Lipid nanoparticle formulations for antisense oligonucleotide delivery

Abstract

The lipid nanoparticles provided herein comprise encapsulated oligonucleotide molecules, which may be single-stranded or double-stranded and have a length of 5 to 500 nucleotides, a neutral lipid content of 20 to 70 mol% relative to the total lipid present in the lipid nanoparticle, an ionizable lipid; a sterol; and, optionally, a hydrophilic polymer-lipid conjugate.

Description

[Technical Field]

[0001] The present disclosure relates to lipid nanoparticle formulations for delivery of oligonucleotide cargo. [Background technology]

[0002] (background) Lipid nanoparticle (LNP) formulations represent a revolution in the field of nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro™, 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 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 essential for the in vitro and in vivo activity of LNP systems, and therefore most research in this area has focused on improving 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 lipids, which promotes fusion with anionic lipids 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, PEG-lipids are well known to improve the circulation lifetime of LNPs, and cholesterol is well known to function as a particle stabiliser, but in general, relatively little attention has been paid to the neutral lipids beyond their structural role.

[0006] Although significant advances have been made in LNP-mediated siRNA delivery, it is widely known that the Onpattro™ four-component LNP formulation accumulates primarily in hepatic (liver) tissue. The ability of LNPs to accumulate in organs and tissues other than the liver would greatly expand the clinical utility of these delivery systems.

[0007] 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 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, 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.

[0008] Thus, there is a need in the art for improved in vivo delivery of nucleic acids, such as RNA, using lipid nanoparticles. (overview)

[0009] In certain advantageous embodiments, the present disclosure provides lipid nanoparticles (LNPs) encapsulating oligonucleotides (herein "oligo-LNPs"). Such lipid components include ionizable lipids, neutral lipids such as phospholipids, sterols (e.g., cholesterol), and, optionally, hydrophilic polymer-lipid conjugates. In particular, the neutral lipids are present at a higher content than in conventional LNPs, e.g., at least 20 mol%, at least 30 mol%, or at least 40 mol% (relative to the total lipid content of the LNP). Such novel oligo-LNP compositions exhibit surprisingly improved biodistribution in tissues and / or organs other than the liver. The ability of the LNPs of the present invention to accumulate in extrahepatic organs and tissues significantly expands the clinical utility of the delivery system.

[0010] Furthermore, the inventors have discovered that in certain examples of the present disclosure, significant improvements in the biodistribution of oligonucleotides, such as siRNA LNPs, can be achieved by adjusting the nitrogen-to-phosphate (N / P) molar charge ratio or nucleic acid weight / micromoles of total lipid of the LNPs herein. In some embodiments, the N / P of the lipid nanoparticles can be 4-15 or 4-10.

[0011] According to one aspect of the present disclosure, there is provided a lipid nanoparticle comprising: (i) an oligonucleotide molecule, the oligonucleotide molecule being single-stranded or double-stranded and having a length of 5 to 500 nucleotides; (ii) a neutral lipid at a content of 30 mol% to 70 mol%; (iii) an ionizable lipid at a content of 5 mol% to 50 mol%; (iv) a sterol selected from cholesterol or its derivatives; and (v) optionally, a hydrophilic polymer-lipid conjugate present at a lipid content of 0.5 mol% to 5 mol%; wherein each lipid content is a relative value relative to the total lipid content of the lipid nanoparticle, and optionally, the phosphatidylcholine lipid content is not egg phosphatidylcholine (EPC).

[0012] According to a further aspect of the present disclosure, there is provided a lipid nanoparticle comprising an encapsulated oligonucleotide molecule, wherein the oligonucleotide molecule is single-stranded or double-stranded and has a length of 5 to 500 nucleotides; a neutral lipid content of 20 to 70 mol% relative to the total lipid present in the lipid nanoparticle, an ionizable lipid; a sterol; and optionally a hydrophilic polymer-lipid conjugate, wherein the lipid nanoparticle exhibits at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin, and / or ear compared to a baseline Onpattro-type formulation of 50 / 10 / 38.5 / 1.5 mol:mol of MC3 / DSPC / cholesterol / PEG lipids encapsulating the oligonucleotide molecule, measured under otherwise identical conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration, and wherein the neutral content includes no more than three types of phosphatidylcholine lipids.

[0013] In another aspect of the present disclosure, there is provided a lipid nanoparticle comprising: (i) oligonucleotide molecules, the oligonucleotide molecules being single-stranded or double-stranded and having a length of 5 to 500 nucleotides; (ii) a neutral lipid at a content of 30 mol% to 70 mol%; (iii) an ionizable lipid at a content of 5 mol% to 50 mol%; (iv) a sterol selected from cholesterol or its derivatives; and (v) a hydrophilic polymer-lipid conjugate present at a lipid content of 0 mol% to 5 mol%; wherein each lipid content is relative to the total lipid content of the lipid nanoparticle, and the lipid nanoparticle comprises a heterogeneous core, the core being at least partially surrounded by an aqueous portion, and the aqueous portion being surrounded by an outer bilayer as visualized by cryo-electron microscopy.

[0014] In one embodiment of any one of the above aspects, the neutral lipid is a phosphatidylcholine lipid that is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), or dipalmitoylphosphatidylcholine (DPPC). In another embodiment, the phosphatidylcholine lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC).

[0015] In another embodiment, the neutral lipid content is 40 mol% to 60 mol%, or 42 mol% to 60 mol%, or 45 mol% to 60 mol%, or 46 mol% to 60 mol%, or 48 mol% to 60 mol%.

[0016] In another embodiment, the ionizable lipid is an amino lipid.

[0017] In a further embodiment, the ionizable lipid is present at less than 40 mol %.

[0018] In another embodiment, the hydrophilic polymer lipid conjugate is a polyethylene glycol lipid conjugate.

[0019] According to a further embodiment, the sterol is present at 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle. In a further embodiment, the sterol is present at 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

[0020] In another embodiment, 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 a baseline Onpattro-type formulation of 50 / 10 / 38.5 / 1.5 mol:mol of MC3 / DSPC / cholesterol / PEG lipids encapsulating oligonucleotide molecules, measured under otherwise identical conditions, and biodistribution quantified in an animal model by detection of labeled lipid 24 hours after administration.

[0021] In a further embodiment, the oligonucleotide is a single-stranded antisense oligonucleotide having a length of 30 to 300 nucleotides.

[0022] In another embodiment, the oligonucleotide is an siRNA.

[0023] In a further aspect, there is provided a method of delivering an oligonucleotide molecule to a mammalian subject in vivo, wherein the oligonucleotide molecule is single-stranded or double-stranded and is between 5 and 500 nucleotides in length, the method comprising administering to the mammalian subject a lipid nanoparticle of any one of the foregoing aspects of the present disclosure or embodiments thereof.

[0024] The present disclosure also provides, in one aspect, a method for delivering an oligonucleotide molecule to a cell, the method comprising contacting a lipid nanoparticle of any one of the preceding aspects or embodiments with a cell in vivo or in vitro.

[0025] In another embodiment, the oligonucleotide is an antisense oligonucleotide molecule that accumulates in the spleen, bone marrow, heart, lungs and / or kidneys of said subject for at least one day after administration.

[0026] In still further embodiments of the above embodiments, the lipid nanoparticles are used to treat a disease or disorder that is an autoimmune disease.

[0027] In another embodiment, the disease or disorder is an infectious disease.

[0028] In still a further embodiment, the disease or disorder is cancer.

[0029] In a further aspect of the present disclosure, there is provided a lipid nanoparticle of any one of the preceding aspects or embodiments thereof for delivery of an oligonucleotide molecule to a mammalian cell in vivo or in vitro.

[0030] According to a further aspect of the present disclosure, there is provided a use of a lipid nanoparticle of any one of its aspects or embodiments for the manufacture of a medicament for in vivo or in vitro delivery of an oligonucleotide molecule to a mammalian cell. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows bar graphs depicting the size (nm), polydispersity index (PDI), and encapsulation efficiency of firefly luciferase siRNA in four-component lipid nanoparticles (LNPs) designated B1-B17, which contain the ionizable lipid MC3 or sulfur-containing ionizable amino lipid (MF019 described herein), various amounts of DSPC ranging from 10 mol% to 55 mol%, cholesterol from 18.9 mol% to 38.5 mol%, and PEG2000-DMG at 1.5 mol%. The mol% and nitrogen / phosphate ratio (N / P) of each lipid component in the LNPs designated B1-B17 are shown in Table 1.

[0032] [Figure 2A]Images of mice treated with 40 mol% DSPC-containing LNPs (B10 LNPs in Table 1) containing MF019 ionizable lipid / DSPC / cholesterol / PEG2000-DMG at 33 / 40 / 25.5 / 1.5 ratios, encapsulating firefly luciferase siRNA and labeled with a DiD lipophilic fluorescent marker, with an N / P ratio of 6. The upper panel shows the mouse before dissection; the middle panel shows the peritoneal cavity of a mouse with intact organs; and the lower panel shows the fluorescence of the kidney (K), spleen (S), lung (Lu), and liver (Li) excised from the mouse.

[0033] [Figure 2B] Images of mice treated with 50 mol% DSPC-containing LNPs (B12 LNPs in Table 1) containing MF019 ionizable lipid / DSPC / cholesterol / PEG2000-DMG at a ratio of 27.4 / 50 / 21.1 / 1.5, encapsulating firefly luciferase siRNA and labeled with a DiD fluorescent marker, with an N / P ratio of 6. The upper panel shows the mouse before dissection; the middle panel shows the peritoneal cavity of a mouse with intact organs; and the lower panel shows the fluorescence of the kidney (K), spleen (S), lung (Lu), and liver (Li) excised from the mouse.

[0034] [Figure 2C] Images of mice treated with 40 mol% DSPC-containing LNPs (B6 LNPs in Table 1) containing MF019 ionizable lipid / DSPC / cholesterol / PEG2000-DMG at a ratio of 33 / 40 / 25.5 / 1.5, encapsulating firefly luciferase siRNA and labeled with a DiD marker, with an N / P ratio of 3. The upper panel shows the mouse before dissection; the middle panel shows the peritoneal cavity of a mouse with intact organs; and the lower panel shows the fluorescence of the kidney (K), spleen (S), lung (Lu), and liver (Li) excised from the mouse.

[0035] [Figure 3]Figure 3 is a bar graph showing the size (nm), polydispersity index (PDI), and encapsulation efficiency of firefly luciferase siRNA in four-component lipid nanoparticles (LNPs) designated A1-A16, which contain the ionizable lipid MC3 or sulfur-containing ionizable amino lipid (MF019 described herein), various amounts of egg sphingomyelin (ESM) ranging from 10 mol% to 55 mol%, cholesterol from 18.9 mol% to 38.5 mol%, and 1.5 mol% PEG2000-DMG. The mol% and nitrogen / phosphate ratio (N / P) of each lipid component in the LNPs, labeled A1-A16, are shown in Table 3.

[0036] [Figure 4A] Plots showing fluorescence intensity per mg of liver homogenate from mice treated with phosphate-buffered saline (PBS) control; and firefly luciferase siRNA-containing LNPs with MF019 / ESM / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B) with an N / P of 6; MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D) with an N / P of 6; and firefly luciferase siRNA-containing LNPs with MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-E) with an N / P of 3. LNPs were labeled with the DiD fluorescent marker.

[0037] [Figure 4B]Plots showing fluorescence intensity per mg of spleen homogenate from mice treated with phosphate-buffered saline (PBS) control; and firefly luciferase siRNA-containing LNPs with MF019 / ESM / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B) with an N / P of 6; MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D) with an N / P of 6; and firefly luciferase siRNA-containing LNPs with MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-E) with an N / P of 3. LNPs were labeled with the DiD fluorescent marker.

[0038] [Figure 4C] Plots showing fluorescence intensity per mg of abdominal skin homogenate from mice treated with phosphate-buffered saline (PBS) control; and firefly luciferase siRNA-containing LNPs with MF019 / ESM / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B) with an N / P of 6; MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D) with an N / P of 6; and firefly luciferase siRNA-containing LNPs with MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-E) with an N / P of 3. LNPs were labeled with the DiD fluorescent marker.

[0039] [Figure 4D]Plots showing fluorescence intensity per mg of dorsal skin homogenate from mice treated with phosphate-buffered saline (PBS) control; and firefly luciferase siRNA-containing LNPs with MF019 / ESM / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B) with an N / P of 6; MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D) with an N / P of 6; and firefly luciferase siRNA-containing LNPs with MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-E) with an N / P of 3. LNPs were labeled with the DiD fluorescent marker.

[0040] [Figure 4E] Plots showing fluorescence intensity per mg of ear homogenate from mice treated with phosphate-buffered saline (PBS) control; and firefly luciferase siRNA-containing LNPs with MF019 / ESM / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B) with an N / P of 6; MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D) with an N / P of 6; and firefly luciferase siRNA-containing LNPs with MF019 / DSPC / cholesterol / PEG2000-DMG molar ratios of 33 / 40 / 25.5 / 1.5 (LNP-E) with an N / P of 3. LNPs were labeled with the DiD fluorescent marker.

[0041] [Figure 5]In vivo fluorescence of DiD in the liver after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type baseline composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro™ and high-DSPC compositions are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0042] [Figure 6] In vivo fluorescence of DiD in the spleen after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type baseline composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro-type baseline and high-DSPC formulations have ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD ratios of 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol, respectively.

[0043] [Figure 7]In vivo fluorescence of DiD in bone marrow (BM) after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro-type baseline and high-DSPC compositions are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0044] [Figure 8] In vivo fluorescence of DiD in the heart after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type baseline composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro-type baseline and high-DSPC compositions are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0045] [Figure 9]In vivo fluorescence of DiD in the lung after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type baseline composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro-type baseline and high-DSPC compositions are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0046] [Figure 10] In vivo fluorescence of DiD in the kidney after treatment with ssRNA encapsulated in DiD-labeled LNPs as a function of DSPC content. LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C have an Onpattro-type baseline composition with 10 mol% DSPC content, while LNPs B and D have a 50 mol% DSPC composition. The Onpattro-type baseline and high-DSPC compositions are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0047] [Figure 11A] Cryo-TEM image of siRNA-containing LNPs encapsulating firefly luciferase siRNA and containing MF019 ionizable lipid / DSPC / cholesterol / PEG2000-DMG at 33 / 40 / 25.5 / 1.5 with an N / P of 6.

[0048] [Figure 11B]Cryo-TEM image of siRNA-containing LNPs encapsulating firefly luciferase siRNA and containing MF019 ionizable lipid / DSPC / cholesterol / PEG2000-DMG at 27.4 / 50 / 21.1 / 1.5 with an N / P of 6.

[0049] [Figure 11C] Cryo-TEM image of siRNA-containing LNPs encapsulating firefly luciferase siRNA and containing MF019 ionizable lipid / ESM / cholesterol / PEG2000-DMG at 27.4 / 50 / 21.1 / 1.5 with an N / P of 6. DETAILED DESCRIPTION OF THE INVENTION

[0050] (Detailed explanation) The lipid nanoparticles described herein provide improved delivery of oligonucleotide cargo compared to conventional four-component LNPs, referred to herein as Onpattro-type LNPs. In particular, the LNPs comprise an ionizable lipid, a neutral lipid, such as a phosphatidylcholine lipid (e.g., DSPC), a sterol, and optionally a hydrophilic polymer-lipid conjugate, wherein the neutral lipid (e.g., phosphatidylcholine lipid) is present at a mol% of at least 20 mol%, at least 25 mol%, or at least 30 mol%, and the ionizable lipid, in some embodiments, is present at less than 40 mol%. As described herein, the inclusion of a neutral lipid, such as phosphatidylcholine, at a mol% higher than that used in conventional nucleic acid delivery formulations allows for selective delivery to extrahepatic tissues compared to Onpattro-type baselines.

[0051] An "oligonucleotide" or "oligonucleotide cargo" is a single- or double-stranded RNA or DNA molecule, 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 shorter double-stranded silencing RNA molecules (siRNAs), 3 to 40 nucleotides in length. neutral lipid

[0052] Neutral lipids are amphipathic lipids that allow for particle formation and generally have no net charge at physiological pH. The term includes zwitterionic lipids, examples of which include phospholipids.

[0053] 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). The phosphatidylcholine lipid component can comprise a mixture of two or more different neutral lipids.

[0054] In one embodiment, the phosphatidylcholine lipid is selected from DSPC, POPC, and mixtures thereof.

[0055] 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.

[0056] 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.

[0057] In one embodiment, the neutral lipid is most advantageously DSPC. In some embodiments of the present disclosure, increased DSPC lipid content improves the biodistribution of LNPs over other neutral phospholipids. In certain embodiments, the DSPC 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.

[0058] In some embodiments, the phosphatidylcholine lipid is a sphingolipid.As used herein, the term "sphingolipid" refers to the lipid that comprises sphingosine backbone and is suitable for incorporation into the LNP herein.Sphingolipids include but are not limited to ceramide, sphingomyelin, cerebroside, ganglioside, or their derivatives, such as their reduced analogs that have no double bond in the sphingosine unit.Sphingolipids have phosphocholine head group and include sphingomyelin.

[0059] In certain embodiments, the sphingolipid 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 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 lipids present in the lipid nanoparticles.

[0060] 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 the LNPs have no detectable sphingomyelin (less than 0.5 mol%), or the LNPs are substantially sphingomyelin-free, meaning that the LNPs have less than 5 mol% or less than 2.5 mol% sphingomyelin.

[0061] 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 lipids are present 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.

[0062] 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 in some embodiments. Thus, in some embodiments, the fusogenic lipid content of the lipid nanoparticles is less than 5 mol%, less than 4 mol%, less than 3 mol%, less than 2 mol%, less than 1 mol%, less than 0.75 mol%, or less than 0.5 mol%. In 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%.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] In some embodiments, the phosphatidylcholine lipid is a phosphatidylcholine-sterol conjugate, such as SPC-cholesterol, OPC-cholesterol, or PPC-cholesterol conjugate.Further phospholipid-sterol conjugates are described in US2011 / 0177156, which is incorporated herein by reference.Examples of suitable phospholipids that are SPC-cholesterol conjugates are described below. [ka] .

[0067] In some embodiments, the transition temperature of the neutral lipid or mixture thereof 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. In one embodiment, the phase transition temperature of the neutral lipid or mixture thereof when incorporated into a lipid nanoparticle is at least 38° C., 39° C., or 40° C.

[0068] The neutral lipid content is determined based on the total amount of lipid (mol:mol) in the lipid nanoparticles containing sterol. Ionized lipids

[0069] The LNPs of the present disclosure have ionizable cationic lipids and include one or a combination of two or more such lipids.

[0070] As used herein, the term "ionizable cationic lipid" refers to a lipid that is electrostatically neutral at a particular pH, such as physiological pH, and that accepts a proton to become electrostatically positively charged at a pH below its pKa. In the electrostatically neutral form, the calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) is greater than 8. In some embodiments, the cationic lipid, when formulated into an LNP, has a pKa of 5.0-7.5 or 6.0-7.5.

[0071] Thus, ionized lipids may be charged at low pH and have virtually no net charge at physiological pH. This allows electrostatic interactions between the lipid and the negatively charged nucleic acid cargo during initial formulation. Because ionized lipids are near-neutral at physiological pH, they reduce 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 anionic lipids in the endosomal membrane, subsequently destabilizing the membrane and releasing the nucleic acid-based therapeutic into the cytoplasm where it exerts its effects.

[0072] In some embodiments, the apparent pKa of the LNPs is between 5.0 and 7.5, 6.5 and 7.5, or 6.8 and 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 em Fluorescence 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.

[0073] In some embodiments, it is desirable to include less than 50 mol% of ionizable lipids in the LNP. That is, the ionizable lipid content, as measured based on the total lipid content of the LNP, 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%. In certain embodiments, the lower limit of the ionizable lipid content can be greater than 5 mol%, greater than 8 mol%, greater than 10 mol%, greater than 12 mol%, greater than 14 mol%, greater than 15 mol%, greater than 16 mol%, greater than 18 mol%, greater than 20 mol%, greater than 25 mol%, or greater than 30 mol%. Any one of the upper limit values ​​can be combined with any one of the lower limit values ​​to obtain a suitable ionizable lipid content in the LNP.

[0074] 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.

[0075] In some embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) head group and two C16-C18 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 may 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 WO2022 / 246555; WO2022 / 246568; WO2022 / 246571; WO2023 / 147657; WO2022 / 155728; PCT / CA2023 / 050644, filed May 11, 2023; and PCT / CA2023 / 0, filed September 27, 2023, each of which is incorporated herein by reference. No. 51272; PCT / CA2023 / 051273, filed September 27, 2023; 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.

[0076] In some embodiments, it is desirable to include less than 50 mol% ionizable cationic lipids in the LNPs, i.e., the ionizable cationic 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%.

[0077] In certain embodiments, the ionized 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.

[0078] In certain embodiments, the ionized amino cationic lipid content is between 15 mol% and 40 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] 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

[0080] In some embodiments, the LNP further comprises a sterol. The term "sterol" refers to a natural or synthetic compound having a gonane backbone with a hydroxyl moiety attached to one of its rings (usually the A ring).

[0081] Examples of sterols include cholesterol, or cholesterol derivatives, the latter of which refers to a cholesterol molecule with a gonane structure and one or more additional functional groups.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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. Hydrophilic polymer lipid conjugates

[0087] In one embodiment, the lipid nanoparticle comprises a hydrophilic polymer-lipid conjugate that can be incorporated into LNP. In some examples, the conjugate comprises a vesicle-forming lipid having a polar head group and a hydrophilic polymer chain covalently attached to the head group. The lipid comprises any moiety having at least a hydrophobic portion. 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.

[0088] 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. In certain embodiments, the hydrophilic polymer lipid conjugate may be present in the nanoparticles 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.

[0089] 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.

[0090] 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.

[0091] In such embodiments, the lipid portion of the hydrophilic polymer-lipid conjugate typically has an acyl chain length of less than 18 and zero to two double bonds in one or both of the acyl 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).

[0092] In one embodiment, the hydrophilic polymer-lipid conjugate is absent or present at low concentrations in the lipid nanoparticles. In some embodiments, the hydrophilic polymer-lipid conjugate 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%.

[0093] 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%.

[0094] 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). Optional Additional Lipid Ingredients

[0095] In some embodiments, lipid nanoparticles for extrahepatic delivery comprise lipids "consisting essentially of" neutral amphipathic lipids, such as phosphocholine, sterols or their derivatives, ionizable cationic lipids, and, optionally, hydrophilic polymer-lipid conjugates, with less than 10 mol% of additional lipid components other than the aforementioned lipid components. That is, in some examples, the additional lipid components are present at 0-20 mol%, 0-15 mol%, 0-10 mol%, 0-8 mol%, or 0-5 mol%, and include any one of a number of charged and / or uncharged lipids. In some embodiments, the additional lipid components consist of one or more neutral lipids rather than charged lipids at physiological pH. Avoiding such charged lipids reduces uptake by the RES system and ensures that the circulatory life of the particles is not impaired. Examples of such optional additional lipids include triacylglycerides, diacylglycerides, monoacylglycerides, zwitterionic lipids, antioxidants, and vitamins.

[0096] Other exemplary phospholipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylserine (DSPS), and sphingomyelins. Examples of sphingomyelins in this context include ceramides, sphingomyelins, cerebrosides, gangliosides, or reduced analogs thereof, in which the sphingosine unit lacks a double bond.

[0097] In embodiments in which an anionic lipid is included in the formulation, the anionic lipid can be a lipid having a hydroxyl head group, including, but not limited to, anionic lipids such as DOPG and A-0001, which are described in U.S. Provisional Patent Application No. 63 / 453,766, filed March 22, 2023 (incorporated herein by reference).

[0098] Further additional components include a hydrophobic moiety, such as a lipid, conjugated to a targeting ligand. The ligand may be a peptide, polypeptide, or protein, including an antibody or a fragment thereof. In one embodiment, the ligand may be a single-chain antibody fragment. The targeting ligand may be used to target a receptor on a cell in vivo. In some embodiments, the targeting ligand may be conjugated to the phospholipid component of the LNP. When a certain amount of hydrophilic polymer-lipid conjugate is present, the targeting ligand may be conjugated to its distal end. In such embodiments, the phospholipid-targeting ligand conjugate is present at less than 10 mol%, less than 5 mol%, or less than 3 mol%. In some embodiments, the absence of a targeting ligand is most advantageous.

[0099] In one embodiment, the lipid nanoparticles contain low (less than 5 mol%) or no permanently charged cationic lipids. In some disclosed examples, the lipid nanoparticles have no net charge at physiological pH, promoting extended circulatory life and thereby facilitating extrahepatic delivery to cells. Examples of permanently charged cationic lipids to avoid include dioctadecyldimethylammonium bromide (DDAB) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Such permanently charged cationic lipids may also impart toxicity to the lipid nanoparticles. Nanoparticle preparation and morphology

[0100] The lipid nanoparticles that incorporate oligonucleotide can be prepared by various suitable methods, such as rapid mixing / ethanol dilution process.The example of preparation method is 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.

[0101] Without being bound by theory, we hypothesize that the mechanism by which lipid nanoparticles containing encapsulated oligonucleotides can be formed using the rapid mixing / ethanol dilution process begins with the formation of a dense region of a hydrophobic RNA-ionized lipid core at low pH (e.g., pH 4) surrounded by a monolayer of neutral lipids / cholesterol that fuses 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 inner hydrophobic core. If the neutral lipid content is sufficiently high, the outer bilayer, which favors neutral lipids, can form a complete bilayer around the inner, confined volume.

[0102] LNPs may contain a "core" region. The core is considered heterogeneous in that it contains electron-dense regions and aqueous regions that surround at least a portion of any electron-dense regions. Electron-dense regions are visualized by cryo-electron microscopy using the procedures described in the "Materials and Methods" section of this specification. As observed, but not limited to, by cryo-TEM, the electron-dense regions in the core may be partially surrounded by aqueous regions within a confined space. The aqueous regions form distinct aqueous compartments within the lipid nanoparticle. In other words, the aqueous regions are not simply hydrated layers.

[0103] In one embodiment, at least about one-fifth of the core (trapped volume) comprises an aqueous portion, and the electron-dense regions are partially adjacent to the lipid layers comprising the bilayer, as determined qualitatively by cryo-electron microscopy. In another embodiment, at least about one-quarter of the core comprises an aqueous portion, and the electron-dense cores are each partially adjacent to the lipid layers comprising the bilayer, as determined qualitatively by cryo-electron microscopy. In a further embodiment, at least about one-third of the core comprises an aqueous portion, and the electron-dense regions are each partially adjacent to the lipid layers comprising the bilayer, as determined qualitatively by cryo-electron microscopy. In another embodiment, at least about half of the core comprises an aqueous portion, and the electron-dense cores are each partially adjacent to the lipid layers comprising the bilayer, as determined qualitatively by cryo-electron microscopy.

[0104] In one embodiment, the electron-dense regions are generally spherical, hi another embodiment, the electron-dense regions are hydrophobic.

[0105] Lipid nanoparticles of the present invention can show particularly high encapsulation efficiency of oligonucleotide.In this specification, the term "encapsulation" regarding the incorporation of oligonucleotide into lipid nanoparticles refers to any association between any lipid component or compartment (including lipophilic part or aqueous part) of lipid nanoparticles and oligonucleotide.In one embodiment, oligonucleotide is present at least in the core of LNP.

[0106] In one embodiment, the encapsulation efficiency is at least 50, 55, 60, 65, 70, 75, 80, 85, 90% or 92%. The encapsulation efficiency of oligonucleotides is determined as described in the Materials and Methods section of the Examples herein.

[0107] Lipid nanoparticles can contain a single bilayer or multiple lipid layers (i.e., multiple layers). One or more lipid layers comprising the 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 embodiment, the lipid layer is a continuous bilayer surrounding the core.

[0108] Thus, in certain embodiments, the electron-dense region of the core is separated from the lipid layer comprising the bilayer by an aqueous portion. 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 partially surrounded by an aqueous portion, and the aqueous portion is partially surrounded by a lipid layer comprising a bilayer as visualized by cryo-electron microscopy.

[0109] 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.

[0110] In one embodiment, the lipid nanoparticle is part of a lipid nanoparticle preparation, wherein at least 20% of the electron-dense region of the lipid nanoparticle is (i) enveloped by aqueous or (ii) partially surrounded by aqueous and adjacent to a lipid layer, as visualized by cryo-electron microscopy in a plane around the electron-dense region.

[0111] 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.

[0112] 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 surrounded or enveloped by an adjacent aqueous space disposed between the lipid layer (e.g., bilayer) and the electron-dense region, as visualized by cryo-electron microscopy.

[0113] LNPs can be visualized by cryo-TEM, as described in the Examples section below.

[0114] In another embodiment, the polydispersity index (PDI) of the LNP preparation is less than 0.3, 0.25, 0.2, 0.15, 0.12, or 0.10.

[0115] In another embodiment, the particle size distribution is such that 90% of the particles in an LNP preparation of the present disclosure have a diameter between 40 nm and 200 nm, between 40 nm and 150 nm, between 40 nm and 140 nm, between 45 nm and 150 nm, between 50 nm and 120 nm, or between 50 nm and 140 nm. In some embodiments, the LNPs herein have a PDI of less than 0.25, less than 0.20, less than 0.18, less than 0.16, less than 0.15, or less than 0.14.

[0116] 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. This can be mathematically expressed as N / P. In one embodiment, the lipid nanoparticles have an N / P ratio of 4-15, or 4.5-10, or 5-10, or 5.5-8.

[0117] In one embodiment, the lipid nanoparticles have an N / P ratio of at least 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 upper and lower limits. N / P is the charge ratio calculated as described in Materials and Methods.

[0118] 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. Oligonucleotide cargo

[0119] Oligonucleotide cargoes include interfering RNA and antisense oligonucleotides, which are described in more detail below. Interfering RNA

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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)

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] In general, ASOs may contain "mismatch motifs" or "mismatch regions," which refer to portions of an 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 may comprise 2, 3, 4, 5, or more nucleotides.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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).

[0141] 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. Improved biodistribution of oligo-LNPs by increasing neutral lipid content

[0142] As described in the Examples section, oligo-LNPs of the present disclosure having elevated neutral lipid content may provide improved biodistribution to a wider range of tissues and / or organs than Onpattro-type baseline formulations as described in the Examples section herein. In further embodiments, the baseline formulation may 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 ratio that is 0.20:1 less than the micromoles of nucleic acid by weight / total lipid ratio 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.

[0143] 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, as described in Materials and Methods) is used to assess the biodistribution of oligo-LNPs in predetermined tissues or organs relative to a baseline. Animal studies are used as a surrogate for assessing biodistribution in subjects, such as patients, in a clinical setting.

[0144] 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.

[0145] 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.

[0146] The oligo-LNPs being compared were prepared using identical materials and methods. In other words, the two formulations contained the same ionizable lipids, PEG-lipids, and sterols and were prepared using the rapid ethanol injection method as described in Materials and Methods.

[0147] 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).

[0148] 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

[0149] In some embodiments, the oligo-LNPs are part of a pharmaceutical composition and are administered to treat and / or prevent a disease state. Treatment can have a preventative, ameliorative, or therapeutic effect. The pharmaceutical composition is administered in any suitable dose.

[0150] The oligo-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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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. (Example) Materials and Methods Preparation of oligonucleotide-containing lipid nanoparticles

[0159] LNPs were prepared by dissolving siRNA firefly luciferase or scrambled antisense oligonucleotides (ASOs) in 25 mM sodium acetate, pH 4.0, while dissolving the indicated mole percent lipid components in absolute ethanol. The lipophilic fluorescent dye DiD perchlorate (2-[5-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1,3-pentadien-1-yl]-3,3-dimethyl-1-octadecyl-3H-indolium perchlorate) was included in the LNPs to assess their biodistribution in vivo. Lipids in ethanol and siRNA or ASOs in buffer were mixed in a 1:3 volumetric ratio using a dual-syringe T-junction. The solution was pumped 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 siRNA- or ASO-containing syringe). Subsequently, to remove ethanol from the formulation, the mixture was dialyzed overnight against approximately 400–1000 volumes of 1× phosphate-buffered saline (pH 7.4) using a Spectro / Por dialysis membrane (molecular weight cutoff 12,000–14,000 Da). Biophysical characterization

[0160] Analysis of lipid nanoparticle size and morphology: LNP size (number-weighted) and polydispersity index (PDI) were determined by dynamic light scattering (DLS) using a Malvern Zetasizer NanoZS™ particle sizer (Worcestershire, UK). Analysis of lipid nanoparticle siRNA or ASO encapsulation efficiency

[0161] The encapsulation efficiency of siRNA or ASO was determined using the Quant-iTRiboGreen™ RNA assay (LifeTechnologies™, Burlington, ON). Briefly, LNP-siRNA or LNP-ASO was incubated at 37°C for 10 minutes in the presence or absence of 1% Triton X-100 (Sigma-Aldrich™, St. Louis, MO), after which RiboGreen™ reagent was added. Fluorescence intensity (Ex / Em: 480 / 520 nm) was measured; Triton X-100-treated samples represent total siRNA or ASO, while untreated samples represent unencapsulated siRNA or ASO. Measurement of N / P ratio of lipid nanoparticles

[0162] The total number of negative charges (P) is first calculated using the average molecular weight of the nucleic acid base pairs (approximately 650 g / mol / bp), the size of the nucleic acid construct in bp, and the number of negative charges (2 / bp). Multiplying this value in mol*negative charges by Avogadro's constant, 6.022E23 / mol, gives the total number of negative charges.

[0163] Similarly, the total number of positive charges on the ionizable lipid can be calculated by first using the molecular weight and the amount of ionizable lipid to determine the number of moles of ionizable lipid used in the formulation. Multiplying this value by the number of positive charges per molecule (1) gives the moles*positive charges, which, when multiplied by Avogadro's constant 6.022E23 / mol, gives the total number of positive charges. The N / P ratio is calculated by taking the ratio of the positive charges on the ionizable lipid to the negative charges on the nucleic acid. Measurement of fluorescence in intact organs / tissues in vivo

[0164] CD-1 mice were intravenously injected with LNP at a concentration of 1.0 mg / kg siRNA, calculated as follows: mouse body weight (grams) x 10 μL. Twenty-four hours after injection, the mice were anesthetized with 5% isofluorane (air flow rate set at 1%), followed by CO2 asphyxiation until the animals lost their reflexes. Blood was collected via cardiac puncture using a 1 mL needle. The animals were then imaged using an In Vivo Imaging System (IVIS™) manufactured by PerkinElmer™.

[0165] After imaging, the animals were cut from the bladder to the rib cage and the skin was pinned down. The spleen (S), lungs (Lu), liver (Li), and kidneys (K) were removed from the abdominal cavity and placed in a plastic dish. The animals with the organs intact were imaged using an IVIS™ imager, after which the organs were removed from the mice and placed in the plastic dish. Tissue homogenate assay

[0166] 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.

[0167] For bone marrow tissue homogenates, the femurs are removed from the mice. The femurs are incised and centrifuged to separate the bone marrow. The bone marrow is then processed in the same way as the tissues described above to prepare homogenates and measure fluorescence. Cryo-TEM

[0168] Prior to cryo-TEM imaging, LNPs were 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, was applied to glow-discharged copper grids and quenched using an FEI Mark IV Vitrobot to generate vitreous ice. These grids were stored in liquid nitrogen until imaging with an FEI Titan Krios or FEI Glacios TEM. The instrument was operated at 200 kV under low-dose conditions, and images were acquired using a bottom-mounted FEI Falcon direct electron detector camera at 47–88,000X magnification with an underfocus of 0.5–2 μm for contrast enhancement. Example 1: Increasing the level of neutral lipids (e.g., DSPC) and decreasing the level of ionized lipids in siRNA-LNPs does not adversely affect biophysical properties

[0169] At the expense of ionizable lipids and cholesterol, siRNA-LNPs (firefly luciferase) with various DSPC contents (10 mol% to 55 mol%) were prepared and their biophysical properties were evaluated. In particular, the following ionizable lipids (MC3 or MF019 as indicated); DSPC; and PEG-lipid (PEG 2000 Four-component siRNA-LNP formulations (reported in mol%) containing cholesterol, with α-DMG held constant at 1.5 mol%, were evaluated for encapsulation efficiency, size, and polydispersity index (PDI) according to Materials and Methods. The N / P of each LNP was 3 or 6, as shown in Table 1. [Table 1] *MF019 is an ionized sulfur lipid described in PCT Patent Application No. PCT / CA2022 / 050042, which is incorporated herein by reference; MC3 refers to DLin-MC3-DMA ionized lipid (Dilinoleyl-methyl-4-dimethylaminobutyrate).

[0170] For each formulation in Table 1 above, the size, polydispersity index (PDI), and encapsulation rate are shown in Figure 1.

[0171] We found that each formulation exhibited acceptable biophysical properties despite increasing levels of DSPC and decreasing ionizable lipid content. While one skilled in the art would expect that a decrease in ionizable lipid would result in a decrease in encapsulation efficiency of siRNA, the results in Figure 1 surprisingly show that encapsulation efficiency remained relatively constant across the formulations tested, even with a decrease in ionizable lipid content (and a concomitant increase in neutral lipid content). Example 2: Increasing the level of neutral lipids (e.g., DSPC) in siRNA-LNPs significantly improves in vivo biodistribution

[0172] The effect of increasing the amount of DSPC from 40 mol% to 50 mol% in four-component LNPs containing an siRNA cargo with an N / P of 6 was evaluated in vivo.

[0173] Formulations B10, B12 and B6 from Table 1 above were selected for in vivo biodistribution studies in CD-1 mice. These formulations are summarized in Table 2 below: [Table 2] *MF019 is an ionizable lipid described in PCT / CA2022 / 050042 (ibid.).

[0174] As seen in Figures 2A, 2B, and 2C, siRNA-LNPs with 50 mol% DSPC exhibited significant fluorescence intensity in both the liver and spleen, as determined by detection of the lipophilic fluorescent dye DiD perchlorate 2-[5-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1,3-pentadien-1-yl]-3,3-dimethyl-1-octadecyl-3H-indolium perchlorate, 24 h after injection. In contrast, LNPs containing only 40 mol% DSPC exhibited fluorescence in the liver but minimal signal in the spleen. Furthermore, LNPs with an N / P ratio of 3 (at 40 mol% DSPC) showed little detectable fluorescence in any of the organs examined. Example 3: Increasing the Level of Sphingomyelin and Decreasing the Level of Ionized Lipid in siRNA-LNPs Does Not Adversely Affect Biophysical Properties

[0175] We prepared siRNA-LNPs with various sphingomyelin contents ranging from 10 mol% to 55 mol%, and evaluated their biophysical properties. LNPs were also prepared using PEG-lipids (PEG 2000 The sphingomyelin content was increased at the expense of both cholesterol and ionizable lipids, as shown in Table 3, and the N / P ratio was either 3 or 6. [Table 3] *MF019 is an ionizable sulfur lipid described in PCT / CA2022 / 050042 (ibid.), and MC3 refers to DLin-MC3-DMA ionizable lipid (dilinoleylmethyl-4-dimethylaminobutyrate).

[0176] For each formulation in Table 3 above, the size, polydispersity index (PDI), and encapsulation rate are shown in FIG.

[0177] We found that despite the increased levels of sphingomyelin and decreased ionized lipid content, each siRNA-LNP formulation generally exhibited acceptable biophysical properties. Example 4: Increasing the Level of Neutral Lipids (e.g., DSPC and Sphingomyelin) in siRNA-LNPs Improves In Vivo Biodistribution as Measured in Tissue Homogenates

[0178] We performed biodistribution studies on various four-component siRNA-LNP formulations containing 40 mol% and 50 mol% DSPC or egg sphingomyelin (ESM). Results showed that in most cases, siRNA-LNPs with 50 mol% DSPC or ESM resulted in significantly higher LNP concentrations in measured organs (tissue homogenates) compared with LNPs with 10 mol% less neutral lipid (40 mol%). The neutral lipid content (DSPC and ESM) increased at the expense of ionized lipids and cholesterol.

[0179] The results also show that siRNA-LNPs with an N / P of 3 and 40 mol% DSPC exhibited significantly reduced fluorescence intensity compared to the same formulation with an N / P of 6.

[0180] The LNPs investigated are listed in Table 4 below. [Table 4] *MF019 is an ionized sulfur lipid described in PCT / CA2022 / 050042 (ibid.).

[0181] All LNP formulations showed high encapsulation efficiency (>90%) at ionized lipid concentrations of 27.4 mol% and 33 mol%.

[0182] The half-maximal inhibitory concentration (IC 50 ) values ​​were within the acceptable range.

[0183] Tissue homogenates were prepared as described in the Materials and Methods section. Fluorescence intensity of the tissue homogenates was measured as described using the DiD fluorescent marker.

[0184] As shown in Figure 4A, the fluorescence intensity of LNPs containing 50 mol% sphingomyelin in liver homogenate increased approximately twice as much as that of LNPs containing only 40 mol% sphingomyelin (LNP-A and LNP-B). Similar increases in fluorescence were observed in LNPs containing 50 mol% DSPC and LNPs containing 40 mol% DSPC (LNP-C and LNP-D). All formulations with an N / P ratio of 6 (LNP A–D) showed increased fluorescence intensity compared to 40 mol% DSP-containing LNPs with an N / P ratio of 3 (LNP-E).

[0185] As shown in Figure 4B, the fluorescence intensity of LNPs containing 50 mol% sphingomyelin in spleen homogenate was similar to that of LNPs containing only 40 mol% sphingomyelin (LNP-A and LNP-B). However, a significant increase in fluorescence intensity was observed for LNPs containing 50 mol% DSPC compared to LNPs containing only 40 mol% DSPC (LNP-C and LNP-D). Furthermore, all formulations with an N / P ratio of 6 (LNP-A–D) showed higher fluorescence intensity compared to 40 mol% DSP-containing LNPs (LNP-E) with an N / P ratio of only 3.

[0186] Figures 4C and 4D show the biodistribution results of the siRNA-LNPs listed in Table 4 in the abdominal and dorsal skin of mice, respectively. As shown in Figure 4C, the fluorescence intensity of LNPs containing 50 mol% egg sphingomyelin (ESM) in the abdominal skin was increased more than that of LNPs containing only 40 mol% ESM (LNP-A vs. LNP-B). A significant increase in fluorescence intensity in the abdominal skin was observed for LNPs containing 50 mol% DSPC compared to LNPs containing only 40 mol% DSPC (LNP-C vs. LNP-D). All DSPC-containing formulations with an N / P ratio of 6 showed higher fluorescence intensity than the 40 mol% DSPC formulation with an N / P ratio of only 3. Similar results were observed in the dorsal skin of mice (see Figure 4D).

[0187] The results of biodistribution in the ear are shown in Figure 4E. As shown, the fluorescence intensity of siRNA-LNPs containing 50 mol% sphingomyelin in the ear was significantly increased compared to that of LNPs containing only 40 mol% sphingomyelin (LNP-A vs. LNP-B). A more than two-fold increase in fluorescence intensity in the ear was observed for LNPs containing 50 mol% DSPC compared to LNPs containing only 40 mol% DSPC (LNP-C and LNP-D). Most formulations with an N / P ratio of 6 showed higher fluorescence intensity than the 40 mol% DSPC formulation with an N / P ratio of only 3. Example 5: Four-component siRNA-LNPs with elevated phosphatidylcholine lipid levels exhibit surprising morphology

[0188] The morphology of the following LNP formulations was determined by cryo-TEM as described in the Materials and Methods section: The cargo was siRNA targeting firefly luciferase. [Table 5]

[0189] The images in Figures 11A, 11B, and 11C show that at high phosphatidylcholine content (e.g., DSPC), siRNA-LNPs exhibit a unique morphology with a core containing electron-dense regions and an aqueous portion, surrounded by a bilayer. Example 6: Four-component ASO-LNPs with high levels of phosphatidylcholine lipids show a surprising increase in extrahepatic organ delivery

[0190] In this example, we investigate the effect of increasing the content of a representative phosphatidylcholine lipid (DSPC) from 10 mol% to 50 mol% on the biodistribution of ASO-LNPs. The DSPC content was increased at the expense of ionizable lipids and cholesterol.

[0191] Lipid nanoparticles encapsulating 50-mer and 221-mer ssRNAs were prepared as described in the Methods section above. The lipid compositions of the investigated ASO-LNPs are shown in Table 6 below. [Table 6] a. The ionizable lipid was MC3 (DLin-MC3-DMA or "MC3" (dilinoleylmethyl-4-dimethylaminobutyrate)). b. The ionizable lipid is MF019, described in PCT / CA2022 / 050042, filed January 12, 2022, published as WO2022 / 155728A1, which is incorporated by reference herein.

[0192] LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C are Onpattro™ formulations with 10 mol% DSPC content, while LNPs B and D are 50 mol% DSPC formulations. The Onpattro™ and high-DSPC formulations are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipid / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0193] The ASOs were non-coding single-stranded RNAs (ssRNAs) 50 nucleotides ("50-mer") and 221 nucleotides ("221-mer") in length. The ssRNAs were randomly generated to prevent complementary binding for biodistribution experiments. The 50-mer and 221-mer were synthesized by Integrated DNA Technologies™ (IDT). The two sequences are shown in Table 7 below. [Table 7]

[0194] As shown in Figures 5-10, ASO-LNPs containing 50 mol% DSPC (LNP B and D) showed remarkable improvement in biodistribution to the spleen, bone marrow, heart, lungs, and kidneys at 24 h after administration compared to lipid nanoparticles containing 10 mol% DSPC (LNP A and C) (Figures 6-10). This remarkable improvement in biodistribution with LNPs B and D was observed for both the 50mer ASO (LNP B) and the 221mer ASO (LNP D). As shown in Figure 5, in the liver, the biodistribution of ASO-LNPs containing 50 mol% DSPC (LNP B) and 50mer ASO was improved compared to ASO-LNPs containing only 10 mol% DSPC (LNP A).

[0195] 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.

Claims

1. A lipid nanoparticle, comprising: (i) an oligonucleotide molecule, said oligonucleotide molecule being single-stranded or double-stranded and having a length of 5 to 500 nucleotides; (ii) a neutral lipid content of 30 mol% to 70 mol%; (iii) an ionizable lipid content of 5 mol% to 50 mol%; (iv) a sterol selected from cholesterol or a derivative thereof; (v) optionally a hydrophilic polymer lipid conjugate present at a lipid content of 0.5 mol % to 5 mol %; Lipid nanoparticles, wherein each lipid content is a relative value to the total lipid content of the lipid nanoparticle, with the proviso that the phosphatidylcholine lipid content is not egg phosphatidylcholine (EPC).

2. 1. A lipid nanoparticle comprising an encapsulated oligonucleotide molecule, the oligonucleotide molecule being single-stranded or double-stranded and 5-500 nucleotides in length; a neutral lipid content of 20-70 mol% relative to the total lipid present in the lipid nanoparticle, an ionizable lipid; a sterol; and optionally a hydrophilic polymer-lipid conjugate, wherein the lipid nanoparticle exhibits at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, dorsal skin and / or ear compared to a baseline Onpattro formulation of 50 / 10 / 38.5 / 1.5 mol:mol of MC3 / DSPC / cholesterol / PEG lipids encapsulating the oligonucleotide molecule, measured under otherwise identical conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipids 24 hours after administration, and wherein the neutral content includes no more than three types of phosphatidylcholine lipids.

3. A lipid nanoparticle, comprising: (i) an oligonucleotide molecule, said oligonucleotide molecule being single-stranded or double-stranded and having a length of 5 to 500 nucleotides; (ii) a phosphatidylcholine lipid content of 30 mol% to 70 mol%; (iii) an ionizable lipid content of 5 mol% to 50 mol%; (iv) a sterol selected from cholesterol or a derivative thereof; (v) a hydrophilic polymer lipid conjugate present at a lipid content of 0 mol% to 5 mol%; Each lipid content is a relative value to the total lipid content of the lipid nanoparticle; The lipid nanoparticles comprise a heterogeneous core having an electron-dense region and an aqueous portion, surrounded by an outer lipid layer, as visualized by cryo-electron microscopy.

4. The lipid nanoparticle of claim 1, 2 or 3, wherein the neutral lipid is a phosphatidylcholine lipid 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 content of the neutral lipid is 40 mol% to 60 mol%.

7. The lipid nanoparticle of claim 6, wherein the neutral lipid content is 42 mol% to 60 mol%.

8. The lipid nanoparticle of claim 7, wherein the neutral lipid content is 45 mol% to 60 mol%.

9. The lipid nanoparticle of claim 8, wherein the neutral lipid content is 46 mol% to 60 mol%.

10. The lipid nanoparticle of claim 9, wherein the neutral lipid content is 48 mol% to 60 mol%.

11. The lipid nanoparticle according to any one of claims 1 to 10, wherein the ionizable lipid is an amino lipid.

12. The lipid nanoparticle of any one of claims 1 to 11, wherein the ionizable lipid is present at less than 40 mol%.

13. The lipid nanoparticle of any one of claims 1 to 12, wherein the hydrophilic polymer lipid conjugate is a polyethylene glycol lipid conjugate.

14. The lipid nanoparticle of any one of claims 1 to 13, wherein the sterol is present in an amount of 15 mol% to 45 mol% based on the total lipid present in the lipid nanoparticle.

15. The lipid nanoparticle of any one of claims 1 to 14, wherein the sterol is present in an amount of 18 mol% to 40 mol% based on the total lipid present in the lipid nanoparticle.

16. 4. The lipid nanoparticle of claim 1 or 3, 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 a baseline Onpattro formulation of 50 / 10 / 38.5 / 1.5 mol:mol of MC3 / DSPC / cholesterol / PEG lipids encapsulating the oligonucleotide molecule, measured under otherwise identical conditions, and wherein the biodistribution is quantified in an animal model by detecting labeled lipid 24 hours after administration.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the oligonucleotide is a single-stranded antisense oligonucleotide having a length of 30 to 300 nucleotides.

18. The lipid nanoparticle of any one of claims 1 to 17, wherein the oligonucleotide is siRNA.

19. 19. A method for in vivo delivery of an oligonucleotide molecule to a mammalian subject, wherein the oligonucleotide molecule is single-stranded or double-stranded and is 5 to 500 nucleotides in length, the method comprising administering to the mammalian subject a lipid nanoparticle described in any one of claims 1 to 18.

20. 20. A method for delivering an oligonucleotide molecule to a cell, said method comprising contacting said cell in vivo or in vitro with a lipid nanoparticle according to any one of claims 1 to 18.

21. 20. The method of claim 19, wherein the oligonucleotide is an antisense oligonucleotide molecule and accumulates in the spleen, bone marrow, heart, lungs and / or kidneys of the mammalian subject for at least one day after administration.

22. The method of any one of claims 19 to 21, wherein the lipid nanoparticles are used to treat a disease or disorder that is an autoimmune disease.

23. The method of any one of claims 19 to 21, wherein the lipid nanoparticles are used to treat a disease or disorder that is an infectious disease.

24. The method of any one of claims 19 to 21, wherein the lipid nanoparticles are used to treat a disease or disorder that is cancer.

25. 20. Use of lipid nanoparticles according to any one of claims 1 to 18 for the in vivo or in vitro delivery of said oligonucleotide molecules to mammalian cells.

26. 20. Use of lipid nanoparticles according to any one of claims 1 to 18 for the manufacture of a medicament for the in vivo or in vitro delivery of said oligonucleotide molecule to mammalian cells.

Images