Ionized lipids, lipid nanoparticles, and uses thereof

Abstract

Provided herein are ionized lipids and lipid nanoparticles comprising the ionized lipids, as well as methods of making and using the lipid nanoparticles to deliver one or more payload agents, such as polypeptides, mRNA (e.g., mRNA encoding a Cas9 nuclease), and guide RNAs that include backbone, sugar, or base modifications, to cells.

Description

[Technical field]

[0001] Prior Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 267,084, filed January 24, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Gene therapy and vaccination rely on effective, specific, and tolerable delivery of therapeutic agents in vivo. Lipid nanoparticles (LNPs) for in vivo delivery have emerged as promising delivery vehicles for many therapeutic applications. However, there remains a need for LNPs with improved therapeutic efficacy and minimal toxicity for applications such as gene editing. Summary of the Invention [Means for solving the problem]

[0003] overview Provided herein are ionizable lipids and LNPs comprising ionizable lipids for in vitro, ex vivo, or in vivo delivery of agents. For example, described herein are ionizable lipids having the formula I [ka] A lipid nanoparticle (LNP) comprising an ionized lipid of In the formula, R1 and R2 are independently [ka] is selected from the group consisting of X1 and X2 are hydrolyzable linkers, and (a) is 2-9 (i.e., 2, 3, 4, 5, 6, 7, 8, or 9 carbons) and (b) is 2-9 (i.e., 2, 3, 4, 5, 6, 7, 8, or 9 carbons). In some embodiments, the hydrolyzable linker in the LNP is an ester. In some embodiments, the LNP has Formula II [ka] Contains ionized lipids.

[0004] In some embodiments, the LNP further comprises cholesterol, a helper phospholipid, and a polyethylene glycol (PEG)-lipid. In some embodiments, about 20% to about 80% of the total lipid content of the LNP is an ionizable lipid of Formula I or Formula II.

[0005] In some embodiments, the LNP comprises: (a) 20%-80% ionizable lipid of formula I; (b) 5%-20% helper lipid; (c) 10%-60% cholesterol; and (d) 0.5%-5% polyethylene glycol (PEG).

[0006] In some embodiments, the LNP further comprises a polypeptide, a nucleic acid, or a combination thereof. In some embodiments, a nucleic acid encoding a heterologous polypeptide is encapsulated in the LNP. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA encodes an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is a Cas9 nuclease. In some embodiments, the LNP comprising an mRNA encoding an RNA-guided endonuclease further comprises a guide RNA (gRNA). The guide RNA may be a modified guide RNA. Optionally, the guide RNA comprises one or more modifications selected from the group consisting of a backbone modification, a sugar modification, and a base modification. By way of example, the sugar modification may be a 2'OMe modification or a 2'F modification. In some embodiments, the backbone modification is a thioester modification. Optionally, the gRNA comprises a nucleic acid sequence of 100 nucleotides, and 69 of the 100 nucleotides are modified.

[0007] The mRNA encoding the RNA-guided endonuclease and the guide RNA are encapsulated separately and then combined to form the LNP. In some embodiments, the mRNA encoding the RNA-guided endonuclease and the guide RNA are co-encapsulated in the LNP. Optionally, the ratio of nitrogen groups from the ionized lipid to phosphate groups in the mRNA and / or gRNA in the LNP is about 4 to about 6. In some embodiments, the LNP further comprises at least one nucleic acid template.

[0008] Also provided are any multiples of the LNPs described herein. Additionally, provided are pharmaceutical compositions comprising one or more of the LNPs described herein.

[0009] Further provided are methods of delivering a heterologous polypeptide to a cell, comprising contacting the cell with one or more LNPs comprising a heterologous polypeptide, or comprising contacting the cell with an LNP or LNPs comprising a nucleic acid encoding a heterologous polypeptide as described herein. Also provided are methods of delivering a heterologous polypeptide to a cell, comprising contacting the cell with a pharmaceutical composition comprising one or more LNPs comprising a heterologous polypeptide, or comprising contacting the cell with a pharmaceutical composition comprising one or more LNPs comprising a nucleic acid encoding a heterologous polypeptide as described herein.

[0010] Also provided is a method of editing the genome of a cell, comprising contacting the cell with one or more LNPs comprising a nucleic acid, the nucleic acid encoding an RNA-guided endonuclease and a guide RNA as described herein, or contacting the cell with one or more LNPs comprising a ribonucleoprotein complex (RNP), the RNA comprising an RNA-guided endonuclease and a guide RNA as described herein. Also provided is a method of editing the genome of a cell, comprising contacting the cell with a pharmaceutical composition, the pharmaceutical composition comprising one or more LNPs comprising a nucleic acid encoding an RNA-guided endonuclease as described herein and a guide RNA, or contacting the cell with a pharmaceutical composition comprising one or more LNPs comprising a ribonucleoprotein complex (RNP), the RNP comprising an RNA-guided endonuclease and a guide RNA as described herein. The cell is optionally edited in vitro, ex vivo, or in vivo. In some embodiments, the cells are human cells.

[0011] Also provided are cells comprising one or more of the LNPs described herein, as well as genetically modified cells produced by any of the methods described herein.

[0012] Description of the drawings This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the present compositions and methods, and to supplement the description of the present compositions and methods. The drawings are not intended to limit the scope of the present compositions and methods, unless expressly indicated as such in the description herein. [Brief description of the drawings]

[0013] [Figure 1] A comparison of formulation 1 (F1) (formulation containing ionizable lipid PCTA-1 (Formula II)), comparative formulation 1 (CF1), and comparative formulation 2 (CF2) is shown. All formulations were packaged with Cas9 mRNA and sgRNA targeting either TTR or PCSK9. When targeting TTR (first three columns from the left), F1 (formulation containing PCTA-1) achieved the highest editing, showing improved editing compared to CF2, and a large (approximately 5-fold higher) improvement compared to CF1. When targeting PCSK9 (first three columns from the right), F1 achieved significantly higher editing (2-3-fold higher) compared to both CF2 and CF1 when using the same sgRNA. [Diagram 2] Shown are TTR sgRNA sequences with selected chemical modifications (guide 1 (SEQ ID NO: 1)), guide 2 (SEQ ID NO: 1), guide 3 (SEQ ID NO: 2), and guide 4 (SEQ ID NO: 3). Bases in capital letters are ribonucleotides. Bases in lower case letters are 2'-OMe ribonucleotides. Guide 2 is SEQ ID NO: 1 ( [ka] ) contains the 2'-deoxy-2'-fluoro-ribonucleotides underlined in the parentheses. [Figure 3A] FIG. 3 shows the results of an in vitro primary mouse hepatocyte screen showing similar editing for all guide RNAs listed in FIG. [Figure 3B]FIG. 3 shows the results of an in vitro primary mouse hepatocyte screen demonstrating reduced cell supernatant protein levels for all guide RNAs described in FIG. [Figure 4A] 1 shows in vivo results for literature nanoparticle formulations. [Figure 4B] A comparison of literature formulations with nanoparticle formulations containing PCTA-1 (Formula II) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description Provided herein are ionizable lipids and lipid nanoparticles (LNPs) comprising the ionizable lipids that can be used to deliver drugs to one or more cells (e.g., delivery of nucleic acids, polypeptides, or combinations thereof to cells). As used herein, an ionizable lipid is a lipid molecule that is neutral at physiological pH, but is protonated and positively charged at low pH. The ionizable lipids described herein use a combination of branched alkyl and linoleyl tails with pKa-tuned ethanolamine head groups to allow efficient loading, delivery, and release of drugs from the LNPs. The ionizable lipids also include hydrolyzable linkers to allow for rapid lipid clearance after LNP destruction (e.g., delivery of LNPs to cells), thereby limiting toxicity.

[0015] As used herein, formula I [ka] wherein R1 and R2 are independently: [ka] is selected from the group consisting of An ionizable lipid is provided, where X1 and X2 are hydrolyzable linkers, (a) is 2 to 9, and (b) is 2 to 9. In some embodiments, the hydrolyzable linker is an ester. In some embodiments, the ionization is performed according to formula II (PCTA-1): [ka] is a lipid.

[0016] Also provided are LNPs comprising any of the ionizable lipids described herein. For example, provided herein are LNPs comprising an ionizable lipid of Formula I or Formula II. The ionizable lipids of Formula I or Formula II have a unique combination of chemical features that allow for successful delivery of payloads (e.g., nucleic acids, polypeptides, small molecules, etc.). The ethanolamine head group has a pKa in the range of 6.2-6.8, such that the amine is uncharged at physiological pH but positively charged at acidic pH. Additionally, the ester bond is hydrolyzable, thus allowing for rapid clearance of the lipid after delivery of the payload. The unsaturation in the linoleyl tail prevents ordered membrane packing, allowing the LNP to release the payload and escape the endosome more easily. The branched alkyl tail serves a similar function. The combination of these two tails with the tuned pKa of the ethanolamino head group allows for efficient payload loading, delivery, and release, while the hydrolyzable linker limits toxicity by allowing for rapid lipid clearance after LNP destruction.

[0017] As used herein, lipid nanoparticles (LNPs) are spherical vesicles in the nanometer range. Generally, lipid nanoparticles are taken up by cells by endocytosis, and the ionizable nature of lipids at low pH allows escape from endosomes, which allows release of cargo into the cytoplasm. In addition, lipid nanoparticles optionally contain helper lipids to promote cell binding, cholesterol to fill gaps between lipids, and / or polyethylene glycol (PEG) to reduce opsonization and reticuloendothelial clearance by serum proteins. LNPs containing ionizable lipids can carry nucleic acids, polypeptides, drugs, or other substances that are encapsulated by or contained within the lipid layer. LNPs can vary in size (i.e., diameter). For example, LNPs can be about 1000 nanometers (nm) or less in size. For example, LNPs can be about 50 nm to about 1000 nm, about 50 nm to about 900 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nm to about 100 nm in size. LNPs described herein include LNPs that include an ionized lipid and one or more payload agents, such as, for example, a nucleic acid (e.g., mRNA, gRNA, siRNA, antisense molecule, or aptamer), a polypeptide, a ribonucleoprotein complex (e.g., an RNA-guided endonuclease complexed with a gRNA), a drug, or a small molecule, to name a few. Combinations of these agents, such as, for example, an mRNA and a guide RNA, can also be present within any of the LNPs described herein. As used throughout, an encapsulated agent refers to an agent that is completely or partially located in the interior aqueous or non-aqueous space of the LNP. For example, in the LNPs described herein, at least about 75%, 80%, 85%, 90%, 95%, or 99% of the agent is entrapped in the interior space of the LNP.

[0018] Pluralities or populations of two or more of the LNPs described herein are also provided. For example, the LNPs may be present in a range of from about 2 to about 1×10 14 A plurality of LNPs may include up to 100 trillion LNPs. For example, a plurality of LNPs may have at least 100, 250, 500, 750, 1000, 5000, 10,000, 25,000, 50,000, 100,000, 500,000, 1 million, or more LNPs. The plurality of LNPs may optionally differ in payload, type of ionizable lipid, and / or size. The LNPs may be produced by any suitable method known to those of skill in the art or may be produced by any suitable method later discovered by those of skill in the art.

[0019] Exemplary methods for preparation of LNPs are described in Parhi and Suresh ("Preparation and characterization of solid lipid nanoparticles-a review," Curr. Drug Discov. Technol. 9(1):2-16 (2012)) and Corrias and Lai ("New methods for lipid nanoparticles preparation," Recent. Pat. Drug. Deliv. Formul. 5(3):201-213 (2011)), which are incorporated by reference in their entireties.

[0020] In some embodiments, about 20% to about 80% of the total lipid content of any of the LNPs described herein is an ionizable lipid, e.g., an ionizable lipid of formula I, or an ionizable lipid of formula II. Optionally, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any percentage between these percentages of the total lipid content of the LNP can be an ionizable lipid of formula I, or an ionizable lipid of formula II. Any of the LNPs that include an ionizable lipid of formula I or formula II can further include one or more additional lipids. In general, a variety of lipid components can be used. These include neutral lipids that exist in either uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.The synthetic derivatives of any of the lipids described herein can also be used to manufacture lipid nanoparticles.Lipid nanoparticles can also include sterols, for example, cholesterol.Lipid nanoparticles can also include cationic lipids that carry a net positive charge near physiological pH.Such cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP.Cl); 3. beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ( "DC-Chol"), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoroacetate ("DOSPA"), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Anionic lipids are also suitable for use in the lipid nanoparticles described herein. These include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids.

[0021] In some examples, the LNP comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG), sphingomyelin, cholesterol, or any combination thereof. In some embodiments, the PEG can be from PEG molecular weight (MW500) to PEG-MW20000.

[0022] In some embodiments, the LNPs comprise an ionizable lipid having Formula I or Formula II, cholesterol, a helper phospholipid, and a polyethylene glycol lipid (e.g., pegylated myristoyl diglyceride (DMG-PEG2000)). In some embodiments, the LNPs comprise an ionizable lipid having Formula I or Formula II, cholesterol, DSPC, and DMG-PEG2000. Optionally, the LNPs comprise about 45%-55% ionizable lipid having Formula I or Formula II, about 35%-45% cholesterol, about 5%-10% DSPC, and about 1%-5% DMG-PEG2000. In some embodiments, the LNPs comprise about 50% ionizable lipid having Formula I or Formula II, about 38% cholesterol, about 10% DSPC, and about 2% DMG-PEG2000.

[0023] Optionally, the LNP comprises about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any percentage between these percentages of an ionizable lipid of Formula I or Formula II; about 5%, 10%, 15%, 20%, or any percentage between these percentages of a helper lipid; about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any percentage between these percentages of cholesterol; and about 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, or any percentage between these percentages of PEG.

[0024] LNPs optionally include a targeting molecule or moiety attached to or incorporated within the lipid layer of the LNP. The targeting molecule or moiety can be used to target the LNP to a specific cell or tissue in vitro, ex vivo, or in vivo to deliver the payload agent encapsulated in the LNP to the cell. As used throughout, a targeting molecule refers to a molecule that has binding affinity (optionally specific binding affinity) for an antigen on a cell, and can include, but is not limited to, an antibody, a polypeptide, a peptide, an aptamer, or a small molecule. As used herein, an antigen on a cell can be, but is not limited to, a protein, a lipid, or a carbohydrate.

[0025] Drug-containing LNP The LNPs described herein include LNPs that include ionizable lipids and payload agents. The agent can be, for example, a nucleic acid (e.g., mRNA, gRNA, siRNA, antisense molecule), a polypeptide, a ribonucleoprotein complex (e.g., an RNA-guided endonuclease complexed with a guide RNA), a drug, or a small molecule that is encapsulated (partially or completely) in the LNP. In some embodiments, the agent is a polypeptide. As used throughout, polypeptide, protein, and peptide are used interchangeably to refer to a polymer of amino acid residues. As used herein, the terms encompass any length of amino acid chain (e.g., full-length protein) in which the amino acid residues are linked by covalent peptide bonds.

[0026] In some embodiments, the encapsulated agent is a nucleic acid. As used throughout, the term nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. In some embodiments, the nucleic acid comprises an exon sequence, an intron sequence, a regulatory sequence (e.g., a promoter, an enhancer, or a silencer), a transcription control sequence, a translation control sequence, a splicing site, or a non-coding sequence. Optionally, the nucleic acid is a nucleic acid that codes for a polypeptide, such as a DNA molecule that codes for a polypeptide, or an RNA molecule (e.g., an mRNA) that codes for a polypeptide. Optionally, the nucleic acid that codes for a polypeptide can be operably linked to one or more of an intron sequence, a regulatory sequence (e.g., a promoter, an enhancer, or a silencer), a transcription control sequence, a translation control sequence, a splicing site, or a non-coding sequence. As used herein, a nucleic acid is operably linked when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence, and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation of the coding sequence.

[0027] Optionally, the mRNA encodes an RNA-guided endonuclease. The CRISPR / Cas system, an RNA-guided nuclease system that uses Cas endonuclease, can be used to edit the genome of a host cell or host organism. The CRISPR / Cas system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease (e.g., Cas9) in a complex with a guide RNA and an activator RNA to recognize and cleave foreign nucleic acids. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual activity guide RNAs are referred to as single guide RNAs (sgRNAs).

[0028] As used herein, the term Cas9 or Cas9 nuclease refers to an RNA-mediated nuclease (e.g., an RNA-mediated nuclease of bacterial or archaeal origin, or an RNA-mediated nuclease derived from a bacterial or archaeal nuclease). Exemplary RNA-mediated nucleases include the aforementioned Cas9 protein and its homologues. Other RNA-mediated nucleases include Cpf1 (see, e.g., Zetsche et al. (2015) Cell163(3):759-771), Cas13-based RNA editors, and their homologues.

[0029] Cas9 homologs have been found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs are described, for example, in Chylinksi et al. (2013) RNA Biol. 10(5):726-737; Makarova et al. (2011) Nat. Rev. Microbiol. 9(6):467-477; Hou et al. (2013) Proc Natl Acad Sci USA 110(39):15644-49; Sampson et al. (2013) Nature 497(7448):254-7; and Jinek et al. (2012) Science 337(6096):816-21. Any variant of the Cas9 nuclease provided herein may be optimized for efficient activity or enhanced stability in a host cell. Thus, engineered Cas9 nucleases are also contemplated. See, e.g., Slaymaker et al. (2016) Rationally engineered Cas9 nucleases with improved specificity, Science 351(6268):84-88.

[0030] Cas9 endonuclease optionally binds to a target nucleic acid and generates a double-stranded break. However, Cas9 protein may be a nickase, so that when it binds to a target nucleic acid as part of a complex with a guide RNA, a single-stranded break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a guide RNA with a different structure, may be targeted to two proximal sites of a target genomic region, so that a pair of proximal single-stranded breaks may be introduced into the target genomic region. A nickase pair may enhance specificity, because off-target effects are more likely to cause a single nick, which is generally repaired by base excision repair mechanisms without damage. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations. See, e.g., Ran et al. (2013), Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity, Cell 154(6):1380-1389.

[0031] Also provided herein is a LNP that comprises one or more guide RNAs or comprises one or more nucleic acids encoding guide RNAs.As used throughout, guide RNA (gRNA) sequence refers to a sequence that interacts with RNA-guided nuclease and specifically binds or hybridizes with a target nucleic acid in the genome of a cell, so that the gRNA and the targeting nuclease co-localize with the target nucleic acid in the genome of the cell.The degree of complementarity between the guide RNA and the genome sequence can be about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

[0032] In some cases, the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence (e.g., a promoter). Each gRNA comprises a DNA targeting sequence or protospacer sequence of about 10-50 nucleotides in length that specifically binds or hybridizes to a target DNA sequence in a genome. For example, the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. Optionally, the gRNA does not include a tracrRNA sequence.

[0033] In some embodiments, the LNP comprises an mRNA encoding an RNA-guided endonuclease and a guide RNA. The mRNA encoding the RNA-guided endonuclease and the guide RNA may be encapsulated in separate LNPs, which are then combined to form a mixed population of LNPs comprising either the mRNA encoding the RNA-guided endonuclease or the guide RNA in different LNPs. In some embodiments, the mRNA encoding the RNA-guided endonuclease and the guide RNA are co-encapsulated in the same LNP. Optionally, the ratio of nitrogen groups of the ionizable lipid of the LNP to phosphate groups in the mRNA and / or gRNA in the LNP is about 4 to about 6.

[0034] The guide RNA is optionally a modified guide RNA. The one or more modifications to the guide RNA may be selected from the group consisting of backbone modifications, sugar modifications, and base modifications. Optionally, the guide RNA has one or more sugar modifications, one or more base modifications, and / or one or more backbone modifications.

[0035] In some guide RNAs, the sugar modification is a modification of the 2' position of the ribose group. In some guide RNAs, the modification of the 2' position is selected from 2'-O-methyl (2'-OMe), which replaces the 2' hydroxyl group with a methyl group, and 2'-fluoro (2'-F), which replaces the 2' hydroxyl group with a fluorine.

[0036] In some guide RNAs, the phosphate backbone modification is a thioester modification (e.g., phosphorothioate linkage). In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the nucleotides in the guide RNA are modified nucleotides. In certain cases, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 1 9, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides are modified. In some embodiments, the guide RNA comprises a nucleic acid sequence of 100 nucleotides, and 69 of the 100 nucleotides are modified using the modification patterns shown below.

[0037] Optionally, the LNP comprises a ribonucleotide protein complex (RNP), the RNP comprises an RNA-guided nuclease (e.g., Cas9 nuclease) and a guide RNA. In other embodiments, the LNP comprises an RNP, the RNP comprises an RNA-guided nuclease (e.g., Cas9 nuclease), a guide RNA, and one or more template nucleic acids for repair or recombination in the genome of a cell. The template may be used to modify a nucleic acid sequence at or near a target site of the RNA-guided endonuclease, or to insert a nucleic acid sequence at or near the target. In some embodiments, the template is a heterologous nucleic acid sequence that encodes a polypeptide, an exon sequence, an intron sequence, a regulatory sequence, a transcription control sequence, a translation control sequence, a splicing site, or a non-coding sequence, etc. Optionally, the template is a single-stranded, double-stranded, or partially double-stranded template. In some embodiments, the template may be used to repair a genomic sequence by homology-directed repair or non-homologous end joining.

[0038] As used herein, the term heterologous refers to something that is not normally found in nature. The term heterologous nucleic acid sequence refers to a nucleic acid sequence that is not normally found in a given cell in nature. Thus, a heterologous nucleic acid sequence can be (a) foreign to its host cell (i.e., exogenous to the cell), (b) naturally found in the host cell (i.e., endogenous) but present in the cell in an unnatural amount (i.e., greater or less than that naturally found in the host cell), or (c) naturally found in the host cell but located outside its natural locus.

[0039] In the LNPs described herein, the template can be 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. In some embodiments, the template can be flanked by homology arms (i.e., nucleic acid sequences that are complementary to sequences located upstream and downstream of an RNA-guided endonuclease cleavage site in the genome of a cell). As used herein, the term complementary or complementarity refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C. In some embodiments, the degree of complementarity between the nucleic acid sequence (e.g., a guide RNA or a homology arm) and the genomic sequence may be about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.

[0040] Also provided herein are one or more cells comprising any of the LNPs or LNPs described herein. Additionally provided are compositions comprising one or more of the LNPs described herein. A pharmaceutical composition, for example, comprises a therapeutically effective amount of any of the LNPs described herein and a pharmaceutical carrier. The term carrier refers to a compound, composition, substance, or structure that, in combination with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for an intended use or purpose. For example, the carrier may be selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebrospinal fluid, dextrose, and water.

[0041] Optionally, a composition comprising one or more LNPs is present in a kit, which includes, for example, a container for the composition or its components, a device for administering the composition, and / or a mixer or mixing device for producing the composition.

[0042] Pharmaceutical compositions containing any of the LNPs described herein may be prepared according to standard techniques, and may further include a pharma- ceutical acceptable carrier. Generally, saline is used as a pharma- ceutical acceptable carrier. Other suitable carriers include, for example, water, buffered water or saline, 0.4% saline, 0.3% glycine, dextrose, and the like, including glycoproteins to enhance stability, such as albumin, lipoproteins, and globulins. These compositions are usually sterile. Pharmaceutical compositions may also include a pharma- ceutical acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce a harmful immune response in the individual to whom the composition is administered, and that may be administered without undue toxicity. These may include pharma- ceutically acceptable salts, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Preparation of pharma- ceutically acceptable carriers, excipients, and formulations containing these substances are described, for example, in Remington: The Science and Practice of Pharmacy, 22nd edition, Loyd V. Allen et al, editors, Pharmaceutical Press (2012).

[0043] The aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition can contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, osmolality adjusters, and the like (e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride). In addition, the LNP suspension can contain lipid protectants that protect lipids from damage caused by free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

[0044] The concentration of LNPs in pharmaceutical formulations can vary widely (i.e., from less than about 0.05% to about 2-5%, or 10-30% by weight). The concentration can be selected primarily based on liquid volume, viscosity, and the particular mode of administration selected. LNPs can also be dried or lyophilized and resuspended to the desired concentration with water or buffer at the time of use.

[0045] method Provided herein is a method for delivering a polypeptide (e.g., a recombinant polypeptide) to a cell. The method includes contacting a cell with one or more of the LNPs comprising a heterologous polypeptide described herein, or contacting a cell with one or more of the LNPs comprising a nucleic acid encoding a heterologous polypeptide described herein. Optionally, the cell can be contacted with a pharmaceutical composition comprising the LNP.

[0046] Similarly, a method of editing the genome of a cell is provided, comprising contacting the cell with a nucleic acid encoding an RNA-guided nuclease and an LNP comprising a guide RNA, where the guide RNA interacts with the RNA-guided nuclease, such that the RNA-guided nuclease specifically binds to and cleaves a target DNA in the genome. As used throughout, the term editing, in the context of editing the genome of a cell, refers to inducing a structural change in the sequence of the genome at a target genomic region. For example, editing or modifying can take the form of inserting a nucleotide sequence (e.g., a nucleic acid template) into the genome of the cell. Such editing can be performed, for example, by inducing a double-stranded break within the target genomic region, or by inducing a pair of single-stranded nicks on opposite strands and flanking the target genomic region. Methods of inducing single-stranded or double-stranded breaks at or within a target genomic region include the use of an RNA-guided endonuclease or derivative thereof and a guide RNA or a pair of guide RNAs, as described herein, directed to the target genomic region. Any of the editing methods described herein may be used to achieve an editing efficiency of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% in a population of cells. Also provided are genetically modified cells produced by any of the methods described herein.

[0047] In any of the delivery methods described herein, the agent(s) encapsulated in the LNP are introduced into the cell after contacting the cell with the LNP. As used herein, introduction in the context of introducing a nucleic acid, a polypeptide, or a complex containing a nucleic acid (e.g., an RNP complex) refers to the transfer of a nucleic acid sequence, a polypeptide, or a complex from outside the cell into the cell. In some cases, introduction refers to the transfer of a nucleic acid or complex from outside the cell into the nucleus of the cell.

[0048] Any of the methods described herein may be used to deliver agents to cells in vitro, ex vivo, or in vivo. For in vivo applications, see, e.g., Skipper (2019), Toward In Vivo Gene Therapy Using CRISPR, Methods Mol. Biol. 1961:293-306, and Gillmore et al. (2021) CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis, N. Engl. J. Med. 385:493-502.

[0049] In any of the methods provided herein, the cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell. One or more LNPs comprising an RNA-guided endonuclease mRNA and a gRNA may be administered to a subject in need. One or more LNPs comprising an RNP complex may be administered to a subject, the RNP comprising an RNA-guided endonuclease, a gRNA, and an optional nucleic acid template. In some embodiments, the LNPs are targeted to one or more target cells or tissues of the subject. For example, the target tissue may be, but is not limited to, the liver, eye, endothelial tissue, lung, blood, muscle, or kidney, or a specific cell type within the liver, eye, endothelial tissue, lung, blood, muscle, or kidney.

[0050] Ex vivo methods include, but are not limited to, obtaining one or more cells from a subject having a disease or disorder, (1) using LNPs comprising an RNA-guided endonuclease mRNA and a gRNA, or (2) using LNPs comprising an RNP complex comprising an RNA-guided endonuclease and a gRNA (optionally with a nucleic acid template), ex vivo editing one or more target nucleotide sequences in the one or more cells, and reintroducing the one or more cells having the edited target nucleotide sequences into the subject having the disease or disorder. In any of the ex vivo methods described herein, the cells may be cultured and / or expanded before or after editing.

[0051] The LNP compositions disclosed herein can be administered to a subject in many ways, depending on whether local or systemic treatment is desired and on the cells / tissues / areas to be treated. The compositions are administered by any of several routes of administration (e.g., orally, intranasally, by inhalation, by nebulizer, parenterally, intravenously, intraperitoneally, intracranially, intraspinal, intrathecal, intracerebroventricularly, intramuscularly, subcutaneously, intracavitary, or transdermally). The pharmaceutical compositions can also be delivered locally to the area in need of treatment, for example, by topical application or local injection. The pharmaceutical compositions can also be delivered by pumps or at the site of surgery. The effective dose for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0052] The amount of LNP administered, or the amount of active agent in the LNP, depends on many factors, such as the disease state of the subject being treated or prevented, and the judgment of the clinician, but generally is about 0.01 to about 150 mg of drug per kilogram of body weight, preferably about 0.1 to about 20 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, or about 0.1 to about 5 mg / kg body weight, and the amount may be administered in a single dose or in the form of individual doses, such as 1 to 4 times per day. Administration may be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days. One of skill in the art may adjust the dosage based on the particular characteristics of the drug and the subject to which the drug is administered.

[0053] Disclosed are materials, compositions, and components that can be used in, in combination with, in the preparation of, or the products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that although specific reference to the various individual and collective combinations and sequences of each of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, when a method is disclosed and discussed, and many modifications that can be made to many molecules that comprise the method are discussed, any and all combinations and sequences of the method, and possible modifications, are specifically contemplated unless specifically indicated to the contrary. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the disclosure, including, but not limited to, steps in a method that uses the disclosed compositions. As such, if there are various additional steps that may be performed, it is understood that each of these additional steps may be performed with any specific method step or combination of method steps of the methods of the present disclosure, and that each such combination or subset of combinations is specifically contemplated and should be considered as disclosed.

[0054] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties. EXAMPLES

[0055] Working Example The following examples are offered by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results.

[0056] Ionized lipid formulations PCTA-1 (Formula II) was used in a formulation containing 50% PCTA-1, 10% distearoylphosphatidylcholine (DSPC), 38% cholesterol, and 2% pegylated myristoyl diglyceride (DMG-PEG2000). Depending on the application, this formulation can be adjusted to contain about 20% to about 80% PCTA-1, the percentage of helper lipid is about 5% to about 20%, the percentage of cholesterol is 10% to 60%, and the percentage of polyethylene glycol (PEG) is about 0.5% to about 5%.

[0057] For applications involving nucleic acid delivery, the nucleic acid (e.g., mRNA, or a combination of mRNA and gRNA, in the case of gene editing) was incorporated at an N:P (nitrogen to phosphate) ratio of 5.5, so that the number of ionizable amines (from PCTA-1, since the amines of DSPC are non-ionizable) was 5.5 times that of the phosphate groups in the nucleic acid. LNPs were generated by combining a lipid solution in ethanol with a nucleic acid solution in a buffer, using a microfluidic mixer to rapidly mix the solutions. This buffer should be at an acidic pH, but can otherwise vary, and the most effective buffer was observed to be a 25 mM citrate buffer at pH 4.5. The concentration of lipid in ethanol can also vary depending on the desired properties, but generally ranges from 1 to 50 mg / mL lipid. The concentration of the nucleic acid is determined by the N:P ratio, the lipid concentration in the buffer, and the ratio of ethanol to buffer. The ratio of ethanol to buffer can also be varied, with ratios of 3:1 buffer:ethanol to 1:1 being effective. Microfluidic mixing can be performed at a variety of flow rates, with flow rates of 1-10 mL / min being most effective. Many mixing structures (e.g., alternating herringbone mixing structures) are also available. In this example, a Dolomite micromixer chip was used for PCTA-1.

[0058] Using PCTA-1 in an LNP formulation, we demonstrated effective delivery of cas9 mRNA and guide RNA to the liver in vivo. High editing efficiency was achieved for two different genes. Mice were administered via the tail vein and the liver was removed on day 5. Editing rates were determined by isolating gDNA from homogenized whole liver and editing events were quantified using next-generation targeted amplicon sequencing.

[0059] As shown in FIG. 1, Formulation 1 (F1) (formulation containing PCTA-1 (Formulation II)) was compared to Comparative Formulations (CF1) and CF2 (CF2) (both formulated according to literature reports) produced using Formula III for CF1 (see, e.g., Finn et al. Cell Reports 22, 2227-2235 (2018)) and Formula IV for CF2 (heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate; see, e.g., Sabnis et al. Molecular Therapy 26(6):1509-1519 (2018)). [ka]

[0060] All formulations were packaged with Cas9 mRNA and sgRNA targeting either TTR (first three columns from the left) or PCSK9 (4-6 columns from the left). All formulations were administered at 0.7 mg / kg in groups of three mice and organs were harvested 5 days later. When targeting TTR, F1 achieved the highest editing, showing improvement over CF2 and approximately 5-fold improvement over CF1. When targeting liver PCSK9, F1 achieved significantly higher editing (2-3-fold) compared to CF2 and CF1 with the same sgRNA.

[0061] Modified guide RNA A number of chemical modification patterns were designed and tested for gene editing in cells and in vivo mouse models. In this design, 69 of the 100 nucleotides found within the sgRNA were modified (Figure 2). 2'Ome and 2'F sugar modifications were used along with thioester modifications to the phosphate backbone to increase nuclease resistance in vitro and improve in vivo delivery of active sgRNAs. Three sgRNAs were designed and compared to previously published modifications (Guide 1). Testing of the sgRNAs in vitro using primary mouse hepatocytes showed similar editing rates and reduced target protein levels in cell supernatants. Briefly, Cas9 mRNA and sgRNA were complexed using MessengerMax transfection reagent and added to seeded mouse hepatocytes, then incubated for 48 hours before isolating and sequencing genomic DNA. Genomic DNA was used as input to amplify the target region of interest around the predicted gene edit and barcoded with unique adapter sequences so that samples could be combined for next generation sequencing. Figure 3A shows the editing rate of the target of interest. All guides showed similar editing in vitro.

[0062] Target protein levels in cell culture supernatants were measured using a commercial ELISA kit according to the manufacturer's instructions (Figure 3B). Results show a similar reduction of all sgRNAs compared to the control.

[0063] All sgRNAs were initially screened in vivo using the CD1 mouse model. Briefly, mice were injected via the tail vein and on day 5, plasma, liver, and spleen were harvested. Editing rates were determined from genomic DNA isolated from homogenized whole liver or whole spleen. Plasma was used for protein determination by ELISA. Results showed that guide 2 with additional engineered chemical modifications showed a 2-fold increase in editing rate using the nanoparticle formulation described in the literature (Figure 4A). Subsequent in vivo studies using the nanoparticle formulation described herein showed that ~60% liver editing was achieved (Figure 4B). This is likely near saturation, as hepatocytes account for approximately 70% of liver cells.

Claims

1. Formula I 【Chemistry 1】 A lipid nanoparticle (LNP) comprising an ionized lipid of In the formula, R １ and R ２ is, independently, 【Chemistry 2】 is selected from the group consisting of X １ and X ２ is a hydrolyzable linker, (a) is 2-9, and (b) is 2-9; LNP.

2. The LNP of claim 1 , wherein the hydrolyzable linker is an ester.

3. The ionization is carried out according to formula II 【Chemistry 3】 The LNP of claim 1 or 2, wherein the lipid is a lipid.

4. The LNP of any one of claims 1 to 3, further comprising cholesterol, a helper phospholipid, and a polyethylene glycol (PEG)-lipid.

5. The LNP of any one of claims 1 to 4, wherein about 20% to about 80% of the total lipid content of the LNP is an ionized lipid of formula I.

6. The LNPs are (a) 20% to 80% of an ionizable lipid of formula I; (b) 5% to 20% helper lipid; (c) 10% to 60% cholesterol; and (d) 0.5% to 5% PEG The LNP of any one of claims 1 to 5, comprising:

7. The LNP of any one of claims 1 to 6, further comprising a polypeptide, a nucleic acid, or a combination thereof.

8. The LNP of claim 7 , wherein a nucleic acid encoding a heterologous polypeptide is encapsulated in the LNP.

9. The LNP of claim 8 , wherein the nucleic acid is mRNA.

10. The LNP of claim 9 , wherein the mRNA encodes an RNA-guided endonuclease.

11. The LNP of claim 10, wherein the RNA-guided endonuclease is a Cas9 nuclease.

12. The LNP of any one of claims 9 to 11, further comprising a guide RNA.

13. The LNP of claim 12, wherein the guide RNA is a modified guide RNA.

14. The LNP of claim 13, wherein the guide RNA comprises one or more modifications selected from the group consisting of a backbone modification, a sugar modification, a base modification, and any combination thereof.

15. 15. The LMP of claim 14, wherein the sugar modification is a 2'OMe or 2'F modification of the 2' ribose group.

16. The LNP of claim 14 or 15, wherein the backbone modification is a thioester modification.

17. The LNP of any one of claims 13 to 16, wherein the guide RNA comprises a nucleic acid sequence of 100 nucleotides, and 69 of the 100 nucleotides are modified.

18. The LNP of any one of claims 10 to 17, wherein the mRNA encoding the RNA-guided endonuclease and the guide RNA are encapsulated separately and then combined to form one or more LNPs comprising the guide-guided endonuclease and the guide RNA.

19. The LNP of claim 18, wherein the mRNA encoding the RNA-guided endonuclease and the guide RNA are co-encapsulated in one or more LNPs.

20. The LNP of claim 19, wherein the ratio of nitrogen groups in the ionizable lipid to phosphate groups in the mRNA and / or gRNA in the LNP is about 4 to about 6.

21. The LNP of any one of claims 10 to 20, further comprising at least one template sequence.

22. A pharmaceutical composition comprising the LNP according to any one of claims 1 to 21.

23. A method for delivering a heterologous polypeptide to a cell, comprising contacting the cell with the LNP of claim 8 or a pharmaceutical composition comprising the LNP of claim 8.

24. A method for editing the genome of a cell, comprising contacting the cell with an LNP according to any one of claims 10 to 21, or a pharmaceutical composition comprising the LNP according to any one of claims 10 to 21.

25. 25. The method of claim 24, wherein the cell is present in vitro, ex vivo, or in vivo.

26. 26. The method of claim 25, wherein the cell is a human cell.

27. A genetically modified cell produced by the method according to any one of claims 24 to 26.

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