Lipid nanoparticle formulations for antisense oligonucleotide delivery

Abstract

Provided herein are lipid nanoparticles comprising an encapsulated oligonucleotide molecule, wherein the oligonucleotide molecule is single-stranded or double-stranded and has a length of 5 to 500 nucleotides; and a neutral lipid content in a molar ratio of 20 to 70% relative to the total lipids present in the lipid nanoparticles, ionizable lipids; a sterol; and optionally, a hydrophilic polymer-lipid conjugate.

Description

Technical Field

[0001] This disclosure relates to lipid nanoparticle formulations for delivering oligonucleotide cargoes.

[0002] background

[0003] Lipid nanoparticle (LNP) formulations represent a revolution in nucleic acid delivery. An early example of a lipid nanoparticle product approved for clinical use is Onpattro developed by Alnylam. TM Onpattro TM This is a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of hereditary thyroxine transporter amyloidosis-induced polyneuropathy. The success of this LNP delivery system paves the way for the clinical development of a primary LNP-based COVID-19 mRNA vaccine.

[0004] Onpattro TM LNP formulations consist of four main lipid components: an ionizable amino lipid (DLin-MC3-DMA or "MC3" (dilinoleyl-methyl-4-dimethylaminobutyrate)), distearate phosphatidylcholine (DSPC), cholesterol, and a polyethylene glycol-conjugated lipid (PEG-lipid), with molar amounts of 50 / 10 / 38.5 / 1.5, respectively. Onpattro TM It is still considered the gold standard for comparison in LNP-mediated efficacy studies, and the design methodology of LNPs currently used in clinical practice is almost unbiased compared to the four-component system.

[0005] Of these four components, ionizable lipids constitute Onpattro TM The main component of the formulation, present at a 50% molar ratio. Ionizable lipids are considered essential for the in vitro and in vivo activity of the LNP system, and therefore much work in this field has focused on improving this lipid component. Ionizable lipids, typically aminolipids, have been carefully engineered to be charged at low and near-neutral physiological pH. This allows for electrostatic interactions between the lipids and negatively charged nucleic acids during initial formulation. Because the ionizable lipids are near-neutral at physiological pH, toxicity and renal clearance are reduced. Following endocytic uptake of cells, the acidic environment of the endosome leads to an increase in the net positive charge of the ionizable aminolipids, which promotes fusion with anionic lipids at the endosome membrane and subsequent membrane instability, as well as the release of nucleic acid-based therapeutic agents into the cytoplasm to exert their effects.

[0006] Regarding the remaining three lipid components, PEG-lipids are well known to improve the cyclic life of LNPs, and cholesterol is used to stabilize particles. However, relatively little attention is typically devoted to studying neutral lipids beyond their structural roles.

[0007] Despite progress in LNP-mediated siRNA delivery, Onpattro is widely known. TM The four-component LNP formulation accumulates significantly in liver tissue. The ability of LNP to accumulate in organs and tissues outside the liver will greatly expand the clinical applications of these delivery systems.

[0008] Studies have found 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 show any significant benefit due to modulating DSPC levels in LNPs to improve extrahepatic siRNA delivery. These studies used Onpattro with DSPC incorporated at molar ratios of 10 and 40%. TM The study examined extrahepatic siRNA gene silencing in vivo using type LNP (MC3 / Chol / DSPC / PEG-DMP) (Ordobadi, 2019, "Lipid Nanoparticles for Delivery of Bioactive Molecules," a dissertation submitted to partially meet the requirements of a PhD degree, The University of British Columbia). Results showed that a 10% molar ratio of DSPC to PEG-DMP... TM The formulation exhibited similar hepatic cumulative and blood circulation lifespan to the 40% molar ratio DSPC formulation (MC3 / Chol / DSPC / PEG-DMG; 18.5 / 40 / 40 / 1.5% molar ratio). Furthermore, the LNP containing 40% molar ratio DSPC siRNA (siRNA-LNP) performed comparable to the 10% molar ratio DSPC formulation only in bone marrow gene silencing.

[0009] Therefore, there is an urgent need in the field to use lipid nanoparticles to improve the in vivo delivery of nucleic acids such as RNA.

[0010] Overview

[0011] In some advantageous embodiments, this disclosure provides lipid nanoparticles (LNPs) encapsulated with oligonucleotides (referred to herein as "oligo-LNPs"). This lipid component comprises ionizable lipids, neutral lipids such as phospholipids, sterols (e.g., cholesterol), and optionally hydrophilic polymer-lipid conjugates. In particular, the neutral lipids are present in higher amounts than conventional LNPs, for example, at least 20%, at least 30%, or at least 40% molar ratio (relative to the total lipid content of the LNP). This novel oligo-LNP composition exhibits surprisingly improved biodistribution in tissues and / or organs outside the liver. The ability of the LNPs of the present invention to accumulate in extrahepatic organs and tissues greatly expands the clinical applications of the delivery system.

[0012] Furthermore, the inventors have discovered that, in certain embodiments of this disclosure, significant improvements in the biodistribution of oligonucleotides (LNPs), such as siRNA, can be achieved by adjusting the molar charge ratio of nitrogen-phosphate (N / P) or the weight of total lipids in the LNPs relative to nucleic acids per micromolar. In some embodiments, the N / P of the lipid nanoparticles can be 4 to 15 or 4 to 10.

[0013] According to one aspect of this disclosure, lipid nanoparticles are provided comprising: (i) oligonucleotide molecules, wherein the oligonucleotide molecules are single-stranded or double-stranded and have a length of 5 to 500 nucleotides; (ii) neutral lipid contents at a molar ratio of 30% to 70%; (iii) ionizable lipid contents at a molar ratio of 5% to 50%; (iv) sterols selected from cholesterol or derivatives thereof; and (v) optionally, a hydrophilic polymer-lipid conjugate present at a lipid content of 0.5% to 5% relative to the total lipid content of the lipid nanoparticles, wherein optionally the phosphatidylcholine lipid contents are not egg yolk lecithin (EPC).

[0014] According to another aspect of this disclosure, lipid nanoparticles are provided comprising encapsulated oligonucleotide molecules, wherein said oligonucleotide molecules are single-stranded or double-stranded and have a length of 5 to 500 nucleotides; and neutral lipid contents, ionizable lipids, sterols, and optionally hydrophilic polymer-lipid conjugates, at a molar ratio of 20 to 70% relative to the total lipids present in the lipid nanoparticles, measured under the same conditions as a baseline 50 / 10 / 38.5 / 1.5 mol:mol ratio of encapsulated oligonucleotide molecules in an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipids, wherein said lipid nanoparticles show at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear, and wherein said biodistribution is quantified in animal models by detecting labeled lipids 24 hours after administration, wherein said neutral contents comprise no more than three different phosphatidylcholine lipids.

[0015] In another aspect of this disclosure, lipid nanoparticles are provided comprising: (i) oligonucleotide molecules, wherein the oligonucleotide molecules are single-stranded or double-stranded and have a length of 5 to 500 nucleotides; (ii) neutral lipid contents of 30% to 70% molar ratio; (iii) ionizable lipid content of 5% to 50% molar ratio; (iv) sterols selected from cholesterol or derivatives thereof; and (v) a hydrophilic polymer-lipid conjugate present at a lipid content of 0% to 5% molar ratio, wherein each lipid content is relative to the total lipid content of the lipid nanoparticles, and wherein, as visible by cryo-electron microscopy, the lipid nanoparticles comprise a non-uniform core having an electron-dense region and an aqueous portion, and are surrounded by an outer lipid layer.

[0016] In one embodiment of any of the foregoing aspects, the neutral lipid is a phosphatidylcholine lipid, which is distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), or dipalmitoylphosphatidylcholine (DPPC). In another embodiment, the phosphatidylcholine lipid is distearylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC).

[0017] In another embodiment, the neutral lipid content is 40% to 60% molar ratio, or 42% to 60% molar ratio, or 45% to 60% molar ratio, or 46% to 60% molar ratio, or 48% to 60% molar ratio.

[0018] In another embodiment, the ionizable lipid is an amino lipid.

[0019] In another embodiment, the ionizable lipid is present at a molar ratio of less than 40%.

[0020] In another embodiment, the hydrophilic polymer-lipid conjugate is a polyethylene glycol-lipid conjugate.

[0021] According to another embodiment, sterols are present in the lipid nanoparticles at a molar ratio of 15% to 45%. In another embodiment, sterols are present in the lipid nanoparticles at a molar ratio of 18% to 40%.

[0022] In another embodiment, the lipid nanoparticles, measured under the same conditions, showed at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear, compared to a baseline formulation of 50 / 10 / 38.5 / 1.5 mol:mol of encapsulated oligonucleotide molecules in an Onpattro-type formulation, and wherein the biodistribution was quantified in animal models by detecting labeled lipids 24 hours after administration.

[0023] In another embodiment, the oligonucleotide is a single-stranded antisense oligonucleotide having a length of 30 to 300 nucleotides.

[0024] In another embodiment, the oligonucleotide is siRNA.

[0025] In another aspect, a method is provided for in vivo delivery of oligonucleotide molecules to a mammalian individual, wherein the oligonucleotide molecules are single-stranded or double-stranded and have a length between 5 and 500 nucleotides, the method comprising: administering to the mammalian individual lipid nanoparticles of any of the foregoing aspects of this disclosure or embodiments thereof.

[0026] On the one hand, this disclosure also provides a method for delivering oligonucleotide molecules to cells, the method comprising contacting lipid nanoparticles of any of the foregoing aspects or embodiments with cells in vivo or in vitro.

[0027] In another embodiment, the oligonucleotide is an antisense oligonucleotide molecule and accumulates in the spleen, bone marrow, heart, lungs and / or kidneys of the individual for at least one day after administration.

[0028] In another embodiment of the above implementation scheme, the lipid nanoparticles are used to treat diseases or conditions of autoimmune diseases.

[0029] In another embodiment, the disease or condition is an infectious disease.

[0030] In another implementation, the disease or condition is cancer.

[0031] In another aspect of this disclosure, lipid nanoparticles are provided for delivering oligonucleotide molecules to mammalian cells in vivo or in vitro, according to any of the foregoing aspects or embodiments thereof.

[0032] According to another aspect of this disclosure, the use of the lipid nanoparticles described in any aspect or embodiment thereof in the preparation of a medicament for the in vivo or in vitro delivery of oligonucleotides to mammalian cells is provided.

[0033] Brief description of the attached figures

[0034] Figure 1 This is a bar chart showing the size (nm), polydispersity index (PDI), and encapsulation percentage of firefly luciferase siRNA in four-component lipid nanoparticles (LNPs) referred to as B1 to B17. These four-component lipid nanoparticles contain ionizable lipids, MC3, or sulfur-containing ionizable amino lipids (such as MF019 described herein), varying amounts of DSPC ranging from 10% to 55% molar, cholesterol ranging from 18.9% to 38.5% molar, and PEG at 1.5% molar. 2000 -DMG. Table 1 lists the % molar ratio and nitrogen / phosphate ratio (N / P) of each lipid component in the LNP labeled B1 to B17.

[0035] Figure 2A Images of mice treated with LNPs (B10LNPs in Table 1) containing 40% molar ratio of DSPC are shown. These LNPs encapsulate firefly luciferase siRNA and have MF019 ionizable lipid / DSPC / cholesterol / PEG ratios of 33 / 40 / 25.5 / 1.5. 2000 -DMG, labeled with a DiD lipophilic fluorescent marker and having an N / P ratio of 6. The top row shows the mouse before dissection; the middle row shows the peritoneum of the mouse with intact organs; the bottom row shows the fluorescence in the kidney (K), spleen (S), lung (Lu), and liver (Li) removed from the mouse.

[0036] Figure 2B Images of mice treated with LNPs containing 50% molar ratio of DSPC (B12 LNPs in Table 1), which encapsulate firefly luciferase siRNA and have MF019 ionizable lipid / DSPC / cholesterol / PEG ratios of 27.4 / 50 / 21.1 / 1.5, are shown. 2000 -DMG, which is labeled with a DiD fluorescent marker and has an N / P ratio of 6. The top row shows the mouse before dissection; the middle row shows the peritoneum of the mouse with intact organs; the bottom row shows the fluorescence in the kidney (K), spleen (S), lung (Lu), and liver (Li) removed from the mouse.

[0037] Figure 2CImages of mice treated with LNPs containing 40% molar ratio of DSPC (B6 LNPs in Table 1), which encapsulate firefly luciferase siRNA and have MF019 ionization properties of 33 / 40 / 25.5 / 1.5, are shown. 2000 -DMG, which is labeled with DiD and has an N / P ratio of 3. The top row shows the mouse before dissection; the middle row shows the peritoneum of the mouse with intact organs; the bottom row shows the fluorescence in the kidney (K), spleen (S), lung (Lu) and liver (Li) removed from the mouse.

[0038] Figure 3 This is a bar chart showing the size (nm), polydispersity index (PDI), and encapsulation percentage of firefly luciferase siRNA in four-component lipid nanoparticles (LNPs) referred to as A1 to A16, which contain either an ionizable lipid MC3 or a sulfur-containing ionizable amino lipid (such as MF019 described herein), varying amounts of sphingomyelin (ESM) ranging from 10% to 55% molar, cholesterol ranging from 18.9% to 38.5% molar, and PEG at 1.5% molar. 2000 -DMG. Table 3 lists the % molar ratio and nitrogen / phosphate ratio (N / P) of each lipid component in the LNPs labeled A1 to A16.

[0039] Figure 4A This figure shows the fluorescence intensity / mg liver homogenate of mice treated with phosphate-buffered saline (PBS) control and LNP containing firefly luciferase siRNA; with molar ratios of MF019 / ESM / cholesterol / PEG of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B). 2000 -DMG and N / P ratio of 6 for LNP; MF019 / DSPC / cholesterol / PEG with 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D). 2000 -DMG and LNP with an N / P ratio of 6; MF019 / DSPC / cholesterol / PEG 2000 -DMG(LNP-E) LNPs with a chromatogram of 33 / 40 / 25.5 / 1.5 and an N / P ratio of 3. The LNPs were labeled with a DiD fluorescent marker.

[0040] Figure 4BThis figure shows the fluorescence intensity / mg spleen homogenate of mice treated with phosphate-buffered saline (PBS) control and LNP containing firefly luciferase siRNA; with molar ratios of MF019 / ESM / cholesterol / PEG of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B). 2000 -DMG and N / P ratio of 6 for LNP; MF019 / DSPC / cholesterol / PEG with 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D). 2000 -DMG and LNP with an N / P ratio of 6; MF019 / DSPC / cholesterol / PEG 2000 -DMG(LNP-E) LNPs with a chromatogram of 33 / 40 / 25.5 / 1.5 and an N / P ratio of 3. The LNPs were labeled with a DiD fluorescent marker.

[0041] Figure 4C This figure shows the fluorescence intensity / mg of abdominal skin homogenate from mice treated with phosphate-buffered saline (PBS) control and LNP containing firefly luciferase siRNA; with molar ratios of MF019 / ESM / cholesterol / PEG of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B). 2000 -DMG and N / P ratio of 6 for LNP; MF019 / DSPC / cholesterol / PEG with 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D). 2000 LNPs with 33 / 40 / 25.5 / 1.5 N / P of MF019 / DSPC / cholesterol / PEG2000-DMG (LNP-E) and a ratio of 33 / 40 / 25.5 / 1.5. LNPs were labeled with DiD fluorescent marker.

[0042] Figure 4D This figure shows the fluorescence intensity / mg of dorsal skin homogenate of mice treated with phosphate-buffered saline (PBS) control and LNP containing firefly luciferase siRNA; with molar ratios of MF019 / ESM / cholesterol / PEG of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B). 2000 -DMG and N / P ratio of 6 for LNP; MF019 / DSPC / cholesterol / PEG with 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D). 2000-DMG and LNP with an N / P ratio of 6; MF019 / DSPC / cholesterol / PEG 2000 -DMG(LNP-E) LNPs with a chromatogram of 33 / 40 / 25.5 / 1.5 and an N / P ratio of 3. The LNPs were labeled with a DiD fluorescent marker.

[0043] Figure 4E This figure shows the fluorescence intensity / mg ear homogenate of mice treated with phosphate-buffered saline (PBS) control and LNP containing firefly luciferase siRNA; with molar ratios of MF019 / ESM / cholesterol / PEG of 33 / 40 / 25.5 / 1.5 (LNP-A) or 27.4 / 50 / 21.1 / 1.5 (LNP-B). 2000 -DMG and N / P ratio of 6 for LNP; MF019 / DSPC / cholesterol / PEG with 33 / 40 / 25.5 / 1.5 (LNP-C) or 27.4 / 50 / 21.1 / 1.5 (LNP-D). 2000 -DMG and LNP with an N / P ratio of 6; MF019 / DSPC / cholesterol / PEG 2000 -DMG(LNP-E) LNPs with a chromatogram of 33 / 40 / 25.5 / 1.5 and an N / P ratio of 3. The LNPs were labeled with a DiD fluorescent marker.

[0044] Figure 5 In vivo fluorescence of DiD in the liver after treatment with LNPs containing DiD-labeled encapsulated ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were the Onpattro-type baseline compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. Onpattro TM The 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 lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0045] Figure 6In vivo fluorescence of DiD in spleen after treatment with DiD-labeled LNPs encapsulating ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were Onpattro-type baseline compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. The Onpattro-type baseline formulation and the high-DSPC composition were 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0046] Figure 7 In vivo fluorescence of DiD in bone marrow (BM) after treatment with DiD-labeled LNPs encapsulating ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were Onpattro-type compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. The baseline and high DSPC compositions for Onpattro-type were 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0047] Figure 8 In vivo fluorescence of DiD in the heart after treatment with DiD-labeled LNPs encapsulating ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were Onpattro-type baseline compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. The Onpattro-type baseline and high DSPC compositions were 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0048] Figure 9In vivo fluorescence of DiD in the lungs after treatment with DiD-labeled LNPs encapsulating ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were Onpattro-type baseline compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. The Onpattro-type baseline and high DSPC compositions were 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0049] Figure 10 In vivo fluorescence of DiD in the kidneys after treatment with DiD-labeled LNPs encapsulating ssRNA as a function of DSPC content. LNPs A and B encapsulated 50-mer ssRNA, and LNPs C and D encapsulated 221-mer ssRNA. LNPs A and C were Onpattro-type baseline compositions with a 10% molar DSPC content, while B and D were compositions with a 50% molar DSPC content. The Onpattro-type baseline and high DSPC compositions were 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0050] Figure 11A Cryo-electron microscopy images of LNPs containing siRNA encapsulating firefly luciferase siRNA, and the LNPs possessing MF019 ionization properties of 33 / 40 / 25.5 / 1.5 for ionsizing lipids / DSPC / cholesterol / PEG. 2000 -DMG and N / P is 6.

[0051] Figure 11B Cryo-electron microscopy image of a firefly luciferase siRNA-encapsulated LNP containing siRNA. The LNP has MF019 ionization properties of 27.4 / 50 / 21.1 / 1.5, which can ionize lipids / DSPC / cholesterol / PEG. 2000 -DMG and N / P is 6.

[0052] Figure 11C Cryo-electron microscopy image of a firefly luciferase siRNA-encapsulated LNP containing siRNA. The LNP has MF019 ionization properties of 27.4 / 50 / 21.1 / 1.5, which can ionize lipids / ESM / cholesterol / PEG. 2000 -DMG and N / P is 6.

[0053] Detailed Explanation

[0054] The lipid nanoparticles described herein are superior to conventional four-component LNPs, referred to herein as Onpattro-type LNPs, in terms of oligonucleotide cargo delivery. Specifically, the LNP comprises ionizable lipids, neutral lipids such as phosphatidylcholine lipids (e.g., DSPC), sterols, and optionally hydrophilic polymer-lipid conjugates, wherein the neutral lipids (e.g., phosphatidylcholine lipids) are present at a % molar ratio of at least 20%, at least 25%, or at least 30%, and wherein, in some instances, the ionizable lipids are present at a less than 40% molar ratio. As described herein, the inclusion of neutral lipids such as phosphatidylcholine at a higher % molar ratio than that used in conventional formulations for nucleic acid delivery provides selective delivery to extrahepatic tissues relative to the Onpattro-type baseline.

[0055] "Oligonucleotides" or "oligonucleotide cargoes" are single-stranded or double-stranded RNA or DNA molecules with a length of 5 to 500 nucleotides. The term includes single-stranded antisense oligonucleotides (ASOs) that are typically 30 to 500 nucleotides or shorter, and double-stranded silent RNA molecules (siRNAs) that are 3 to 40 nucleotides in length.

[0056] neutral lipids

[0057] Neutral lipids are amphiphilic lipids that allow for particle formation and typically do not carry a net charge at physiological pH. The term includes zwitterionic lipids and examples include phospholipids.

[0058] In some embodiments, the neutral lipid is a phosphatidylcholine lipid. The phosphatidylcholine lipid may be selected from distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The phosphatidylcholine lipid component may include a mixture of two or more different types of neutral lipids.

[0059] In one embodiment, the phosphatidylcholine lipid is selected from DSPC, POPC, and mixtures thereof.

[0060] In some embodiments, the phosphatidylcholine content is greater than 20%, 25%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, or 50%. In some embodiments, the upper limit for the neutral lipid content is 70%, 65%, 60%, 55%, 50%, or 45%. This disclosure also covers sub-ranges of any combination of the aforementioned upper and lower limits.

[0061] For example, in some embodiments, the phosphatidylcholine lipid content is 20% to 80% of the total lipids present in the lipid nanoparticles, or 25% to 60% of the total lipids, or 30% to 60% of the total lipids, or 35% to 60% of the total lipids, or 40% to 60% of the total lipids, or 42% to 58% of the total lipids, or 43% to 57% of the total lipids, or 44% to 56% of the total lipids, or 45% to 55% of the total lipids.

[0062] In one embodiment, the neutral lipid is most advantageously DSPC. In certain instances of this disclosure, DSPC lipids improve the biodistribution of LNPs relative to other neutral phospholipids when elevated. In some embodiments, the DSPC lipid content is 20% to 80% molar ratio, or 25% to 60% molar ratio, or 30% to 60% molar ratio, or 35% to 60% molar ratio, or 40% to 60% molar ratio, or 42% to 60% molar ratio, or 43% to 60% molar ratio, or 44% to 60% molar ratio, or 45% to 60% molar ratio, or 46% to 60% molar ratio, or 48% to 60% molar ratio, of the total lipids present in the lipid nanoparticles.

[0063] In some embodiments, the phosphatidylcholine lipid is a sphingolipid. As used herein, the term "sphingolipid" refers to a lipid comprising a sphingosine backbone suitable for formulation in the LNPs herein. Sphingolipids include ceramides, sphingomyelins, cerebrosides, gangliosides, or derivatives, such as, but not limited to, their reduced analogs lacking a double bond in the sphingosine unit. The sphingolipid has a phosphocholine head group and comprises sphingomyelin.

[0064] In some embodiments, the sphingolipid content is 20% to 80% of the total lipids present in the lipid nanoparticles, or 25% to 60% of the total lipids, or 30% to 60% of the total lipids, or 35% to 60% of the total lipids, or 40% to 60% of the total lipids, or 42% to 60% of the total lipids, or 43% to 60% of the total lipids, or 44% to 60% of the total lipids, or 45% to 60% of the total lipids, or 46% to 60% of the total lipids, or 48% to 60% of the total lipids.

[0065] In some embodiments, the sphingomyelin content of the lipid nanoparticles is less than 5% molar, less than 4% molar, less than 3% molar, less than 2% molar, less than 1% molar, less than 0.75% molar, or less than 0.5% molar. In some embodiments, the LNP is "sphingomyelin-free," meaning that there is no detectable sphingomyelin in the LNP (less than 0.5% molar), or the LNP is substantially sphingomyelin-free, meaning that there is less than 5% molar or 2.5% molar of sphingomyelin in the LNP.

[0066] LNPs can contain other neutral lipids besides phosphatidylcholine lipids. For example, LNPs can contain other lipids that have a net positive or negative charge at physiological pH. In another instance, LNPs may also contain small amounts of one or more fusion lipids (as opposed to phosphatidylcholine lipids), such as DOPEs, which are cone-shaped and thus facilitate fusion with the cell membrane. Typically, these lipids will be present in a molar ratio of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% relative to the total lipids present in the LNP.

[0067] Fusion lipids containing substances such as dioleoylphosphatidylethanolamine (DOPE) are thought to facilitate the delivery of nucleic acids in vitro or in vivo. However, in some embodiments, this disclosure generally does not favor the inclusion of such lipids. Therefore, in some instances, the fusion lipid content of the lipid nanoparticles is less than 5% molar, less than 4% molar, less than 3% molar, less than 2% molar, less than 1% molar, less than 0.75% molar, or less than 0.5% molar. In some embodiments, the LNP is "fusion lipid-free," meaning that there is no detectable amount of fusion lipid (less than 0.5% molar) in the LNP, or the LNP is substantially free of fusion lipids, meaning that the measured fusion lipid content is less than 5% molar relative to the total lipid content in the LNP.

[0068] In some instances, the DOPE content of the lipid nanoparticles is less than 10% molar, less than 8% molar, 5% molar, less than 4% molar, less than 3% molar, less than 2% molar, less than 1% molar, less than 0.75% molar, or less than 0.5% molar. In some embodiments, the LNP is "DOPE-free," meaning that there is no detectable DOPE (less than 0.5% molar) in the LNP, or that the LNP is substantially DOPE-free, meaning that the measured DOPE is less than 5% molar relative to the total lipid content in the LNP.

[0069] In other embodiments, it may be advantageous to include a mixture of different phosphatidylcholine lipids in the LNP. However, in some instances, the phosphatidylcholine lipid contents include fewer than 5, 4, or 3 different phosphatidylcholine lipids.

[0070] In some embodiments, the egg yolk lecithin (EPC) content of the lipid nanoparticles is less than 5% molar, less than 4% molar, less than 3% molar, less than 2% molar, less than 1% molar, less than 0.75% molar, or less than 0.5% molar. In some embodiments, the LNP is "EPC-free," meaning that there is no detectable EPC (less than 0.5% molar) in the LNP, or the LNP is substantially EPC-free, meaning that the measured EPC in the lipid nanoparticles is less than 5% molar relative to the total lipid content in the LNP.

[0071] In some embodiments, the phosphatidylcholine lipid is a phosphatidylcholine-sterol conjugate, such as an SPC-cholesterol, OPC-cholesterol, or PPC-cholesterol conjugate. Other phosphatidylcholine-sterol conjugates are described in US2011 / 0177156, which is incorporated herein by reference. Examples of suitable phospholipids are the SPC-cholesterol conjugates shown below:

[0072]

[0073] In some embodiments, the transition temperature of the neutral lipids or mixtures 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 lipids or mixtures thereof when incorporated into lipid nanoparticles is at least 38, 39, or 40°C.

[0074] The content of neutral lipids was determined based on the total amount of lipids (mol:mol) in lipid nanoparticles, including sterols.

[0075] Ionizable lipids

[0076] The LNP disclosed herein has ionizable cationic lipids, comprising one or more combinations of such lipids.

[0077] As used herein, the term "ionizable cationic lipid" refers to a lipid that is electrostatically neutral and accepts protons at a given pH, such as physiological pH, and thus becomes positively charged at pH below its pKa. The electrostatically neutral form has a calculated logarithm (i.e., cLogP) greater than 8 for the partition coefficient between water and 1-octanol. In some embodiments, when formulated in LNP, the cationic lipid has a pKa of 5.0 to 7.5 or 6.0 to 7.5.

[0078] Therefore, ionizable lipids can be charged at low pH and have virtually no net charge at physiological pH. This allows for electrostatic interactions between the lipids and negatively charged nucleic acid cargo during initial formulation. Since ionizable lipids are near neutral at physiological pH, toxicity and renal clearance are reduced. Unbound from theory, the acidic environment of the endosome after endocytic uptake leads to an increase in the net positive charge of ionizable amino lipids, which promotes fusion with anionic lipids at the endosome membrane and subsequent membrane destabilization and release of nucleic acid-based therapeutic agents into the cytoplasm to exert their effects.

[0079] In some embodiments, the LNP has an apparent pKa of 5.0 to 7.5, 6.5 to 7.5, or 6.8 to 7.3. The apparent pKa is measured using a modified 6-(p-toluidine)-2-naphthalenesulfonic acid (TNS) assay, derived from previous studies in other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; and Jayaraman et al., 2012, Angew. Chem Int. Ed., 51:8529-8533, which are incorporated herein by reference for the purpose of determining the apparent pKa). According to this method, a series of buffer solutions spanning a pH range of 2 to 11 are prepared in 0.5 pH increments, each increment consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM HEPES in 0.15 to 0.2 mM LNP. Subsequently, 0.06 mM TNS solution was mixed in triplicate with 175 μL LNP at each buffer pH in a black polystearin 96-well plate to produce final concentrations of 6.25 and 6 μM lipids and TNS, respectively, in each well. SpectraMax was then used for further analysis. TM M5 microplate reader at λ ex =321nm, λ emFluorescence was measured at 445 nm. Then, it was analyzed using Prism... TM The S-shaped curve fitting plots fluorescence relative to pH, where pKa is determined to be the pH value at which the maximum fluorescence intensity is 50%.

[0080] In some embodiments, it is desirable to contain less than 50% molar of ionizable lipids in the LNP. That is, based on measurements of the total lipid content of the LNP, the ionizable lipid content can be less than 50% molar, less than 45% molar, less than 40% molar, less than 35% molar, less than 30% molar, less than 25% molar, less than 20% molar, less than 15% molar, less than 10% molar, or less than 5% molar. In some embodiments, the lower limit of the ionizable lipid content can be greater than 5% molar, greater than 8% molar, greater than 10% molar, greater than 12% molar, greater than 14% molar, greater than 15% molar, greater than 16% molar, greater than 18% molar, greater than 20% molar, greater than 25% molar, or greater than 30% molar. Any of the upper limits can be combined with any of the lower limits to obtain a suitable ionizable lipid content in the LNP.

[0081] In some embodiments, the ionizable lipid content is 5% to 50% of the total lipids present in the lipid nanoparticles, or 8% to 47% of the total lipids, or 10% to 50% of the total lipids, or 15% to 45% of the total lipids, or 15% to 35% of the total lipids.

[0082] In some embodiments, the cationic lipid has an amino group. In some cases, the cationic lipid comprises a protonable tertiary amine (e.g., pH-titrile) head group and two C16 to C18 alkyl chains having 0 to 3 double bonds. Such lipids include, but are not limited to, thiolipins, such as MF019 and DODMA described herein. Other lipids that can be used to implement this disclosure include MC3-type and KC2-type lipids, which are well known to those skilled in the art. In other embodiments, the ionizable lipid is selected from WO 2022 / 246555; WO 2022 / 246568; WO 2022 / 246571; WO 2023 / 147657; WO The following lipids described herein are each incorporated by reference: PCT / CA2023 / 050644, filed May 11, 2023; PCT / CA2023 / 051272, filed September 27, 2023; 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.

[0083] In some embodiments, it is desirable to include less than 50% molar of ionizable cationic lipids in the LNP. That is, the content of ionizable cationic lipids can be less than 50% molar, less than 45% molar, less than 40% molar, less than 35% molar, less than 30% molar, less than 25% molar, less than 20% molar, less than 15% molar, less than 10% molar, or less than 5% molar.

[0084] In some embodiments, the content of ionizable cationic lipids is 5% to 50% of the total lipids present in the lipid nanoparticles, or 8% to 47% of the total lipids, or 10% to 50% of the total lipids, or 15% to 45% of the total lipids, or 15% to 35% of the total lipids.

[0085] In some embodiments, the content of ionizable amino-cationic lipids is 15% to 40% molar ratio, 8% to 47% molar ratio, 10% to 50% molar ratio, 15% to 45% molar ratio, or 15% to 35% molar ratio of the total lipids present in the lipid nanoparticles.

[0086] In some embodiments, the ionizable cationic lipid is not a lipid structure, including but not limited to C12-200 (see Khare et al., 2022, AAPS Journal, 24:8, incorporated herein by reference) and related structures known to those skilled in the art.

[0087] Sterols

[0088] In some embodiments, the LNP also includes sterols. The term "sterol" refers to a naturally occurring or synthetic compound having a sterane skeleton and a hydroxyl moiety attached to one of its rings, typically an A-ring.

[0089] Examples of sterols include cholesterol or cholesterol derivatives, the latter referring to cholesterol molecules having a sterane structure and one or more other functional groups.

[0090] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmasterol, dihydrocholesterol, α-cholesterol, epi-cholesterol, sterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, 3β[N-(N,N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxapropylcyclosterol, 23-oxapropylcyclosterol, 24-oxapropylcyclosterol, cycloaltiol, 22-ketosterol, 20- Hydroxysterols, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholest-3β-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanosterol, lupeosterol, sitocalciferol, calcipotriol, coccosterol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassosterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, yeast sterol, diosgenin, fucosterol, coccosterol or their salts or esters.

[0091] In one embodiment, based on the total lipids present in the lipid nanoparticles, sterols are present in a molar ratio of 15% to 50%, 18% to 45%, 20% to 45%, 25% to 45%, or 30% to 45%.

[0092] In another embodiment, based on the total lipids present in the lipid nanoparticles, the sterol is cholesterol and is present in a molar ratio of 15% to 50%, 18% to 45%, 20% to 45%, 25% to 45%, or 30% to 45%.

[0093] In another embodiment, based on the total lipids present in the lipid nanoparticles, the sterols are cholesterol derivatives and are present in a molar ratio of 15% to 50%, 18% to 45%, 20% to 45%, 25% to 45%, or 30% to 45%.

[0094] In one embodiment, based on the total lipids present in the lipid nanoparticles, the combination comprises (i) sterol content (e.g., cholesterol or cholesterol derivatives thereof); and (ii) phosphatidylcholine lipid content of at least 50% molar ratio, at least 55% molar ratio, at least 60% molar ratio, at least 65% molar ratio, at least 70% molar ratio, at least 75% molar ratio, at least 80% molar ratio, or at least 85% molar ratio.

[0095] Hydrophilic polymer-lipid conjugate

[0096] In one embodiment, the lipid nanoparticles comprise a hydrophilic polymer lipid conjugate capable of incorporating into the LNP. In some instances, the conjugate comprises a vesicle-forming lipid, hydrophilic polymer chain having a polar head group and covalently linked to the head group. The lipid comprises any portion having at least one hydrophobic portion. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyethylene methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyl methacrylate, polyethyl methacrylate, polyethyl methacrylate, polyhydroxypropyl methacrylate, polycarboxylic acid, polyethyl methacrylate, polyhydroxypropyl methacrylate, polysarcosine, and polyasparagine. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer lipid conjugate may also be a naturally occurring or synthetic oligosaccharide-containing molecule, such as monosialotetrahexosylganglioside (G... M1 The ability of a given hydrophilic-polymer lipid conjugate to enhance the cycle life of the LNP described herein can be readily determined by those skilled in the art using known methods.

[0097] The hydrophilic polymer lipid conjugate may be present in the nanoparticles at a molar ratio of 0.5% to 5%, 0.5% to 3%, 0.5% to 2.5%, 0.5% to 2.0%, or 0.5% to 1.8% of the total lipids. In some embodiments, the hydrophilic polymer lipid conjugate may be present in the nanoparticles at a molar ratio of 0% to 5%, 0% to 3%, 0% to 2.5%, 0% to 2.0%, or 0% to 1.8% of the total lipids.

[0098] In another embodiment, the PEG-lipid conjugate is present in the nanoparticles at a molar ratio of 0.5% to 5%, or 0.5% to 3%, or 0.5% to 2.5%, or 0.5% to 2.0%, or 0.5% to 1.8% of the total lipids. In some embodiments, the PEG-lipid conjugate may be present in the nanoparticles at a molar ratio of 0% to 5%, or 0% to 3%, or 0% to 2.5%, or 0% to 2.0%, or 0% to 1.8% of the total lipids.

[0099] In one embodiment, the hydrophilic polymer-lipid is selected based on its exchangeability with lipid nanoparticles. This property can enhance in vivo efficacy because the hydrophilic polymer-lipid conjugate is at least partially lost from the LNP when it reaches the target site in vivo.

[0100] In such embodiments, the lipid moiety of the hydrophilic polymer-lipid conjugate typically has an acyl chain length of less than 18 and contains 0 to 2 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 dimyristoyl phosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoyl phosphatidylethanolamine-PEG (DPPE-PEG), dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG), dipalmitoyl phosphatidylethanolamine-PEG (DPPE-PEG), dimyristoyl diglyceride-PEG (DMG-PEG), or cholesterol-PEG (Chol-PEG).

[0101] In one embodiment, the hydrophilic polymer-lipid conjugate is absent or present at a low concentration in the lipid nanoparticles. In some embodiments, the hydrophilic polymer-lipid conjugate content is less than 0.5% molar ratio, 0.45% molar ratio, 0.40% molar ratio, 0.35% molar ratio, 0.30% molar ratio, 0.25% molar ratio, 0.20% molar ratio, or 0.15% molar ratio.

[0102] In one embodiment, the hydrophilic polymer lipid conjugate is not DSPE-PEG. In other embodiments, the DSPE-PEG content is less than 0.5% molar, 0.45% molar, 0.40% molar, 0.35% molar, 0.30% molar, 0.25% molar, 0.20% molar, or 0.15% molar.

[0103] In another embodiment, in the hydrophilic polymer lipid conjugate, a cleavable linker is present between the lipid moiety and the hydrophilic polymer. This linker can be cleaved by exposure to low pH, reducing agents, or proteases present in vivo. Examples of cleavable linkers include esters, ethers, aminophosphates, hydrazones, β-thiopropionates, disulfide groups, and peptides (Romberg et al., 2008, Pharmaceutical Research, 25:55-71, incorporated herein by reference).

[0104] Optional additional lipid components

[0105] In some embodiments, lipid nanoparticles for extrahepatic delivery comprise lipids “substantially composed of” neutral amphiphilic lipids such as phosphocholine, sterols or derivatives thereof, ionizable cationic lipids, and optionally hydrophilic polymer-lipid conjugates, and have an additional lipid component in a molar ratio of less than 10% in addition to the aforementioned lipid components. That is, in some instances, the additional lipid component is present in a molar ratio of 0 to 20%, 0 to 15%, 0 to 10%, 0 to 8%, or 0 to 5%, and includes any of a number of charged and / or uncharged lipids. In some embodiments, the additional lipid component consists of one or more neutral lipids, rather than lipids charged at physiological pH. Avoiding such charged lipids reduces uptake by the RES system, thereby not impairing the cycle life of the particles. Examples of such optional additional lipids include triglycerides, diglycerides, monoglycerides, zwitterionic lipids, antioxidants, and vitamins.

[0106] Other examples of phospholipids include phosphatidylglycerols, such as dioleoylphosphatidylglycerol (DOPG), distearylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), distearylphosphatidylserine (DSPS), and sphingomyelin. Examples of sphingomyelins in this article include ceramides, sphingomyelins, cerebrosides, gangliosides, or their reduced analogs lacking a double bond in the sphingosine unit.

[0107] In those embodiments in which the formulation comprises anionic lipids, the anionic lipids may be lipids having a hydroxyl head group. This includes, but is not limited to, anionic lipids such as DOPG and A-0001, described in U.S. Provisional Patent Application No. 63 / 453,766, filed March 22, 2023 (incorporated herein by reference).

[0108] Other additional components include hydrophobic portions, such as lipids conjugated to a targeting ligand. The ligand includes peptides, polypeptides, or proteins, and may include antibodies or fragments thereof. In one embodiment, the ligand may be a single-chain antibody fragment. The targeting ligand can be used to target receptors on cells in vivo. In some embodiments, the targeting ligand may be conjugated to the phospholipid component of the LNP. If a certain amount of a 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 a molar ratio of less than 10%, less than 5%, or less than 3%. In some embodiments, the targeting ligand is most advantageously absent.

[0109] In one embodiment, the lipid nanoparticles have low levels (less than 5% molar ratio) or no permanently charged cationic lipid contents. In certain instances of this disclosure, the lipid nanoparticles promote extended cycle life due to the lack of net charge at physiological pH, thereby facilitating extrahepatic delivery to cells. Examples of permanently charged cationic lipids best avoided include bis(octadecyldimethylammonium bromide) (DDAB) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Such permanently charged cationic lipids may also confer toxicity to the lipid nanoparticles.

[0110] Nanoparticle preparation and morphology

[0111] Lipid nanoparticles incorporating oligonucleotides can be prepared using a variety of suitable methods, such as rapid mixing / ethanol dilution methods. Examples of preparation methods are described in Jeffs, LB, et al., Pharm Res, 2005, 22(3):362-72; and Leung, AK, et al., The Journal of Physical Chemistry.C, Nanomaterials and Interfaces, 2012, 116(34):18440-18450, which are each incorporated herein by reference in their entirety.

[0112] Without being bound by theory, the mechanism by which lipid nanoparticles containing encapsulated oligonucleotides can be formed using a rapid mixing / ethanol dilution method can be hypothesized as follows: at low pH (e.g., pH 4), a dense region of a hydrophobic RNA-ionizable lipid core begins to form, surrounded by a neutral lipid / cholesterol monolayer. As pH increases, the neutral lipid / cholesterol monolayer fuses with smaller empty vesicles due to the conversion of ionizable cationic lipids to a neutral form. With an increasing proportion of neutral lipids in the bilayer, the bilayer gradually forms vesicles, and ionizable lipids migrate to the inner hydrophobic core. At sufficiently high neutral lipid contents, the outer bilayer of neutral lipids preferably forms a complete bilayer around the internal cutoff volume.

[0113] LNPs can include a “core” region. The core can be considered inhomogeneous because it comprises an electron-dense region and, optionally, an aqueous portion that at least partially surrounds the electron-dense region. The electron-dense region is observed by cryo-electron microscopy using the methods described in the Materials and Methods section of this document. Without limitation, the electron-dense region within the core can be partially surrounded by an aqueous portion within an enclosed space, as observed by cryo-electron microscopy. The aqueous portion forms distinct aqueous compartments within the lipid nanoparticle. In other words, the aqueous portion is not merely a hydration layer.

[0114] In one embodiment, at least about one-fifth of the nucleus (retained volume) contains an aqueous portion, and the electron-dense region is partially adjacent to a lipid bilayer, as determined qualitatively by cryo-electron microscopy. In another embodiment, at least about one-quarter of the nucleus contains an aqueous portion, and the electron-dense nucleus is partially adjacent to a lipid bilayer, as determined qualitatively by cryo-electron microscopy. In another embodiment, at least about one-third of the nucleus contains an aqueous portion, and the electron-dense region is partially adjacent to a lipid bilayer, as determined qualitatively by cryo-electron microscopy. In yet another embodiment, at least about half of the nucleus contains an aqueous portion, and the electron-dense nucleus is partially adjacent to a lipid bilayer, as determined qualitatively by cryo-electron microscopy.

[0115] In one embodiment, the electron-dense region is typically spherical. In another embodiment, the electron-dense region is hydrophobic.

[0116] The lipid nanoparticles described herein exhibit exceptionally high oligonucleotide encapsulation efficiency. As used herein, the term "encapsulation" in relation to the incorporation of oligonucleotides into lipid nanoparticles refers to any association between the oligonucleotide and any lipid component or compartment of the lipid nanoparticle, including lipophilic or aqueous portions. In one embodiment, the oligonucleotide is present at least in the core of the LNP.

[0117] In one embodiment, the encapsulation efficiency is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 92%. The encapsulation efficiency of the oligonucleotide is determined as described in the Materials and Methods section of the embodiments herein.

[0118] Lipid nanoparticles may comprise a single bilayer or multiple lipid layers (i.e., multilayers). One or more lipid layers comprising a bilayer may form a continuous layer surrounding a core, or they may be discontinuous. In some embodiments, the lipid layer may be a combination of a bilayer and a monolayer. In one embodiment, the lipid layer is a continuous bilayer surrounding a core.

[0119] Therefore, in some embodiments, the electron-dense region of the core is separated from the lipid bilayer by an aqueous portion. For example, this disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, wherein, as determined by cryo-electron microscopy, at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles have a core having an electron-dense region partially surrounded by an aqueous portion, and wherein, as observed by cryo-electron microscopy, the aqueous portion is partially surrounded by a lipid bilayer.

[0120] In another embodiment, and not limitingly, this disclosure provides lipid nanoparticle formulations comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles have an elongated shape (e.g., typically elliptical), as determined qualitatively by cryo-electron microscopy. In a later embodiment, as observed by cryo-electron microscopy, the electron-dense region of the nucleus may be partially surrounded by an aqueous space.

[0121] In one embodiment, the lipid nanoparticles are part of a lipid nanoparticle formulation, and at least 20% of the electronically dense regions of the lipid nanoparticles are either (i) encapsulated by an aqueous portion or (ii) partially surrounded by an aqueous portion, and wherein, as observed by cryo-electron microscopy, a portion of the periphery of the electronically dense regions is adjacent to the lipid layer.

[0122] In some embodiments, this disclosure provides lipid nanoparticle formulations comprising a plurality of lipid nanoparticles, wherein, as determined by cryo-electron microscopy, typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles have a core, as observed by cryo-electron microscopy, which has an electron-dense region adjacent to a lipid bilayer.

[0123] In another embodiment, and not limitingly, this disclosure provides a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, wherein typically at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the particles have a core, as observed by cryo-electron microscopy, said core comprising an electron-dense region surrounded or enclosed by a continuous aqueous space disposed between a lipid layer (e.g., a bilayer) and an electron-dense region.

[0124] LNPs can be observed using cryo-electron microscopy as described in the following examples section.

[0125] In another embodiment, the polydispersity index (PDI) of the LNP formulation is less than 0.3, 0.25, 0.2, 0.15, 0.12, or 0.10.

[0126] In another embodiment, the particle size distribution is such that 90% of the particles in the LNP formulation of this disclosure have a diameter of 40 nm to 200 nm, 40 nm to 150 nm, 40 nm to 140 nm, 45 nm to 150 nm, 50 nm to 120 nm, or 50 nm to 140 nm. In some embodiments, the LNPs of this invention 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.

[0127] Embodiments of this disclosure also provide lipid nanoparticles described based on the molar ratio between the positively charged amine group (N) of the amine lipid and the negatively charged phosphate group (P) of the oligonucleotide to be encapsulated. This can be mathematically represented by the equation N / P. In one embodiment, the N / P ratio of the lipid nanoparticles is 4 to 15, or 4.5 to 10, or 5 to 10, or between 5.5 and 8.

[0128] In one embodiment, the N / P ratio of the lipid nanoparticles is 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 can be 15, 14, 13, 12, 11, 10, 9, or 8. This disclosure also includes any combination of any two of the upper and lower limits. N / P is the charge ratio calculated as described in the Materials and Methods section.

[0129] In one embodiment, the lipid nanoparticles have a weight ratio of nucleic acid to total lipids 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 weight ratio of nucleic acid to total lipids. In another embodiment, the upper limit is 0.80:1, 0.82:1, 0.84:1, 0.86:1, 0.88:1, 0.90:1, 0.92:1, 0.94:1, 0.96:1, or 0.98:1 weight nucleic acid / micromolar total lipids. This disclosure also includes any combination of any two of the upper and lower limits.

[0130] Oligonucleotide cargo

[0131] Oligonucleotide cargoes include interfering RNA and antisense oligonucleotides, which are described in more detail below.

[0132] Interfering RNA

[0133] In one embodiment, the oligonucleotide is a “short interfering RNA” or “siRNA,” which is an RNA molecule capable of reducing or inhibiting the expression of a target gene or nucleic acid sequence in a cell. In one embodiment, the short interfering RNA can mediate the degradation of the target mRNA, as measured in vitro or in vivo. In such an embodiment, the siRNA functions by pairing with complementary sequence bases of the target mRNA (when single-stranded) and inducing mRNA cleavage.

[0134] siRNA is double-stranded and can have various lengths, but is typically less than 35 nucleotides. In some embodiments, siRNA has a length such as 1 to 35 nucleotides, 15 to 30 nucleotides, or 20 to 25 nucleotides.

[0135] In embodiments where 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 mismatch regions (i.e., mismatch motifs). The sequence of the siRNA may correspond to the full-length target sequence or a subsequence of it.

[0136] The double-stranded siRNA encapsulated in the LNP can include double-stranded RNA, such as double-stranded small interfering RNA, asymmetric interfering RNA (aiRNA), or premiRNA, or a hybrid molecule containing RNA and DNA. In one embodiment, the double-stranded RNA is self-complementary. In such an embodiment, the siRNA may form a stem-loop or hairpin structure at one end.

[0137] The siRNAs covered by the embodiments of this disclosure can be used to inhibit the expression of a wide range of target polynucleotides. siRNA molecules targeting specific polynucleotides for any therapeutic, preventative, or diagnostic application can be readily prepared according to methods known in the art. siRNA target sites can be selected, and the corresponding interfering RNA can be chemically synthesized, produced by in vitro transcription, or expressed from a vector or PCR product.

[0138] As described above, the siRNA described herein may contain "mismatch motifs" or "mismatch regions," which refer to a portion of the siRNA sequence that is not 100% complementary to its target sequence. The siRNA may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be continuous or 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 two, three, four, five, or more nucleotides.

[0139] The nucleotides of siRNA may or may not be chemically modified. Examples of optional modifications include, but are not limited to, 2'-O-Me or 2'-O-methoxyethyl modifications, 2'-O-alkyl modifications, and 2'-halogen modifications such as 2'-fluorine modifications. In other embodiments, the siRNA comprises one, two, three, four, or more 2'-deoxynucleotides, for example, in the sense and / or antisense strands of the double-stranded region. Alternatively, in some embodiments, the siRNA comprises a phosphate backbone modification.

[0140] Within siRNA, the antisense and sense strands can be programmed to either anneal without overhangs at either end of the duplex when they form a doublet due to complementary base pairing, resulting in blunt ends, or to have overhangs at one or more of the 3' end of the sense strand, the 3' end of the antisense strand, the 5' end of the sense strand, and the 5' end of the antisense strand. In some embodiments, there are no 5' overhangs and no 3' antisense overhangs, but there are 3' sense overhangs. In others, there are no 5' overhangs, but there are both 3' antisense and 3' sense overhangs. The overhangs may contain T or U nucleotides.

[0141] In some embodiments, siRNA covalently binds to one or more other moieties to form conjugates. In some embodiments, conjugates are selected based on their ability to facilitate siRNA delivery to an organism or cell. siRNA may bind to a nucleotide at, for example, the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or at a position not at the 3' or 5' end of either strand.

[0142] Examples of conjugates include, but are not limited to, one or more antibodies or fragments thereof, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell-penetrating peptides, polymers, and glycosides, including glycomonomers, oligosaccharides, and modifications thereof. In a non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc).

[0143] Antisense oligonucleotides (ASO)

[0144] 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 mismatch regions (i.e., mismatch motifs). The sequence of the ASO may correspond to the full-length target sequence or a subsequence of it.

[0145] ASO 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, ASO forms part of a gene-editing complex for directing nucleases to target sites for site-specific cleavage of DNA.

[0146] In embodiments where ASO reduces the expression of a target gene or sequence through complementary base pairing and mRNA degradation, ASO functions by pairing with the complementary sequence of the target mRNA and inducing mRNA cleavage. ASO can prevent or reduce the translation of the complementary RNA strand by binding to RNA. ASO can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, the target sequence can be degraded by the enzyme RNase H, which is present in the cell nucleus and / or cytoplasm. In one embodiment, ASO is a “gapmer” sequence containing 2 to 5 chemically modified nucleotides at each end of a central gap region of DNA (e.g., an 8 to 10 base “gap”). The chemically modified nucleotides reduce nuclease degradation and increase the affinity of ASO for the target sequence. The gap allows for the formation of hybrid sequences that can be cleaved by RNase H. Furthermore, oligonucleotides can be chemically modified using known methods to recruit RNase H.

[0147] In another implementation, ASO can bind to target mRNA and block gene expression. ASOs that work by blocking gene expression are called "steric blockers" and block ribosome binding, thereby preventing or reducing the translation of the target nucleotide sequence.

[0148] In another implementation, ASO can modulate the splicing of pre-mRNA sequences. ASO can be programmed to target sequences within the pre-mRNA to influence splicing and increase the production of desired isotypes. ASO can be used, for example, to remove mutant exons, thereby restoring the appropriate reading frame and producing a more functional protein product.

[0149] In one embodiment, the ASO comprises about 15 to about 500 nucleotides, about 20 to about 300 nucleotides, about 25 to about 200 nucleotides, or about 30 to about 150 nucleotides.

[0150] Typically, an ASO (Active Sequence of Occurrence) may contain "mismatch motifs" or "mismatch regions," which refer to portions of the ASO sequence that are not 100% complementary to its target sequence. An ASO may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be continuous or 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 two, three, four, five, or more nucleotides.

[0151] The nucleotides of the ASO may or may not be chemically modified. Modifications can improve the stability of the ASO, for example, by increasing nuclease resistance in addition to the protection provided by LNPs. Furthermore, in some instances of this 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'-fluorine modifications. In other embodiments, the ASO comprises one, two, three, four or more 2'-deoxynucleotides, for example, in the sense and / or antisense strands of the double-stranded region. Alternatively, in some embodiments, the ASO comprises a phosphate backbone modification, such as a phosphate thioester backbone modification. Additional 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 favorable for binding to the complementary nucleic acid sequence. The associated bridging modification is a bridged nucleic acid (BNA). Other examples include ASO and CpG oligomers with a peptide backbone (PNA), as well as other oligomers known to those skilled in the art.

[0152] The ASO encapsulated in the LNP is typically monochain. However, in some instances of this disclosure, the ASO has a self-complementary sequence. In such embodiments, the ASO may form one or more stem loops or hairpin structures within the chain.

[0153] In some embodiments, ASO is covalently bound to one or more other moieties to form a conjugate. In some embodiments, conjugates are selected based on their ability to facilitate ASO delivery to an organism or cell. ASO may bind to a nucleotide at, for example, the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or at a position not at the 3' end or 5' end of either strand.

[0154] Examples of conjugates include, but are not limited to, one or more antibodies or fragments thereof, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell-penetrating peptides, polymers such as hydrophilic polymers of polyethylene glycol, and sugar groups including sugar monomers, oligosaccharides, and modifications thereof. In a non-limiting example, the conjugate is N-acetylgalactosamine (GalNAc).

[0155] Methods for designing antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific to a given target sequence can be based on analysis of the selected target sequence and its secondary structure, T... m The binding energy and relative stability of ASO sequences can be determined through computational design or experimental methods.

[0156] Improved biodistribution of oligomeric LNPs with increased neutral lipid content

[0157] As described in the Examples section, the oligo-LNPs of this disclosure with elevated neutral lipid content can provide improved biodistribution to a wider range of tissues and / or organs than the Onpattro-type baseline formulations described in the Examples section herein. In another embodiment, the baseline formulation may be (a) an LNP that is otherwise identical in all respects with a 10% molar ratio of lower levels of the same neutral lipids; (b) an LNP that is otherwise identical in all respects with an N / P ratio of 1 or 3 when the N / P ratio of the LNP is equal to or greater than 4; and / or (c) an LNP that is otherwise identical in all respects with a total lipid weight nucleic acid / micromolar total lipid ratio of 0.20:1 when the lipid nanoparticles have a total lipid weight nucleic acid / micromolar total lipid ratio of 0.05:1 to 1:1, which is smaller than the total lipid weight nucleic acid / micromolar total lipid ratio of the lipid nanoparticles of this disclosure.

[0158] In one embodiment, the LNPs of this disclosure exhibit increased biodistribution in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear in a specified mouse model compared to a relevant baseline. In another embodiment, this includes increased biodistribution in extrahepatic tissues selected from the spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear relative to a relevant baseline. In yet another embodiment, this includes increased biodistribution in extrahepatic tissues selected from the spleen or bone marrow relative to a relevant baseline. Whether the LNP-encapsulated oligonucleotide oligo-LNP formulation exhibits such enhanced biodistribution in one or more given tissues or organs relative to baseline is determined by biodistribution studies in in vivo mouse models as detailed in the Examples section. Fluorescent lipid markers (DiD, as described in the Materials and Methods section) are used to assess the biodistribution of oligo-LNPs in a given tissue or organ relative to baseline. Animal studies are used as proxies to assess biodistribution in individuals such as patients in a clinical setting.

[0159] In one embodiment, relative to any of the aforementioned baselines, the biodistribution of the lipid nanoparticles in the liver, spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear of mice shows an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%, wherein the biodistribution in the mouse model is measured by detecting lipid markers at 1, 4, 10, and / or 24 hours after administration. Measurements are performed on tissue homogenates of one or more of the aforementioned tissues or organs, as described in the Examples section.

[0160] The percentage increase in fluorescence relative to the relevant baseline is determined by comparing the fluorescence signal of the LNP evaluated in the relevant tissue and / or organ / mg tissue homogenate with the fluorescence signal of the tissue homogenate generated by administering baseline LNP.

[0161] The compared oligo-LNPs were prepared using the same materials and methods. In other words, the two formulations compared had the same ionizable lipids, PEG-lipids, and sterols, and were prepared using rapid ethanol injection as described in the Materials and Methods section.

[0162] Biodistribution was assessed at the same time points (1, 4, 10 and / or 24 hours) after administration to the same mice, and marker lipids were measured using the same analytical techniques (see Materials and Methods section).

[0163] In those embodiments in which the baseline formulation has a neutral lipid (e.g., DSPC or sphingomyelin) that is 10% lower in molar ratio than the LNP of this disclosure, the neutral lipids may be reduced at the baseline at the expense of cholesterol and ionizable lipids in equal proportions, but the ionizable lipids:cholesterol (mol:mol) ratio remains constant between the baseline and LNP of this disclosure.

[0164] Clinical and non-clinical uses of LNP in this article

[0165] In some embodiments, oligo-LNPs are part of the pharmaceutical composition and are administered to treat and / or prevent disease states. Treatment may provide preventive (avoidant), ameliorative, or therapeutic benefits. The pharmaceutical composition will be administered at any suitable dose.

[0166] The oligo-LNPs described herein can be used to treat and / or prevent any disease, symptom, or disease state in a mammalian individual. This includes diseases, symptoms, or disease states such as cancer; infectious diseases such as bacterial, viral, fungal, or parasitic infections; inflammatory and / or autoimmune conditions, including treatments that induce immune tolerance; and cardiovascular diseases such as hypertension, arrhythmias, and restenosis.

[0167] Examples of cancers include lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small bowel cancer, stomach cancer (gastric cancer), esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), central nervous system cancers, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteosarcoma, and hematologic malignancies. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis from certain other non-hepatocellular carcinoma types), and hepatoblastoma.

[0168] Non-limiting examples of other diseases, conditions, or disease states that can be treated by the oligo-LNPs described in this article and can be at least partially attributed to immune disorders include colitis, Crohn's disease, allergic encephalitis, allogeneic graft transplantation / graft-versus-host disease (GVHD), diabetes, and multiple sclerosis.

[0169] The LNP described herein can also be used in applications other than treating and / or preventing diseases or conditions. LNPs can be used to treat disease states such as aging, as preventative medicine, and / or as part of a personalized medicine regimen. In other embodiments, LNPs are used in diagnostic applications.

[0170] In one embodiment, the LNP is part of a parenteral drug composition, i.e., administered intra-arterially, intravenously, subcutaneously, or intramuscularly. In another embodiment, the drug composition is administered intratumorally. In yet another embodiment, the drug composition is administered intranasally, intravitreally, subretinally, intrathecally, or via other local routes. In some embodiments, the oligo-LNP is applied or administered to the skin.

[0171] Pharmaceutical compositions contain pharmaceutically acceptable salts and / or excipients. As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt derived from a variety of organic and inorganic counterions well known in the art, and includes, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and, when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochlorides, hydrobromic acids, tartrates, methanesulfonates, acetates, maleates, and oxalates. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

[0172] As used herein, the term "excipient" refers to a substance used to formulate an active pharmaceutical ingredient (API) into a pharmaceutical preparation. Non-limiting examples include mannitol, Starch, magnesium stearate, sodium saccharin, talc, cellulose, 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 those skilled in the art.

[0173] The compositions described herein may be administered to individuals. As used herein, "individual" includes human or non-human individuals, including mammals. Oligo-LNPs may be administered as part of prophylactic treatment, and therefore individuals are not limited to patients.

[0174] The examples are intended to illustrate the preparation and properties of specific lipid nanoparticle oligomer formulations, but are by no means intended to limit the scope of the invention. Example

[0175] Materials and methods

[0176] Preparation of lipid nanoparticles containing oligonucleotides

[0177] LNPs were prepared by dissolving siRNA firefly luciferase or out-of-order antisense oligonucleotides (ASO) in 25 mM sodium acetate (pH 4.0) while simultaneously dissolving a specific percentage molar ratio of lipid components in anhydrous ethanol. The LNPs included the lipophilic fluorescent dye DiD perchlorate (2-[5-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indolyl-2-ylidene)-1,3-pentadien-1-yl]-3,3-dimethyl-1-octadecyl-3H-indolium perchlorate) to assess in vivo biodistribution. The lipids in ethanol and siRNA or ASO in the buffer were combined at a 1:3 volume ratio using a three-way connector with dual syringes. The solution was propelled through the three-way connector at a combined 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). The mixture was then dialyzed overnight with approximately 400 to 1000 volumes of 1× phosphate-buffered saline (pH 7.4) using a Spectro / Por dialysis membrane (molecular weight cutoff 12,000 to 14,000 Da) to remove ethanol from the formulation.

[0178] Biophysical characterization

[0179] Analysis of lipid nanoparticle size and morphology: using Malvern Zetasizer NanoZS via dynamic light scattering (DLS). TM Particle size analyzer (Worcestershire, UK) measures LNP size (number-weighted) and polydispersity index (PDI).

[0180] Analysis of encapsulation efficiency of lipid nanoparticles with siRNA or ASO

[0181] Using Quant-iT RiboGreen TM RNA detection (Life Technologies) TM The efficiency of siRNA or ASO encapsulation was determined using Triton X-100 (Sigma-Aldrich) in the presence or absence of 1% Triton X-100 (Sigma-Aldrich). TM In the case of St. Louis, MO, incubate LNP-siRNA or LNP-ASO at 37°C for 10 minutes, then add RiboGreen. TM Reagents. Fluorescence intensity was measured (Ex / Em: 480 / 520 nm), and samples treated with Triton X-100 represented total siRNA or ASO, while untreated samples represented unencapsulated siRNA or ASO.

[0182] Determination of the N / P ratio of lipid nanoparticles

[0183] First, the total number of negative charges (P) is 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). This value (mol * negative charge) is then multiplied by Avogadro's constant 6.022E23 / mol to obtain the total number of negative charges.

[0184] Similarly, the total number of positive charges on ionizable lipids can be calculated first using the molecular weight and amount of the ionizable lipids to provide the moles of ionizable lipids used in the formulation. This value, multiplied by the number of positive charges per molecule (1), provides a value in mol* of positive charge, which is then multiplied by Avogadro's constant 6.022E23 / mol to obtain the total number of positive charges. The N / P ratio is calculated by taking the ratio of positive charges on ionizable lipids to negative charges on nucleic acids. Measurement of fluorescence in intact organs / tissues in vivo.

[0185] In CD-1 mice, LNP was administered intravenously (iv) at a concentration of 1.0 mg / kg siRNA in a volume of 10 μL (grams per mouse). Twenty-four hours post-injection, mice were anesthetized with 5% isoflurane (set to 1% airflow) and then asphyxiated with CO2 until the animals lost their reflexes. Cardiac puncture was performed using a 1 mL needle and blood was collected. The animals were then transferred to a PerkinElmer... TM Manufacturing in vivo imaging system (IVIS) TM Imaging on the surface.

[0186] After imaging, the animal was cut from the bladder to the thorax, and the skin was pinned back. The spleen (S), lung (Lu), liver (Li), and kidney (K) were removed from the abdominal cavity and placed on a plastic dish. The animal with intact organs was then placed in IVIS. TM Imaging was performed on an imaging device, and organs were subsequently removed from the mice and placed in plastic dishes.

[0187] Tissue homogenate assay

[0188] Tissue was removed from mice and placed in 2 mL tubes and rapidly frozen in liquid nitrogen. The tissue was then frozen at -80°C. Appropriate volumes of Promega... TM GLO TM Add lysis buffer to each tube, ensuring the sample is frozen before adding the lysis buffer. Place the sample in FastPrep. TM In the homogenizer, the homogenizer was operated and repeated twice, for a total of three rounds. The homogenized sample was centrifuged at room temperature, and then the homogenized product 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.

[0189] If analyzing bone marrow tissue homogenate, a femur is removed from a mouse. The femur is cut and then centrifuged to separate the bone marrow. The bone marrow is then processed as described above for tissue analysis to prepare a homogenate and measure fluorescence.

[0190] Cryo-electron microscopy

[0191] Prior to cryo-electron microscopy imaging, LNPs were concentrated to an estimated total lipid concentration of 15 to 25 mg / mL. A determined volume, e.g., 2 to 4 μL, of the resulting LNP solution was added to glow-discharged copper grids and impregnated with FEI Mark IV Vitrobot to produce vitreous ice. These grids were stored in liquid nitrogen until imaging via FEI Titan Krios or FEI GlaciosTEM. The instrument was operated at 200 kV under low-dose conditions, and the resulting images were acquired at underfocus of 0.5 to 2 μm using a bottom-mounted FEI Falcon direct electron detector camera at magnifications of 47 to 88,000 × 1000 to enhance contrast.

[0192] Example 1: Elevated levels of neutral lipids (e.g., DSPC) and decreased levels of ionizable lipids in siRNA-LNP do not negatively affect biophysical properties.

[0193] At the expense of ionizable lipids and cholesterol, a variety of siRNA-LNPs (firefly luciferases) with DSPC content ranging from 10% to 55% molar ratio were prepared to evaluate their biophysical properties. Specifically, according to the Materials and Methods section, the following were evaluated: ionizable lipids (such as MC3 or MF019 as shown); DSPC; and PEG-lipids (PEG-LNPs) maintained at a constant molar ratio of 1.5%. 2000 Encapsulation efficiency, size, and polydispersity index (PDI) of a four-component siRNA-LNP formulation of cholesterol (DMG) (reported as a percentage molar ratio). As shown in Table 1, the N / P ratio for each LNP is 3 or 6.

[0194] Table 1: Distearate phosphatidylcholine (DSPC) LNP formulations tested for biophysical properties

[0195]

[0196]

[0197] *MF019 is an ionizable thiolipin described in PCT patent application No. PCT / CA2022 / 050042, which is incorporated herein by reference; and MC3 represents DLin-MC3-DMA ionizable lipid (dilinoleyl-methyl-4-dimethylaminobutyrate).

[0198] The size, polydispersity index (PDI), and encapsulation percentage of each formulation in Table 1 above are shown in Table 1. Figure 1 middle.

[0199] The inventors discovered that despite increased DSPC levels and decreased ionizable lipid content, each formulation exhibited acceptable biophysical properties. While those skilled in the art would expect that the encapsulation efficiency of siRNA would decrease with decreasing ionizable lipids, however… Figure 1 The results showed that even with a decrease in the content of ionizable lipids (accompanied by an increase in the content of neutral lipids), the encapsulation efficiency in the tested formulations remained relatively constant.

[0200] Example 2: Elevated levels of neutral lipids (e.g., DSPC) in siRNA-LNP significantly improved in vivo biodistribution.

[0201] The effect of increasing the amount of DSPC from 40% molar ratio to 50% molar ratio in a four-component LNP containing siRNA cargo with an N / P ratio of 6 was evaluated in vivo.

[0202] 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:

[0203] Table 2: Selection of DSPC LNP formulations for in vivo biodistribution

[0204]

[0205]

[0206] *MF019 is an ionizable lipid as described in PCT / CA2022 / 050042 (same as above).

[0207] like Figure 2A , 2B As shown in 2C, siRNA-LNPs with a 50% molar ratio of 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-indolyl-2-ylidene)-1,3-pentadien-1-yl]-3,3-dimethyl-1-octadecyl-3H-indolium perchlorate 24 hours post-injection. Conversely, LNPs with only a 40% molar ratio of DSPC showed fluorescence in the liver but exhibited minimal signal in the spleen. Furthermore, almost no detectable fluorescence was observed in organs where LNPs with an N / P ratio of 3 (40% molar ratio of DSPC) were detected.

[0208] Example 3: Increased sphingomyelin levels and decreased ionizable lipid levels in siRNA-LNP do not negatively affect biophysical properties.

[0209] A variety of siRNA-LNPs with sphingomyelin contents ranging from 10% to 55% were prepared to evaluate their biophysical properties. The LNPs also contained varying molar percentages of ionizable lipids and PEG-lipids (PEG-LNPs). 2000 -DMG) was kept constant at a cholesterol molar ratio of 1.5% (as shown). Encapsulation efficiency, size, and polydispersity index (PDI) of siRNA-LNP were evaluated as described in the Materials and Methods section. Sphingomyelin content was increased at the expense of cholesterol and ionizable lipids, and N / P was 3 or 6 as shown in Table 3.

[0210] Table 3: Sphingomyelin LNP formulations tested for biophysical properties

[0211]

[0212]

[0213] *MF019 is the ionizable sulfur lipid described in PCT / CA2022 / 050042 (same as above); and MC3 represents DLin-MC3-DMA ionizable lipid (dilinolenic-methyl-4-dimethylaminobutyrate).

[0214] The size, polydispersity index (PDI), and encapsulation percentage of each formulation in Table 3 above are shown in Table 3. Figure 3 middle.

[0215] The inventors have found that, despite increased sphingomyelin levels and decreased ionizable lipid content, each siRNA-LNP formulation typically exhibits acceptable biophysical properties.

[0216] Example 4: Elevated levels of neutral lipids (e.g., DSPC and sphingomyelin) in siRNA-LNP improved in vivo biodistribution measured in tissue homogenates.

[0217] Biodistribution studies were conducted on various four-component siRNA-LNP formulations containing 40 and 50% molar ratios of DSPC or egg yolk sphingomyelin (ESM). Results showed that siRNA-LNPs with 50% molar ratios of DSPC or ESM generally exhibited significantly higher LNP concentrations in the organs (tissue homogenates) measured, compared to LNPs with less than 10% molar ratios of neutral lipids (40% molar ratio). This increase in neutral lipid content (DSPC and ESM) occurred at the expense of ionizable lipids and cholesterol.

[0218] The results also showed that, compared with the same formulation with an N / P ratio of 6, siRNA-LNP with an N / P ratio of 3 and a 40% molar ratio of DSPC had significantly reduced fluorescence intensity.

[0219] The LNPs examined are listed in Table 4 below.

[0220] Table 4: DSPC and sphingomyelin siRNA-LNP formulations examined in tissue homogenate rat biodistribution studies.

[0221]

[0222]

[0223] *MF019 is an ionizable sulfur lipid described in PCT / CA2022 / 050042 (same as above).

[0224] The LNP formulation exhibited high encapsulation efficiency (>90%) at both 27.4% and 33% molar concentrations of ionizable lipids.

[0225] The half-maximal inhibitory concentration (IC50) of most LNPs 50 The value is within an acceptable range.

[0226] Tissue homogenates were prepared as described in the Materials and Methods section. The fluorescence intensity of the tissue homogenates was measured using a DiD fluorescent marker as described.

[0227] like Figure 4A As shown, the fluorescence intensity of LNPs containing 50% molar sphingomyelin in liver homogenate was approximately 2-fold higher than that of LNPs containing only 40% molar sphingomyelin (LNP-A and LNP-B). A similar increase in fluorescence was observed for LNPs containing 50% molar sphingomyelin compared to 40% molar DSPCs (LNP-C and LNP-D). Formulations with an N / P ratio of 6 (LNP AD) all showed increased fluorescence intensity compared to 40% molar DSPC LNPs (LNP-E) with an N / P ratio of 3.

[0228] like Figure 4B As shown, the fluorescence intensity of LNPs containing 50% molar sphingomyelin in spleen homogenate was similar to that of LNPs containing only 40% molar sphingomyelin (LNP-A and LNP-B). However, a significant increase in fluorescence intensity was observed in LNPs containing 50% molar DSPC compared to LNPs containing only 40% molar DSPC (LNP-C and LNP-D). The formulation with an N / P ratio of 6 (LNP AD) again showed higher fluorescence intensity compared to the 40% molar DSPC LNP (LNP-E) with an N / P ratio of only 3.

[0229] Figure 4C and 4D The biodistribution of siRNA-LNP in the abdominal and dorsal skin of mice, as shown in Table 4, is presented respectively. Figure 4C As shown, the fluorescence intensity of LNPs containing 50% molar ratio of egg yolk sphingomyelin (ESM) in abdominal skin was increased compared to that of LNPs containing only 40% molar ratio of ESM (LNP-A and LNP-B). A significant increase in fluorescence intensity was observed in the 50% molar ratio of DSPC in abdominal skin compared to LNPs containing only 40% molar ratio of DSPC (LNP-C and LNP-D). All DSPC-containing formulations with an N / P ratio of 6 showed higher fluorescence intensity compared to a 40% molar ratio DSPC formulation with an N / P ratio of only 3. Similar results were observed in the dorsal skin of mice (see...). Figure 4D ).

[0230] The results of the biodistribution of the ear are shown in Figure 4E In the figure, the fluorescence intensity of siRNA-LNP containing 50% molar sphingomyelin in the ear was significantly higher than that of LNP containing only 40% molar sphingomyelin (LNP-A and LNP-B). Compared to LNPs containing only 40% molar DSPC (LNP-C and LNP-D), LNPs containing 50% molar DSPC showed a more than two-fold increase in fluorescence intensity in the ear. Most formulations with an N / P ratio of 6 exhibited higher fluorescence intensity than the 40% molar DSPC formulation with an N / P ratio of only 3.

[0231] Example 5: The four-component siRNA-LNP with elevated levels of phosphatidylcholine lipids exhibited a surprising morphology.

[0232] The morphology of the following LNP formulation was determined by cryo-electron microscopy as described in the Materials and Methods section. The cargo is siRNA targeting firefly luciferase.

[0233] Table 5: LNP formulations evaluated by cryo-electron microscopy

[0234]

[0235] Figure 11A , 11B Images from 11C show that siRNA-LNP exhibits a unique morphology at elevated phosphatidylcholine levels (e.g., DSPC), with an electron-dense region and an aqueous portion of the nucleus, which are subsequently surrounded by a double layer.

[0236] Example 6: The four-component ASO-LNP with elevated levels of phosphatidylcholine lipids showed a surprising increase in extrahepatic organ delivery.

[0237] This example investigated the effect of increasing the content of representative phosphatidylcholine lipids (DSPC) from 10% molar ratio to 50% molar ratio on the biodistribution of ASO-LNP. The increase in DSPC content came at the cost of ionizable lipids and cholesterol.

[0238] Lipid nanoparticles encapsulating 50-mer and 221-mer ssRNA were prepared as described in the method section above. The lipid composition of the studied ASO-LNPs is provided in Table 6 below:

[0239] Table 6: ASO-LNP formulations studied in biodistribution studies

[0240]

[0241] a. The ionizable lipid is MC3 (DLin-MC3-DMA or "MC3" (dilinoleyl-methyl-4-dimethylaminobutyrate)).

[0242] b. The ionizable lipid is MF019, which is described in PCT / CA2022 / 050042 filed on January 12, 2022, and disclosed as WO2022 / 155728A1, and is incorporated herein by reference.

[0243] LNPs A and B encapsulate 50-mer ssRNA, while LNPs C and D encapsulate 221-mer ssRNA. LNPs A and C are Onpattros with a 10% molar ratio of DSPC content. TM The composition, wherein B and D are compositions having a 50% molar ratio of DSPC. Contains Onpattro TM The compositions with high DSPC are 50 / 10 / 37.75 / 1.5 / 0.75 and 27.4 / 50 / 20.35 / 1.5 / 0.75 mol / mol ionizable lipids / DSPC / cholesterol / PEG-DMG / DiD, respectively.

[0244] ASOs are non-coding single-stranded RNAs (ssRNAs) of length 50 nucleotides (“50mer”) and 221 nucleotides (“221mer”). ssRNAs are generated randomly, ensuring that no complementary binding occurs for biodistribution experiments. The 50mer and 221mer are produced via Integrated DNA Technologies. TM (IDT) synthesis. The two sequences are listed in Table 7 below.

[0245] Table 7: ASO Sequences

[0246]

[0247] like Figures 5 to 10 As shown, ASO-LNP containing 50% molar ratio of DSPC (LNP B and D) exhibited a surprising improvement in biodistribution to the spleen, bone marrow, heart, lung, and kidney compared to lipid nanoparticles containing 10% molar ratio of DSPC (LNP A and C) 24 hours after administration. Figures 6 to 10 A surprising improvement in the biodistribution of both LNP B and LNP D was observed using 50-mer ASO (LNP B) and 221-mer ASO (LNP D). Figure 5 As shown, in the liver, the biodistribution of ASO-LNP with 50% molar ratio DSPC (LNP B) and 50% molar ratio ASO was improved compared to ASO-LNP with only 10% molar ratio DSPC (LNP A).

[0248] This specification is intended to illustrate embodiments and examples of the invention, but is in no way intended to limit the scope of the invention as defined by the appended claims.

Claims

1. Lipid nanoparticles, comprising: (i) an oligonucleotide molecule, wherein the oligonucleotide molecule is single-stranded or double-stranded and has a length of 5 to 500 nucleotides; (ii) Neutral lipid contents in a 30% to 70% molar ratio; (iii) Ionizable lipid contents in a molar ratio of 5% to 50%; (iv) Sterols selected from cholesterol or its derivatives; and (v) Optionally, a hydrophilic polymer-lipid conjugate present at a lipid content of 0.5% to 5% molar ratio. Each lipid content is relative to the total lipid content of the lipid nanoparticles, provided that the phosphatidylcholine lipid contents are not egg yolk lecithin (EPC).

2. Lipid nanoparticles comprising encapsulated oligonucleotide molecules, wherein the oligonucleotide molecules are single-stranded or double-stranded and have a length of 5 to 500 nucleotides; and neutral lipid contents, ionizable lipids, at a molar ratio of 20 to 70% relative to the total lipids present in the lipid nanoparticles; sterols; and optionally, hydrophilic polymer-lipid conjugates, measured under the same conditions as a baseline 50 / 10 / 38.5 / 1.5 mol:mol ratio of encapsulated oligonucleotide molecules in an Onpattro-type formulation of MC3 / DSPC / cholesterol / PEG-lipids, wherein the lipid nanoparticles show at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear, and wherein the biodistribution is quantified in animal models by detecting labeled lipids 24 hours after administration, wherein the neutral contents comprise no more than three different phosphatidylcholine lipids.

3. Lipid nanoparticles, comprising: (i) an oligonucleotide molecule, wherein the oligonucleotide molecule is single-stranded or double-stranded and has a length of 5 to 500 nucleotides; (ii) Phosphatidylcholine lipid contents in a 30% to 70% molar ratio; (iii) Ionizable lipid contents in a molar ratio of 5% to 50%; (iv) Sterols selected from cholesterol or its derivatives; and (v) Hydrophilic polymer-lipid conjugates present with lipid content ranging from 0% to 5% molar ratio. Each lipid content is relative to the total lipid content of the lipid nanoparticles. Furthermore, as can be seen by cryo-electron microscopy, the lipid nanoparticles contain a non-uniform core with an electron-dense region and an aqueous portion, and are surrounded by an outer lipid layer.

4. The lipid nanoparticles according to claim 1, 2 or 3, wherein the neutral lipid is a phosphatidylcholine lipid selected from distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC).

5. The lipid nanoparticles of claim 4, wherein the phosphatidylcholine lipid is distearate phosphatidylcholine (DSPC) or dioleoyl phosphatidylcholine (DOPC).

6. The lipid nanoparticles according to any one of claims 1 to 5, wherein the neutral lipid content is 40% molar ratio to 60% molar ratio.

7. The lipid nanoparticles of claim 6, wherein the neutral lipid content is from 42% to 60% molar ratio.

8. The lipid nanoparticles of claim 7, wherein the neutral lipid content is from 45% to 60% molar ratio.

9. The lipid nanoparticles of claim 8, wherein the neutral lipid content is from 46% to 60% molar ratio.

10. The lipid nanoparticles of claim 9, wherein the neutral lipid content is from 48% to 60% molar ratio.

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

12. The lipid nanoparticles according to any one of claims 1 to 11, wherein the ionizable lipids are present in a molar ratio of less than 40%.

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

14. The lipid nanoparticles according to any one of claims 1 to 13, wherein the sterols are present in a molar ratio of 15% to 45% based on the total lipids present in the lipid nanoparticles.

15. The lipid nanoparticles according to any one of claims 1 to 14, wherein the sterols are present in a molar ratio of 18% to 40% based on the total lipids present in the lipid nanoparticles.

16. The lipid nanoparticles of claim 1 or 3, wherein, measured under the same conditions, the lipid nanoparticles show at least a 10% increase in biodistribution in the spleen, bone marrow, heart, lung, kidney, abdominal skin, back skin, and / or ear, compared to a baseline Onpattro formulation of MC3 / DSPC / cholesterol / PEG-lipids encapsulated with oligonucleotide molecules at a ratio of 50 / 10 / 38.5 / 1.5 mol:mol, and wherein the biodistribution is quantified in an animal model by detecting labeled lipids 24 hours after administration.

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

19. A method for in vivo delivery of an oligonucleotide molecule into a mammalian individual, wherein the oligonucleotide molecule is single-stranded or double-stranded and has a length of 5 to 500 nucleotides, the method comprising: The lipid nanoparticles of any one of claims 1 to 18 are administered to the mammalian individual.

20. A method for delivering oligonucleotide molecules to cells, the method comprising contacting the cells with lipid nanoparticles as claimed in any one of claims 1 to 18 in vivo or in vitro.

21. 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 a mammalian individual 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 diseases or conditions of autoimmune diseases.

23. The method of any one of claims 19 to 21, wherein the lipid nanoparticles are used to treat diseases or conditions of infectious diseases.

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

25. Use of the lipid nanoparticles of any one of claims 1 to 18 for delivering oligonucleotide molecules into mammalian cells in vivo or in vitro.

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

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