or between 35 mol% and 55 mol%, or between 38 mol% and 55 mol%, or between 40 mol% and 55 mol%, or between 42 mol% and 55 mol%, or between 43 mol% and 55 mol%, or between 44 mol% and 55 mol%, or between 45 mol% and 55 mol% of the total lipid present in the lipid nanoparticle.

[0062] In one embodiment, the neutral lipid is DOPC. In some embodiments of the present disclosure, increased DOPC lipid content improves the biodistribution of LNPs over other neutral phospholipids. In certain embodiments, the DOPC lipid content is 20 mol% to 80 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or 42 mol% to 60 mol%, or 43 mol% to 60 mol%, or 44 mol% to 60 mol%, or 45 mol% to 60 mol%, or 46 mol% to 60 mol%, or 48 mol% to 60 mol% of the total lipid present in the lipid nanoparticle.

[0063] In one embodiment, the neutral lipid is DPPC. In some embodiments of the present disclosure, increased DPPC lipid content improves the biodistribution of LNPs over other neutral phospholipids. In certain embodiments, the DPPC lipid content is 20 mol% to 80 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or 42 mol% to 60 mol%, or 43 mol% to 60 mol%, or 44 mol% to 60 mol%, or 45 mol% to 60 mol%, or 46 mol% to 60 mol%, or 48 mol% to 60 mol% of the total lipid present in the lipid nanoparticle.

[0064] Neutral lipids can also include sphingolipids such as ceramides, sphingomyelins, cerebrosides, gangliosides, or derivatives thereof, including but not limited to reduced analogs thereof, in which the sphingosine unit lacks a double bond. In some embodiments, sphingolipids are present at 20 mol% to 80 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or 42 mol% to 58 mol%, or 43 mol% to 57 mol%, or 44 mol% to 56 mol%, or 45 mol% to 55 mol% of the total lipid present in the lipid nanoparticles. In some embodiments, sphingomyelin is present at 20 mol% to 80 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or 42 mol% to 58 mol%, or 43 mol% to 57 mol%, or 44 mol% to 56 mol%, or 45 mol% to 55 mol% of the total lipid present in the lipid nanoparticle.

[0065] In some embodiments, the lipid nanoparticles have a sphingomyelin 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 some embodiments, the LNPs are "sphingomyelin-free," meaning that there is no detectable sphingomyelin in the LNPs (less than 0.5 mol%), or the LNPs are substantially sphingomyelin-free, meaning that there is less than 5 mol% sphingomyelin in the LNPs.

[0066] In addition to phosphatidylcholine lipids, LNPs can contain additional neutral lipids. For example, LNPs can contain other lipids that have a net positive or negative charge at physiological pH. In another example, LNPs can further contain a smaller amount (relative to the phosphatidylcholine lipids) of one or more fusogenic lipids, such as DOPE, which are conical in shape and thereby promote fusion with cell membranes. Typically, such non-phosphatidylcholine lipids are present in LNPs at less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, or less than 5 mol% of the total lipids present in the LNP.

[0067] The inclusion of fusogenic lipids, such as dioleoylphosphatidylethanolamine (DOPE), is believed to facilitate nucleic acid delivery in vitro or in vivo. However, the present disclosure generally discourages the inclusion of such lipids. Thus, in some embodiments, the fusogenic lipid content of 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 some embodiments, the LNPs are "fusogenic lipid-free," meaning that the LNPs have no detectable amount of fusogenic lipids (less than 0.5 mol%), or the LNPs are substantially fusogenic lipid-free, meaning that the fusogenic lipid content, measured relative to the total lipid content of the LNPs, is less than 5 mol%.

[0068] In some embodiments, the lipid nanoparticles have a DOPE content of 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 some embodiments, the LNPs are "DOPE-free," meaning that there is no detectable DOPE in the LNPs (less than 0.5 mol%), or the LNPs are substantially DOPE-free, meaning that there is less than 5 mol% DOPE measured relative to the total lipid content in the LNPs.

[0069] In further embodiments, it may be advantageous to include a mixture of different phosphatidylcholine lipids in the LNP, although in some examples the phosphatidylcholine lipid content includes less than five, four, or three different phosphatidylcholine lipids.

[0070] In some embodiments, the lipid nanoparticle egg phosphatidylcholine (EPC) content 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 some embodiments, the LNPs are "EPC-free," meaning that there are no detectable EPCs in the LNPs (less than 0.5 mol%), or the LNPs are substantially EPC-free, meaning that there is less than 5 mol% EPC in the lipid nanoparticles as measured relative to the total lipid content in the LNP.

[0071] In another embodiment, the structural lipid, neutral lipid, zwitterionic lipid, or non-cationic lipid content of the lipid nanoparticles is composed of less than 20, 10, or 5 mol% (measured relative to the total phosphatidylcholine, structural lipid, or neutral lipid content) of non-phosphatidylcholine lipids, such as POPC.

[0072] In some embodiments, the transition temperature of the structured lipid, neutral lipid, zwitterionic lipid, or non-cationic lipid, e.g., a phospholipid with a choline 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. LNPs often include hydrophilic polymer-lipid conjugates to prevent particle fusion and aggregation. Without intending to be limited to a particular theory, it is believed that fusion and aggregation of lipid nanoparticles that do not contain (or have low levels of) hydrophilic polymer-lipid conjugates during particle formation using the mixing method described in commonly owned and co-pending U.S. Provisional Patent Application No. 63 / 588,167, filed October 5, 2023, which is incorporated herein by reference, can be avoided by selecting structured, neutral, zwitterionic, or non-cationic lipids that are in a gel phase rather than a disordered liquid crystalline phase at room temperature or above. Inclusion of such structured, neutral, zwitterionic, or non-cationic lipids in lipid nanoparticles may also improve blood stability after injection.

[0073] In one embodiment, the phase transition temperature of the neutral lipid or mixture thereof incorporated into the lipid nanoparticles is at least 38°C, 39°C, or 40°C.

[0074] The neutral lipid content is determined based on the total amount of lipid (mol:mol) in the lipid nanoparticles containing sterol. Ionized lipids

[0075] The LNPs of the present disclosure comprise ionized lipids, which are charged at low pH and may have substantially no net charge at physiological pH. This allows for electrostatic interactions between the lipid and the negatively charged nucleic acid cargo upon initial formulation. The ionized lipids are near-neutral at physiological pH, thereby reducing toxicity and renal clearance. Without being limited by theory, after endocytosis, the acidic environment of the endosome increases the net positive charge of the ionized amino lipid, which promotes fusion with the anionic lipid in the endosomal membrane, subsequently destabilizing the membrane and releasing the nucleic acid-based therapeutic into the cytoplasm where it exerts its effects.

[0076] In some embodiments, the apparent pKa of the LNPs is 5.0-8.5, 5.0-8.0, 5.0-7.5, 6.5-7.5, or 6.6-7.3. The apparent pKa is measured using a 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay modified from previous work 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 purposes of determining apparent pKa). According to this method, a series of buffers spanning a pH range of 2 to 11 in 0.5 pH unit increments is prepared, consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM HEPES, resulting in 0.15 to 0.2 mM LNP. Next, a 0.06 mM TNS solution is mixed with 175 μL of LNP at each of the three buffer pHs in a black polystyrene 96-well plate, so that the final lipid and TNS concentrations in each well are 6.25 μM and 6 μM, respectively. The λ values ​​are then measured using a SpectraMax™ M5 microplate reader. ex = 321 nm, λ emFluorescence is then measured at 445 nm. Fluorescence is then plotted against pH using sigmoidal curve fitting with Prism™, and the pKa is determined as the pH value at 50% of maximum fluorescence intensity.

[0077] In some embodiments, it is desirable to include less than 50 mol% ionizable lipids in the LNP, i.e., the ionizable lipid content can be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, or less than 5 mol%, as measured based on the total lipid content of the LNP.

[0078] In certain embodiments, the ionized lipid content is between 5 mol% and 50 mol%, or between 8 mol% and 47 mol%, or between 10 mol% and 50 mol%, or between 15 mol% and 45 mol%, or between 15 mol% and 35 mol% of the total lipid present in the lipid nanoparticle.

[0079] 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 particular pH, such as physiological pH, and can accept a proton at a lower pH to become electrostatically positively charged, and whose 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 pKa of the cationic lipid is between 5.0 and 7.0.

[0080] In some embodiments, the cationic lipid has an amino group. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) head group and two alkyl chains with 0-3 double bonds. Such lipids include, but are not limited to, sulfur lipids such as MF019 and DODMA, as described herein. Other lipids that can be used in the practice of the present disclosure include MC3-type lipids and KC2-type lipids, which are well known to those skilled in the art. In further embodiments, the ionizable lipid is selected from the group consisting of WO2022 / 246555; WO2022 / 246568; WO2022 / 246571; WO2023 / 147657; WO2022 / 155728; WO2023 / 215989; PCT / CA2023 / 051272, filed September 27, 2023; and PCT / CA2023 / 051272, filed September 27, 2023, each of which is incorporated herein by reference. The lipids are selected from one or more of the lipids described in U.S. Provisional Patent Application No. PCT / CA2023 / 051273, filed September 27, 2020; U.S. Provisional Patent Application No. 63 / 434,506, filed December 22, 2022; PCT / CA2023 / 051274, filed September 27, 2023; and U.S. Provisional Patent Application No. 63 / 445,854, filed February 15, 2023.

[0081] In one embodiment, the ionizable cationic lipid has a protonatable amino head group, at least two lipophilic moieties, the amino head group having a central nitrogen or carbon atom to which each of the two lipophilic moieties is directly attached, each lipophilic chain having a total of 15-40 carbon atoms, and the lipid has (i) a pK of 6-8.0. a (e.g., when formulated), and (ii) has a logP of at least 11.

[0082] Optionally, at least one of the lipophilic moieties attached 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 (see, for example, U.S. Provisional Patent Application No. 63 / 434,506, filed December 22, 2022, which is incorporated herein by reference). In one embodiment, the ionized cationic lipid has at least one lipophilic moiety of the following formula: [ka] .

[0083] In one embodiment, R 1 and R 2 are independently linear, cyclic or branched, optionally substituted, C3-C6 alkylene alkyls with various degrees of unsaturation. 20 alkyl, and n is 2 to 8 or 4 to 8.

[0084] In some embodiments, it is desirable to include less than 50 mol% cationic lipids in the LNPs, i.e., the ionizable lipid content can be less than 50 mol%, less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, or less than 5 mol%.

[0085] In certain embodiments, the cationic lipid content is between 5 mol% and 50 mol%, or between 8 mol% and 47 mol%, or between 10 mol% and 50 mol%, or between 15 mol% and 45 mol%, or between 15 mol% and 35 mol% of the total lipid present in the lipid nanoparticle.

[0086] The inclusion of permanently positively charged lipids, such as dimethyldioctadecylammonium bromide (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), and cholesterol imidazolium (CHIM), in LNPs is believed to facilitate in vitro or in vivo nucleic acid transfection. Such permanently charged lipids contain non-ionizable quaternary amines, which are permanently charged. However, the present disclosure generally discourages the inclusion of such permanently charged lipids. Thus, in some embodiments, 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 mol%. In some embodiments, the LNPs are "permanently charged cationic lipid-free," meaning that there are no detectable amounts of permanently charged cationic lipids in the LNPs (less than 0.5 mol%), or the LNPs are substantially free of permanently charged cationic lipids, meaning that the permanently charged cationic lipid content, measured relative to the total lipid content in the LNPs, is less than 5 mol% or less than 3 mol%.

[0087] In some embodiments, the DDAB 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 some embodiments, the LNPs are "DDAB-free," meaning that there is no detectable DDAB in the LNPs (less than 0.5 mol%), or the LNPs are substantially DDAB-free, meaning that DDAB is less than 5 mol% measured relative to the total lipid content in the LNPs.

[0088] In some embodiments, the DOTMA 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 some embodiments, the LNPs are "DOTMA-free," meaning that there is no detectable DOTMA in the LNPs (less than 0.5 mol%), or the LNPs are substantially DOTMA-free, meaning that DOTMA is less than 5 mol% measured relative to the total lipid content in the LNPs.

[0089] In some embodiments, the DOTAP 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 some 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 there is less than 5 mol% DOTAP measured relative to the total lipid content in the LNPs.

[0090] In some embodiments, the lipid nanoparticles have a DOSPA content of 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 some embodiments, the LNPs are "DOTAP-free," meaning that there is no detectable DOSPA in the LNPs (less than 0.5 mol%), or the LNPs are substantially DOSPA-free, meaning that DOSPA is less than 5 mol% measured relative to the total lipid content in the LNPs.

[0091] In some embodiments, the lipid nanoparticles have a CHIM content of 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 some embodiments, the LNPs are "CHIM-free," meaning that there is no detectable CHIM in the LNP (less than 0.5 mol%), or the LNPs are substantially CHIM-free, meaning that CHIM is less than 5 mol% as measured relative to the total lipid content in the LNP.

[0092] Ionized lipid components can include ionized anionic lipids as part of ionized lipid content.One example of such lipid is cholesteryl hemisuccinate (CHEMS).Further examples of ionized anionic lipids are described in co-pending and co-owned U.S. Provisional Patent Application No. 63 / 453,766, entitled "Ionized Anionic Lipids," filed on March 22, 2023, and is incorporated herein by reference in its entirety.

[0093] In some embodiments, the ionizable cationic lipid is not a lipid-like structure, including but not limited to C12-200 (see Khare et al., 2022, AAPS Journal, 24:8, incorporated by reference) and related structures known to those skilled in the art. sterols

[0094] The term "sterol" means a natural or synthetic steroid. This term includes cholesterol, plant sterols, zoosterols and their derivatives.

[0095] The term "sterol derivative" refers to modified sterols or precursors thereof, including triterpenes.

[0096] The term "cholesterol" refers to a natural or synthetic compound with a gonane skeleton and a hydroxyl attached to one of its rings (usually the A ring).

[0097] LNPs may also include "cholesterol derivatives," which may be naturally occurring or synthetic and include, but are not limited to, cholesterol molecules having a gonane structure and one or more additional functional groups, including derivatization of the terminal hydroxyl group.

[0098] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol or stigmastanol, dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol)], 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, Examples of the fatty acids include cholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctan-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, and salts or esters thereof.

[0099] In one embodiment, the sterol is present at 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol%, or 30 mol% to 45 mol%, based on the total lipid present in the lipid nanoparticle.

[0100] In another embodiment, the sterol is cholesterol and is present at 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol%, or 30 mol% to 45 mol% based on the total lipids present in the lipid nanoparticles.

[0101] In another embodiment, the sterol is a cholesterol derivative and is present at 15 mol% to 50 mol%, 18 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 45 mol%, or 30 mol% to 45 mol%, based on the total lipids present in the lipid nanoparticles.

[0102] In one embodiment, the sum of (i) the sterol content (e.g., cholesterol or a cholesterol derivative thereof); and (ii) the phosphatidylcholine lipid content is at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, or at least 85 mol%, based on the total lipids present in the lipid nanoparticle.

[0103] In one embodiment, the molar ratio of sterol:ionizable lipid is between 0.70 and 1.30, or any range therebetween. Hydrophilic polymer lipid conjugates

[0104] In one non-limiting example, the lipid nanoparticle comprises a hydrophilic polymer lipid conjugate that can be incorporated into LNP. The conjugate comprises a lipophilic portion (e.g., a lipid portion) and a hydrophilic polymer chain, optionally with a linker (e.g., succinate) between the lipophilic portion and the polymer chain. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyaspartamide. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate is a monosialoganglioside (G M1 The ability of a particular hydrophilic polymer-lipid conjugate to extend the circulatory lifetime of the LNPs described herein can be readily determined by one of skill in the art using known methodologies.

[0105] The hydrophilic polymer lipid conjugate may be present in the nanoparticles at 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid.

[0106] 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 in an amount of 0 mol% to 5 mol%, or 0 mol% to 3 mol%, or 0 mol% to 2.5 mol%, or 0 mol% to 2.0 mol%, or 0 mol% to 1.8 mol% of the total lipid.

[0107] In another embodiment, the PEG-lipid conjugate may be present in the nanoparticle at 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, or 0 mol% to 3 mol%, or 0 mol% to 2.5 mol%, or 0 mol% to 2.0 mol%, or 0 mol% to 1.8 mol% of the total lipid.

[0108] In one embodiment, the hydrophilic polymeric lipid is selected based on its exchangeability from the lipid nanoparticle, which may facilitate in vivo efficacy by at least partially losing the hydrophilic polymer-lipid conjugate from the LNP when the LNP reaches its target site in vivo.

[0109] In such embodiments, the lipid portion of the hydrophilic polymer-lipid conjugate has a lipophilic chain length of less than 18 carbon atoms and 0-2 double bonds in one or both chains. In some embodiments, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate selected from dimyristoylphosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), dioleylphosphatidylethanolamine-PEG (DOPE-PEG), dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), dimyristoyldiglyceride-PEG (DMG-PEG), or cholesterol-PEG (Chol-PEG).

[0110] In one embodiment, the hydrophilic polymer lipid conjugate is not DSPE-PEG. In a further embodiment, 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%.

[0111] In another embodiment, there is a cleavable linker between the lipid moiety and the hydrophilic polymer in the hydrophilic polymer lipid conjugate.This linker can be cleaved by being exposed to low pH, reducing agent, or protease present in vivo.Examples of cleavable linkers include ester, ether, phosphoramidate, hydrazone, β-thiopropionate, disulfide group, and peptide (Romberg et al., 2008, Pharmaceutical Research, 25:55-71, incorporated herein by reference). Additional lipid ingredients

[0112] In addition to the above lipid components (neutral lipids, cholesterol, ionizable cationic lipids, and any hydrophilic polymer-lipid conjugates), LNPs may contain additional lipid components. Without limitation, such additional lipid components may be present at less than 10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol%, 1 mol%, or 0.5 mol% (relative to the total lipids in the LNP). Such additional lipids include other lipid components, such as lipids containing targeting moieties, charged lipids (cationic or anionic lipids that are charged at physiological pH), or vitamins (e.g., tocopherol). In some embodiments, the LNPs consist essentially of neutral lipids, cholesterol, ionizable cationic lipids, and any hydrophilic polymer-lipid conjugates, meaning that the additional lipids, measured relative to the total lipids in the LNP, are present at less than 5 mol%.

[0113] In one embodiment, the LNP lacks a ligand-lipid conjugate for targeting stem or progenitor cells. In such an embodiment, the ligand-lipid conjugate is undesirable because it may induce an immune response. Instead, targeting can be achieved by the inherent long-circulating properties of lcLNP™ due to the increased phosphatidylcholine content. Thus, in some embodiments, the ligand-lipid conjugate is present at less than 1 mol, less than 0.5 mol%, or 0 mol%.

[0114] Additional components may include anionic phospholipids such as phosphatidylserine, and / or ionizable anionic lipids. One example of such lipids is cholesteryl hemisuccinate (CHEMS). Further examples of ionizable anionic lipids are described in a co-pending and co-owned U.S. provisional patent entitled "Ionizable Anionic Lipids," filed March 23, 2023, the entire contents of which are incorporated herein by reference.

[0115] Alternatively or additionally, additional lipid components may include permanently charged cationic lipids, including lipids containing quaternary ammonium cations (e.g., DOTMA, DOSPA, DDAB, CHIM, and DOTAP), or permanently charged anionic lipids, such as phosphatidylserine. Such permanently charged lipids are, in some embodiments, most advantageously present at less than 10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol%, 1 mol%, 0.5 mol%, or 0.25 mol% of the total lipid content. Nanoparticle preparation and morphology

[0116] Nucleic acid-incorporated delivery vehicle can be prepared using various suitable methods, such as rapid mixing / ethanol dilution process.Examples of preparation methods are disclosed 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.

[0117] Without being bound by theory, we hypothesize that the mechanism by which lipid nanoparticles containing encapsulated nucleic acids can be formed using the rapid mixing / ethanol dilution process begins with the formation of a dense region of a hydrophobic nucleic acid-ionized lipid core at low pH (e.g., pH 4) surrounded by a monolayer of neutral lipids / sterols (e.g., cholesterol) that fuse with smaller empty vesicles as the pH increases due to the conversion of ionized cationic lipids to their neutral forms. As the proportion of neutral lipids in the bilayer increases, the bilayer lipids gradually form blebs, and the ionized lipids migrate to the interior hydrophobic core. If the neutral lipid content is sufficiently high, the outer bilayer, which favors neutral lipids, can form a complete bilayer around the interior confined volume.

[0118] LNPs may contain a "core" region. Surprisingly, it has been observed that the core visualized by cryo-electron microscopy is not homogeneous, as it contains both electron-dense regions and aqueous portions or compartments. As observed, but not limited to, by cryo-TEM, the electron-dense region within the core may be partially surrounded by an aqueous compartment within a confined space. The aqueous portion forms a distinct aqueous region or compartment within the lipid nanoparticle. In other words, in some embodiments, the aqueous portion is not simply a hydration layer. Further, such particles are described in co-owned and co-pending WO 2023 / 184038, the contents of which are incorporated herein by reference. Figure 5 herein is a reproduction of an LNP having an electron-dense region and an aqueous portion from Figure 16 of co-owned and co-pending WO 2023 / 184038.

[0119] In one embodiment, at least about one-fifth of the core (trapped volume) comprises an aqueous compartment, and the electron-dense regions are partially adjacent to a 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 compartment, and the electron-dense cores are each partially adjacent to a lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy. In a further embodiment, at least about one-third of the core comprises an aqueous compartment, and the electron-dense regions are each partially adjacent to a 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 compartment, and the electron-dense cores are each partially adjacent to a lipid layer comprising a bilayer, as qualitatively determined by cryo-electron microscopy.

[0120] In one embodiment, the electron-dense regions are generally spherical, hi another embodiment, the electron-dense regions are hydrophobic.

[0121] In another embodiment, the electron-dense regions of the LNP surprisingly appear to be completely surrounded by aqueous matter, as visualized by cryo-TEM microscopy. This morphology is observed in a single plane, and although some of the electron-dense regions are adjacent to the bilayer, they cannot be visualized because they are not in the plane being visualized.

[0122] Lipid nanoparticles can contain a single bilayer or multiple lipid layers (i.e., multiple layers). One or more lipid layers comprising a bilayer can form a continuous layer surrounding the core or can be discontinuous. In some embodiments, the lipid layer can be a combination of a bilayer and a monolayer. In one non-limiting example, the lipid layer is a continuous bilayer surrounding the core.

[0123] 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 preparation comprising a plurality of lipid nanoparticles, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles, as determined by cryo-electron microscopy, have a core with an electron-dense region and an aqueous portion, the aqueous portion being partially surrounded by a lipid layer comprising a bilayer as visualized by cryo-electron microscopy.

[0124] In another, non-limiting embodiment, the present disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles, typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles having an elongated shape (e.g., generally elliptical) as qualitatively determined by cryo-electron microscopy. In this latter embodiment, a core electron-dense region may be partially surrounded by an aqueous space as visualized by cryo-electron microscopy.

[0125] In one embodiment, the lipid nanoparticle is part of a lipid nanoparticle preparation, wherein at least 20% of the electron-dense regions of the lipid nanoparticle are (i) enveloped by aqueous or (ii) partially surrounded by aqueous, and a portion of the periphery of the electron-dense regions is adjacent to the lipid layer, as visualized by cryo-electron microscopy in a single plane.

[0126] In certain embodiments, the present disclosure provides lipid nanoparticle preparations comprising a plurality of lipid nanoparticles, typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles as determined by cryo-electron microscopy have a core with an electron-dense region adjacent to a lipid layer comprising a bilayer as visualized by cryo-electron microscopy.

[0127] In another embodiment, without limitation, the present disclosure provides a lipid nanoparticle preparation comprising a plurality of lipid nanoparticles, typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles having a core comprising an electron-dense region that appears to be surrounded or enveloped by an adjacent aqueous space disposed between the lipid layer (e.g., bilayer) and the electron-dense region, as visualized in a single plane by cryo-electron microscopy.

[0128] LNPs are visualized by cryo-TEM as described in Materials and Methods in the Examples section.

[0129] In another embodiment, the polydispersity index (PDI) of the LNP preparation is less than 0.2, 0.15, 0.12, or 0.10.

[0130] In another embodiment, the particle size distribution is such that at least 90% of the particles in an LNP preparation of the present disclosure have a diameter between 40 and 150 nm, 40 and 140 nm, 45 and 150 nm, 50 and 150 nm, 50 and 120 nm, or 50 and 140 nm.

[0131] Lipid nanoparticles of the present invention can show particularly high encapsulation efficiency of nucleic acid.In this specification, the term "encapsulation" regarding the incorporation of nucleic acid into lipid nanoparticles refers to any association between any lipid component or compartment (including lipophilic part or aqueous part) of lipid nanoparticles and nucleic acid.In one embodiment, nucleic acid is present at least in the core of LNP.

[0132] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%. Nucleic acid encapsulation efficiency is determined as described in the Materials and Methods section of the Examples herein.

[0133] Embodiments of the present disclosure also provide lipid nanoparticles described according to the molar ratio of the positively charged amine groups (N) of the amine lipids (N) to the negatively charged phosphate groups (P) of the encapsulated oligonucleotides, which can be mathematically expressed as N / P. In one embodiment, the lipid nanoparticles have an N / P ratio of 3-15, 4-15, 4.5-10, 5-10, or 5.5-8.

[0134] In one embodiment, the lipid nanoparticles have an N / P ratio of 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 is 15, 14, 13, 12, 11, 10, 9, or 8. The present disclosure also includes any two combinations of the upper and lower limits.

[0135] In one embodiment, the lipid nanoparticles have a nucleic acid weight / total lipid micromoles ratio 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 nucleic acid weight / total lipid micromoles. 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 by weight of nucleic acid per micromoles of total lipid. The present disclosure also includes any two combinations of upper and lower limits.

[0136] In one embodiment, the mRNA copy number / LNP is 1-10 or 4-8. target area

[0137] The lipid nanoparticles include an associated targeting moiety that facilitates binding and entry of the LNP into target cells via endocytosis. The targeting moiety is any molecule or fragment thereof on the surface of the LNP that binds to target cells, such as via a cell surface receptor or epitope present on the target cells. In some embodiments, the targeting moiety is selected to recognize a specific cell subset, such as a malignant cell or a pathological cell, such as an infectious pathogen. In some embodiments, the targeting moiety is referred to as a ligand.

[0138] The binding affinity of a targeting moiety for a target cell can be detected by any means known in the art, e.g., any standard in vitro assay, such as ELISA, flow cytometry, immunocytochemistry, surface plasmon resonance, etc. Fragments of targeting moieties are considered targeting moieties as used herein and may be used in certain embodiments of the present disclosure, provided that the fragment is capable of binding to the appropriate cell surface epitope.

[0139] Examples of targeting moieties include proteins, including but not limited to antibodies, nanobodies, 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.

[0140] Non-limiting binding pairs include antibody-antigen, nanobody-antigen, DARPin-receptor, hormone-receptor, enzyme-substrate, nutrient (such as a vitamin)-transport protein, growth factor-growth factor receptor, and carbohydrate-lectin.

[0141] In one embodiment, the targeting moiety is a protein or peptide that comprises the antigen-binding sequence of an immunoglobulin, such as an antibody or a fragment thereof. In a further embodiment, the targeting moiety is an antigen-binding antibody fragment that lacks the Fc sequence of an immunoglobulin. ab The antibody fragments may be enzymatically or recombinantly produced, such as F(ab)2 fragments of immunoglobulins, Fv antibody fragments, or single-chain Fv antibody fragments (scFv).

[0142] In another embodiment, the targeting moiety may be a nanobody, which is a heavy chain antibody having a VHH. Nanobodies may be desirable in certain instances of the present disclosure because they may be easier to manufacture on a large scale than polyclonal antibodies and / or may have improved stability.

[0143] In one embodiment, the targeting moiety is a moiety that forms a binding pair with a tyrosine kinase growth factor receptor that is overexpressed on the cell surface of many tumors. Exemplary tyrosine kinase growth factors include VEGF receptor, FGF receptor, PDGF receptor, IGF receptor, EGF receptor, TGF-α receptor, TGF-β receptor, HB-EGF receptor, ErbB2 receptor, ErbB3 receptor, and ErbB4 receptor. EGF receptor vIII and ErbB2 (HER2) receptor are particularly preferred for cancer treatment using the lipid nanoparticles described herein because they are specific to cancer cells, such as malignant cells. Alternatively, the targeting moiety is selected to recognize cells that require gene correction or genetic modification by introducing beneficial genes, such as epithelial cells, endocrine cells of genetically deficient organisms, in vitro germ cells, germ cells, stem cells, or germ cells.

[0144] A non-limiting example of a surface-modified LNP for cancer therapy is an anti-HER ScFv that targets HER2 expressed on breast cancer cells. Binding to the HER2 extracellular domain can inhibit its activity. In another embodiment, ankyrin repeat proteins (DARPins) can be used to inhibit HER2 activity in breast cancer cells. According to such an embodiment, LNPs can be modified with biparatopic antitumor DARPins (bipDARPins), which have two binding moieties that recognize the two extracellular domains of HER2. Both targeting moieties can act to capture and stabilize the inactive conformation of HER2. This is particularly effective in promoting apoptosis of HER2-dependent tumor cells. (See Stuber et al., 2021, Communications Biology 4(762); incorporated herein by reference.)

[0145] In another example, the surface of the LNPs can be modified to incorporate nucleic acid cargo and allow binding to T cells. In some examples, targeted T cells include CD5+ or CD4+ T cells. A non-limiting example includes delivering a nucleic acid encoding a chimeric antigen receptor to T cells. In such therapy, chimeric antigen receptor T cells (i.e., CART cells) are generated that are genetically engineered to generate an artificial T cell receptor specific for a desired target antigen. The resulting CAR T cells can be used to target antigens present on the surface of a subset of cell types. Upon binding to the surface antigen, the CAR T cells become activated and exert a desired therapeutic and / or prophylactic effect on the target cells in vivo. This may include stimulating cell proliferation, cytotoxicity, and / or cell destruction through increased secretion of factors that can affect other cells, including, but not limited to, cytokines, interleukins, and / or growth factors. Such CAR T therapy can be used to treat or prevent symptoms of various diseases, such as cancer, immune disorders, or cardiovascular conditions. For example, such an approach can be used to treat cardiac injury by delivering mRNA encoding an anti-fibrotic CAR to T lymphocytes in vivo. The mRNA is formulated in LNPs modified with a CD5 targeting moiety. Such an 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.) The use of targeted LNPs with improved biodistribution in the liver or extrahepatic tissues / organs can deliver mRNA encoding a chimeric antigen receptor to various target cells to treat various diseases or conditions.

[0146] Another non-limiting example of using LNPs to target a subset of cells is conjugating a CD4 antibody to the LNP to target CD4, including T cells. +This type of LNP targeting can be used to deliver nucleic acids to T cells in vivo, and can be used for immunotherapy, such as treating symptoms of HIV or other diseases (Tombacz et al., 2021, Mol Ther, 29(11):3293-3304, incorporated herein by reference).

[0147] Antibody-conjugated LNPs can target receptors present on stem and progenitor cells, such as HSPCs. Examples include CD117, CD49d, CD44, and IL-6R receptors expressed on HSPCs. Therefore, LNPs can contain anti-CD49d, CD44, and IL-6R antibodies.

[0148] In some embodiments, the lipid nanoparticle comprises two or more different targeting moieties.

[0149] The ligand can be bound to the LNP by any suitable method available in the art. The binding can be performed covalently or non-covalently, such as by adsorption or complex formation. The binding preferably includes a lipophilic molecular moiety that can be conjugated to the ligand by forming a covalent or non-covalent bond. The lipophilic molecular moiety can be called an "anchor." The anchor is partitioned into a lipophilic environment, such as a bilayer, thereby binding the ligand to the LNP. Methods for binding the ligand via a lipophilic moiety are known in the art.

[0150] A particularly suitable mode of ligand attachment to LNPs is the use of ligands conjugated to lipophilic anchors via intermediate polymer linkers, such as, but not limited to, hydrophilic polymers. Targeting moieties conjugated to lipophilic anchors, such as lipids, via hydrophilic polymer intermediate linkers advantageously stably associate with the LNPs of the present disclosure.

[0151] The size of the linker varies depending on the ligand. In one embodiment, the size of the linker varies between 0.50 kDa and 20 kDa, 1 and 10 kDa, or 1.5 and 8 kDa. The polymer can be functionalized with end groups that selectively react with functional groups on the ligand. Typically, the linker is polyethylene glycol (PEG), although other polymer linkers of various lengths known to those skilled in the art can also be used.

[0152] The targeting moiety can also be directly conjugated 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 that contain a lipophilic region.

[0153] Targeting moieties conjugated to lipophilic anchors (directly or via a linker) can be incorporated into 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, lipids conjugated to targeting moieties directly (e.g., glycolipids) or via a linker (e.g., a polymer such as PEG) can be added to an ethanolic lipid solution (containing lipid components including ionizable lipids, phosphatidylcholine, sterol, and hydrophilic polymer-lipid conjugates), which can then be mixed with a buffered solution of nucleic acid to form LNP particles in a T-junction mixer. The pH of the external solution can then be raised above the pKa of the ionizable lipid.

[0154] In another example, LNPs are modified with targeting moieties conjugated to lipophilic anchors after formulation using post-insertion techniques. Such methods can be utilized when the targeting moiety is sensitive to conditions used during formulation (e.g., proteins such as peptides or antibodies), such as the high ethanol concentrations used in the ethanol rapid mixing technique. As an example, LNP particles can be conjugated to proteins or peptides such as antibodies using lipid-PEG-maleimide conjugates. Such lipid-PEG-maleimide conjugates can be attached to antibodies functionalized with N-succinimidyl S-acetylthioacetate via sulfhydryl groups on the antibody. Other post-insertion techniques can also be used in the practice of the present invention to introduce targeting moieties onto the surface of LNPs that are sensitive to formulation conditions.

[0155] In another example, the targeting moiety (directly or indirectly attached 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 mol% relative to the total lipid content of the LNP.

[0156] In another example, the targeting moiety (directly or indirectly attached to the lipophilic moiety via a linker) is present at 0.25-3 mol %, 0.30-1.5 mol %, or 0.35-1.25 mol %. Nucleic Acid Cargo

[0157] Lipid nanoparticles contain nucleic acid cargo.As used herein, the term "encapsulation" with respect to incorporating nucleic acid into nanoparticles refers to any association between nucleic acid and any component or compartment of lipid nanoparticles.In one embodiment, nucleic acid is incorporated into the core of lipid nanoparticles (visualized by cryo-electron microscope).In another embodiment, nucleic acid is incorporated between two adjacent lipid layers.

[0158] Nucleic acids include, but are not limited to, oligonucleotides, vector DNA, or mRNA. Oligonucleotide cargo

[0159] Oligonucleotide cargoes include interfering RNA and antisense oligonucleotides, which are described in more detail below. An "oligonucleotide" or "oligonucleotide cargo" is a single-stranded or double-stranded RNA or DNA molecule, and is 5 to 500 nucleotides in length. This term includes antisense oligonucleotides (ASOs), which are single-stranded and generally 30 to 500 nucleotides in length, or double-stranded silencing RNA molecules (siRNAs), which are 3 to 40 nucleotides in length or shorter. Small interfering RNA

[0160] In one embodiment, the oligonucleotide is " short interfering RNA " or " siRNA ", which is an RNA molecule that can reduce or inhibit the expression of target gene or nucleic acid sequence in cells.In one embodiment, the short interfering RNA can mediate the degradation of target mRNA, as measured in vitro or in vivo.In this embodiment, the siRNA functions through base pairing (when single-stranded) with the complementary sequence of target mRNA, and can induce the cleavage of mRNA.

[0161] siRNAs are double-stranded and can vary in length, but are typically less than 35 nucleotides in length, in some embodiments, the length of the siRNA is 1-35 nucleotides, or 15-30 nucleotides, or 20-25 nucleotides, etc.

[0162] In embodiments in which siRNA reduces the expression of a target gene or sequence through complementary base pairing and mRNA degradation, the siRNA may have substantial or complete identity with the gene encoding the target sequence, or may contain mismatched regions (i.e., mismatch motifs). The sequence of the siRNA may correspond to the full-length target sequence or a subsequence thereof.

[0163] The double-stranded siRNA encapsulated in LNP can include double-stranded RNA such as double-stranded small interfering RNA, asymmetric interfering RNA (aiRNA), or pre-miRNA, or a hybrid molecule that contains both RNA and DNA.In one embodiment, the double-stranded RNA is self-complementary.In such an embodiment, the siRNA can form a stem-loop or hairpin structure at one end.

[0164] The siRNA included in the embodiments of the present disclosure can be used to inhibit the expression of a wide range of target polynucleotides.The siRNA molecule that targets specific polynucleotides for any therapeutic, preventive or diagnostic use can be easily prepared according to the procedures known in the art.The siRNA target site can be selected, and the corresponding interfering RNA can be chemically synthesized, produced by in vitro transcription, or expressed from vector or PCR product.

[0165] As indicated, the siRNAs described herein may contain "mismatch motifs" or "mismatch regions," which refer to portions of the siRNA sequence that are not 100% complementary to its target sequence. An siRNA may have at least 1, 2, 3, 4, 5, 6, or more mismatch regions. The 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 contain a single nucleotide, or may contain 2, 3, 4, 5, or more nucleotides.

[0166] The nucleotide of siRNA may or may not be chemically modified.Examples of any modification include, but are not limited to, 2'-O-alkyl modification, such as 2'-O-Me or 2'-O-methoxyethyl modification, and 2'-halogen modification, such as 2'-fluoro modification.In yet another embodiment, siRNA comprises one, two, three, four or more 2'-deoxynucleotides, for example, in the sense strand and / or antisense strand of double-stranded region.Alternatively or additionally, in some embodiments, siRNA comprises phosphate backbone modification.

[0167] In siRNA, when antisense strand and sense strand form a duplex by complementary base pairing, they can be designed to anneal without overhang to form blunt ends at both ends of the duplex, or can anneal with 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 some embodiments, there is no 5' overhang, no 3' antisense overhang, but there is a 3' sense overhang.In other embodiments, there is no 5' overhang, but there is a 3' antisense overhang and a 3' sense overhang.The overhang can comprise T or U nucleotide.

[0168] In some embodiments, siRNA is covalently linked with one or more other moieties to form conjugate.In some embodiments, conjugate is selected based on its ability to promote the delivery of siRNA to organism or cell.For example, siRNA can be linked to the 5'-end of antisense strand, the 3'-end of antisense strand, the 5'-end of sense strand, the 3'-end of sense strand, or the nucleotide at the position that is not the 3'-end or 5'-end of either strand.

[0169] Examples of conjugates include, but are not limited to, one or more of an antibody or fragment thereof, a peptide, an amino acid, an aptamer, a phosphate group, a cholesterol moiety, a lipid, a cell-penetrating peptide, a polymer, and a sugar group, including sugar monomers, oligosaccharides, and modifications thereof. In one non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc). Antisense oligonucleotides (ASOs)

[0170] 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 may have substantial or complete identity with the gene encoding the target sequence, or may contain a mismatch region (i.e., a mismatch motif). The sequence of the ASO may correspond to the full-length target sequence or a subsequence thereof.

[0171] ASOs can reduce or inhibit the expression of target genes or nucleic acid sequences in cells through a variety of mechanisms, some of which are described below: In one embodiment, the ASO forms part of a gene editing complex to direct a nuclease to a target site for site-specific cleavage of DNA.

[0172] In embodiments in which an ASO reduces expression of a target gene or sequence through complementary base pairing and mRNA degradation, the ASO exerts its effect through base pairing with a complementary sequence in the target mRNA, inducing mRNA cleavage. By binding to RNA, an ASO can inhibit or reduce translation of the complementary RNA strand. ASOs can be used to target specific complementary (coding or non-coding) RNA. Upon binding, the target sequence can be degraded by the enzyme RNase H, which is present in the cell's nucleus and / or cytoplasm. In one embodiment, the ASO is a "gapmer" sequence, containing two to five chemically modified nucleotides at each end that blank a central gap region (e.g., an 8- to 10-base "gap") in the DNA. The chemically modified nucleotides reduce degradation by nucleases and increase the affinity of the ASO for the target sequence. The gap forms a hybrid sequence that is cleavable by RNase H. Additionally, oligonucleotides can be chemically modified using known methods to recruit RNase H.

[0173] In another embodiment, ASOs can bind to target mRNA and inhibit gene expression. ASOs that function by inhibiting gene expression are known as "steric inhibitors" and prevent or reduce translation of the target nucleotide sequence by preventing it from binding to ribosomes.

[0174] In a further embodiment, ASOs can regulate the splicing of pre-mRNA sequences. ASOs can be designed to target sequences within the pre-mRNA to affect splicing and increase the production of desired isoforms. ASOs can be used, for example, to remove mutated exons, thereby restoring the proper reading frame and producing a more functional protein product.

[0175] 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.

[0176] In general, ASOs may contain "mismatch motifs" or "mismatch regions," which refer to portions of the ASO sequence that are not 100% complementary to its target sequence. An ASO may have at least 1, 2, 3, 4, 5, 6, or more mismatch regions. The mismatch regions may be contiguous or 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 it may comprise 2, 3, 4, 5, or more nucleotides.

[0177] The nucleotides of an ASO may or may not be chemically modified. In addition to protection by LNPs, modifications may improve the stability of the ASO, such as increasing nuclease resistance. Furthermore, in some embodiments of the present disclosure, chemical modifications improve potency and / or selectivity by increasing the binding affinity of the ASO to its complementary sequence. 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 yet another embodiment, the ASO contains one, two, three, four, or more 2'-deoxynucleotides, for example, in the sense and / or antisense strands of the double-stranded region. Alternatively or additionally, in some embodiments, the ASO contains a phosphate backbone modification, such as a phosphorothioate backbone modification. Further backbone modifications include backbone analogs, such as locked nucleic acids (LNAs). A non-limiting example is a structure containing a methylene bridge between the 2' and 4' positions of the ribose, which "locks" the ribose ring in a conformation that facilitates binding to a complementary nucleic acid sequence. A related bridge modification is bridged nucleic acid (BNA). Further examples include ASOs with peptide backbones (PNAs), CpG oligomers, etc., which are known to those skilled in the art.

[0178] ASOs encapsulated in LNPs are generally single-stranded. However, in some 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.

[0179] In some embodiments, ASO is covalently linked with one or more other moieties to form conjugate.In some embodiments, conjugate is selected based on its ability to promote the delivery of ASO to organism or cell.ASO can be, for example, linked to the 5'-end of antisense strand, the 3'-end of antisense strand, the 5'-end of sense strand, the 3'-end of sense strand, or the nucleotide at the position that is not the 3'-end or 5'-end of either strand.

[0180] Examples of conjugates include, but are not limited to, one or more of an antibody or fragment thereof, a peptide, an amino acid, an aptamer, a phosphate group, a cholesterol moiety, a lipid, a cell-penetrating peptide, a polymer, e.g., a hydrophilic polymer such as polyethylene glycol, and a sugar group, including sugar monomers, oligosaccharides, and modifications thereof. In one non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc).

[0181] 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 determined by analysis of the selected target sequence, secondary structure, T m The sequence of an ASO can be derived by computational design or experimentation. Vector DNA

[0182] The lipid nanoparticles described herein may contain encapsulated DNA vectors. As used herein, the term "DNA vector" refers to a circular or linear polynucleotide that encodes at least one peptide, polypeptide, or protein.

[0183] DNA vectors can replicate autonomously or can be inserted into the genome of a host cell and replicated using methods well known in the art. Autonomously replicating vectors contain an origin of replication or an autonomously replicating sequence (ARS) that functions in the host cell. DNA vectors can be used in multiple host cells, for example, E. coli for cloning and construction, and mammalian cells for expression.

[0184] The DNA vector can be administered to a subject to restore, enhance, inhibit, or reduce the expression of a cellular protein or peptide. Thus, the nucleotide polymer can be a nucleotide sequence comprising genomic DNA, cDNA, or RNA.

[0185] As those skilled in the art will understand, vectors can encode promoter regions, operator regions, or structural regions.DNA vectors can comprise double-stranded DNA or can be composed of DNA-RNA hybrids.Non-limiting examples of double-stranded DNA include structural genes, genes containing operator control and termination regions, and self-replicating systems such as vector DNA.

[0186] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triplex-forming oligonucleotides. To enhance activity, single-stranded nucleic acids preferably have some or all of the nucleotide bonds replaced with stable non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkylphosphotriester linkages.

[0187] DNA vectors may contain nucleic acids in which one or more sugar moieties and / or one or more pyrimidine or purine bases have been modified. Such sugar modifications may include replacing one or more hydroxyl groups with halogens, alkyl groups, amines, azides, or functionalizing them as ethers or esters. In another embodiment, the entire sugar may be replaced with a sterically and electronically similar structure, including azasugars and carbocyclic sugar analogs. Modifications of purine or pyrimidine base moieties include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substituents known to those skilled in the art.

[0188] In certain embodiments, DNA vectors can be modified with modifying molecules such as peptides, proteins, steroids, or sugar moieties. Modifying DNA vectors with such molecules can facilitate delivery to the desired target site. In some embodiments, such modifications allow the DNA vector to translocate beyond the nucleus of the target cell. As an example, modifiers can be attached to specific portions of the DNA vector (usually portions that do not encode the gene of interest), but they can also contain peptides or other modifiers with nuclear homing effects, such as nuclear localization signals. A non-limiting example of a modifier 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.

[0189] DNA vectors can contain sequences encoding various proteins or peptides. Promoters, enhancers, stress- or chemically-regulated promoters, antibiotic- or nutrient-sensitive regions, and therapeutic protein-encoding sequences can be optionally included. DNA vectors can also contain non-encoding sequences.

[0190] The nucleic acids used in the present method can be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid-phase methods. Solid-phase synthesis is generally preferred. Detailed descriptions of procedures for solid-phase synthesis of nucleic acids using phosphite triester, phosphate triester, and H-phosphonate chemistries are widely available.

[0191] In one embodiment, the DNA vector is double-stranded DNA and comprises at least 700 base pairs, at least 800 base pairs, at least 900 base pairs, or at least 1000 base pairs.

[0192] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0193] 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 for diagnostic purposes. mRNA

[0194] Lipid nanoparticles described herein can contain messenger RNA cargo.As used herein, the term " messenger RNA " or " mRNA " refers to the polynucleotide that encodes and expresses at least one peptide, polypeptide or protein.This term is intended to include, but is not limited to, circular or linear mRNA, as well as small activating RNA (saRNA) and trans-amplifying RNA (taRNA).

[0195] The concentration of mRNA in the LNP can range from 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.

[0196] As used herein, mRNA includes both 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, or can be produced using recombinant expression systems and optionally purified, or can be chemically synthesized.

[0197] In embodiments in which mRNA is chemically synthesized, the mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the mRNA contains natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxyuridine, C5-methyl ... zaadenosine, 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).

[0198] The mRNA of the present invention can be synthesized according to any of various known methods.For example, in certain embodiments, the mRNA can be synthesized by in vitro transcription (IVT).Briefly, IVT is usually carried out using a linear or circular DNA template, which contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that can contain DTT and magnesium ions, and a suitable RNA polymerase (for example, T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0199] In some embodiments, in vitro synthesized mRNA may be purified prior to encapsulation to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0200] The present disclosure can be used to formulate and encapsulate mRNAs of various lengths, in some embodiments, the present disclosure can be used to formulate and encapsulate in vitro synthesized mRNAs ranging in length from 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.

[0201] In these embodiments where the mRNA is linear, synthesis involves adding a "cap" to the 5' end and a "tail" to the 3' end. The presence of a cap may confer resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from degradation by exonucleases.

[0202] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation (e.g., an iron-responsive element). In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0203] In some embodiments, the 3' untranslated region includes a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides or more in length.

[0204] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5' and 3' ends, making it less susceptible to exonuclease degradation and therefore more stable in vivo. Circular mRNA can be prepared by any known method, including any one of the methods described in Deviatkin et al., 2023, "Cap-Independent Circular mRNA Translation Efficiency," Vaccines, 11(2), 238, which is incorporated herein by reference. Circular mRNA is translated by a cap-independent translation initiation mechanism.

[0205] In certain embodiments, mRNA provided from an in vitro transcription reaction may be desirable, although other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0206] The mRNA sequence may contain a reporter gene sequence, although inclusion of the reporter gene sequence in the pharmaceutical formulation for administration is optional and is typically omitted. Such sequences are incorporated into the mRNA for in vivo studies in animal models to assess biodistribution. Edit Cargo

[0207] In one embodiment, the LNP-encapsulated cargo edits cells to produce a desired modification to treat, prevent, or ameliorate a disease or condition.

[0208] As used herein, the term "editing cargo" includes protein and / or nucleic acid-based cargo that causes cellular modification at a specific locus or group of loci to produce the desired modification to treat, prevent, or ameliorate a disease or condition.

[0209] As used herein, the term "nucleic acid editor" includes protein- and / or nucleic acid-based systems that cause the modification of any nucleic acid in a cell at a specific locus or group of loci to produce the desired modification to treat, prevent, or ameliorate a disease or condition.

[0210] The cargo may include 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, a protein- and / or nucleic acid-based system in which nucleic acids are inserted, deleted, modified (e.g., epigenetically edited), or replaced at a site-specific location in the genetic material of an organism.

[0211] Nucleic acid editing complexes can be used for genetic modification of cells, including post-translational modifications.

[0212] Alternatively or additionally, the cargo comprises a peptide or protein that is part of an editor or forms an editing complex.

[0213] 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 acting on RNA (ADARs), prime editors, base editors, epigenetic, transposase, meganucleases, ARCUS gene editing cargoes, or any variant or combination thereof. These nucleic acid editing cargoes are exemplary and include any cargo that can modify the genetic material of a cell (including RNA transcripts and non-coding regions) to treat, prevent, or ameliorate a disorder or disease. Gene editing cargoes may include, but are not limited to, those designed by a process known to those skilled in the art as directed nuclease editing (DNE).

[0214] Cas-based editing cargoes include CRISPR and non-CRISPR gene editing cargoes. Additionally, editing cargoes include those that cleave DNA and epigenetic editing cargoes that modify nucleic acid markers, as described below.

[0215] The CRISPR gene editing cargo most advantageously comprises a nucleic acid (e.g., mRNA) encoding one or more proteins of the class II Cas nuclease family and a guide RNA. The nuclease encoded by the nucleic acid is an enzyme with DNA endonuclease activity and can be directed by an appropriate guide RNA to cleave a desired nucleic acid target. The nuclease and the guide RNA form a complex called a ribonucleoprotein (RNP). In some embodiments, the nuclease is a class II CRISPR enzyme, which is further classified into types II, V, and VI. According to one embodiment, the mRNA encodes a Cas protein that is part of the type II CRISPR / Cas system, such as a Cas9 protein or a Cpf1 protein.

[0216] 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 Cas13a, which is an RNA endonuclease that cleaves single-stranded RNA.

[0217] The guide RNA can guide the Cas nuclease to a target sequence on the target nucleic acid molecule, where the guide RNA hybridizes to the target sequence and the Cas nuclease cleaves or modulates the sequence. In some embodiments, the guide RNA binds to a class 2 nuclease, thereby providing specificity for cleavage.

[0218] The guide RNA of the CRISPR / Cas9 nuclease system includes CRISPR RNA (crRNA) or tracr RNA (tracr). In some embodiments, the crRNA can include a target sequence that is complementary to and hybridizes with a target sequence on a target nucleic acid molecule. The crRNA can also include a flagpole that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA can correspond to the structure of a natural crRNA transcribed from a bacterial CRISPR locus, where the target sequence functions as a spacer in the CRISPR / Cas9 system. The flagpole corresponds to the portion of the repeat sequence adjacent to the spacer above the CRISPR locus.

[0219] The guide RNA of the RNP can target any sequence of interest through the target sequence of the crRNA. In some embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule can be 100% complementary. In other embodiments, the target sequence of the guide RNA and the target sequence on the target nucleic acid molecule can contain at least one mismatch.

[0220] The length of the target sequence may vary depending on the RNP system and components used. For example, different Cas proteins in different bacterial species have various optimal target sequence lengths. Thus, target sequences 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 in length may be included. In some embodiments, the target sequence may be 18-24 nucleotides in length. In some embodiments, the target sequence may be 19-21 nucleotides in length. In some embodiments, the target sequence may be 20 nucleotides in length.

[0221] In some embodiments, the editing system includes Cas1, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csy1, 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 versions thereof.

[0222] As mentioned above, Cas-based gene editing cargoes other than CRISPR are also included in the embodiments of the present disclosure. Cas-based editing systems can include a Cas enzyme fused to a deaminase (Luo et al., 2020, Microbial Cell Factories, 19(93)). One example is a cytosine or adenine base editor generated by fusing the endonuclease Cas to the cytosine deaminase pmCDA1 or the heterodimeric adenine deaminase TadA-TadA. A further non-limiting example includes a Cas fused to a reverse transcriptase (Mohr et al., 2018, Mol Cell., 72(4):700-714).

[0223] Fanzor is a eukaryotic RNA-guided endonuclease that can function as a gene editor in certain embodiments described herein. (See Saito et al., 2023, Nature 620:660-668, which is incorporated herein by reference.) In some embodiments, Fanzor proteins use RNA as a guide to precisely target DNA and can be engineered to edit cells using the LNPs described herein. In some examples, compact Fanzor cargoes may be capable of facilitating improved delivery over CRISPR-Cas cargoes.

[0224] In these 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 one embodiment, the system comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In one embodiment, the peptide having nuclease activity is a type II restriction enzyme type 1-like endonuclease, e.g., FokI endonuclease.

[0225] In these embodiments in which the cargo is a ZFN, the nucleic acid can encode a zinc finger DNA binding domain, a fragment or variant thereof, and / or a peptide having nuclease activity, e.g., endonuclease activity. In embodiments, the zinc finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, or more zinc fingers. In one embodiment, the peptide having nuclease activity is a type II restriction enzyme type 1-like endonuclease, e.g., FokI endonuclease.

[0226] Adenosine deaminase (ADAR) acting on RNA is another editing cargo included in the embodiment 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 it to inosine (see Song et al., 2022, PMC, 13(1):e1665, which is incorporated herein by reference).

[0227] Meganucleases are enzymes of the endonuclease family that can induce homologous recombination, generate mutations, and alter the reading frame. Meganucleases include homing endonucleases, which are intron or intein endonucleases. In one embodiment, the meganuclease is from the LAGLIDADG family, GIY-YIG endonucleases, HNH endonucleases, His-Cys box endonucleases, or PD-(D / E)XK endonucleases. Meganucleases can be combined with components of other gene editing systems. In one embodiment, the DNA-binding domain of a transcription activator-like (TAL) effector is combined with the meganuclease to generate a "megaTAL." In another embodiment, meganucleases can be fused to DNA end-processing enzymes to promote error-prone non-homologous end joining.

[0228] ARCUS nuclease is a gene editing system based on I-CreI, a homing endonuclease that evolved in the alga Chlamydomonas reinhardtii. In some embodiments, the nuclease can inactivate itself after gene editing, thereby reducing off-target effects. In some embodiments, ARCUS nuclease can generate a unique cleavage site with a 4-base pair 3' overhang, allowing for gene insertion, gene excision, gene repair, or a combination thereof.

[0229] Epigenetic editing is also included in the present disclosure. Such editing of genetic material does not cut nucleic acids, but rather alters epigenomic marks that "decorate" DNA. Altering a cell's epigenetic signature can modify the cell's epigenetic signature and alter its transcriptional profile. In some embodiments, the epigenetic editing system can target and edit one or more methylation sites in a nucleic acid sequence. In some embodiments, genome-homing proteins with artificially engineered or naturally occurring nuclease functions for gene editing can be mutated and adapted to function solely as a delivery system. In one embodiment, an epigenetic modification enzyme or domain can be fused to the homing protein, which can alter local epigenetic modifications upon protein recruitment. Targeting proteins that recognize DNA sequences can be linked to effector proteins that alter epigenomic marks, such as methylation. Examples of targeting 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 demethylation of cytosines at CpG sites; LSD1, which induces demethylation of H3K4me1 / 2 and indirectly causes deacetylation of H3K27; and CIB1 / CRY2, a cryptochrome / blue light-activated complex that modifies chromatin upon light irradiation.

[0230] Further 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 polycomb repressive complexes or components thereof, such as PRC1 or PRC2, or PR-DUBs, or fragments (e.g., biologically active fragments) or variants thereof.

[0231] In embodiments, the epigenetic editor comprises a molecule that modifies chromatin structure and / or a molecule that modifies histones. In embodiments, the epigenetic modulator is a molecule that modifies chromatin structure, such as a SWI / SNF remodeling complex or a component thereof. In embodiments, the epigenetic modulator is a molecule that modifies histones, such as a molecule that methylates and / or acetylates histones, such as a histone modifying enzyme or a fragment (e.g., a biologically active fragment) thereof or a variant thereof, such as an HMT, HDM, HAT, or HDAC. Improved targeting and biodistribution of ligand-LNPs by increasing neutral lipid content

[0232] As described in the Examples section, targeting moiety-LNPs of the present disclosure with increased neutral lipid content can improve biodistribution to a wider range of tissues and / or organs than Onpattro™-type formulations described herein or baseline formulations. In further embodiments, the baseline formulation can be (a) an otherwise identical LNP with 10 mol% lower levels of the same neutral lipids; (b) an otherwise identical LNP with an N / P of 1 or 3, when the N / P of the LNP is 4 or greater; and / or (c) an otherwise identical LNP with a micromoles of nucleic acid by weight / total lipid that is 0.20:1 less than the micromoles of nucleic acid by weight / total lipid of the lipid nanoparticles of the present disclosure, when the lipid nanoparticles have a micromoles of nucleic acid by weight / total lipid ratio of 0.05:1 to 1:1.

[0233] In one embodiment, the LNPs of the present disclosure exhibit increased biodistribution in the liver, spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin, and / or ears in a specific mouse model compared to a relevant baseline. In another embodiment, this includes increased biodistribution to extrahepatic tissues selected from the spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin, and / or ears compared to a relevant baseline. In another embodiment, this includes increased biodistribution to extrahepatic tissues selected from the spleen or bone marrow compared to a relevant baseline. Whether oligonucleotide-encapsulated LNPs exhibit such enhanced biodistribution to one or more predetermined tissues or organs compared to a baseline oligo-LNP formulation is determined by biodistribution testing in an in vivo mouse model, as detailed in the Examples section. A fluorescent lipid marker (DiD, described in Materials and Methods) is used to assess the biodistribution of oligo-LNPs in predetermined tissues or organs relative to baseline.

[0234] In one embodiment, the lipid nanoparticles exhibit at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% increase in biodistribution measured in vivo in the liver, spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin, and / or ears of mice compared to any one of the relevant baselines described above, where biodistribution is measured by detection of lipid markers at 1, 4, 10, and / or 24 hours post-administration in a mouse model. Measurements are performed on tissue homogenates of one or more of the aforementioned tissues or organs, as described in the Examples section.

[0235] The percentage increase in fluorescence relative to the relevant baseline is determined by comparing the fluorescence signal of the LNP being assessed in the relevant tissue and / or organ, per mg of tissue homogenate, with the fluorescence signal of the tissue homogenate resulting from administration of the baseline LNP.

[0236] The oligo-LNPs being compared were prepared using identical materials and methods. In other words, the two formulations compared contained the same ionizable lipids, PEG-lipids, (if present), and sterols, and were prepared using the rapid ethanol injection method as described in Materials and Methods.

[0237] Biodistribution is assessed in the same mice at the same time points (1, 4, 10, and / or 24 h) after administration, using the same analytical techniques to measure marker lipids (see Materials and Methods).

[0238] In these embodiments where the baseline formulation has 10 mol% less neutral lipid (e.g., DSPC or sphingomyelin) than the LNPs of the present disclosure, the neutral lipids can be reduced at baseline at the expense of both cholesterol and ionized lipids equally, while the ionized lipid:cholesterol (mol:mol) is kept constant between the baseline and the LNPs of the present disclosure. Clinical and Non-Clinical Uses of LNPs Herein

[0239] In some embodiments, the LNP-encapsulated nucleic acid is part of a pharmaceutical composition and is administered to treat and / or prevent a disease state. Treatment can have a prophylactic, ameliorative, or therapeutic effect. The pharmaceutical composition is administered in any suitable dose.

[0240] The nucleic acid-LNPs described herein can be used to treat and / or prevent any disease, disorder, or condition in a mammal, including cancer, infectious diseases such as bacterial, viral, fungal, or parasitic infections, inflammatory and / or autoimmune diseases (including treatments that induce immune tolerance), and cardiovascular diseases such as hypertension, arrhythmia, and restenosis.

[0241] Examples of cancer include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteosarcoma, 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 other non-liver cancer cell types), and hepatoblastoma.

[0242] Non-limiting examples of other diseases, disorders, or conditions treatable by the oligo-LNPs herein and which may be at least partially due to immune disease include colitis, Crohn's disease, allergic encephalitis, allograft / graft-versus-host disease (GVHD), diabetes, and multiple sclerosis.

[0243] The LNPs herein may be used for purposes other than the treatment and / or prevention of a disease or disorder. The LNPs may be used to treat symptoms such as aging, preventative medicine, and / or as part of a personalized medical plan. In further embodiments, the LNPs are used for diagnostic purposes.

[0244] In one embodiment, the LNPs are part of a pharmaceutical composition that is administered parenterally, i.e., intraarterially, intravenously, subcutaneously, or intramuscularly. In yet another embodiment, the pharmaceutical composition is for intratumoral administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinally, intrathecally, or via other topical routes. In some embodiments, the oligo-LNPs are spread or applied to the skin.

[0245] Pharmaceutical compositions include pharmaceutically acceptable salts and / or excipients.The term " pharmaceutically acceptable salts " used herein refers to pharmaceutically acceptable salts derived from various organic and inorganic counterions well known in the art, including sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; if the molecule contains a basic functional group, includes the salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate.Suitable salts include those listed in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

[0246] As used herein, the term "excipient" refers to a substance used to formulate an active pharmaceutical ingredient (API) into a pharmaceutical formulation. Non-limiting examples include mannitol, Captisol®, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, croscarmellose 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 one of ordinary skill in the art.

[0247] The compositions described herein can be administered to a subject.The term "subject" as used herein includes human or non-human subjects, including mammals.The subject is not limited to a patient, as oligo-LNPs may also be administered as part of preventive treatment.

[0248] These examples are intended to illustrate the preparation of specific lipid nanoparticle oligo preparations and their properties and are not intended to limit the scope of the invention.

[0249] As used herein, the articles "a" or "an" are meant to include both the singular and the plural of the term or phrase referred to herein, unless otherwise stated. [Example]

[0250] Methods and Materials Preparation of surface-modified lipid nanoparticles containing nucleic acids Unless otherwise specified, LNPs were prepared by dissolving mRNA or plasmid DNA (pDNA) in 25 mM sodium acetate (pH 4.0), with the lipid components at the indicated mol% dissolved in absolute ethanol. Lipids in ethanol and nucleic acid cargo in buffer were mixed at a 1:3 volumetric ratio using a dual-syringe T-junction. The solution was extruded through the T-junction at a total flow rate of 20 mL / min (5 mL / min for the lipid-containing syringe and 15 mL / min for the mRNA-containing syringe). The mixture was then dialyzed overnight against at least approximately 100x the volume of 1x phosphate-buffered saline (PBS), pH 7.4, using a Spectro / Por™ dialysis membrane (molecular weight cutoff 12,000–14,000 Da). LNPs were concentrated as needed using Amicon Ultra™ 10,000 MWCO (molecular weight cutoff) regenerated cellulose concentrators. The lipophilic targeting moiety was a DSPE-PEG lipid conjugated with 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).

[0251] The encapsulation efficiency was determined by the mRNA-LNP(F i The unencapsulated mRNA content was determined by measuring the fluorescence upon addition of RiboGreen™ to the PBS solution, and this value was then adjusted to 2% Triton X-100 (F t) and calculated relative to the total mRNA content obtained by dissolving the LNPs: % encapsulation = (F t -F i ) / F t ×100.

[0252] Particle size and polydispersity index (PDI) were characterized using a Zetasizer Nano ZS™. In vitro analysis of A7 astrocyte cells

[0253] A7 astrocyte cells 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 medium was aspirated and replaced with medium containing LNP diluted at relevant concentrations ranging from 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). Tissue homogenate assay

[0254] Tissues were removed from mice, placed in 2 mL tubes, and flash-frozen in liquid nitrogen. They were then frozen at -80°C. An appropriate volume of Promega™ GLO™ Lysis Buffer was added to each tube to ensure the samples remained frozen before adding the lysis buffer. The samples were placed in a FastPrep™ Homogenizer, and the homogenizer was run twice for a total of three rounds. The homogenized samples were centrifuged at room temperature, after which the homogenate was added to a black plate. The plate was transferred to a plate reader, and fluorescence was read at 640 nm excitation / 720 nm emission. Cryo-TEM

[0255] Below, we describe a method for evaluating LNP morphology. Prior to cryo-TEM imaging, LNPs are concentrated to an estimated total lipid content of 15–25 mg / mL. A defined volume of the resulting LNP solution, e.g., 2–4 μL, is applied to glow-discharged copper grids and quenched using an FEI Mark IV Vitrobot to generate vitreous ice. These grids are stored in liquid nitrogen until imaging with an FEI Titan Krios or FEI Glacios TEM. The instrument is operated at 200 kV under low-dose conditions, and images are obtained using a bottom-mounted FEI Falcon direct electron detector camera at 47–88,000X magnification with an underfocus of 0.5–2 μm for contrast enhancement. Example 1: Particle properties of neutral lipid-enriched and targeting moiety-modified LNPs

[0256] The encapsulation efficiency, PDI, and particle size of the following lipid nanoparticles, A-E, containing varying amounts of RGD-PEG-lipid and different types of sterols (cholesterol and beta-sitosterol) were measured (see Materials and Methods). All formulations tested contained elevated levels of neutral lipid (40 mol% DSPC). Formulations A-D contained cholesterol as the sterol and various mol% of targeting moiety lipid conjugates (RGD-modified). Formulation E contained beta-sitosterol as the sterol and 1.5 mol% of targeting moiety lipid conjugates (RGD-modified). The ionizable lipid was norMC3 (described in WO2022 / 246571). [Table 1]

[0257] The results are shown in Figure 1 and suggest that in non-limiting embodiments of the present disclosure, a targeting moiety-lipophilic moiety content of less than 2 mol% in the particles achieves adequate PDI, particle size, and encapsulation efficiency. Example 2: Dose-dependent in vitro activity of neutral lipid-elevating, targeting moiety-modified LNPs

[0258] Lipid nanoparticles G, H, and I in Table 2 below were analyzed for dose-dependent activity in A7 astrocyte cells according to Materials and Methods. [Table 2]

[0259] The results are shown in Figure 2 and indicate that, in a non-limiting embodiment of the present disclosure, target moiety-modified LNPs exhibited enhanced transfection as measured in astrocyte cells. Example 3: Tissue expression of mRNA-LNPs with targeting moieties and enriched neutral lipid content

[0260] The in vivo expression of various amounts of RGD-PEG lipids in lipid nanoparticles B and C (see Example 1) in Table 1 was measured in mice (see Materials and Methods).

[0261] All formulations tested contained high levels of neutral lipids (40 mol% DSPC) and 1 and 2 mol% RGD-PEG lipids. Formulations included nMC3 37.9%:40% DSPC:Chol 21.1%:DSPE-PEG2k-RGD 1% (LNP B) and nMC3 36.9%:40% DSPC:Chol 21.1%:DSPE-PEG2k-RGD 2%.

[0262] Liver and bone marrow tissue homogenates were analyzed, and the results are shown in Figures 3A and 3B, respectively.

[0263] Of particular note is the increased mRNA expression in bone marrow compared to liver for both formulations B and C. These results indicate that LNPs with increased neutral lipid and targeting moieties at both 1 mol% and 2 mol% show enhanced extrahepatic expression as measured in vivo. Example 4: Expression of mRNA-LNPs with increased neutral lipid content and targeting moieties for cell surface markers of hematopoietic stem cells and myeloid progenitor cells

[0264] LNPs containing 50 mol% DSPC were modified with an antibody against CD117 to evaluate the effect of the modified LNPs on the expression of the cargo in bone marrow hematopoietic stem and / or progenitor cells (HSPCs) in vivo. As a negative control, the same LNPs were prepared using an antibody against a cell surface marker for T cells (anti-CD5). The model used was C57Bl / 6 mice.

[0265] In particular, the following eGFP mRNA formulations (reported in mol%) containing nMC3 ionizable lipid (WO2022 / 246571), DSPC, cholesterol, PEG lipid, and antibody-conjugated lipid were compared in this example: [Table 3]

[0266] The gating scheme was as follows: [Table 4]

[0267] In Figures 4A and 4B, statistics were determined by two-way ANOVA compared to the LNPC (nMC3 lcLNP™) control group. In Figure 4C, statistics were determined by one-way ANOVA compared to the LNPC (nMC3 lcLNP™) control group.

[0268] The results show that formulation D (aCD117-lcLNP™), which contains an antibody against the CD117 cell surface marker in hematopoietic cells, showed higher eGFP expression compared to both Onpattro, lcLNP without antibody, and aCD5 against the T cell marker (Figure 4A-C).

[0269] This specification is intended to illustrate embodiments and examples of the present invention and is not intended to limit the scope of the invention, which is defined by the appended claims.

[0270] As used herein, the articles "a" or "an" are intended to include both the singular and the plural, unless otherwise specified.

Claims

1. A lipid nanoparticle, comprising: (i) a nucleic acid; (ii) a neutral lipid content of greater than 35 mol%; (iii) an ionized cationic lipid in a content of 5 mol% to 50 mol%; (iv) with a sterol or a derivative thereof; (v) a targeting moiety bound to a lipophilic moiety present in the lipid layer of the nanoparticle, the targeting moiety optionally being bound to the lipophilic moiety via a linker; Each mol% is a relative value to the total lipid content of the lipid nanoparticles; Optionally, the lipid nanoparticle comprises a core, the core comprising an electron-dense region and an aqueous portion, the core being at least partially surrounded by the lipid layer as visualized by cryo-electron microscopy.

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

3. 3. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle is prepared by ethanol injection, which includes a step of lowering the pH of a solution outside the nanoparticle after formation of the nanoparticle, thereby generating the 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 selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), or dipalmitoylphosphatidylcholine (DPPC).

5. The lipid nanoparticle of claim 4, wherein the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC).

6. The lipid nanoparticle according to any one of claims 1 to 5, wherein the phosphatidylcholine content is 38 mol% to 60 mol%.

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

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

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

10. The lipid nanoparticle of claim 9, wherein the phosphatidylcholine content is 46 mol% to 60 mol%.

11. The lipid nanoparticle of claim 10, wherein the phosphatidylcholine content is 48 mol% to 60 mol%.

12. The lipid nanoparticle according to 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 at less than 20 mol%.

14. The lipid nanoparticle of any one of claims 1 to 13, further comprising a hydrophilic polymer-lipid conjugate having 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 an amount of 15 mol% to 45 mol% 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 an amount of 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

17. 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, lungs, kidneys, abdominal skin, dorsal skin and / or ears compared to an otherwise identical baseline formulation that does not contain the targeting moiety and / or an Onpattro-type formulation that encapsulates the nucleic acid but measured under an otherwise identical set of conditions, and wherein the biodistribution is quantified in an animal model by detection of labeled lipid 24 hours after administration.

18. 17. 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 formulation encapsulating the nucleic acid, and biodistribution is quantified in an animal model by detection of labeled lipid 24 hours after administration.

19. 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 spleen, bone marrow, heart, lungs, kidneys, abdominal skin, dorsal skin and / or ears compared to an otherwise identical baseline formulation not containing the targeting moiety and / or an Onpattro-type formulation encapsulating the nucleic acid but measured under an otherwise identical set of conditions, and wherein the biodistribution is quantified in an animal model by detection of labeled lipid 24 hours after administration.

20. 17. 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 the targeting moiety and / or an Onpattro formulation encapsulating the nucleic acid, and biodistribution is quantified in an animal model by detection of labeled lipid 24 hours after administration.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present at less than 2 mol%.

22. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present at less than 1.8 mol%.

23. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present at less than 1.5 mol%.

24. The lipid nanoparticle of any one of claims 1 to 20, wherein the targeting moiety is present at less than 1.2 mol%.

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

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

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

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

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

30. 27. The method of claim 25 or 26, wherein the cell is a stem cell.

31. 31. The method of claim 30, wherein the stem cells are hematopoietic stem cells or hematopoietic progenitor cells.

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

33. Use of lipid nanoparticles according to any one of claims 1 to 24 for the in vivo or in vitro delivery of said nucleic acid to mammalian cells.

34. Use of lipid nanoparticles according to any one of claims 1 to 24 for the manufacture of a medicament for the in vivo or in vitro delivery of said nucleic acid to mammalian cells.

35. 1. A lipid nanoparticle encapsulating a nucleic acid and comprising at least 38 mol % of a neutral lipid, a sterol or a derivative thereof, and a targeting moiety immobilized thereon via a lipophilic moiety, wherein a linker is optionally present between the lipophilic moiety and the targeting moiety, the lipid nanoparticle comprising a core, the core comprising an electron-dense region and an aqueous portion, the core being at least partially surrounded by a lipid layer as visualized by cryo-electron microscopy.

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