Base editing of the transthyretin gene

Abstract

Provided herein are compositions for genetic modification involving base editor systems and methods of using the same to treat or prevent conditions associated with the extracellular deposition of amyloid fibrils formed by aggregation of misfolded transthyretin (TTR) protein in various tissues, including, but not limited to, hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis cardiomyopathy (hATTR-CM), both of which are associated with autosomal dominant mutations in the TTR gene and an age-related cardiomyopathy (also known as senile cardiac amyloidosis) associated with the wild-type TTR protein (ATTRwt).

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 383,394, filed November 11, 2022, the entire contents of which are incorporated herein by reference. Sequence Listing

[0002]

[0001] This application contains a Sequence Listing that has been filed electronically in XML format, the entire contents of which are incorporated herein by reference. The XML file of the Sequence Listing, created on November 10, 2023, is named 180802-046902PCT_SL.xml and is 5,147,724 bytes in size. [Background technology]

[0003] Transthyretin (TTR) is a 55-kDa transport protein for both thyroxine (T4) and retinol-binding protein (RBP), which circulates in soluble form in the serum and cerebrospinal fluid (CSF) of healthy individuals. Under normal conditions, the TTR protein circulates as a homotetramer. Hereditary transthyretin amyloidosis (hATTR) is a disease caused by mutations in the gene encoding TTR. Autosomal dominant mutations destabilize the TTR tetramer and enhance its dissociation into monomers, leading to misfolding, aggregation, and subsequent extracellular deposition of TTR amyloid fibrils at various sites. This multisystemic extracellular amyloid deposition leads to the dysfunction of various organs and tissues. In particular, transthyretin amyloidosis-associated polyneuropathy (ATTR-PN) and cardiomyopathy (ATTR-CM) are severe diseases associated with significant morbidity and mortality.

[0004] Because TTR is primarily produced in the liver, the initial treatment for hATTR amyloidosis was liver transplantation. Other treatments include the administration of oral drugs that act as dynamic stabilizers of the TTR tetramer (e.g., tafamidis and diflunisal), and the suppression of TTR protein synthesis using gene-silencing drugs such as small interfering RNA (siRNA) (patisiran) and antisense oligonucleotides (inotersen).

[0005] The present invention recognizes that a gene editing approach for the treatment of transthyretin amyloidosis, including both polyneuropathy and cardiomyopathy, may provide a one-time treatment with superior results over existing treatments. Summary of the Invention

[0006] Provided herein are compositions for gene modification or editing, and methods of using them to treat or prevent conditions associated with the extracellular deposition of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) protein in various tissues. Such conditions include, but are not limited to, hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis cardiomyopathy (hATTR-CM), both of which are associated with autosomal dominant mutations in the TTR gene and an age-related cardiomyopathy (also known as senile cardiac amyloidosis) associated with the wild-type TTR protein (ATTRwt). Disclosed are compositions and methods aimed at editing the TTR gene using editing systems, such as those comprising base editors and guide RNAs.

[0007] In some aspects, provided herein is a base editor system for modifying a target transthyretin (TTR) gene, comprising a guide RNA comprising a sequence defined by: TIFF2025540623000001.tif24165 where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, the nucleotides shown in bold are linked by phosphorothioate (PS) backbone bonds, A base editor system is provided in which a guide RNA directs the base editor system to make a nucleobase change in the TTR gene.

[0008] In some aspects, provided herein is a guide RNA comprising a sequence defined by: TIFF2025540623000002.tif26165, wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, and the nucleotides represented in bold are linked by phosphorothioate (PS) backbone bonds; A guide RNA is provided, which guides the base editor system to make a nucleobase change in the TTR gene.

[0009] 1. An engineered, non-natural base editing system for modifying a target transthyretin (TTR) gene, comprising: (a) a guide RNA molecule having a sequence defined by: TIFF2025540623000003.tif25165 and (b) a codon-optimized nucleic acid encoding a Cas9 protein fused to a deaminase, wherein the Cas9 protein fusion is capable of binding to a guide RNA and editing a target TTR sequence complementary to the guide RNA.

[0010] In some embodiments, the deaminase comprises a cytosine deaminase or an adenine deaminase.

[0011] In some embodiments, the Cas9 protein is a catalytically impaired Cas9 protein.

[0012] In some embodiments, the Cas9 protein is a dead Cas9 or a nickase Cas9.

[0013] In some embodiments, the cytosine or adenine deaminase is a deoxycytosine or deoxyadenosine deaminase.

[0014] In some embodiments, Cas9 is fused to ABE8.8.

[0015] In some embodiments, provided herein are lipid nanoparticles (LNPs) comprising the systems described herein.

[0016] In some embodiments, the LNP comprises an ionizable amino lipid, a neutral helper lipid, a PEG lipid, a sterol lipid, and / or a GalNAc lipid.

[0017] In some embodiments, the ionizable amino lipid is VL422 or LP-01, the neutral helper lipid is DSPC, and the PEG lipid is PEG 2000 -DMG, the sterol lipid is cholesterol, and the GalNAc lipid is DSG-PEG-Lys-tris(GalNAc).

[0018] In some embodiments, the ionizable lipid is LP-01 (or CIN16645), which is defined by the following structure: [ka]

[0019] In some embodiments, the LNPs comprise an N:P ratio of about 1:40 to about 1:1.

[0020] In some embodiments, the LNPs comprise an N:P ratio of about 1:6.

[0021] In some embodiments, pharmaceutical compositions comprising the LNPs described herein are provided.

[0022] In some embodiments, provided herein are methods for editing a TTR gene in a cell, comprising: activating the cell with (a) a guide RNA molecule having a sequence defined by: TIFF2025540623000005.tif24165

[0023] (b) a base editor system comprising a codon-optimized nucleic acid encoding a Cas9 protein fused to a deaminase, wherein the Cas9 protein fusion is capable of binding to a guide RNA and editing a target nucleic acid sequence complementary to the guide RNA.

[0024] In some embodiments, provided herein are methods of treating a disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0025] In some embodiments, the disease or disorder is hereditary transthyretin amyloidosis, cardiomyopathy, polyneuropathy, or senile cardiac amyloidosis.

[0026] In some embodiments, the pharmaceutical composition is administered by a route selected from intravenous, intradermal, transdermal, intranasal, intramuscular, subcutaneous, transmucosal, or oral.

[0027] In some embodiments, the LNPs are delivered to the liver. [Brief explanation of the drawings]

[0028] [Figure 1A] FIG. 1A shows a general schematic diagram of a gene editor complexed with a gRNA targeting a gene of interest. The Cas9 protein, guide RNA, spacer sequence, protospacer sequence, and PAM (protospacer adjacent motif) are identified (FIG. 1A). FIG. 1A discloses SEQ ID NO: 5760. Additionally, a schematic diagram of the general principle of base editing using a cytosine base editor (CBE) (FIG. 1B) and an adenine base editor (ABE) (FIG. 1C) is shown. [Figure 1B] FIG. 1A shows a general schematic diagram of a gene editor complexed with a gRNA targeting a gene of interest. The Cas9 protein, guide RNA, spacer sequence, protospacer sequence, and PAM (protospacer adjacent motif) are identified (FIG. 1A). FIG. 1A discloses SEQ ID NO: 5760. Additionally, a schematic diagram of the general principle of base editing using a cytosine base editor (CBE) (FIG. 1B) and an adenine base editor (ABE) (FIG. 1C) is shown. [Figure 1C] FIG. 1A shows a general schematic diagram of a gene editor complexed with a gRNA targeting a gene of interest. The Cas9 protein, guide RNA, spacer sequence, protospacer sequence, and PAM (protospacer adjacent motif) are identified (FIG. 1A). FIG. 1A discloses SEQ ID NO: 5760. Additionally, a schematic diagram of the general principle of base editing using a cytosine base editor (CBE) (FIG. 1B) and an adenine base editor (ABE) (FIG. 1C) is shown.

[0029] [Figure 2] Alteration of splice donor sites by base editing. The top panel shows normal splicing of RNA transcribed from a gene. The bottom panel shows the splicing that can occur when transcribing a gene with a splice site disrupted by editing.

[0030] [Figure 3] A map of the human TTR gene (hTTR gene) showing the locations of various restriction enzyme recognition sites, exons 1 to 4, and single guide RNAs GA457, GA459, GA460, and GA461, as designated in Table 1.

[0031] [Figure 4] The nucleotide sequence of the human TTR gene (UniProtKB-P02766 (TTHY_HUMAN)) from the reference human genome (GRCh38) is shown, indicating the regions on the gene where guides GA457, GA459, GA460, and GA461 are located. SEQ ID NO: 5761 is disclosed.

[0032] [Figure 5] Schematic diagram showing the TTR guide and editing locations of GA457 (A), GA460 (B), and GA461 (C). Human genomic DNA (gDNA) sequence is labeled in black. The guide sequence is highlighted in gray above. Genomic exon sequences are in uppercase, and intron sequences are in lowercase. The main locations targeted by ABE editing are labeled with black arrows. SEQ ID NOs: 1, 5762, 4, 5763, 5, and 5764 are disclosed in order of appearance, respectively.

[0033] [Figure 6] Figure 1 shows a graph showing the percentage of splice editing in human hepatocytes using ABE editing with single guide RNAs GA457, GA459, GA460, and GA461. The three TTR guide RNAs GA457, GA460, and GA461 show high activity in human hepatocytes. Each guide uses the same tracr sequence and differs only in the RNA spacer sequence that corresponds to a given DNA protospacer sequence on the target TTR gene.

[0034] [Figure 7] FIG. 1 is a flowchart of the ONE-seq protocol for determining candidate off-target sites.

[0035] [Figure 8] 1 is a schematic diagram of a comparison of GA519 and GA457 hybridized to NHP and human TTR exon 1. Disclosed in order of appearance are SEQ ID NOs: 1, 5765, 2, and 5766, respectively.

[0036] [Figure 9] FIG. 1 is a schematic diagram showing a comparison of GA520 and GA460 hybridized to NHP and human TTR exon 3. Disclosed in order of appearance are SEQ ID NOs: 5767-5768 and 5767-5768, respectively.

[0037] [Figure 10] 1 is a bar graph showing hepatic editing of the TTR gene by LNP1 and LNP2 in non-human primates (NHPs) as described in the Examples.

[0038] [Figure 11] 1 is a bar graph showing the change in serum TTR protein, as measured by ELISA, in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0039] [Figure 12] 1 is a bar graph showing changes in serum TTR protein as measured by mass spectrometry in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0040] [Figure 13] 1 is a bar graph showing serum alanine aminotransferase (ALT) levels (A) and serum aspartate aminotransferase (AST) levels (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0041] [Figure 14] 1 is a bar graph showing serum lactate dehydrogenase (LDH) levels (A) and serum glutamate dehydrogenase (GDH) levels (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0042] [Figure 15]1 is a bar graph showing serum gamma-glutamyltransferase (GGT) levels (A) and serum alkaline phosphatase (AP) levels (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0043] [Figure 16] 1 is a bar graph showing serum total bilirubin concentrations in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0044] [Figure 17] 1 is a bar graph showing serum creatine kinase levels in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0045] [Figure 18] 1 shows bar graphs of serum cytokine concentrations (MCP-1, top left panel; IL-6, top right panel; IP-10, bottom left panel; and IL-1RA, bottom right panel) over time for NHPs treated with LNP1 and LNP2 as described in the Examples.

[0046] [Figure 19] 1A and 1B are plots of the plasma pharmacokinetic profiles of iLipid (A) and PEG-Lipid (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0047] [Figure 20] 1 is a bar graph showing hepatic editing of the TTR gene by LNP3 in NHPs, as described in the Examples.

[0048] [Figure 21] 1 is a plot showing the change in serum TTR protein, as measured by ELISA, in NHPs treated with LNP3 as described in the Examples.

[0049] [Figure 22] 1 is a plot showing the change in serum TTR protein as measured by liquid chromatography-mass spectrometry in NHPs treated with LNP3 as described in the Examples.

[0050] [Figure 23] 1 is a bar graph showing serum alanine aminotransferase (ALT) levels (A) and serum aspartate aminotransferase (AST) levels (B) in NHPs treated with LNP3 as described in the Examples.

[0051] [Figure 24] 1 is a bar graph showing serum lactate dehydrogenase (LDH) levels (A) and serum glutamate dehydrogenase (GDH) levels (B) in NHPs treated with LNP3 as described in the Examples.

[0052] [Figure 25] 1A-B are bar graphs showing serum gamma-glutamyltransferase (GGT) levels (A) and serum alkaline phosphatase (AP) levels (B) in NHPs treated with LNP3 as described in the Examples.

[0053] [Figure 26] 1 is a bar graph showing serum total bilirubin concentrations in NHPs treated with LNP2 as described in the Examples.

[0054] [Figure 27] 1 is a bar graph showing serum creatine kinase levels in NHPs treated with LNP3 as described in the Examples.

[0055] [Figure 28] 1A and 1B are plots of the plasma pharmacokinetic profiles of iLipid (A) and PEG-Lipid (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0056] [Figure 29] Figure 1 shows the percentage of base editing in primary human hepatocytes at various doses of total RNA (ng / TA / ml) when GA521 was used as a guide RNA. GA521 demonstrated sustained base editing of over 40% in primary human hepatocytes. DETAILED DESCRIPTION OF THE INVENTION

[0057] Provided herein are compositions for gene modification or editing, and methods of using them to treat or prevent conditions associated with the extracellular deposition of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) protein in various tissues. Such conditions include, but are not limited to, hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis cardiomyopathy (hATTR-CM), both of which are associated with autosomal dominant mutations in the TTR gene and an age-related cardiomyopathy (also known as senile cardiac amyloidosis) associated with the wild-type TTR protein (ATTRwt). Disclosed are compositions and methods aimed at editing the TTR gene using editing systems, such as those comprising base editors and guide RNAs.

[0058] definition The following provides definitions of several terms that are provided throughout this disclosure. In some cases, terms are defined in places herein other than this "Definitions" section.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be understood that the term "or" is generally used in a sense that includes "and / or" unless the content clearly dictates otherwise. As used herein, the terms "and / or" and "any combinations thereof," as well as their grammatical equivalents, can be used interchangeably. These terms may mean that any and all combinations are specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combinations thereof" may mean "A alone; B alone; A and B; B and C; A and C; and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the content specifically dictates disjunctive use.

[0060] The term "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of 5, more preferably within 2, of a value. When a particular value is described in this application and claims, unless otherwise specified, it means within an acceptable error range for the particular value.

[0061] As used in the specification and claim(s), "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0062] An article, composition, method, etc. that comprises one or more elements may be composed of or consist essentially of one or more elements. As used in this specification and claim(s), "consisting of" (and any form of consisting of, e.g., "consists of" and "consist of") means including and limited to. As used in this specification and claim(s), an article, composition, method, etc. "consisting essentially of" (and any form of consisting essentially of, e.g., "consists essentially of" and "consist essentially of") means that the article, composition, method, etc. includes the specifically recited elements, e.g., components, compounds, materials, steps, etc., and may include additional elements that do not materially affect the basic and novel characteristics of the article, composition, method, etc.

[0063] References herein to "some embodiments," "an embodiment," "one embodiment," "embodiments," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with an embodiment is included in at least some, but not necessarily all, embodiments of the present disclosure.

[0064] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0065] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single- or double-stranded form, including DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked via the phosphate group. "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines (which include, but are not limited to, modifications that introduce new reactive groups, such as amines, alcohols, thiols, carboxylates, and alkyl halides). Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages or modified sugar residues, or non-standard / chemically modified nucleobases and combinations thereof, which are synthetic, natural, and unnatural, and which have binding properties similar to those of standard nucleic acids. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0066] The term "nucleic acid" includes any oligonucleotide or polynucleotide; fragments containing up to 60 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphate at the sugar's 5' and 3' carbons to form alternating, unbranched polymers. DNA can be in the form of, for example, antisense molecules, plasmid DNA, precondensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose. Thus, the terms "polynucleotide" and "oligonucleotide" can refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" can also include polymers or oligomers containing non-naturally occurring monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and stability in the presence of nucleases. It should be understood that the terms "polynucleotide" and "oligonucleotide" can also include polymers or oligomers comprising a combination of both deoxy- and ribonucleotides, in combination with backbone modifications, as described herein, or variants thereof.

[0067] A "nucleic acid" as used herein may contain one or more nucleotide variants, such as non-standard nucleotide(s), unnatural nucleotide(s), nucleotide analog(s), and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl eosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl Cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, etc. In some cases, the nucleotide may contain modifications of the phosphate moiety, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (eg, phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (eg, α-thiotriphosphate and β-thiotriphosphate).

[0068] The nucleic acids described herein may be modified at the base moiety (e.g., one or more atoms normally available to form hydrogen bonds with a complementary nucleotide and / or one or more atoms normally not capable of forming hydrogen bonds with a complementary nucleotide), the sugar moiety, or the phosphate backbone. Backbone modifications may include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, phosphoroamidate, and phosphorodiamidate linkages. Phosphorothioate linkages replace non-bridging oxygen atoms in the phosphate backbone with sulfur atoms, retarding nuclease degradation of oligonucleotides. Phosphorodiamidate linkages (N3'→P5') prevent nuclease recognition and degradation. Backbone modifications can also include peptide bonds in place of phosphorus in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are reviewed in Micklefield, Curr. Med. Chem., 8 (10): 1157-79, 2001, and Lyer et al., Curr. Opin. Mol. Ther., 1 (3): 344-358, 1999. The nucleic acid molecules described herein can contain sugar moieties including ribose or deoxyribose, as present in natural nucleotides, or modified sugar moieties or sugar analogs. Modified sugar moieties include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars. The 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-form or RNA-like structure of the oligonucleotide, increasing its binding affinity to RNA and its nuclease resistance.Modified sugar moieties can also include an extra bridge (e.g., a methylene bridge connecting the 2'-O atom and the 4'-C atom of the ribose of a locked nucleic acid) or a sugar analog such as a morpholine ring (e.g., in phosphorodiamidate morpholino).

[0069] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly stated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)).

[0070] The present disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions containing those molecules. As used herein, an "isolated" or "purified" DNA or RNA molecule is a DNA or RNA molecule that exists away from its native environment. An isolated DNA or RNA molecule can exist in purified form or can exist in a non-native environment, such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule, or a biologically active portion thereof, will be substantially free of other cellular material or culture medium if produced recombinantly, or substantially free of chemical precursors or other chemicals if chemically synthesized. In one embodiment, an "isolated" nucleic acid will be free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0071] As used herein, the terms "protein," "polypeptide," and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and may consist of two or more polypeptide chains. The terms "polypeptide," "protein," and "peptide" refer to a polymer of at least two amino acid monomers linked via amide bonds. The amino acids may be the L or D optical isomer. More specifically, the terms "polypeptide," "protein," and "peptide" refer to a molecule composed of two or more amino acids in a specific order, e.g., an order determined by the base sequence of nucleotides in a gene or RNA encoding the protein. Proteins are essential for the structure, function, and regulation of cells, tissues, and organs of the body, and each protein has a unique function. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a portion of a protein, e.g., a domain, subdomain, or motif of a protein. In some cases, a protein may be a variant (or mutation) of a protein in which one or more amino acid residues are inserted, deleted, and / or substituted in the naturally occurring (or at least known) amino acid sequence of the protein. The protein or its variant can be naturally occurring or recombinant. Methods for detecting and / or measuring polypeptides in biological materials are well known in the art and include, but are not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomic techniques. An exemplary method for measuring or detecting polypeptides is an immunoassay such as ELISA. This type of protein quantification can be based on an antibody capable of capturing a specific antigen and a secondary antibody capable of detecting the captured antigen.

[0072] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees, and other ape and monkey species; domestic animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals such as rodents, including rats, mice, and guinea pigs.

[0073] A "subject in need thereof" refers to an individual who has a disease, a symptom of a disease, or a predisposition to a disease, and for whom the goal is to cure, eliminate, alleviate, mitigate, alter, treat, ameliorate, improve, or affect the disease, symptom of a disease, or predisposition to a disease. In some embodiments, the subject has hereditary transthyretin amyloidosis (hATTR). In some embodiments, the subject has transthyretin amyloidosis cardiomyopathy (ATTR-CM). In some embodiments, the subject has transthyretin amyloidosis polyneuropathy (ATTR-PN). In some embodiments, the subject has wild-type ATTR (ATTRwt), age-related deposits of wild-type TTR protein (formerly known as senile amyloidosis).

[0074] As used herein, "administering" and its grammatical equivalents can refer to providing one or more replication-competent recombinant adenoviruses or pharmaceutical compositions described herein to a subject or patient. By way of example and not limitation, "administering" can be performed by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, intramuscular (im) injection, intravascular injection, intracerebroventricular (icv) injection, intrathecal (it) injection, infusion (inf.), oral route (po.), topical (top.) administration, or rectal (pr.) administration. One or more such routes can be used. One or more such routes can be used.

[0075] The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraventricular, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration can be performed, for example, by bolus injection or gradual perfusion over time. Additionally, it can be administered to a subject via an injectable depot administration route, for example, using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0076] As used herein, the terms "treat," "treating," or "treatment," and their grammatical equivalents, can include alleviating, reducing, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, e.g., arresting the progression of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating symptoms caused by a disease or condition, or halting the symptoms of a disease or condition, prophylactically and / or therapeutically. "Treating" can refer to administering a composition containing nanoparticles, such as lipid nanoparticles (LNPs), to a subject after the onset or suspected onset of a disease or condition. "Treating" encompasses the concept of "alleviating," which refers to reducing the frequency of occurrence or recurrence or severity of any symptoms or other adverse effects associated with a disease or condition, and / or side effects associated with a disease or condition. The term "treating" also encompasses the concept of "managing," which refers to reducing the severity of a patient's particular disease or disorder or delaying its recurrence, e.g., extending the period of remission in a patient suffering from a disease. The term "treating" further encompasses the concepts of "preventing," "preventing," and "prophylaxis." Although not excluded, it is understood that treating a disorder or condition does not require that the associated disorder, condition, or symptoms be completely eliminated.

[0077] As used herein, the terms "prevent," "preventing," "prevention," and the like refer to not developing the disease or condition, but reducing the likelihood of developing the disease or condition in a subject who is at risk of or susceptible to it.

[0078] As used herein, the term "ameliorate" can refer to reducing, suppressing, attenuating, decreasing, arresting, or stabilizing the onset or progression of a disease.

[0079] As used herein, "delaying" the onset of a disease means delaying, hindering, slowing, inhibiting, stabilizing, and / or postponing the progression of the disease. This delay can be of varying duration, depending on the history of the disease and / or the individual being treated. A method that "delays" or reduces the onset of a disease or delays the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame compared to not using the method. Such comparisons are usually based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0080] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. Disease onset can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of the condition. "Onset" includes occurrence, recurrence, and onset.

[0081] As used herein, "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[0082] The term "therapeutic agent" can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect. A therapeutic agent can also be referred to as an "active substance" or "active drug." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0083] As used herein, the term "pharmaceutical composition" and its grammatical equivalents may refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or therapeutic agents to be administered to a subject, e.g., a human in need thereof.

[0084] As used herein, the term "pharmaceutically acceptable" and its grammatical equivalents can refer to the attributes of a material useful in preparing a pharmaceutical composition that is generally safe, non-toxic, not biologically or otherwise undesirable, and acceptable for veterinary and human pharmaceutical use. "Pharmaceutically acceptable" can refer to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, i.e., such a substance can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition it is contained in.

[0085] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, which does not destroy its pharmacological activity and which is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0086] A "pharmaceutically acceptable salt" may be a salt of an acid or base generally considered in the art to be suitable for use in contact with the tissues of humans or animals without undue toxicity, irritation, allergic reaction, or other problem or complication. Those skilled in the art will recognize from this disclosure and knowledge in the art that additional pharmaceutically acceptable salts include those set forth in: Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).

[0087] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, payload, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) to be delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0088] Ranges provided herein are understood to be abbreviations for all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 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, or 50, as well as all decimal numbers between the recited integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either endpoint of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0089] Numbers expressing amounts of components, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. The doctrine of equivalents is not intended to be a limitation on the scope of the claims, and each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0090] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements.

[0091] As used herein, a spacer sequence of a guide nucleic acid is considered "homologous" to a protospacer sequence of a target nucleic acid if a base editor of a base editor system that includes the spacer sequence is capable of making a modification to a base in the target nucleic acid. A spacer sequence that is homologous to a protospacer sequence can be identical or substantially identical to the protospacer sequence.

[0092] As used herein, a nucleic acid sequence that is "substantially identical" to another nucleic acid sequence is a nucleotide sequence that has 70% or greater sequence identity with the other nucleic acid sequence.

[0093] For purposes of percent sequence identity between an RNA sequence (e.g., a spacer) and a DNA sequence (e.g., a target gene protospacer), a uracil base in the RNA should be considered identical to a thymine base in the DNA sequence.

[0094] As used herein, "sequence identity" refers to the degree to which two optimally aligned nucleic acid sequences are consistent across the entire window of alignment of components, e.g., nucleotides. "Identity" can be easily calculated by known methods, including but not limited to, those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0095] As used herein, the term "percent sequence identity" or "percent identity" refers to the proportion of identical nucleotides in a linear polynucleotide sequence of a reference ("query") nucleic acid (or its complementary strand) compared to a test ("subject") nucleic acid (or its complementary strand) when the two sequences are optimally aligned. Percent sequence identity can be determined when the sequences being compared are aligned for maximum correspondence, as measured using sequence comparison algorithms described below and as known in the art, or by visual inspection.

[0096] In sequence comparison, one sequence usually serves as a reference sequence to be compared with the test sequence.When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are designed, and the parameters of the sequence algorithm program are designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) to the reference sequence based on the designated program parameters.The optimal alignment of sequences to align the comparison window is well known to those skilled in the art, and can be performed using tools such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman similarity search method, or optionally, computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). The "percent identity" of an aligned segment of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences divided by the total number of elements in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. Percent sequence identity is expressed as the percentage of identity multiplied by 100.

[0097] "Percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. Extension of the word hits in each direction is halted when the cumulative alignment score drops by an amount X from the maximum achieved, when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below 0, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands.

[0098] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence versus the reference nucleotide sequence is less than about 0.1 to less than about 0.001.

[0099] In some embodiments, a first nucleotide sequence homologous to a second nucleotide sequence can hybridize to the complementary sequence of the second nucleotide sequence under stringent or highly stringent conditions. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization are sequence-dependent and vary under different environmental parameters. An extensive guide to nucleic acid hybridization can be found in: Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide containing 1 mg heparin at 42°C, with hybridization carried out overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent washing conditions is a 0.2xSSC wash at 65°C for 15 minutes (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3 for a description of SSC buffer). rd(ed., Cold Spring Harbor Laboratory Press, 2001 for a description of SSC buffer). Often, a low-stringency wash is performed before a high-stringency wash to remove background probe signal. For example, a moderate-stringency wash for a double-stranded probe of more than 100 nucleotides is 1x SSC at 45°C for 15 minutes. For example, a low-stringency wash for a double-stranded probe of more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a pH of 7.0-8.3, a salt concentration of less than about 1.0 M Na ion, typically about 0.01-1.0 M Na ion (or other salt), and a temperature of at least about 30°C. Stringent conditions can also be achieved by adding a destabilizing agent, such as formamide.

[0100] In several places throughout this application, guidance is provided through examples; these examples, including specific embodiments, can be used in various combinations and are the subject of the claims. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0101] For any method disclosed herein including discrete steps, the steps may be performed in any order possible, and any combination of two or more steps may be performed simultaneously, if desired.

[0102] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

[0103] Transthyretin protein and gene Transthyretin (TTR), originally known as prealbumin, is a 55 kDa transport protein for both thyroxine (T4) and retinol-binding protein, which circulates in soluble form in the serum and cerebrospinal fluid (CSF) of healthy humans. TTR is understood to be synthesized primarily in the liver. Under normal conditions, TTR circulates as a homotetramer with a central channel. The wild-type TTR monomer is 147 amino acids in length and has the following amino acid sequence: MASHRLLLLC LAGLVFVSEA GPTGTGESKC PLMVKVLDAV RGSPAINVAV HVFRKAADDT WEPFASGKTS ESGELHGLTT EEEFVEGIYK VEIDTKSYWK ALGISPFHEH AEVVFTANDS GPRRYTIAAL LSPYSYSTTA VVTNPKE (SEQ ID NO: 23).

[0104] The TTR gene consists of four exons and is located on chromosome 18 at 18q12.1. The complete sequence of the human TTR gene is shown in Figure 4 and is also available at UniProtKB-P02766 (TTHY_HUMAN). Over 120 TTR variants have been identified to date, the majority of which are pathogenic. The most common pathogenic variant consists of a point mutation resulting in the substitution of valine with methionine at position 30 of the mature protein. This Val30Met mutation is the cause of hATTR amyloidosis and is the most frequent amyloidogenic mutation worldwide, accounting for approximately 50% of TTR variants.

[0105] Hereditary transthyretin amyloidosis (hATTR) is a disease caused by mutations in the TTR gene. Autosomal dominant mutations destabilize the TTR tetramer and enhance its dissociation into monomers, leading to misfolding, aggregation, and subsequent extracellular deposition of TTR amyloid fibrils at various tissue sites. This multisystem extracellular amyloid deposition (amyloidosis) leads to dysfunction of various organs and tissues. In particular, transthyretin amyloidosis-associated polyneuropathy (ATTR-PN) and transthyretin amyloidosis-associated cardiomyopathy (ATTR-CM) are severe diseases associated with significant morbidity and mortality.

[0106] If there is clinical suspicion of hATTR-PN, diagnosis is usually made by tissue biopsy with amyloid staining, amyloid typing (using immunohistochemistry or mass spectrometry), and / or TTR gene sequencing. If there is clinical suspicion of ATTR-CM, important diagnostic tools are either intracardiac muscle biopsy (using tissue staining and amyloid typing by immunohistochemistry or mass spectrometry) or technetium-99m pyrophosphate scanning. Both of these approaches can provide a diagnosis of ATTR-CM. TTR gene sequencing can be used to distinguish between hATTR-CM (mutation-positive) and ATTRwt-CM (mutation-negative).

[0107] The compositions described herein include a spacer having a nucleotide sequence that serves as a guide to direct a gene editing protein (e.g., a base editor) to alter the TTR gene, for example, by introducing one or more nucleic acid base changes into the TTR gene. These point mutations can be used to disrupt gene function by introducing missense mutation(s) that result in the production of a hypofunctional or non-functional protein, thereby silencing the TTR gene. Alternatively, it is contemplated herein that correction of one or more point mutations can be performed using a gene editing protein to modify the mutated gene to correct the mutation responsible for causing TTR gene dysfunction or otherwise alleviate the dysfunction of the gene.

[0108] Gene editing / gene modification As used herein, the term "gene editing" or "genetic modification" and its grammatical equivalents refer to genetic manipulation in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a naturally occurring nuclease or an artificially engineered nuclease). Genetic modification can include introducing a double-strand break, a nonsense mutation, a frameshift mutation, a splice site change, or an inversion in a polynucleotide sequence, e.g., a target polynucleotide sequence. Figure 1A shows the crispr Cas9 protein, an RNA-guided endonuclease that can be used to create a double-strand break at a site-specific location in DNA or a gene. Genetic modification can also be performed using other editors, such as base editors.

[0109] Base Editor A base editor (BE) or nucleobase editor (NBE) refers to an agent comprising a polypeptide capable of making modifications to bases (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). A base editor may comprise a macromolecule or macromolecular complex capable of converting (e.g., transitioning or transverting) a nucleobase in a polynucleic acid sequence to another nucleobase at one or more positions within a base editing window. A base editor may comprise a combination of (a) a nucleotide, nucleoside, or nucleobase-converting enzyme and (b) a nucleic acid-binding protein that can be programmed to bind to a specific nucleic acid sequence. The nucleic acid-binding protein can be catalytically inactivated or impaired so as not to cleave a single-stranded nucleic acid target or to nick or cleave at most one strand of a double-stranded nucleic acid target.

[0110] The base editor may comprise a polynucleotide programmable DNA-binding domain fused or linked to a domain with base-editing activity, resulting in a base editor fusion protein. The base editor fusion protein may comprise one or more linkers, e.g., peptide linkers between the domains. In some embodiments, the domain with base editing activity is linked to the guide RNA (e.g., via an RNA-binding motif on the guide RNA and an RNA-binding domain fused to a deaminase).

[0111] In some embodiments, base editors are a class of modular programmable proteins that contain a deaminase domain fused to a catalytically impaired CRISPR-Cas enzyme. Through hydrolytic deamination and subsequent cellular processing, adenine base editors (ABEs) convert A:T base pairs to G:C base pairs, and cytosine base editors (CBEs) convert C:G base pairs to T:A base pairs, without generating double-stranded DNA breaks. Each deaminase of the base editors is targeted to a site of interest by a guide RNA (gRNA) within D10A nickase Cas9 (nCas9). The cytidine deaminase enzyme of the CBE induces the conversion of cytosine to uridine, resulting in a C→T (or G→A) substitution (see Figure 1B). "Cytidine deaminase" is used herein to refer to a deaminase enzyme that acts on deoxycytidine, cytidine, or both deoxycytidine and cytidine to convert cytosine to uridine. Cytidine deaminase and cytosine deaminase may be used interchangeably herein. When the goal is to disrupt a gene in vivo for therapeutic purposes, cytosine base editors (CBEs) could potentially directly introduce stop codons into the coding sequence of a gene (nonsense mutations) by altering specific codons for glutamine (CAG → TAG, CAA → TAA), arginine (CGA → TGA), and tryptophan (TGG → TAG / TAA / TGA, editing the cytosine on the antisense strand).

[0112] In comparison, the adenosine deaminase enzyme of ABE induces the conversion of adenosine to inosine, resulting in an A→G (or T→C) substitution (see FIG. 1B). "Adenosine deaminase" is used herein to refer to a deaminase enzyme that acts on deoxyadenosine, adenosine, or both deoxyadenosine and adenosine to convert adenine to hypoxanthine or adenosine to inosine. Because the structure of inosine is similar to that of guanosine (inosine does not contain the exocyclic amino group of guanosine), inosine tends to behave like guanosine. Inosine is ultimately replaced by guanosine through subsequent cellular processing. Thus, adenosine deaminase results in an A→G (or T→C) substitution. Adenosine deaminase and adenine deaminase may be used interchangeably herein. Adenine base editors (ABEs) cannot directly introduce stop codons because they lack the A→G change that causes nonsense mutations.

[0113] Adenine base editors can be used to disrupt gene function, for example, by editing the start codon from ATG to GTG or from ATG to ACG. A second way in which adenine base editors can disrupt gene function is by editing the splice donor at the 5' end of an intron or the splice acceptor at the 3' end of an intron. Disruption of a splice site can result in the inclusion of intronic sequences in the messenger RNA (mRNA), which can introduce nonsense, frameshift, or in-frame mutations. These mutations can result in premature stop codons, or insertions / deletions of amino acids that inhibit protein activity, or the exclusion of exon sequences that can introduce nonsense, frameshift, or in-frame indel mutations.

[0114] As shown in Figure 2, the canonical splice donor contains the DNA sequence GT on the sense strand, while the canonical splice acceptor contains the DNA sequence AG. Alterations in the sequences disrupt normal splicing. The splice donor can be disrupted by adenine base editing of the complementary base at the second position of the antisense strand (GT → GC), and the splice acceptor can be disrupted by adenine base editing at the first position of the sense strand (AG → GG).

[0115] Adenine base editors (ABEs) include, but are not limited to, ABE8.8 (Gaudelli et al., Nat Biotechnol. 2020 Jul; 38(7): 892-900. doi:10.1038 / s41587-020-0491-6. Epub 2020 Apr 13)).

[0116] In embodiments, the adenine base editor is encoded by an mRNA comprising the sequence of MA004 mRNA shown in Table 11. In embodiments, the adenine base editor has 50% or more sequence identity with a MA004 mRNA shown in Table 11, 60% or more sequence identity with a MA004 mRNA shown in Table 11, 70% or more sequence identity with a MA004 mRNA shown in Table 11, 75% or more sequence identity with a MA004 mRNA shown in Table 11, 80% or more sequence identity with a MA004 mRNA shown in Table 11, 85% or more sequence identity with a MA004 mRNA shown in Table 11, 90% or more sequence identity with a MA004 mRNA shown in Table 11, 95% or more sequence identity with a MA004 mRNA shown in Table 11, 96% or more sequence identity with a MA004 mRNA shown in Table 11, 97% or more sequence identity with a MA004 mRNA shown in Table 11, 98% or more sequence identity with a MA004 mRNA shown in Table 11, or It is encoded by mRNA containing a sequence that has 99% or more sequence identity with the mRNA.

[0117] ABE7.10 (Gaudelli et al., Nature. 2017 Nov 23; 551 (7681): 464-471. doi:10.1038 / nature24644), and other ABE variants containing Streptococcus pyogenes Cas9. The CRISPR Journal, Volume 4, Number 2, 2021, pp. 169-177, and Supplementary Figures S1-S9, Supplementary Data S4-S6, and Supplementary Tables S1-S2 disclose additional inlaid base editor (IBE) variants.

[0118] In some embodiments, a base editor can convert a C:G base pair to a G:C base pair. Examples of such base editors are disclosed in: (a) Chen et al. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat Commun 12, 1384 (2021). doi: 10.1038 / s41467-021-21559-9. Epub 2021-March-02; (b) Kurt, IC, Zhou, R., Iyer, S. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 39, 41-46 (2021). doi: 10.1038 / s41587-020-0609-x. Epub 2020-July-20; and (c) Zhao, D., Li, J., Li, S. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39, 35-40 (2021). doi: 10.1038 / s41587-020-0592-2. Epub 2020-July-20. Such base editors may include Cas9 nickase and cytidine deaminase. Such base editors may further include the DNA binding and ligase domains of uracil-DNA glycosylase, DNA repair proteins such as XRCC1, DNA ligase S, or DNA polymerase b.

[0119] In some embodiments, the base editor can convert a C:G base pair to an A:T base pair. Examples of such base editors are disclosed in: Zhao, D., Li, J., Li, S. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39, 35-40 (2021). doi: 10.1038 / s41587-020-0592-2. Epub 2020-July-20. Such base editors may include a Cas9 nickase and a cytidine deaminase. Such base editors may further include a uracil-DNA glycosylase.

[0120] The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In various embodiments, the base editor system comprises: (1) a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9); (2) a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) for deaminating the nucleobase; and (3) one or more guide polynucleotides (e.g., guide RNAs). In some embodiments, the base editor system comprises a base editor fusion protein comprising (1) and (2), or a polynucleotide (e.g., mRNA) encoding a base editor fusion protein comprising (1) and (2). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is an adenine base editor or an adenosine base editor (ABE). In some embodiments, the base editor is a cytosine base editor (CBE).

[0121] Genome editing systems include clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems. Exemplary guide nucleotide sequence-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), saCas9 (e.g., saCas9d, saCas9d, saKKHCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Argonaute, and any other suitable protein described herein, or a suitable variant thereof.

[0122] Through the use of guide RNAs (gRNAs) with sequences homologous to sequences of DNA in the target genome (known as protospacers) adjacent to specific protospacer adjacent motifs (PAMs) containing the sequence NGG (where N is any standard base), Cas9 can be used to create double-strand breaks (DSBs) in target sequences. Non-homologous end joining (NHEJ) at the DSBs can create indels and potentially knock out genes at the locus. Similarly, homology-directed repair (HDR) using introduced template DNA can insert genes or modify target sequences.

[0123] In recent years, various Cas9-based tools have been developed. Methods using guide nucleotide sequence programmable DNA binding proteins such as Cas9 for site-specific cleavage (for example, for genome modification) have been described (see, for example, Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Hwang et al., Nature Biotechnology 31, 227-229 (2013); Jinek et al., eLife 2, e00471 (2013); Dicarlo et al., Nucleic Acids Research (2013); and Jiang et al., Nature Biotechnology 31, 233-239 (2013)).

[0124] In 2016, Komor et al. described the use of CRISPR-Cas9 to convert cytosine bases to thymine bases without the introduction of a template DNA strand and without the need for DSBs (Komor et al., Nature, 2016, 533: 420-4). After the cytidine deaminase domain of rat APOBEC1 was fused to the N-terminus of catalytically inactive Cas9 (dCas9) using the linker XTEN (resulting in a fusion protein called base editor 1 or BE1), cytosine-to-uracil conversion was observed between positions 4 and 8 within the 20-nt protospacer region of DNA (or, alternatively, 13–17 nucleotides upstream of the PAM). Notably, cytosine bases within this “window” were susceptible to editing, leading to variable results depending on how many and which cytosines were edited. After DNA replication or repair, each uracil was replaced with a thymine, completing the C to T base editing.

[0125] The next version of the base editor (BE2) incorporated a uracil glycosylase inhibitor (UGI) fused to the C-terminus of dCas9 to help inhibit base excision repair of uracil bases resulting from cytidine deaminase activity (which would otherwise act to restore the original cytosine base); this improved the efficiency of C→T base editing.

[0126] The next version of the base editor (BE3) used the Cas9 nickase instead of dCas9. The nickase cleaves the unedited strand opposite the edited C→T base, stimulating removal of the opposite guanidine through eukaryotic mismatch repair. BE2 and BE3 base editing were observed in both human and mouse cell lines. Figure 1B shows a diagram of BE3. The specificity of base editing was further improved by adding mutations to the Cas9 nickase; in a similar manner, Cas9 was mutated to narrow the editing window from approximately 5 nucleotides to only 1–2 nucleotides (Rees et al., Nat Commun, 2017, 8: 15790, Kim et al., Nat Biotechnol, 2017, 35: 371–376).

[0127] Fusing the Escherichia coli adenine tTNA deaminase TadA (ecTadA) to dCas9 and mutagenesis of the ecTadA domain along with selection for editing activity revealed that the A106V and D108N mutations generated a base editor (termed ABE7.10) capable of editing adenine to guanine in DNA (Gaudelli et al. Nature, 2017, 551: 464-71).

[0128] ABE8.8-m (also referred to herein as ABE8.8) uses its core Streptococcus pyogenes nickase Cas9 (nSpCas9) protein in conjunction with a guide RNA (gRNA) to engage a double-stranded protospacer DNA sequence flanked at its 3' end by an NGG protospacer adjacent motif (PAM) sequence. The protospacer sequence is specified by hybridization of the first 20 bases of the gRNA with a complementary sequence on the "target" DNA strand, leaving a portion of the remaining ("non-target") strand exposed in a single-stranded structure (called an R-loop). Unlike Cas9 and Cas12, ABE8.8 does not make a double-stranded break within the target DNA sequence. Rather, as shown in Figure 1C, ABE8.8 uses an evolved deoxyadenosine deaminase domain fused to nSpCas9 to chemically modify the adenosine nucleoside contained in the single-stranded DNA portion of the R-loop to inosine, creating a nick in the target DNA strand within the DNA:RNA heteroduplex of the R-loop. This nick biases the DNA repair mechanism to use the newly deaminated strand as a template, enabling highly efficient base transfer mutagenesis at the target site. The activity window of ABE8.8 is typically 12–18 base pairs 5′ to the NGG PAM (positions 21–23) and ranges from positions 3–9 of the gRNA-specified protospacer DNA sequence, with peak editing observed at position 6 of the protospacer (Gaudelli et al., Nat Biotechnol. 2020 Jul;38(7):892–900).

[0129] In some embodiments, the nucleic acid encoding the base editor fusion protein is mRNA. In some embodiments, the mRNA, when translated in the target cell or subject after administration, produces the base editor fusion protein. In some embodiments, the base editor fusion protein forms a ribonucleoprotein (RNP) complex in the target cell or subject.

[0130] It should be understood that the fusion proteins of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein may include a cytoplasmic localization sequence, a transport sequence such as a nuclear transport sequence, or other localization sequence, and a sequence tag useful for solubilizing, purifying, or detecting the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag (also known as histidine tag or His tag), maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Sof tag (e.g., Softag 1, Softag 3), strep tag, biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Additional suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein comprises one or more His tags.

[0131] Protospacer As used herein, the terms "protospacer" or "target sequence" and their grammatical equivalents may refer to a PAM-adjacent nucleic acid sequence. A protospacer can be a nucleotide sequence within a gene, genome, or chromosome targeted by a gRNA. In its natural state, a protospacer is adjacent to a PAM (protospacer adjacent motif). A cleavage site by an RNA-guided nuclease is located within the protospacer sequence. For example, as shown in Figure 1A, when a gRNA targets a specific protospacer, a Cas protein generates a double-stranded break within the protospacer sequence, thereby cleaving the protospacer. After cleavage, disruption of the protospacer can result in non-homologous end joining or homology-directed repair. Disruption of the protospacer can result in deletion of the protospacer. Additionally or alternatively, disruption of the protospacer can result in insertion of an exogenous nucleic acid sequence into the protospacer or replacement of the protospacer.

[0132] In this disclosure, we identified protospacer sequences within the nucleic acid sequence of the human TTR gene to be used as guide sequences to cause ABE8.8 (and other ABE variants containing Streptococcus pyogenes Cas9, such as ABE7.10, or another Cas protein that can use NGG PAM) to destroy a start codon or a splice site, regardless of donor or acceptor, via A→G editing within the editing window (approximately positions 4-7 of the 20-nt protospacer region of DNA). Four of the sequences shown in Table 1 were identified within the human TTR gene. An alignment of these four protospacer sequences on the map of the human TTR gene is shown in Figure 3.

[0133] The protospacer corresponding to guide RNA GA457 has the sequence 5'-GCCATCCTGCCAAGAATGAG-3' (SEQ ID NO: 24) and is located at 34,879 to 34,898 bp of the human TTR gene.

[0134] The protospacer corresponding to guide RNA GA459 has the sequence 5'-GCAACTTACCCAGAGGCAAA-3' (SEQ ID NO: 25) and is located at 36,007 to 36,026 bp of the human TTR gene.

[0135] The protospacer corresponding to guide RNA GA460 has the sequence 5'-TATAGGAAAACCAGTGAGTC-3' (SEQ ID NO: 26) and is located at bp 38,106 to 38,125 of the human TTR gene.

[0136] The protospacer corresponding to guide RNA GA461 has the sequence 5'-TACTCACCTCTGCATGCTCA-3' (SEQ ID NO: 27) and is located at positions 38,234 to 38,253 of the human TTR gene.

[0137] The protospacer corresponding to guide RNA GA458 has the sequence 5'-GCCATCCTGCCAAGAACGAG-3' (SEQ ID NO: 28) and represents the sequence within the cynomolgus TTR gene that corresponds to the human protospacer sequence corresponding to guide RNA GA459.

[0138] The guide nucleic acid (e.g., guide RNA) is approximately 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides complementary to the target protospacer sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to a standard PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to a standard PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

[0139] In some embodiments, the guide polynucleotide is DNA. In some embodiments, the guide polynucleotide is RNA, also referred to herein as guide RNA or gRNA. In some embodiments, the guide polynucleotide is a modified artificial polynucleotide.

[0140] In some embodiments, a guide polynucleotide, including but not limited to a gRNA, can be synthesized. The guide polynucleotide may include a spacer sequence configured to hybridize, for example, under intracellular conditions, to the complement of a protospacer sequence shown in Table 1. The guide polynucleotide may include a spacer sequence that is homologous to a protospacer sequence shown in Table 1. In some embodiments, the guide polynucleotide includes a guide RNA that includes a spacer having a sequence homologous to a protospacer listed in Table 1. In some embodiments, the guide RNA may have a sequence that includes a guide RNA (gRNA) sequence listed in Table 1.

[0141] The present disclosure includes guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457). The present disclosure includes guide polynucleotides having the sequence 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457).

[0142] The present disclosure includes modified guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), where GCC is modified by methylation (C is modified to 2'-O-methyl cytidine and G is modified to 2'-O-methyl guanosine). The present disclosure includes modified guide polynucleotides having the sequence 5'-mGsmCsmCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA521), where mC: 2'-O-methyl cytidine, mG: 2'-O-methyl guanosine, and s: phosphorothioate (PS) backbone linkage.

[0143] The present disclosure includes guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO:2) (GA458). The present disclosure includes guide polynucleotides having the sequence 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO:2) (GA458).

[0144] The present disclosure includes guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459). The present disclosure includes guide polynucleotides having the sequence 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459).

[0145] The present disclosure includes guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460). The present disclosure includes guide polynucleotides having the sequence 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460).

[0146] The present disclosure includes guide polynucleotides having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO: 5) (GA461). The present disclosure includes guide polynucleotides having the sequence 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO: 5) (GA461).

[0147]

[0023] In some aspects, provided herein is a guide RNA comprising a sequence defined by: mG*mC*mC*AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU* Provided is a guide RNA that is mU(GA521) (SEQ ID NO: 11), where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, and mU* is 2'-O-methyl uridine, and the nucleotides represented in bold are linked by phosphorothioate (PS) backbone linkages.

[0148] Alternatively, GA521 is represented as:

[0149] mG*smC*smC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmsGsmCsmUsmAsGsmAsmAsmAsmUsmAsmGsmCssmAsmAsGsUsUsUsmAsAsmAsAsmUsAsmAs mGsmGsmCsmUsmAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAsmGsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsm UsmGsmCsmU*smU*smU*smU (GA521) (SEQ ID NO: 11), where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, and the nucleotides represented in bold are linked by phosphorothioate (PS) backbone linkages represented by the letter "s."

[0150] In some embodiments, mG*mC*mC*AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (GA521, SEQ ID NO: 11) , where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, and the nucleotides shown in bold are linked by phosphorothioate (PS) backbone linkages.

[0151] Alternatively, GA521 is represented as: mG*mC*mC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmsGsmCsmUsmAsGsmAsmAsmAsmUsmAsmGsmCssmAsmAsGsUsUsmAsAsAsAsAsmUsAsmAsmGsmGsmCsmU smAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAsmGsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsmUsmGsmCsmU*mU*mU*mU (GA521) (SEQ ID NO: 11), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, and nucleotides represented in bold are linked by phosphorothioate (PS) backbone linkages represented by the letter "s."

[0152] In some embodiments, at least two or more of the tracrRNA nucleotides are linked by phosphorothioate (PS) backbone linkages. In some embodiments, all of the tracrRNA nucleotides are linked by phosphorothioate (PS) backbone linkages. In some embodiments, only the tracrRNA nucleotides are linked by phosphorothioate (PS) backbone linkages.

[0153] In some embodiments, only the methyl-modified nucleotides are linked by phosphorothioate (PS) backbone linkages.

[0154] A guide polynucleotide may contain at least three regions: a first region (spacer region) at the 5' end that may be homologous to a target site in a chromosomal sequence, a second internal region that may form a stem-loop structure, and a 3' region that may be single-stranded. The second and third regions are considered the tracr sequence or region of the guide RNA and function as a binding scaffold for base editors or CRISPR / Cas proteins, while the spacer region functions to guide the protein to a specific target site. The acronym tracr stands for trans-activating CRISPR.

[0155] The second region of the gRNA may form a secondary structure. In some embodiments, the secondary structure formed by the gRNA may comprise a stem (or hairpin) and a loop. The lengths of the loop and stem may vary. In some embodiments, the loop may range from about 3 to about 10 nucleotides in length. In some embodiments, the stem may range from about 6 to about 20 nucleotides in length. The stem may include one or more bulges of 1 to 10 nucleotides or about 10 nucleotides in length. In some embodiments, the total length of the second region may range from about 16 to 60 nucleotides in length. In some embodiments, the loop may be about 4 nucleotides in length. In some embodiments, the stem may be about 12 nucleotides in length.

[0156] The third region at the 3' end of the gRNA can be single-stranded. In some embodiments, the third region is not complementary to any chromosomal sequence in the cell of interest, nor is it complementary to the remainder of the gRNA. The third region can have any suitable length. For example, the third region can be 3 nucleotides or more, or 4 nucleotides or more. In some embodiments, the length of the third region can vary from about 5 to about 60 nucleotides.

[0157] In some embodiments, the guide polynucleotide comprises a spacer sequence that is homologous to a protospacer sequence of the TTR gene shown in Table 1 with 0, 1, 2, 3, 4, or 5 mismatches. In some embodiments, the guide polynucleotide comprises a spacer sequence that is homologous to a protospacer sequence of the TTR gene shown in Table 1 with no mismatches.

[0158] In some embodiments, the length of the guide polynucleotide depends on the CRISPR / Cas components of the base editor system and the components used. For example, different Cas proteins from different bacterial species have various optimal targeting sequence lengths. Thus, the targeting sequence may comprise 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, or 50 or more nucleotides in length. In some embodiments, the targeting sequence comprises 18-24 nucleotides in length. In some embodiments, the targeting sequence comprises 19-21 nucleotides in length. In some embodiments, such as those described in Table 1, the targeting sequence comprises 20 nucleotides in length.

[0159] In some embodiments, the guide polynucleotide includes a spacer sequence and otherwise conforms to the standard 100 nt Streptococcus pyogenes CRISPR gRNA sequence.

[0160] In some embodiments, the guide RNA is chemically modified. Chemically modified gRNAs may have increased stability when transfected into mammalian cells. For example, gRNAs can be chemically modified to include a combination of 2'-O-methylribosugar and phosphorothioate backbone modifications in at least one 5' nucleotide and at least one 3' nucleotide of each gRNA. In some cases, the three terminal 5' nucleotides and the three terminal 3' nucleotides are chemically modified to include a combination of 2'-O-methylribosugar and phosphorothioate modifications.

[0161] The gRNAs described herein can be synthesized chemically, enzymatically, or a combination thereof. For example, gRNAs can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, gRNAs can be synthesized in vitro by operably linking DNA encoding the gRNA to a promoter regulatory sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include, but are not limited to, T7, T3, and SP6 promoter sequences, or variations thereof. In some embodiments, the gRNA comprises two separate molecules (e.g., crRNA (including a spacer) and tracrRNA). One molecule (e.g., crRNA) can be chemically synthesized, and the other molecule (e.g., tracrRNA) can be enzymatically synthesized.

[0162] therapeutic use The guide polynucleotides and compositions described herein can be administered in therapeutically effective amounts to target cells or to a subject in need thereof to prevent or treat conditions associated with transthyretin amyloidosis. In some embodiments, the subject has hereditary transthyretin amyloidosis (hATTR). In some embodiments, the subject has transthyretin amyloidosis cardiomyopathy (ATTR-CM). In some embodiments, the subject has transthyretin amyloidosis polyneuropathy (ATTR-PN). In some embodiments, the subject has wild-type ATTR (ATTRwt), age-related deposits of wild-type TTR protein (formerly known as senile amyloidosis).

[0163] Upon such administration, the guide polynucleotide induces the editor system (e.g., the ABE editor system) to impart a nucleic acid base change to the subject's TTR gene, editing the TTR gene to reduce or eliminate the amount of full-length, functional protein produced, thereby treating the condition. In some embodiments, the base change occurs in cells of the subject's liver (hepatocytes).

[0164] For example, a gRNA and an adenosine base editor protein can be expressed in a cell in which target gene editing is desired (e.g., a hepatocyte), thereby allowing contact of the target gene with the gRNA and adenosine base editor protein. In some embodiments, binding of the adenosine base editor protein to its target polynucleotide sequence within the target gene is guided by a guide RNA, and the spacer sequence of the gRNA hybridizes to a target polynucleotide sequence within the target gene, e.g., a sequence complementary to the protospacer. Thus, the guide RNA guides the adenosine base editor protein to edit the target polynucleotide sequence (e.g., the protospacer sequence) within the target gene. In some embodiments, the guide RNA is co-introduced into a cell in which editing is desired along with the adenosine base editor protein or a nucleic acid encoding the adenosine base editor protein.

[0165] In certain embodiments, adenine base editors can be used to disrupt gene function and / or expression by modifying the nucleobases of splice sites of target genes. In some embodiments, the adenosine nucleobase editors described herein can be used to disrupt a splice donor site at the 5' end of an intron or a splice acceptor site at the 3' end of an intron. In some embodiments, splice site disruption results in the inclusion of intronic sequences in messenger RNA (mRNA), which can introduce nonsense, frameshift, or in-frame indel mutations. These mutations result in premature stop codons, or insertions / deletions of amino acids that disrupt protein activity, or the exclusion of exon sequences that can also introduce nonsense, frameshift, or in-frame indel mutations.

[0166] The canonical splice donor comprises the DNA sequence GT on the sense strand, while the canonical splice acceptor comprises the DNA sequence AG. In some embodiments, base editors such as the adenosine nucleobase editors described herein can be used to generate sequence changes that disrupt normal splicing. In some embodiments, the adenosine base editor destroys the complementary A on the antisense strand of the splice donor, resulting in a GT→GC edit. In some embodiments, the adenosine base editor destroys the A at the splice acceptor site on the sense strand, resulting in an AG→GG edit.

[0167] In some embodiments, the methods and compositions disclosed herein reduce or eliminate the expression and / or function of the transthyretin protein encoded by the TTR gene. For example, the methods and compositions disclosed herein may reduce transthyretin expression and / or function by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, or at least ten-fold compared to a control.

[0168] In some embodiments, as described herein, a method for treating or preventing a condition in a subject in need thereof comprises administering to the subject (i) a guide polynucleotide and (ii) a nucleic acid encoding a base editor fusion protein.

[0169] In some embodiments, as described herein, a method of treating or preventing a condition in a subject in need thereof comprises administering a lipid nanoparticle (LNP) encapsulating (i) a guide polynucleotide or a nucleic acid encoding a guide polynucleotide, and / or (ii) a base editor fusion protein comprising a programmable DNA-binding domain and a deaminase or a nucleic acid encoding the same. In some aspects, (i) the guide polynucleotide or a nucleic acid encoding the same, and (ii) the base editor fusion protein comprising a programmable DNA-binding domain and a deaminase or a nucleic acid encoding the same are encapsulated in the same LNP. In some aspects, they are encapsulated in separate LNPs.

[0170] Pharmaceutical Composition In some aspects, provided herein are pharmaceutical compositions comprising the base editor system provided herein and a pharmaceutically acceptable carrier or excipient. In some aspects, provided herein are pharmaceutical compositions for genetic modification comprising a guide RNA and a base editor fusion protein or a nucleic acid sequence encoding a base editor fusion protein described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions are formulated in conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compound into a pharmaceutically usable preparation. Formulations and delivery methods suitable for use in the present disclosure are generally well known in the art. Suitable formulations depend on the selected route of administration. Summary summaries of the pharmaceutical compositions described herein can be found, for example, in: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0171] A pharmaceutical composition can be a mixture of a guide RNA or a nucleic acid sequence encoding a guide RNA described herein and a base editor fusion protein or a nucleic acid sequence encoding a base editor fusion protein, together with one or more other chemical components (i.e., pharmaceutically acceptable components), such as carriers, excipients, binders, fillers, suspending agents, flavorings, sweeteners, disintegrants, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, osmotic agents, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. The pharmaceutical composition facilitates administration to an organism or subject in need thereof.

[0172] The pharmaceutical compositions of the present disclosure can be administered to a subject using any suitable method known in the art. The pharmaceutical compositions described herein can be administered to a subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, intracolonally, intrarectally, or intraperitoneally. In some embodiments, the pharmaceutical composition can be administered to a subject by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally.

[0173] In some embodiments, the pharmaceutical composition for genetic modification includes an additional therapeutic agent. The additional therapeutic agent may modulate various aspects of the disease, disorder, or condition being treated, providing a greater overall benefit than administration of the therapeutic agent alone. The therapeutic agent includes, but is not limited to, a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapeutic agent, and / or an immunotherapeutic agent. In some embodiments, the therapeutic agent may be a radiotherapeutic agent. In some embodiments, the therapeutic agent may be a hormonal therapeutic agent. In some embodiments, the therapeutic agent may be an immunotherapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. The preparation and administration schedule of the additional therapeutic agent can be used according to the manufacturer's instructions or as empirically determined by a skilled physician.

[0174] Lipid Nanoparticle (LNP) Composition The pharmaceutical compositions for genetic modification described herein can be encapsulated in lipid nanoparticles (LNPs). As used herein, a "lipid nanoparticle (LNP) composition" or "nanoparticle composition" refers to a composition containing one or more of the described lipids. LNP compositions or formulations contemplated herein are typically submicrometer in size and may contain a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions or formulations contemplated herein may be liposomes with a diameter of 500 nm or less and a lipid bilayer. The LNPs described herein can have an average diameter of about 1 nm to about 2500 nm, about 10 nm to about 1500 nm, about 20 nm to about 1000 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 50 nm to 90 nm, about 55 nm to 85 nm, about 55 nm to 75 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. The LNPs described herein can have an average diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or more. In one embodiment, the average diameter of the LNPs is about 70 nm ± 20 nm, 70 nm ± 10 nm, or 70 nm ± 5 nm. The LNPs described herein can be substantially non-toxic.

[0175] Lipid nanoparticles (LNPs) use a nonviral drug delivery mechanism that can penetrate blood vessels and reach hepatocytes [Am. J. Pathol. 2010, 176, 14-21]. Apolipoprotein E (ApoE) protein can bind to LNPs after PEG lipid diffusion from the nearly neutrally charged LNP surface in the bloodstream, thereby functioning as an endogenous ligand for hepatocytes expressing the low-density lipoprotein receptor (LDLr) [Mol. Ther., 2010, 18, 1357-1364]. Control of efficient liver delivery of LNPs involves: 1) effective PEG lipid shedding from the LNP surface in serum and 2) binding of ApoE to LNPs. The endogenous ApoE-mediated LDLr-dependent LNP delivery pathway is unavailable or less effective for achieving LNP-based hepatic gene delivery in LDLr-deficient patient populations.

[0176] Efficient delivery to cells requires specific targeting and substantial protection from the extracellular environment, especially serum proteins. One way to achieve specific targeting is to conjugate a targeting moiety to a pharmaceutical effector, such as an active drug or a nucleic acid drug, thereby directing the active drug or pharmaceutical effector to a specific cell or tissue depending on the specificity of the targeting moiety. One way that a targeting moiety can improve delivery is through receptor-mediated endocytosis activity. This uptake mechanism involves the transfer of nucleic acid substances bound to membrane receptors into the interior of a membrane-enclosed region through invagination of a membrane structure or fusion of the delivery system with the cell membrane. This process is initiated through the activation of cell surface or membrane receptors after specific ligands bind to the receptor. Receptor-mediated endocytosis systems include those that recognize sugars, such as galactose, mannose, mannose-6-phosphate, peptides and proteins, such as transferrin, asialoglycoprotein, vitamin B12, insulin, and epidermal growth factor (EGF). Lipophilic moieties such as cholesterol or fatty acids, when attached to highly hydrophilic molecules such as nucleic acids, can significantly enhance plasma protein binding and therefore extend circulatory half-life. Lipophilic conjugates can also be used in combination with targeting ligands to improve intracellular trafficking in targeted delivery approaches.

[0177] The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor abundant in hepatocytes. ASGP-R exhibits a 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than D-Gal. LNPs containing receptor-targeting conjugates can be used to facilitate targeted delivery of drug substances described herein. LNPs may contain one or more receptor-targeting moieties on or around the particle at specific or engineered surface densities ranging from relatively low to relatively high. The receptor-targeting conjugate may comprise a targeting moiety (or ligand), a linker, and a lipophilic moiety attached to the targeting moiety. In some embodiments, the receptor-targeting moiety (or ligand) targets a lectin receptor. In some embodiments, the lectin receptor is the asialoglycoprotein receptor (ASGPR). In some embodiments, the receptor-targeting moiety is GalNAc or a derivative of GalNAc that targets the ASGPR. In one aspect, the receptor-targeting conjugate comprises a GalNAc moiety or a derivative thereof. In another aspect, the receptor-targeting conjugate comprises two different GalNAc moieties or derivatives thereof. In another aspect, the receptor-targeting conjugate comprises three different GalNAc moieties or derivatives thereof. In another aspect, the receptor-targeting conjugate is lipophilic. In some embodiments, the receptor-targeting conjugate comprises one or more GalNAc moieties and one or more lipid moieties, i.e., GalNAc-lipids. In some embodiments, the receptor-targeting conjugate is a GalNAc-lipid.

[0178] Described herein are (i) LNP compositions comprising an amino lipid, a phospholipid, a PEG-lipid, cholesterol or a derivative thereof, a payload, or any combination thereof, and (ii) LNP compositions comprising an amino lipid, a phospholipid, a PEG-lipid, cholesterol, a GalNAc-lipid or a derivative thereof, a payload, or any combination thereof, each of which is described in detail below.

[0179] To prepare an LNP composition containing the excipients amino lipid, phospholipid, PEG-lipid, and cholesterol, the four excipients are dissolved in a water-miscible organic solvent, such as ethanol, in the desired molar ratio. The homogeneous lipid solution is then rapidly mixed in-line with an aqueous buffer solution containing a nucleic acid payload, typically an acidic pH range of 4-6.5, to form lipid nanoparticles (LNPs) encapsulating the nucleic acid payload(s). After rapid in-line mixing, the LNPs thus formed undergo further downstream processing, including concentration and buffer exchange, to obtain a final LNP pharmaceutical composition with a near-neutral pH for administration to cell lines or animal disease models for evaluation, or to human subjects.

[0180] For the preparation of GalNAc-LNP pharmaceutical compositions, GalNAc-lipids are mixed with four lipid excipients in a water-miscible organic solvent prior to preparation of GalNAc-LNP. Preparation of the GalNAc-LNP pharmaceutical composition then follows the same steps as described for the LNP pharmaceutical composition. The mole percent of GalNAc-lipids in the GalNAc-LNP preparation ranges from 0.001 to 2.0 of the total excipients.

[0181] In both LNP and GalNAc-LNP preparations, the payload comprises a guide RNA targeting the TTR gene and an mRNA encoding a base editor protein. In some embodiments, the weight ratio of guide RNA to mRNA in the acidic aqueous buffer and in the final formulation is 6:1, 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:5, or 1:6. In some embodiments, the mRNA encodes an adenosine base editor protein. In some other embodiments, the mRNA encodes a cytosine or cytidine base editor protein.

[0182] In some embodiments, LNP compositions may be prepared as described below: U.S. Patent Application No. 17 / 192,709, entitled "COMPOSITIONS AND METHODS FOR TARGETED RNA DELIVERY," filed March 4, 2021, which claims the benefit of U.S. Provisional Patent Application Nos. 62 / 984,866 (filed March 4, 2020) and 63 / 078,982 (filed September 16, 2020), naming Kallanthottathil G. Rajeev as inventor and Verve Therapeutics, Inc. as applicant (which application is incorporated herein by reference in its entirety).

[0183] Amino lipids In some embodiments, the LNP composition comprises an amino lipid. In some embodiments, the cationic lipid is an ionizable lipid. In some embodiments, the amino lipid (e.g., an ionizable lipid) is a cationic lipid. In some embodiments, the amino lipid (e.g., an ionizable / cationic lipid) comprises one or more nitrogen atoms. Exemplary, non-limiting amino lipids suitable for the compositions described herein include those described herein.

[0184] Formula (I) In one aspect, disclosed herein is an amino lipid having the structure of formula (I), or a pharmaceutically acceptable salt or solvate thereof: [ka] (In the formula, R 1 and R 2 Each of the C3-C 22 Alkyl, C3-C 22 Alkenyl, C3-C8 cycloalkyl, -C2-C 10 Alkylene-LR 6 ,or [ka] wherein each of alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted; Each of X, Y, and Z is independently —C(═O)NR 4 -, -NR 4 C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C(=O)NR 4 -, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4 C(=S)-, -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4 C(=S)NR 4 -, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-, -SC(=O)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -O, S, or a bond, Each L is independently —C(═O)NR 4 -, -NR 4 C(=O)-, -C(=O)O-, -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C(=O)NR 4 -, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4C(=S)-, -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4 C(=S)NR 4 -, -C(=O)S-, SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-, -SC(=O)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, O, S, -C1-C 10 Alkylene-O-, -C1-C 10 Alkylene -C(=O)O-, -C1-C 10 alkylene -OC(=O)-, or a bond, wherein alkylene is substituted or unsubstituted; R 3 is -C0-C 10 Alkylene-NR 7 R 8 , -C0-C 10 Alkylene-heterocycloalkyl, or -C0-C 10 alkylene-heterocycloaryl, wherein alkylene, heterocycloalkyl, and heterocycloaryl are independently substituted or unsubstituted; R 4 are each independently hydrogen or substituted or unsubstituted C1-C6 alkyl; R 5 is hydrogen or substituted or unsubstituted C1-C6 alkyl; R 6 each independently being a substituted or unsubstituted C-C 22 Alkyl, or substituted or unsubstituted C3-C 22 is alkenyl, R 7 and R 8 are each independently hydrogen, or substituted or unsubstituted C1-C6 alkyl, or R7 and R 8 together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl; p is an integer from 1 to 10, Each of n, m, and q is independently 0, 1, 2, 3, 4, or 5.

[0185] In some embodiments of Formula (I), when the structure has two or more asymmetric C atoms, each asymmetric C atom independently represents a racemic isomer, a chirally pure R and / or a chirally pure S isomer, or a combination thereof.

[0186] In some embodiments, each of n, in, and q of Formula (I) is independently 0, 1, 2, or 3. In some embodiments, each of n, m, and q of Formula (I) is 1. Formula (Ia)

[0187] In some embodiments, the compound of Formula (I) has the structure of Formula (Ia), or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof: [ka] (In the formula, R 1 and R 2 Each of the C3-C 22 Alkyl, C3-C 22 Alkenyl, C3-C8 cycloalkyl, -C 2- C 10 Alkylene-LR 6 ,or [ka] wherein each of alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted; Each of X, Y, and Z is independently C(═O)NR 4 -, -NR 4C(D)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C=O)NR 4 -, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4 C(=S)-, -C(E)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4 C(=S)NR 4 -, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-, -SC(=O)NR 4 --C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, O, S, -C1-C 10 alkylene-O-, or a bond, wherein alkylene is substituted or unsubstituted; Each L is independently —C(═O)NR 4 -, -NR 4 C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C(=O)NR 4 -, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4 C(=S)-, -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4C(=S)NR 4 -, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-, -SC(=O)NR 4 --C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, O, S, -C1-C 10 Alkylene-O-, -C1-C 10 Alkylene -C(=O)O-, -C1-C 10 alkylene -OC(=O)-, or a bond, wherein alkylene is substituted or unsubstituted; R 3 is -C0-C 10 Alkylene-NR 7 R 8 , -C0-C 10 Alkylene-heterocycloalkyl, or -C0-C 10 alkylene-heterocycloyl, wherein the alkylene, heterocycloalkyl, and heterocycloaryl are independently substituted or unsubstituted; R 4 are each independently hydrogen or substituted or unsubstituted C1-C6 alkyl; R 5 is hydrogen or substituted or unsubstituted C1-C6 alkyl; R 6 each independently being a substituted or unsubstituted C-C 22 Alkyl, or substituted or unsubstituted C3-C 22 is alkenyl, R 7 and R 8 are each independently hydrogen, or substituted or unsubstituted C1-C6 alkyl, or R 7 and R 8 together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl; and p is an integer from 1 to 10.

[0188] In some embodiments of Formula (Ia), when the structure has two or more asymmetric C atoms, each asymmetric C atom independently represents a racemic isomer, a chirally pure R isomer and / or a chirally pure S isomer, or a combination thereof.

[0189] Variations of Formulas (I) and (Ia) In some embodiments, R of Formula (I) and Formula (Ia) 1 and R 2 independently, C3-C 22 Alkyl, C3-C 22 Alkenyl, -C2-C 10 Alkylene-LR 6 ,or [ka] and each of alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) 1 and R 2 independently, C 10 -C 20 Alkyl, C 10 -C 20 Alkenyl, -C8-C7 alkylene-LR 6 ,or [ka] and each of alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) 1 teeth, [ka] is.

[0190] In some embodiments, each L in Formula (I) and Formula (Ia) is independently O, S, —C—C 10Alkylene-O-, -C1-C 10 Alkylene -C(=O)O-, -C1-C 10

[0023] In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, -C-C alkylene-O-, -C-C alkylene-C(=O)O-, -C-C alkylene-O-C(=O)-, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, -C-C alkylene-O-, -C-C alkylene-C(=O)O-, -C-C alkylene-O-C(=O)-, or a bond, wherein the alkylene is a straight or branched unsubstituted alkylene.

[0191] In some embodiments, R of Formula (I) and Formula (Ia) 6 each independently being a substituted or unsubstituted linear C3- C22 Alkyl or substituted or unsubstituted linear C3-C 22 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 6 each independently represents a substituted or unsubstituted C-C 20 Alkyl, or substituted or unsubstituted C3-C 20 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 6 each independently represents a substituted or unsubstituted C-C 10 Alkyl, or substituted or unsubstituted C3-C 10 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 6 each of which is independently a substituted or unsubstituted C-C 10 In some embodiments, R in Formula (I) and Formula (Ia) is alkyl. 6 each of which is independently a substituted or unsubstituted straight chain C-C 10 In some embodiments, R in Formula (I) and Formula (Ia) is alkyl. 6Each of R is independently substituted or unsubstituted n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl. 6 In some embodiments, each of R in Formula (I) and Formula (Ia) is independently substituted or unsubstituted n-octyl. 6 Each of is n-octyl.

[0192] In some embodiments, each L in Formula (I) and Formula (Ia) is independently —C(═O)O—, —OC(═O)—, —C-C 10 In some embodiments, each L in Formula (I) and Formula (Ia) is -C1-C3 alkylene-O-, or O. In some embodiments, each L in Formula (I) and Formula (Ia) is O. In some embodiments, each L in Formula (I) and Formula (Ia) is -C1-C3 alkylene-O-. In some embodiments, p in Formula (I) and Formula (Ia) is 1, 2, 3, 4, or 5. In some embodiments, p in Formula (I) and Formula (Ia) is 2.

[0193] In some embodiments, R of Formula (I) and Formula (Ia) 1 is as follows: [ka]

[0194] In some embodiments, R of Formula (I) and Formula (Ia) 1 is R 2 is.

[0195] In some embodiments, R of Formula (I) and Formula (Ia) 4 is independently H or substituted or unsubstituted C1-C4 alkyl. In some embodiments, R 4 Each of R is independently a substituted or unsubstituted linear C1-C4 alkyl. In some embodiments, R in Formula (I) and Formula (Ia) 4is H. In some embodiments, each of R in Formula (I) and Formula (Ia) 4 is independently H, -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R in Formula (I) and Formula (Ia) 4 Each of R is independently H or —CH. In some embodiments, R in Formula (I) and Formula (Ia) 4 Each of is -CH3.

[0196] In some embodiments, X in Formula (I) and Formula (Ia) is -C(=O)O- or -OC(=O)-. In some embodiments, X in Formula (I) and Formula (Ia) is -C(=O)NR 4 -or-NR 4 In some embodiments, X in Formula (I) and Formula (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In some embodiments, X in Formula (I) and Formula (Ia) is -C(=O)NH-, -C(=O)N(CH3)-, -OC(=O))-, -NHC(=O)-, -N(CH3)C(=O))-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -N(CH3)C(=O)O-, -OC(=O))NH-, -OC(=O)N(CH3)-, -NHC(=O)NH-, -N(CH3)C(=O) NH-, -NHC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, NHC(=NH)NH-, -N(CH3)C(=NH)NH-, -NHC(=NH)N(CH3)-, -N(CH3 )C(=NH)N(CH3)-, NHC(=NMe)NH-, -N(CH3)C(=NMe)NH-, -NHC(=NMe)N(CH3)-, or -N(CH3)C(=NMe)N(CH3)-.

[0197] In some embodiments, R of Formula (I) and Formula (Ia) 2 is C3-C 22 Alkyl, C3-C 22 Alkenyl, -C2-C 10 Alkylene-LR 6,or [ka] and each of alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) 2 is a substituted or unsubstituted C7-C 22 Alkyl, or substituted or unsubstituted C3-C 22 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 2 is a substituted or unsubstituted linear C7-C 22 Alkyl or substituted or unsubstituted linear C3-C 22 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 2 is a substituted or unsubstituted C 10 -C 20 Alkyl, or substituted or unsubstituted C 10 -C 20 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 2 is the unsubstituted C 10 -C 20 In some embodiments, R in Formula (I) and Formula (Ia) is alkyl. 2 is the unsubstituted C 10 -C 20 In some embodiments, R in Formula (I) and Formula (Ia) is alkenyl. 2 is -C2-C 10 Alkylene-LR 6 In some embodiments, R in Formula (I) and Formula (Ia) 2 is -C2-C 10 Alkylene-C(=O)OR 6 or -C2-C 10 Alkylene-OC(=O)-R 6 is.

[0198] In some embodiments, R of Formula (I) and Formula (Ia) 2 is as follows: [ka]

[0199] In some embodiments, R of Formula (I) and Formula (Ia) 1 is R 1 is.

[0200] In some embodiments, Y in Formula (I) and Formula (Ia) is -C(=O)O- or -OC(=O)-. In some embodiments, Y in Formula (I) and Formula (Ia) is -C(=O)NR 4 -or-NR 4 In some embodiments, Y in Formula (I) and Formula (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In some embodiments, Y in Formula (I) and Formula (Ia) is -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, or -NR 4 C(=O)NR 4 In some embodiments, Y of Formula (I) and Formula (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-, -OC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, or -N(CH3)C(=O)NH-. In some embodiments, Y of Formula (I) and Formula (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0201] In some embodiments, R of Formula (I) and Formula (Ia) 3 is -C0-C 10 Alkylene-NR 7 R 8 or -C0-C 10 In some embodiments, R in Formula (I) and Formula (Ia) is an alkylene-heterocycloalkyl, where the alkylene and heterocycloalkyl are independently substituted or unsubstituted. 3 is -C0-C 10 Alkylene-NR7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C1-C6 alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C1-C4 alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C1-alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C2-alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C3-alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C4-alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C5-alkylene-NR 7 R 8 In some embodiments, R in Formula (I) and Formula (Ia) 3 is -C0-C 10 In some embodiments, R of Formula (I) and Formula (Ia) is alkylene-heterocycloalkyl. 3 is —C1-C6 alkylene-heterocycloalkyl, where the heterocycloalkyl contains 1 to 3 nitrogens and 0 to 2 oxygens. In some embodiments, R 3 is -C1-C6 alkylene-heterocycloaryl.

[0202] In some embodiments, R of Formula (I) and Formula (Ia) 7 and R 8is independently hydrogen or substituted or unsubstituted C-C alkyl. In some embodiments, R 7 and R 8 is independently hydrogen or substituted or unsubstituted C-C alkyl. In some embodiments, R 7 and R 8 Each of R is independently substituted or unsubstituted C-C alkyl. 7 and R 8 Each of R and R is independently -CH, -CHCH, -CHCHCH, or -CH(CH). 8 Each of R is CH. 7 and R 8 Each of is -CH2CH3.

[0203] In some embodiments, R of Formula (I) and Formula (Ia) 7 and R 8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocyclyl. In some embodiments, R 7 and R 8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted C2-C6 heterocycloalkyl. In some embodiments, R 7 and R 8 taken together with the nitrogen to which they are attached form a substituted or unsubstituted 3- to 7-membered heterocycloalkyl.

[0204] In some embodiments, R of Formula (I) and Formula (Ia) 3 is as follows: [ka]

[0205] In some embodiments, R of Formula (I) and Formula (Ia) 3 is as follows: [ka]

[0206] In some embodiments, R of Formula (I) and Formula (Ia) 3 is as follows: [ka]

[0207] In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)O— or —OC(═O)—.

[0208] In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)NR 4 -or-NR 4 C(=O)-.

[0209] In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)N(CH)—, —N(CH)C(═O)—, —C(═O)NH—, or —NHC(═O)—.

[0210] In some embodiments, Z in Formula (I) and Formula (Ia) is —OC(═O)O—, —NR 4 C(=O)O-, -OC(O)NR 4 -, or -NR 4 C(=O)NR 4 -It is.

[0211] In some embodiments, Z in Formula (I) and Formula (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-, -OC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, -NHC(=O)N(CH3)-, or -N(CH3)C(=O)NH-.

[0212] In some embodiments, Y of Formula (I) and Formula (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0213] In some embodiments, R of Formula (I) and Formula (Ia) 5 is hydrogen, or substituted or unsubstituted C1-C3 alkyl.

[0214] In some embodiments, R of Formula (I) and Formula (Ia) 5 is H, -CH3, -CH-)CH3, -CH2CH2CH3, or -CH(CH3)2.

[0215] In some embodiments, R of Formula (I) and Formula (Ia) 5 is H.

[0216] Exemplary lipids of WO2022140252 In another aspect, the amino lipid is according to any formula or structure set forth in International Publication No. WO2022140252, which is incorporated herein by reference in its entirety, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the amino lipid has a structure according to any of the following formulae A', A, I", I', I, II", II', II, III', III, I"-a, I'-a, Ia, I"-ai, I"-a-ii, I"-a-iii, I"-b, I'-b, Ib, I"-bi, I"-b-ii, I"-b-iii, I"-c, I'-c, Ic, I"-ci, I"-c-ii, I"-c-iii, I'-d, Id, I'-di, II-a, II-ai, III-a, and III-ai of WO2022140252, or a pharmaceutically acceptable salt or solvate thereof. Exemplary amino lipids also include any of the lipids in Table 1 of WO2022140252, including any of the lipids represented by Examples 7-1 to 7-253 and Examples 8-1 to 8-106, or a pharmaceutically acceptable salt or solvate thereof.

[0217] In some embodiments, the amino lipid has Formula A' of WO2022140252: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein: L 1 is absent, C 1-6 Alkylenyl, or C 2-6 is heteroalkylenyl, Each L 2 are independently optionally substituted C2-15 alkylenyl, or optionally substituted C 3-15 is heteroalkylenyl, L is C 1-10 Alkylenyl, or C 2-10 is heteroalkylenyl, X 2 is -OC(O)-, -C(O)O-, or -OC(O)O-, X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-; R" is hydrogen, [ka] or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered alicyclic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; R and R a each independently represents hydrogen or C 6-20 an optionally substituted group selected from aliphatic, 3- to 12-membered alicyclic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L 3 and L 3a each of which is independently optionally substituted 1-10 alkylenyl, or optionally substituted C 2-10 is heteroalkylenyl, R 1is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered alicyclic, optionally substituted 3- to 7-membered heterocyclyl containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR 2 , -C(O)OR 2 , -C(O)SR 2 , -OC(O)R 2 , -OC(O)OR 2 , -CN, -N(R 2 )2, -C(O)N(R 2 )2, -S(O)2N(R 2 )2, -NR 2 C(O)R 2 , -OC(O)N(R 2 )2, -N(R 2 )C(O)OR 2 , -NR 2 S(O)2R 2 , -NR 2 C(O)N(R 2 )2, -NR 2 C(S)N(R 2 )2, -NR 2 C(NR 2 )N(R 2 )2, -NR 2 C(CHR 2 )N(R 2 )2, -N(OR 2 )C(O)R 2 , -N(OR 2 )S(O)2R 2 , -N(OR 2 )C(O)OR 2 , -N(OR 2 )C(O)N(R 2 )2, -N(OR 2 )C(S)N(R 2 )2, -N(OR 2 )C(NR 2 )N(R 2 )2, -N(OR 2)C(CHR 2 )N(R 2 )2, -C(NR 2 )N(R 2 )2, -C(NR 2 )R 2 , -C(O)N(R 2 ) OR 2 , -C(R 2 )N(R 2 )2C(O)OR 2 , -CR 2 (R 3 )2, -OP(O)(OR 2 )2, or -P(O)(OR 2 )2, or R 1 teeth, [ka] or a ring selected from 3- to 7-membered alicyclic and 3- to 7-membered heterocyclyl containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and the alicyclic or heterocyclyl ring is selected from 1 to 4 R 2 Or R 3 optionally substituted with a group, Each R 2 are independently hydrogen, oxo, -CN, -NO2, -OR 4 , -S(O)2R 4 , -S(O)2N(R 4 )2, -(CH2) n -R 4 , or C 1-6 aliphatic, phenyl, 3- to 7-membered alicyclic, 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or R 2 two occurrences of together with the atom(s) to which they are attached form an optionally substituted 4-7 membered heterocyclyl containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 3are independently -(CH2) n -R 4 or R 3 two occurrences of together with the atom(s) to which they are attached form an optionally substituted 5-6 membered heterocyclyl containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 4 are independently hydrogen, -OR 5 , -N(R 5 )2, -OC(O)R 5 , -OC(O)OR 5 , -CN, -C(O)N(R 5 )2, -NR 5 C(O)R 5 , -OC(O)N(R 5 )2, -N(R 5 )C(O)OR 5 , -NR 5 S(O)2R 5 , -NR 5 C(O)N(R 5 )2, -NR 5 C(S)N(R 5 )2, -NR 5 C(NR 5 )N(R 5 )2, or [ka] and Each R 5 are independently hydrogen or optionally substituted C 1-6 is aliphatic, R 5 two occurrences of together with the atom(s) to which they are attached form an optionally substituted 4-7 membered heterocyclyl containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 6 independently, C 4-12 is aliphatic, Each n is independently 0 to 4.

[0218] In some embodiments, the amino lipid has formula III-a of WO2022140252: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein R, R 1 , L, L 1 , L 2 , L 3 are as defined therein for Formulas A', A, III', and III, both alone and in combination, as described above and in classes and subclasses herein. In an embodiment of Formula III-a, R, R 1 , L, L 1 , L 2 , L 3 each of which is as defined herein for formula A' above.

[0219] In some embodiments, the amino lipid has formula III-ai of WO2022140252: [ka] or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein R, R 1 , L, L 1 , and L 2 are as defined therein for Formulas A', A, III', and III, both alone and in combination, as described above and in classes and subclasses herein. In embodiments of Formula III-ai, R, R 1 , L, L 1 , and L 2 each of which is as defined herein for formula A' above.

[0220] In some embodiments, the amino lipid is selected from any of the lipids listed in Table 1 of WO2022140252, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is selected from the group consisting of: TIFF2025540623000025.tif156170TIFF2025540623000026.tif184170TIFF2025540623000027.tif199170TIFF2025540623000028.tif214170TIFF2025540623000029.tif166170

[0221] In some embodiments, the amino lipid is Example 7-1 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-2 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-19 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-20 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-22 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-24 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-25 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-1 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-2 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-3 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-4 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-5, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-13, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-14, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-17, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-18, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-19, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-20, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-55, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-57, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-58, or a pharmaceutically acceptable salt thereof.In some embodiments, the amino lipid is Example 8-59, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-60, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-61, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-62, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-63, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-232, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-233, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-234, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-235, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-236, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-237 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-238 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-239 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-67 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-68 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-69 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-70 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-71 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 8-72 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-243 or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-244, or a pharmaceutically acceptable salt thereof.In some embodiments, the amino lipid is Example 7-245, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 7-246, or a pharmaceutically acceptable salt thereof.

[0222] Exemplary lipids of WO2022159472 In another aspect, the amino lipid is according to any formula or structure set forth in International Publication No. WO2022159472, the entirety of which is incorporated herein by reference, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the amino lipid has a structure according to any of Formulas I, II, III, IIIA, IIIB, IIIC, IV, V, VA, VI, VIA, VII, and VII of WO2022159472, or a pharmaceutically acceptable salt or solvate thereof. Exemplary amino lipids also include any of the lipids in Table 1 of WO2022159472, including any of the lipids represented by Examples 4-1 to 4-86, or a pharmaceutically acceptable salt or solvate thereof.

[0223] In some embodiments, the amino lipid has Formula I of WO2022159472: [ka] or a pharmaceutically acceptable salt thereof, wherein: L 1 is a covalent bond, -C(O)-, or -OC(O)-, L 2 is a covalent bond, an optionally substituted divalent saturated or unsaturated, straight or branched C1 to C12 hydrocarbon chain, or [ka] and Cy A is an optionally substituted ring selected from phenylene and 3- to 7-membered saturated or partially unsaturated carbosilene; each m is independently 0, 1, or 2; L3 is a covalent bond, —C(O)—, —C(O)O—, —OC(O)—, —O—, or —OC(O)O—; R 1 teeth, [ka] Optionally substituted saturated or unsaturated, straight-chain or branched alkyl groups in which 1 to 3 methylene units are optionally and independently replaced by -O- or -NR-. C1~C 20 hydrocarbon chains, or [ka] and Cy B is a 3- to 12-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, [ka] an optionally substituted ring selected from sterolyl, and phenyl; p is 0, 1, 2, or 3; Each L 4 are independently a divalent saturated or unsaturated, straight or branched C1 to C6 hydrocarbon chain; Each A 1 and A 2 are independently optionally substituted C1-C 20 Aliphatic or -L 5 -R 5 Or or A 1 and A 2 together with their intervening atoms form an optionally substituted ring: [ka] may be formed, During the ceremony, x is selected from 1 or 2; # is L 4 represents the point of attachment to Each L 5 is independently a divalent saturated or unsaturated, straight or branched C1-C alkyl group in which 1 to 3 methylene units are optionally and independently replaced by -O- or -NR- 20 is a hydrocarbon chain, Each R 5 is independently an optionally substituted group selected from a 5- to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring; X 1 is a covalent bond, -O-, or -NR-, X 2 is a covalent bond or one to three methylene units are optionally and independently -O-, -NR-, or -Cy C optionally substituted divalent saturated or unsaturated, straight or branched C1-C 12 is a hydrocarbon chain, Cy C is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbosilene, a phenylene, a 3- to 7-membered saturated or partially unsaturated heterocyclene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroarylene having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; X 3 is an optionally substituted ring selected from hydrogen, a 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; each R is independently hydrogen or an optionally substituted C1-C6 aliphatic; However, L 3 is a covalent bond, R 1 teeth, [ka] It must be.

[0224] In some embodiments, the amino lipid has Formula VI of WO2022159472: [ka] or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3, or 4; 2 , R 1 , A 1 , A 2 , X 1 , X 2 , and X 3 is as defined therein for formula I and is also set forth in classes and subclasses therein, both alone and in combination. In an embodiment, L 2 , R 1 , A 1 , A 2 , X 1 , X 2 , and X 3 is as defined herein for formula I above.

[0225] In some embodiments, the amino lipid has formula VIA of WO2022159472: [ka] or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3, or 4; 2 , R 1 , A 1 , A 2 , X 2 , and X 3 is as defined therein for formula I and is also set forth in classes and subclasses therein, both alone and in combination. In an embodiment, L 2 , R 1 , A 1 , A 2 , X 2 , and X 3 is as defined herein for formula I above.

[0226] In some embodiments, the amino lipid is selected from any of the lipids listed in Table 1 of WO2022159472, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0227] In some embodiments, the amino lipid is Example 4-62, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-63, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-64, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-65, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-66, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-67, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-68, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-69, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-70, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-71, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-72, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-73, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-74, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-75, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-76, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-77, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-78, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-79, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-80, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-81, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-82, or a pharmaceutically acceptable salt thereof.In some embodiments, the amino lipid is Example 4-83, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-84, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-85, or a pharmaceutically acceptable salt thereof. In some embodiments, the amino lipid is Example 4-86, or a pharmaceutically acceptable salt thereof.

[0228] LNP compositions containing different amino lipids In some embodiments, LNPs contain multiple amino lipids with different formulas. For example, LNP compositions can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino lipids. In another example, LNP compositions can contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 20 amino lipids. In yet another example, LNP compositions can contain up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 9, up to 10, up to 20, or up to 30 amino lipids.

[0229] In some embodiments, the LNP composition comprises a first amino lipid. In some embodiments, the LNP composition comprises a first amino lipid and a second amino lipid. In some embodiments, the LNP composition comprises a first amino lipid, a second amino lipid, and a third amino lipid. In some embodiments, the LNP composition comprises a first amino lipid, a second amino lipid, a third amino lipid, and a fourth amino lipid. In some embodiments, the LNP composition does not comprise a fourth amino lipid. In some embodiments, the LNP composition does not comprise a third amino lipid. In some embodiments, the molar ratio of the first amino lipid to the second amino lipid is about 0.1 to about 10. In some embodiments, the molar ratio of the first amino lipid to the second amino lipid is about 0.20 to about 5. In some embodiments, the molar ratio of the first amino lipid to the second amino lipid is about 0.25 to about 4. In some embodiments, the molar ratio of the first amino lipid to the second amino lipid is about 0.25, about 0.33, about 0.5, about 1, about 2, about 3, or about 4.

[0230] In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 4:1:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 1:1:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 2:1:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 2:2:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 3:2:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 3:1:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 5:1:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 3:3:1. In some embodiments, the molar ratio of the first amino lipid:second amino lipid:third amino lipid is about 4:4:1.

[0231] Additional Amino Lipid Embodiments In some embodiments, the LNP composition comprises one or more amino lipids. In some embodiments, the one or more amino lipids comprise about 40 mol% to about 65 mol% of the total lipids present in the particle. In some embodiments, the one or more amino lipids comprise about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, or about 65 mol% of the total lipids present in the particle. In some embodiments, the first amino lipid comprises about 1 mol% to about 99 mol% of the total amino lipids present in the particle. In some embodiments, the first amino lipid comprises from about 16.7 mol% to about 66.7 mol% of the total amino lipid present in the particle, hi some embodiments, the first amino lipid comprises from about 20 mol% to about 60 mol% of the total amino lipid present in the particle.

[0232] In some embodiments, the amino lipid is an ionizable lipid. The ionizable lipid may contain one or more ionizable nitrogen atoms. In some embodiments, at least one of the one or more ionizable nitrogen atoms is positively charged. In some embodiments, at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, 95 mol%, or 99 mol% of the ionizable nitrogen atoms in the LNP composition are positively charged. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, an imine, an amide, a guanidine moiety, a histidine residue, a lysine residue, an arginine residue, or any combination thereof. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In some embodiments, the amino lipid comprises a tertiary amine.

[0233] In some embodiments, the amino lipid (e.g., an ionizable lipid) is a cationic lipid. In some embodiments, the cationic lipid is an ionizable lipid. In some embodiments, the amino lipid comprises one or more nitrogen atoms. In some embodiments, the amino lipid comprises one or more ionizable nitrogen atoms. Exemplary cationic and / or ionizable lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N142-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC), 3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β )-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)).

[0234] In some embodiments, the amino lipids described herein can be in the form of salts, such as pharmaceutically acceptable salts.All pharmaceutically acceptable salts of amino lipids are included in the present disclosure.As used herein, the term " amino lipid " also includes its pharmaceutically acceptable salts, and its diastereomeric, enantiomeric and epimeric forms.

[0235] In some embodiments, the amino lipids described herein have one or more stereocenters, and each stereocenter exists independently in either the R or S configuration. The lipids provided herein include all diastereomeric, enantiomeric, and epimeric forms, as well as appropriate mixtures thereof. The lipids provided herein include all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers, as well as appropriate mixtures thereof. In certain embodiments, the lipids described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds / salts, separating the diastereomers, and recovering the optically pure enantiomers. In some embodiments, resolution of the enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In other embodiments, the diastereomers are separated using a separation / resolution technique based on differences in solubility. In other embodiments, separation of stereoisomers is carried out by chromatography, or by formation of diastereomeric salts and separation by recrystallization, or by separation by chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley and Sons, Inc., 1981. In one aspect, stereoisomers are obtained by stereoselective synthesis.

[0236] In some embodiments, lipids such as amino lipids are substituted based on the structure disclosed herein.In some embodiments, lipids such as amino lipids are not substituted.In other embodiments, lipids described herein are labeled isotopically (e.g., with a radioisotope) or by other means, including but not limited to, the use of a chromophore or fluorescent moiety, a bioluminescent label, or a chemiluminescent label.

[0237] The lipids described herein include isotopically labeled compounds that are identical to those listed in the various formulas and structures presented herein, except that one or more atoms are replaced with atoms that have different atomic masses or mass numbers than those that are normally found in nature.Examples of isotopes that can be incorporated into the lipids of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, and chlorine, for example: 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 In one embodiment, the isotope-labeled lipids described herein, for example, 3 H and 14 Lipids incorporating radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium offers certain therapeutic advantages due to increased metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0238] In some embodiments, the asymmetric carbon atom of amino lipid exists in enantiomerically enriched form.In certain embodiments, the asymmetric carbon atom of amino lipid has at least 50% enantiomeric excess of (S) configuration or (R) configuration, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.

[0239] In some embodiments, the disclosed amino lipids can be converted to N-oxides. In some embodiments, N-oxides are formed by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid and / or hydrogen peroxide). Thus, when valence and structure permit, NO or N + -O - Disclosed herein are N-oxide compounds of the described amino lipids, which can be represented as follows: In some embodiments, the nitrogen in the disclosed compounds can be converted to N-hydroxy or N-alkoxy. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as ra-CPBA. All nitrogen-containing compounds shown are also contemplated. Accordingly, N-hydroxy and N-alkoxy (e.g., N-OR, where R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3- to 14-membered carbocyclic ring, or 3- to 14-membered heterocyclic ring) derivatives of the described amino lipids are also disclosed herein.

[0240] In some embodiments, the one or more amino lipids comprise from about 40 mol % to about 65 mol % of the total lipid present in the particle.

[0241] PEG lipids In some embodiments, the described LNP compositions comprise one or more PEG-lipids. As used herein, "PEG lipid" or "PEG-lipid" refers to a lipid containing a polyethylene glycol moiety. Examples of suitable PEG-lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the one or more PEG-lipids may comprise PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, or combinations thereof.

[0242] In some embodiments, the PEG-lipid comprises about 0.1 mol % to about 10 mol % of the total lipid present in the particle.

[0243] phospholipids In some embodiments, the described LNP compositions comprise one or more phospholipids.

[0244] In some embodiments, the phospholipid comprises about 5 mol % to about 15 mol % of the total lipid present in the particle.

[0245] cholesterol In some embodiments, the LNP composition comprises cholesterol or a derivative thereof.

[0246] GalNAc lipids In some embodiments, the LNP composition comprises a receptor-targeting conjugate comprising a compound of formula (V): [ka] (In the formula, the plurality of A groups collectively comprise a receptor-targeting ligand; Each L 1 , L 2 , L 3 , L 4 , L 5 , L6 , L 7 , L 8 , L 9 , L 10 , and L 12 are independently substituted or unsubstituted C-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C-C 12 Alkenylene, substituted or unsubstituted C-C 12 Alkynylene, -(CH2CH2O) m -, -(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)NR 1 -, -NR 1 S(=O)2-, -NR 1 -, or -N(OR 1 )- and L 11 is a substituted or unsubstituted -(CH2CH2O) n -, substituted or unsubstituted -(OCH2CH2) n - or substituted or unsubstituted -(CH2) n - and Each R 1 are independently H or substituted or unsubstituted C1-C6 alkyl; R is a lipid, a nucleic acid, an amino acid, a protein, or a lipid nanoparticle; m is an integer selected from 1 to 10; and n is an integer selected from 1 to 200.

[0247] In some embodiments, each L 1 , L 4 , and L 7 are independently substituted or unsubstituted C-C 12 In some embodiments, each L 1 , L 4 , and L 7 is independently a substituted or unsubstituted C-C alkylene. In some embodiments, each L 1 , L 4 , and L 7 is a C4 alkylene. In some embodiments, each L 2 , L 5 , and L 8 are independently -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR1C(=O)O-, -NR1C(=O)NR1-, or -C(=O)NR1C(=O)-. In some embodiments, each L 2 , L 5 , and L 8 are independently -C(=O)NR 1 - または In some embodiments, each L 2 , L 5 , and L 8 is —C(═O)NH—. In some embodiments, each L 3 , L 6 , and L 9 are independently substituted or unsubstituted C-C 12 In some embodiments, each L 3 is a substituted or unsubstituted C2-C6 alkylene. In some embodiments, L 3 is a C4 alkylene. In some embodiments, each L 6 and L 9 are independently substituted or unsubstituted C-C 10 In some embodiments, each L 6 and L 9 is independently a substituted or unsubstituted C-C alkylene. In some embodiments, each L6 and L 9 is a C3 alkylene. In some embodiments, A binds to a lectin. In some embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In some embodiments, A is N-acetylgalactosamine (GalNAc) or [ka] or its derivatives. A is N-acetylgalactosamine (GalNAc) [ka] or a derivative thereof.

[0248] In some embodiments, the receptor-targeting conjugate comprises from about 0.001 mol % to about 20 mol % of the total lipid content present in the nanoparticle composition.

[0249] Phosphate Charge Neutralizer In some embodiments, the LNPs described herein comprise a phosphate charge neutralizing agent. In some embodiments, the phosphate charge neutralizing agent comprises arginine, asparagine, glutamine, lysine, histidine, a cationic dendrimer, a polyamine, or a combination thereof. In some embodiments, the phosphate charge neutralizing agent comprises one or more nitrogen atoms. In some embodiments, the phosphate charge neutralizing agent comprises a polyamine.

[0250] Suitable phosphate charge neutralizing agents for use in the LNP formulations shown below include, but are not limited to, spermidine and 1,3-propanediamine.

[0251] antioxidants In some embodiments, the LNPs described herein comprise one or more antioxidants. In some embodiments, the one or more antioxidants function to reduce degradation of the cationic lipid, the payload, or both. In some embodiments, the one or more antioxidants comprise hydrophilic antioxidants. In some embodiments, the one or more antioxidants are chelating agents such as ethylenediaminetetraacetic acid (EDTA) and citrate. In some embodiments, the one or more antioxidants comprise lipophilic antioxidants. In some embodiments, the lipophilic antioxidant comprises a vitamin E isomer or a polyphenol. In some embodiments, the one or more antioxidants are present in the LNP composition at a concentration of at least 1 mM, at least 10 mM, at least 20 mM, at least 50 mM, or at least 100 mM. In some embodiments, the one or more antioxidants are present in the particles at a concentration of about 20 mM.

[0252] Other lipids In some embodiments, the disclosed LNP compositions may include a helper lipid. In some embodiments, the disclosed LNP compositions include a neutral lipid. In some embodiments, the disclosed LNP compositions include a stealth lipid. In some embodiments, the disclosed LNP compositions include an additional lipid. The neutral lipid may function to stabilize and improve LNP processing.

[0253] "Helper lipid" may refer to a lipid that enhances transfection (e.g., transfection of nanoparticles (LNPs) comprising a composition provided herein that includes a biologically active agent). The mechanism by which a helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity. Helper lipids may include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure may include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some embodiments, the helper lipid is cholesterol. In some embodiments, the helper lipid may be cholesterol hemisuccinate.

[0254] "Stealth lipid" may refer to a lipid that modifies the length of time that nanoparticles can exist in vivo (e.g., in blood). Stealth lipids may aid in the formulation process, for example, by reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate the pharmacokinetic properties of LNPs. Stealth lipids suitable for use in the lipid compositions of the present disclosure may include, but are not limited to, stealth lipids having a hydrophilic head group attached to the lipid moiety. Information regarding stealth lipids suitable for use in the lipid compositions of the present disclosure and the biochemistry of such lipids can be found in: Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pp. 55-71 and I-Toekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, for example, in WO2006 / 007712.

[0255] In some embodiments, the stealth lipid is a PEG lipid. In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly(N-(2-hydroxypropyl)methacrylamide)-based polymers. The stealth lipid may comprise a lipid moiety. The stealth lipid may comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from a diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, the chain optionally comprising one or more functional groups, e.g., amide or ester. The dialkylglycerol or dialkylglycamide group may further comprise one or more substituted alkyl groups.

[0256] The structures and properties of helper lipids, neutral lipids, stealth lipids, and / or other lipids are further described in WO2017173054A1, WO2019067999A1, US20180290965A1, US20180147298A1, US20160375134A1, US8236770, US8021686, US8236770B2, US7371404B2, US7780983B2, US7858117B2, US20180200186A1, US20070087045A1, WO2018119514A1, and WO2019067992A1, all of which are incorporated herein by reference in their entireties.

[0257] LNP formulation Specific formulations of nanoparticle compositions comprising one or more of the described lipids are described herein.

[0258] The described nanoparticle compositions can deliver therapeutic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within a mammalian body. The physicochemical properties of the nanoparticle composition can be modified to enhance selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of different organs. The therapeutic agents included in the nanoparticle composition can also be selected based on the desired delivery target(s). For example, therapeutic agents can be selected for a particular indication, symptom, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems or groups thereof. In certain embodiments, the nanoparticle composition can contain mRNA encoding a polypeptide of interest that can be translated to produce the polypeptide of interest (e.g., a base editor) within the cell. Such compositions can have specificity or affinity for a specific organ or cell type, such as the liver or hepatocytes, to facilitate delivery of the drug substance to them.

[0259] The amount of therapeutic or drug substance (e.g., mRNA encoding base editor and guide RNA) in the LNP composition can depend on the size, composition, desired target and / or use, or other characteristics of the nanoparticle composition. For example, the amount of RNA included in the nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of lipid component to therapeutic agent in the nanoparticle composition can be about 5:1 to about 60:1, e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the weight / weight ratio of lipid component to therapeutic agent can be from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1. The amount of therapeutic agent in the nanoparticle composition can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0260] In some embodiments, the LNP formulation comprises one or more nucleic acids, such as RNA. In some embodiments, the one or more RNAs, lipids, and amounts thereof can be selected to provide a particular N / P ratio. The N / P ratio can be selected from about 1 to about 30. The N / P ratio can be selected from about 2 to about 12. In some embodiments, the N / P ratio is about 0.1 to about 50. In some embodiments, the N / P ratio is about 2 to about 8. In some embodiments, the N / P ratio is about 2 to about 15, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 15, about 3 to about 10, about 3 to about 8, about 3 to about 6, about 4 to about 15, about 4 to about 10, about 4 to about 8, or about 4 to about 6. In some embodiments, the N / P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, or about 10. In some embodiments, the N / P ratio is about 4 to about 6. In some embodiments, the NIP ratio is about 4, about 4.5, about 5, about 5.5, or about 6.

[0261] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms in the lipid(s) to phosphate groups in the nucleic acid molecular entity(ies), e.g., in a physiological pH range, e.g., in a nanoparticle composition comprising a lipid component and RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH ranges of different cellular compartments (e.g., organs, tissues, and cells) and bodily fluids (e.g., blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain embodiments, the physiological pH range refers to the pH range of mammalian blood, e.g., about 7.35 to about 7.45. Similarly, for phosphate charge neutralizing agents having one or more ionizable nitrogen atoms, the N / P ratio can refer to the molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizing agent to phosphate groups in the nucleic acid. In some embodiments, the ionizable nitrogen atoms refer to nitrogen atoms that are ionizable within a pH range of 5 to 14.

[0262] For payloads that do not contain phosphate groups, the N / P ratio can refer to the molar ratio of ionizable nitrogen atoms in the lipid to the total negative charges in the payload. For example, the N / P ratio of an LNP composition can refer to the molar ratio of the total ionizable nitrogen atoms in the LNP composition to the total negative charges in the payload present in the composition.

[0263] In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%, about 70% to about 90%, or about 90% to about 100%. In some embodiments, LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 98%.

[0264] In another aspect, provided herein are lipid nanoparticles (LNPs) comprising the compositions provided herein. As used herein, a "lipid nanoparticle (LNP) composition" or "nanoparticle composition" is a composition comprising one or more of the described lipids. LNP compositions are typically on the order of micrometers or less and may comprise a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, LNPs refer to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, the nanoparticles may range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 40 to 100 nm, 50 to 100 nm, 50 to 90 nm, 50 to 80 nm, 50 to 70 nm, 55 to 95 nm, 55 to 80 nm, 55 to 75 nm, 60 to 100 nm, 60 to 90 nm, 60 to 80 nm, 60 to 70 nm, 25 to 100 nm, 25 to 80 nm, or 40 to 80 nm.

[0265] In some embodiments, LNPs can be made from cationic, anionic, or neutral lipids. In some embodiments, LNPs may include a neutral lipid, such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as a helper lipid to enhance transfection activity and nanoparticle stability. In some embodiments, LNPs may include hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or lipid combination known in the art can be used to generate LNPs. Examples of lipids used to generate LNPs include, but are not limited to, DOTMA (N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOSPA (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1-propaniminium pentahydrochloride), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propaniminium bromide), DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), DOTAP-cholesterol, GAP-DMORIE-DPyP E, and GL67A-DOPE-DMPE (,2-bis(dimethylphosphino)ethane)-polyethylene glycol (PEG). Examples of cationic lipids include, but are not limited to, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include, but are not limited to, DPSC, DPPC (dipalmitoylphosphatidylcholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE, and SM (sphingomyelin). Examples of PEG-modified lipids include, but are not limited to, PEG-DMG (dimyristoylglycerol), PEG-CerC14, and PEG-CerC20.In some embodiments, lipids can be combined in any number of molar ratios to produce LNPs, hi some embodiments, polynucleotides can be combined with lipid(s) in a wide range of molar ratios to produce LNPs.

[0266] The definitions of terms in the following eight paragraphs apply only to compounds of formulae (I), (Ia), and (V) above.

[0267] The term "substituted," unless otherwise indicated, refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioallcyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, aiylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic group, and aliphatic group. It is understood that the substituent may be further substituted. Exemplary substituents include amino, alkylamino, and the like.

[0268] As used herein, the term "substituent" refers to a positional variable on an atom of a core molecule that is substituted at a designated atomic position, replacing one or more hydrogens on the designated atom, provided that the substitution does not exceed the normal valence of the designated atom, and the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. Those of skill in the art should note that any carbon and heteroatom with valences that appear unsatisfied as described or depicted herein are assumed to have a sufficient number of hydrogen atoms to satisfy the valences described or depicted. In certain cases, one or more substituents having a double bond (e.g., "oxo" or "=O") as a point of attachment may be described, depicted, or listed herein within a substituent group, and the structure may show only a single bond as the point of attachment to the core structure of Formula (I). Those of skill in the art will understand that a double bond is intended for those substituents, even if only a single bond is depicted.

[0269] The term "alkyl" refers to a straight or branched hydrocarbon chain radical having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. Alkyl containing up to 10 carbon atoms is C1-C 10 Similarly, when an alkyl is expressed as an alkyl containing up to 6 carbon atoms, for example, it is a C1-C6 alkyl. Alkyl containing other numbers of carbon atoms (and other moieties defined herein) are expressed similarly. Alkyl groups include C1-C 10Examples of alkyl groups include, but are not limited to, alkyl, C1-C9 alkyl, and C1-C8 alkyl. Examples include, but are not limited to, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethylpropyl, and the like. In some embodiments, alkyl is methyl or ethyl. In some embodiments, alkyl is —CH(CH3)2 or —C(CH3)3. Unless otherwise specifically stated herein, alkyl groups may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical group. In some embodiments, alkylene is -CH-, -CHCH-, or -CHCHCH-. In some embodiments, alkylene is -CH-. In some embodiments, alkylene is -CHCH-. In some embodiments, alkylene is -CHCHCH-. In some embodiments, alkylene is -CHCHCH-.

[0270] The term "alkenyl" refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula -C(R)=CR 2 and R refers to the remainder of the alkenyl group, which may be the same or different. In some embodiments, R is H or alkyl. In some embodiments, alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of alkenyl groups include -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2.

[0271] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetranyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-onespiro[2.2]pentyl, norbornyl, and bicycle[1.1.1]pentyl. Unless otherwise specifically stated herein, cycloalkyl groups may be optionally substituted. Depending on the structure, cycloalkyl groups can be monovalent or divalent (i.e., cycloalkylene groups).

[0272] The term "heterocycle" or "heterocyclic" refers to heteroaromatic rings (also known as heteroaryls) and heterocycloalkyls (also known as heteroalicyclic groups) containing at least one heteroatom selected from nitrogen, oxygen, and sulfur, each heterocyclic group having 3 to 12 atoms in its ring system, provided that no ring contains two adjacent O or S atoms. A "heterocyclyl" is a monovalent group formed by removing a hydrogen atom from any ring atom of a heterocyclic compound. In some embodiments, the heterocycle is a monocyclic, bicyclic, polycyclic, spirocyclic, or bridged compound. Non-aromatic heterocyclic groups (also known as heterocycloalkyls) contain rings having 3 to 12 atoms in their ring system, and aromatic heterocyclic groups contain rings having 5 to 12 atoms in their ring system. Heterocyclic groups include benzo-fused ring systems. Examples of non-aromatic heterocyclic groups are: pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl , dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 1,3-azabicyclo[4.1.0]heptanyl, 3h-indolyl, indolin-2-onyl, isoindolin-1-onyl, isoindolin-1,3-dionyl, 3,4-dihydroisoquinolin-1(2H)-onyl, 3,4-dihydroquinolin-2(1H)-onyl, isoindolin-1,3-dithionyl, benzo[d]oxazol-2(3H)-onyl, 1H-benzo[d]imidazol-2(3H)-onyl, benzo[d]thiazol-2(3H)-onyl, and quinolizinyl.Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, butyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups are either C-attached (or C-linked) or N-attached where possible. For example, groups derived from pyrrole include both pyrrol-1-yl (N-linked) and pyrrol-3-yl (C-linked). Furthermore, groups derived from imidazole include imidazol-1-yl or imidazol-3-yl (both N-linked) or imidazol-2-yl, imidazol-4-yl, or imidazol-5-yl (all C-linked). Heterocyclic groups include benzo-fused ring systems. Non-aromatic heterocycles are optionally substituted with one or two oxo (=O) moieties, such as pyrrolidin-2-one. In some embodiments, at least one of the two rings of a bicyclic heterocycle is aromatic. In some embodiments, both rings of a bicyclic heterocycle are aromatic.

[0273] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically stated in the specification, a heterocycloalkyl radical can be a monocyclic or bicyclic ring system, which can include fused (when fused to an aryl or heteroyl ring, the heterocycloalkyl is attached through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocycloalkyl radical can be partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes carbohydrates of all ring forms, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Unless otherwise specified, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 1 or 2 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 3 or 4 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2 to 12 carbons, 1 to 3 N atoms, 0 to 1 O atoms, and 0 to 1 S atoms within the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) comprising the heterocycloalkyl (i.e., the skeletal atoms of the heterocycloalkyl ring). Unless otherwise specifically stated in the specification, a heterocycloalkyl group may be optionally substituted. As used herein, the term "tetracycloalkylene" may refer to a divalent heterocycloalkyl group.

[0274] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Heteroaryls are monocyclic or bicyclic. Examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, heteroaryl contains 0 to 6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group is partially reduced to form a heterocycloalkyl group as defined herein. In some embodiments, the heteroaryl group is fully reduced to form a heterocycloalkyl group as defined herein.

[0275] The definitions of terms in the following 25 paragraphs apply only to compounds of Formulae A', III-a, and III-aI, 1, VI, and VIA above.

[0276] The terms "aliphatic" or "aliphatic group," as used herein, mean a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "carbocyclic," "alicyclic," or "cycloalkyl") and has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms. In some embodiments, aliphatic groups contain 1-3 carbon atoms, and in some embodiments, aliphatic groups contain 1-2 carbon atoms. In some embodiments, "carbocyclic" (or "alicyclic" or "carbocycle" or "cycloalkyl") refers to an optionally substituted C3-C8 hydrocarbon, or an optionally substituted C6-C8 hydrocarbon, that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. 12"Cycloalkyl" refers to a bicyclic hydrocarbon. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0277] As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds. In some embodiments, the term "alkenyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one double bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched hydrocarbon chain having an alkyl group. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

[0278] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below as substituted aliphatic groups.

[0279] As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1 to 100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has 1 to 20 carbon atoms in its backbone (e.g., C1 to C6 for straight chain). 20 , C2 to C for branched chains 20 ), and alternatively have about 1-10 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure, and such rings are monocyclic or bicyclic, and alternatively have about 5, 6, or 7 carbons in the ring structure. In some embodiments, alkyl groups can be lower alkyl groups, where the lower alkyl group contains 1-4 carbon atoms (e.g., for a straight chain lower alkyl, C1 ~ C4 ).

[0280] The term "alkenyl" or "alkylene" refers to a divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) that is straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the above monovalent alkyl groups can become alkylenyl by abstraction of a second hydrogen atom from the alkyl. In some embodiments, "alkylenyl" refers to a polymethylene group, i.e., -(CH2) n -, where n is a positive integer, preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, or 4 to 8. A substituted alkylenyl is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below as substituted aliphatic groups.

[0281] As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds. In some embodiments, the term "alkynyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one triple bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched chain hydrocarbon group having an alkynyl group. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0282] The term "aryl" refers to a group having a total of 6 to 14 ring members (e.g., C 6-14 "aryl" refers to monocyclic and bicyclic ring systems having at least one ring in the system that is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Unless otherwise specified, "aryl" groups are hydrocarbons.

[0283] As used herein, the term "divalent" refers to a chemical moiety that has two points of attachment. For example, a "divalent C 1-8 (or C 1-6 ) saturated or unsaturated, straight or branched hydrocarbon chain" means straight or branched divalent alkylene, alkenylene, and alkynylene chains as defined herein.

[0284] As used herein, the term "bridged bicyclic" refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms, or an atom, or a valence bond connecting two bridgeheads, and a "bridgehead" is any skeletal atom of the ring system (excluding hydrogen) that is connected to three or more skeletal atoms. In some embodiments, bridged bicyclic groups have 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include the groups described below, in which each group is attached to the remainder of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, bridged bicyclic groups may be substituted with one or more substituents described for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to, the following: [ka]

[0285] The terms "carbocyclyl," "carbocycle," and "carbocyclic ring," as used herein, refer to a saturated or partially unsaturated cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein, having 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a "carbocyclyl" (or "alicyclic") refers to an optionally substituted C3-C8 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the remainder of the molecule, or an optionally substituted C6-C8 hydrocarbon. 12It refers to a bicyclic hydrocarbon. The term "cycloalkyl" refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, the cycloalkyl group has 3 to 6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0286] The term "haloaliphatic" refers to an aliphatic group substituted with one or more halogen atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 halo, e.g., fluoro, iodo, bromo, or chloro). In some embodiments, a haloaliphatic group contains 1-7 halogen atoms. In some embodiments, a haloaliphatic group contains 1-5 halogen atoms. In some embodiments, a haloaliphatic group contains 1-3 halogen atoms.

[0287] The term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 halo, e.g., fluoro, iodo, bromo, or chloro). In some embodiments, a haloalkyl group contains 1 to 7 halogen atoms. In some embodiments, a haloalkyl group contains 1 to 5 halogen atoms. In some embodiments, a haloalkyl group contains 1 to 3 halogen atoms.

[0288] The terms "heteroalkylenyl" or "heteroalkylene," as used herein, refer to an optionally substituted, straight-chain (i.e., unbranched) or branched, divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) having, in addition to carbon atoms, 1 to 5 heteroatoms. The term "heteroatom" is explained below. In some embodiments, a heteroalkylenyl group contains 2 to 10 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, a heteroalkylenyl group contains 2 to 8 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, a heteroalkylenyl group contains 4 to 8 carbon atoms, with 1 to 3 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 2 to 5 carbon atoms, with 1 to 2 carbon atoms optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroalkylenyl groups contain 1 to 3 carbon atoms, with 1 carbon atom optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroalkylenyl groups include, but are not limited to, -CHO-, -(CH)O-, -CHOCH-, -O(CH)-, -(CH)O-, -(CH)OCH-, -CHO(CH)-, -O(CH)-, -(CH)O-, -(CH)OCH-, -CHO(CH)-, -(CH)O(CH)-, and -O(CH)-. Unless otherwise specified, C x Heteroalkylenyl refers to a heteroalkylenyl having x carbon atoms prior to replacement with the heteroatom.

[0289] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl), sharing 6, 10, or 14 pi-electrons in the ring, and having 1 to 5 heteroatoms in addition to carbon atoms. Exemplary heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring (i.e., bicyclic heteroaryl rings having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and benzisoxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include rings that are optionally substituted.

[0290] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon, the quaternized form of any basic nitrogen, or a heterocyclic ring, e.g., N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (including a substitutable nitrogen, as in N-substituted pyrrolidinyl).

[0291] The terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably herein and refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated and has one or more, e.g., 1-4, heteroatoms in addition to carbon atoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl). The heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and [ka] Heterocyclyl groups can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions can independently be optionally substituted. Bicyclic heterocyclic rings also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or alicyclic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocyclic rings can also be spirocyclic ring systems (e.g., 7-11 membered spirocyclic fused heterocyclic rings, as defined above, having one or more heteroatoms (e.g., 1, 2, 3, or 4 heteroatoms) in addition to carbon atoms). Bicyclic heterocyclic rings can also be bridged ring systems (e.g., 7-11 membered bridged heterocyclic rings having 1, 2, or 3 bridging atoms).

[0292] As used herein, the term "linker" is used to refer to the portion of a multi-component agent that connects different components to one another. For example, one skilled in the art will understand that polypeptides having a structure comprising two or more functional or organizational domains often include a stretch of amino acids between such domains that connects them to one another. In some embodiments, polypeptides comprising a linker element "L'" have an overall structure of the general type S1-L'-S2, where S1 and S2 may be the same or different and represent two domains joined together by the linker. In some embodiments, the linker is at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide. A variety of different linker elements that may be suitably used in engineering polypeptides (e.g., fusion polypeptides) are known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1 121-1123).

[0293] The term "sterolyl," as used herein, refers to a 17-membered fused polycyclic ring moiety that is either saturated or partially unsaturated, substituted with at least one hydroxyl group, and has a single point of attachment to the remainder of the molecule at any substitutable carbon or oxygen atom. In some embodiments, the sterolyl group is a cholesterolyl group, or a variant or derivative thereof. In some embodiments, the cholesterolyl group is modified. In some embodiments, the cholesterolyl group is an oxidized cholesterolyl group (e.g., oxidized on the beta ring structure or hydrocarbon tail structure). In some embodiments, the cholesterolyl group is an esterified cholesterolyl group. In some embodiments, the sterolyl group is a phytosterolyl group. Exemplary sterolyl groups include, but are not limited to, 25-hydroxycholesterolyl (25-OH), 20α-hydroxycholesterolyl (20α-OH), 27-hydroxycholesterolyl, 6-keto-5α-hydroxycholesterolyl, 7-ketocholesterolyl, 7β-hydroxycholesterolyl, 7α-hydroxycholesterolyl, 7β-25-dihydroxycholesterolyl, β-sitosterolyl, stigmasterolyl, brassicasterolyl, and campesterolyl.

[0294] As described herein, compounds of the present disclosure may be described as "substituted" or "optionally substituted." That is, compounds may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been replaced with a suitable substituent. "Substituted" applies to one or more hydrogens explicitly or implicitly shown from the structure (e.g., [ka] At least [ka] refers to, [ka] At least [ka] (Refers to "optionally substituted"). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be either the same or different at all positions. Combinations of substituents contemplated by the present disclosure are preferably those that result in the formation of stable or chemically suitable compounds. The term "stable," as used herein, refers to a compound that remains substantially unchanged when subjected to conditions that permit the compound's production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as "substituted" preferably have 1 to 4 substituents, more preferably 1 to 2 substituents. Groups described as "optionally substituted" may be unsubstituted or "substituted" as described above.

[0295] Suitable monovalent substituents include halogen, -(CH2) 0-4 R°, -(CH2) 0-4 OR°, -O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR°, -(CH2) 0-4 CH(OR°)2, -(CH2) 0-4 Ph (optionally substituted with R°), —(CH2) 0-4 O(CH2) 0-1 Ph (optionally substituted with R°), -CH=CHPh (optionally substituted with R°), -(CH2) 0-4 O(CH2) 0-1 -pyridyl (optionally substituted with R°), -NO2, -CN, -N3, -(CH2) 0-4 N(R°)2, -(CH2) 0-4N(R°)C(O)R°, -N(R°)C(S)R°, -(CH2) 0-4 N(R°)C(O)NR°2, -N(R°)C(S)NR°2, -(CH2) 0-4 N(R°)C(O)OR°, -N(R°)N(R°)C(O)R°, -N(R°)N(R°)C(O)NR°2, -N(R°)N(R°)C(O)OR°, -(CH2) 0-4 C(O)R°, -C(S)R°, -(CH2) 0-4 C(O)OR°, -(CH2) 0-4 C(O)SR°, -(CH2) 0-4 C(O)OSiR°3, -(CH2) 0-4 OC(O)R°, -OC(O)(CH2) 0-4 SR°-, -SC(S)SR°, -(CH2) 0-4 SC(O)R°, -(CH2) 0-4 C(O)NR°2, -C(S)NR°2, -C(S)SR°, -SC(S)SR°, -(CH2) 0-4 OC(O)NR°2, -C(O)N(OR°)R°, -C(O)C(O)R°, -C(O)CH2C(O)R°, -C(NOR°)R°, -(CH2) 0-4 SSR°, -(CH2) 0-4 S(O)2R°, -(CH2) 0-4 S(O)2OR°, -(CH2) 0-4 OS(O)2R°, -S(O)2NR°2, -(CH2) 0-4 S(O)R°, -N(R°)S(O)2NR°2, -N(R°)S(O)2R°, -N(OR°)R°, -C(NH)NR°2, -P(O)2R°, -P(O)R°2, -OP(O)R°2, -OP(O)(OR°)2, -SiR°3, -OSiR°3, -(C 1-4 linear or branched alkylene)ON(R°)2, or -(C 1-4 and alkylene)C(O)ON(R°), where each R° may be optionally substituted as defined below and independently represents hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1Ph, -CH2- (a 5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, notwithstanding the above definitions, two independent occurrences of R° taken together with their intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be optionally substituted as defined below.

[0296] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with their intervening atoms) are independently halogen, —(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2, -O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 C(O)NH2, -(CH2) 0-2 C(O)NHR ● , -(CH2) 0-2 C(O)NR ● 2, -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● 、 -(C 1-4Linear or branched alkylene)C(O)OR ● , or -SSR ● and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0~1 or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0297] Suitable divalent substituents include the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S- is mentioned, R * Each independent occurrence of may be hydrogen, optionally substituted as defined below. 1-6 A 5-6 membered, saturated, partially unsaturated, or aryl ring is selected from aliphatic or unsubstituted rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to a vicinal substitutable carbon of an "optionally substituted" group include -O(CR * 2) 2-3 O- and R * Each independent occurrence of is hydrogen, C1 which may be substituted as defined below -6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0298] R * Suitable substituents on the aliphatic group include halogen, -R● ,-(Halo R ● ), -OH, -OR ● , -O(halo ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0299] In some embodiments, suitable substituents on a substitutable nitrogen include -R † , -NR † 2. -C(O)R † , -C(O)OR † , C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † are listed, and each R † are independently hydrogen, optionally substituted as defined below, C 1-6 an aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definitions, R † two independent occurrences of, taken together with their intervening atom(s), form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0300] R† Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(halo ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0301] As used herein, an amino lipid may contain at least one primary, secondary, or tertiary amine moiety that is protonatable (or ionizable) in the pH range of 4 to 14. In some embodiments, the amine moiety(s) function as the hydrophilic head group of the amino lipid. When most of the amine moieties of the amino lipid(s) in a nucleic acid-lipid nanoparticle formulation are protonated at physiological pH, the nanoparticles can be referred to as cationic lipid nanoparticles (cLNPs). When most of the amine moieties of the amino lipid(s) in a nucleic acid-lipid nanoparticle formulation are not protonated at physiological pH but can be protonated at acidic pH, e.g., endosomal pH, the nanoparticles can be referred to as ionizable lipid nanoparticles (iLNPs). The amino lipids that make up cLNPs can generally be referred to as cationic amino lipids (cLipids). The amino lipids that make up iLNPs can be referred to as ionizable amino lipids (iLipids). The amino lipids can be iLipids or cLipids at physiological pH.

[0302] As used herein, LNP compositions or formulations typically have sizes on the order of micrometers or less and may contain a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (lipid vesicles), and lipoplex nanoparticle compositions (liposomes with a lipid bilayer and a diameter of 500 nm or less). The LNPs described herein may have an average diameter of about 1 nm to about 2500 nm, about 10 nm to about 1500 nm, about 20 nm to about 1000 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. The LNPs described herein can have an average diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or more. The LNPs described herein can be substantially non-toxic.

[0303] As used herein, "phospholipid" may refer to a lipid comprising a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may comprise one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion with a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of a phospholipid with a membrane may allow one or more components of the LNP to cross the membrane (i.e., delivery of one or more components to a cell).

[0304] payload The LNPs described herein can be designed to deliver a payload, such as one or more therapeutic or drug substances, to a target cell or organ of interest. In some embodiments, the LNPs described herein encapsulate one or more components of the base editor systems described herein. For example, the LNPs may encapsulate one or more of a guide RNA, a nucleic acid encoding a guide RNA, a vector encoding a guide RNA, a base editor fusion protein, a nucleic acid encoding a base editor fusion protein, a programmable DNA-binding domain, a nucleic acid encoding a programmable DNA-binding domain, a deaminase, a nucleic acid encoding a deaminase, or all or any combination thereof. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, e.g., mRNA and / or guide RNA. In some embodiments, the nucleic acid(s) are chemically modified.

[0305] In some embodiments, the payload comprises one or more nucleic acids (i.e., one or more nucleic acid molecular entities). In some embodiments, the nucleic acid is a single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid is DNA. In some embodiments, the single-stranded nucleic acid is RNA. In some embodiments, the nucleic acid is a double-stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is DNA. In some embodiments, the double-stranded nucleic acid is RNA. In some embodiments, the double-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments, the nucleic acid is messenger RNA (mRNA), microRNA, asymmetric interfering RNA (aiRNA), short hairpin RNA (shRNA), antisense oligonucleotide, or Dicer substrate dsRNA. In some embodiments, the single-stranded nucleic acid forms a secondary structure, e.g., one or more stem-loops. In some other embodiments, the single-stranded nucleic acid comprises one or more stem-loop and single-stranded regions within the molecule.

[0306] kit It is contemplated herein that the therapeutic or drug substances disclosed herein may be part of the kits described herein. Accordingly, one aspect of the present disclosure relates to a kit comprising the single guide RNA provided herein, the base editor system and complex provided herein, the composition provided herein, and / or the composition comprising the lipid nanoparticle formulation provided herein for treating or preventing a condition. The kit may further comprise one or more additional therapeutic regimens or drugs for treating or preventing a condition.

[0307] In certain embodiments, kits and articles of manufacture for use in one or more of the methods described herein are also disclosed herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers, e.g., vials, tubes, etc., each of which contains one of the separate elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0308] The articles of manufacture provided herein contain packaging materials, including, but not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material appropriate for the selected formulation and intended mode of administration and treatment.

[0309] For example, the container(s) contain a composition described herein and, optionally, further contain a therapeutic regimen or agent disclosed herein. Such kits optionally include identifying instructions or labels or instructions for their use in the methods described herein.

[0310] The kit typically includes a label describing the contents and / or instructions for use, and a package insert with instructions for use. A set of instructions is also typically included.

[0311] In an embodiment, the label is on the container or is associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other characters that form the label are attached, molded, or etched on the container itself; when the label is present in the container or carrier that holds the container, it is associated with the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents are used for a specific therapeutic purpose. The label also indicates how to use the contents, such as in the methods described herein.

[0312] Administration Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, and the subject's general characteristics (including the subject's health, sex, weight, and / or age, as well as other diseases present), can affect the dosage and frequency of administration required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or can include a series of treatments. It will also be understood that the effective dosage of the disclosed composition used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage may result or become apparent from the results of the diagnostic assays described herein. A therapeutically effective dosage generally depends on the patient's condition at the time of administration. The precise amount can be determined by routine experimentation but may ultimately depend on the judgment of the clinician, e.g., monitoring the patient for signs of disease and adjusting treatment accordingly.

[0313] Dosage frequency can be determined and adjusted over the course of therapy and will generally, but not necessarily, be based on treating and / or suppressing and / or ameliorating and / or delaying the disease. Alternatively, sustained continuous release formulations of the polypeptide or polynucleotide may be appropriate.

[0314] Dosage regimens (including compositions disclosed herein) can vary over time. The particular dosing regimen, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history, as well as the characteristics of the polypeptide or polynucleotide (e.g., the half-life of the polypeptide or polynucleotide, and other considerations well known in the art).

[0315] The appropriate therapeutic dose of the compositions described herein will depend on the particular agent (or composition thereof) used, the formulation and route of administration, the type and severity of the disease, whether the polypeptide or polynucleotide is being administered for prophylactic or therapeutic purposes, previous therapy, the subject's clinical history and response to the antagonist, and the judgment of the attending physician. Typically, the clinician will administer the polypeptide until a dosage is reached that achieves the desired result.

[0316] Administration of one or more compositions can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the compositions can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either before, during, or after the onset of disease.

[0317] The methods and compositions of the present disclosure described herein (including embodiments thereof) can be administered with one or more additional therapeutic regimens, agents, or treatments that can be co-administered to a mammal. "Co-administering" means administering one or more additional therapeutic regimens, agents, or treatments and a composition of the present disclosure close enough in time to enhance the effect of the one or more additional therapeutic agents (or vice versa). In this regard, the compositions of the present disclosure described herein can be administered simultaneously with one or more additional therapeutic regimens, agents, or treatments, at different times, or on entirely different treatment schedules (e.g., a first treatment can be daily, while an additional treatment is weekly). For example, in embodiments, a second therapeutic regimen, agent, or treatment is administered simultaneously with, before, or after a composition of the present disclosure.

[0318] In embodiments, a polynucleotide encoding a base editor fusion protein and a guide RNA are administered to a subject. In embodiments, the polynucleotide encoding the base editor fusion protein is mRNA. In embodiments, an LNP comprising such an amount of a polynucleotide encoding a base editor fusion protein and a guide RNA is administered to a subject. In embodiments, the subject is a primate. In embodiments, the subject is a non-human primate. In embodiments, the non-human primate is a cynomolgus monkey.

[0319] In embodiments, administration of a polynucleotide encoding a guide RNA and a base editor fusion protein to a non-human primate, such as a cynomolgus monkey, results in base changes (as measured by next-generation sequencing) in 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more of total hepatocytes. In embodiments, such percentages of base changes are achieved when a subject is administered a combined dose of a polynucleotide encoding a guide RNA and a base editor fusion protein. In embodiments, such doses are administered in LNP. In embodiments, such administration results in a reduction in serum TTR levels.

[0320] The present invention is illustrated in the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. [Example]

[0321] Example 1 A guide to adenine base editing in the TTR gene In this example, we identified gRNA sequences that cause ABE8.8 (and other ABE variants containing Streptococcus pyogenes Cas9, such as ABE7.10, or another Cas protein that can use the NGG PAM) to either 1) destroy the start codon or 2) destroy a splice site (donor or acceptor) via A→G editing within the editing window (approximately positions 4–7 of the 20-nt protospacer region of DNA). Five sequences were identified across the entire human TTR gene (Table 1). We synthesized gRNAs that matched each of the protospacer sequences and separately matched the 100-nt standard Streptococcus pyogenes CRISPR gRNA sequence, with each gRNA molecule having minimal chemical modifications (as specified in Table 1). Each gRNA was co-transfected into primary human hepatocytes using MessengerMax reagent (Lipofectamine) at various dilutions (2500, 1250, 625 ng / RNA / mL) with an equal amount of in vitro transcribed ABE8.8 mRNA (molecular weight ratio 1:1) to assess the editing activity of various concentrations of the test substance. [Table 1-1] [Table 1-2]

[0322] Each gRNA was also transfected into primary cynomolgus monkey hepatocytes with equal amounts of ABE8.8 mRNA (1:1 molecular weight ratio) at 5000, 2500, 1250, 625, 312.5, and 156.25 ng / RNA / mL, using the orthogonal protospacer sequence of the corresponding cynomolgus monkey TTR gene sequence. The ABE8.8 (MA004) mRNA and corresponding amino acid sequence used are listed in Table 11 below. Three days after transfection, genomic DNA was collected from the hepatocytes and evaluated for base editing using next-generation sequencing of PCR amplicons generated around the target splice site. Some sites showed high editing efficiency. In particular, GA457 (GA458 is the cynomolgus monkey equivalent), GA460, and GA461 showed high editing activity in both human and cynomolgus monkey primary hepatocytes. See Figures 5A-5C, Figure 6, and Tables 2-3. [Table 2] [Table 3]

[0323] It should be understood that the results presented in Tables 2 and 3 are representative of results that may be achieved in accordance with the teachings provided herein. Compositions for editing the TTR gene provided herein may produce editing activities that differ by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more from the activities set forth in Table 2 or Table 3. In some embodiments, the compositions provide editing activities that are within 100%, within 90%, within 80%, within 70%, within 60%, within 50%, within 40%, within 30% or more, within 20% or more, or within 10% of the activities set forth in Table 2 or Table 3.

[0324] Example 2 Off-target analysis To establish the safety of base editing therapy to knockdown TTR in the human liver in vivo, off-target mutation analysis was evaluated in primary human hepatocytes. Off-target editing in human hepatocytes was assessed according to the ONE-seq procedure detailed in PCT / US19 / 27788 ("Highly Sensitive in vitro Assays to Define Substrate Preferences and Sites of Nucleic-Acid Binding, Modifying, and Cleaving Agents"). A simplified flowchart of the off-target analysis using the ONE-seq procedure is shown in Figure 7. Using the in vitro biochemical assay ONE-seq, a list of potential off-target sites was generated and the nature of ribonucleoproteins containing the ABE8.8 base editor protein and each of the three protospacer guide sequences (GA457, GA460, and GA461) cleaved oligonucleotides in the library was determined. The results of ONE-seq analysis of libraries generated against GA457, GA460, and GA461, along with the potential off-target sites, are shown in Tables 8–10.

[0325] The ONE-seq methodology is as follows: ONE-seq library design begins with the computational identification of sites in the reference genome that have sequence homology to the on-target. For the human ONE-seq library, the reference human genome (GRCh38, Ensemblv98, chromosome ftp: / / ftp.ensembl.org / pub / release-98 / fasta / homo_sapiens / dna / Homo_sapiens.GRCh38.dna.chromosome.{1-22,X,Y,MT}.fa and ftp: / / ftp.ensembl.org / pub / release-98 / fasta / homo_sapiens / dna / Homo_sapiens.GRCh38.dna.nonchromosomal.fa) was searched for potential off-target sites with up to six mismatches to the protospacer, as well as sites with up to four mismatches and up to two DNA or RNA bulges, using Cas-Designer v1.2 (http: / / www.rgenome.net / cas-designer / ).

[0326] Sites with up to six mismatches and no bulges are referred to using the code X<number of mismatches><number of bulges>. Thus, an on-target site is labeled X00; a site with one mismatch to the on-target and no bulge is labeled X10, and so on. Sites with DNA bulges are referred to by a similar nomenclature: DNA<number of mismatches><number of bulges>. Thus, a site with four mismatches to the on-target and two DNA bulges is labeled DNA42. The same nomenclature is used for RNA bulges, but they are coded as RNA<number of mismatches><number of bulges>.

[0327] The identified protospacer sequences were extended by 10 nucleotides (nt) on either side of flanking sequences from the respective reference genomes (these regions are referred to herein as genomic contexts). These extended sequences were then padded with additional sequences, including six predefined constant regions of different nucleotide composition and sequence length; two copies of a 14-nt site-specific barcode (one on each side of the central protospacer sequence); and two different 11-nt unique molecular identifiers (UMIs) (one on each side of the central protospacer sequence), up to a final length of approximately 200 nt. The UMIs were used to correct for bias due to PCR amplification, while the barcodes allowed unambiguous identification of each site during analysis. Barcodes were selected from an initial list of 668,420 barcodes that did not contain CC or GG sequences, and each barcode had a Hamming distance of 2 from any other barcode. A custom Python script was used to design the final library.

[0328] The final oligonucleotide libraries were synthesized by a commercial vendor (Agilent Technologies). Each library was PCR amplified and subjected to 1.25x AMPure XP bead purification (Beckman Coulter). After incubation in CutSmart buffer (New England Biolabs) for 10 minutes at 25°C, RNPs containing 769 nM recombinant ABE8.8-m protein and 1.54 μM gRNA were mixed with 100 ng of purified library and incubated at 37°C for 8 hours. The RNP dose was derived from analyses demonstrating that it was a supersaturating dose, i.e., a dose exceeding the dose that achieved the maximum amount of on-target editing in biochemical assays.

[0329] The reaction was stopped by the addition of proteinase K (New England Biolabs) at 37°C for 45 minutes, followed by 2x AMPure XP bead purification. The reaction was then sequentially incubated with EndoV (New England Biolabs) for 30 minutes at 37°C, Klenow Fragment (New England Biolabs) for 30 minutes at 37°C, and NEBNext Ultra II End Prep Enzyme Mix (New England Biolabs) for 30 minutes at 20°C, followed by 30 minutes at 65°C, with 2x AMPure XP bead purification after each incubation. The reaction was ligated with annealed adapter oligonucleotide duplexes at 20°C for 1 hour to facilitate PCR amplification of the cleaved library products, followed by 2x AMPure XP bead purification. Size selection of the ligation reaction was performed with the PippinHT system (Sage Sciences) to isolate 150–200 bp DNA on a 3% agarose gel cassette, followed by two rounds of PCR amplification to generate barcoded libraries, which were subjected to paired-end sequencing on an Illumina MiSeq system as described above.

[0330] In ONE-seq experiments, two cleavage products are obtained: the PROTO side contains the part of the oligonucleotide upstream of the cleavage site, and the PAM side contains the part of the oligonucleotide downstream of the cleavage site. In ABE experiments, only the PROTO side is informative of editing activity (A → G substitution), and therefore only this side is sequenced.

[0331] Paired-end reads were adapter-trimmed using trimmomatic v0.39 (Bolger et al., 2014) with custom Nextera adapters (PrefixPE / 1: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 30); PrefixPE / 2: 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 31); as specified in the file) and the parameters "ILLUMINACLIP:NEB_custom.fa:2:30:10:1:true LEADING:0 TRAILING:0 SLIDINGWINDOW:4:30 MINLEN:36". For the experiment with low sequencing quality (VOL014), these parameters were set to "ILLUMINACLIP NEB_custom.fa:2:30:10:1:true LEADING:2 TRAILING:0 SLIDINGWINDOW:30:30 MINLEN:36." Reads were then merged using FLASH v1.2.11 (Magoc and Salzberg, 2011) with the parameters "max mismatch density = 0.25 max overlap = 160." Merged reads were scanned for constant sequences, barcodes, and protospacer sequences specific to each site and filtered in an edit window for evidence of an A → G substitution (defined as positions 1–10 of the most PAM-distal portion of the protospacer). Duplicate reads were discarded.

[0332] For each site, the total number of edited reads was normalized to the total number of edited reads assigned to the on-target site, and this ratio defines the site's ONE-seq score. Sites were ranked by ONE-seq score, and sites with a score of 0.001 or higher were selected for validation. Scores of 0.001 or higher include sites with up to 1000-fold lower editing activity in biochemical assays compared to on-target site editing. This threshold is based on the premise that, in cells, 100% on-target editing translates to less than 1 / 1000 of editing activity converting to less than 0.1% off-target editing (below the lower limit of editing detection by NGS). Oligonucleotides with a larger number of sequences reflect a higher propensity for Cas9 / gRNA cleavage in vitro and, therefore, a higher likelihood of off-target mutagenesis in cells.

[0333] We analyzed several off-target site candidates for off-target editing in primary human hepatocytes. Table 4 shows the results of examining 47 off-target site candidates for guide RNA GA457 in primary human hepatocytes co-transfected with gRNA and an equal amount of in vitro transcribed ABE8.8 mRNA (molecular weight ratio 1:1) using MessengerMax reagent (Lipofectamine). While the on-target site showed high editing efficiency, all off-target sites showed little to no editing (net editing <0.4%). [Table 4-1] [Table 4-2]

[0334] GA459, GA460, and GA461 were similarly evaluated for off-target editing, as shown in Tables 5, 6, and 7, respectively. While the on-target site of each guide shows higher editing efficiency in the treated group compared to the control group, little to no off-target editing is observed at potential off-target sites. [Table 5] [Table 6] [Table 7]

[0335] Table 8 provides some results of off-target editing using the GA457 guide.

[0336] Table 9 provides some results of off-target editing using the GA460 guide.

[0337] Table 10 provides some results of off-target editing using the GA461 guide.

[0338] It should be understood that the results presented in Tables 4, 6, 7, 8, 9, and 10 are representative of results that may be achieved in accordance with the teachings provided herein. Compositions for editing a TTR gene according to the present invention may produce total off-target editing activity that differs from the activity described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461. For example, a composition may produce total off-target editing activity that differs by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more from the activity described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461, for one or more off-target sites described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461. In some embodiments, the compositions provide a total off-target editing activity that is within 100%, within 90%, within 80%, within 70%, within 60%, within 50%, within 40%, within 30% or more, within 20% or more, or within 10% of the activity described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461, for one or more sites described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461. In some embodiments, the compositions result in an off-target editing activity that is equal to or less than the activity described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461, for one or more sites described in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461. In some embodiments, the composition does not produce off-target editing activity for one or more sites listed in Tables 4, 6, 7, 8, 9, or 10, or discussed with respect to GA457, 460, or 461. [Table 8]

[0339] Additional examples of GA457 off-target sites are provided in U.S. Provisional Patent Application No. 63 / 322,182, filed March 21, 2022. The GA457 off-target site may include any one of SEQ ID NOs: 92-1073. [Table 9]

[0340] Additional examples of GA460 off-target sites are provided in U.S. Provisional Patent Application No. 63 / 322,182, filed March 21, 2022. The GA460 off-target site may include any one of SEQ ID NOs: 1074-3725. [Table 10]

[0341] Additional examples of GA461 off-target sites are provided in U.S. Provisional Patent Application No. 63 / 322,182, filed March 21, 2022. The GA461 off-target site may include any one of SEQ ID NOs: 3726-5745. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0342] Other ABE variants can be used to edit the human TTR gene. Examples of such ABE variants are described in International Patent Application No. PCT / US21 / 26729, filed April 9, 2021, entitled "BASE EDITING OF PCSK9 AND METHODS OF USING SAME FOR TREATMENT OF DISEASE," and assigned to Verve Therapeutics, Inc.

[0343] Example 3 In vivo non-human primate (NHP) base editing of the TTR gene In this example, NHP surrogate sgRNAs (GA519 and GA520) corresponding to the above-described human GA457 and GA460 sgRNAs were prepared, co-formulated with the above-described ABE8.8 mRNA, encapsulated in lipid nanoparticles (LNPs), and administered intravenously to NHPs. This study included two distinct aspects.

[0344] The first aspect of the in vivo study in NHPs involved the evaluation of LNP1 and LNP2, which differed only in that LNP2 was formulated to encapsulate GA520 and ABE8.8 mRNAs, while LNP1 was formulated to encapsulate GA519 and ABE8.8 mRNAs. The second aspect of the study involved the formulation and evaluation of a third LNP (LNP3). Like LNP1, LNP3 was formulated to encapsulate GA519 and ABE8.8 mRNAs. However, LNP3 differed from LNP1 in that it contained a GalNAc moiety. In each aspect of the study, base editing efficiency, TTR protein expression, safety profile, and pharmacokinetics were assessed multiple times after injection of NHPs, as described in further detail below and shown in the accompanying figures.

[0345] Part A: In vivo NHP evaluation of GA519 and GA520 using non-GalNAc LNPs Preparation of LNPs In this first aspect of the NHP study, two LNPs (LNP1 and LNP2) were formulated, with LNP1 encapsulating GA519 and ABE8.8 mRNAs, and LNP2 encapsulating GA520 and ABE8.8 mRNAs. The components of each LNP consisted of an ionizable amino lipid (iLipid), a neutral helper lipid, a PEG lipid, and a sterol lipid, as described in Table 12 below and in the ratios shown therein. [Table 12]

[0346] It should be understood that the lipids in Table 12 can be substituted with other suitable lipids of the described class. In some embodiments, for example, the LNP comprises the amino lipid VL422, described in International Published Patent Application No. WO2022 / 060871A1. For example, the amino lipid can be VL422, or a pharmaceutically acceptable salt or solvate thereof: [ka]

[0347] It should be further understood that the mole percents of lipids in Table 12 may be adjusted, and that the mole percents included in Table 12 are target percentages of excipients for LNPs, are intended to represent the total mole percents of all LNPs formulated in a given batch, and that particular LNPs within a batch may have varying mole percents. Accordingly, it is contemplated herein that the mole percents of one or more or all of the LNP components listed in Table 12 may be adjusted, e.g., by ±1-5%, ±5-10%, or ±10-20%. It is further contemplated herein that for particular LNPs formulated in a given batch of LNPs formulated according to desired target excipient percentages, the mole percents of one or more or all of the LNP components listed in Table 12 may differ from the target mole percents, e.g., by ±1-5%, ±5-10%, ±10-20%, or even by more than ±20%. It should be further understood that additional LNP components, including non-lipid components, can be added to the LNP components listed in Table 12. As shown in Table 13, LNP1 was formulated with sgRNA GA519, and LNP2 was formulated with GA520, which correspond to the above-mentioned sgRNAs GA457 and GA460, respectively. GA519 and GA520 were chemically synthesized, and the sequences and chemical modifications of GA519 and GA520 are shown in Table 13. [Table 13]

[0348] Notably, compared to GA457, GA519 hybridizes between positions 50,681,581 and 50,681,603 in exon 1 of the reference cynomolgus monkey genome (macFas5) and edits the adenosine at position 50,681,584, thereby converting the methionine amino acid to a threonine amino acid, resulting in disruption of the full-length TTR protein sequence and inhibiting protein translation (Figure 8). GA519 is a cynomolgus monkey surrogate for the human GA457 gRNA and, as described above, maps to the analogous region of the human TTR locus in Figure 4. The cynomolgus monkey GA519 gRNA differs from GA457 by a single nucleotide at position 17 of the protospacer, highlighted by an underline in the protospacer column in Table 13. Furthermore, GA519 and GA457 differ from each other in that the tracr region of GA519 incorporates a chemical modification (detailed in Table 13). Chemical modifications are designed for, or can improve, in vivo stability.

[0349] Similarly, compared to GA460, GA520 hybridizes between positions 50,678,305 and 50,678,327 in exon 3 of the reference cynomolgus monkey genome (macFas5) and edits the adenosine at position 50,678,324, resulting in a splice acceptor disruption that generates a truncated, non-functional TTR protein (Figure 9). The protospacer region of GA520 is identical to that of human GA460 and, as described above, maps to the analogous region of the human TTR locus as shown in Figure 4. GA520 and GA460 differ in the tracr region and incorporate chemical modifications designed for, or capable of, improved in vivo stability, as detailed in the table above.

[0350] For reference, the target nucleotides for base editing are highlighted in bold in Figures 8 and 9. Figures 8 and 9 also identify the location of the GA519 and GA520 spacers relative to the TTR gene, as described above.

[0351] LNP1 and LNP2 were formulated using ABE8.8 mRNA and GA519 and GA520 at a 1:1 sgRNA:mRNA weight ratio, respectively. In other words, the LNPs were formulated with an equal weight of guide RNA to the mRNA. The resulting LNPs encapsulating the sgRNA and ABE8.8 mRNA were filtered using a 0.2 micron filter and frozen at -80°C. The physical properties of the formulated LNPs are summarized in Table 14. [Table 14] Those skilled in the art will understand that the average LNP size, PDI, and RNA capture values ​​set forth in Table 14 are subject to measurement error or precision. It is also contemplated herein that the LNP size, PDI, and RNA capture values ​​set forth in Table 14 may vary by ±1-5%, ±5-10%, or ±10%-20%.

[0352] NHP research design In this aspect of the study, female cynomolgus monkeys of Cambodian origin were used as study animals. A premedication regimen containing dexamethasone and H1 and H2 antihistamines was administered to all animals on Day -1 (approximately 24 hours prior to dosing) and Day 1 (pre-dose), 30-60 minutes prior to administration of the test article dose. Three monkeys received LNP1 and three monkeys received LNP2 on Day 1 of the study via a single IV infusion, at a combined sgRNA and mRNA dose level of 3 mg / kg (animal body weight) and a dose volume of 6 mL / kg (n=3 / group).

[0353] Blood samples were collected from all animals before dosing for baseline measurements and at various time points from days 1 to 15 after dosing to assess biomarkers, cytokines, plasma iLipid and PEG-lipid pharmacokinetics, and serum safety parameters.

[0354] Necropsies were performed on all animals on day 16. Liver biopsies were collected to assess TTR gene editing.

[0355] Editing Efficiency Analysis The amount of gene editing in the liver was assessed by next-generation sequencing (NGS) of targeted polymerase chain reaction (PCR) amplicons at the TTR target site derived from genomic DNA extracted from the animal's liver using methods described above (Musunuru et al., Nature 593, no. 7859 (May 2021): 429-34. https: / / doi.org / 10.1038 / s41586-021-03534-y). Percent editing was reported as the percentage of all reads containing the non-reference allele at the target adenine.

[0356] Figure 10 shows the TTR editing efficiency of LNP1 compared to LNP2. Notably, as shown in Figure 10, the mean liver TTR editing efficiency is higher in NHPs treated with LNP1 (52%) compared to LNP2 (29%).

[0357] Quantification of TTR protein expression in serum For TTR protein analysis, serum was collected from all animals on days -10, -7, and -5 before injection and on days 7 and 14 after LNP injection. Two methods were used to quantify serum TTR. TTR protein levels were first quantified using a custom TTR sandwich ELISA, with the data from that analysis presented in Figure 11. Values ​​on days -10, -7, and -5 were averaged to obtain a baseline value. Notably, animals treated with LNP1 showed greater liver TTR editing and greater plasma TTR reduction (-63% change from baseline at day 14) compared with animals treated with LNP2 (3% change from baseline at day 14). TTR protein collected from serum was also quantified using liquid chromatography-mass spectrometry (LC-MS). Four unique serum TTR peptide fragments were quantified from each sample time point, and the average of the results is reported. LC-MS serum TTR quantification analysis using LC-MS is shown in Figure 12 and was consistent with the data obtained from ELISA quantification, particularly in that LNP1 also demonstrated greater plasma TTR reduction (-73% change from baseline at day 14) when compared to LNP2 (-21% change from baseline at day 14).

[0358] Thus, as shown in Figures 10, 11, and 12, injection of LNP1 and LNP2 into NHPs resulted in editing of the TTR gene in the liver, with LNP1 showing greater editing than LNP2. The greater editing in LNP1 NHPs corresponded to a corresponding increase in the reduction of serum TTR concentrations in serum.

[0359] Safety analysis For safety analysis, serum was collected from all animals on days -10, -7, and -5 before infusion, and at 6, 24, 48, 96, 168, 240, and 336 hours after LNP infusion, specifically to observe changes in liver enzyme and cytokine levels. Serum chemistry parameters were measured directly from serum samples using a Beckman Coulter AU680 analyzer. Baseline values ​​were obtained by averaging values ​​on days -10, -7, and -5. Both LNP1- and LNP2-treated animals showed a transient elevation in alanine aminotransferase, which peaked 48 hours after the end of infusion and returned to baseline levels 168 hours after the end of infusion (Figure 13A). As shown in Figure 13B, aspartate aminotransferase levels were also elevated by both LNP1 and LNP2 treatment, peaking 6 hours after the end of infusion and returning to baseline levels 96 hours after the end of infusion. Serum lactate dehydrogenase (Figure 14A) and glutamate dehydrogenase (Figure 14B) concentrations were also found to briefly increase after administration of either LNP1 or LNP2 and return to baseline levels 96 to 168 hours after the end of the infusion. Infusion of either LNP1 or LNP2 did not alter serum γ-glutamyltransferase (Figure 15A) or alkaline phosphatase (Figure 15B). Furthermore, as shown in Figure 16, LNP1 and LNP2 treatment did not affect serum total bilirubin concentrations. As shown in Figure 17, animals administered LNP1 and LNP2, respectively, also exhibited elevated serum creatine kinase concentrations, which in both cases peaked 6 hours after the end of the infusion and returned completely to baseline levels by 168 hours.

[0360] For serum cytokine analysis, serum was collected from all animals on days -10, -7, and -5 before treatment and at 24, 168, and 336 hours after LNP infusion. Cytokines were measured using a multiplex sandwich immunoassay. Four cytokines (MCP-1, IL-6, IP-10, and IL-1RA) were simultaneously quantified from serum samples using the U-PLEX Biomarker Group 1 (Monkey) assay from Meso Scale Diagnostics (Rockville, MD). Baseline values ​​were obtained by averaging values ​​from days -10, -7, and -5. As shown in Figure 18, both animals administered LNP1 and LNP2 showed increases in serum IL-6 concentrations, which peaked at 6 hours after the end of the infusion and returned to baseline by 24 hours. As further shown in Figure 18, both animals administered LNP1 and LNP2 showed increases in serum IL-1RA, which peaked at 6 hours and returned completely to baseline by 336 hours. Also, as shown in FIG. 18, neither LNP1 nor LNP2 had any measurable significant effect on serum MCP-1 or IP-10 concentrations.

[0361] Overall, analysis of the above parameters showed that infusion of either LNP1 or LNP2 into monkeys resulted in a transient increase in liver enzymes and cytokines that rapidly disappeared.

[0362] Pharmacokinetic (PK) evaluation Blood samples were collected (K2EDTA) for plasma pharmacokinetic analysis and determination of the concentrations of iLipid and PEG lipid excipients comprising LNP1 and LNP2. After the end of the infusion, plasma samples were collected at 0.25, 2, 6, 24, 48, 96, 168, 240, and 336 hours after LNP infusion. Concentrations of iLipid and PEG lipid were measured using a qualified LC-MS assay, as shown in Figure 19A. Time points at which lipids fell below the limit of quantitation (LLOQ) are not included in the figure. As shown in Figure 19A, serum iLipid concentrations in animals administered LNP1 and LNP2 decreased continuously until they approached the lower limit of quantitation (LLOQ) 96 hours after LNP infusion. Similarly, as shown in Figure 19B, serum PEG lipid concentrations in animals administered LNP1 and LNP2 also rapidly decreased, reaching the LLOQ 24 hours after the end of the infusion.

[0363] Part B: In vivo NHP evaluation of GA519 using GalNAc LNPs In further evaluation of GA519, an additional LNP (LNP3) was formulated to encapsulate the same GA519 and ABE8.8 mRNA at a 1:1 weight ratio and administered intravenously to NHPs as described above. LNP3 differs from LNP1 in that it was formulated with an additional GalNAc ligand excipient, as described in more detail below.

[0364] Preparation of LNPs The GalNAc LNP (LNP3) formulated for this aspect of the study was composed of the same iLipid, neutral helper lipid, PEG lipid, and sterol lipid described for LNP1 / LNP2, but unlike LNP1 / LNP2, LNP3 also contains a GalNAc conjugate lipid. The molar ratios of each component of LNP3 are listed in Table 15. [Table 15]

[0365] It should be understood that the lipids in Table 15 can be substituted with other suitable lipids of the classes described. For example, the amino lipid can be the following amino lipid or a salt thereof: [ka]

[0366] It should be understood that the mole percents of lipids in Table 13 may be adjusted, and that the mole percents included in Table 13 are intended to be target percentages of excipients for LNPs and represent the total mole percents of all LNPs formulated in a given batch, and that particular LNPs within a batch may have varying mole percents. Accordingly, it is contemplated herein that the mole percents of one or more or all of the LNP components listed in Table 13 may be adjusted, e.g., by ±1-5%, ±5-10%, or ±10-20%. It is further contemplated herein that for particular LNPs formulated in a given batch of LNPs formulated according to desired target excipient percentages, the mole percents of one or more or all of the LNP components listed in Table 13 may differ from the target mole percents, e.g., by ±1-5%, ±5-10%, ±10-20%, or even by more than ±20%. Furthermore, it should be understood that additional LNP components, including non-lipid components, may be added to the LNP components set forth in Table 13.

[0367] When formulating LNP3, GalNAc-lipid was premixed with other LNP excipients referenced in Table 15 before in-line mixing with GA519sgRNA and ABE8.8 mRNA (1:1 weight ratio) to form LNP3. Rajeev et al., WO2021178725, contains a description of the synthesis and characterization of GalNAc-lipid. Similar to LNP1 / LNP2, the resulting GalNAc-LNP, LNP3, was filtered using a 0.2 micron filter and frozen at -80°C. The physical properties of formulated LNP3 are summarized in Table 16. [Table 16]

[0368] Those skilled in the art will understand that the average LNP size, PDI, and RNA capture values ​​set forth in Table 16 are subject to measurement error or precision. It is also contemplated herein that the LNP size, PDI, and RNA capture values ​​set forth in Table 16 may vary by ±1-5%, ±5-10%, or ±10%-20%.

[0369] NHP research design This study involved male cynomolgus monkeys of Cambodian origin. A premedication regimen containing dexamethasone and H1 and H2 antihistamines was administered to all animals on Day -1 (approximately 24 hours prior to dosing) and Day 1 (pre-dose), 30–60 minutes prior to administration of the test article dose. The LNP3 dosage formulation was administered once on Day 1 of the study via IV infusion to two groups of three monkeys at the following dose levels: (i) for Group 1 of three monkeys, 2 mg / kg (animal body weight) of the sgRNA and mRNA combination and a dose volume of 6 mL / kg (n=3 / group); and (ii) for Group 2 of three monkeys, 3 mg / kg (animal body weight) of the sgRNA and mRNA combination and a dose volume of 6 mL / kg (n=3 / group).

[0370] Blood samples were collected from all animals before dosing for baseline measurements and at various time points from days 1 to 35 post-infusion to assess biomarkers, plasma iLipid and PEG pharmacokinetics, and serum safety parameters. Necropsies were performed on day 36. Liver tissue samples were collected from all animals to assess TTR gene editing in the liver.

[0371] Editing Efficiency Analysis The amount of gene editing in the liver was assessed by next-generation sequencing (NGS) of targeted polymerase chain reaction (PCR) amplicons at the TTR target site derived from genomic DNA extracted from the liver as described above (Musunuru et al., Nature 593, no. 7859 (May 2021): 429-34. https: / / doi.org / 10.1038 / s41586-021-03534-y). Percent editing was reported as the percentage of all reads containing the non-reference allele at the target adenine.

[0372] As shown in Figure 20, LNP3 resulted in similar levels of liver TTR editing efficiency in monkeys dosed at 2 mg / kg (60%) compared to monkeys dosed at 3 mg / kg (63%).

[0373] Quantification of TTR protein expression in serum For TTR protein analysis, serum was collected on days −10, −7, and −5 before infusion and 7, 14, 21, 28, and 35 after the end of infusion. Serum TTR was initially quantified using a custom TTR sandwich ELISA, with the data obtained from that analysis presented in Figure 21. The values ​​on days −10, −7, and −5 were averaged to obtain baseline values. As shown in Figure 21, both groups of animals administered LNP3 showed significant reductions in serum TTR protein at the first time point after administration (day 7). These reductions were maintained throughout the study, reaching maximum reductions of −84% and −91% change from baseline at day 28 for the 2 mg / kg and 3 mg / kg monkey groups, respectively. To confirm the ELISA results, TTR protein was also quantified by LC-MS. Four unique TTR peptide fragments were quantified in serum at each time point, and the average of the four results is reported. LC-MS serum TTR quantification, shown in Figure 22, confirmed that TTR was reduced at the first time point after infusion in animals on day 7 and maintained until necropsy on day 35. In animals administered 2 mg / kg LNP3, the maximum reduction in TTR protein was reached on day 35 (-82% change from baseline), while the maximum in the 3 mg / kg group of TTR protein was reached on day 28 (-87% change from baseline).

[0374] Thus, as described above and shown in the reference figures above, NHPs administered both 2 mg / kg and 3 mg / kg of LNP3 resulted in significant and relatively rapid liver TTR gene editing and a corresponding reduction in serum TTR protein levels.

[0375] Safety analysis For safety analysis, serum was collected from each test animal on days -10, -7, and -5 before the infusion, and on days 6, 24, 48, 96, 168, and 336 hours, 21, 28, and 35 after the end of the infusion, specifically to observe changes in liver enzyme and cytokine levels. Serum chemistry parameters were measured directly from serum samples using a Beckman Coulter AU680 analyzer. Baseline values ​​were obtained by averaging values ​​on days -10, -7, and -5. As shown in Figure 23A, animals administered LNP3 showed a dose-dependent, transient increase in alanine aminotransferase, which peaked 24 to 48 hours after the end of the infusion and returned to baseline levels 336 hours after the end of the infusion. As shown in Figure 23B, aspartate aminotransferase levels increased to a similar extent at both the 2 mg / kg and 3 mg / kg LNP3 doses, peaked 6 hours after the end of the infusion, and returned to baseline levels 168 hours after the end of the infusion. As shown in Figure 24A, both 2 mg / kg and 3 mg / kg LNP3 doses increased serum lactate dehydrogenase concentrations, which returned to baseline levels by 168 hours after the end of the infusion. As shown in Figure 24B, LNP3 also dose-dependently increased glutamate dehydrogenase concentrations, peaking at 24 hours and returning to baseline levels 336 hours after the end of the infusion. Serum concentrations of γ-glutamyltransferase and alkaline phosphatase, shown in Figures 25A and 25B, respectively, did not change significantly with either LNP dose. Furthermore, as shown in Figure 26, LNP3 treatment did not significantly affect serum total bilirubin concentrations. As shown in Figure 27, LNP3 increased serum creatine kinase concentrations, peaking 6 hours after the end of the infusion and then returning to baseline levels by 168 hours after the end of the infusion.

[0376] Analysis of the above-described safety parameters in this aspect of the in vivo NHP study was consistent with previous aspects of the study in that they showed that both doses of LNP3 produced transient increases in liver enzymes that rapidly resolved within two weeks of administration to subjects. Pharmacokinetic (PK) evaluation Blood samples were collected from all animals for plasma pharmacokinetic analysis and determination of the concentrations of the ionizable amino lipid (iLipid) and PEG-lipid components of LNP3 (K2EDTA). After the end of the infusion, plasma samples were collected at 0.25, 2, 6, 24, 48, 96, 168, 240, and 336 hours after LNP3 infusion. Concentrations of iLipid and PEG-lipid were measured using a qualified LC-MS assay. As shown in Figure 28A, dose-dependent iLipid plasma exposure was observed, falling below the LLOQ by 96 hours after the end of the infusion. As shown in Figure 28B, dose-dependent PEG-lipid plasma exposure was also observed, reaching the LLOQ by 24 hours after the end of the infusion.

[0377] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, e.g., nucleotide sequence submissions to GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq), are incorporated by reference. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of a document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to those skilled in the art will be within the scope of the invention as defined by the claims.

[0378] Example 4 TTR gene editing with GA521 guide RNA This example demonstrates gene editing with an exemplary modified guide RNA, GA521.

[0379] An exemplary guide RNA, GA521, was transfected into primary human hepatocytes using MessengerMax transfection. GA521 disrupts the start codon AUG of the TTR gene by editing it to ACG using an A-to-G base editor (e.g., ABE8.8; ABE8.8-m).

[0380] Three days after transfection, genomic DNA was harvested from hepatocytes and assessed for base editing using next-generation sequencing of PCR amplicons generated around the targeted splice sites.

[0381] The human TTR locus primers used for NGS analysis are listed below:

[0382] With NGS adapter: Forward (F): TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATAAGCAGCCTAGCTCAGGAGA (SEQ ID NO: 5769) Reverse (R): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGCCAGCCTCAGACACAAA (SEQ ID NO: 5770)

[0383] Without NGS adapter: Forward (F): GATAAGCAGCCTAGCTCAGGAGA (SEQ ID NO: 5771) Reverse (R): GGGCCAGCCTCAGACACAAA (SEQ ID NO: 5772)

[0384] Figure 29 illustrates the dose response of human gRNA GA521 in primary human hepatocytes. Percent base editing at various doses (ng / ml) of total RNA was determined from NGS analysis. GA521 was the guide RNA.

[0385] Overall, GA521 showed increased base editing with increasing doses (ng / ml) of total RNA in human cells, and sustained editing activity of over 40%.

[0386] Other embodiments From the foregoing description, it will be apparent that variations and modifications to the disclosure described herein may be made to adapt it to various usages and conditions. Such embodiments also fall within the scope of the following claims.

[0387] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment, any portion of an embodiment, or in combination with any other embodiment or any portion thereof.

[0388] As described herein, it will be understood that the disclosure includes specific examples and embodiments, individually and in combination, describing specific embodiments and examples of base editing systems for effecting nucleobase changes in genes, and methods of using them to treat disease, including compositions comprising such base editing systems, designs, and modifications thereof; and the synthesis, manufacture, use, and efficacy of the foregoing, including as pharmaceutical compositions for treating disease and for in vivo and in vitro delivery of active agents to mammalian cells under the conditions described.

[0389] While specific examples and numerous embodiments have been provided to illustrate aspects and combinations of aspects of the foregoing, it should be recognized and understood that any aspect or combination of aspects of an exemplary or disclosed embodiment can be excluded therefrom to form another embodiment, without limitation, and that any such embodiment can be considered to form a separate, independent claim. Similarly, it should be recognized and understood that any aspect or combination of aspects of one or more embodiments can also be included or combined with any aspect or combination of aspects of one or more embodiments, and that all such combinations are contemplated herein to be within the scope of this disclosure and can be presented as separate, independent claims, without limitation. Thus, it should be recognized that any feature presented in one claim can be included in another claim; any feature presented in one claim can be deleted from a claim that would otherwise constitute a claim without that feature; and any feature presented in one claim can be combined with any feature of another claim, each of which is contemplated herein. [Table 17] [Table 18] [Table 19] [Table 20]

[0390] It will be understood by reviewing this disclosure that it is contemplated that one or more aspects or features presented in a clause or group of related clauses may also be included in other clauses or may be combined with one or more aspects or features of other clauses.

Claims

1. 1. A base editor system for modifying a target transthyretin (TTR) gene, comprising a guide RNA comprising a sequence defined by: where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyl adenosine, mC* is 2'-O-methyl cytidine, mG* is 2'-O-methyl guanosine, mU* is 2'-O-methyl uridine, and the nucleotides represented in bold are linked by phosphorothioate (PS) backbone linkages; The base editor system, wherein the guide RNA guides the base editor system to make a nucleobase change in the TTR gene.

2. 1. An engineered, non-natural base editing system for modifying a target transthyretin (TTR) gene, comprising: (a) a guide RNA molecule having a sequence defined by: (b) a codon-optimized nucleic acid encoding a Cas9 protein fused to a deaminase; wherein the Cas9 protein fusion is capable of binding to the guide RNA and editing the sequence of the target TTR that is complementary to the guide RNA.

3. 3. The composition of claim 2, wherein the deaminase comprises a cytosine deaminase or an adenine deaminase.

4. 4. The composition of claim 2 or 3, wherein the Cas9 protein is a catalytically impaired Cas9 protein.

5. 5. The composition of claim 4, wherein the Cas9 protein is an inactive Cas9 or a nickase Cas9.

6. The composition of claim 3 , wherein the cytosine or adenine deaminase is deoxycytosine or deoxyadenosine deaminase.

7. 4. The composition of claim 3, wherein the Cas9 is fused to ABE8.

8.

8. A lipid nanoparticle (LNP) comprising the system according to any one of claims 2 to 7.

9. The LNP of claim 8 , wherein the LNP comprises an ionizable amino lipid, a neutral helper lipid, a PEG lipid, a sterol lipid, and / or a GalNAc lipid.

10. The ionizable amino lipid is VL422 or LP-01, the neutral helper lipid is DSPC, and the PEG lipid is PEG 2000 10. The LNP of claim 9, wherein the sterol lipid is -DMG, the sterol lipid is cholesterol, and the GalNAc lipid is DSG-PEG-Lys-tris(GalNAc).

11. 11. The LNP of claim 10, wherein the ionizable lipid is LP-01 (alternatively CIN16645), which is defined by the following structure: 【Chemistry 1】

12. 10. The LNP of any one of the preceding claims, wherein the LNP comprises an N:P ratio of about 1:40 to about 1:

1.

13. 13. The LNP of claim 12, wherein the LNP comprises an N:P ratio of about 1:

6.

14. A pharmaceutical composition comprising the LNP of any one of claims 9 to 13.

15. 1. A method for editing the TTR gene in a cell, comprising: (a) a guide RNA molecule having a sequence defined by: (b) a base editor system comprising a codon-optimized nucleic acid encoding a Cas9 protein fused to a deaminase; wherein the Cas9 protein fusion is capable of binding to the guide RNA and editing the target nucleic acid sequence complementary to the guide RNA.

16. 15. A method of treating a disease or disorder, comprising administering to a subject in need thereof the pharmaceutical composition of claim 14.

17. 17. The method of claim 16, wherein the disease or disorder is hereditary transthyretin amyloidosis, cardiomyopathy, polyneuropathy, or senile cardiac amyloidosis.

18. 18. The method of claim 16 or 17, wherein the pharmaceutical composition is administered by a route selected from intravenous, intradermal, transdermal, intranasal, intramuscular, subcutaneous, transmucosal, or oral.

19. The method of any one of claims 16 to 18, wherein the LNP is delivered to the liver.

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