Transthyretin gene base editing

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 age-related cardiomyopathy associated with wild-type TTR protein (ATTRwt), also known as senile cardiac amyloidosis.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 191,458, filed May 21, 2021, and U.S. Provisional Patent Application No. 63 / 322,182, filed March 21, 2022.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 20, 2022, is named 0650_000001WO01_SL.txt and is 946,110 bytes in size. [Background technology]

[0003] Transthyretin (TTR) is a 55 kDa transport protein for both thyroxine (T4) and retinol binding protein, which circulates in a soluble form in serum and cerebrospinal fluid (CSF) of healthy humans. Under normal conditions, 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 TTR tetramers and enhance dissociation into monomers, which leads to misfolding, aggregation, and subsequent extracellular deposition of TTR amyloid fibrils at various sites. This multisystem 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 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 inhibition of TTR protein synthesis using gene silencing drugs such as small interfering RNA (siRNA) (patisiran) and antisense oligonucleotides (inotersen).

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

[0006] Provided herein are compositions for gene modification or editing and methods of using same to treat or prevent conditions associated with extracellular deposition of amyloid fibrils in various tissues formed by aggregation of misfolded transthyretin (TTR) protein. 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 age-related cardiomyopathy (also known as senile cardiac amyloidosis) associated with wild-type TTR protein (ATTRwt). Disclosed are compositions and methods aimed at editing the TTR gene using editing systems, such as those including base editors and guide RNAs.

[0007] In a first aspect, an isolated polynucleotide or a nucleic acid encoding the same is described. The polynucleotide comprises a 5'-spacer sequence comprising about 17 to about 23 nucleotides that is homologous to a target protospacer sequence in a gene encoding transthyretin (TTR) adjacent to a NGG protospacer adjacent motif (PAM) sequence in the genome. The isolated polynucleotide serves as a guide polynucleotide that directs a base editor system to effect a nucleobase change in the TTR gene.

[0008] The protospacer sequence may comprise the start codon or a splice site of the TTR gene. The nucleobase changes affecting the TTR gene may comprise a disruption of the start codon or a disruption of an intron exon splice site. The isolated polynucleotide, or a polynucleotide encoded by a nucleic acid encoding it, may comprise a spacer sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identical to: 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA519), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA458), 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459), 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460 / GA520), or 5'-UACUCACCUCUGCAUGCUCA-3' (sequence number 5) (GA461).

[0009] The isolated polynucleotide, or the polynucleotide encoded by the nucleic acid encoding it, may comprise a guide RNA.

[0010] In a second aspect, a composition is described. The composition comprises a polynucleotide or a nucleic acid according to the first aspect. The composition may comprise a nucleic acid encoding a base editor fusion protein. The base editor fusion protein may comprise a programmable DNA binding domain and a deaminase. The deaminase may comprise a cytosine deaminase or an adenine deaminase. The deaminase may comprise ABE8.8. The programmable DNA binding domain may comprise a Cas9 protein. The Cas9 protein, for example, a Streptococcus pyogenes Cas9 protein, is modified (altered) such that its cleavage activity is partially or completely abolished. Such modified Cas9 proteins are referred to herein as "catalytically impaired" Cas9 proteins.

[0011] In a third aspect, a pharmaceutical composition is described. The pharmaceutical composition may comprise a composition according to the second aspect, or may comprise a polynucleotide or nucleic acid according to the first aspect.

[0012] In a fourth aspect, a lipid nanoparticle (LNP) is described. The LNP may comprise a pharmaceutical composition according to the third aspect, a composition according to the second aspect, or a polynucleotide or nucleic acid according to the first aspect. The LNP may comprise cholesterol.

[0013] In a fifth aspect, a pharmaceutical composition comprising the LNP is described.

[0014] In a sixth aspect, a method of generating one or more nucleobase changes in a TTR gene in a cell is described, the method comprising contacting the cell with a polynucleotide or nucleic acid according to the first aspect, a composition according to the second aspect, a pharmaceutical composition according to the third or fifth aspect, or an LNP according to the fourth aspect.

[0015] In a seventh aspect, a method of generating one or more nucleobase changes in the transthyretin (TTR) gene in a subject is described. The method comprises administering to the subject a polynucleotide or nucleic acid according to the first aspect, a composition according to the second aspect, a pharmaceutical composition according to the third or fifth aspect, or an LNP according to the fourth aspect. In some embodiments, one or more alleles of the TTR gene are silenced. The subject may be a human. The subject may be a subject in need thereof. The subject may be a subject suffering from or at risk of hereditary transthyretin amyloidosis (hATTR) due to one or more mutations in the TTR gene. The subject may be a subject suffering from or at risk of cardiomyopathy (hATTR-CM) and / or polyneuropathy (hATTR-PN). The subject may be a subject suffering from or at risk of senile cardiac amyloidosis characterized by a wild-type allele (ATTRwt) of the TTR gene.

[0016] The polynucleotide or nucleic acid according to the first aspect, the composition according to the second aspect, the pharmaceutical composition according to the third or fifth aspect, or the LNP according to the fourth aspect may be administered to a subject in a therapeutically effective amount. The polynucleotide or nucleic acid according to the first aspect, the composition according to the second aspect, the pharmaceutical composition according to the third or fifth aspect, or the LNP according to the fourth aspect may be administered intravenously.

[0017] In an eighth aspect, a composition for editing the TTR gene is described. The composition includes (a) an mRNA encoding a base editor protein having an editing window, and (b) a guide RNA including a tracr sequence that functions as a binding scaffold for the base editor protein and a spacer sequence that functions to guide the base editor protein to a protospacer on the TTR gene. The spacer sequence is at least partially complementary to a splice site or start codon of the sense or antisense strand of the TTR gene.

[0018] The base editor protein may comprise a cytidine deaminase or an adenosine deaminase. The cytidine deaminase may be a deoxycytidine deaminase. The adenosine deaminase may be a deoxyadenosine deaminase. The base editor protein may comprise a fusion protein comprising a nickase and a cytidine deaminase or an adenosine deaminase. The base editor protein may comprise a fusion protein comprising a D10A nickase Cas9 and a cytidine deaminase or an adenosine deaminase. The base editor protein may be comprised of a fusion protein comprising the adenine base editor ABE8.8.

[0019] The spacer sequence may be homologous to a protospacer sequence selected from Table 1 or Table 13. The spacer sequence may be selected from the following tables: TIFF2024519922000002.tif80165

[0020] (wherein A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyl adenosine; c is 2'-O-methyl cytidine; g is 2'-O-methyl guanosine, u is 2'-O-methyl uridine; and s is a phosphorothioate (PS) backbone linkage).

[0021] The spacer sequence may have greater than 80% sequence identity with the spacer sequences provided in the table below: TIFF2024519922000003.tif67165

[0022] (wherein A is a modified or unmodified adenosine; C is a modified or unmodified cytidine; G is a modified or unmodified guanosine; and U is a modified or unmodified uridine).

[0023] The guide RNA may be selected from the following table: TIFF2024519922000004.tif136165

[0024] (wherein A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyl adenosine; c is 2'-O-methyl cytidine; g is 2'-O-methyl guanosine, u is 2'-O-methyl uridine; s is a phosphorothioate (PS) backbone linkage; the bold characters represent spacer sequences).

[0025] The spacer sequence may have greater than 80% sequence identity with a guide RNA sequence selected from the following table: TIFF2024519922000005.tif129165

[0026] The composition can be capable of producing the editing activity described in Table 2 (except GA459) or Table 3. The composition can be capable of producing minimal or no off-target editing activity described in Table 4, 6, 7, 8, 9, or 10. The composition can be encapsulated in lipid nanoparticles. The composition can be administered to a subject in vivo. [Brief description of the drawings]

[0027] [Figure 1] General schematic diagram of gene editor complexed with gRNA targeting a gene of interest. Cas9 protein, guide RNA, spacer sequence, protospacer sequence, and PAM (protospacer adjacent motif) are specified (Figure 1A). Figure 1A discloses SEQ ID NO: 5760. Additionally, a schematic diagram of the general principle of base editing using a cytosine base editor (CBE) (Figure 1B) and an adenine base editor (ABE) (Figure 1C) is shown.

[0028] [Diagram 2] Altering a splice donor site by base editing. The top panel shows normal splicing of RNA transcribed from a gene. The bottom panel shows the splicing that can occur upon transcription of a gene with a splice site disrupted by editing.

[0029] [Diagram 3] A map of the human TTR gene (hTTR gene) showing the location of various restriction enzyme recognition sites, exons 1-4, and single guide RNAs GA457, GA459, GA460, and GA461, as designated in Table 1.

[0030] [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 the guides GA457, GA459, GA460, and GA461 are located. Figure 4 discloses SEQ ID NO: 5761.

[0031] [Diagram 5] Schematic diagram showing the TTR guide and editing positions of GA457 (FIG. 5A), GA460 (FIG. 5B), and GA461 (FIG. 5C). Human genomic DNA (gDNA) sequences are labeled in black. Guide sequences are highlighted in grey above. Genomic exon sequences are in uppercase and intron sequences are in lowercase. Primary positions targeted with ABE editing are labeled with black arrows. FIG. 5 discloses SEQ ID NOs: 1, 5762, 4, 5763, 5, and 5764, respectively, in order of appearance.

[0032] [Figure 6] Graph showing splice editing rate (%) in human hepatocytes using ABE editing with single guide RNA GA457, GA459, GA460, and GA461 guide RNAs. The three TTR guide RNAs GA457, GA460, and GA461 show high activity in human hepatocytes. Each of the guides 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.

[0033] [Figure 7]FIG. 1 is a flow chart of the ONE-seq protocol for determining candidate off-target sites.

[0034] [Figure 8] 1 is a schematic diagram of a comparison of GA519 and GA457 hybridized to NHP and human TTR exon 1. In order of appearance, SEQ ID NOs: 1, 5765, 2, and 5766, respectively, are disclosed.

[0035] [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.

[0036] [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.

[0037] [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.

[0038] [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.

[0039] [Figure 13] 1 is a bar graph showing serum alanine aminotransferase (ALT) (A) and aspartate aminotransferase (AST) (B) concentrations in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0040] [Figure 14]1 is a bar graph showing serum lactate dehydrogenase (LDH) levels (A) and serum glutamate dehydrogenase (GDH) levels (B) of NHPs treated with LNP1 and LNP2 as described in the Examples.

[0041] [Figure 15] 1 is a bar graph showing serum gamma-glutamyltransferase (GGT) levels (A) and serum alkaline phosphatase (AP) levels (B) of NHPs treated with LNP1 and LNP2 as described in the Examples.

[0042] [Figure 16] 1 is a bar graph showing serum total bilirubin concentrations in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0043] [Figure 17] 1 is a bar graph showing serum creatine kinase concentrations in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0044] [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.

[0045] [Figure 19] 1 is a plot of the plasma pharmacokinetic profiles of iLipid (A) and PEG-Lipid (B) in NHPs treated with LNP1 and LNP2 as described in the Examples.

[0046] [Figure 20] 1 is a bar graph showing hepatic editing of the TTR gene by LNP3 in NHPs, as described in the Examples.

[0047] [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.

[0048] [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.

[0049] [Diagram 23] 1 is a bar graph showing serum alanine aminotransferase (ALT) (A) and serum aspartate aminotransferase (AST) (B) concentrations in NHPs treated with LNP3 as described in the Examples.

[0050] [Figure 24] 1 is a bar graph showing serum lactate dehydrogenase (LDH) levels (A) and serum glutamate dehydrogenase (GDH) levels (B) of NHPs treated with LNP3 as described in the Examples.

[0051] [Diagram 25] 1 is a bar graph showing serum gamma-glutamyltransferase (GGT) levels (A) and serum alkaline phosphatase (AP) levels (B) of NHPs treated with LNP3 as described in the Examples.

[0052] [Figure 26] 1 is a bar graph showing serum total bilirubin concentrations in NHPs treated with LNP2 as described in the Examples.

[0053] [Figure 27] 1 is a bar graph showing serum creatine kinase levels in NHPs treated with LNP3 as described in the Examples.

[0054] [Figure 28]1 is a plot of the plasma pharmacokinetic profiles of iLipid (A) and PEG-Lipid (B) in NHPs treated with LNP1 and LNP2 as described in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Provided herein are compositions for gene modification or editing and methods of using same to treat or prevent conditions associated with extracellular deposition of amyloid fibrils in various tissues formed by aggregation of misfolded transthyretin (TTR) protein. 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 age-related cardiomyopathy (also known as senile cardiac amyloidosis) associated with wild-type TTR protein (ATTRwt). Disclosed are compositions and methods aimed at editing the TTR gene using editing systems, such as those including base editors and guide RNAs.

[0056] 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 in this "Definitions" section.

[0057] As used herein and in 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 the sense of including "and / or" unless the content clearly dictates otherwise. As used herein, the terms "and / or" and "any combination thereof," as well as their grammatical equivalents, can be used interchangeably. These terms may mean that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination 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 otherwise.

[0058] The term "about" or "approximately" may 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" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean within 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 may mean within an order of magnitude, within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the application and claims, unless otherwise indicated, the term "about" means that the particular value is deemed to be within an acceptable error range, unless otherwise indicated.

[0059] 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") are inclusive or open-ended and do 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. Further, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0060] An article, composition, method, etc. that includes one or more elements may be composed of one or more elements or may consist essentially of one or more elements. As used in this specification and claim(s), "consisting of" (as well as any form of consisting of, e.g., "consists of" and "consist of") means inclusive and limited. As used in this specification and claim(s), an article, composition, method, etc. that "consisting essentially of" (as well as 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 properties of the article, composition, method, etc.

[0061] References herein to "some embodiments," "an embodiment," "one embodiment," "embodiments," or "other embodiments" mean that a 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.

[0062] The words "preferred" and "preferably" refer to embodiments of the invention that may provide 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.

[0063] The term "nucleic acid" as used herein 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 through the phosphate group. "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place 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, that have similar binding properties as 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).

[0064] The term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides, and longer fragments referred to as polynucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. DNA can be in the form of, for example, an antisense molecule, a plasmid DNA, a precondensed DNA, a PCR product, a vector, an expression cassette, a chimeric sequence, a 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 a polymer or oligomer 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 containing a combination of both deoxy and ribonucleotides, in combination with backbone modifications, as described herein, or variants thereof.

[0065] A "nucleic acid" as described herein may include one or more nucleotide variants, such as non-standard nucleotide(s), non-natural 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-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 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), wybutoxocine, 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 include 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).

[0066] 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 available to form hydrogen bonds with a complementary nucleotide), at the sugar moiety, or at 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 oxygens of the phosphate backbone with sulfur atoms, retarding nuclease degradation of oligonucleotides. Phosphorodiamidate linkages (N3'→P5') prevent nuclease recognition and degradation. Backbone modifications may 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 may 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 binding affinity to RNA and increasing nuclease resistance.Modified sugar moieties may also include an extra bridge bond (e.g., a methylene bridge connecting the 2'-O and 4'-C atoms of the ribose of a locked nucleic acid) or a sugar analog such as a morpholine ring (e.g., in phosphorodiamidate morpholinos).

[0067] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly stated. 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)).

[0068] 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 apart from its native environment. An isolated DNA or RNA molecule may exist in purified form or may 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 is substantially free of other cellular materials 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 is 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).

[0069] As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked through peptide bonds and may be composed of two or more polypeptide chains. The terms "polypeptide", "protein", and "peptide" refer to a polymer of at least two amino acid monomers linked through amide bonds. The amino acids may be L or D optical isomers. 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 that encodes the protein. Proteins are essential for the structure, function, and control of the body's cells, tissues, and organs, 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 into the naturally occurring (or at least known) amino acid sequence of the protein. The protein or variants thereof may 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 may be based on an antibody capable of capturing a specific antigen and a second antibody capable of detecting the captured antigen.

[0070] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, such as rodents, such as rats, mice, and guinea pigs.

[0071] 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 who is intended to cure, eliminate, alleviate, mitigate, change, treat, ameliorate, improve, or affect the disease, the symptom of a disease, or the predisposition to a disease. In some embodiments, the subject is afflicted with hereditary transthyretin amyloidosis (hATTR). In some embodiments, the subject is afflicted with transthyretin amyloidosis cardiomyopathy (ATTR-CM). In some embodiments, the subject is afflicted with transthyretin amyloidosis polyneuropathy (ATTR-PN). In some embodiments, the subject is afflicted with wild-type ATTR (ATTRwt), age-related deposition of wild-type TTR protein (formerly known as senile amyloidosis).

[0072] As used herein, "administering" and its grammatical equivalents may refer to providing one or more of the replication-competent recombinant adenoviruses or pharmaceutical compositions described herein to a subject or patient. By way of example and not limitation, "administering" may 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 may be used.

[0073] 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 by gradual perfusion over time. Additionally, it can be administered to a subject via an injectable depot route of administration, for example, using 1, 3, or 6 month depot injectable or biodegradable materials and methods.

[0074] The terms "treat", "treating" or "treatment" as used herein, and grammatical equivalents thereof, may include alleviating, reducing or improving 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, relieving a condition caused by a disease or condition, or halting a symptom of a disease or condition, prophylactically and / or therapeutically. "Treatment" may refer to administering a composition comprising nanoparticles, such as lipid nanoparticles (LNPs), to a subject after onset or suspected onset of a disease or condition. "Treatment" includes the concept of "alleviating", which refers to reducing the frequency of occurrence or recurrence, or the 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 "treat" also encompasses the concept of "managing", which refers to reducing the severity of a particular disease or disorder in a patient, or delaying its recurrence, e.g., extending the period of remission in a patient who has been affected by 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 disorder, condition, or symptoms associated therewith be completely eliminated.

[0075] 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 or susceptible to it.

[0076] As used herein, the term "ameliorate" can refer to decreasing, suppressing, attenuating, reducing, arresting, or stabilizing the onset or progression of a disease.

[0077] As used herein, "delaying" the onset of a disease means delaying, impeding, slowing down, inhibiting, stabilizing, and / or postponing the progression of a disease. This delay can be of various durations, depending on the history of the disease and / or the individual being treated. A method of "delaying" or reducing the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of a 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.

[0078] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease 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 a condition. "Onset" includes occurrence, recurrence, and onset.

[0079] As used herein, "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[0080] The term "therapeutic agent" may refer to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. A therapeutic agent may 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.

[0081] 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 pharma- ceutically acceptable excipients, carriers, and / or therapeutic agents to be administered to a subject, e.g., a human in need thereof.

[0082] As used herein, the term "pharmaceutical acceptable" and its grammatical equivalents can refer to the attributes of a material useful in the preparation of 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 abolish the biological activity or properties of a compound and is relatively non-toxic, i.e., such a substance may 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 in which it is contained.

[0083] A "pharmaceutical 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 is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0084] A "pharmaceutically acceptable salt" may be a salt of an acid or base that is generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications. Those skilled in the art will recognize from this disclosure and the knowledge of the art that additional pharmaceutically acceptable salts include those described in: Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).

[0085] 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 is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0086] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges in 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 fractional 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 opposite direction.

[0087] The 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 on the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the claims to the contrary, the doctrine of equivalents should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0088] 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 deviation found in their respective testing measurements.

[0089] As used herein, a spacer sequence of a guide nucleic acid is considered to be "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.

[0090] 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.

[0091] For purposes of percent sequence identity between an RNA sequence (eg, a spacer) and a DNA sequence (eg, a target gene protospacer), a uracil base in the RNA should be considered to be identical to a thymine base in the DNA sequence.

[0092] As used herein, "sequence identity" refers to the degree to which two optimally aligned nucleic acid sequences are invariant over 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).

[0093] 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.

[0094] In sequence comparison, one sequence usually serves as a reference sequence to be compared with a test sequence. When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designed if necessary, and parameters of sequence algorithm program are specified. Sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on the specified program parameters. Optimal alignment of sequences for aligning comparison window is well known to those skilled in the art, and can be performed with tools such as Smith and Waterman local homology algorithm, Needleman and Wunsch homology alignment algorithm, Pearson and Lipman similarity search method, and optionally computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of 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.

[0095] "Percent identity" may 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-valued threshold score T. T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is 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 falls off an amount X from the maximum achieved, or when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below zero, 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 wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands.

[0096] 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 minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the minimum sum probability in the comparison of the test nucleotide sequence versus the reference nucleotide sequence is less than about 0.1 to less than about 0.001.

[0097] In some embodiments, a first nucleotide sequence that is 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 are different 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). In general, 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 with 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 Russel, Molecular Cloning: A laboratory Manual, 3 rded., Cold Spring Harbor Laboratory Press, 2001 for a description of SSC buffer). Often, a low stringency wash is performed before the high stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex, e.g., greater than 100 nucleotides, is 1×SSC for 15 minutes at 45° C. An example of a low stringency wash for a duplex, e.g., greater than 100 nucleotides, is 4-6×SSC for 15 minutes at 40° C. 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 concentration (or other salt), and a temperature of typically at least about 30° C. Destabilizing agents, such as formamide, can also be added to achieve stringent conditions.

[0098] In several places throughout this application, guidance is provided through examples, which 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 should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0099] For any method disclosed herein including distinct steps, the steps may be performed in any order practicable, and, where appropriate, any combination of two or more steps may be performed simultaneously.

[0100] 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.

[0101] 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 a soluble form in serum and cerebrospinal fluid (CSF) in 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).

[0102] 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). More than 120 TTR variants have been identified so far, the majority of which are pathogenic. The most common pathogenic variant consists of a point mutation leading to the substitution of valine by 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.

[0103] 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, which leads to misfolding, aggregation, and subsequent extracellular deposition of TTR amyloid fibrils at various tissue sites. This multisystem extracellular amyloid deposition (amyloidosis) leads to the dysfunction of various organs and tissues. In particular, transthyretin amyloidosis-associated polyneuropathy (ATTR-PN) and transthyretin amyloidosis-associated cardiomyopathy (ATTR-CM) are severe diseases with significant morbidity and mortality.

[0104] If there is clinical suspicion of hATTR-PN, the 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, the key diagnostic tools are either intracardiac muscle biopsy (with tissue staining and amyloid typing by immunohistochemistry or mass spectrometry) or technetium 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).

[0105] The compositions described herein include a spacer having a nucleotide sequence that serves as a guide to guide a gene editing protein (e.g., a base editor) to change the TTR gene, for example, by introducing one or more nucleobase 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 nonfunctional protein, thereby silencing the TTR gene. Alternatively, it is contemplated herein that corrections to one or more point mutations can be made using a gene editing protein that modifies the mutated gene to correct the mutation responsible for causing the dysfunction of the TTR gene or otherwise alleviate the dysfunction of the gene.

[0106] 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. Genetic editing can be performed using nucleases (e.g., naturally occurring nucleases or artificially engineered nucleases). Genetic modification can include introducing double-strand breaks, nonsense mutations, frameshift mutations, splice site changes, or inversions 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 impart double-strand breaks at site-specific locations in DNA or genes. Genetic modification can also be performed using other editors, such as base editors.

[0107] Base Editor A base editor (BE) or nucleobase editor (NBE) refers to an agent that includes a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). A base editor may include a macromolecule or macromolecular complex that can convert (e.g., transition or transversion) a nucleobase in a polynucleic acid sequence to another nucleobase at one or more positions within a base editing window. A base editor may include a combination of (a) a nucleotide, nucleoside, or nucleobase conversion 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 may 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.

[0108] The base editor may comprise a programmable DNA binding domain of a polynucleotide 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., a peptide linker between the domains. In some embodiments, the domain with base editing activity is linked to a guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to a deaminase).

[0109] 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 directed to the site of interest by a guide RNA (gRNA) in D10A nickase Cas9 (nCas9). The cytidine deaminase enzyme of the CBE directs the conversion of cytosine to uridine, resulting in a C→T (or G→A) substitution (see FIG. 1B). "Cytidine deaminase" is used herein to refer to a deaminase enzyme that acts on deoxycytidine, on cytidine, or on both deoxycytidine and cytidine to convert cytosine to uridine. Cytidine deaminase and cytosine deaminase may be used interchangeably herein. If 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).

[0110] In comparison, the adenosine deaminase enzyme of ABE directs 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, on adenosine, or on both deoxyadenosine and adenosine to convert adenine to hypoxanthine or adenosine to inosine. Since the structure of inosine is similar to guanosine (inosine does not contain the exocyclic amino group of guanosine), inosine tends to behave like guanosine. Inosine is eventually 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 would cause a nonsense mutation.

[0111] Adenine base editors can be used to disrupt gene function, for example, by editing the start codon, either ATG→GTG or ATG→ACG. A second way in which adenine base editors can disrupt gene function is by editing a splice donor at the 5' end of an intron or a 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) where nonsense, frameshift, or in-frame mutations can be introduced, which can result in premature stop codons, or insertions / deletions of amino acids that inhibit the activity of the protein, or the exclusion of exonic sequences that can introduce nonsense, frameshift, or in-frame indel mutations.

[0112] 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 an 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 an adenine base editing at the first position of the sense strand (AG → GG).

[0113] 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)).

[0114] 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 MA004 mRNA shown in Table 11, 60% or more sequence identity with MA004 mRNA shown in Table 11, 70% or more sequence identity with MA004 mRNA shown in Table 11, 75% or more sequence identity with MA004 mRNA shown in Table 11, 80% or more sequence identity with MA004 mRNA shown in Table 11, 85% or more sequence identity with MA004 mRNA shown in Table 11, 90% or more sequence identity with MA004 mRNA shown in Table 11, 95% or more sequence identity with MA004 mRNA shown in Table 11, 96% or more sequence identity with MA004 mRNA shown in Table 11, 97% or more sequence identity with MA004 mRNA shown in Table 11, 98% or more sequence identity with MA004 mRNA shown in Table 11, or a sequence identical to that shown in Table 11. It is encoded by an mRNA that contains a sequence that has 99% or more sequence identity to the mRNA.

[0115] 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.

[0116] 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 uracil-DNA glycosylase, DNA repair proteins such as XRCC1, DNA ligase S, or the DNA binding and ligase domains of DNA polymerase β.

[0117] In some embodiments, the base editor may 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.

[0118] The term "base editor system" refers to a system for editing a nucleobase 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 for deaminating the nucleobase (e.g., adenosine deaminase or cytidine deaminase); and (3) one or more guide polynucleotides (e.g., guide RNA). 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 programmable nucleotide binding domain of the polynucleotide is a programmable DNA binding domain of the polynucleotide. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is a cytosine base editor (CBE).

[0119] 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.

[0120] Through the use of a guide RNA (gRNA) with a sequence homologous to a sequence of DNA in the target genome (known as a protospacer) adjacent to a specific protospacer adjacent motif (PAM) containing the sequence NGG (where N is any standard base) in the DNA, Cas9 can be used to create a double-stranded break (DSB) in the target sequence. Non-homologous end joining (NHEJ) at the DSB 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 the target sequence.

[0121] 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 (e.g., for genome modification) have been described (see, for example, Cong et al., Science 339, 819-823 (2013); Mali et al., Science339, 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)).

[0122] 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 dead 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, expressed alternatively, 13-17 nucleotides upstream of the PAM). Notably, cytosine bases within this "window" were susceptible to editing, which led to a variety of outcomes depending on how many and which cytosines were edited. After DNA replication or repair, each uracil is replaced with a thymine, completing the C to T base editing.

[0123] 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.

[0124] The next version of the base editor (BE3) used the Cas9 nickase rather than dCas9. The nickase cleaves the unedited strand opposite the edited C→T base, stimulating removal of the opposing 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 the base editing was further improved by adding mutations to the Cas9 nickase; in a similar manner, Cas9 was mutated to narrow the width of the editing window from about 5 nucleotides to only 1-2 nucleotides (Rees et al., Nat Commun, 2017, 8: 15790, Kim et al., Nat Biotechnol, 2017, 35: 371-6).

[0125] 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 (called ABE7.10) capable of editing adenines to guanines in DNA (Gaudelli et al. Nature, 2017, 551: 464-71).

[0126] Adenine 8.8-m (also referred to herein as ABE8.8) uses its core Streptococcus pyogenes nickase Cas9 (nSpCas9) protein together with a guide RNA (gRNA) to engage double-stranded protospacer DNA sequences flanked at the 3' end by a 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 form (called an R-loop). Unlike Cas9 and Cas12, ABE8.8 does not make a double-stranded break within the targeted 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, nicking the target DNA strand within the DNA:RNA heteroduplex of the R-loop. This nick biases the DNA repair machinery to use the newly deaminated strand as a template, allowing for highly efficient base transfer mutations 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 protospacer DNA sequence specified by the gRNA, with peak editing observed at position 6 of the protospacer (Gaudelli et al., Nat Biotechnol. 2020 Jul;38(7):892-900).

[0127] In some embodiments, the nucleic acid encoding the base editor fusion protein is an mRNA. In some embodiments, the mRNA, when translated in a 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 a target cell or subject.

[0128] 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 called 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.

[0129] Protospacer The term "protospacer" or "target sequence" as used herein and its grammatical equivalents may refer to a PAM-adjacent nucleic acid sequence. A protospacer may be a nucleotide sequence within a gene, genome, or chromosome targeted by a gRNA. In the natural state, a protospacer is adjacent to a PAM (protospacer adjacent motif). A cleavage site by an RNA-guided nuclease is within the protospacer sequence. For example, as shown in FIG. 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 may result in non-homologous end joining or homology-directed repair. Disruption of the protospacer may result in deletion of the protospacer. Additionally or alternatively, disruption of the protospacer may result in insertion of an exogenous nucleic acid sequence into the protospacer or replacement of the protospacer.

[0130] In this disclosure, we have 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, including Streptococcus pyogenes Cas9, such as ABE7.10, or another Cas protein that may use NGG PAM) to destroy a start codon or destroy a splice site (donor or acceptor) via A→G editing within an editing window (approximately positions 4-7 of the 20 nt protospacer region of DNA). Within the human TTR gene, we have identified four of the sequences shown in Table 1. An alignment of these four protospacer sequences on the map of the human TTR gene is shown in FIG. 3.

[0131] The protospacer corresponding to guide RNA GA457 has the sequence 5'-GCCATCCTGCCAAGAATGAG-3' (SEQ ID NO: 24) and is located at bp 34,879 to 34,898 of the human TTR gene.

[0132] The protospacer corresponding to guide RNA GA459 has the sequence 5'-GCAACTTACCCAGAGGCAAA-3' (SEQ ID NO: 25) and is located at bp 36,007 to 36,026 of the human TTR gene.

[0133] The protospacer corresponding to the 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.

[0134] 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.

[0135] The protospacer corresponding to guide RNA GA458 has the sequence 5'-GCCATCCTGCCAAGAACGAG-3' (SEQ ID NO:28) and represents the sequence within the cyno TTR gene that corresponds to the human protospacer sequence corresponding to guide RNA GA459.

[0136] The guide nucleic acid (e.g., guide RNA) is about 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 the canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to the canonical 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.

[0137] 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.

[0138] In some embodiments, a guide polynucleotide may be synthesized, including but not limited to a gRNA. The guide polynucleotide may include a spacer sequence configured to hybridize, for example, under conditions within a cell, to a complementary sequence 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.

[0139] The present disclosure includes a guide polynucleotide having a sequence that is 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 a guide polynucleotide having the sequence 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO:1) (GA457).

[0140] The present disclosure includes a guide polynucleotide having a sequence that is 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 a guide polynucleotide having the sequence 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO:2) (GA458).

[0141] The disclosure includes a guide polynucleotide having a sequence that is 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 disclosure includes a guide polynucleotide having the sequence 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459).

[0142] The present disclosure includes guide polynucleotides having a sequence that is 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) (GA4560).The present disclosure includes guide polynucleotides having the sequence 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA4560).

[0143] The present disclosure includes a guide polynucleotide having a sequence that is 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) (GA4561).The present disclosure includes a guide polynucleotide having the sequence 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO:5) (Ga4561).

[0144] A guide polynucleotide may contain at least three regions: a first region at the 5' end (spacer region) 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, which serves as a binding scaffold for the base editor or CRISPR / Cas protein, and the spacer region serves to guide the protein to a specific target site. The acronym tracr stands for trans-activating CRISPR.

[0145] The second region of the gRNA may form a secondary structure. In some embodiments, the secondary structure formed by the gRNA may include a stem (or hairpin) and a loop. The length 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.

[0146] 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 in length.

[0147] 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.

[0148] 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 more than 50 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.

[0149] In some embodiments, the guide polynucleotide comprises a spacer sequence and is otherwise compatible with the standard 100 nt Streptococcus pyogenes CRISPR gRNA sequence.

[0150] 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-methyl ribosugar 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-methyl ribosugar and phosphorothioate modifications.

[0151] The gRNA described herein can be synthesized chemically, enzymatically, or a combination thereof. For example, the gRNA can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, the gRNA can be synthesized in vitro by operably linking the DNA encoding the gRNA to a promoter control sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include, but are not limited to, T7, T3, 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.

[0152] therapeutic use The guide polynucleotides and compositions described herein may be administered to a target cell or a subject in need thereof in a therapeutically effective amount to prevent or treat a condition associated with transthyretin amyloidosis. In some embodiments, the subject is afflicted with hereditary transthyretin amyloidosis (hATTR). In some embodiments, the subject is afflicted with transthyretin amyloidosis cardiomyopathy (ATTR-CM). In some embodiments, the subject is afflicted with transthyretin amyloidosis polyneuropathy (ATTR-PN). In some embodiments, the subject has wild-type ATTR (ATTRwt), age-related deposition of wild-type TTR protein (formerly known as senile amyloidosis).

[0153] Upon such administration, the guide polynucleotide induces the editor system (e.g., ABE editor system) to impart a nucleobase 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 the cells of the subject's liver (hepatocytes).

[0154] For example, the gRNA and adenosine base editor protein may be expressed in a cell in which target gene editing is desired (e.g., a hepatocyte, etc.), 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 in the target gene is guided by a guide RNA, and the spacer sequence of the gRNA hybridizes to a target polynucleotide sequence in the target gene, e.g., a complementary sequence to the protospacer. Thus, the guide RNA guides the adenosine base editor protein to edit the target polynucleotide sequence (e.g., the protospacer sequence) in the target gene. In some embodiments, the guide RNA is co-introduced into the cell in which editing is desired with the adenosine base editor protein or a nucleic acid encoding the adenosine base editor protein.

[0155] In certain embodiments, adenine base editors may be used to disrupt gene function and / or expression by modifying the nucleobases of splice sites of target genes. In some embodiments, adenosine nucleobase editors described herein may 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 the messenger RNA (mRNA), which may introduce nonsense, frameshift, or in-frame indel mutations, which result in premature stop codons, or insertions / deletions of amino acids that disrupt protein activity, or exclusion of exon sequences that may also introduce nonsense, frameshift, or in-frame indel mutations.

[0156] 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, a base editor, such as an adenosine nucleobase editor described herein, can be used to generate a sequence change that disrupts 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 of the splice acceptor site on the sense strand, resulting in an AG→GG edit.

[0157] In some embodiments, the methods and compositions disclosed herein reduce or eliminate 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 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to a control.

[0158] In some embodiments, a method for treating or preventing a condition in a subject in need of such treatment or prevention as described herein comprises administering to the subject (i) a guide polynucleotide and (ii) a nucleic acid encoding a base editor fusion protein.

[0159] In some embodiments, a method of treating or preventing a condition in a subject in need of such treatment or prevention as described herein 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.

[0160] Pharmaceutical Compositions In some aspects, provided herein is a pharmaceutical composition comprising the base editor system provided herein and a pharma- ceutically acceptable carrier or excipient. In some aspects, provided herein is a pharmaceutical composition for gene modification comprising a guide RNA and a base editor fusion protein or a nucleic acid sequence encoding a base editor fusion protein as described herein, and a pharma- ceutically acceptable carrier. The pharmaceutical composition is formulated in a conventional manner using one or more pharma- ceutically acceptable inactive ingredients that facilitate the processing of the active compound into a pharma- ceutical usable preparation. Formulations and delivery methods suitable for use in the present disclosure are generally well known in the art. The appropriate formulation depends on the selected route of administration. Summary summaries of 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, HA 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).

[0161] A pharmaceutical composition can be a mixture of a guide RNA or a nucleic acid sequence encoding a guide RNA and a base editor fusion protein or a nucleic acid sequence encoding a base editor fusion protein as described herein, together with one or more of other chemical components (i.e., pharma- ceutically acceptable components), such as carriers, excipients, binders, fillers, suspending agents, flavorings, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetrants, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. A pharmaceutical composition facilitates administration to an organism or subject in need thereof.

[0162] The pharmaceutical composition of the present disclosure can be administered to a subject using any suitable method known in the art.The pharmaceutical composition 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, intramuscular, subcutaneous, or intravenous injection.In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally.

[0163] 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. Preparation and administration schedules of the additional therapeutic agent may be used according to the manufacturer's instructions or as empirically determined by the skilled physician.

[0164] Lipid Nanoparticle (LNP) Composition The pharmaceutical compositions for genetic modification described herein may be encapsulated in lipid nanoparticles (LNPs). As used herein, a "lipid nanoparticle (LNP) composition" or "nanoparticle composition" is a composition that includes one or more of the described lipids. The LNP compositions or formulations contemplated herein are typically on the order of micrometers or less in size and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, the nanoparticle compositions or formulations contemplated herein may be liposomes with a diameter of 500 nm or less that have 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 may 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, 70 nm ± 5 nm. The LNPs described herein may be substantially non-toxic.

[0165] Lipid nanoparticles (LNPs) use a non-viral drug delivery mechanism that can cross 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 near-neutral charged LNP surface in the bloodstream, thereby serving as an endogenous ligand for hepatocytes expressing the low-density lipoprotein receptor (LDLr) [Mol. Ther., 2010, 18, 1357-1364.]. Control of efficient hepatic 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.

[0166] 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 the 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 the targeting moiety can improve delivery is through receptor-mediated endocytosis activity. This uptake mechanism involves the transfer of nucleic acid material bound to a membrane receptor into the interior of a membrane-encased region, either through invagination of a membrane structure or by fusion of the delivery system with the cell membrane. This process is initiated through activation of a cell surface or membrane receptor after a specific ligand binds 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 conjugated to highly hydrophilic molecules such as nucleic acids, can greatly enhance plasma protein binding and thus extend circulatory half-life. Lipophilic conjugates can also be used in combination with targeting ligands to improve intracellular trafficking in targeted delivery approaches.

[0167] The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor that is abundant on hepatocytes. ASGP-R exhibits 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 the surface or periphery of the particle at specific or engineered surface densities ranging from relatively low to relatively high surface densities. The receptor-targeting conjugates may include 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 GalNAc that targets the ASGPR. In one aspect, the receptor-targeting conjugate includes one GalNAc moiety or a derivative thereof. In another aspect, the receptor-targeted conjugate comprises two different GalNAc moieties or derivatives thereof. In another aspect, the receptor-targeted conjugate comprises three different GalNAc moieties or derivatives thereof. In another aspect, the receptor-targeted conjugate is lipophilic. In some embodiments, the receptor-targeted conjugate comprises one or more GalNAc moieties and one or more lipid moieties, i.e., GalNAc-lipids. In some embodiments, the receptor-targeted conjugate is a GalNAc-lipid.

[0168] 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.

[0169] In preparing an LNP composition containing the excipients amino lipids, phospholipids, PEG lipids, and cholesterol, the four excipients in the desired molar ratio are dissolved in a water-miscible organic solvent, e.g., ethanol. The homogenous lipid solution is then rapidly mixed in-line with an aqueous buffer in the acidic pH range of 4-6.5 containing the nucleic acid payload to form lipid nanoparticles (LNPs) that encapsulate 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 for administration to human subjects.

[0170] For the preparation of GalNAc-LNP pharmaceutical compositions, GalNAc-lipids are mixed with the four lipid excipients in a water-miscible organic solvent prior to preparation of GalNAc-LNP. Preparation of GalNAc-LNP pharmaceutical compositions then follows the same steps as described for LNP pharmaceutical compositions. The mole % of GalNAc-lipids in GalNAc-LNP preparations ranges from 0.001 to 2.0 of the total excipients.

[0171] 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.

[0172] In some embodiments, the 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 No. 62 / 984,866, filed March 4, 2020, and U.S. Provisional Patent Application No. 63 / 078,982, filed September 16, 2020, naming Kallanthottathil G. Rajeev as the inventor and Verve Therapeutics, Inc. as the applicant (which application is incorporated by reference in its entirety into this specification).

[0173] Amino lipids Formula (I) In some embodiments, the LNP composition comprises an amino lipid. In one aspect, disclosed herein is an amino lipid having the structure of formula (I), or a pharma- ceutically 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 the 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 -, -NR4 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 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 4C(=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, where alkylene, heterocycloalkyl, and heterocycloaryl are independently substituted or unsubstituted; R 4 each is independently hydrogen, or substituted or unsubstituted C1-C6 alkyl; R 5 is hydrogen or substituted or unsubstituted C1-C6 alkyl; R 6 each independently is a substituted or unsubstituted C-C 22 Alkyl, or substituted or unsubstituted C3-C 22 alkenyl, R 7 and R 8 Each of is independently hydrogen, or a 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; p is an integer from 1 to 10; Each of n, m, and q is independently 0, 1, 2, 3, 4, or 5.

[0174] 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.

[0175] 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.

[0176] Formula (Ia) In some embodiments, the compound of Formula (1) has the structure of Formula (Ia), or a pharma- ceutically acceptable salt or a pharma- ceutically 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 the 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(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 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-heterocycloyl, where the alkylene, heterocycloalkyl, and heterocycloaryl are independently substituted or unsubstituted; R 4 each is independently hydrogen, or a substituted or unsubstituted C1-C6 alkyl; R 5 is hydrogen or substituted or unsubstituted C1-C6 alkyl; R 6 each independently is a substituted or unsubstituted C-C 22 Alkyl, or substituted or unsubstituted C3-C 22 alkenyl, R 7 and R 8 Each of R is independently hydrogen, or a substituted or unsubstituted C1-C6 alkyl, or 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.

[0177] 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.

[0178] Variations of Formulas (I) and (Ia) In some embodiments, R of Formula (I) and Formula (Ia) 1 and R 2 is independently 22 Alkyl, C3-C 22 Alkenyl, -C2-C 10 Alkylene-LR 6 ,or [ka] and each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) is 1 and R 2 is independently 10 -C 20 Alkyl, C 10 -C 20 Alkenyl, -C8-C7 alkylene-LR 6 ,or [ka] and each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) is 1 teeth, [ka] It is.

[0179] In some embodiments, each L in Formula (I) and Formula (Ia) is independently O, S, -C 10 Alkylene-O-, -C1-C 10 Alkylene-C(=O)O-, -C1-C 10In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, -C1-C3 alkylene-O-, -C1-C3 alkylene-C(=O)O-, -C1-C3 alkylene-OC(=O)-, or a bond, where the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, -C1-C3 alkylene-O-, -C1-C3 alkylene-C(=O)O-, -C1-C3 alkylene-OC(=O)-, or a bond, where the alkylene is a straight or branched unsubstituted alkylene.

[0180] In some embodiments, R of Formula (I) and Formula (Ia) 6 each independently represents 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 C3-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 Each of R is independently substituted or unsubstituted n-octyl. In some embodiments, R 6 Each of is n-octyl.

[0181] 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 of L in Formula (I) and Formula (Ia) is O. In some embodiments, each of 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.

[0182] In some embodiments, R of Formula (I) and Formula (Ia) 1 is: [ka]

[0183] In some embodiments, R of Formula (I) and Formula (Ia) 1 is R 2 It is.

[0184] In some embodiments, R of Formula (I) and Formula (Ia) 4 Each of is independently H, or a 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 4Each of is H. In some embodiments, R 4 Each of is independently H, -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R in Formula (I) and Formula (Ia) is 4 Each of is independently H or -CH3. In some embodiments, R 4 Each of is -CH3.

[0185] 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)-.

[0186] 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 the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R in Formula (I) and Formula (Ia) is 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 unsubstituted C 10 -C 20 In some embodiments, R in Formula (I) and Formula (Ia) is alkyl. 2 is 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 of formula (I) and formula (Ia) is 2 is -C2-C 10 Alkylene-C(=O)OR 6 or -C2-C 10 Alkylene-OC(=O)-R 6 It is.

[0187] In some embodiments, R of Formula (I) and Formula (Ia) 2 teeth, [ka] It is.

[0188] In some embodiments, R of Formula (I) and Formula (Ia) 1 is R 1 It is.

[0189] 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-.

[0190] 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 10Alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C1-C6 alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C1-C4 alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C1-alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C2-alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C3-alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C4-alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C5-alkylene-NR 7 R 8 In some embodiments, R of formula (I) and formula (Ia) is 3 is -C0-C 10 In some embodiments, R of Formula (I) and Formula (Ia) is an 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.

[0191] In some embodiments, R of Formula (I) and Formula (Ia) 7 and R 8Each of is independently hydrogen or substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 7 and R 8 Each of is independently hydrogen or substituted or unsubstituted C1-C3 alkyl. In some embodiments, R 7 and R 8 Each of is independently a substituted or unsubstituted C1-C3 alkyl. In some embodiments, R 7 and R 8 Each of is independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R and R 8 Each of is CH. In some embodiments, R 7 and R 8 Each of is -CH2CH3.

[0192] 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 together with the nitrogen to which they are attached form a substituted or unsubstituted 3- to 7-membered heterocycloalkyl.

[0193] In some embodiments, R of Formula (I) and Formula (Ia) 3 is: [ka]

[0194] In some embodiments, R of Formula (I) and Formula (Ia) 3 is: [ka] In some embodiments, R of Formula (I) and Formula (Ia) 3 is:

[0195] [ka]

[0196] In some embodiments, Z in formula (I) and formula (Ia) is -C(=O)O- or -OC(=O)-.

[0197] In some embodiments, Z in formula (I) and formula (Ia) is -C(=O)NR 4 -OR-NR 4 C(=O)-.

[0198] In some embodiments, Z in formula (I) and formula (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-.

[0199] 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.

[0200] 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-.

[0201] In some embodiments, Y of formula (I) and formula (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0202] In some embodiments, R of Formula (I) and Formula (Ia) 5 is hydrogen, or substituted or unsubstituted C1-C3 alkyl.

[0203] In some embodiments, R of Formula (I) and Formula (Ia) 5 is H, -CH3, -CH-)CH3, -CH2CH2CH3, or -CH(CH3)2.

[0204] In some embodiments, R of Formula (I) and Formula (Ia) 5 is H.

[0205] LNP Compositions Comprising Different Amino Lipids In some embodiments, the LNP comprises multiple amino lipids with different formulas. For example, the LNP composition may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino lipids. In another example, the LNP composition may comprise 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, the LNP composition may comprise 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.

[0206] 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.

[0207] 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: the second amino lipid: the third amino lipid is about 3:3:1. In some embodiments, the molar ratio of the first amino lipid: the second amino lipid: the third amino lipid is about 4:4:1.

[0208] 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 lipid 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 lipid present in the particle. In some embodiments, the first amino lipid comprises about 1 mol% to about 99 mol% of the total amino lipid 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. In some embodiments, the first amino lipid comprises from about 20 mol% to about 60 mol% of the total amino lipid present in the particle.

[0209] In some embodiments, the amino lipid is an ionizable lipid. The ionizable lipid may include 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 includes 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 includes a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In some embodiments, the amino lipid includes a tertiary amine.

[0210] In some embodiments, the amino lipid is a cationic lipid. In some embodiments, the amino 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)).

[0211] 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, as well as its diastereomeric, enantiomeric and epimeric forms.

[0212] In some embodiments, the amino lipids described herein have one or more stereocenters, each of which 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 diastereomeric compounds / salts, separating the diastereomers, and recovering the optically pure enantiomers. In some embodiments, the resolution of the enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, the diastereomers are separated using a separation / resolution technique based on differences in solubility. In other embodiments, separation of stereoisomers is accomplished 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.

[0213] 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 isotopically (e.g., with radioisotopes) or by other means, including but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0214] 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 an atom having an atomic mass or mass number different from the atomic mass or mass number 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, e.g., 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 provides certain therapeutic advantages due to increased metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0215] In some embodiments, the asymmetric carbon atom of amino lipid is present 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.

[0216] 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. Thus, N-hydroxy and N-alkoxy (e.g., N-OR (wherein 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.

[0217] 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.

[0218] PEGylated lipids In some embodiments, the LNP compositions described include one or more PEG lipids. As used herein, "PEG lipid" or "PEG-lipid" refers to a lipid that includes 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 include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, or combinations thereof.

[0219] In some embodiments, the PEG lipid comprises from about 0.1 mol % to about 10 mol % of the total lipid present in the particle.

[0220] Phospholipids In some embodiments, the LNP compositions described comprise one or more phospholipids.

[0221] In some embodiments, the phospholipid comprises about 5 mol % to about 15 mol % of the total lipid present in the particle.

[0222] cholesterol In some embodiments, the LNP composition comprises cholesterol or a derivative thereof.

[0223] GalNAc lipids In some embodiments, the LNP composition comprises a receptor-targeting conjugate comprising a compound of formula (V): [ka] (In the formula, the A groups collectively comprise a receptor-targeting ligand; Each L 1 , L 2 , L 3 , L 4 , L5 , L 6 , 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 C2-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)2NR 1 -, -NR 1 S(=O)2-, -NR 1 - or -N 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.

[0224] 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 C2-C6 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 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 - or -C(=O)NR 1 In some embodiments, each L 2 , L 5 , and L 8 are independently -C(=O)NR 1 -OR-NR 1 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 C2-C 10 In some embodiments, each L 6 and L9 is independently a substituted or unsubstituted C2-C6 alkylene. In some embodiments, each L 6 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.

[0225] 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.

[0226] 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.

[0227] Suitable phosphate charge neutralizing agents for use in the LNP formulations set forth below include, but are not limited to, for example, spermidine and 1,3-propanediamine.

[0228] Antioxidants In some embodiments, the LNPs described herein include 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 include a hydrophilic antioxidant. 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 include a lipophilic antioxidant. In some embodiments, the lipophilic antioxidant includes 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.

[0229] 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 processing of the LNP.

[0230] "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 helper lipids enhance transfection includes enhancing particle stability. In some embodiments, helper lipids enhance 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.

[0231] "Stealth lipid" may refer to a lipid that modifies the length of time that a nanoparticle may reside 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 as 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 that have 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, pg. 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.

[0232] 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. In some embodiments, the lipid moiety of the stealth lipid may be derived from a diacylglycerol or diacylglycamide, including those that comprise a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, the chain may comprise one or more functional groups, e.g., amide or ester. The dialkylglycerol or dialkylglycamide group may further comprise one or more substituted alkyl groups.

[0233] The structures and properties of helper lipids, neutral lipids, stealth lipids, and / or other lipids are further described in W02017173054A1, W02019067999A1, US20180290965A1, US20180147298A1, US20160375134A1, US8236770, US8021686, US8236770B2, US7371404B2, US7780983B2, US7858117B2, US20180200186A1, US20070087045A1, W02018119514A1, and W02019067992A1, all of which are incorporated by reference in their entireties.

[0234] LNP formulation Described herein are specific formulations of nanoparticle compositions that include one or more of the described lipids.

[0235] 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 altered to enhance selectivity for specific body targets. For example, particle size can be adjusted based on fenestration size of different organs. The therapeutic agent included in the nanoparticle composition can also be selected based on the desired delivery target(s). For example, the therapeutic agent can be selected for a particular indication, condition, disease, or disorder and / or for delivery (e.g., localized delivery, or specific delivery) to a specific cell, tissue, organ, or system or groups thereof. In certain embodiments, the nanoparticle composition can include an 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 particular organ or cell type, e.g., the liver or hepatocytes, to facilitate delivery of the drug substance to them.

[0236] 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 properties of the nanoparticle composition. For example, the amount of RNA included in the nanoparticle composition can depend on the size, sequence, and other properties of the RNA. The relative amounts of therapeutic 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-Vis spectroscopy).

[0237] 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 may be selected to provide a particular N / P ratio. The N / P ratio may be selected from about 1 to about 30. The N / P ratio may 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.

[0238] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms (e.g., in the physiological pH range) in the lipid(s) to phosphate groups in the nucleic acid molecular entity(s), 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 range 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 5-14.

[0239] In the case of a payload that does not contain a phosphate group, the N / P ratio can refer to the molar ratio of the 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.

[0240] In some embodiments, the 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, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 98%.

[0241] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising a composition 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 submicrometer in size and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, LNP refers 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 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 40-100 nm, 50-100 nm. 50-90 nm, 50-80 nm, 50-70 nm, 55-95 nm, 55-80 nm, 55-75 nm, 60-100 nm, 60-90 nm, 60-80 nm, 60-70 nm, 25-100 nm, 25-80 nm, or 40-80 nm.

[0242] In some embodiments, LNPs can be made from cationic, anionic, or neutral lipids. In some embodiments, LNPs may include neutral lipids such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol as helper lipids 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 combination of lipids known in the art can be used to generate LNPs. Examples of lipids used to generate LNPs include, but are not limited to, the following: 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)-2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), Examples of cationic lipids include N,N-dimethyl-2,3-bis(tetradecyloxy-1-propanaminium bromide), DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE (,2-bis(dimethylphosphino)ethane)-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMAC ... 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.

[0243] 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, arylaminocarbonyl, alkyl ... laminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, aiylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic groups, and aliphatic groups. It is understood that the substituents may be further substituted. Exemplary substituents include amino, alkylamino, and the like.

[0244] The term "substituent" as used herein means 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 results in a stable compound, and does not exceed the normal valence of the designated atom. Combinations of substituents and / or variables are permitted only if such combinations result in a stable compound. Those skilled in the art should note that any carbon and heteroatom that has a valence that appears to be unsatisfied as described or depicted herein is assumed to have a sufficient number of hydrogen atoms (or 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 a point of attachment to the core structure of formula (I). Those skilled in the art will understand that a double bond is intended for those substituents, even if only a single bond is depicted.

[0245] 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, an alkyl group may be represented as an alkyl group having, for example, up to 6 carbon atoms, which is a C1-C6 alkyl group. Alkyl groups having other numbers of carbon atoms (and other moieties defined herein) may be represented similarly. 10Examples of alkyl groups include, but are not limited to, alkyl, C1-C9 alkyl, C1-C8 alkyl, 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-ethyl-propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is -CH(CH3)2 or -C(CH3). Unless otherwise specifically stated herein, the alkyl group may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the remainder 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-.

[0246] 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, the 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.

[0247] 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 (where 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)-one, spiro[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).

[0248] 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, with each heterocyclic group having 3-12 atoms in the 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) include rings having 3-12 atoms in the ring system, and aromatic heterocyclic groups include rings having 5-12 atoms in the 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, furaz.anyl, benzofuraz.anyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The above 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) or 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 the bicyclic heterocycle is aromatic. In some embodiments, both rings of the bicyclic heterocycle are aromatic.

[0249] 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 may be a monocyclic or bicyclic system, which may include fused (when fused with 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 may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical may 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 all ring forms of carbohydrates, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Unless otherwise stated, 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 2-12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in 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) that make up the heterocycloalkyl (i.e., the skeletal atoms of the heterocycloalkyl ring). Unless otherwise specifically stated herein, a heterocycloalkyl group may be optionally substituted. As used herein, the term "tetracycloalkylene" may refer to a divalent heterocycloalkyl group.

[0250] 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. Illustrative 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 0-6 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.

[0251] 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. If the majority of the amine moiety(s) of the amino lipid (or amino lipids) in the nucleic acid-lipid nanoparticle formulation are protonated at physiological pH, the nanoparticles can be referred to as cationic lipid nanoparticles (cLNPs). If the majority of the amine moiety(s) of the amino lipid (or amino lipids) in the nucleic acid-lipid nanoparticle formulation are not protonated at physiological pH, but can be protonated at acidic pH, e.g., endosomal pH, they can be referred to as ionizable lipid nanoparticles (iLNPs). The amino lipids that make up a cLNP can generally be referred to as cationic amino lipids (cLipids). The amino lipids that make up an iLNP can be referred to as ionizable amino lipids (iLipids). The amino lipids can be iLipids or cLipids at physiological pH.

[0252] As used herein, an LNP composition or formulation is typically of a size on the order of micrometers or less and may contain a lipid bilayer. Nanoparticle compositions include 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 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.

[0253] As used herein, "phospholipid" may refer to a lipid that includes a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may include one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of an LNP to cross the membrane (i.e., delivery of one or more elements to a cell).

[0254] 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 system 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 a guide RNA. In some embodiments, the nucleic acid(s) are chemically modified.

[0255] 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), small 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 loops and single-stranded regions within the molecule.

[0256] kit It is contemplated herein that the therapeutic or pharmaceutical agent disclosed herein is part of the kit 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.

[0257] 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 carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, etc., each of the container(s) containing 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.

[0258] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0259] 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 an identifying instruction or label or instructions for its use in the methods described herein.

[0260] 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 will also typically be included.

[0261] In an embodiment, the label is on the container or 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; the label is associated with the container when it is in the container or carrier that holds the container, for example, as a package insert.In one embodiment, the label is used to indicate that the contents should be used for a specific therapeutic purpose.The label also indicates the instructions for using the contents in the methods described herein, etc.

[0262] 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, general characteristics of the subject (including the subject's health condition, sex, weight and / or age, and other diseases present), can affect the dosage and frequency of administration required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or, preferably, a series of treatments. It will also be understood that the effective dosage of the disclosed composition used for treatment can increase or decrease during the course of a particular treatment. Changes in dosage may result or become evident from the results of the diagnostic assays described herein. Therapeutically effective dosages generally depend on the condition of the patient at the time of administration. The exact amount can be determined by routine experimentation, but may ultimately depend on the judgment of the clinician, for example, monitoring the patient for signs of disease and adjusting treatment accordingly.

[0263] The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the disease. Alternatively, a sustained continuous release formulation of the polypeptide or polynucleotide may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art. In some embodiments, the dosage is every day, every 2nd day, every 3rd day, every 4th day, every 5th day, or every 6th day. In some embodiments, the frequency of administration is once a week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks, or once a month, once every 2 months, or once every 3 months, or more. The progress of this treatment is easily monitored by conventional techniques and assays.

[0264] Dosage regimens (including compositions disclosed herein) may vary over time. In some embodiments, it is contemplated that a dose ranging from about 0.01 to 1000 mg / kg may be administered for a normal weight adult subject. In some embodiments, the dose is 1 to 200 mg. In some embodiments, the dose may be 0.03 mg / kg to 3 mg / kg or any amount therebetween. The particular dosing regimen, i.e., dose, 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).

[0265] The appropriate therapeutic dosage 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 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.

[0266] Administration of one or more compositions may 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 the practitioner. Administration of the compositions may be essentially continuous over a preselected period of time, or may be in a series of spaced doses, for example, either before, during, or after the onset of disease.

[0267] The disclosed methods and compositions (including embodiments thereof) described herein can be administered with one or more additional therapeutic regimens or agents or treatments that can be administered simultaneously to a mammal. "Co-administered" means administering one or more additional therapeutic regimens or agents or treatments and the disclosed compositions close enough in time to enhance the effect of the one or more additional therapeutic agents (or vice versa). In this regard, the disclosed compositions described herein can be administered simultaneously with one or more additional therapeutic regimens or 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 an embodiment, a second therapeutic regimen or agent or treatment is administered simultaneously with, before, or after the disclosed compositions.

[0268] In an embodiment, a polynucleotide encoding a base editor fusion protein and a guide RNA are administered to a subject. In an embodiment, the polynucleotide encoding a base editor fusion protein is an mRNA. In an embodiment, the dose of the combined polynucleotide encoding a base editor fusion protein and guide RNA is 0.01 mg / kg to 10 mg / kg, such as 0.5 mg / kg to 5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, etc. In an embodiment, a LNP comprising such an amount of a polynucleotide encoding a base editor fusion protein and a guide RNA is administered to a subject. In an embodiment, the subject is a primate. In an embodiment, the subject is a non-human primate. In an embodiment, the non-human primate is a cynomolgus monkey.

[0269] 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 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 liver cells as measured by next generation sequencing. In embodiments, such percentages of base changes are achieved when a subject is administered a combined dose of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, or 3 mg / kg 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.

[0270] The present invention is illustrated in the following examples. It should 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 described herein. EXAMPLES

[0271] Example 1 A guide to adenine base editing in the TTR gene In this example, gRNA sequences were identified that would cause ABE8.8 (and other ABE variants containing Streptococcus pyogenes Cas9, such as ABE7.10, or another Cas protein that may use the NGG PAM) to 1) destroy the start codon or 2) destroy a splice site (donor or acceptor) via A → G editing within an editing window (approximately positions 4-7 of the 20 nt protospacer region of DNA). Five sequences were identified across the human TTR gene (Table 1). gRNAs were synthesized that matched each of the protospacer sequences and separately fit the 100 nt standard Streptococcus pyogenes CRISPR gRNA sequence, with each gRNA molecule having minimal chemical modifications (specified in Table 1). Each of the gRNAs was co-transfected with an equal amount of in vitro transcribed ABE8.8 mRNA (molecular weight ratio 1:1) into primary human hepatocytes via MessengerMax reagent (Lipofectamine) using various dilutions (2500, 1250, 625 ng / RNA / mL) to evaluate the editing activity at various concentrations of the test substance. [Table 1]

[0272] Each gRNA was also transfected into primary cynomolgus 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, with the orthogonal protospacer sequence of the corresponding cynomolgus TTR gene sequence. The ABE8.8 (MA004) mRNA and corresponding amino acid sequences used are shown in Table 11 below. Three days after transfection, genomic DNA was harvested 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 equivalent), GA460, and GA461 showed high editing activity in both human and cynomolgus primary hepatocytes. See Figures 5A-5C, Figure 6, and Tables 2-3. [Table 2] [Table 3]

[0273] 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 according to the invention may result in an 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 set forth in Table 2 or Table 3. In some embodiments, the compositions provide an 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 set forth in Table 2 or Table 3.

[0274] Example 2 Off-target analysis Off-target mutation analysis in primary human hepatocytes is evaluated with the goal of establishing the safety of base editing therapy to knockdown TTR in human liver in vivo. Off-target editing in human hepatocytes was evaluated 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 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 the 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 are shown in Tables 8-10 with potential off-target sites noted.

[0275] The ONE-seq methodology is as follows: ONE-seq library design begins with 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 genomes (GRCh38, Ensemblv98, chromosomes 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) were 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 / ).

[0276] Sites with up to six mismatches and no bulges are referred to using the X<number of mismatches><number of bulges> code. Thus, an on-target site is labelled X00; a site with one mismatch to the on-target and no bulge is labelled X10, etc. 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 labelled DNA42. The same nomenclature is used for RNA bulges, but these are coded as RNA<number of mismatches><number of bulges>.

[0277] The identified protospacer sequences were extended by 10 nucleotides (nt) on either side of the 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 either side of the central protospacer sequence); and two different 11 nt unique molecular identifiers (UMIs) (one on either 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 allow for unambiguous identification of each site during analysis. The barcodes were selected from an initial list of 668,420 barcodes that did not contain CC or GG in the sequence, and each barcode has a Hamming distance of 2 from any other barcode. A custom Python script was used to design the final library.

[0278] The final oligonucleotide library is synthesized by a commercial vendor (Agilent Technologies). Each library is PCR amplified and subjected to 1.25x AMPure XP bead purification (Beckman Coulter). After 10 min incubation at 25°C in CutSmart buffer (New England Biolabs), RNPs containing 769 nM recombinant ABE8.8-m protein and 1.54 μM gRNA are mixed with 100 ng of purified library and incubated at 37°C for 8 h. The RNP dose is derived from an analysis demonstrating that it is a supersaturating dose, i.e., a dose that exceeds the dose that achieves the maximum amount of on-target editing in a biochemical assay.

[0279] Proteinase K (New England Biolabs) was added to stop the reaction at 37°C for 45 min, followed by 2x AMPure XP bead purification. The reaction was then sequentially incubated with EndoV (New England Biolabs) at 37°C for 30 min, Klenow Fragment (New England Biolabs) at 37°C for 30 min, NEBNext Ultra II End Prep Enzyme Mix (New England Biolabs) at 20°C for 30 min, and then at 65°C for 30 min, followed by 2x AMPure XP bead purification after each incubation. The reaction was ligated with annealed adaptor oligonucleotide duplexes at 20°C for 1 h 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.

[0280] 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.

[0281] Paired-end reads were trimmed of sequencing adapters 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 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 as follows: "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 unique to each site and filtered in an edit window for those with evidence of A → G substitutions (defined as positions 1-10 of the most PAM-distal position of the protospacer). Duplicate reads were discarded.

[0282] 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, when there is 100% on-target editing, less than 1 / 1000th of editing activity is converted to less than 0.1% off-target editing (below the lower limit of editing detection by NGS). Oligonucleotides with a higher number of sequences reflect a higher propensity for Cas9 / gRNA cleavage in vitro and therefore a higher probability of off-target mutagenesis in cells.

[0283] For off-target editing in human primary hepatocytes, several off-target site candidates were analyzed. Table 4 shows the results of validating 47 off-target site candidates of guide RNA GA457 from cells co-transfected with gRNA and an equal amount of in vitro transcribed ABE8.8 mRNA (molecular weight ratio 1:1) into primary human hepatocytes using MessengerMax reagent (Lipofectamine). The on-target site shows high editing efficiency, but all off-target sites show little to no editing (net editing less than 0.4%). [Table 4-1] [Table 4-2]

[0284] GA459, GA460, and GA461 were similarly evaluated for off-target editing, as shown in Tables 5, 6, and 7, respectively. While the on-target sites of each guide show higher editing efficiency in the treated groups compared to the control groups, little to no off-target editing is observed at potential off-target sites. [Table 5] [Table 6] [Table 7]

[0285] Table 8 provides some results of off-target editing using the GA457 guide.

[0286] Table 9 provides some results of off-target editing using the GA460 guide.

[0287] Table 10 provides some results of off-target editing using the GA461 guide.

[0288] 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. A composition for editing a TTR gene according to the present invention may produce a 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 a 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 one or more off-target sites described in Tables 4, 6, 7, 8, 9, or 10, 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. 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]

[0289] Additional examples of GA457 off-target sites are provided in U.S. Provisional Patent Application No. 63 / 322,182, filed March 21, 2022. The off-target site of GA457 may include any one of SEQ ID NOs: 92-1073. [Table 9]

[0290] 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]

[0291] 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] [Table 11-7] [Table 11-8] [Table 11-9]

[0292] 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 designated Verve Therapeutics, Inc. as the applicant.

[0293] 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 human GA457 and GA460 sgRNAs described above were prepared, co-formulated with the ABE8.8 mRNA described above, encapsulated in lipid nanoparticles (LNPs), and administered intravenously to NHPs. This study included two distinct aspects.

[0294] The first aspect of the NHP in vivo study involved the evaluation of LNP1 and LNP2, which differed only in that LNP2 was formulated to encapsulate GA520 and ABE8.8 mRNA, while LNP1 was formulated to encapsulate GA519 and ABE8.8 mRNA. 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 mRNA. However, LNP3 differed from LNP1 in that LNP3 contained a GalNAc moiety. In each aspect of the study, base editing efficiency, TTR protein expression, safety profile, and pharmacokinetics were evaluated multiple times after injection of NHPs, as described in more detail below and shown in the accompanying figures.

[0295] 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, LNP1 encapsulating GA519 and ABE8.8 mRNA, and LNP2 encapsulating GA520 and ABE8.8 mRNA. The components of each LNP are composed 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]

[0296] It should be understood that the lipids in Table 12 may be substituted with other suitable lipids of the classes described. 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 may be VL422, or a pharma- ceutically acceptable salt or solvate thereof: [ka]

[0297] It should be understood that the mole percent of lipids in Table 12 may be adjusted, and that the mole percents included in Table 12 are intended to represent the total mole percent of all LNPs formulated in a given batch, and that a particular LNP within a batch may have a different mole percent. Thus, it is contemplated herein that the mole percent of one or more or all of the LNP components listed in Table 12 may be adjusted, for example, by ±1-5%, ±5-10%, or ±10%-20%. It is further contemplated herein that for a particular LNP formulated in a given batch of LNPs formulated according to a desired target excipient percentage, the mole percent of one or more or all of the LNP components listed in Table 12 may vary from the target mole percent, for example, by ±1-5%, ±5-10%, or ±10%-20%, or even greater than ±20%. It should further be understood that additional LNP components, including non-lipid components, may be added to the LNP components described in Table 12. As shown in Table 13, LNP1 is formulated with sgRNA GA519 and LNP2 is formulated with GA520, which correspond to sgRNAs GA457 and GA460, respectively, described above. GA519 and GA520 were chemically synthesized, and the sequences and chemical modifications of GA519 and GA520 are shown in Table 13. [Table 13]

[0298] Notably, compared to GA457, GA519 hybridizes at positions 50,681,581-50,681,603 in exon 1 of the reference cynomolgus genome (macFas5) and edits the adenosine at position 50,681,584, which results in disruption of the full-length TTR protein sequence by converting the methionine amino acid to a threonine amino acid, inhibiting protein translation (Figure 8). GA519 is a cynomolgus surrogate for the human GA457 gRNA and, as described above, maps to the analogous region of the human TTR locus as in Figure 4. The cynomolgus GA519 gRNA differs from GA457 by a single nucleotide at position 17 of the protospacer, highlighted by underline in the protospacer column of Table 13. Additionally, GA519 and GA457 differ from each other in that the tracr region of GA519 incorporates a chemical modification (detailed in Table 13). The chemical modifications may be designed or capable of improving in vivo stability.

[0299] Similarly, compared to GA460, GA520 hybridizes at positions 50,678,305-50,678,327 in exon 3 of the reference cynomolgus genome (macFas5) and edits the adenosine at position 50,678,324, resulting in a splicing acceptor disruption that generates a truncated non-functional TTR protein (Figure 9). The protospacer region of GA520 is identical to human GA460 and maps to the analogous region of the human TTR locus as in Figure 4, as described above. GA520 and GA460 differ in the tracr region and incorporate chemical modifications designed for or capable of improving in vivo stability, as detailed in the table above.

[0300] 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.

[0301] ABE8.8 mRNA and GA519 and GA520 were used in a 1:1 weight ratio of sgRNA:mRNA to formulate LNP1 and LNP2, respectively. In other words, LNPs were formulated with an equal weight of guide RNA to mRNA. The resulting LNPs encapsulating 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] One of skill 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%.

[0302] NHP research design In this aspect of the study, female cynomolgus monkeys of Cambodian origin were used as study animals. A premedication regimen including 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 dose of test article. Three monkeys were administered LNP1 and three monkeys were administered LNP2 on day 1 of the study by 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).

[0303] 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 evaluate biomarkers, cytokines, plasma iLipid and PEG lipid pharmacokinetics, and serum safety parameters.

[0304] Necropsies were performed on all animals on day 16. Liver biopsies were collected to assess TTR gene editing.

[0305] 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 of the animals using methods 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 percent of all reads containing the non-reference allele at the targeted adenine.

[0306] Figure 10 shows the TTR editing efficiency of LNP1 compared to LNP2. Notably, as shown in Figure 10, the average liver TTR editing efficiency is higher in NHPs treated with LNP1 (52%) compared to LNP2 (29%).

[0307] Quantification of TTR protein expression in serum Serum was collected from all animals on days -10, -7, -5 prior to injection and on days 7 and 14 after LNP injection for TTR protein analysis. Two methods were used to quantify serum TTR. TTR protein levels were first quantified using a custom TTR sandwich ELISA with the data obtained from that analysis presented in Figure 11. The values ​​on days -10, -7, and -5 were averaged to obtain baseline values. Notably, animals treated with LNP1 showed greater hepatic TTR editing and also greater plasma TTR reduction (-63% change from baseline on day 14) when compared to animals treated with LNP2 (3% change from baseline on 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, notably 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).

[0308] 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.

[0309] Safety analysis For safety analysis, serum was collected from all animals on days -10, -7, and -5 before infusion, and 6, 24, 48, 96, 168, 240, and 336 hours after LNP infusion, specifically to observe changes in liver enzymes and cytokine levels. Serum chemistry parameters were measured directly from serum samples on a Beckman Coulter AU680 analyzer. Values ​​on days -10, -7, and -5 were averaged to obtain baseline values. Both LNP1 and LNP2-treated animals showed a transient elevation of 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 concentrations, shown in FIG. 14A, and glutamate dehydrogenase concentrations, shown in FIG. 14B, were also found to be briefly elevated after administration of either LNP1 or LNP2, returning to baseline levels 96-168 hours after the end of the infusion. Infusion of either LNP1 or LNP2 did not alter serum concentrations of gamma-glutamyltransferase, shown in FIG. 15A, or alkaline phosphatase, shown in FIG. 15B. Furthermore, as shown in FIG. 16, LNP1 and LNP2 treatment did not affect serum total bilirubin concentrations. As shown in FIG. 17, animals administered LNP1 and LNP2, respectively, also showed 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.

[0310] For serum cytokine analysis, serum was collected from all animals on days -10, -7, -5 before treatment, and 24, 168, and 336 hours after LNP infusion. Cytokines were measured using a multiplex sandwich immunoassay. Four cytokines (MCP-1, IL-6, IP-10, IL-1RA) were simultaneously quantified from serum samples using the U-PLEX Biomarker Group 1 (Monkey) assay from Meso Scale Diagnostics (Rockville, MD). The values ​​on days -10, -7, and -5 were averaged to obtain baseline values. As shown in Figure 18, both animals administered LNP1 and LNP2 showed an increase in serum IL-6 concentrations, which peaked at 6 hours after the end of infusion, to a similar extent, and returned to baseline by 24 hours. As further shown in Figure 18, both animals administered LNP1 and LNP2 showed an increase 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.

[0311] Overall, analysis of the above parameters showed that infusion of either LNP1 or LNP2 into monkeys produced a transient increase in liver enzymes and cytokines that quickly disappeared.

[0312] Pharmacokinetic (PK) evaluation Blood samples were collected (K2EDTA) for plasma PK analysis and for determination of the concentrations of iLipid and PEG lipid excipients that make up 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. A qualified LC-MS assay was used to measure the concentrations of iLipid and PEG lipids. This is shown in Figure 19A. The time points at which lipids fell below the limit of quantification are not included in the figure. As shown in Figure 19A, the serum iLipid concentrations of animals administered LNP1 and LNP2 decreased continuously until approaching the lower limit of quantification (LLOQ) 96 hours after LNP infusion. Similarly, as shown in Figure 19B, the serum PEG lipid concentrations of animals administered LNP1 and LNP2 also decreased rapidly, reaching the LLOQ 24 hours after the end of the infusion.

[0313] Part B: In vivo NHP evaluation of GA519 with 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.

[0314] Preparation of LNPs The GalNAc LNP formulated for this aspect of the study (LNP3) 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]

[0315] It should be understood that the lipids in Table 15 may be substituted with other suitable lipids of the classes described. For example, the amino lipid may be the following amino lipids or salts thereof: [ka]

[0316] 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 the LNPs and represent the total mole percents of all LNPs formulated in a given batch, and that particular LNPs within a batch may have different mole percents. Thus, 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, for example, by ±1-5%, ±5-10%, or ±10%-20%. It is further contemplated herein that for a particular LNP formulated in a given batch of LNPs formulated according to a desired target excipient percentage, the mole percents of one or more or all of the LNP components listed in Table 13 may vary from the target mole percents, for example, by ±1-5%, ±5-10%, or ±10%-20%, or even greater than ±20%. Moreover, it should be understood that additional LNP components, including non-lipid components, can be added to the LNP components set forth in Table 13.

[0317] When formulating LNP3, GalNAc lipids were premixed with other LNP excipients referenced in Table 15 prior to in-line mixing with GA519 sgRNA and ABE8.8 mRNA (weight ratio 1:1) to form LNP3. Rajeev et al., WO2021178725 contains a description of the synthesis and characterization of GalNAc lipids. 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]

[0318] One of skill 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%.

[0319] NHP research design In this aspect of the study, male cynomolgus monkeys of Cambodian origin were used. A premedication regimen including 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 dose of test article. The LNP3 dose formulation was administered once on day 1 of the study by IV infusion to two groups of three monkeys at the following dose levels: (i) for the first group of three monkeys, 2 mg / kg (animal body weight) of sgRNA and mRNA combination and 6 mL / kg dose volume (n=3 / group), and (ii) for the second group of three monkeys, 3 mg / kg (animal body weight) of sgRNA and mRNA combination and 6 mL / kg dose volume (n=3 / group).

[0320] Blood samples were collected from all animals before dosing for baseline measurements and at various time points from days 1 to 35 after injection to evaluate 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.

[0321] Editing Efficiency Analysis The amount of gene editing in 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 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 percent of all reads containing the non-reference allele at the targeted adenine.

[0322] As shown in FIG. 20, LNP3 resulted in similar levels of hepatic TTR editing efficiency in monkeys dosed at 2 mg / kg (60%) compared to monkeys dosed at 3 mg / kg (63%).

[0323] Quantification of TTR protein expression in serum For TTR protein analysis, serum was collected on days -10, -7, -5 prior to 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 data 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 a significant reduction in serum TTR protein at the first time point after dosing (day 7). These reductions were maintained over the course of the study, reaching a maximum reduction 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, quantifying four unique TTR peptide fragments in serum at each time point, with the average of the four results being reported. LC-MS serum TTR quantification, shown in Figure 22, confirmed that TTR was reduced at the earliest time point post-injection 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).

[0324] Thus, as described above and shown in the reference figures above, NHPs administered both 2 mg / kg and 3 mg / kg LNP3 resulted in significant and relatively rapid hepatic TTR gene editing and a corresponding reduction in serum TTR protein concentrations.

[0325] Safety analysis For safety analysis, serum was collected from each of the test animals on days -10, -7, and -5 prior to infusion, and on days 6, 24, 48, 96, 168, 336 hours, 21 days, 28 days, and 35 days after the end of infusion, specifically to observe changes in liver enzyme and cytokine levels. Serum chemistry parameters were measured directly from serum samples on a Beckman Coulter AU680 analyzer. Values ​​on days -10, -7, and -5 were averaged to obtain baseline values. Animals administered LNP3 showed a dose-dependent, transient elevation of alanine aminotransferase, as shown in Figure 23A, which peaked 24-48 hours after the end of infusion and returned to baseline levels 336 hours after the end of infusion. As shown in Figure 23B, aspartate aminotransferase levels were elevated to a similar extent at both 2 mg / kg and 3 mg / kg LNP3 doses, peaking 6 hours after the end of infusion and returning to baseline levels 168 hours after the end of infusion. As shown in FIG. 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 FIG. 24B, LNP3 also dose-dependently increased glutamate dehydrogenase concentrations, which peaked at 24 hours and returned to baseline levels by 336 hours after the end of the infusion. Serum concentrations of gamma-glutamyltransferase and alkaline phosphatase, shown in FIG. 258A and 25B, respectively, did not change significantly at either LNP dose. Furthermore, as shown in FIG. 26, LNP3 treatment did not significantly affect serum total bilirubin concentrations. As shown in FIG. 27, LNP3 increased serum creatine kinase concentrations, which peaked 6 hours after the end of the infusion and then returned to baseline levels by 168 hours after the end of the infusion.

[0326] Analysis of the above-mentioned 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 2 weeks following administration to subjects.

[0327] Pharmacokinetic (PK) evaluation Blood samples were collected from all animals for plasma PK analysis and determination of the concentrations of ionizable amino lipids (iLipid) and PEG lipids that make up 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 plasma exposure of PEG lipid was also observed, reaching the LLOQ by 24 hours after the end of the infusion.

[0328] 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.

[0329] 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 prevail. The above detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art will fall within the scope of the present invention as defined by the claims.

[0330] Other embodiments From the above 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.

[0331] 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.

[0332] As described herein, it will be understood that the disclosure includes specific examples and embodiments describing, individually and in combination, specific embodiments and examples of base editing systems for effecting nucleobase changes in genes, as well as 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, as well as for in vivo and in vitro delivery of active agents to mammalian cells, under the conditions described.

[0333] Although specific examples and multiple embodiments are provided to illustrate aspects and combinations of aspects of the above, it should be recognized and understood that any aspect or combination of the exemplary or disclosed embodiments may be removed therefrom to form another embodiment without limitation, and that any such embodiment may 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 may 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 may be presented as separate independent claims without limitation. Thus, it should be recognized that any feature presented in a claim may be included in another claim; any feature presented in a claim may be deleted from a claim that constitutes a claim without that feature; and any feature presented in a claim may be combined with any feature of another claim, each of which is contemplated herein. The following enumerated clauses are further illustrative examples of combinations of aspects and aspects of the above-mentioned embodiments and examples.

[0334] The following are examples of enumerated clauses: 1. An isolated polynucleotide or a nucleic acid encoding same, said polynucleotide comprising a 5'-spacer sequence comprising about 17 to about 23 nucleotides that is homologous to a target protospacer sequence in a gene encoding transthyretin (TTR) adjacent to a NGG protospacer adjacent motif (PAM) sequence in a genome, said isolated polynucleotide acting as a guide polynucleotide that directs a base editor system to make a nucleobase change in said TTR gene. 2. The isolated polynucleotide of clause 1 or a nucleic acid encoding same, further comprising a tracrRNA domain 3' of the 5' spacer, wherein the tracrRNA is configured to bind to a base editor protein. 3. The isolated polynucleotide or nucleic acid encoding same of clause 1 or 2, wherein said protospacer sequence comprises the start codon or splice site of said TTR gene. 4. The isolated polynucleotide or nucleic acid encoding same of any one of the preceding clauses, wherein the nucleobase change introduced into the TTR gene comprises a disruption of the start codon or a disruption of an intron exon splice site. 5. The isolated polynucleotide or nucleic acid encoding same of any one of the preceding clauses, wherein the nucleobase changes effected in the TTR gene comprise a disruption of an intron exon splice site. 6. The isolated polynucleotide or nucleic acid encoding same of any one of the preceding clauses, wherein the polynucleotide encoded by the isolated polynucleotide or nucleic acid encoding same comprises a spacer sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to: 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA519), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA458), 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459), 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460 / GA520), or 5'-UACUCACCUCUGCAUGCUCA-3' (sequence number 5) (GA461). 7. The isolated polynucleotide or nucleic acid encoding same according to clause 6, wherein the polynucleotide encoded by said isolated polynucleotide or said nucleic acid encoding same comprises a spacer sequence having one of the following sequences: 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA519), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA458), 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459), 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460 / GA520), or 5'-UACUCACCUCUGCAUGCUCA-3' (sequence number 5) (GA461). 8. The isolated polynucleotide or nucleic acid encoding same according to any one of the preceding clauses, wherein the polynucleotide encoded by the isolated polynucleotide or nucleic acid encoding same comprises a guide RNA. 9. A composition comprising an isolated polynucleotide according to any one of the preceding clauses or a nucleic acid encoding same. 10. A composition comprising the isolated polynucleotide of any one of the preceding clauses or a nucleic acid encoding same, and a nucleic acid encoding a base editor fusion protein. 11. The composition of clause 10, wherein the base editor fusion protein comprises a programmable DNA binding domain and a deaminase. 12. The composition described in clause 11, wherein the deaminase comprises cytosine deaminase or adenine deaminase. 13. The composition of any one of clauses 10 to 12, wherein the programmable DNA-binding domain comprises a catalytically impaired Cas9 protein. 14. The composition of clause 13, wherein the catalytically impaired Cas9 protein comprises a catalytically impaired Streptococcus pyogenes Cas9 protein. 15. The composition of any one of clauses 11 to 14, wherein the deaminase comprises ABE8.8. 16. The composition of any one of clauses 11-14, wherein the deaminase is encoded by an mRNA comprising the MA004 mRNA sequence of Table 11. 17. The composition of any one of clauses 11 to 14, wherein the deaminase is encoded by an mRNA comprising a sequence having 95% or more sequence identity to the MA004 mRNA shown in Table 11. 18. The composition of any one of clauses 11 to 14, wherein the deaminase is encoded by an mRNA comprising a sequence having 96% or more sequence identity to MA004 mRNA shown in Table 11. 19. The composition of any one of clauses 11 to 14, wherein the deaminase is encoded by an mRNA comprising a sequence having 97% or more sequence identity to the MA004 mRNA shown in Table 11. 20. The composition of any one of clauses 11 to 14, wherein the deaminase is encoded by an mRNA comprising a sequence having 98% or more sequence identity to MA004 mRNA shown in Table 11. 21. The composition of any one of clauses 11 to 14, wherein the deaminase is encoded by an mRNA comprising a sequence having 99% or greater sequence identity to MA004 mRNA shown in Table 11. 22. A pharmaceutical composition comprising the isolated polynucleotide according to any one of clauses 1 to 8 or a nucleic acid encoding same, or the composition according to any one of clauses 9 to 21. 23. A lipid nanoparticle (LNP) comprising an isolated polynucleotide according to any one of claims 1 to 8 or a nucleic acid encoding same, a composition according to any one of clauses 9 to 21, or a pharmaceutical composition according to clause 22. 24. The LNP of clause 23, comprising an amino lipid having the following structure, or a pharma- ceutically acceptable salt or solvate thereof: [ka] 25. The LNP of clause 23, comprising an amino lipid having the following structure, or a pharma- ceutically acceptable salt or a pharma- ceutically acceptable solvate thereof: [ka] 26. The LNP of clause 24 or 25, further comprising a neutral helper lipid, a PEG lipid, and a sterol lipid. 27. The LNP according to clause 24 or 25, comprising a component as set out in Table 12. 28. The LNP of clause 27, wherein the components set forth in Table 12 are present in the LNP at a mole percent (mol %) within 10% to 20% of the mole percent set forth in Table 12. 29. The LNP of clause 27, wherein the component set forth in Table 12 is present in the LNP at a mole percent (mol %) within 5% to 10% of the mole percent set forth in Table 12. 30. The LNP of clause 27, wherein the components set forth in Table 12 are present in the LNP at a mole percent (mol %) within 1% to 5% of the mole percent set forth in Table 12. 31. The LNP of clause 27, wherein the components set forth in Table 12 are present in the LNP in the mole percent (mol %) set forth in Table 12. 32. The LNP of any one of clauses 27-31, wherein the LNP comprises a receptor-targeting conjugate comprising a compound of formula (V): [ka] (In the formula, Multiple A's are N-acetylgalactosamine (GalNAc) or [ka] or [ka] or a derivative thereof, Each L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , 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 C2-C 12 Alkenylene, substituted or unsubstituted C2-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)2NR 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, m is an integer selected from 1 to 10; and n is an integer selected from 1 to 200. 33. A pharmaceutical composition comprising an LNP according to any one of clauses 23 to 32. 34. A method for generating one or more nucleobase changes in a TTR gene in a cell, the method comprising contacting the cell with a polynucleotide or nucleic acid according to any one of clauses 1-8, a composition according to any one of clauses 9-21, a pharmaceutical composition according to claim 22 or 33, or an LNP according to any one of clauses 23-32. 35. The method of clause 34, wherein one or more alleles of the TTR gene are silenced. 36. A method for producing one or more nucleobase changes in the transthyretin (TTR) gene in a subject, the method comprising administering to the subject a polynucleotide or nucleic acid described in any one of clauses 1 to 8, a composition described in any one of clauses 9 to 21, a pharmaceutical composition described in clause 22 or 33, or a lipid nanoparticle described in any one of clauses 23 to 32. 37. The method of claim 36, wherein the base change occurs in 25% or more of the total liver cells in the subject, as measured by next-generation sequencing. 38. The method of claim 36, wherein the base change occurs in 40% or more of the total liver cells in the subject, as measured by next-generation sequencing. 39. The method of claim 36, wherein the base change occurs in 50% or more of the total liver cells in the subject, as measured by next-generation sequencing. 40. The method of any one of clauses 36 to 39, wherein the base change results in a decrease in serum TTR levels. 41. The method of any one of clauses 36 to 40, wherein one or more alleles of the TTR gene are silenced. 42. The method of any one of clauses 36 to 41, wherein the subject is a non-human primate. 43. The method of any one of clauses 36 to 41, wherein the subject is a human. 44. The method of clause 43, wherein the subject to whom the polynucleotide or nucleic acid, composition, pharmaceutical composition, or LNP is administered is a subject in need thereof. 45. The method of clause 44, wherein administering the polynucleotide or nucleic acid, composition, pharmaceutical composition, or LNP comprises administering a therapeutically effective amount of the polynucleotide, composition, pharmaceutical composition, or LNP. 46. ​​The method of clause 45, wherein the subject is affected by or at risk for hereditary transthyretin amyloidosis (hATTR) due to one or more mutations in the TTR gene. 47. The method of clause 46, wherein the subject is suffering from or at risk for cardiomyopathy (hATTR-CM) and / or polyneuropathy (hATTR-PN). 48. The method of clause 45, wherein the subject is suffering from or at risk for senile cardiac amyloidosis characterized by a wild-type allele (ATTRwt) of the TTR gene. 49. The method of any one of clauses 36 to 48, wherein the polynucleotide or nucleic acid, the composition, the pharmaceutical composition, or the LNP is administered intravenously. 50. A composition for editing the TTR gene, comprising: (a) an mRNA encoding a base editor protein having an editing window; and (b) a guide RNA comprising a tracr sequence that functions as a binding scaffold for the base editor protein and a spacer sequence that functions to guide the base editor protein to a protospacer sequence on the TTR gene; The composition, wherein the spacer sequence is at least partially complementary to a splice site or start codon of the TTR gene. 51. The composition of clause 50, wherein the base editor protein comprises cytidine deaminase or adenosine deaminase. 52. The composition of clause 50, wherein the base editor protein comprises a fusion protein comprising a nickase and a cytidine deaminase or an adenosine deaminase. 53. The composition of clause 50, wherein the base editor protein comprises a fusion protein comprising D10A nickase Cas9 and a cytidine deaminase or an adenosine deaminase. 54. The composition of any one of clauses 51 to 53, wherein the cytidine deaminase is deoxycytidine deaminase. 55. The composition of any one of clauses 51 to 53, wherein the adenosine deaminase is deoxyadenosine deaminase. 56. The composition of clause 50, wherein the base editor protein comprises a fusion protein comprising the adenine base editor ABE8.8. 57. The composition of any one of clauses 60 to 56, wherein the spacer sequence is homologous to a protospacer sequence selected from Table 1. 58. The composition according to any one of clauses 50 to 56, wherein the spacer sequence is selected from the following table: [Table 17] (wherein A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyl adenosine; c is 2'-O-methyl cytidine; g is 2'-O-methyl guanosine, u is 2'-O-methyl uridine; and s is a phosphorothioate (PS) backbone linkage). 59. The composition according to any one of clauses 50 to 56, wherein the spacer sequence has more than 80% sequence identity with a spacer sequence provided in the following table: [Table 18] (wherein A is a modified or unmodified adenosine; C is a modified or unmodified cytidine; G is a modified or unmodified guanosine; and U is a modified or unmodified uridine). 60. The composition according to any one of clauses 50 to 56, wherein the guide RNA is selected from the following table: [Table 19] (wherein A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyl adenosine; c is 2'-O-methyl cytidine; g is 2'-O-methyl guanosine, u is 2'-O-methyl uridine; s is a phosphorothioate (PS) backbone linkage; the bold characters represent spacer sequences). 61. The composition according to any one of clauses 50 to 60, wherein the spacer sequence has more than 80% sequence identity with a guide RNA sequence selected from the following table: [Table 20] 62. The composition of any one of clauses 50 to 61, wherein the composition is capable of producing an editing activity within 50% of the editing activity listed in Table 2 excluding GA459, or is capable of producing an editing activity within 50% of the editing activity listed in Table 3. 63. The composition of any one of clauses 50-62, wherein the composition is capable of producing within 50% of the total off-target editing activity, or less than the observed off-target editing activity, or no off-target editing activity at one or more potential off-target sites listed in Tables 4, 6, 7, 8, 9, or 10. 64. The composition of any one of clauses 50 to 63, wherein the composition is encapsulated in a lipid nanoparticle. 65. The composition of any one of clauses 50 to 64, wherein the composition is administered to a subject in vivo.

[0335] It will be understood by review of 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 combined with one or more aspects or features of other clauses.

Claims

1. An isolated polynucleotide or nucleic acid encoding the same, wherein the polynucleotide comprises a 5'-spacer sequence comprising about 17 to about 23 nucleotides that are homologous to a target spacer sequence within a gene encoding transthyretin (TTR) adjacent to an NGG protospacer adjacent motif (PAM) sequence in the genome, and the isolated polynucleotide or nucleic acid encoding the same functions as a guide polynucleotide that induces a base editor system to cause a change in the nucleic acid bases in the TTR gene.

2. a) the spacer sequence comprises the start codon or splice site of the TTR gene; and / or b) the change in the nucleic acid bases brought about in the TTR gene comprises disruption of the start codon or disruption of an intron-exon splice site, The isolated polynucleotide or nucleic acid encoding the same according to Claim 1.

3. The isolated polynucleotide or nucleic acid encoding the same according to Claim 1, wherein the change in the nucleic acid bases brought about in the TTR gene comprises disruption of an intron-exon splice site.

4. The isolated polynucleotide, or the polynucleotide encoded by the nucleic acid encoding the same, comprises a spacer sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the following sequences: 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA519), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA458), 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459), 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460 / GA520), or 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO: 5) (GA461); In particular, the polynucleotide encoded by the isolated polynucleotide or the nucleic acid encoding the same has the following sequence: 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 1) (GA457), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA519), 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 2) (GA458), 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 3) (GA459), 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 4) (GA460 / GA520), or 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO: 5) (GA461) containing a spacer sequence having one of them, The isolated polynucleotide according to claim 1, or a nucleic acid encoding the same.

5. The isolated polynucleotide according to claim 1, or a nucleic acid encoding the same, wherein the polynucleotide encoded by the isolated polynucleotide or the nucleic acid encoding the same contains a guide RNA.

6. The composition comprising the isolated polynucleotide according to claim 1 or a nucleic acid encoding the same, optionally further comprising a nucleic acid encoding a base editor fusion protein, the base editor fusion protein comprising a programmable DNA binding domain and a cytosine deaminase or an adenine deaminase. Composition.

7. The composition according to claim 6, wherein the programmable DNA binding domain comprises a Cas9 protein with impaired catalytic activity, and optionally, the Cas9 protein with impaired catalytic activity comprises a Streptococcus pyogenes Cas9 protein with impaired catalytic activity.

8. The composition according to claim 6 or 7, wherein the base editor fusion protein comprises ABE8.

8.

9. A pharmaceutical composition comprising the isolated polynucleotide according to claim 1 or a nucleic acid encoding the same.

10. A lipid nanoparticle (LNP) comprising the isolated polynucleotide according to claim 1 or a nucleic acid encoding the same.

11. A pharmaceutical composition comprising the LNP according to claim 10.

12. An in vitro or ex vivo method of causing one or more nucleic acid base changes in the intracellular TTR gene, the method comprising contacting the cell with the polynucleotide or nucleic acid according to claim 1, the composition according to claim 6, the pharmaceutical composition according to claim 9, or the LNP according to claim 10, respectively.

13. The pharmaceutical composition according to claim 9 or 11 for use in a method of causing one or more nucleobase changes in the transthyretin (TTR) gene in a subject, said method comprising administering said pharmaceutical composition to said subject, said pharmaceutical composition.

14. a) the subject is human; and / or b) the subject is a subject suffering from or at risk of developing hereditary transthyretin amyloidosis (hATTR) due to one or more mutations in the TTR gene, in particular, the subject is suffering from or at risk of developing cardiomyopathy (hATTR-CM) and / or polyneuropathy (hATTR-PN); or, the subject is suffering from or at risk of developing senile cardiac amyloidosis characterized by the wild-type allele (ATTRwt) of the TTR gene, The pharmaceutical composition according to claim 13.

15. The method according to claim 13, wherein one or more alleles of the TTR gene are silenced.

16. A composition for editing the TTR gene, comprising (a) an mRNA encoding a base editor protein having an editing window and comprising a cytidine deaminase or adenosine deaminase, and (b) a guide RNA comprising a tracr sequence that functions as a binding scaffold for the base editor protein and a spacer sequence that functions to direct the base editor protein to a protospacer sequence on the TTR gene, wherein the spacer sequence is at least partially complementary to a splice site or start codon of the TTR gene, said composition.

17. The composition according to claim 16, wherein the base editor protein further comprises a fusion protein comprising D10A nickase Cas9.

18. The composition according to claim 16, wherein the base editor protein comprises a fusion protein comprising adenine base editor ABE8.

8.

19. a) the spacer sequence is homologous to a protospacer sequence selected from Table 1, or b) the spacer sequence is the following table: (In the sequence, A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyladenosine; c is 2'-O-methylcytidine; g is 2'-O-methylguanosine; u is 2'-O-methyluridine; s is a phosphorothioate (PS) backbone linkage), or c) the spacer sequence is the following table: (In the sequence, A is modified or unmodified adenosine; C is modified or unmodified cytidine; G is modified or unmodified guanosine; U is modified or unmodified uridine) has more than 80% sequence identity to the spacer sequence presented, or d) the guide RNA is the following table: (In the sequence, A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2'-O-methyladenosine; c is 2'-O-methylcytidine; g is 2'-O-methylguanosine; u is 2'-O-methyluridine; s is a phosphorothioate (PS) backbone linkage; bold represents the spacer sequence) selected from The composition according to claim 16. **Claim 20** The composition according to claim 16, wherein the composition is encapsulated within lipid nanoparticles.

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