Compositions and methods for altering nucleobases in transthyretin polynucleotides

Abstract

Disclosed are compositions for modifying or editing polynucleotides (e.g., genes), and methods of using the compositions to treat or prevent disorders associated with extracellular deposition of amyloid fibrils formed by aggregation of misfolded transthyretin (TTR) protein in various tissues. Such disorders include, but are not limited to, hereditary transthyretin amyloidosis-associated polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis-associated 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. Also disclosed are compositions and methods of using base editor systems to edit TTR polynucleotides.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 624,030, filed January 23, 2024, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application contains a sequence list, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The sequence list XML file was created on January 13, 2025, named 180802-056202PCT_SL.xml, and has a size of 1,550,753 bytes. Background Technology

[0003] Amyloidosis is a disease characterized by the abnormal deposition of amyloid proteins in the body's organs and tissues. These protein deposits can occur in the peripheral nervous system, which consists of nerves connecting the brain and spinal cord to muscles and sensory cells (which detect sensations such as touch, pain, heat, and sound). Deposits in these nerves can lead to loss of sensation in the limbs (peripheral neuropathy). The autonomic nervous system, which controls involuntary bodily functions such as blood pressure, heart rate, and digestion, can also be affected by amyloidosis. In some cases, the brain and spinal cord (central nervous system) are also affected. Mutations in the transthyretin (TTR) gene are associated with transthyretin amyloidosis. Furthermore, patients expressing wild-type TTR may also develop amyloidosis. Liver transplantation remains the gold standard for treating transthyretin amyloidosis. However, the number of organ donors is limited, and patients may have to wait years for a usable organ. Therefore, there is a need for compositions and methods for treating amyloidosis. Invention Overview This document provides compositions for modifying or editing polynucleotides (e.g., DNA, RNA, genomic DNA, genes), and methods for using said compositions to treat or prevent conditions associated with extracellular deposition of amyloid fibers formed by the aggregation of misfolded transthyretin (TTR) in various tissues. Such conditions include, but are not limited to, hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis cardiomyopathy (hATTR-CM), both associated with autosomal dominant mutations in the TTR gene, and age-related cardiomyopathy (ATTRwt) associated with wild-type TTR protein, also known as senile cardiac amyloidosis. This document discloses compositions and methods for editing TTR polynucleotides using a base editor system.

[0005] In one aspect, this disclosure relates to a lipid nanoparticle (LNP) comprising components A), B), and C). Component A) is a guide RNA or a polynucleotide encoding said guide RNA, wherein said guide RNA comprises a spacer sequence comprising a nucleotide sequence selected from those sequences listed in GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472) and any of Tables 1B, 1C, 1D, 1E, 2A, 2B, 2D, or 2E. Component B) is an mRNA molecule or a polynucleotide encoding said mRNA molecule, wherein said mRNA molecule encodes a base editor polypeptide comprising a nucleic acid programmable DNA-binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain comprises an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence, or a truncated version thereof that is missing only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase further comprises a combination of amino acid alterations relative to the amino acid sequence of TadA*7.10, the combination being selected from one or more of the following: a) I76Y, V82T, Y123H, Y147R, and Q154R; b) V82T, Y123H, D147R, and Q154R; c) V82T, Y123H, D147T, and Q154S; d) V82T and Q154R; e) V82T, Y147T, and Q154S; and f) I76Y, V82T, Y123H, Y147T, and Q154S. Component C) is an ionizable lipid according to any of the following formulas, or a pharmaceutically acceptable salt thereof: i) Compounds of formula (Ih): (Ih) in: L 1 and L 1’ Each can be independently -C(O)- or -OC(O)-; L 2 and L 2’ Each is independently a divalent saturated or unsaturated, straight-chain or branched C, which can be optionally substituted. 1-12 hydrocarbon chain; L 3It is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; R 1 C is an optional substitute 1-20 Aliphatic groups, or ; L CyA It is a covalently bonded or optionally substituted divalent saturated or unsaturated, straight-chain or branched C 1-12 hydrocarbon chain; Cy A The rings are optionally substituted with the following: 3 to 7 saturated or partially unsaturated carbocyclic groups, 1-adamantyl, 2-adamantyl, sterol, and phenyl; L Ra It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-12 hydrocarbon chain; R a and R 1’ Each of the Cs can be substituted independently. 1-20 Aliphatic groups; Y 1 It is -C(O)- or -C(O)O-; Y 2 It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-6 hydrocarbon chain; Y 3 C is an optional substitute 1-20 Aliphatic groups; X 1 It is a covalent bond, -O-, or -NR-; X 2 It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-12 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; X 3 It is a hydrogen-containing or optionally substituted ring selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and Each R is independently hydrogen or an optionally substituted C. 1-6 Aliphatic groups; ii) Compounds of formula (Ia): Ia in: R 1 It is C9-C 20Alkyl groups or C9-C groups having 1-3 unsaturated units 20 alkenyl; X 1 and X 2 Each of them does not exist independently or is selected from –O–, –NR 2 -and , where each R 2 Independently hydrogen or C1-C6 alkyl; Each 'a' is an independent integer from 1 to 6; X 3 and X 4 Each of the following is independently absent or selected from one or more of the following: 4- to 8-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups; 5- to 6-membered heteroaryl groups optionally substituted with 1 or 2 C1-C6 alkyl groups; 5- to 6-membered aryl groups optionally substituted with 1 or 2 C1-C6 alkyl groups; 4- to 7-membered cycloalkyl groups optionally substituted with 1 or 2 C1-C6 alkyl groups; –O– and –NR. 3 – where each R 3 Independently a hydrogen atom or a C1-C6 alkyl group, wherein X 1 -X 2 -X 3 -X 4 It does not contain any oxygen-oxygen, oxygen-nitrogen, or nitrogen-nitrogen bonds; X 5 It is –(CH2) b – where b is an integer from 0 to 6; X 6 It is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl group optionally substituted with 1 or 2 C1-C6 alkyl groups, or –NR 4 R 5 , where R 4 and R 5 Each is independently hydrogen or C1-C6 alkyl; or, R 4 and R 5 Together with the nitrogen to which they are attached, they form 4- to 7-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic groups optionally contain additional heteroatoms selected from oxygen, sulfur and nitrogen; Each X 7 Independently hydrogen, hydroxyl, or –NR 6 R 7 , where R 6 and R 7 Each is independently hydrogen or C1-C6 alkyl; or, R 6 and R 7 Together with the nitrogen to which they are attached, they form 4- to 7-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic groups optionally contain additional heteroatoms selected from oxygen, sulfur and nitrogen; X 1 X 2 X 3 X 4 and X 5 At least one of them exists; A 1 and A 2 Each is independently selected from: C5-C 12 Haloalkyl, C5-C 12 alkenyl, C5-C 12 alkynyl group, (C5-C) 12 Alkyl group (CH2) n2 – optionally substituted with one or two halogen, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy rings (C5-C 10 (aryl)-(CH2) n3 – and (C3-C8 cycloalkyl)-(CH2) optionally substituted with one or two C1-C6 alkyl groups. n4 –; or, A 1 and A 2 They are connected together with the atoms they are attached to form one or two C4-C atoms. 10 Alkyl group-substituted 5- to 6-membered cyclic acetals; n1, n2, and n3 are each independent integers from 1 to 4; and n4 is an integer from 0 to 4; iii) Compounds of formula (Ib): Ib in: R 1 It is C9-C 20 Alkyl groups or C9-C groups having 1-3 unsaturated units 20 alkenyl; X 1 and X 2 Each of them does not exist independently or is selected from –O–, NR 2 and , where R 2 It is a C1-C6 alkyl group, and X is therein. 1 and X 2 Different from –O– or NR at the same time 2 ; a is an integer from 1 to 6; X 3 and X 4Each of the following is independently absent or selected from one or more of the following: 4- to 7-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups, 5- to 6-membered heteroaryl groups optionally substituted with 1 or 2 C1-C6 alkyl groups, and –NR 3 – where each R 3 It consists of hydrogen atoms or C1-C6 alkyl groups; X 5 For –(CH2) b – where b is an integer from 0 to 6; X 6 It is hydrogen, C1-C6 alkyl, 5- to 6-membered heteroaryl group optionally substituted with 1 or 2 C1-C6 alkyl groups, or –NR 4 R 5 , where R 4 and R 5 Each is independently hydrogen or C1-C6 alkyl; or, R 4 and R 5 Together with the nitrogen to which they are attached, they form 4- to 7-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic groups optionally contain additional heteroatoms selected from oxygen, sulfur and nitrogen; X 7 For hydrogen or –NR 6 R 7 , where R 6 and R 7 Each is independently hydrogen or C1-C6 alkyl; or, R 6 and R 7 Together with the nitrogen to which they are attached, they form 4- to 7-membered heterocyclic groups optionally substituted with 1 or 2 C1-C6 alkyl groups, wherein the heterocyclic groups optionally contain additional heteroatoms selected from oxygen, sulfur and nitrogen; X 1 X 2 X 3 X 4 and X 5 At least one of them exists; and The condition is: when X 1 or X 2 When it is –O–, X 3 and X 4 None of them are And when X 1 or X 2 When it is –O–, R 4 and R 5 Not both ethyl; iv) Compounds of formula (Ic): Ic or its N-oxide, or its salt, wherein L1 is a C1-6 alkylene group, or C 2-6 Heteroalkyl; Each L 2 Independently for C 2-10 alkylene, or C 3-10 Heteroalkyl; L does not exist, or it is C. 1-10 Alkylene or C 2-10 Heteroalkyl; L 3 It does not exist, or it is C. 1-10 Alkylene or C 2-10 Heteroalkyl; X does not exist, or is -OC(O)-, -C(O)O-, or -OC(O)O-; Each R is independently hydrogen, Or optionally substituted with groups selected from the following: C 6-20 Aliphatic groups, C 6-20 Halogenated aliphatic groups, 3 to 7-membered alicyclic rings, 1-adamantyl, 2-adamantyl, sterol, and phenyl; R 1 It is a hydrogen, 3 to 7 membered alicyclic ring, a 3 to 7 membered heterocycle containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, -OR 2 -C(O)OR 2 -C(O)SR 2 -OC(O)R 2 -OC(O)OR 2 -CN, -N(R) 2 )2、-C(O)N(R 2 )2、-NR 2 C(O)R 2 -OC(O)N(R) 2 )2、-N(R 2 )C(O)OR 2 -NR 2 S(O)2R 2 -NR 2 C(O)N(R 2 )2、-NR 2 C(S)N(R 2 )2、-NR 2 C(NR 2 )N(R 2 )2、-NR 2 C(CHR 2 )N(R 2 2. -N(OR) 2 )C(O)R 2 -N(OR) 2)S(O)2R 2 -N(OR) 2 )C(O)OR 2 -N(OR) 2 )C(O)N(R 2 2. -N(OR) 2 )C(S)N(R 2 2. -N(OR) 2 )C(NR 2 )N(R 2 2. -N(OR) 2 )C(CHR 2 )N(R 2 )2、-C(NR 2 )N(R 2 )2、-C(NR 2 )R 2 -C(O)N(R) 2 OR 2 -C(R) 2 )N(R 2 )2C(O)OR 2 , -CR 2 (OR 2 )R 3 , ,or ,or ; Each R 2 Independently, -CN, -NO2, -OR 4 -S(O)2R 4 -S(O)2N(R) 4 2、-(CH2) n R 4 Or optionally substituted with groups selected from the following: C 1-6 Aliphatic groups, 3- to 7-membered alicyclic rings, and 3- to 7-membered heterocycles containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or Two Rs 2 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocycle containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 3 Independently -(CH2) n R 4 ,or Two Rs 3 Together with one or more atoms to which it is attached, it forms an optional substituted 5- to 6-membered heterocycle containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 4Independently hydrogen, -OR 5 -N(R) 5 )2、-OC(O)R 5 -OC(O)OR 5 -CN, -C(O)N(R) 5 )2、-NR 5 C(O)R 5 -OC(O)N(R) 5 )2、-N(R 5 )C(O)OR 5 -NR 5 S(O)2R 5 -NR 5 C(O)N(R 5 )2、-NR 5 C(S)N(R 5 )2、-NR 5 C(NR 5 )N(R 5 )2, or ; Each R 5 Independently hydrogen, optionally substituted C 1-6 Aliphatic groups, or Two Rs 5 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocycle containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; Each R 6 Independently for C 4-12 Aliphatic groups; and n is between 0 and 4; Compounds of formula (Id) v): Id or its N-oxide, or its pharmaceutically acceptable salt, wherein L1 is absent, or is either a C1-6 alkylene or a C2-6 heteroalkylene; Each L2 is independently a C that can be substituted. 2-15 Alkylene, or optionally substituted C 3-15 Heteroalkyl; L 3 C does not exist, or is an optional substitution. 1-10 Alkylene, or optionally substituted C 2-10 Heteroalkyl; X does not exist, or is -OC(O)-, -C(O)O-, or -OC(O)O-; Each R' is independently a optionally substituted group selected from the following: C 4-12Aliphatic groups, 3 to 12-membered alicyclic groups, 7 to 12-membered bridged bicyclic groups containing 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl; R is hydrogen, Or optionally substituted with groups selected from the following: C 6-20 Aliphatic groups, 3 to 12-membered alicyclic groups, 7 to 12-membered bridged bicyclic groups containing 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl; R 1 The following groups are optionally substituted: hydrogen, phenyl, 3- to 7-membered alicyclic groups, 3- to 7-membered heterocyclic groups, 5- to 6-membered monocyclic heteroaryl groups, 8- to 10-membered bicyclic heteroaryl groups, and -OR. 2 -C(O)OR 2 -C(O)SR 2 -OC(O)R 2 -OC(O)OR 2 -CN, -N(R) 2 )2、-C(O)N(R 2 )2、-S(O)2N(R 2 )2、-NR 2 C(O)R 2 -OC(O)N(R) 2 )2、-N(R 2 )C(O)OR 2 -NR 2 S(O)2R 2 -NR 2 C(O)N(R 2 )2、-NR 2 C(S)N(R 2 )2、-NR 2 C(NR 2 )N(R 2 )2、-NR 2 C(CHR 2 )N(R 2 2. -N(OR) 2 )C(O)R 2 -N(OR) 2 )S(O)2R 2 -N(OR) 2 )C(O)OR 2 -N(OR) 2 )C(O)N(R2 2. -N(OR) 2 )C(S)N(R 2 2. -N(OR) 2 )C(NR 2 )N(R 2 2. -N(OR) 2 )C(CHR 2 )N(R 2 )2、-C(NR 2 )N(R 2 )2、-C(NR 2 )R 2 -C(O)N(R) 2 OR 2 -C(R) 2 )N(R 2 )2C(O)OR 2 -CR 2 (R 3 )2、-OP(O)(OR 2 )2 or -P(O)(OR 2 )2; or R 1 for Or, a ring selected from 3 to 7-membered alicyclic groups and comprising 1 to 3 heterocyclic groups independently selected from nitrogen, oxygen, and sulfur, wherein the alicyclic group or heterocyclic ring is optionally surrounded by 1 to 4 R atoms. 2 or R 3 Group substitution; Each R 2 Independently, it can be hydrogen, oxo, -CN, -NO2, or -OR. 4 -S(O)2R 4 -S(O)2N(R) 4 2、-(CH2) n -R 4 Or optionally substituted with groups selected from the following: C 1-6 Aliphatic groups, phenyl groups, 3- to 7-membered alicyclic groups, 5- to 6-membered monocyclic heteroaryl groups comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclic groups comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or Two Rs 2 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur; Each R 3 Independently -(CH2) n -R 4 ;or Two Rs 3Together with one or more atoms to which it is attached, it forms a 5- to 6-membered heterocyclic group with 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur, which may be substituted. Each R 4 Independently hydrogen, -OR 5 -N(R) 5 )2、-OC(O)R 5 -OC(O)OR 5 -CN, -C(O)N(R) 5 )2、-NR 5 C(O)R 5 -OC(O)N(R) 5 )2、-N(R 5 )C(O)OR 5 -NR 5 S(O)2R 5 -NR 5 C(O)N(R 5 )2、-NR 5 C(S)N(R 5 )2、-NR 5 C(NR 5 )N(R 5 )2, or ; Each R 5 Independently hydrogen, or optionally substituted C 1-6 Aliphatic groups; or Two Rs 5 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur; Each R 6 Independently for C 4-12 Aliphatic groups; and Each n is independently between 0 and 4; vi) Compounds of formula (Ie): (Ie) Or its pharmaceutically acceptable salt, wherein: L 1 It is a covalent bond, -C(O)- or -OC(O)-; L 2 It is a covalent bond, optionally substituted divalent saturated or unsaturated, straight-chain or branched C1-C 12 hydrocarbon chain, or ; Cy A It is optionally substituted with a ring selected from the following: phenylene and 3 to 7 membered saturated or partially unsaturated carbocyclic groups; Each m is independently 0, 1, or 2; L 3 It is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; R 1 for Or optionally substituted saturated or unsaturated, straight or branched C1-C 20 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by –O- or –NR-; Cy B The substituted ring is selected from the following: 3 to 12-membered saturated or partially unsaturated carbocyclic groups, 1-adamantyl, 2-adamantyl, , sterol group and phenyl group; p is 0, 1, 2 or 3; X 1 For covalent bonds, –O– or –NR–; X 2 C1-C bonds that are covalently bonded or optionally substituted, divalent saturated or unsaturated, straight or branched 12 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently converted by -O-, -NR- or –Cy C -replace; Cy C It is an optional substituted ring selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. X 3 The ring is hydrogen or optionally substituted with one of the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered saturated or partially unsaturated heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. Each R is independently hydrogen or an optionally substituted C1-C6 aliphatic group; Z 1 It is a covalent bond or -O-; Z 2 The optional substituted group is selected from the following: 4 to 12 saturated or partially unsaturated carbocyclic groups, phenyl, 1-adamantyl and 2-adamantyl; Z 3 It is hydrogen, or optionally substituted with a group selected from the following: C1-C 10 Aliphatic groups, and 4 to 12 saturated or partially unsaturated carbocyclic groups; and d can be 0, 1, 2, 3, 4, 5, or 6; The condition is: when L 3 When it is a covalent bond, then R 1 Must be ; vii) Compounds of formula (If): (If) Or its pharmaceutically acceptable salt, wherein: L 1 and L 1’ Each is independently -C(O)- or -C(O)O-; L 2 and L 2’ Each is an independently covalently bonded, optionally substituted divalent saturated or unsaturated, straight-chain or branched C1-C bond. 12 hydrocarbon chain, or ; Each Cy A The ring is independently selected from the group consisting of phenylene or 3 to 7 saturated or partially unsaturated carbocyclic groups; Each m is independently 0, 1, or 2; L 3 and L 3’ Each can be independently a covalent bond, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; R 1 and R 1’ Each of the optional substituted groups is selected from the following: saturated or unsaturated, straight-chain or branched C1-C groups. 20 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-, 3 to 12 saturated or partially unsaturated carbocyclic rings, 1-adamantyl, 2-adamantyl, sterol, phenyl, and ; Each L 4 Independently, it is a divalent saturated or unsaturated, straight-chain or branched C1-C6 hydrocarbon chain; A 1 and A 2 Each of the C1-Cs can be substituted independently. 20 Aliphatic groups or -L 5 -R 5 ; Or A 1 and A 2 Together with the atoms between them, they can form rings with optional substitutions: ; in x is selected from 1 or 2; and # indicates the relationship with L 4The connection point; Each L 5 Independently divalent, saturated or unsaturated, straight-chain or branched C1-C 20 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; Each R 5 The groups that are independently substituted are selected from the following: 5- to 10-membered aromatic rings and 3- to 8-membered carbon rings; X 1 For covalent bonds, –O– or –NR–; X 2 It is a covalently bonded or optionally substituted divalent, saturated or unsaturated, straight or branched C1-C bond. 12 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; X 3 It is hydrogen or -Cy B ; Cy B It is an optionally substituted ring selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered saturated or partially unsaturated heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and Each R is independently hydrogen or an optionally substituted C1-C6 aliphatic group; The condition is: when X 3 When it is hydrogen, R 1 or R 1’ At least one of them is ; viii) Compounds of formula (Ig): (Ig) Or its pharmaceutically acceptable salt, wherein: L 1 and L 1’ Each can be independently a covalent bond, -C(O)- or -OC(O)-; L 2 and L 2’ Each is an independently covalently bonded, optionally substituted divalent saturated or unsaturated, straight-chain or branched C1-C bond. 12 hydrocarbon chain, or ; Each Cy A The ring is independently selected from the group consisting of phenylene or 3 to 7 saturated or partially unsaturated carbocyclic groups; Each m is independently 0, 1, or 2; L3 and L 3’ Each can be independently a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; R 1 and R 1’ Each of the optional substituted groups is selected from the following: saturated or unsaturated, straight-chain or branched C1-C groups. 20 A hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by –O- or –NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterol, phenyl, or ; Each L 4 Independently divalent, saturated or unsaturated, straight-chain or branched C1-C 20 hydrocarbon chain; A 1 and A 2 Each of the C1-Cs can be substituted independently. 20 Aliphatic groups or –L 5 -R 5 , Or A 1 and A 2 Together with the atoms between them, they can form rings with optional substitutions: , in x is selected from 1 or 2; and # indicates the relationship with L 4 The connection point; Each L 5 Independently divalent, saturated or unsaturated, straight-chain or branched C1-C 20 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; Each R 5 The group independently selected as an optional substituted group is a 6- to 10-membered aromatic ring or a 3- to 8-membered carbon ring; Y 1 It is a covalent bond, –C(O)- or –C(O)O-; Y 2 It is a divalent saturated or unsaturated, straight-chain or branched C1-C6 hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced by cyclopropyl, -O- or –NR-. Y 3 The substituted group is selected from the following: saturated or unsaturated, straight-chain or branched C1-C groups. 14 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by –O- or –NR-, 3 to 7 saturated or partially unsaturated carbon rings, 1-adamantyl, 2-adamantyl, or phenyl; X 1 For covalent bonds, –O– or –NR-; X 2 It is an optional substituted divalent saturated or unsaturated, straight-chain or branched C1-C 12 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently converted by –O-, -NR- or –Cy. B -replace; Each Cy B The ring is independently selected from the following rings that are optionally substituted: 3 to 7-membered saturated or partially unsaturated carbocyclic groups, phenylene groups, 3 to 7-membered heterocyclic groups having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or 5 to 6-membered heteroaryl groups having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. X 3 The ring is hydrogen or optionally substituted, selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and Each R is independently hydrogen or an optionally substituted C1-C6 aliphatic group; Compounds of formula A' (ix): A' or its N-oxide, or its pharmaceutically acceptable salt, wherein L 1 It does not exist, so it is C. 1-6 alkylene, or C 2-6 Heteroalkyl; Each L 2 C can be substituted independently. 2-15 Alkylene, or optionally substituted C 3-15 Heteroalkyl; L is C 1-10 alkylene, or C 2-10 Heteroalkyl; X 2 is -OC(O)-, -C(O)O- or -OC(O)O-; X does not exist; it is -OC(O)-, -C(O)O-, or -OC(O)O-. "R" represents hydrogen, , Or optionally substituted with groups selected from the following: C 6-20 Aliphatic groups, 3 to 12-membered alicyclic groups, 7 to 12-membered bridged bicyclic groups containing 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl; R and R a Each group is independently hydrogen, or optionally substituted with a group selected from the following: C 6-20 Aliphatic groups, 3 to 12-membered alicyclic groups, 7 to 12-membered bridged bicyclic groups containing 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl; L 3 and L 3a Each of them does not exist independently, and is an optional substitution of C. 1-10 Alkylene, or optionally substituted C 2-10 Heteroalkyl; R 1 The following groups are optionally substituted: hydrogen, phenyl, 3- to 7-membered alicyclic groups, 3- to 7-membered heterocyclic groups, 5- to 6-membered monocyclic heteroaryl groups, 8- to 10-membered bicyclic heteroaryl groups, and -OR. 2 -C(O)OR 2 -C(O)SR 2 -OC(O)R 2 -OC(O)OR 2 -CN, -N(R) 2 )2、-C(O)N(R 2 )2、-S(O)2N(R 2 )2、-NR 2 C(O)R 2 -OC(O)N(R) 2 )2、-N(R 2 )C(O)OR 2 -NR 2 S(O)2R 2 -NR 2 C(O)N(R 2 )2、-NR 2 C(S)N(R 2 )2、-NR 2 C(NR 2 )N(R 2 )2、-NR 2 C(CHR 2 )N(R 2 2. -N(OR) 2 )C(O)R 2 -N(OR) 2 )S(O)2R 2 -N(OR) 2 )C(O)OR 2 -N(OR)2 )C(O)N(R 2 2. -N(OR) 2 )C(S)N(R 2 2. -N(OR) 2 )C(NR 2 )N(R 2 2. -N(OR) 2 )C(CHR 2 )N(R 2 )2、-C(NR 2 )N(R 2 )2、-C(NR 2 )R 2 -C(O)N(R) 2 OR 2 -C(R) 2 )N(R 2 )2C(O)OR 2 -CR 2 (R 3 )2、-OP(O)(OR 2 )2 or -P(O)(OR 2 )2; or R 1 for Or a ring selected from 3 to 7-membered alicyclic groups and 3 to 7-membered heterocyclic groups comprising 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein the alicyclic group or heterocyclic ring is optionally surrounded by 1 to 4 R atoms. 2 or R 3 Group substitution; Each R 2 Independently, it can be hydrogen, oxo, -CN, -NO2, or -OR. 4 -S(O)2R 4 -S(O)2N(R) 4 2、-(CH2) n -R 4 , or optionally substituted with a group selected from the following: C 1-6 Aliphatic groups, phenyl groups, 3- to 7-membered alicyclic groups, 5- to 6-membered monocyclic heteroaryl groups comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclic groups comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or Two Rs 2 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur; Each R 3 Independently -(CH2) n -R 4 ;or Two Rs3 Together with one or more atoms to which it is attached, it forms a 5- to 6-membered heterocyclic group with 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur, which may be substituted. Each R 4 Independently hydrogen, -OR 5 -N(R) 5 )2、-OC(O)R 5 -OC(O)OR 5 -CN, -C(O)N(R) 5 )2、-NR 5 C(O)R 5 -OC(O)N(R) 5 )2、-N(R 5 )C(O)OR 5 -NR 5 S(O)2R 5 -NR 5 C(O)N(R 5 )2、-NR 5 C(S)N(R 5 )2、-NR 5 C(NR 5 )N(R 5 )2, or ; Each R 5 Independently hydrogen, or optionally substituted C 1-6 Aliphatic groups; or Two Rs 5 Together with one or more atoms to which it is attached, it forms an optional substituted 4- to 7-membered heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen and sulfur; Each R 6 Independently for C 4-12 Aliphatic groups; and Each n is independently between 0 and 4; or x) Compounds of Formula I: (I) Or its pharmaceutically acceptable salt, wherein: L 1 It is a covalent bond, -C(O)- or -OC(O)-; L 2 It is a covalent bond, optionally substituted divalent saturated or unsaturated, straight-chain or branched C1-C 12 hydrocarbon chain, or ; Cy A It is optionally substituted with a ring selected from the following: phenylene and 3 to 7 membered saturated or partially unsaturated carbocyclic groups; Each m is independently 0, 1, or 2; L 3 It is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; R 1 yes Optionally substituted saturated or unsaturated, straight-chain or branched C1-C 20 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by –O- or –NR-, or ; Cy B The substituted ring is selected from the following: 3 to 12-membered saturated or partially unsaturated carbocyclic groups, 1-adamantyl, 2-adamantyl, , sterol group and phenyl group; p is 0, 1, 2 or 3; Each L 4 Independently, it is a divalent saturated or unsaturated, straight-chain or branched C1-C6 hydrocarbon chain; A 1 and A 2 Each of the C1-Cs can be substituted independently. 20 Aliphatic groups or -L 5 -R 5 ; Or A 1 and A 2 Together with the atoms between them, they can form rings with optional substitutions: ; in, x is selected from 1 or 2; and # indicates the relationship with L 4 The connection point; Each L 5 Independently divalent, saturated or unsaturated, straight-chain or branched C1-C 20 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; Each R 5 The group independently selected as an optional substituted group is a 5- to 10-membered aromatic ring or a 3- to 8-membered carbon ring; X 1 For covalent bonds, –O– or –NR–; X 2 C1-C bonds that are covalently bonded or optionally substituted, divalent saturated or unsaturated, straight or branched 12 Hydrocarbon chain, wherein 1-3 methylene units are optionally and independently converted by -O-, -NR- or –Cy C -replace; CyC It is an optional substituted ring selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. X 3 The ring is hydrogen or optionally substituted, selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered saturated or partially unsaturated heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and Each R is independently hydrogen or an optionally substituted C1-C6 aliphatic group; The condition is: when L 3 When it is a covalent bond, then R 1 Must be .

[0006] In another aspect, this disclosure relates to a lipid nanoparticle (LNP) comprising components A), B), and C). Component A) is a guide RNA or a polynucleotide encoding said guide RNA, wherein said guide RNA is capable of directing a base editor polypeptide to alter nucleotides in a transthyretin (TTR) polynucleotide. Component B) is an mRNA molecule or a polynucleotide encoding said mRNA molecule, wherein said mRNA molecule encodes a base editor polypeptide comprising a nucleic acid programmable DNA-binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain comprises i) an amino acid sequence having at least 90% identity with the following TadA*7.10 amino acid sequence, or a truncated version thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain further comprises a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence, the combination being selected from one or more of the following: a) I76Y, V82T, Y123H, Y147R, and Q154R; b) V82T, Y123H, D147R, and Q154R; c) V82T, Y123H, D147T, and Q154S; d) V82T and Q154R; e) V82T, Y147T, and Q154S; f) I76Y, V82T, Y123H, Y147T, and Q154S; g) Y123H, Y147R, and Q154R; h) I76Y, Y147R, and Q154R; i) Y147R, Q154R, and T166R; j) Y147T and Q154R; k) Y147T and Q154S; l) I76Y, Y123H, Y147R, and Q145R; m) I76Y and V82S; n) V82S and Y147R; o) V82S, Y123H, and Y147R; p) V82S and Q154R; q) V82S, Y123H, and Q154R; r) V82S, Y123H, Y147R, and Q154R; s) I76Y, V82S, Y123H, Y147R, and Q154R. Component C) is an ionizable lipid of formula (I), or a pharmaceutically acceptable salt thereof: (Ih) in: L 1 and L1’ Each can be independently -C(O)- or -OC(O)-; L 2 and L 2’ Each is independently a divalent saturated or unsaturated, straight-chain or branched C, which can be optionally substituted. 1-12 hydrocarbon chain; L 3 It is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; R 1 C is an optional substitute 1-20 Aliphatic groups, or ; L CyA It is a covalently bonded or optionally substituted divalent saturated or unsaturated, straight-chain or branched C 1-12 hydrocarbon chain; Cy A The rings are optionally substituted with the following: 3 to 7 saturated or partially unsaturated carbocyclic groups, 1-adamantyl, 2-adamantyl, sterol, and phenyl; L Ra It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-12 hydrocarbon chain; R a and R 1’ Each of the Cs can be substituted independently. 1-20 Aliphatic groups; Y 1 It is -C(O)- or -C(O)O-; Y 2 It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-6 hydrocarbon chain; Y 3 C is an optional substitute 1-20 Aliphatic groups; X 1 It is a covalent bond, -O- or -NR-; X 2 It is a divalent saturated or unsaturated, straight-chain or branched C that can be optionally substituted. 1-12 Hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; X 3 The ring is hydrogen or optionally substituted, selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a phenyl group, a 3- to 7-membered heterocyclic group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and Each R is independently hydrogen or an optionally substituted C.1-6 Aliphatic groups.

[0007] On the other hand, this disclosure relates to a lipid nanoparticle (LNP) comprising components A), B) and C). Component A) is a guide RNA having the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmGmUmGGmCmAmCmGmAmGmUmGmGmGmGmGmGmGmUs ... Component B) is a base editor polypeptide, or a polynucleotide encoding the base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA-binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain comprises an amino acid sequence having at least 90% identity with the following amino acid sequence, or a truncated version thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain further comprises a combination of the following amino acid changes relative to the TadA*7.10 amino acid sequence: I76Y, V82T, Y123H, Y147R, and Q154R. Component C) is an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof: (IZ4).

[0008] In another aspect, this disclosure relates to a base editor system comprising components A) and B). Component A) is a guide RNA, or a polynucleotide encoding said guide RNA, wherein said guide RNA is capable of directing a base editor polypeptide to alter nucleotides in a transthyretin (TTR) polynucleotide. Component B) is a base editor polypeptide, or one or more polynucleotides encoding said base editor polypeptide, wherein said base editor polypeptide comprises a nucleic acid programmable DNA-binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain comprises an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence, or a truncated version thereof that is missing only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain further comprises a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence, the combination being selected from one or more of the following: a) V82T, Y123H, D147R, and Q154R; b) V82T, Y123H, D147T, and Q154S; c) V82T and Q154R; d) V82T, Y147T, and Q154S; e) I76Y, V82T, Y123H, Y147R, and Q154R; and f) I76Y, V82T, Y123H, Y147T, and Q154S.

[0009] On the other hand, this disclosure relates to a polynucleotide or polynucleotide set that encodes a base editor system for any aspect of this disclosure, or an embodiment thereof.

[0010] In another aspect, this disclosure relates to a cell that comprises a base editor system or polynucleotide or polynucleotide group, or an embodiment thereof, of any aspect of this disclosure.

[0011] In another aspect, this disclosure relates to a pharmaceutical composition comprising lipid nanoparticles, base editor systems, polynucleotides or polynucleotide groups, or cells, or embodiments thereof, and pharmaceutically acceptable carriers or excipients, according to any aspect of this disclosure.

[0012] In another aspect, this disclosure relates to a kit comprising lipid nanoparticles, base editor systems, polynucleotides or polynucleotide groups, cells, or pharmaceutical compositions, or embodiments thereof, and a container, according to any aspect of this disclosure.

[0013] In another aspect, this disclosure relates to a method for modifying target nucleobases in thyroxine transporter (TTR) polynucleotides in cells. The method includes contacting the cells with lipid nanoparticles or a base editor system according to any aspect of this disclosure, or embodiments thereof, thereby modifying the target nucleobases in the TTR polynucleotide.

[0014] On the other hand, this disclosure relates to a method for treating a disease in a subject in need. The disease is associated with a pathogenic mutation in a transthyretin (TTR) polynucleotide in the subject. The method includes administering to the subject lipid nanoparticles or a base editor system, or an embodiment thereof, from any aspect of this disclosure to alter a target nucleobase in the TTR polynucleotide.

[0015] In any aspect of this disclosure or in any embodiment thereof, the lipid nanoparticles comprise an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof: (IZ4).

[0016] In any aspect of this disclosure or in any embodiment thereof, the lipid nanoparticles comprise an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof: (IZ3).

[0017] In any aspect of this disclosure or any embodiment thereof, the molar ratio (N:P ratio) of the ionizable nitrogen atoms in the ionizable lipid of the LNP to the total negative charge in the load is about 30:1 to about 1:1. In any aspect of this disclosure or any embodiment thereof, the LNP has an N:P ratio of about 6:1.

[0018] In any aspect of this disclosure or any embodiment thereof, the guide RNA comprises a scaffold having the following nucleotide sequence: gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcuususus (SEQ ID NO: 478), wherein 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; and s is a phosphate thioester (PS) backbone linker. In any aspect of this disclosure or any embodiment thereof, the guide RNA comprises a spacer sequence having the following nucleotide sequence: GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In any aspect of this disclosure or in any embodiment thereof, the guide RNA comprises the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmGmUmGGmCmAmCmGmAmGmUmGmGmGmGmGmGmUs ... In any aspect of this disclosure or in any embodiment thereof, the guide RNA comprises a spacer sequence selected from the spacer sequences listed in any of Tables 1B, 1C, 1D, 1E, 2A, 2B, 2D, or 2E. In any aspect of this disclosure or in any embodiment thereof, the guide RNA comprises 2-5 consecutive 2'-O-methylated nucleotides at its 3' and 5' ends. In any aspect of this disclosure or in any embodiment thereof, the guide RNA comprises 2-5 consecutive nucleotides at its 3' and 5' ends, the nucleotides comprising phosphate-thioester nucleotide links.

[0019] In any aspect of this disclosure or in any embodiment thereof, the napDNAbp domain comprises a Cas9 polypeptide. In any aspect of this disclosure or in any embodiment thereof, the napDNAbp is a nickase.

[0020] In any aspect of this disclosure or in any embodiment thereof, the base editor polypeptide comprises an amino acid sequence having at least about 90% identity with the following amino acid sequence: ABE9.51

[0021] In any aspect of this disclosure or in any implementation thereof, the cells are located within the subject's body.

[0022] In any aspect of this disclosure or in any embodiment thereof, the ionizable lipids of the lipid nanoparticles have a half-life of less than 14 days in the liver of a subject.

[0023] In any aspect of this disclosure or any embodiment thereof, the target nucleobase is altered in the cells and / or the liver of the subject with an editing efficiency of at least about 50% or 60%. In any aspect of this disclosure or any embodiment thereof, alteration of the target nucleobase results in a decrease in the level of the TTR peptide in the cells and / or the subject. In any aspect of this disclosure or any embodiment thereof, the TTR peptide level is reduced by at least about 80% or 90%. In any aspect of this disclosure or any embodiment thereof, the method is associated with only a transient increase or no increase in the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and / or aspartate aminotransferase (AST) in the subject. In any aspect of this disclosure or any embodiment thereof, the method is not associated with an increase in the ALP level in the subject.

[0024] In any aspect of this disclosure or in any implementation thereof, the disease is selected from one or more of the following: amyloidosis, cardiomyopathy, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), familial transthyretin amyloidosis (FTA), senile systemic amyloidosis (SSA), and transthyretin amyloidosis.

[0025] In any aspect of this disclosure or in any embodiment thereof, the average diameter of the LNP is 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 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In any aspect of this disclosure or any embodiment thereof, the average diameter of the LNP is 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 greater. In any aspect of this disclosure or any embodiment thereof, the average diameter of the LNP is about 70 nm + / - 10 nm, 70 nm + / - 5 nm, 65 nm + / - 10 nm, 65 nm + / - 5 nm, 60 nm + / - 10 nm or 60 nm + / - 5 nm.

[0026] In any aspect of this disclosure or an embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount from about 30 mol percent to about 70 mol percent of the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or an embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount from about 33 mol percent to about 60 mol percent of the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or an embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount from about 34 mol percent to about 55 mol percent of the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or an embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount from about 33 mol percent to about 51 mol percent of the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or an embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount from about 34.7 mol percent of the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or any embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount of about 50 mol percent based on the total molar number of the lipid nanoparticle components. In any aspect of this disclosure or any embodiment thereof, the ionizable lipid is present in the lipid nanoparticles (LNPs) in an amount of about 40 mol percent to about 60 mol percent or about 45 mol percent to about 55 mol percent based on the total molar number of the lipid nanoparticle components.

[0027] In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 0 mol percent to about 5 mol percent of conjugate-linker lipids. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 1.5 mol percent of conjugate-linker lipids. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 2.0 mol percent of conjugate-linker lipids. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 2.5 mol percent of conjugate-linker lipids. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 3 mol percent of conjugate-linker lipids. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 3.5 mol percent of conjugate-linker lipids.

[0028] In any aspect of this disclosure or any embodiment thereof, the LNP contains about 5 mol percent to 25 mol percent of phospholipids. In any aspect of this disclosure or any embodiment thereof, the LNP contains about 5 mol percent to 15 mol percent of phospholipids. In any aspect of this disclosure or any embodiment thereof, the LNP contains about 9 mol percent to 11 mol percent of phospholipids. In any aspect of this disclosure or any embodiment thereof, the LNP contains about 10 mol percent of phospholipids.

[0029] In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 25 mole percent to about 45 mole percent of sterols. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 30 mole percent to about 45 mole percent of sterols. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 40 mole percent of sterols. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 35.75 mole percent of sterols. In any aspect of this disclosure or any embodiment thereof, the LNP comprises about 32.5 mole percent of cholesterol.

[0030] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 30 mol percent to about 70 mol percent of ionizable lipids, about 5 mol percent to about 25 mol percent of phospholipids, about 25 mol percent to about 45 mol percent of sterols, and about 0 mol percent to about 5 mol percent of conjugate-linker lipids.

[0031] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 47.5 mol percent of ionizable lipids, about 10 mol percent of phospholipids, about 40 mol percent of sterols, and about 2.5 mol percent of conjugate-linker lipids.

[0032] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 51.25 mol percent of ionizable lipids, about 10 mol percent of phospholipids, about 35.75 mol percent of sterols and about 3 mol percent of conjugate-linker lipids.

[0033] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 55 mol percent of ionizable lipids, about 10 mol percent of phospholipids, about 32.5 mol percent of sterols, and about 2.5 mol percent of conjugate-linker lipids.

[0034] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 45 mol percent to about 60 mol percent of any of the provided compounds of an ionizable lipid, about 9 mol percent to about 11 mol percent of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and about 1 mol percent to about 5 mol percent of PEG. 2000 -DMG, and approximately 30 mole percent to approximately 45 mole percent of cholesterol, based on the total moles of these four components.

[0035] In any aspect of this disclosure or in any implementation thereof, the LNP is formulation LNP1, LNP2, LNP3 or LNP4 in Table 10A.

[0036] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 47.5 mol% of an ionizable lipid IZ1, about 10 mol% of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and about 2.5 mol% of PEG. 2000 -DMG, and approximately 40 molar percentages of cholesterol, with an N / P ratio of 6 based on the total moles of these four components, and a payload comprising an mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain having an amino acid alteration V82T or a combination of amino acid alterations V82T, Y147T, and Q154S, and a guide RNA comprising a spacer sequence containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table 1B.

[0037] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 55 mol% of an ionizable lipid IZ2, about 10 mol% of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and about 2.5 mol% of PEG. 2000 -DMG, and approximately 32.5 mol% cholesterol, with an N / P ratio of 6 based on the total moles of these four components, and a payload comprising an mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain having an amino acid alteration V82T or a combination of amino acid alterations V82T, Y147T, and Q154S, and a guide RNA comprising a spacer sequence containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table 1B.

[0038] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 51.25 mol% of ionizable lipid IZ3, about 10 mol% of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and about 3 mol% of PEG. 2000 -DMG, and approximately 35.75 mol% cholesterol, with an N / P ratio of 6 based on the total moles of these four components, and a payload comprising an mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain having an amino acid alteration V82T or a combination of amino acid alterations V82T, Y147T, and Q154S, and a guide RNA comprising a spacer sequence containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table 1B.

[0039] In any aspect of this disclosure or in any embodiment thereof, the LNP comprises about 47.5 mol% of ionizable lipid IZ4, about 10 mol% of 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and about 2.5 mol% of PEG. 2000 -DMG, and approximately 40 molar percentages of cholesterol, with an N / P ratio of 6 based on the total moles of these four components, and a payload comprising an mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain having an amino acid alteration V82T or a combination of amino acid alterations V82T, Y147T, and Q154S, and a guide RNA comprising a spacer sequence containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table 1B.

[0040] In any aspect of this disclosure or in any embodiment thereof, the N / P ratio of the lipid nanoparticles is from about 1 to about 30. In any aspect of this disclosure or in any embodiment thereof, the N / P ratio of the lipid nanoparticles is from about 2 to about 15, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 15, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 4 to about 15, from about 4 to about 10, from about 4 to about 8, or from about 4 to about 6. In any aspect of this disclosure or in any embodiment thereof, the N / P ratio of the lipid nanoparticles is from about 2, from about 2.5, from about 3, from about 3.5, from about 4, from about 4.5, from about 5, from about 5.5, from about 6, from about 6.5, from about 7, from about 7.5, from about 8, from about 9, or from about 10. In any aspect of this disclosure or in any embodiment thereof, the N / P ratio of the lipid nanoparticles is from about 5 to about 7. In any aspect of this disclosure or in any embodiment thereof, the N / P ratio of the lipid nanoparticles is from about 6.

[0041] In any aspect or implementation thereof provided herein, the method is not a process for modifying human germline genetic identity.

[0042] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide general definitions for many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms have the meanings assigned to them hereinafter.

[0043] "TadA*8.8 polypeptide" refers to an adenosine deaminase domain with deaminase activity, containing alterations Y123H, Y147R, and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity with the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some implementations, TadA*8.8 includes further changes relative to the reference sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more changes).

[0044] "TadA*8.8 polynucleotide" refers to the polynucleotide that encodes the TadA*8.8 polypeptide.

[0045] "TadA*8.13 polypeptide" refers to an adenosine deaminase domain with deaminase activity, containing alterations I76Y, Y123H, Y147R, and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity with the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some implementations, TadA*8.13 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more total changes relative to the reference sequence.

[0046] "TadA*8.13 polynucleotide" refers to the polynucleotide that encodes the TadA*8.13 polypeptide.

[0047] "TadA*8.17 polypeptide" refers to an adenosine deaminase domain with deaminase activity, containing alterations V82S and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity with the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some implementations, TadA*8.17 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more total changes relative to the reference sequence.

[0048] "TadA*8.17 polynucleotide" refers to the polynucleotide that encodes the TadA*8.17 polypeptide.

[0049] "TadA*8.20 polypeptide" refers to an adenosine deaminase domain with deaminase activity, containing alterations I76Y, V82S, Y123H, Y147R, and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity with the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some implementations, TadA*8.20 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more total changes relative to the reference sequence.

[0050] "TadA*8.20 polynucleotide" refers to the polynucleotide that encodes the TadA*8.20 polypeptide.

[0051] In various embodiments, any adenosine deaminase domain provided herein (e.g., TadA*8.8, TadA*8.17, or TadA*8.20, and variants thereof) contains the following changes relative to the TadA*7.10 reference sequence: V82T; or V82T, Y147T, and Q154S.

[0052] "ABE9.51 polypeptide" refers to an adenosine deaminase base editor comprising a TadA*8.20 adenosine deaminase polypeptide variant with amino acid alteration V82T, and a nucleic acid programmable DNA-binding protein (napDNAbp) domain. In some embodiments, ABE9.51 comprises an amino acid sequence having at least about 85% sequence identity with the following polypeptide sequence, or a functional fragment thereof capable of deaminating adenosine nucleotides:

[0053] In some embodiments, ABE9.51 contains one or more uracil glycosylation inhibitor (UGI) domains. In some embodiments, ABE9.51 contains two UGI domains.

[0054] "ABE9.51 polynucleotide" refers to the polynucleotide that encodes the ABE9.51 polypeptide.

[0055] "Adenine" or "9H-purine-6-amine" refers to a purine nucleobase with the molecular formula C5H5N5, and its structure is... This corresponds to CAS number 73-24-5.

[0056] "Adenosine" or "4-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]pyrimidin-2(1H)-one" refers to an adenine molecule linked to a ribose via a glycosidic bond, with the following structure: This corresponds to CAS number 65-46-3. Its molecular formula is C2. 10 H 13 N5O4.

[0057] "Adenosine deaminase" or "adenine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or catalyzes the hydrolytic deamination of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism (e.g., eukaryotes, prokaryotes), including but not limited to algae, bacteria, fungi, plants, invertebrates (e.g., insects), and vertebrates (e.g., amphibians, mammals). In some embodiments, the adenosine deaminase is a variant of adenosine deaminase with one or more modifications and capable of deaminating both adenine and cytosine in target polynucleotides (e.g., DNA, RNA), and may be referred to as a "bifunctional deaminase". Non-limiting examples of bifunctional deaminases include those described in PCT / US22 / 22050. In some embodiments, the target polynucleotide is single-stranded or double-stranded. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant is capable of deaminating both adenine and cytosine in RNA. In embodiments, the adenosine deaminase variant is selected from those described in PCT / US2020 / 018192, PCT / US2020 / 049975, PCT / US2017 / 045381, and PCT / US2020 / 028568, the entire contents of which are incorporated herein by reference for all purposes.

[0058] "Adenosine deaminase activity" refers to the activity that catalyzes the deamination of adenine or adenosine in polynucleotides into guanine.

[0059] "Adenosine base editor (ABE)" refers to a base editor that contains adenosine deaminase.

[0060] "Adenosine base editor (ABE) polynucleotide" refers to the polynucleotide that encodes ABE.

[0061] "Adenosine base editor 8 (ABE8) polypeptide" or "ABE8" refers to a base editor as defined herein, comprising an adenosine deaminase or a variant of an adenosine deaminase comprising one or more alterations listed in Table 5B, a combination of alterations listed in Table 5B, or alterations at one or more amino acid positions listed in Table 5B, such alterations being relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or relative to the corresponding position in another adenosine deaminase. In an embodiment, ABE8 comprises alterations at amino acids 82 and / or 166 of SEQ ID NO: 1. In some implementations, ABE8 includes further modifications relative to the reference sequence, as described herein.

[0062] "Adenosine base editor 8 (ABE8) polynucleotide" refers to a polynucleotide that encodes the ABE8 polypeptide.

[0063] "Administration" herein means providing one or more of the compositions described herein to a patient or subject. By way of example and not limitation, administration of the composition (e.g., injection) may be performed via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more of these routes may be used. Parenteral administration may be performed, for example, by rapid bolus injection or gradual perfusion over time. In some embodiments, parenteral administration includes intravascular, intravenous, intramuscular, intra-arterial, intrathecal, intratumoral, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, and intrasternal infusion or injection. Alternatively, or concurrently, administration may be performed via oral route.

[0064] The term "pharmaceutical" means any small molecule compound, antibody, nucleic acid molecule, lipid nanoparticle, peptide, peptide complex, or fragment thereof. In some embodiments, the pharmaceutical agent is a lipid nanoparticle of this disclosure. In some embodiments, the lipid nanoparticle comprises a base editor system of this disclosure. In some cases, the pharmaceutical agent is a base editor system of this disclosure or a component thereof. The base editor system comprises a base editor, or a polynucleotide (e.g., an mRNA molecule) encoding the base editor, and a guide RNA, or a polynucleotide encoding the guide RNA.

[0065] As used herein, the terms “aliphatic” or “aliphatic group” mean a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic (also referred to herein as “carbocyclic,” “carbocyclic,” “aliphatic,” or “cycloalkyl”) having a single connection point to the rest of the molecule. Unless otherwise stated, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-5 carbon atoms. In some embodiments, the aliphatic group contains 1-4 carbon atoms. In some embodiments, the aliphatic group contains 1-3 carbon atoms, and in some embodiments, the aliphatic group contains 1-2 carbon atoms. In some embodiments, “carbocyclic” (or “aliphatic,” “carbocyclic,” or “cycloalkyl”) means an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C6-C8 hydrocarbon. 12 A bicyclic hydrocarbon, which is fully saturated or contains one or more unsaturated units but is not aromatic, has a single connection point to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, ynyl and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0066] As used herein, the term "alkenyl" refers to an alkyl group as defined herein that has one or more double bonds. In some embodiments, the term "alkenyl," used alone or as part of a larger group, refers to an optionally substituted straight-chain or branched hydrocarbon chain that has at least one double bond and has (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-20 C 2-18 C 2-16 C 2-14 C 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 Exemplary alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

[0067] "Alkenyl" refers to a divalent alkenyl group. A substituted alkenyl chain is a polymethylene group containing at least one double bond, wherein one or more hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0068] As used herein, the term "alkyl" has its common meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl, branched alkyl, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups; in some embodiments, the alkyl group has 1 to 100 carbon atoms; in some embodiments, the straight-chain or branched alkyl group has about 1 to 20 carbon atoms in its main chain (e.g., the straight chain is C1-C1). 20 The branch is C2-C. 20 The alkyl group has about 3 to 10 carbon atoms in its ring structure, or has about 5, 6 or 7 carbon atoms in its ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group contains 1 to 4 carbon atoms (e.g., a straight-chain lower alkyl group is C1-C4).

[0069] The term "alkylene" (alkylenyl or alkylene) refers to a divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) that is straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any monovalent alkyl group can become an alkylene group by abstracting a second hydrogen atom from the alkyl group. In some embodiments, "alkylene" is a polymethylene group, i.e., –(CH2). n – where n is a positive integer, preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, or 4 to 8. The substituted alkylene group is a polymethylene group, wherein one or more methylene hydrogen atoms are replaced by a substituent. Suitable substituents include those described below with respect to substituted aliphatic groups.

[0070] As used herein, the term "alkynyl" refers to an alkyl group as defined herein that has one or more triple bonds. In some embodiments, the term "alkynyl," used alone or as part of a larger structural portion, refers to an optionally substituted straight-chain or branched hydrocarbon group that has at least one triple bond and has (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-20 C 2-18 C 2-16 C 2-14 C 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 Exemplary alkynyl groups include ethynyl, propynyl, butynyl, penynyl, hexynyl, and heptynyl.

[0071] "Alteration" refers to a change in the level, structure, or activity of an analyte, gene, or peptide detected by standard methods known in the art, such as those described herein. As used herein, alteration includes changes in expression levels (e.g., increases or decreases). In embodiments, the increase or decrease in expression levels is 10%, 25%, 40%, 50%, or greater. In some embodiments, alteration includes the insertion, deletion, or substitution of nucleobases or amino acids (e.g., through genetic engineering). In some embodiments, the base editor described herein alters the sequence of the TTR polynucleotide.

[0072] "Improvement" means reducing, inhibiting, weakening, mitigating, preventing, or stabilizing the development or progression of a disease. In the implementation plan, the disease is amyloidosis, and the base editor system described herein reduces the accumulation of amyloid protein in the subject's tissues.

[0073] "Analogous molecules" are molecules that are not entirely identical but have similar functions or structural features. For example, peptide analogs retain the biological activity of the corresponding naturally occurring peptide while possessing certain biochemical modifications that enhance the function of the analog relative to the naturally occurring peptide. Such biochemical modifications can improve the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may contain non-natural amino acids.

[0074] The term "aryl" refers to an aryl group having a total of six to fourteen ring members (e.g., C14, C24, C34, C44, C54, C6 ... 6-14 A monocyclic and bicyclic ring system, wherein at least one ring in the system is aromatic, and each ring in the system contains three to seven ring members. The term "aryl" is used interchangeably with the term "aromatic ring." In some embodiments, "aryl" refers to an aromatic ring system, including but not limited to phenyl, naphthyl, anthracene, etc., which may have one or more substituents. Unless otherwise stated, the "aryl" group is a hydrocarbon.

[0075] "Base editor (BE)" or "nucleobase editor polypeptide (NBE)" refers to a substance that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide-binding domain (e.g., Cas9 or Cpf1). Representative nucleic acid and protein sequences of the base editor include those sequences that have approximately or at least about 85% sequence identity with any base editor sequence provided in the sequence listing (e.g., those corresponding to SEQ ID NOs: 2-11).

[0076] "BE4 cytidine deaminase (BE4) polypeptide" refers to a base editor comprising a nucleic acid programmable DNA-binding protein (napDNAbp) domain, a cytidine deaminase domain, and two uracil glycosylation inhibitor domains (UGIs). In an embodiment, the napDNAbp is a Cas9n (D10A) polypeptide. Non-limiting examples of the cytidine deaminase domain include rAPOBEC, ppAPOBEC, RrA3F, AmAPOBEC1, and SsAPOBEC3B.

[0077] "BE4 cytidine deaminase (BE4) polynucleotide" refers to a polynucleotide that encodes the BE4 polypeptide.

[0078] "Base editing activity" refers to the action of chemically altering a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting a target C•G to T•A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A•T to G•C.

[0079] The term "base editor system" or "base editing system" refers to an intermolecular complex used to edit nucleobases in a target nucleotide sequence. In various embodiments, a base editor (BE) system comprises (1) a polynucleotide-programmable nucleotide-binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in a target nucleotide sequence, and (2) one or more guide polynucleotides (e.g., guide RNA) that bind to said polynucleotide-programmable nucleotide-binding domain. In various embodiments, a base editor (BE) system comprises a nucleobase editor domain selected from adenosine deaminase or cytidine deaminase, and a domain having nucleic acid sequence-specific binding activity. In some embodiments, a base editor system comprises (1) a base editor (BE) comprising a polynucleotide-programmable DNA-binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs that bind to said polynucleotide-programmable DNA-binding domain. In some embodiments, said polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine or cytosine base editor (CBE). In some embodiments, the base editor system (e.g., a base editor system comprising cytidine deaminase) comprises a uracil glycosylase inhibitor or other agent or peptide that inhibits the inosine base excision repair system (e.g., a uracil-stabilizing protein as provided in WO2022015969, the entire disclosure of which is incorporated herein by reference for all purposes).

[0080] As used in this article, the term "divalent" refers to a chemical moiety with two connection points. For example, "divalent C..." 1-8 (or C) 1-6 "Saturated or unsaturated, straight or branched hydrocarbon chains" refers to straight or branched divalent alkylene, alkenylene, and ynylene chains as defined herein.

[0081] As used herein, the term "bridged bicyclic" refers to any bicyclic ring system, i.e., a carbocyclic or heterocyclic ring, saturated or partially unsaturated, having at least one bridge. According to the IUPAC definition, a "bridge" is an unbranched chain of atoms, an atom, or a valence bond connecting two bridgehead atoms, wherein a "bridgehead atom" is any skeletal atom in the system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups listed below, wherein each group is attached to the remainder of the molecule at any substituted carbon or nitrogen atom. Unless otherwise stated, the bridged bicyclic group is optionally substituted with one or more substituents as described for aliphatic groups. Additionally or alternatively, any substituted nitrogen atom of the bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to: As used herein, the terms “carbocyclic,” “carbocyclic,” and “carbocyclic ring” refer to a saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring system having 3 to 14 members, as described herein, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, “carbocyclic” (or “alicyclic”) refers to an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C6-C… 12 A bicyclic hydrocarbon is a fully saturated or non-aromatic hydrocarbon containing one or more unsaturated units and having a single connection point to the rest of the molecule. The term "cycloalkyl" refers to an optionally substituted saturated ring system having about 3 to about 10 ring carbon atoms. In some embodiments, the cycloalkyl group has 3-6 carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0082] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing the active, inactive, or partially active DNA-cutting domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also called casnl nucleases or CRISPR (clustered regularly spaced short palindromic repeats)-associated nucleases.

[0083] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another, which shares common characteristics. One functional approach to defining common characteristics among amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins from homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analyses, amino acid groups can be defined where amino acids within a group preferentially exchange with each other, thus having the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, ibid.). Non-restrictive examples of conserved mutations include amino acid substitutions, such as lysine replacing arginine and vice versa, to maintain a positive charge; glutamic acid replacing aspartic acid and vice versa, to maintain a negative charge; serine replacing threonine to maintain a free –OH group; and glutamine replacing asparagine to maintain a free –NH2 group.

[0084] Amino acids can generally be classified into several categories based on the following common side chain properties: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, He; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

[0085] In some embodiments, conservative substitution may involve exchanging a member of one of these categories for another member of the same category. In some embodiments, non-conservative amino acid substitution may involve exchanging a member of one of these categories for another.

[0086] As used interchangeably in this document, the terms "coding sequence" or "protein-coding sequence" refer to a segment of a polynucleotide that encodes a protein. A coding sequence may also be called an open reading frame. This region or sequence is defined by a start codon closer to the 5′ end and a stop codon closer to the 3′ end. Stop codons that can be used with the base editor described herein include the following: TAG, TAA, and TGA.

[0087] A “complex” refers to a combination of two or more molecules interacting through intermolecular forces. Non-limiting examples of intermolecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonds, ionic bonds, halogen bonds, hydrophobic bonds, van der Waals forces (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces), and the π effect. In one embodiment, the complex comprises a polypeptide, a polynucleotide, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, the complex comprises one or more polypeptides that bind to form a base editor (e.g., a base editor comprising a nucleic acid programmable DNA-binding protein (such as Cas9) and a deaminase) and a polynucleotide (e.g., guide RNA). In one embodiment, the complex is bound together by hydrogen bonds. It should be understood that one or more components of the base editor (e.g., a deaminase or a nucleic acid programmable DNA-binding protein) may be covalently or non-covalently bound. As an example, the base editor may include a deaminase covalently linked to a nucleic acid programmable DNA-binding protein (e.g., via a peptide bond). Alternatively, the base editor may include a non-covalently bound deaminase and a nucleic acid-programmable DNA-binding protein (e.g., wherein one or more components of the base editor are provided in a trans form and bind directly or through another molecule, such as a protein or nucleic acid). In one embodiment, one or more components of the complex are linked together by hydrogen bonds.

[0088] "Cytosine" or "4-aminopyrimidine-2(1H)-one" refers to a purine nucleobase with the molecular formula C4H5N3O, whose structure is... And it corresponds to CAS number 71-30-7.

[0089] "Cytidine" refers to a cytosine molecule linked to ribose via a glycosidic bond, and its structure is as follows: It corresponds to CAS number 65-46-3. Its molecular formula is C9H. 13 N3O5.

[0090] "Cytidine base editor" refers to a base editor that includes cytidine deaminase.

[0091] "Cytidine base editor (CBE) polynucleotide" refers to a polynucleotide that encodes CBE.

[0092] "Cytidine deaminase" or "cytosine deaminase" refers to a polypeptide or fragment thereof capable of deaminating cytidine or cytosine. In embodiments, the cytidine or cytosine is present in a polynucleotide. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. The terms "cytidine deaminase" and "cytosine deaminase" are used interchangeably throughout the application. (The text then abruptly shifts to a seemingly unrelated topic: sea lamprey...) Petromyzon marinusCytosine deaminase 1 (PmCDA1) (SEQ ID NO: 13-14), activation-inducible cytidine deaminase (AICDA) (SEQ ID NOs: 15-21), and APOBEC (SEQ ID NOs: 12-61) are exemplary cytidine deaminases. Further exemplary cytidine deaminase (CDA) sequences are provided in the sequence listing as SEQ ID NOs: 62-66 and SEQ ID NOs: 67-189. Non-limiting examples of cytidine deaminases include those described in PCT / US20 / 16288, PCT / US2018 / 021878, 180802-021804 / PCT, PCT / US2018 / 048969, and PCT / US2016 / 058344.

[0093] "Cytosine deaminase activity" refers to the catalytic deamination reaction of cytosine or cytidine. In one embodiment, a polypeptide having cytosine deaminase activity converts an amino group to a carbonyl group. In one embodiment, a cytosine deaminase converts cytosine to uracil (i.e., C to U) or converts 5-methylcytosine to thymine (i.e., 5mC to T). In some embodiments, the cytosine deaminases provided herein have increased cytosine deaminase activity relative to a reference cytosine deaminase (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more).

[0094] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or fragment thereof that catalyzes a deamination reaction.

[0095] "Disease" refers to any condition or symptom that impairs or interferes with the normal function of cells, tissues, or organs. Exemplary diseases include those suitable for treatment using the methods and / or compositions of this disclosure, and non-limiting examples include amyloidosis, cardiomyopathy, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), familial transthyretin amyloidosis (FTA), senile systemic amyloidosis (SSA), and transthyretin amyloidosis. The disease can be any disease associated with a mutation in the transthyretin (TTR) polynucleotide sequence.

[0096] "Dual editing activity" or "dual deaminase activity" refers to the presence of both adenosine deaminase and cytidine deaminase activity. In one embodiment, a base editor with dual editing activity possesses both A→G and C→T activities, wherein the two activities are approximately equal or differ from each other by about 10% or 20%. In another embodiment, the A→G activity of the dual editor does not exceed about 10% or 20% of its C→T activity. In yet another embodiment, the A→G activity of the dual editor is not less than about 10% or 20% of its C→T activity. In some embodiments, the adenosine deaminase variant primarily possesses cytosine deaminase activity and has little to no (if any) adenosine deaminase activity. In some embodiments, the adenosine deaminase variant possesses cytosine deaminase activity but does not have significant or detectable adenosine deaminase activity.

[0097] "Effective amount" refers to the amount of the agent described herein (e.g., base editor, cells) that is required to improve disease symptoms relative to an untreated patient or disease-free individual (i.e., a healthy individual), or a dose sufficient to elicit the desired biological response. The effective amount of the active compound used to practice embodiments of this disclosure to therapeutically treat a disease varies depending on the route of administration, the subject's age, weight, and overall health condition. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as the "effective" amount. In one embodiment, the effective amount is sufficient to introduce an altered base editor of this disclosure into a target gene in cells (e.g., in vitro or in vivo cells). In one embodiment, the effective amount is the amount of base editor required to achieve a therapeutic effect. Such a therapeutic effect does not require alteration of the pathogenic gene in all cells of a subject, tissue, or organ, but only alteration of the pathogenic gene present in approximately 1%, 5%, 10%, 25%, 50%, 75%, or more cells of a subject, tissue, or organ. In one embodiment, the effective amount is sufficient to improve one or more symptoms of the disease. In one implementation, the effective amount of the base editor is sufficient to alter the TTR polynucleotide sequence in 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the cells in the tissue.

[0098] The term "encapsulated" is used herein to refer to a substance that is completely surrounded by another material. In one embodiment, an mRNA molecule encoding a base editor and / or guide polynucleotide is encapsulated by lipid nanoparticles of this disclosure.

[0099] A “fragment” refers to a portion of a polypeptide or nucleic acid molecule. This portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. In some embodiments, the fragment is a functional fragment.

[0100] "Guide polynucleotide" refers to a polynucleotide or polynucleotide complex that is specific to a target sequence and capable of forming a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNA can exist as a complex of two or more RNA molecules or as a single RNA molecule.

[0101] The term "haloaliphatic" refers to an aliphatic group substituted with one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halogens, such as fluorine, iodine, bromine, or chlorine). In some embodiments, the haloaliphatic group contains 1-7 halogen atoms. In some embodiments, the haloaliphatic group contains 1-5 halogen atoms. In some embodiments, the haloaliphatic group contains 1-3 halogen atoms.

[0102] The term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halogens, such as fluorine, iodine, bromine, or chlorine). In some embodiments, the haloalkyl group contains 1-7 halogen atoms. In some embodiments, the haloalkyl group contains 1-5 halogen atoms. In some embodiments, the haloalkyl group contains 1-3 halogen atoms.

[0103] As used herein, the term "heteroalkylenyl" or "heteroalkylene" refers to an optionally substituted straight-chain (i.e., unbranched) or branched divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) having one to five heteroatoms in addition to carbon atoms. The term "heteroatom" is described below. In some embodiments, the heteroalkyl group contains 2–10 carbon atoms, wherein 1–3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroalkyl group contains 2–8 carbon atoms, wherein 1–3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroalkyl group contains 4–8 carbon atoms, wherein 1–3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroalkyl group contains 2–5 carbon atoms, wherein 1–2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In other embodiments, the heteroalkyl group contains 1–3 carbon atoms, wherein one carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroalkyl groups include, but are not limited to, -CH2O-, -(CH2)2O-, -CH2OCH2-, -O(CH2)2-, -(CH2)3O-, -(CH2)2OCH2-, -CH2O(CH2)2-, -O(CH2)3-, -(CH2)4O-, -(CH2)3OCH2-, -CH2O(CH2)3-, -(CH2)2O(CH2)2-, and -O(CH2)4-. Unless otherwise stated, C x Heteroalkyl refers to a heteroalkyl group that has x carbon atoms before being replaced by heteroatoms.

[0104] The terms “heteroaryl” and “heteroaryl–” (e.g., “heteroarylalkyl” or “heteroarylalkoxy”) used alone or as part of a larger structural part refer to a monocyclic or bicyclic cyclic group having 5 to 10 ring atoms (e.g., a 5 to 6-membered monocyclic heteroaryl or a 9 to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π electrons shared in the cyclic array; and having one to five heteroatoms in addition to carbon atoms. Exemplary heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, pyridinyl, pyridonel, pyridazinyl, pyrimidinyl, indoleazinyl, purine, naphridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thiophenezopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroary-” as used herein also include groups in which a heteroaryl ring is fused with one or more aryl, alicyclic, or heterocyclic rings, wherein the linking group or linking point is on the heteroaryl ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, inzolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, terpineyl, phthalazinyl, quinazolinyl, quinoxolinyl, 4H-quinazinyl, carbazolyl, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3–b]–1,4–oxazin–3(4H)–one, and benzoisooxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaromatic ring,” “heteroaryl group,” or “heteroaromatic,” any of which includes an optionally substituted ring.

[0105] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of basic nitrogen; or a nitrogen that can be substituted on a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolealkyl), or NR). + (e.g., in N-substituted pyrroleyl groups).

[0106] The terms “heterocycle,” “heterocyclic group,” “heterocyclic group,” and “heterocyclic ring” are used interchangeably herein to refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (e.g., one to four) heteroatoms as defined above, in addition to a carbon atom. When referring to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolidinyl), or NR.+ (e.g., in N-substituted pyrrolidinyl groups). Heterocyclic rings can be attached to their side groups at any heteroatom or carbon atom, provided a stable structure is produced, and any ring atom can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, azirrobutyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolyl, piperazinyl, tetrahydropyranyl, dioxalyl, dioxopentyl, diazaheptanyl, oxazaheptanyl, thiazaheptanyl, morpholinyl, thiomorpholinyl, and... The heterocyclic group can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. The term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl and heterocyclic portions are optionally substituted independently. Bicyclic heterocyclic rings also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl, or alicyclic rings. Exemplary bicyclic heterocyclic groups include indololinyl, isoindololinyl, benzodioxanepentyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocyclic rings can also be spirocyclic systems (e.g., 7- to 11-membered spirocyclic fused heterocyclic rings having one or more heteroatoms as defined above (e.g., one, two, three, or four heteroatoms) in addition to a carbon atom). Bicyclic heterocyclic rings can also be bridged ring systems (e.g., 7- to 11-membered bridged heterocyclic rings having one, two, or three bridge atoms).

[0107] "Hybridization" refers to the hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that pair up by forming hydrogen bonds.

[0108] "Increase" refers to a positive change of at least 10%, 25%, 50%, 75%, or 100%, or approximately 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, or 100 times.

[0109] "Base repair inhibitors," "IBR," or their grammatical equivalents refer to proteins that can inhibit the activity of nucleic acid repair enzymes (such as base excision repair enzymes).

[0110] The terms "isolated," "purified," or "biologically pure" refer to materials that are, to a certain extent, free from components that are normally present in their native state. "Isolated" indicates the degree of separation from the original source or surrounding environment. "Purified" indicates a degree of separation beyond that of isolated. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the protein's biological properties or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced using recombinant DNA technology, or substantially free of chemical precursors or other chemicals during chemical synthesis. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can also mean that the nucleic acid or protein produces essentially a single band in the electrophoresis gel. For proteins that can be modified (e.g., phosphorylation or glycosylation), different modifications may produce different isolated proteins that can be purified separately.

[0111] "Separated polynucleotide" refers to a nucleic acid molecule that does not contain a gene flanking the gene in the naturally occurring genome of the organism from which the nucleic acid molecule originates. Therefore, this term includes, for example, recombinant DNA integrated into a vector; recombinant DNA integrated into an autonomously replicating plasmid or virus; or recombinant DNA integrated into prokaryotic or eukaryotic genomic DNA; or recombinant DNA existing as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). Furthermore, this term includes RNA molecules transcribed from DNA molecules, and recombinant DNA as part of a heterozygous gene encoding an additional polypeptide.

[0112] "Isolated polypeptide" refers to the polypeptide of this disclosure, which has been separated from its naturally occurring accompanying components. Generally, a polypeptide is considered isolated when it contains at least 60% by weight of proteins and naturally occurring organic molecules naturally associated with it. In embodiments, the preparation is at least 75%, at least 90%, or at least 99% by weight of the polypeptide of this disclosure. The isolated polypeptide of this disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0113] As used herein, the term "linker" refers to a molecule that connects two parts. In one embodiment, the term "linker" refers to a covalent linker (e.g., a covalent bond) or a non-covalent linker. For example, those skilled in the art will understand that polypeptides whose structures comprise two or more functional or tissue domains typically include an amino acid sequence between these domains, linking them to each other. In some embodiments, polypeptides containing the linker element "L'" have an overall structure of the general formula S1-L'-S2, where S1 and S2 may be the same or different and represent two domains associated with each other by the linker. In some embodiments, the length of the peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized by its tendency to provide flexibility to the peptide rather than a rigid three-dimensional structure. A variety of different linker elements known in the art can be suitably used for engineered peptides (e.g., fusion peptides) (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1 121-1123).

[0114] As used herein, “lipid nanoparticle (LNP) composition” refers to a nanoparticle composition comprising one or more ionizable lipids. Exemplary LNP compositions include, but are not limited to, phospholipids, conjugated-linked lipids (e.g., PEG-lipids), sterols, and ionizable lipids (e.g., IZ1, IZ4).

[0115] As used herein, the term “mutation” refers to the substitution of one residue in a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by specifying the original residue, followed by its position in the sequence, and then the newly substituted residue. Various methods for preparing the amino acid substitutions (mutations) presented herein are well known in the art and are provided, for example, by Green and Sambrook in *Molecular Cloning: A Laboratory Manual* (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0116] The “N / P ratio” used herein refers to the molar ratio of ionizable nitrogen atoms in lipids to phosphate groups in nucleic acid molecular entities. In some embodiments, the nitrogen atoms are ionizable within a physiological pH range. In some embodiments, the molecular entities in the nanoparticle composition include lipid components and RNA. Ionizable nitrogen atoms may include, for example, nitrogen atoms that are protonable at, for example, pH 1, pH 2, pH 3, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, or pH 8 or higher. The physiological pH range may include, for example, the pH range of different cellular compartments (such as organs, tissues, and cells) and body fluids (such as blood, cerebrospinal fluid, gastric juice, breast milk, bile, saliva, tears, and urine). In some specific embodiments, the physiological pH range refers to the pH range of mammalian blood, for example, from about 7.35 to about 7.45. Similarly, for phosphate charge neutralizers having one or more ionizable nitrogen atoms, the N / P ratio can refer to the molar ratio of ionizable nitrogen atoms to phosphate groups in the nucleic acid. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable in the pH range of 5 to 14.

[0117] For loadings that do not contain phosphate groups, the N / P ratio can refer to the molar ratio of ionizable nitrogen atoms in the lipid to the total negative charge in the loading. 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 charge in the loading present in the composition.

[0118] As used herein, the term "nanoparticle" refers to a particle with a diameter of less than 1000 nanometers (nm). In some embodiments, the nanoparticles have a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, the nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, the nanoparticles are micelles because they contain enclosed compartments that are separated from the bulk solution by a micelle membrane, which is typically composed of an amphiphilic entity that surrounds and encloses a space or compartment (e.g., to define an inner cavity). In some embodiments, the micelle membrane contains at least one polymer, such as a biocompatible and / or biodegradable polymer. In some embodiments, the lipid nanoparticles described herein may have an average hydrodynamic diameter of about 30 to about 170 nm. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein may be 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, 155 nm, 160 nm, 165 nm, 170 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range defined by any two of the foregoing values ​​as endpoints. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 50 nm to 100 nm. For example, in some embodiments, the compositions, formulations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of 50 nm to 90 nm. In some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of about 60 to about 80 nm. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 50 nm to 70 nm.

[0119] As used herein, the term "nanoparticle composition" refers to a composition containing at least one nanoparticle and at least one additional pharmaceutical agent or ingredient. In some embodiments, the nanoparticle composition contains a substantially uniform aggregate of nanoparticles as described herein.

[0120] As used herein, the terms “nucleic acid” and “nucleic acid molecule” refer to compounds comprising nucleobases and an acidic moiety (e.g., nucleosides, nucleotides, or nucleotide polymers). Typically, polymeric nucleic acids (e.g., nucleic acid molecules comprising three or more nucleotides) are linear molecules in which adjacent nucleotides are linked together by phosphodiester bonds. In some embodiments, “nucleic acid” refers to a single nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more single nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” are used interchangeably to refer to a nucleotide polymer (e.g., a chain having at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, for example, in the context of genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, granules, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or molecules comprising non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having a structure other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, generated using a recombinant expression system and optionally purified, chemically synthesized, etc. Where appropriate, such as in the case of chemically synthesized molecules, nucleic acids contain nucleoside analogs, such as analogs having chemically modified bases or sugars, and backbone modifications. Unless otherwise stated, nucleic acid sequences are presented in a 5′ to 3′ orientation. In some embodiments, nucleic acids are or contain natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylur ... Cytidine, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g. methylated bases); intercalated bases; modified sugars (e.g. 2′-fluororibose, ribose, 2′-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g. thiophosphates and 5′-N-phosphoramide bonds).

[0121] The terms “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refer to an amino acid sequence that facilitates protein entry into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al.’s international PCT application PCT / EP2000 / 011690 (filed November 23, 2000, published May 31, 2001 as WO / 2001 / 038547), the disclosure of which regarding exemplary nuclear localization sequences is incorporated herein by reference. In other embodiments, the NLS is an optimized NLS, such as that described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some implementations, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 190), KRPAATKKAGQAKKKK (SEQ ID NO: 191), KKTELQTTNAENKTKKL (SEQ ID NO: 192), KRGINDRNFWRGENGRKTR (SEQ ID NO: 193), RKSGKIAAIVVKRPRK (SEQ ID NO: 194), PKKKRKV (SEQ ID NO: 195), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 196), PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328), or RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 329).

[0122] The terms “nucleobase,” “nitrogenous base,” or “base” are used interchangeably herein to refer to nitrogen-containing biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to form base pairs and stack on top of each other directly results in long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are referred to as primary or classical bases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other modified (non-primary) bases. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced in the presence of mutagens, both through deamination (replacing the amino group with a carbonyl group). Hypoxanthine can be derived from adenine. Xanthine can be derived from guanine. Uracil can be produced by deamination of cytosine. A "nucleoside" consists of a nucleobase and a pentose sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a pentose sugar (ribose or deoxyribose), and at least one phosphate group. Non-limiting examples of chemically modified nucleobases that may be included include: pseudouridine, 5-methylcytosine, 2′-O-methyl-3′-phosphonoacetate, 2′-O-methylthio-PACE (MSP), 2′-O-methyl-PACE (MP), 2′-fluoroRNA (2′-F-RNA), constrained ethyl (S-cEt), 2′-O-methyl ('M'), 2′-O-methyl-3′-thiophosphate ('MS'), 2′-O-methyl-3′-thiophosphonoacetate ('MSP'), 5-methoxyuridine, thiophosphate, and N1-methylpseudouridine.

[0123] The terms "nucleic acid programmable DNA-binding protein" or "napDNAbp" are used interchangeably with "polynucleotide programmable nucleotide-binding domain" and refer to a protein that binds to a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), which guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide-binding domain is a polynucleotide programmable DNA-binding domain. In some embodiments, the polynucleotide programmable nucleotide-binding domain is a polynucleotide programmable RNA-binding domain. In some embodiments, the polynucleotide programmable nucleotide-binding domain is a Cas9 protein. The Cas9 protein can bind to a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ (Cas12j / Casphi).Non-restricted examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, and Cse5e. Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, their homologs, or modified or engineered versions thereof. Other nucleic acid-programmable DNA-binding proteins are also within the scope of this disclosure, although they may not be specifically listed herein. See, for example, Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct;1:325-336. doi:10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cassystems” Science. 2019 Jan 4; 363(6422):88-91. doi: 10.1126 / science.aav7271, the full contents of each of which are incorporated herein by reference. Exemplary nucleic acid-programmable DNA-binding proteins and nucleic acid sequences encoding them are provided in the sequence listing as SEQ ID NO: 197-245, 254-260 and 378.In some embodiments, the napDNAbp is a Cas9 endonuclease (CRISPR-related system), such as Cas9 (Csnl) from Streptococcus pyogenes (e.g., SEQ ID NO: 197), Cas9 (NmeCas9; SEQ ID NO: 208) from Neisseria meningitidis, Nme2Cas9 (SEQ ID NO: 209), Cas9 (ScoCas9) from Streptococcus constellatus, or derivatives thereof (e.g., sequences having at least about 85% sequence identity with Cas9 (such as Nme2Cas9 or spCas9)).

[0124] As used herein, the terms "nucleobase editing domain" or "nucleobase editing protein" refer to proteins or enzymes capable of catalyzing nucleobase modifications in RNA or DNA, such as the conversion of cytosine (or cytidine) to uracil (or uridine) or thymidine (or thymidine), the deamination of adenine (or adenosine) to hypoxanthine (or inosine), and the addition and insertion of non-template-based nucleotides. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase; or cytidine deaminase or cytosine deaminase).

[0125] As used herein, “obtaining”, such as “obtaining an agent”, includes synthesizing, purchasing, or otherwise acquiring the agent.

[0126] "Patients in need" or "subjects in need" in this article refers to patients who are diagnosed with, at risk of having, pre-determined to have, or suspected of having a certain disease or condition.

[0127] The terms "pathogenic mutation," "pathogenic variant," "pathogenic mutation," "pathogenic agent," "harmful mutation," or "susceptibility mutation" refer to a genetic alteration or mutation that is associated with or increases an individual's susceptibility or predisposition to a disease or condition. In some embodiments, a pathogenic mutation includes the substitution of at least one wild-type amino acid in a protein encoded by a gene for at least one pathogenic amino acid. In some embodiments, the pathogenic mutation is located in a stop region (e.g., a stop codon). In some embodiments, the pathogenic mutation is located in a non-coding region (e.g., an intron, a promoter, etc.).

[0128] As used herein, the term "pharmaceutically acceptable salt" means a salt of the compound used in the pharmaceutical context, that is, a salt suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. have described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0129] As used herein, "phospholipid" can refer to a lipid comprising a phosphate ester moiety and one or more carbon chains (e.g., unsaturated fatty acid chains). Phospholipids may contain one or more multiple bonds (e.g., double or triple bonds). In some embodiments, phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with the membrane can allow one or more elements of the LNP to cross the membrane, i.e., deliver the one or more elements into the cell.

[0130] The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably in this document to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0131] The term "fusion protein" in this document refers to a hybrid polypeptide that contains protein domains derived from at least two different proteins.

[0132] As used herein, the term "recombinant" in the context of proteins or nucleic acids refers to a protein or nucleic acid that is not found in nature but is a product of human engineering. For example, in some embodiments, the recombinant protein or nucleic acid molecule contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in its amino acid or nucleotide sequence compared to any naturally occurring sequence.

[0133] "Reduction" refers to a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0134] “Reference” means a standard or control condition. In one embodiment, the reference is wild-type or healthy cells. In other embodiments, and not limited thereto, the reference is untreated cells not subjected to the test condition, or cells receiving a placebo or saline, culture medium, buffer, and / or a control vector not containing the target polynucleotide. The reference may be cells or subjects having pathogenic mutations in the transthyretin (TTR) polynucleotide sequence and / or the transthyretin (TTR) polypeptide sequence. The reference may be subjects or cells with amyloidosis (e.g., transthyretin amyloidosis), or subjects or cells without amyloidosis. In one embodiment, the level of amyloidosis in cells or tissues treated with the base editor system described herein is compared with the level of amyloidosis in corresponding untreated cells or tissues.

[0135] A “reference sequence” is a defined sequence used as a benchmark for sequence comparison. The reference sequence can be a subset or the entirety of a specific sequence; for example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For peptides, the reference peptide sequence will typically be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids in length. For nucleic acids, the reference nucleic acid sequence will typically be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides in length, or any integer close to or between these values. In some embodiments, the reference sequence is the wild-type sequence of the target protein. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0136] As used herein, the term "sterol group" refers to a 17-membered fused polycyclic moiety that is saturated or partially unsaturated, substituted with at least one hydroxyl group, and has a single connection point to the remainder of the molecule at any substituted carbon or oxygen atom. In some embodiments, the sterol group is a cholesterol group, or a variant or derivative thereof. In some embodiments, the cholesterol group is modified. In some embodiments, the cholesterol group is an oxidized cholesterol group (e.g., oxidized on a β-ring structure or a hydrocarbon tail structure). In some embodiments, the cholesterol group is an esterified cholesterol group. In some embodiments, the sterol group is a phytosterol group. Exemplary sterol groups include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, β-sitosterol, stigmasterol, brassicasterolyl, and campesterolyl.

[0137] The term "subject" or "patient" refers to a mammal, including but not limited to human or non-human mammals. In one embodiment, the mammal is a cow, horse, dog, sheep, rabbit, rodent, non-human primate, or cat. In one embodiment, "patient" refers to a mammalian subject with a higher than average probability of developing a disease or condition. Exemplary patients may be humans, non-human primates, cats, dogs, pigs, cows, horses, camels, alpacas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female.

[0138] As described herein, the compounds disclosed may be described as “substituted” or “optionally substituted.” That is, the compounds may contain optionally substituted and / or substituted moieties. Generally, regardless of whether the term “optionally” is preceding it, the term “substituted” means that one or more hydrogens of the specified moieties are replaced by suitable substituents. “Substituted” applies to one or more hydrogens that are explicitly or implicitly present in the structure (e.g., At least refers to ;and At least refers to , , or Unless otherwise stated, an "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure may be substituted by more than one substituent selected from the specified group. The combinations of substituents included in this disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" means a compound that remains substantially unchanged when subjected to conditions permissible for its preparation, detection, and, in some embodiments, its recovery, purification, and use for one or more of the purposes described herein. Groups described as "substituted" preferably have 1 to 4 substituents, more preferably 1 or 2 substituents. Groups described as "optionally substituted" may be unsubstituted or "substituted" as described above.

[0139] Suitable monovalent substituents include halogens; –(CH2) 0–4 R°;–(CH2) 0–4 OR°;–O(CH2) 0-4 R o ,–O–(CH2) 0–4 C(O)OR°;–(CH2) 0–4 CH(OR°)2;–(CH2) 0–4 Ph, which can be replaced by R°; -(CH2) 0–4 O(CH2)0–1 Ph, which can be substituted by R°; –CH=CHPh, which can be substituted by R°; –(CH2) 0–4 O(CH2) 0–1 -pyridyl group, which can be substituted by R°; –NO2; –CN; –N3; -(CH2) 0–4 N(R°)2;–(CH2) 0–4 N(R°)C(O)R°; –N(R°)C(S)R°; –(CH2) 0–4 N(R°)C(O)NR°2; -N(R°)C(S)NR°2; –(CH2) 0–4 N(R°)C(O)OR°; –N(R°)N(R°)C(O)R°; –N(R°)N(R°)C(O)NR°2; –N(R°)N(R°)C(O)OR°; –(CH2) 0–4 C(O)R°; –C(S)R°; –(CH2) 0–4 C(O)OR°;–(CH2) 0–4 C(O)SR°;(CH2) 0–4 C(O)OSiR°3;–(CH2) 0–4 OC(O)R°;–OC(O)(CH2) 0–4 SR°–,–SC(S)SR°;-(CH2) 0–4 SC(O)R°;–(CH2) 0–4 C(O)NR°2; –C(S)NR°2; –C(S)SR°; –SC(S)SR°; –(CH2) 0–4 OC(O)NR°2; -C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; –C(NOR°)R°; (CH2) 0–4 SSR°;–(CH2) 0–4 S(O)2R°;–(CH2) 0–4 S(O)₂OR°;–(CH₂) 0–4 OS(O)2R°; –S(O)2NR°2; -(CH2) 0–4 S(O)R°; –N(R°)S(O)2NR°2; –N(R°)S(O)2R°; –N(OR°)R°; –C(NH)NR°2; –P(O)2R°; –P(O)R°2; –OP(O)R°2; –OP(O)(OR°)2; –SiR°3; –OSiR°3; –(C 1–4 (linear or branched alkylene)O–N(R°)2; or –(C 1–4(straight-chain or branched alkylene)C(O)O–N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C 1–6 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, -CH2- (5-6 membered heteroaromatic ring), or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independently occurring R° together with one or more atoms between them to form a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

[0140] A suitable monovalent substituent at R° (or a ring formed by two independently occurring R° atoms together with one or more atoms in between) is independently a halogen, –(CH2). 0–2 R ● –(halogenated R) ● ), –(CH2) 0–2 OH, –(CH2) 0–2 OR ● –(CH2) 0–2 CH(OR ● )2、–O(halogenated R ● –CN, –N3, –(CH2) 0–2 C(O)R ● –(CH2) 0–2 C(O)OH, –(CH2) 0–2 C(O)OR ● –(CH2) 0–2 C(O)NH2、–(CH2) 0–2 C(O)NHR ● –(CH2) 0–2 C(O)NR ● 2. –(CH2) 0–2 SR ● –(CH2) 0–2 SH, –(CH2) 0–2 NH2、–(CH2) 0–2 NHR ● –(CH2) 0–2 NR ● 2, –NO2, –SiR ● 3. –OSiR ● 3. -C(O)SR ● 、–(C 1–4 (straight-chain or branched alkylene)C(O)OR ● 、or –SSR ● , where each R● It is either unsubstituted or, when preceded by "halogenated", substituted by only one or more halogens, and independently selected from C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 5–6 membered saturated, partially unsaturated, or aromatic ring having 0–4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on the saturated carbon atom at R° include =O and =S.

[0141] Suitable divalent substituents include the following: =O, =S, =NNR * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * 、 –O(C(R) * 2)) 2–3 O–, or –S(C(R) * 2)) 2–3 S–, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1–6 Aliphatic group, or an unsubstituted 5–6 membered saturated, partially unsaturated, or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the ortho-substituted carbon atom of the "optionally substituted" group include: –O(CR * 2) 2–3 O–, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1–6 Aliphatic group, or unsubstituted 5–6-membered saturated, partially unsaturated or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0142] R * Suitable substituents on aliphatic groups include halogens, -R ● 、(halogenated R) ● –OH, –OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, when preceded by "halogenated", substituted by only one or more halogens, and independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1Ph, or a 5–6 saturated, partially unsaturated or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0143] In some embodiments, suitable substituents that can replace nitrogen include –R † –NR † 2. –C(O)R † –C(O)OR † –C(O)C(O)R † –C(O)CH2C(O)R † –S(O)2R † –S(O)2NR † 2. –C(S)NR † 2. –C(NH)NR † 2 or –N(R) † )S(O)2R † ; where each R † Independently, hydrogen, and C that can be substituted as defined below. 1–6 Aliphatic groups, unsubstituted –OPh, or unsubstituted 5–6-membered saturated, partially unsaturated, or aromatic rings having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, despite the above definition, two independently occurring R groups. † Together with one or more atoms therebetween, they form unsubstituted 3–12 saturated, partially unsaturated, or aromatic monocyclic or bicyclic rings with 0–4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0144] R † Suitable substituents on the aliphatic group can be halogens, -R ● 、(halogenated R) ● –OH, –OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, when preceded by "halogenated", substituted by only one or more halogens, and independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 5–6 saturated, partially unsaturated or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0145] When applied to the compositions described herein, the term “stable” means that the composition retains one or more aspects of its physical structure (e.g., particle size range and / or distribution) over a period of time. In some embodiments, a stable nanoparticle composition is a composition that, under specified conditions, retains an average particle size, a maximum particle size, a particle size range, and / or a particle size distribution (i.e., the percentage of particles exceeding a specified size and / or exceeding a specified size range) over a period of time. In some embodiments, the provided stable composition is a composition that retains biologically relevant activity over a period of time. In some embodiments, the time period is at least about one hour; in some embodiments, the time period is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the time period is in the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. For example, if a group of nanoparticles is subjected to long-term storage, temperature changes and / or pH changes, and most of the nanoparticles in the composition maintain a diameter within the range stated, then the nanoparticle composition is stable. In some embodiments, the stable composition is stable under environmental conditions. In some embodiments, the stable composition is stable under biological conditions (i.e., 37 °C in phosphate-buffered saline).

[0146] "Substantial identity" means a polypeptide or nucleic acid molecule that has at least 50% identity with a reference amino acid sequence. In one embodiment, the reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, the reference sequence is any of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence has at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99% identity with the sequence used for comparison at the amino acid or nucleic acid level.

[0147] Sequence identity is typically measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conserved substitutions typically include those within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0148] Nucleic acid molecules that can be used in the methods of this disclosure include any nucleic acid molecule encoding a polypeptide or a functional fragment thereof of the present disclosure. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. Polynucleotides having “substantial identity” with an endogenous sequence are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” means, under various stringent conditions, the pairing of complementary polynucleotide sequences (e.g., the gene described herein) or portions thereof to form a double-stranded molecule. (See, for example, Wahl, GM and SL Berger (1987) MethodsEnzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0149] The term "target site" refers to a target nucleotide sequence or nucleobase within a nucleic acid molecule that is being modified. In embodiments, the modification is a base deamination. The deaminase may be a cytidine deaminase or an adenine deaminase. Fusion proteins or base editing complexes containing deaminases may comprise dCas9-adenine deaminase fusion proteins, Cas12b-adenine deaminase fusions, or base editors disclosed herein.

[0150] "Transthyretin (TTR) polypeptide" refers to a polypeptide or fragment thereof having at least about 95% amino acid sequence identity with the amino acid sequence or fragment thereof provided in NCBI reference sequence number NP_000362.1 and binding to an anti-TTR antibody. In some embodiments, the TTR polypeptide or fragment thereof has all-trans retinol-binding protein (RBP) and / or thyroxine (T4) transport activity. In embodiments, TTR is capable of forming a tetramer. An exemplary TTR polypeptide sequence is shown below (signal peptide sequences are shown in bold; therefore, a mature TTR polypeptide corresponds to amino acids 21 to 147 of the following sequence): MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE (SEQ ID NO: 464).

[0151] "Transthyretin (TTR) polynucleotide" refers to a nucleic acid molecule encoding a TTR polypeptide, as well as introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences, or fragments thereof, associated with its expression. In embodiments, the regulatory sequence is a promoter region. In embodiments, the TTR polynucleotide is a genomic sequence, cDNA, mRNA, or gene associated with and / or required for TTR expression. Exemplary TTR polynucleotide sequences (corresponding to Common Coding Sequence (CCDS) number 11899.1) are provided below. Further exemplary TTR polynucleotide sequences include the gene Ensembl ID: ENSG00000118271 and the transcript Ensembl ID: ENST00000237014.8.

[0152] ATGGCTCTCATCGTCTGCTCCTCCTCTGCCTTGCTGGACTGGTATTTGTGTCTGAGGCTGGCCCTACGGGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTG CATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATGA (SEQ ID NO: 465).

[0153] Another exemplary TTR polynucleotide sequence is provided in NCBI reference sequence number NG_009490.1 and is shown below (where exons encoding the TTR polypeptide are shown in bold, introns in italics, and exemplary promoter regions are indicated by a combination of underlined and bold underlined text (promoter positions -1 to -177) and bold underlined text (promoter positions -106 to -176): AGACAAGGTTCATATTTGTATGGGTTACTTATTCTCTCTTTGTTGACTA AGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAGCCTAGCTCAGGAGAAGTGAGTATA AAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAGG ATGGCTTCTCATCGTCTGCTCCTCCTCTGCCTTGCTGGACTGGTATTTGTGTCTGAGGCTGGCCCTACG GTGAGTGTTTCTGTGACATCCCATTCCTACA TTTAAGATTCACGCTAAATGAAGTAGAAGTGACTCCTTCCAGCTTTGCCAACCAGCTTTTATTACTAGGGCAAGGG TACCCAGCATCTATTTTTAATATAATTAATTCAAACTTCACAAGAATGAAGTTCCACTGAGCTTACTGAGCTGGG ACTTGAACTCTGAGCATTCTACCTCATTGCTTTGGTGCATTAGGTTTGTAATATCTGGTACCTCTGTTTCCTCAGA TAGATGATAGAAATAAAGATATGATATTAAGGAAGCTGTTAATACTGAATTTTCAGAAAAGTATCCCTCCATAAAA TGTATTTGGGGGACAAACTGCAGGAGATTATATTCTGGCCCTATAGTTATTCAAAACGTATTTATTGATTAATCTT TAAAAGGCTTAGTGAACAATATTCTAGTCAGATATCTAATTCTTAAATCCTCTAGAAGAATTAACTAATACTATAA AATGGGTCTGGATGTAGTTCTGACATTATTTTATAACAACTGGTAAGAGGGAGTGACTATAGCAACAACTAAAATG ATCTCAGGAAAACCTGTTTGGCCCTATGTATGGTACATTACATCTTTTCAGTAATTCCACTCAAATGGAGACTTTT AACAAAGCAACTGTTCTCAGGGGACCTATTTTTCTCCCTTAAAATTCATTATACACATCCCTGGTTGATAGCAGTGT GTTCTGGAGGCAGAAACCATTCTTGCTTTGGAAACAATTACGTCTTGTGTTATACTGAGTAGGGAAGCTCATTAATTG TCGACACTTACGTTCCTGATAATGGGATCAGTGTGTAATTCTTGTTTCGCTCCAGATTTCTAATACCACAAAGAAT AAATCCTTTCACTCTGATCAATTTTGTTAACTTCTCACGTGTCTTCTCTACACCCAG GGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGG GTAAGTTGCCAAAGAACCCTCCCACAGGACTTGGTTTTAT CTTCCCGTTTGCCCCTCACTTGGTAGAGAGAGGCTCACATCATCTGCTAAAGAATTTACAAGTAGATTGAAAAACG TAGGCAGAGGTCAAGTATGCCCTCTGAAGGATGCCCTCTTTTTGTTTTGCTTAGCTAGGAAGTGACCAGGAACCTG AGCATCATTTAGGGGCAGACAGTAGAGAAAAGAAGGAATCAGAACTCCTCTCCTCTAGCTGTGGTTTGCAACCCTT TTGGGTCACAGAACACTTTATGTAGGTGATGAAAAGTAAACATTCTATGCCCAGAAAAAATGCACAGATACACACA CATACAAAATCATATATGTGATTTTAGGAGTTTCACAGATTCCCTGGTGTCCCTGGGTAACACCAAAGCTAAGTGT CCTTGTCTTAGAATTTTAGGAAAAGGTATAATGTGTATTAACCCATTAACAAAAGGAAAGGAATTCAGAAATATTA TTAACCAGGCATCTGTCTGTAGTTAATATGGATCACCCAAAAACCCAAGGCTTTTGCCTAATGAACACTTTGGGGCA CCTACTGTGTGCAAGGCTGGGGGCTGTCAAGCTCAGTTAAAAAAAAAAAGATAGAAGAGATGATCCATGAGGCAA AGTACAGCCCCAGGCTAATCCCACGATCACCCGACTTCATGTCCAAGAGTGGCTTCTCACCTTCATTAGCCAGTTC ACAATTTTCATGGAGTTTTTCTACCTGCACTAGCAAAAACTTCAAGGAAAATACATATTAATAAATCTAAGCAAAG TGACCAGAAGACAGAGCAATCAGGAGACCCTTTGCATCCAGCAGAAGAGGAACTGCTAAGTATTTACATCTCCACA GAGAAGAATTTCTGTTGGGTTTTAATTGAACCCCAAGAACCACATGATTCTTCAACCATTATTGGGAAGATCATTT TCTTAGGTCTGGTTTTAACTGGCTTTTTATTTGGGAATTCATTTATGTTTATATAAAATGCCAAGCATAACATGAA AAGTGGTTACAGGACTATTCTAAGGGAGAGACAGAATGGACACCAAAAATATTCCAATGTTCTTGTGAATCTTTTC CTTGCACCAGGACAAAAAAAAAAAGAAGTGAAAAGAAGAAAGGAGGAGGGGCATAATCAGAGTCAGTAAAGACAAC TGCTATTTTTATCTATCGTAGCTGTTGCAGTCAAATGGGAAGCAATTTCCAACATTCAACTATGGAGCTGGTACTT ACATGGAAATAGAAGTTGCCTAGTGTTTGTTGCTGGCAAAGAGTTATCAGAGAGGTTAAATATATAAAAGGGAAAA GAGTCAGATACAGGTTCTTCTTCCTACTTTAGGTTTTCCACTGTGTGTGCAAATGATACTCCCTGGTGGTGTGCAG ATGCCTCAAAGCTATCCTCACACCACAAGGGAGAGGAGCGAGATCCTGCTGTCCTGGAGAAGTGCAGAGTTAGAAC AGCTGTGGCCACTTGCATCCAATCATCAATCTTGAATCACAGGGACTCTTTCTTAAGTAAACATTATACCTGGCCG GGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGATGCCAAAGTGGGCATATCATCTGAGGTCAGGAGTTCA AGACCAGCCTGGCCAACATGGCAAAACTCCGTCTTTATGAAAAATACAAAAATTAGCCAGGCATGGTGGCAGGCGC CTGTAATCCCAGCTAATTGGGAGGCTGAGGCTGGAGAATCCCTTGAATCTAGGAGGCAGAGGTTGCAGTGAGCTGA GATCGTGCCATTGCACTCCAGCCTGGGTGACAAGAGTAAAACTCTGTCTCAAAAAAAAAAAATTATACCTACATTC TCTTCTTATCAGAGAAAAAAATCTACAGTGAGCTTTTCAAAAAGTTTTTACAAACTTTTTGCCATTTAATTTCAGT TAGGAGTTTTCCCTACTTCTGACTTAGTTGAGGGGAAATGTTCATAACATGTTTATAACATGTTTATGTGTGTTAG TTGGTGGGGGTGTATTACTTTGCCATGCCATTTGTTTCCTCCATGCGTAACTTAATCCAGACTTTCACACCTTATA G GAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAG GTGAGTATACAGACC TTCGAGGGTTGTTTTGGTTTTGGTTTTTGCTTTTGGCATTCCAGGAAATGCACAGTTTTACTCAGTGTACCACAGA AATGTCCTAAGGAAGGTGATGAATGACCAAAGGTTCCCTTTCCTATTATACAAGAAAAAATTCACAACACTCTGAG AAGCAAATTTCTTTTTGACTTTGATGAAAATCCACTTAGTAACATGACTTGAACTTACATGAAACTACTCATAGTC TATTCATTCCACTTTATATGAATATTGATGTATCTGCTGTTGAAATAATAGTTTATGAGGCAGCCCTCCAGACCCC ACGTAGAGTGTATGTAACAAGAGATGCACCATTTTATTTCTCGAAAACCCGTAACATTCTTCATTCCAAAACACAT CTGGCTTCTCGGAGGTCTGGACAAGTGATTCTTGGCAACACATACCTATAGAGACAATAAAATCAAAGTAATAATG GCAACACAATAGATAACATTTACCAAGCATACACCATGTGGCAGACACAATTATAAGTGTTTTCCATATTTAACCT ACTTAATCCTCAGGAATAAGCCACTGAGGTCAGTCCTATTATTATCCCCATCTTATAGATGAAGAAAATGAGGCAC CAGGAAGTCAAATAACTTGTCAAAGGTCACAAGACTAGGAAATACACAAGTAGAAATGTTTACAATTAAGGCCCAG GCTGGGTTTGCCCTCAGTTCTGCTATGCCTCGCATTATGCCCCAGGAAACTTTTTCCCTTGTGAAAGCCAAGCTTA AAAAAAGAAAAGCCACATTTGTAACGTGCTCTGTTCCCCTGCCTATGGTGAGGATCTTCAAACAGTTATACATGGA CCCAGTCCCCCTGCCTTCTCCTTAATTTCTTAAGTCATTTGAAACAGATGGCTGTCATGGAAATAGAATCCAGACA TGTTGGTCAGAGTTAAAGATCAACTAATTCCATCAAAAATAGCTCGGCATGAAAGGGAACTATTCTCTGGCTTAGT CATGGATGAGACTTTCAATTGCTATAAAGTGGTTCCTTTATTAGACAATGTTACCAGGGAAACAACAGGGGTTTGT TTGACTTCTGGGGCCCACAAGTCAACAAGAGAGCCCCATCTACCAAGGAGCATGTCCCTGACTACCCCTCAGCCAG CAGCAAGACATGGACCCCAGTCAGGGCAGGAGCAGGGTTTCGGCGGCGCCCAGCACAAGACATTGCCCCTAGAGTC TCAGCCCCTACCCTCGAGTAATAGATCTGCCTACCTGAGACTGTTGTTTGCCCAAGAGCTGGGTCTCAGCCTGATG GGAACCATATAAAAAGGTTCACTGACATACTGCCCACATGTTGTTCTCTTTCATTAGATCTTAGCTTCCTTGTCTG CTCTTCATTCTTGCAGTATTCATTCAACAAACATTAAAAAAAAAAAAAAGCATTCTATGTGTGGAACACTCTGCTA GATGCTGTGGATTTAGAAATGAAAATACATCCCGACCCTTGGAATGGAAGGGAAAGGACTGAAGTAAGACAGATTA AGCAGGACCGTCAGCCCAGCTTGAAGCCCAGATAAATACGGAGAACAAGAGAGAGCGAGTAGTGAGAGATGAGTCC CAATGCCTCACTTTGGTGACGGGTGCGTGGTGGGCTTCATGCAGCTTCTTCTGATAAATGCCTCCTTCAGAACTGG TCAACTCTACCTTGGCCAGTGACCCAGGTGGTCATAGTAGATTTACCAAGGGAAAATGGAAACTTTTATTAGGAGC TCTTAGGCCTCTTCACTTCATGGATTTTTTTTTCCTTTTTTTTTGAGATGGAGTTTTGCCCTGTCACCCAGGCTGG AATGCAGTGGTGCAATCTCAGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCG AGTAGCTGGGACTACAGGTGTGCGCCACCACACCAGGCTAATTTTTGTATTTTTTGTAAAGACAGGTTTTCACCAC GTTGGCCAGGCTGGTCTGAACTCCAGACCTCAGGTGATTCACCTGTCTCAGCCTCCCAAAGTGCTGGGATTACAGG TGTGAGCCACCGTGCCCGGCTACTTCATGGATTTTTGATTACAGATTATGCCTCTTACAATTTTTAAGAAGAATCA AGTGGGCTGAAGGTCAATGTCACCATAAGACAAAAGACATTTTTATTAGTTGATTCTAGGGAATTGGCCTTAAGGG GAGCCCTTTCTTCCTAAGAGATTCTTAGGTGATTCTCACTTCCTCTTGCCCCAGTATTATTTTTTGTTTTTGGTATG GCTCACTCAGATCCTTTTTTCCTCCTATCCCTAAGTAATCCGGGTTTCTTTTTCCCATATTTAGAACAAAATGTAT TTATGCAGAGTGTGTCCAAACCTCAACCCAAGGCCTGTATACAAAATAAATCAAATTAAACACATCTTTACTGTCT TCTACCTCTTTCCTGACCTCAATATATCCCAACTTGCCTCACTCTGAGAACCAGGCTGTCCCAGCACCTGAGTCG CAGATATTCTACTGATTTGACAGAACTGTGTGACTATCTGGAACAGCATTTTGATCCACAATTTGCCCAGTTACAA AGCTTAAATGAGCTCTAGTGCATGCATATATATTTCAAAATTCCACCATGATCTTCCACACTCTGTATTGTAAATA GAGCCCTGTAATGCTTTTACTTCGTATTTCATTGCTTGTTATACATAAAAATATACTTTTCTCTTCATGTTAGAA AATGCAAAGAATAGGAGGGGTGGGGGAATCTCTGGGCTTGGAGACAGGAGACTTGCCTTCCTACTATGGTTCCATCA GAATGTAGACTGGGACAATACAATAATTCAAGTCTGGTTTGCTCATCTGTAAATTGGGAAGAATGTTTCCAGCTCC AGAATGCTAAATCTCTAAGTCTGTGGTTGGCAGCCACTATTGCAGCAGCTCTTCAATGACTCAATGCAGTTTTGCA TTTCCCCTACCTTTTTTTTCTAAAACCAATAAAATAGATACAGCCTTTAGGCTTTCTGGGATTTCCCTTAGTCAAG CTAGGGGTCATCCTGACTTTCGGCGTGAATTTGCAAAACAAGACCTGACTCTGTACTCCTGCTCTAAGGACTGTGCA TGGTTCCAAAGGCTTAGCTTGCCAGCATATTTGAGCTTTTCCTTCTGTTCAAACTGTTCCAAAATATAAAAGAAT AAAATTAATTAAGTTGGCACTGGACTTCCGGTGGTCAGTCATGTGTGTCATCTGTCACGTTTTTCGGGGCTCTGGTG GAAATGGATCTGTCTGTCTTCTCTCATAG

[0154] In the TTR polynucleotide sequence provided by NCBI reference sequence number NG_009490.1, the exons encoding the TTR polypeptide correspond to the union of nucleotides 5137..5205, 6130..6260, 8354..8489, and 11802..11909, while the spacer sequence corresponds to the intron sequence. The union of nucleotides 5137..5205, 6130..6260, 8354..8489, and 11802..11909 corresponds to Common Coding Sequence (CCDS) number 11899.1.

[0155] "Transthyretin amyloidosis" refers to a disease associated with the accumulation of amyloid protein in the tissues of a subject.

[0156] As used herein, the term "treatment" and similar terms refer to reducing or improving a condition and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a condition or symptom does not require the complete elimination of the condition, symptom, or its associated symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, alleviates, eliminates, relieves, mitigates, or cures the disease and / or unpleasant symptoms attributable to the disease, or reduces their intensity. In some embodiments, the effect is preventative, i.e., the effect protects against or prevents the occurrence or recurrence of the disease or symptom. For this purpose, the methods of this disclosure include administering a therapeutically effective amount of the composition as described herein.

[0157] "Uracil glycosylase inhibitor" or "UGI" refers to an agent that inhibits the uracil excision repair system. A base editor containing a cytidine deaminase converts cytosine to uracil, which is then converted to thymine via DNA replication or repair. In various embodiments, uracil DNA glycosylase (UGI) prevents base excision repair, which converts U back to C. In some cases, contacting cells and / or polynucleotides with a UGI and a base editor can prevent base excision repair, which converts U back to C. An exemplary UGI contains the following amino acid sequence: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylation inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 231).

[0158] In some implementations, the agent that inhibits the uracil excision repair system is uracil stable protein (USP). See, for example, WO 2022015969 A1, which is incorporated herein by reference.

[0159] As used in this article, the term "vector" refers to the means of introducing nucleic acid molecules into cells, thereby leading to cell transformation. Vectors include plasmids, transposons, bacteriophages, viruses, liposomes, lipid nanoparticles, and episomes.

[0160] The ranges provided in this document are understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 is understood to include any value, combination of values, or subrange selected from the groups 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.

[0161] The enumeration of chemical groups listed in any definition of a variable herein includes the definition of the variable as any single group or combination of the listed groups. The enumeration of embodiments of a variable or aspect herein includes the embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0162] All terms are intended to be understood in the manner that would be known to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0163] In this application, unless otherwise expressly stated, the use of the singular form includes the plural form. It must be noted that, as used in the specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly specifies otherwise. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0164] The terms “comprising” (and any form of “comprising” such as “comprise” and “comprises”), “having” (and any form of “having” such as “have” and “has”), “including” (and any form of “including” such as “includes” and “include”), or “containing” (and any form of “containing” such as “contains” and “contain”) as used in this specification and claims are inclusive or open-ended. This wording indicates the presence of a specified element, feature, component, and / or method step, but does not exclude the presence of other elements, features, components, and / or method steps. Any embodiment specified as “comprising” a particular component or element may, in some embodiments, also include “consisting of said particular component or element” or “consisting substantially of said particular component or element.” It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0165] The term “about” or “approximately” refers to a range of acceptable error for a particular value, as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system.

[0166] References to “some implementations,” “implementation,” “one implementation,” or “other implementation” in the specification mean that a particular feature, structure, or characteristic described in connection with the implementation is included in at least some of the implementations of this disclosure, but not necessarily in all of the implementations of this disclosure. Brief description of the attached diagram Figure 1A and 1B Figures and graphs are provided showing that, under similar conditions, a base editor system containing a guide RNA (i.e., human gRNA or "human") targeting human TTR polynucleotide sequences for base editing and administered to primary human hepatocytes is associated with higher levels of base editing compared to a base editor system containing a guide RNA (i.e., cynomolgus monkey gRNA or "cynomolgus monkey") targeting non-human primate TTR polynucleotide sequences and administered to primary cynomolgus monkey hepatocytes. The base editor system contains mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to as "ABE9.51" throughout the figures). Figure 1AGraphs and charts are provided showing the base editing rate measured during base editing of synthetic polynucleotide sequences (“AAVS1 cassettes”) in human embryonic kidney (HEK293) cells containing polynucleotides with target sites for both human gRNA and cynomolgus monkey gRNA. The base editor system was delivered to HEK293T cells using the commercially available lipid transfection reagent Lipofectamine® MessengerMAX mRNA transfection reagent (LipoMM). Figure 1B Figures and graphs are provided showing the base editing rates measured at the TTR gene (left panel) or the ALAS1 gene control (right panel) in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH). The ALAS1 gene (right panel) was edited using a base editor system comprising a control guide RNA sg23, known to be effective for targeting base editors to alter the ALAS1 gene kernel bases, and an mRNA molecule encoding a base editor containing a TadA*8.8 adenosine deaminase domain. Figure 1B The base editor system in this study utilizes lipid nanoparticles containing ionizable lipid IZ1 for delivery. Figure 1A and 1B In this context, the terms PHH-A, PHH-B, and PHH-C represent three different human donors from whom primary human hepatocytes (PHH) were obtained; "cTTR" represents the cynomolgus monkey TTR gene; and "Conserved" represents the ALAS1 gene.

[0168] Figure 2A and 2B Figures and graphs are provided showing that, under similar conditions, a base editor system comprising a guide RNA (i.e., human gRNA or "human") targeting human TTR polynucleotide sequences for base editing and administered to primary human hepatocytes (PHH) is associated with a higher level of base editing compared to a base editor system comprising a guide RNA (i.e., cynomolgus monkey gRNA or "cynomolgus monkey") targeting non-human primate TTR polynucleotide sequences and administered to primary cynomolgus monkey hepatocytes (PCH). The base editor system is administered to cells using lipid nanoparticles containing ionizable lipid IZ4. Figure 2AGraphs and charts showing the base editing rate at the TTR gene in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) are provided. The base editor system administered to PHH contains guide RNA gRNA2944, while the base editor system administered to PCH contains guide RNA gRNA2945. The base editor system contains mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to as "ABE9.51" throughout the figure). Figure 2B Graphs and charts showing the base editing rate at the ALAS1 gene measured in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) are provided. The ALAS1 gene was edited using a base editor system comprising a control guide RNA sg23 known to be effective for targeting the base editor to alter the ALAS1 gene nucleus bases, and an mRNA molecule encoding the base editor containing the TadA*8.8 adenosine deaminase domain. Figure 2A and 2B In this text, the terms PHH1, PHH2, and PHH3 represent primary human hepatocytes (PHHs) from three different human donors. Throughout the figure, the term "surrogacy" refers to the assessment or use of base editing measurements of non-human primate polynucleotide sequences or cells to infer the rate of base editing achieved in human polynucleotide sequences or cells under similar or analogous conditions (e.g., using a base editor system containing guide RNA that targets human polynucleotides rather than non-human primate polynucleotides for base editing).

[0169] Figures 3A to 3F A bar chart is provided showing the results of administering a specified dose of lipid nanoparticles containing lipid IZ1 and the male base editor system. Figures 3A-3C ) and female ( Figure 3D-3F Data on liver enzymes in non-human primates, wherein the base editor system comprises a guide RNA targeting the TTR gene for base editing at the HM17 target site and an mRNA molecule encoding an adenosine deaminase base editor containing a variant of the TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration. Figures 3A to 3F The bar chart from left to right shows the groups corresponding to control, 0.5 mg / kg, 1 mg / kg, and 2 mg / kg, respectively. Figures 3A to 3F In this context, the term "AST" stands for "aspartate aminotransferase," the term "ALT" stands for "alanine aminotransferase," and the term "ALP" stands for "alkaline phosphatase." Figures 3A to 3FThe x-axis represents the number of days after lipid nanoparticle administration (day 0). This experiment demonstrates that the LNP-administered base editor system has no persistent harmful effects.

[0170] Figure 4 A bar chart is provided showing the results from donor PHH donor 1 ( Figure 2A and 2B PHH1) and PHH donor 2 ( Figure 2A and 2B The targeted (TTR gene A>G nucleotide alteration) and non-targeted (EIF3E gene A>G nucleotide alteration) base editing rates were measured in primary human hepatocytes of PHH2, which were administered lipid nanoparticles containing guide RNA targeting the TTR gene for base editing at the HM17 target site and mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to as "ABE9.51" in the entire figure) or a TadA*8.20 adenosine deaminase domain with altered V82T, Y147T, and Q154S amino acids (referred to as "ABE9.52" in the entire figure). For Figure 4 For each pair of bars, the left bar corresponds to "TTR, A>G" and the right bar represents "EIF3E, A>G".

[0171] Figure 5A and 5B Images of primary human hepatocytes were provided. Figure 5A Images of primary cynomolgus monkey hepatocytes that were not administered via any base editor system are provided. Figure 5B Images of primary cynomolgus hepatocytes are provided one day after administration of lipid nanoparticles containing a base editor system comprising a guide RNA that targets a non-human primate TTR gene for base editing and an mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to as “ABE9.51” throughout the figure).

[0172] Figure 6A and 6B Figures and graphs are provided showing that, under similar conditions, a base editor system comprising a guide RNA (i.e., human gRNA or "human") targeting a human TTR polynucleotide sequence for base editing and administered to primary human hepatocytes (PHH) is associated with a higher level of base editing compared to a base editor system comprising a guide RNA (i.e., cynomolgus monkey gRNA or "cynomolgus monkey") targeting a non-human primate TTR polynucleotide sequence and administered to primary cynomolgus monkey hepatocytes (PCH). The base editor system is administered to cells using lipid nanoparticles containing ionizable lipid IZ4. Figure 6A Graphs and charts showing the base editing rate at the TTR gene in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) are provided. The base editor system administered to PHH contains guide RNA gRNA2944, while the base editor system administered to PCH contains guide RNA gRNA2945. The base editor system contains mRNA encoding the base editor, which contains a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to as "ABE9.51" throughout the figure). Figure 6B Graphs and charts showing the base editing rate at the ALAS1 gene measured in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) are provided. The ALAS1 gene was edited using a base editor system comprising a control guide RNA sg23 known to be effective for targeting the base editor to alter the ALAS1 gene nucleus bases, and an mRNA molecule encoding the base editor containing the TadA*8.8 adenosine deaminase domain. Figure 6A and 6B In this context, the terms PHH1, PHH2, and PHH3 refer to primary human hepatocytes (PHH) from three different human donors, wherein the three donors are related to... Figure 2A and 2B The donors are the same. Invention Details This document provides compositions for the modification or editing of polynucleotides (e.g., genes), and methods for treating or preventing conditions associated with the extracellular deposition of amyloid fibers formed by the aggregation of misfolded transthyretin (TTR) in various tissues using said compositions. Such conditions include, but are not limited to, hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) and hereditary transthyretin amyloidosis cardiomyopathy (hATTR-CM), both associated with autosomal dominant mutations in the TTR gene, and age-related cardiomyopathy (ATTRwt) associated with wild-type TTR protein, also known as senile cardiac amyloidosis. This document discloses compositions and methods for editing TTR polynucleotides using an editing system, such as a system comprising a base editor and guide RNA.

[0174] This invention is based at least in part on the discovery that a base editor system can be used to disrupt the expression of transthyretin (TTR) peptides or edit pathogenic mutations in TTR peptides, and that the base editor system can be efficiently delivered to cells using the lipid nanoparticles disclosed herein. In one embodiment, this disclosure provides a guide RNA sequence that, when used in conjunction with a base editing system, can effectively edit the transthyretin (TTR) gene sequence to disrupt splicing or correct pathogenic mutations. In another embodiment, this invention provides a guide RNA sequence that targets the Cas12b nuclease to edit the TTR gene sequence, thereby disrupting TTR peptide expression.

[0175] Therefore, this disclosure provides guide RNA sequences suitable for use with ABE and / or BE4 to disrupt the transthyretin (TTR) gene splice site, and guide RNA sequences suitable for use with bhCas12b nuclease to disrupt the TTR gene. In embodiments, the compositions and methods of the present invention can be used to edit the TTR gene in hepatocytes. The methods provided herein may include reducing or eliminating TTR expression in hepatocytes to treat amyloidosis.

[0176] Transthyretin and its gene Transthyretin (TTR), originally called prealbumin, is a 55-kDa transporter for thyroxine (T4) and retinol-binding proteins, circulating in soluble form in the serum and cerebrospinal fluid (CSF) of healthy individuals. TTR is understood to be primarily synthesized in the liver. Under normal conditions, TTR circulates as a homotetramer with a central channel. An exemplary wild-type TTR monomer is 147 amino acids in length and has the following amino acid sequence: MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE (SEQ ID NO: 464).

[0177] The TTR gene consists of four exons and is located at 18q12.1 on chromosome 18. The complete sequence of the human TTR gene is available in UniProtKB-P02766 (TTHY_human). To date, more than 120 TTR variants have been identified, the vast majority of which are pathogenic. The most common pathogenic variant is a point mutation that replaces valine at position 30 of the mature protein with methionine. This Val30Met mutation is the cause of hATTR amyloidosis and is the most common amyloidogenic mutation worldwide, accounting for approximately 50% of TTR variants.

[0178] Hereditary transthyretin amyloidosis (hATTR) is a disease caused by mutations in the TTR gene. Autosomal dominant mutations destabilize the TTR tetramer and enhance its dissociation into monomers, leading to misfolding, aggregation, and subsequent extracellular deposition of TTR amyloid fibrils in various tissue sites. This multisystemic extracellular deposition of amyloid protein (amyloidosis) results in dysfunction in different organs and tissues. In particular, transthyretin amyloidosis-induced polyneuropathy (ATTR-PN) and transthyretin amyloidosis-induced cardiomyopathy (ATTR-CM) are serious diseases associated with significant morbidity and mortality.

[0179] When hATTR-PN is clinically suspected, diagnosis is typically made via tissue biopsy, including amyloid staining, amyloid typing (using immunohistochemistry or mass spectrometry), and / or TTR gene sequencing. When ATTR-CM is clinically suspected, the key diagnostic tools are endocardial myocardial biopsy (with tissue staining and amyloid typing using immunohistochemistry or mass spectrometry) or 99mTc-pyrophosphate scanning. Both methods can provide a diagnosis of ATTR-CM. TTR gene sequencing can be used to differentiate between hATTR-CM (mutation-positive) and ATTRwt-CM (mutation-negative).

[0180] In some embodiments, the compositions described herein comprise a guide polynucleotide containing a spacer sequence having the function of a guide to direct a gene-editing protein (e.g., a base editor) to alter the nucleotide sequence of the TTR gene, for example, by introducing one or more nucleobase alterations into the TTR gene. These point mutations can be used to disrupt gene function by introducing one or more missense mutations that result in the production of a weakened or nonfunctional protein, thereby silencing the TTR gene. Alternatively, it is envisioned herein that a gene-editing protein can be used to correct one or more point mutations to alter the mutated gene, thereby correcting a potential mutation causing TTR gene dysfunction, or otherwise mitigating gene dysfunction.

[0181] amyloidosis Amyloidosis is a disease involving the extracellular deposition of amyloid proteins in organs or tissues, such as the liver. Amyloidosis can occur when mutant transthyretin polypeptides aggregate (e.g., as fibrils). Amyloidosis caused by mutations in the transthyretin gene can be termed "transthyretin amyloidosis." Some forms of transthyretin amyloidosis are not associated with mutations in the transthyretin gene. Non-limiting examples of mutations in the mature transthyretin (TTR) protein that can lead to amyloidosis include alterations to T60A, V30M, V30A, V30G, V30L, V122I, V122A, and V122(-). One approach to treating transthyretin amyloidosis involves disrupting the expression or activity of transthyretin in subject cells (optionally hepatocytes). Therefore, this document provides methods for reducing or eliminating transthyretin expression in cells. The transthyretin in the cells can be a pathogenic variant. The expression of transthyretin in cells can be disrupted by disrupting the splicing of the transthyretin transcript.

[0182] Transthyretin amyloidosis (THA) is a progressive disease characterized by the accumulation of protein deposits in organs and / or tissues. These protein deposits can occur in the peripheral nervous system, which consists of nerves connecting the brain and spinal cord to muscles and sensory cells (which detect sensations such as touch, pain, heat, and sound). Protein deposits in these nerves can lead to loss of sensation in the extremities (peripheral neuropathy). The autonomic nervous system, which controls involuntary bodily functions such as blood pressure, heart rate, and digestion, can also be affected by amyloidosis. In some cases, the brain and spinal cord (i.e., the central nervous system) are also affected. Other areas affected by amyloidosis include the heart, kidneys, eyes, liver, and gastrointestinal tract. Symptoms can begin to appear between the ages of 20 and 70.

[0183] There are three main forms of transthyretin amyloidosis, which are distinguished according to their symptoms and the systems of the body affected: neuropathic, leptomeningeal, and cardiac.

[0184] The neuropathy form of transthyretin amyloidosis primarily affects the peripheral and autonomic nervous systems, leading to peripheral neuropathy and difficulty controlling bodily functions. Impaired bodily functions may include impotence, diarrhea, constipation, urinary problems, and a sharp drop in blood pressure upon standing (orthostatic hypotension). Some people may also experience heart and kidney problems. Various eye problems may occur, such as clear gel-like opacities filling the eye (vitreous opacities), dry eyes, increased intraocular pressure (glaucoma), or an irregular or "fan-shaped" pupil appearance. Some people with this form of transthyretin amyloidosis develop carpal tunnel syndrome, which can involve numbness, tingling, and weakness in the hands and fingers.

[0185] The leptomeningeal form of transthyretin amyloidosis primarily affects the central nervous system. In this type, amyloidosis occurs in the pia mater, the two thin layers of tissue that cover the brain and spinal cord. The accumulation of protein in this tissue can lead to stroke and cerebral hemorrhage, fluid buildup in the brain (hydrocephalus), difficulty coordinating movements (ataxia), muscle stiffness and weakness (spastic paralysis), seizures, and intellectual disability (dementia). Eye problems similar to those in the neuropathic form can also occur. When people with leptomeningeal transthyretin amyloidosis also have eye problems, they are said to have ocular leptomeningeal amyloidosis.

[0186] Cardiac transthyretin amyloidosis affects the heart. People with cardiac amyloidosis may experience abnormal heartbeats (arrhythmias), an enlarged heart (cardiomegaly), or orthostatic hypertension. These abnormalities can lead to progressive heart failure and death. Occasionally, people with cardiac transthyretin amyloidosis may experience mild peripheral neuropathy.

[0187] Mutations in the transthyretin (TTR) gene lead to transthyretin amyloidosis. TTR is responsible for transporting vitamin A (retinol) and a hormone called thyroxine throughout the body. Contrary to popular belief, in order to transport retinol and thyroxine, TTR must form a tetramer. TTR is primarily produced in the liver (i.e., hepatocytes). Small amounts of TTR are also produced in areas of the brain called the choroid plexus and in the retina.

[0188] TTR gene mutations can alter the structure of transthyretin protein, impairing its ability to bind to other transthyretin proteins. These TTR gene mutations can be autosomal dominant.

[0189] splice site Gene splice sites and splice site motifs are well known in the art, and identifying splice sites in sequences is within the skill range of practitioners (see, for example, Sheth et al., “Comprehensive splice-site analysis using comparative genomics”). Nucleic Acids Research , 34:3955-3967 (2006); Dogan et al., “AplicePort – an interactive splice-site analysis tool”, Nucleic Acids Research, 35:W285-W291 (2007); and Zuallaert et al., “SpliceRover: interpretable convolutional neural networks for improved splice site prediction”, Bioinformatics , 34:4180-4188 (2018)).

[0190] The classic splice donor contains the DNA sequence GT on the sense strand, while the classic splice acceptor contains the DNA sequence AG. Sequence alterations disrupt normal splicing. The splice donor can be disrupted by editing the complementary base at the second site on the antisense strand using adenine bases (GT→GC), while the splice acceptor can be disrupted by editing the first site on the sense strand using adenine bases (AG→GG).

[0191] Editing of target genes To edit the transthyretin (TTR) gene, cells (e.g., hepatocytes) are contacted with guide RNA and a nucleobase editor polypeptide containing a nucleic acid-programmable DNA-binding protein (napDNAbp) and either cytidine deaminase or adenosine deaminase to edit bases in the gene sequence. Base editing can result in disruption of splice sites (e.g., by altering splice site motif nucleobases). Base editing can result in the replacement of pathogenic variant amino acids with non-pathogenic variant amino acids. As a non-limiting example, base editing can result in the replacement of T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(-) alterations in a mature transthyretin (TTR) polypeptide with non-pathogenic variant or wild-type valine residues. The cytidine deaminase may be BE4 (e.g., saBE4). The adenosine deaminase may be ABE (e.g., saABE.8.8). In some embodiments, multiple target sites are edited simultaneously. In some embodiments, the TTR gene is edited by contacting cells with a nuclease and a guide RNA to introduce an indel into the gene sequence. The indel may be associated with a decrease or elimination of gene expression. The nuclease may be Cas12b (e.g., bhCas12b). The cells may be edited in vivo or in vitro. The guide RNA may be single-guide or dual-guide. In some embodiments, the cell to be edited is contacted with at least one nucleic acid, wherein the at least one nucleic acid encodes a guide RNA, or two or more guide RNAs, and a nucleobase editor polypeptide comprising a nucleic acid-programmable DNA-binding protein (napDNAbp) and a deaminase (e.g., adenosine or cytidine deaminase). In some embodiments, the gRNA comprises a nucleotide analog. These nucleotide analogs can inhibit the degradation of the gRNA by cellular processes. Exemplary single-guide RNA (sgRNA) sequences are provided in Tables 1A to 1E, and exemplary spacer sequences and target sequences (e.g., prototype spacer sequences) are provided in Tables 1B, 1C, and 2A to 2E.

[0192] According to this disclosure, a guide polynucleotide for targeting a base editor to alter the nuclear bases of the human TTR gene and a spacer sequence suitable therein are provided, the alteration being performed by A→G editing within an editing window (roughly corresponding to positions 4 to 7 in the 20-nt prototype spacer sequence of the guide polynucleotide spacer sequence) to disrupt the start codon or disrupt the splice site (whether donor or recipient).

[0193] The prototype spacer, corresponding to guide RNA GA457, has the sequence 5'-GCCATCCTGCCAAGAATGAG-3' (SEQ ID NO: 467) and is located at positions 34,879 to 34,898 bp in the human TTR gene.

[0194] The prototype spacer sequence, corresponding to guide RNA GA459, is 5'-GCAACTTACCCAGAGGCAAA-3' (SEQ ID NO: 468), located at positions 36,007 to 36,026 bp of the human TTR gene.

[0195] The prototype spacer sequence, corresponding to guide RNA GA460, is 5'-TATAGGAAAACCAGTGAGTC-3' (SEQ ID NO: 469), located at 38,106-38,125 bp of the human TTR gene.

[0196] The prototype spacer sequence, corresponding to guide RNA GA461, has the sequence 5'-TACTCACCTCTGCATGCTCA-3' (SEQ ID NO: 470) and is located at positions 38,234-38253 of the human TTR gene.

[0197] The prototype spacer sequence, corresponding to guide RNA GA458, has the sequence 5'-GCCATCCTGCCAAGAACGAG-3' (SEQ ID NO: 471), representing the human prototype spacer sequence corresponding to guide RNA GA459 within the cynomolgus macaque TTR gene.

[0198] This disclosure includes a guide polynucleotide having a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 472) (GA457).

[0199] This disclosure includes a modified guide polynucleotide having a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with sequence 5'-GCCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 472), wherein the nucleotide GCC shown in bold is methylated (GA521) (C is modified to 2'-O-methylcytidine, and G is modified to 2'-O-methylguanosine). This disclosure includes a modified guide polynucleotide having the sequence 5'-mGsmCsmCAUCCUGCCAAGAAUGAG-3' (SEQ ID NO: 472) (GA521), wherein mC: 2'-O-methylcytidine, mG: 2'-O-methylguanosine, and s: phosphate thioester (PS) backbone.

[0200] This disclosure includes a guide polynucleotide having a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with the sequence 5'-GCCAUCCUGCCAAGAACGAG-3' (SEQ ID NO: 473) (GA458).

[0201] This disclosure includes a guide polynucleotide having a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with the sequence 5'-GCAACUUACCCAGAGGCAAA-3' (SEQ ID NO: 474) (GA459).

[0202] This disclosure includes a guide polynucleotide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the sequence 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 475) (GA460). This disclosure includes a guide polynucleotide having the sequence 5'-UAUAGGAAAACCAGUGAGUC-3' (SEQ ID NO: 475) (GA460).

[0203] This disclosure includes a guide polynucleotide having a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with 5'-UACUCACCUCUGCAUGCUCA-3' (SEQ ID NO: 476) (GA461).

[0204] In some respects, this article provides a guide RNA comprising a sequence defined as follows: mG*mC*mC*AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmGmUmGGmCmAmCmGmAmGmUmCmGmGmUmGmCmCmU*mU*mU*mU (GA521, (SEQ ID NO: 477)), where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyladenosine, mC* is 2'-O-methylcytidine, mG* is 2'-O-methylguanosine, mU* is 2'-O-methyluridine, and the nucleotides represented in bold are linked by a phosphate thioester (PS) backbone.

[0205] Alternatively, GA521 is represented as mG*smC*smC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmGsmCsmUsmAsGsmAsmAsmAsmUsmAsmGsmCssmAsmAsGsUsUsmAsAsmAsAsmUsAsmAsmGsmGsmCsmUs mAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAsmGsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsmUsmGsmCsmU*smU*smU*smU (GA521, SEQ ID NO: 477), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyladenosine, mC* is 2'-O-methylcytidine, mG* is 2'-O-methylguanosine, mU* is 2'-O-methyluridine, and wherein the nucleotides are linked by a phosphate thioester (PS) backbone represented by the letter 's'.

[0206] In some implementations, mG*mC*mC*AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmGmUmGGmCmAmCmGmAmGmUmCmGmGmUmGmGmGmCmCmU*mU*mU*mU (GA521, SEQ ID NO: 477), where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyladenosine, mC* is 2'-O-methylcytidine, mG* is 2'-O-methylguanosine, mU* is 2'-O-methyluridine, and the nucleotides represented in bold are linked by phosphate thioester (PS) backbone bonds.

[0207] Alternatively, GA521 is represented as mG*mC*mC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmsGsmCsmUsmAsGsmAsmAsmAsmUsmAsmGsmCssmAsmAsGsUsUsmAsAsmAsAsmUsAsmAsmGsmGsmCsmU smAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAsmGsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsmUsmGsmCsmU*mU*mU*mU (GA521, SEQ ID NO: 477), where A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2'-O-methyladenosine, mC* is 2'-O-methylcytidine, mG* is 2'-O-methylguanosine, mU* is 2'-O-methyluridine, and the nucleotides represented in bold are linked by phosphate thioester (PS) backbone bonds represented by the letter 's'.

[0208] Exemplary guide RNAs, spacer sequences, and target sequences are provided in Tables 1A to 1E and Tables 2A to 2E.

[0209] In various cases, it is advantageous for the spacer sequence to contain a 5′ and / or 3′ “G” nucleotide. In some cases, for example, any spacer sequence or guide polynucleotide provided herein contains or further contains a 5′ “G”, wherein, in some embodiments, the 5′ “G” is complementary to or not complementary to the target sequence. In some embodiments, the 5′ “G” is added to a spacer sequence that does not already contain a 5′ “G”. For example, when the guide RNA is expressed under the control of the U6 promoter, it may be advantageous for the guide RNA to contain a 5′ “G”, because the U6 promoter prefers a “G” at the transcription start site (see Cong, L. et al., “Multiplex genome engineering using CRISPR / Cas systems. Science 339:819-823(2013) doi: 10.1126 / science.1231143”). In some cases, a 5′ “G” is added to the guide polynucleotide that will be expressed under promoter control, but optionally not if the guide polynucleotide is not expressed under promoter control or when the guide polynucleotide is not expressed under promoter control.

[0210] This invention covers variants of the spacer sequence provided herein, which include 1, 2, 3, 4, or 5 nucleobase changes. For example, variations in the target polynucleotide sequence within a population (e.g., single nucleotide polymorphisms) may require changes to the spacer sequence to enable it to better bind to variants of the target sequence in the subject.

[0211] In the implementation scheme, the guide RNA contains a sequence complementary to the promoter region of the TTR polynucleotide sequence. In the implementation scheme, the activation sub-region spans from positions +10, +5, +1, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -15, -20, -25, -30, -35, -40, -45, -50, -55, -60, -65, -70, -75, -80, -85, -90, -95, -100, -105, -110, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, -200, -250, or -300 to positions +5, +1, - 1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -15, -20, -25, -30, -35, -40, -45, -50, -55, -60, -65, -70, -75, -80, -85, -90, -95, -100, -105, -110, -115, -120, -125, -130, -135, -140, -145, -150, -155, -160, -165, -170, -175, -180, -185, -190, -195, -200, -250, -300, or -400, where position +1 corresponds to the first A in the start codon (ATG) of the TTR polynucleotide sequence.

[0212] In various embodiments of this disclosure, the guide RNA comprises the following polynucleotide sequence, wherein said sequence can be modified by adding or removing one or more "N"s (i.e., spacer sequences, shown in bold, which can be extended or truncated, for example, by 1, 2, 3, 4, or 5 nucleotides): nsnsnsNNNNNNNNNNNNNNNNNgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcuususus (SEQ ID NO: 1235). Wherein, "n" is a, c, g or u; "N" is A, C, G or U; 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, and s is a phosphate thioester (PS) backbone linker, where bold indicates spacer sequences.

[0213] Table 1A. Exemplary guide RNAs for editing transthyretin (TTR) splice sites and / or introducing insertions / deletions into the TTR gene (e.g., using bhCas12b).

[0214] The lowercase letter 'm' indicates a 2'-O-methylated nucleobase (e.g., mA, mC, mG, mU), while 's' indicates a thiophosphate.

[0215] Table 1B. Exemplary guide RNAs for editing TTR polynucleotides (spacer sequences within the gRNA sequence are shown in bold).

[0216] Letters in the sequence: - A: adenosine; C: cytidine; G: guanosine; U: uridine; a or mA: 2'-O-methyladenosine; c or mC: 2'-O-methylcytidine; g or mG: 2'-O-methylguanosine; u or mU: 2'-O-methyluridine; s or *: phosphate thioester (PS) backbone linker. Bold text in the gRNA sequence indicates the spacer sequence corresponding to the prototype spacer sequence. GA460 and GA520 have the same prototype spacer sequence but different chemical modifications in their gRNA sequences.

[0217] 1 In both humans and non-human primates, the TTR gene region targeted by this base editor is known as "HM17".

[0218] Table 1C. Exemplary guide RNAs for editing TTR polynucleotides (spacer sequences within the gRNA sequence are shown in bold).

[0219] Letters in the sequence: A = adenosine; C = cytidine; G = guanosine; U = uridine; a = 2'-O-methyladenosine; c = 2'-O-methylcytidine; g = 2'-O-methylguanosine; u = 2'-O-methyluridine; s = phosphate thioester (PS) backbone linker. C = Nucleotides that differ from the human TTR sequence at NHP. Bold text in the gRNA sequence indicates the spacer sequence corresponding to the prototype spacer sequence.

[0220] Table 1D. Exemplary guide RNA sequences (spacer sequences are shown in bold)

[0221] 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 phosphate thioester (PS) backbone linker, where bold text indicates spacer sequences.

[0222] Table 1E. Exemplary guide RNA sequences (spacer sequences are shown in bold)

[0223] Table 2A. Exemplary spacer sequences and target site sequences 2 .

[0224] 2 Those skilled in the art will understand that some target site sequences correspond to the inverse complementary sequences of the transthyretin protein polynucleotide sequences provided above; that is, the target sequences can correspond to any strand of the dsDNA molecule encoding the transthyretin protein polynucleotide. Furthermore, it should be understood that the C base can be targeted by cytidine deaminase, while the A base can be targeted by adenine deaminase.

[0225] Table 2B. Exemplary spacer sequences and target site sequences.

[0226] Table 2C. Exemplary human TTR target site sequence and base editor + guide RNA combination.

[0227] Table 2C (continued)

[0228] Table 2D. Exemplary gRNA spacer sequences with PS linkage at the 5' end

[0229] A represents adenosine; C represents cytidine; G represents guanosine; U represents uridine; a represents 2'-O-methyladenosine; c represents 2'-O-methylcytidine; g represents 2'-O-methylguanosine; u represents 2'-O-methyluridine; s represents phosphate thioester (PS) backbone linkage.

[0230] Table 2E. Exemplary gRNA spacer sequences without PS linkage

[0231] A is adenosine, whether modified or unmodified; C is cytidine, whether modified or unmodified; G is guanosine, whether modified or unmodified; and U is uridine, whether modified or unmodified.

[0232] This disclosure uses guide polynucleotide sg23 as a control guide RNA, which is known to effectively target base editor systems to alter nucleobases in the ALAS1 gene. The sg23 guide RNA comprises the following spacer sequence: CAGGAUCCGCACAGACUCCA (SEQ ID NO: 480). The sg23 guide RNA targets the following sequence for base editing, wherein the PAM sequence is shown in bold: CAGGATCCGCACAGACTCCAGGG (SEQ ID NO: 481).

[0233] The spacer sequences corresponding to sgRNA_361, sgRNA_362, sgRNA_363, sgRNA_364, sgRNA_365, sgRNA_366, and sgRNA_367 in Table 2A can be used to target base editors to alter the nucleobases of the transthyretin (TTR) polynucleotide splicing site. The spacer sequences corresponding to sgRNA_368, sgRNA_369, sgRNA_370, sgRNA_371, sgRNA_372, sgRNA_373, and sgRNA_374 in Table 2A can be used to target endonucleases to the TTR polynucleotide sequence. The three spacer sequences corresponding to sgRNA_375, sgRNA_376, and sgRNA_377 in Table 2A can be used to alter the nucleobases of the TTR polynucleotide. The change in nucleobases can result in a change from isoleucine (I) to valine (V) (e.g., to correct the V122I mutation in the transthyretin polypeptide encoded by the transthyretin polynucleotide). In the implementation scheme, the following combinations of base editors and sgRNA sequences can be used to edit transthyretin protein polynucleotides (see Tables 1A and 2A): ABE8.8 and sgRNA_361; ABE8.8 and sgRNA_362; ABE8.8-VRQR and sgRNA_363; BE4-VRQR and sgRNA_363; BE4-VRQR and sgRNA_364; saABE8.8 and sgRNA_365; saBE4 and sgRNA_365; saBE4-KKH and sgRNA_366; ABE-bhCas12b and sgRNA_367; spCas9-ABE and sgRNA_375; spCas9-VRQR-ABE and sgRNA_376; or saCas9-ABE and sgRNA_377. The PAM sequence of spCas9-ABE can be AGG. The PAM sequence for spCas9-VRQR-ABE can be GGA. The PAM sequence for saCas9-ABE can be AGGAAT.

[0234] In some embodiments, the fusion proteins provided herein include one or more features that enhance the base-editing activity of the fusion protein. For example, any fusion protein provided herein may include a Cas9 domain with reduced nuclease activity. In some embodiments, any fusion protein provided herein may have a Cas9 domain without nuclease activity (dCas9), or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule, referred to as a Cas9 nickase (nCas9). Without being bound by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 in cleaving the non-edited (e.g., unmethylated) strand relative to the targeted nucleobase. Mutations in the catalytic residue (e.g., D10 to A10) prevent cleavage of the edited strand containing the target A residue. Such Cas9 variants can generate single-stranded DNA breaks (nicks) at specific locations based on a target sequence defined by gRNA, leading to the repair of the non-edited strand and ultimately resulting in nucleobase changes on the non-edited strand.

[0235] Nucleoside base editor Nucleobase editors that can be used in the methods and compositions described herein are nucleobase editors for editing, modifying, or altering the target nucleotide sequence of a polynucleotide. Nucleobase editors described herein typically include a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain (e.g., adenosine deaminase, cytidine deaminase, or dideaminase). When bound to a guide polynucleotide (e.g., gRNA), the polynucleotide-programmable nucleotide-binding domain can specifically bind to the target polynucleotide sequence, thereby positioning the base editor onto the target nucleic acid sequence to be edited.

[0236] Multinucleotide programmable nucleotide binding domain The polynucleotide programmable nucleotide-binding domain binds to polynucleotides (e.g., RNA, DNA). The polynucleotide programmable nucleotide-binding domain in the base editor may itself contain one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide-binding domain contains an endonuclease or an exonuclease.

[0237] This document discloses a base editor comprising a polynucleotide programmable nucleotide-binding domain comprising all or a portion (e.g., a functional portion) of a CRISPR protein (i.e., a base editor comprising all or a portion (e.g., a functional portion) of a CRISPR protein (e.g., a Cas protein) as a domain, also referred to as a “CRISPR protein-derived domain” of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural version of the CRISPR protein. The CRISPR protein-derived domain may contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to the wild-type or natural version of the CRISPR protein.

[0238] The Cas proteins used in this paper include classes 1 and 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, and Csm4. Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1 (e.g., SEQ) CRISPR enzymes include ID NO: 232, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ, CARF, DinG, their homologs, or modified versions thereof. CRISPR enzymes can be directed to cleave one or both strands at a target sequence, such as within the target sequence and / or within its complementary sequence. For example, a CRISPR enzyme can be directed to cleave one or both strands approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0239] Vectors encoding CRISPR enzymes mutated relative to the corresponding wild-type enzyme can be used, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. Cas proteins (e.g., Cas9, Cas12) or Cas domains (e.g., Cas9, Cas12) can refer to polypeptides or domains having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with wild-type exemplary Cas polypeptides or Cas domains. Cas (e.g., Cas9, Cas12) can refer to wild-type or modified forms of Cas proteins, said modified forms comprising amino acid alterations, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0240] In some implementations, the CRISPR protein-derived domain of the base editor may include all or a portion (e.g., functional portions) of Cas9 from the following species: *Corynebacterium ulcerans* (NCBI reference sequences: NC_015683.1, NC_017317.1); *Corynebacterium diphtheria* (NCBI reference sequences: NC_016782.1, NC_016786.1); *Spiroplasma syrphidicola* (NCBI reference sequence: NC_021284.1); *Prevotella intermedia* (NCBI reference sequence: NC_017861.1); *Spiroplasma taiwanense* (NCBI reference sequence: NC_021846.1); and *Streptococcus*. *Iniae* (NCBI reference sequence: NC_021314.1); *Belliella baltica* (NCBI reference sequence: NC_018010.1); *Psychroflexus torquis* (NCBI reference sequence: NC_018721.1); *Streptococcus thermophilus* (NCBI reference sequence: YP_820832.1); *Listeria innocua* (NCBI reference sequence: NP_472073.1); *Campylobacter jejuni* (NCBI reference sequence: YP_002344900.1); *Neisseria meningitidis* (NCBI reference sequence: YP_002342100.1); *Streptococcus pyogenes*, or *Staphylococcus aureus*. aureus).

[0241] Some aspects of this disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains are known in the art and described, for example, in “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016) by Kleinstiver, BP, et al.; and in “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015) by Slaymaker, IM, et al.; the entire contents of each of these references are incorporated herein by reference. An exemplary high-fidelity Cas9 domain is provided in the sequence listing as SEQ ID NO: 233.

[0242] In some embodiments, any Cas9 fusion protein or complex provided herein contains one or more of the mutations D10A, N497X, R661X, Q695X and / or Q926X, or the corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid.

[0243] Typically, Cas9 proteins, such as Cas9 (spCas9) from *Streptococcus pyogenes*, require a "prototype spacer adjacent motif (PAM)" or PAM-like motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of an NGG PAM sequence is required to bind specific nucleic acid regions, where "N" in "NGG" stands for adenosine (A), thymidine (T), or cytidine (C), and "G" stands for guanosine. In some embodiments, any fusion protein or complex provided herein may contain a Cas9 domain capable of binding nucleotide sequences that do not contain a classic (e.g., NGG) PAM sequence. Cas9 domains that bind non-classical PAM sequences have been described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-classical PAM sequences have been described in Kleinstiver, BP, et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the full text of each of these references is cited in this paper.

[0244] In some implementations, the napDNAbp is a circular substitution mutant (e.g., SEQ ID NO: 238).

[0245] In some embodiments, the polynucleotide programmable nucleotide-binding domain includes a nicking enzyme domain. The term "nicking enzyme" as used herein refers to a polynucleotide programmable nucleotide-binding domain containing a nuclease domain capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., DNA). For example, when the polynucleotide programmable nucleotide-binding domain includes a Cas9-derived nicking enzyme domain, the Cas9-derived nicking enzyme domain may include a D10A mutation and a histidine residue at position 840. In another example, the Cas9-derived nicking enzyme domain includes an H840A mutation, while the amino acid residue at position 10 remains D.

[0246] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain; that is, the Cas9 is a cleavage enzyme, referred to as an "nCas9" protein (representing "cleavage enzyme" Cas9; SEQ ID NO: 201). The Cas9 cleavage enzyme can be a Cas9 protein capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 cleavage enzyme comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with any of the Cas9 cleavage enzymes provided herein. Other suitable Cas9 cleavage enzymes will be apparent to those skilled in the art based on this disclosure and knowledge of the art, and are within the scope of this disclosure.

[0247] This document also provides base editors that include a polynucleotide programmable nucleotide-binding domain that is catalytically inactivated (i.e., cannot cleave the target polynucleotide sequence). For example, in the case of a base editor including a Cas9 domain, the Cas9 may contain both a D10A mutation and an H840A mutation. In a further embodiment, the catalytically inactivated polynucleotide programmable nucleotide-binding domain includes a point mutation (e.g., D10A or H840A) and the deletion of all or part (e.g., the functional portion) of the nuclease domain. The dCas9 domain is known in the art and is described, for example, in Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference.

[0248] The term "prototype spacer adjacent motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by a nucleic acid programmable DNA-binding protein. In some embodiments, the PAM may be a 5′ PAM (i.e., upstream of the 5′ end of the prototype spacer sequence). In other embodiments, the PAM may be a 3′ PAM (i.e., downstream of the 5′ end of the prototype spacer sequence). The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNGATT, NNAGAAW, or NAAAAAC. Y is pyrimidine; N is any nucleotide base; W is A or T.

[0249] The base editor provided by this invention can contain a domain derived from a CRISPR protein, which can bind nucleotide sequences containing classical or non-classical prototype spacer adjacent motif (PAM) sequences.

[0250] In some implementations, the PAM is “NRN” PAM, where “N” in “NRN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and R is adenine (A) or guanine (G); or the PAM is “NYN” PAM, where “N” in “NYN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and Y is cytidine (C) or thymine (T), for example, as described by RT Walton et al. in Science, 2020, 10.1126 / science.aba8853 (2020), the entire contents of which are incorporated herein by reference.

[0251] The following are some of the PAM variants described in Table 3.

[0252] Table 3. Cas9 protein and corresponding PAM sequence. N represents A, C, T, or G; V represents A, C, or G.

[0253] In some embodiments, the PAM is an NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM Cas9 variant contains one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) of D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively, “MQKFRAER”), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) of D1135V, G1218R, R1335Q, and T1337R (collectively, VRQR), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) of D1135V, G1218R, R1335E, and T1337R (collectively, VRER), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from saCas9 (SEQ ID No: 218) of E782K, N968K, and R1015H (collectively, KHH). In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) of D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively, MQKSER), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from spCas9 (SEQ ID No: 197) of D1135M, S1136Q, G1218K, E1219S, R1335E and T1337R (collectively, “MQKSER”), or a corresponding mutation in another Cas9.

[0254] In some embodiments, the CRISPR protein-derived domain of the base editor comprises all or a portion (e.g., the functional portion) of a Cas9 protein having a classical PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may employ a non-classical PAM sequence. Such sequences have been described in the art and will be apparent to those skilled in the art. For example, Cas9 domains binding to non-classical PAM sequences have been described in the following literature: Kleinstiver, BP, et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); RTWalton et al., “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126 / science.aba8853 (2020); Hu et al., “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr. 5, 556(7699), 57-63; Miller et al., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat. Biotechnol., 2020 Apr;38(4):471-481; The full content of each reference is cited in this article.

[0255] A fusion protein or complex containing NapDNAbp and cytidine deaminase and / or adenosine deaminase Some aspects of this disclosure provide fusion proteins or complexes comprising a Cas9 domain or other nucleic acid-programmable DNA-binding protein (e.g., Cas12) and one or more cytidine deaminase, adenosine deaminase, or cytidine adenosine deaminase domains. It should be understood that the Cas9 domain can be any Cas9 domain or Cas9 protein provided herein (e.g., dCas9 or nCas9). In some embodiments, any Cas9 domain or Cas9 protein provided herein (e.g., dCas9 or nCas9) can be fused with any cytidine deaminase and / or adenosine deaminase provided herein. The domains of the base editors disclosed herein can be arranged in any order.

[0256] In some embodiments, the fusion protein or complex comprising cytidine deaminase or adenosine deaminase with napDNAbp (e.g., a Cas9 or Cas12 domain) does not include a linker sequence. In some embodiments, a linker exists between the cytidine deaminase or adenosine deaminase and napDNAbp. In some embodiments, the cytidine deaminase or adenosine deaminase is fused to napDNAbp via any linker provided herein. For example, in some embodiments, the cytidine deaminase or adenosine deaminase is fused to napDNAbp via any linker provided herein.

[0257] It should be understood that the fusion proteins or complexes of this disclosure may include one or more additional features. For example, in some embodiments, the fusion protein or complex may include inhibitors, cytoplasmic localization sequences, export sequences such as nuclear export sequences, or other localization sequences, and sequence tags that can be used for the solubilization, purification, or detection of the fusion protein or complex. Suitable protein tags provided herein include, but are not limited to, biotinylate carboxyl carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags (also known as histidine tags or His tags), maltose-binding protein (MBP) tags, nus tags, glutathione S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softags (e.g., Softag 1, Softag 3), strep tags, biotin ligase tags, FLASH tags, V5 tags, and SBP tags. Other suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein or complex includes one or more His tags.

[0258] Exemplary but non-limiting fusion proteins are described in international PCT applications PCT / US2017 / 045381, PCT / US2019 / 044935 and PCT / US2020 / 016288, each of which is incorporated herein by reference in its entirety.

[0259] Fusion proteins or complexes with internal insertions This document provides fusion proteins or complexes comprising a heterologous polypeptide fused to a nucleic acid-programmable nucleic acid-binding protein (e.g., napDNAbp). The heterologous polypeptide may be fused to the C-terminus of napDNAbp, the N-terminus of napDNAbp, or inserted into an internal location within napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine or adenosine deaminase) or a functional fragment thereof. For example, the fusion protein may comprise a deaminase flanked by the N-terminal and C-terminal fragments of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide.

[0260] The deaminase can be a circularly arranged mutant deaminase. In some embodiments, the deaminase is a circularly arranged mutant TadA, which is circularly arranged at amino acid residues numbered 116, 136, or 65 in the TadA reference sequence.

[0261] The fusion protein or complex may contain more than one deaminase. The fusion protein or complex may contain, for example, 1, 2, 3, 4, 5, or more deaminases. The deaminase in the fusion protein or complex may be adenosine deaminase, cytidine deaminase, or a combination thereof.

[0262] In some embodiments, the napDNAbp in the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide may be a variant of the Cas9 polypeptide. The Cas9 polypeptide may be a cyclically arranged Cas9 protein.

[0263] Heterogeneous polypeptides (e.g., deaminases) can be inserted into suitable locations within napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)) such that the napDNAbp retains its ability to bind target polynucleotides and direct nucleic acids. Deaminases (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase (dimetases)) can be inserted into napDNAbp without impairing the function of the deaminase (e.g., base editing activity) or the function of the napDNAbp (e.g., its ability to bind target nucleic acids and direct nucleic acids).

[0264] In some embodiments, a deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted into a region of the Cas9 polypeptide that has a higher than average B factor (e.g., a higher B factor compared to the total protein or a protein domain containing a disordered region). Locations of the Cas9 polypeptide containing a higher than average B factor may include, for example, residues numbered 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 in the aforementioned Cas9 reference sequence. Regions of the Cas9 polypeptide containing a higher than average B factor may include, for example, residues numbered 792-872, 792-906, and 2-791 in the aforementioned Cas9 reference sequence.

[0265] In some embodiments, a heterologous polypeptide (e.g., a deaminase) is inserted into a flexible loop of the Cas9 polypeptide. The flexible loop portion may be selected from amino acid residues numbered 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 in the aforementioned Cas9 reference sequence, or corresponding amino acid residues in another Cas9 polypeptide. The flexible loop portion may also be selected from amino acid residues numbered 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 in the aforementioned Cas9 reference sequence, or corresponding amino acid residues in another Cas9 polypeptide.

[0266] Heterogeneous polypeptides (e.g., adenine deaminase) can be inserted into the Cas9 polypeptide region corresponding to amino acid residues numbered 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077 in the aforementioned Cas9 reference sequence, or corresponding to the corresponding amino acid residues in another Cas9 polypeptide.

[0267] A heterologous polypeptide (e.g., adenine deaminase) can be inserted into the site of a deletion region of the Cas9 polypeptide. The deletion region may correspond to the N-terminal or C-terminal portion of the Cas9 polypeptide. Exemplary internal fusion base editors are provided in Table 4A below: Table 4A: Insertion sites in the Cas9 protein

[0268] Heterogeneous peptides (e.g., deaminases) can be inserted into the structural or functional domains of a Cas9 peptide. Heterogeneous peptides (e.g., deaminases) can be inserted to replace a structural or functional domain of the Cas9 peptide, for example, after that domain has been removed from the Cas9 peptide. The structural or functional domains of the Cas9 peptide may include, for example, RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH.

[0269] The fusion protein may include a linker between the deaminase and the napDNAbp polypeptide. The linker may be a peptide linker or a non-peptide linker. For example, the linker may be XTEN, (GGGS) n (SEQ ID NO: 246), SGGSSGGS (SEQ ID NO: 330), (GGGGS) n (SEQ ID NO: 247), (G) n , (EAAAK)n (SEQ ID NO:248), (GGS) n SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are linked to the deaminase via linkers. In some embodiments, the N-terminal and C-terminal fragments are linked to the deaminase domain without a linker. In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase, but not between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase, but not between the N-terminal Cas9 fragment and the deaminase.

[0270] In some embodiments, the napDNAbp in the fusion protein or complex is a Cas12 polypeptide, such as Cas12b / C2c1, or a functional fragment thereof capable of associating with a nucleic acid (e.g., gRNA) that guides Cas12 to a specific nucleic acid sequence. The Cas12 polypeptide may be a variant of the Cas12 polypeptide. In other embodiments, the N- or C-terminal fragment of the Cas12 polypeptide contains a nucleic acid-programmable DNA-binding domain or a RuvC domain. In other embodiments, the fusion protein contains a linker between the Cas12 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 250) or GSSGSETPGTSESATPESSG (SEQ ID NO: 251). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the connection base is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

[0271] In other embodiments, the fusion protein or complex contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 261). In other embodiments of the above aspects, the nuclear localization signal is encoded by the following sequence: ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 262). In other embodiments, the Cas12b peptide contains a mutation that silences the catalytic activity of the RuvC domain. In other embodiments, the Cas12b peptide contains D574A, D829A, and / or D952A mutations.

[0272] In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., a Cas12-derived domain) having an internally fused nucleobase editing domain (e.g., all or part of a deaminase domain (e.g., a functional portion), such as an adenosine deaminase domain). In some embodiments, the napDNAbp is Cas12b. In some embodiments, the base editor comprises a BhCas12b domain having an internally fused TadA*8 domain inserted at the site provided in Table 4B below.

[0273] Table 4B: Insertion sites in the Cas12b protein

[0274] In some embodiments, the base editing system described herein is an ABE, wherein TadA is inserted into Cas9. The polypeptide sequence of the relevant ABE for inserting TadA into Cas9 is provided in the accompanying sequence listing as SEQ ID NOs: 263-308.

[0275] Exemplary but non-limiting fusion proteins are described in international PCT application PCT / US2020 / 016285 and U.S. provisional applications 62 / 852,228 and 62 / 852,224, the contents of which are incorporated herein by reference in their entirety.

[0276] A to G editors In some embodiments, the base editor described herein comprises an adenosine deaminase domain. This adenosine deaminase domain of the base editor can facilitate the editing of adenine (A) nucleosides to guanine (G) nucleosides by deaminating adenine (A) to form inosine (I), wherein inosine exhibits the base-pairing properties of G. In some embodiments, the A-to-G base editor also comprises an inosine base excision repair inhibitor, such as a uracil glycosidase inhibitor (UGI) domain or a catalytically inactivating inosine-specific nuclease. Without being bound by any particular theory, the UGI domain or the catalytically inactivating inosine-specific nuclease can inhibit or prevent base excision repair of deamination adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0277] A base editor containing an adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In one embodiment, the adenosine deaminase domain of the base editor contains all or a portion (e.g., a functional portion) of ADAT, which contains one or more mutations that enable ADAT to deaminate target A in DNA. For example, the base editor may contain all or a portion (e.g., a functional portion) of ADAT (EcTadA) from Escherichia coli, which contains one or more mutations of D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase. Exemplary ADAT homologous polypeptide sequences are provided in the sequence listing as SEQ ID NOs: 1 and 309-315.

[0278] The adenosine deaminase can be derived from any suitable organism (e.g., *Escherichia coli*). In some embodiments, the adenosine deaminase is derived from *Escherichia coli*, *Staphylococcus aureus*, *Salmonella typhi*, *Shewanella putrefaciens*, *Haemophilus influenzae*, *Caulobacter crescentus*, or *Bacillus subtilis*. In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase containing one or more mutations corresponding to any mutations provided herein (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified, for example, by sequence alignment and determination of homologous residues. Mutations corresponding to any mutations described herein (e.g., any mutations identified in ecTadA) in any naturally occurring adenosine deaminase (e.g., homologous to ecTadA) can be generated accordingly.

[0279] In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with any amino acid sequence of any adenosine deaminase provided herein. It should be understood that the adenosine deaminases provided herein may include one or more mutations (e.g., any mutations provided herein). This disclosure provides any deaminase domain having a specific percentage of identity plus any mutations described herein or a combination thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 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 mutations compared to a reference sequence or any adenosine deaminase provided herein.

[0280] It should be understood that any mutations provided herein (e.g., based on a TadA reference sequence, such as TadA*7.10 (SEQ ID NO: 1)) can be introduced into other adenosine deaminases, such as Escherichia coli TadA (ecTadA), Staphylococcus aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). In some embodiments, the TadA reference sequence is TadA*7.10 (SEQ ID NO: 1). It will be apparent to those skilled in the art that other deaminases can be similarly aligned to identify homologous amino acid residues that can be mutated as described herein. Therefore, any mutations identified in the TadA reference sequence can be carried out in other adenosine deaminases (e.g., ecTadA) having homologous amino acid residues. It should also be understood that any mutations provided herein can be carried out alone or in any combination in the TadA reference sequence or other adenosine deaminases.

[0281] In some embodiments, the adenosine deaminase comprises a selection of those alterations or sets of alterations listed in Tables 5A-5E below: Table 5A. Adenosine deaminase variants. The residue positions shown are in the E. coli TadA variant (TadA*).

[0282] Table 5B. TadA*8 adenosine deaminase variants. The residue positions shown are in the *E. coli* TadA variant (TadA*). Changes are referenced to TadA*7.10 (first row).

[0283] Table 5C. TadA*9 adenosine deaminase variants. Changes are referenced to TadA*7.10. Additional details about TadA*9 adenosine deaminase are described in international PCT application PCT / US2020 / 049975, which is incorporated herein by reference in its entirety for all purposes.

[0284] In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising amino acid alterations of F149Y. In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising amino acid alterations of R147D, F149Y, T166I, and D167N (TadA*8.10+). In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising amino acid alterations of S82T and F149Y (TadA*9v1). In some embodiments, the adenosine deaminase comprises a TadA*8.20 adenosine deaminase variant further comprising amino acid alterations of Y147D, F149Y, T166I, D167N, and S82T (TadA*9v2).

[0285] In some embodiments, the adenosine deaminase comprises one or more of the following mutations: M1I, M1S, S2A, S2E, S2H, S2R, S2L, E3L, V4D, V4E, V4M, V4K, V4S, V4T, V4A, E5K, F6S, F6G, F6H, F6Y, F6I, F6E, S7K, H8E, H8Y, H8H, H8Q, H8E, H8G, H8S, E9Y, E9K, E9V, E9E, Y10F, Y10W, Y10Y, M12S, M12L, M12R , M12W, R13H, R13I, R13Y, R13R, R13G, R13S, H14N, A15D, A15V, A15L, A15H, T17T, T17A, T17W, T17L, T17F, T17R, T17S, L 18A, L18E, L18N, L18L, L18S, A19N, A19H, A19K, A19A, A19D, A19G, A19M, R21N, K20K, K20A, K20R, K20E, K20G, K20C, K20Q R21A, R21R, R21N, R21Y, R21CG22P, A22W, A22R, W23D, R23H, W23G, W23Q, W23L, W23R, W23H W23D W23M, W23W, W23I, D24E, D24G, D24W, D24D, D24R, E25F, E25M, E25D, E25A, E25G, E25R, E25E, E25H E25V, E25S, E25Y, R26D, R26E, R26G, R26N, R26Q, R26C, R26L, R26K, R26W, R26C, R26P, R26R, R26A, R26H, E27E, E27Q, E27H, E27C, E27G, E2 7K, E27S, E27P, E27R, E27L, E27V, E27D, V28V, V28A, V28C, V28G, V28P, V28S, V28T, P29V, P29P, P29A, P29G, P29K, P29L, V30V, V30I, V30L, V30F, V30G, V30A, V30M, L34S, L34V, L34L, L34M, L34W, L34G, H36E, H36V, L36H, H36L, H36N, N37N, N37H, N37R, N37T, N37S, N38G, N38R, N3 8N, N38E, V40I, W45A, W45W, W45R, W45L, W45N, N46N, N46M, N46P, N46G, N46L, N46R, N46V, R46W, R46F, R46Q, R46M, R47A, R47Q, R47F, R47K,R47P、R47W、R47M、R47R、R47G、R47S、R47V、R47H、P48T、P48L、P48A、P48I、P48S、P48R、P48K、P48D、P48E、P48H、P48G、P48P、P48N、I49G、I49H、I49V、I49F、I49H、I49I、I49M、I49N、I49K、I49Q、I49T、G50L、G50S、G50R、G50G、R51H、R51L、R51N、L51W、R51Y、R51G、R51V、R51R、H52D、H52Y、H52I、H52H、D53D、D53E、D53G、D53P、P54C、P54T、P54P、P54E、A55H、T55A、T55I、T55V、T55G、T55T、A56A、A56H、A56W、A56E、A56S、H57P、H57A、H57H、H57N、A58G、A58E、A58A、A58R、E59A、E59G、E59I、E59Q、E59W、E59E、E59T、E59H、E59P、M61A、M61I、M61L、M61V、M61P、M61G、M61I、L63S、L63V、L63T、L63R、L63H、L63A、R64A、R64Q、R64R、R64D、Q65V、Q65H、Q65G、Q65P、Q65F、Q65Q、Q65R、G66V、G66E、G66T、G66G、G66C、G67G、G67W、G67I、G67A、G67D、G67L、G67V、L68Q、L68M、L68V、L68H、L68L、L68G、V69A、V69M、V69V、M70V、M70L、E70A、M70A、M70M、M70E、M70T、M70v、Q71M、Q71N、Q71L、Q71R、Q71Q、Q71I、N72A、N72K、N72S、N72D、N72Y、N72N、N72H、N72G、N72M、Y73G、Y73I、Y73K、Y73R、Y73S、Y73Y、Y73H、Y73A、R74A、R74Q、R74G、R74K、R74L、R74N、R74G、R74K、R74R、I76H、I76R、I76W、I76Y、I76V、I76Q、I76L、I76D、I76F、I76I、I76N、I76T、I76Y、D77G、D77D、D77A、D77Q、A78Y、A78T、A78G、A78A、A78I、T79M、T79R、T79L、T79T、L80M、L80Y、L80I、L80V、L80L、Y81D、Y81V、Y81Y、Y81M、V82A、V82S、V82G、V82T、V82V、V82Q、V82Y、T83L、T83F、T83T、T83N、L84E、L84F、L84Y、L84I、L84L、L84M、L84A、L84T、L84S、E85K、E85G、E85P、E85S、E85E、E85F、E85V、E85R、P86T、P86C、P86P、P86L、P86N、P86K、P86H、C87M、C87I、C87S、C87N、C87P、S87C、S87L、S87V、V88A、V88M、V88V、V88T、V88E、V88D、V88S、C90S、C90P、C90A、C90T、C90M、A91A、A91G、A91S、A91V、A91T、A91C、A91L、G92T、G92M、G92A、G92Y、G92G、A93I、A93C、A93M、A93V、A93A、M94M、M94T、M94A、M94V、M94L、M94I、M94H、I95S、I95G、I95L、I95H、I95V、H96A、H96L、H96R、H96S、H96H、H96N、H96E、S97C、S97G、S97I、S97M、S97R、S97S、S97P、R98K、R98I、R98N、R98Q、R98G、R98H、R98C、R98L、R98R、G100R、G100V、G100K、G100A、G100S、G100M、G100I、R101V、R101R、R101S、R101C、V102A、V102F、V102I、V102V、D103A、V103A、V103G、V103F、V103V、F104G、D104N、F104V、F104I、F104L、F104A、F104F、F104R、G105V、G105W、G105G、G105M、G105A、A106T、V106Q、V106F、V106W、V106M、A106A、A106Q、A106F、A106G、A106W、A106M、A106V、A106R、A106L、A106S、A106B、A106I、R107C、R107G、R107P、R107K、R107A、R107N、R107W、R107H、R107S、R107R、R107F、D108N、D108F、D108G、D108V、D108A、D108Y、D108H、D108I、D108K、D108L、D108M、D108Q、N108Q、N108F、N108W、N108M、N108K、D108K、D108F、D108M、D108Q、D108R、D108W、D108S、D108E、D108T、D108R、D108D、A109H、A109K、A109R、A109S、A109T、A109V、A109A、A109D、K110G、K110H、K110I、K110R、K110T、K110K、K110A、K110l、T111A、T111G、T111H、T111R、T111T、T111K、G112A、G112G、G112H、G112T、G112R、A113N、A114G、A114H、A114V、A114C、A114S、A114A、G115S、G115G、G115M、G115L、G115A、G115F、L117M、L117L、L117W、L117A、L117S、L117N、L117V、M118D、M118G、M118K、M118N、M118V、M118M、M118L、M118R、D119L、D119N、D119S、D119V、D119D、V120H、V120L、V120V、V120T、V120A、V120E、V120G、V120D、L121D、L121M、L121N、L121K、L121L、H122H、H122N、H122P、H122R、H122S、H122Y、H122G、H122T、H122L、H123C、H123G、H123P、H123V、H123Y、Y123H、H123Y、H123H、P124P、P124H、P124A、P124Y、P124D、P124G、P124I、P124L、P124W、G125H、G125I、G125A、G125M、G125K、G125G、G125P、M126D、M126H、M126K、M126I、M126N、M126O、M126S、M126Y、M126M、M126G、N127H、N127S、N127D、N127K、N127R、N127N、N127I、N127P、N127M、H128R、H128N、H128L、H128H、R129H、R129Q、R129V、R129I、R129E、R129V、R129R、R129M、R129P、V130R、V130V、V130E、V130D、E131E、E131I、E131V、E131K、I132I、I132F、I132T、I132L、I132V、I132E、T133V、T133E、T133G、T133K、T133T、T133A、T133H、T133F、T133I、E134A、E134E、E134G、E134I、E134H、E134K、E134T、G135G、G135V、G135I、G135P、G135E、I136G、I136L、I136T、I136I、l137A、l137D、l137E、L137M、l137S、L137L、L137I、A138D、A138E、A138G、S138A、A138N、A138S、A138T、A138V、A138Y、A138A、A138M、A138L、D139E、D139I、D139C、D139L、D139M、D139D、D139G、D139H、D139A、E140A、E140C、E140L、E140R、E140K、E140E、E140D、C141S、C141A、C141C、C141V、C141E、A142N、A142D、A142G、A142A、A142L、A142S、A142T、A142N、A142S、A142V、A142E、A142C、A143D、A143E、A143G、A143D、A143G、A143E、A143L、A143W、A143M、A143S、A143Q、A143R、A143A、A143I、L144S、L144L、L144T、L144A、L145A、L145F、L145G、L145D、L145L、L145C、L145E、L145s、C146R、S146A、S146C、S146D、S146F、S146R、S146T、S146D、S146G、S146S、S146L、D147D、D147L、D147F、D147G、D147Y、Y147T、Y147R、Y147D、D147R、D147Y、D147A、D147T、D147H、D147F、D147U、D147V、D147I、D147C、F148L、F148F、F148R、F148Y、F148A、F148T、F149C、F149M、F149R、F149Y、F149N、F149F、F149A、F149T、F149V R150R、R150M、R150D、R150F、M151F、M151P、M151R、M151V、M151M、M151E、R152C、R152F、R152H、R152P、R152R、R152P、R152Q、R152M、R152O, R153C, R153Q, R153R, R153V, R153E, R153A, R153P, Q154E, Q154H, Q154M, Q154R, Q154L, Q154S, Q154V, Q154Q, Q154F, Q154I, Q15 4A, Q154K, E155F, E155G, E155I, E155K, E155P, E155V, E155D, E155E, E155L, E155Q, I156V, I156A, I156I, I156L, I156F, I156D, I156K, I 156N, I156R, I156Y, E157A, E157F, E157I, E157P, E157T, E157V, N157K, K157N, K157V, K157P, K157I, K157F, K157F, K157T, K157A, K157 S, K157R, A158Q, A158K, A158V, A158A, A158D, A158S, A158T, A158N, Q159S, Q159Q, Q159A, Q159F, Q159K, Q159L, Q159N, K160A, K160S, K1 60E, K160K, K160N, K160F, K160Q, K161T, K161K, K161R, K161I, K161A, K161N, K161Q, K161S, K161T, A162D, A162Q, R162H, R162P, A162S , A162A, A162N, A162M, A162K, Q163G, Q163S, Q163Q, Q163A, Q163H, Q163N, Q163R, S164F, S164S, S164Q, S164I, S164R, S164Y, S165S, S16 5P, S165Q, S165A, S165D, S165I, S165T, S165Y, T166T, T166Q, T166E, T166S, T166D, T166K, T166I, T166N, T166P, T166R, D167SD167D, D167I, D167G, D167T, D167A and / or D167N, wherein the mutation is located in the TadA reference sequence (e.g., TadA*7.10, ecTadA or TadA8e), and any substitution mutation at the corresponding position, or one or more corresponding mutations in another adenosine deaminase. Other mutations are described in U.S. Patent Application Publication 2022 / 0307003 A1, U.S. Patent 11,155,803, and International Patent Application Publication WO 2023 / 288304 A2 and PCT / CN2022 / 143408.The contents of WO 2018 / 027078 A1, WO 2021 / 158921 A1 and WO 2023 / 034959 A2 are incorporated herein by reference in their entirety for all purposes.

[0286] In some embodiments, this disclosure provides TadA variants comprising V82T, Y147T, and / or Q154S mutations. In some embodiments, this disclosure provides TadA variants comprising V82T, Y147T, and / or Q154S mutations. In some embodiments, this disclosure provides TadA*8.8 further comprising V82T mutations. In some embodiments, this disclosure provides TadA*8.8 further comprising V82T, Y147T, and Q154S mutations. In some embodiments, this disclosure provides TadA*8.17 further comprising V82T mutations. In some embodiments, this disclosure provides TadA*8.17 further comprising V82T, Y147T, and Q154S mutations. In some embodiments, this disclosure provides TadA*8.20 further comprising V82T mutations. In some embodiments, this disclosure provides TadA*8.20 further comprising V82T, Y147T, and Q154S mutations.

[0287] In the implementation, variants of TadA*7.10 include one or more changes selected from any of those changes provided herein.

[0288] In a particular embodiment, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from: Staphylococcus aureus TadA, Bacillus subtilis TadA, Salmonella typhimurium TadA, Shewanella putrefaciens TadA, Haemophilus influenzae F3031 TadA, C. crescentus TadA, G. sulfurreducens TadA, or TadA*7.10.

[0289] In some embodiments, the TadA*8 is a variant as shown in Table 5D. Table 5D shows the position numbers of certain amino acids in the TadA amino acid sequence and the amino acids present at these positions in the TadA-7.10 adenosine deaminase. Table 5D also shows the amino acid changes in the TadA variants relative to TadA-7.10 after phage-assisted discontinuous evolution (PANCE) and phage-assisted continuous evolution (PACE), as described in M. Richter et al., 2020, Nature Biotechnology, doi.org / 10.1038 / s41587-020-0453-z, the entire contents of which are incorporated herein by reference. In some embodiments, the TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, the TadA*8 is TadA*8e. In one embodiment, the adenosine deaminase is TadA*8, which comprises SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity, or is substantially composed of SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity.

[0290] Table 5D. Selected TadA*8 variants

[0291] In some embodiments, the TadA variant is one of the variants shown in Table 5E. Table 5E shows the position numbers of certain amino acids in the TadA amino acid sequence and the amino acids present at these positions in TadA*7.10 adenosine deaminase. In some embodiments, the TadA variant is MSP605, MSP680, MSP823, MSP824, MSP825, MSP827, MSP828, or MSP829. In some embodiments, the TadA variant is MSP828. In some embodiments, the TadA variant is MSP829.

[0292] Table 5E. TadA variants

[0293] In certain embodiments, the fusion protein or complex comprises a single (e.g., provided as a monomer) TadA* (e.g., TadA*8 or TadA*9). Throughout this disclosure, adenosine deaminase base editors comprising a single TadA* domain are referred to by the terms ABEm or ABE#m, where "#" is an identification number (e.g., ABE8.20m) and "m" signifies "monomer". In some embodiments, the TadA* is linked to a Cas9 nickase. In some embodiments, the fusion protein or complex of this disclosure comprises a heterodimer of wild-type TadA (TadA(wt)) linked to TadA*. Throughout this disclosure, adenosine deaminase base editors comprising a single TadA* domain and a TadA(wt) domain are referred to by the terms ABEd or ABE#d, where "#" is an identification number (e.g., ABE8.20d) and "d" signifies "dimer". In other embodiments, the fusion protein or complex of this disclosure comprises a heterodimer of TadA*7.10 linked to TadA*. In some embodiments, the base editor is an ABE8 comprising a TadA* variant monomer. In some embodiments, the base editor is an ABE comprising a TadA* and a TadA(wt) heterodimer. In some embodiments, the base editor is an ABE comprising a TadA* and a TadA*7.10 heterodimer. In some embodiments, the base editor is an ABE comprising a TadA* heterodimer. In some embodiments, the TadA* is selected from Tables 5A-5E.

[0294] In some embodiments, the adenosine deaminase is expressed in monomeric form. In other embodiments, the adenosine deaminase is expressed in heterodimer form. In some embodiments, the deaminase or other polypeptide sequence lacks a methionine, for example, when included as a component of a fusion protein. This may alter the positional numbering. However, those skilled in the art will understand that such corresponding mutations refer to the same mutation.

[0295] Any mutations provided in this invention, as well as any additional mutations (e.g., based on the ecTadA amino acid sequence), can be introduced into any other adenosine deaminase. Any mutations provided in this invention can be introduced, alone or in any combination, into the TadA reference sequence or into another adenosine deaminase (e.g., ecTadA).

[0296] Detailed descriptions of A-to-G nucleobase editing proteins can be found in international PCT application PCT / US2017 / 045381 (WO2018 / 027078) and Gaudelli, NM et al., “Programmable base editing of A•T to G•C ingenomic DNA without DNA cleavage”, Nature, Vol. 551, pp. 464-471 (2017), the entire contents of which are incorporated herein by reference.

[0297] C to T editor In some embodiments, the base editor disclosed herein comprises a fusion protein or complex containing a cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), the uridine (U) having a thymine base-pairing property. In some embodiments, for example in the case that the polynucleotide is double-stranded (e.g., DNA), the uridine base may subsequently be substituted by a thymidine base (e.g., through cellular repair mechanisms), resulting in a C:G to T:A conversion. In other embodiments, the base editor's deamination of C to U in nucleic acids may not be accompanied by U to T substitution.

[0298] The deamination of a target C in a polynucleotide to produce a U is a non-limiting example of one type of base editing that the base editor described herein can perform. In another example, a base editor containing a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a guanine (G) base. For example, the U in a polynucleotide produced by deamination of cytidine by the cytidine deaminase domain of a base editor can be removed from the polynucleotide via a base excision repair mechanism (e.g., via a uracil DNA glycosylase (UDG) domain), producing a base-free site. The nucleobase opposite the base-free site can then be substituted with another base such as C (e.g., via a base repair mechanism), for example, via a trans-damage polymerase. Although the nucleobase opposite the base-free site is typically replaced with C, other substitutions (e.g., A, G, or T) may also occur.

[0299] Therefore, in some embodiments, the base editor described herein includes a deamination domain (e.g., a cytidine deaminase domain) capable of deaminating a target C in a polynucleotide to U. Furthermore, as described below, the base editor may include additional domains that, in some embodiments, facilitate the conversion of the deamination-generated U to T or G. For example, a base editor containing a cytidine deaminase domain may further include a uracil glycosylase inhibitor (UGI) domain to mediate the substitution of U for T, thereby completing the C-to-T base editing event. In another instance, the base editor may include a uracil-stabilizing protein as described herein. In yet another instance, the base editor may be incorporated into a transdamage polymerase to enhance the efficiency of C-to-G base editing, since the transdamage polymerase can promote the incorporation of C at a base-free site relative to the base (i.e., resulting in the incorporation of G at a base-free site, thereby completing the C-to-G base editing event).

[0300] A base editor containing cytidine deaminase as a domain can deaminate target C in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids.

[0301] In some implementations, the base editor's cytidine deaminase comprises all or a portion (e.g., the functional portion) of apolipoprotein B mRNA editing complex (APOBEC) family of deaminases. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is the zinc-dependent cytidine deaminase domain and is essential for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminases.

[0302] According to aspects of this disclosure, other exemplary deaminases that can be fused to Cas9 are provided below. In embodiments, the deaminase is an activation-induced deaminase (AID). It should be understood that in some embodiments, active domains of their respective sequences may be used, for example, domains that do not contain localization signals (nuclear localization sequences, domains that do not contain nuclear output signals, cytoplasmic localization signals).

[0303] Some aspects of this disclosure are based on the understanding that modulating the catalytic activity of the deaminase domain in any fusion protein or complex described herein, for example by making point mutations in the deaminase domain, affects the processability of the fusion protein (e.g., a base editor) or complex. For example, a mutation that reduces, but does not eliminate, the catalytic activity of the deaminase domain in a base-editing fusion protein or complex can reduce the likelihood that the deaminase domain catalyzes deamination of residues adjacent to the target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent undesired deamination of residues adjacent to the target-specific residue, which can reduce or prevent off-target effects.

[0304] In some embodiments, the APOBEC deaminase incorporated into the base editor may contain one or more mutations selected from the following: H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; and any substitution mutations at the corresponding positions, or one or more corresponding mutations in another APOBEC deaminase.

[0305] A variety of modified cytidine deaminases are commercially available, including but not limited to SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, and 85177). In some embodiments, the deaminase incorporated into the base editor comprises all or a portion (e.g., the functional portion) of the APOBEC1 deaminase.

[0306] In some embodiments, the fusion protein or complex of this disclosure comprises one or more cytidine deaminase domains. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine in DNA. The cytidine deaminases can be derived from any suitable organism. In some embodiments, the cytidine deaminases are naturally occurring cytidine deaminases containing one or more mutations corresponding to any mutations provided herein. Those skilled in the art will be able to identify the corresponding residues in any homologous protein by, for example, sequence alignment and determination of homologous residues. Therefore, those skilled in the art will be able to generate mutations corresponding to any mutations described herein in any naturally occurring cytidine deaminase. In some embodiments, the cytidine deaminases are derived from prokaryotes. In some embodiments, the cytidine deaminases are derived from bacteria. In some embodiments, the cytidine deaminases are derived from mammals (e.g., humans).

[0307] In some embodiments, the cytidine deaminase comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with any of the cytidine deaminases described herein. It should be understood that the cytidine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). Some embodiments provide polynucleotide molecules encoding any of the foregoing aspects or cytidine deaminase nucleobase editor polypeptides as described herein. In some embodiments, the polynucleotide is codon-optimized.

[0308] In the implementation scheme, the fusion protein of this disclosure comprises two or more nucleic acid editing domains.

[0309] Detailed descriptions of the C-to-T nucleotide base editing protein can be found in international PCT application PCT / US2016 / 058344 (WO2017 / 070632) and Komor, AC, et al., “Programmable editing of a target basein genomic DNA without double-stranded DNA cleavage”, Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference.

[0310] Cytidine Adenosine Base Editor (CABE) In some embodiments, the base editor described herein comprises an adenosine deaminase variant with enhanced cytidine deaminase activity. Such base editors may be referred to as “cytidine adenosine base editors (CABE)” or “cytosine base editors derived from TadA* (CBE-Ts)”, and their corresponding deaminase domains may be referred to as “TadA* (T*-Ts) acting on DNA cytosine.” AD C) domain. In some cases, adenosine deaminase variants possess both adenine and cytosine deaminase activities (i.e., are bifunctional deaminases). In some embodiments, the adenosine deaminase variant deaminates both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant deaminates both adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant deaminates both adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant primarily deaminates cytosine in DNA and / or RNA (e.g., more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deaminase reactions catalyzed by the adenosine deaminase variant are cytosine deaminases, or the number of cytosine deaminases catalyzed by the variant is approximately or at least approximately the number of adenine deaminases catalyzed by the variant). The number of purine deaminations can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 500, or 1,000 times. In some embodiments, the adenosine deaminase variant has substantially equal cytosine and adenosine deaminase activities (e.g., the two activities differ from each other by about 10% or 20%). In some embodiments, the adenosine deaminase variant has predominantly cytosine deaminase activity and little (if any) adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity and no significant or detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in vitro or in vivo cells. In some embodiments, the cells are bacterial, yeast, fungal, insect, plant, or mammalian cells.

[0311] In some embodiments, the CABE comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the CABE comprises a truncated TadA deaminase variant. In some embodiments, the CABE comprises a fragment of a TadA deaminase variant. In some embodiments, the CABE comprises a TadA*8.20 variant.

[0312] In some embodiments, the adenosine deaminase variant of this disclosure is a TadA adenosine deaminase comprising one or more alterations that increase cytosine deaminase activity (e.g., at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) while maintaining adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). In some cases, the adenosine deaminase variant comprises one or more alterations of cytosine deaminase activity that increase the activity relative to a reference adenosine deaminase (e.g., at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more), and comprises undetectable adenosine deaminase activity or adenosine deaminase activity less than 30%, 20%, 10%, or 5% of the reference adenosine deaminase activity. In some embodiments, the reference adenosine deaminase is TadA*8.20 or TadA*8.19.

[0313] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising two or more alterations at amino acid positions selected from amino acid positions 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167, or corresponding alterations in another deaminase, said amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher identity with SEQ ID NO:1.

[0314] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising one or more alterations to the amino acid sequence selected from the following: S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49 T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P, or a corresponding change in another deaminase, wherein the amino acid sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher identity with SEQ ID NO: 1.

[0315] In some embodiments, the adenosine deaminase variant is an adenosine deaminase that comprises an amino acid alteration or combination of amino acid alterations selected from any of the lists in Tables 6A-6F.

[0316] The residue identities of exemplary adenosine deaminase variants capable of deamination of adenine and / or cytidine in target polynucleotides (e.g., DNA) are provided in Tables 6A-6F below. Further examples of adenosine deaminase variants include variants of the following 1.17 (see Table 6A): 1.17+E27H; 1.17+E27K; 1.17+E27S; 1.17+E27S+I49K; 1.17+E27G; 1.17+I49N; 1.17+E27G+I49N; and 1.17+E27Q. In some embodiments, any amino acid changes provided herein are replaced with conserved amino acid substitutions. Other mutations known in the art may be further added to any adenosine deaminase variants provided herein.

[0317] In some embodiments, the base editor system comprising the CABE provided herein has at least about 30%, 40%, 50%, 60%, 70% or more C-to-T editing activity in the target polynucleotide (e.g., DNA). In some embodiments, the base editor system comprising the CABE provided herein has increased C-to-T base editing activity (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more) relative to a reference base editor system comprising a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).

[0318] Table 6A. Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20. “S” indicates “surface”, and “NAS” indicates “near active site”.

[0319] Table 6A (continued). Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20. “I” indicates “internal”, “S” indicates “surface”, and “NAS” indicates “near active site”.

[0320] Table 6B. Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20.

[0321] Table 6C. Adenosine deaminase variants. Mutations are represented as in variant 1.2 (Table 6A).

[0322] Table 6C. (Continued)

[0323] Table 6D. Adenosine deaminase variants. Mutations are represented by TadA*8.20.

[0324] Table 6E. Heterozygous constructs. Mutations are represented by reference TadA*7.10.

[0325] Table 6F. Base editor variants. Mutations are represented in reference to TadA* 8.19 / 8.20.

[0326] Guide polynucleotides When bound to a guide polynucleotide (e.g., gRNA), the polynucleotide programmable nucleotide-binding domain is able to specifically bind to the target polynucleotide sequence (i.e., through complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby positioning the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0327] In one embodiment, the guide polynucleotide described herein may be RNA or DNA. In one embodiment, the guide polynucleotide is gRNA.

[0328] In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, the guide polynucleotide comprises two or more individual polynucleotides that can interact with each other, for example, through complementary base pairing (e.g., dual guide polynucleotide, dual gRNA). For example, the guide polynucleotide may comprise CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), or may comprise one or more trans-activating CRISPR RNAs (tracrRNA).

[0329] Guide polynucleotides may include natural or non-natural (or non-naturally occurring) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some cases, the target region length of the guide nucleic acid sequence (e.g., spacer sequence) may be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0330] In some embodiments, the methods described herein may utilize engineered Cas proteins. Guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence necessary for Cas binding and a user-defined spacer sequence of about 20 nucleotides defining a genomic target to be modified. Exemplary gRNA scaffold sequences are provided in the sequence listing as SEQ ID NOs: 317-327 and 425. Therefore, those skilled in the art can modify the genomic target of the Cas protein, the specificity of which depends in part on the degree of specificity of the gRNA targeting sequence relative to the rest of the genome. In embodiments, the spacer sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides in length. The spacer sequence of the gRNA may be or may be about 19, 20 or 21 nucleotides in length.

[0331] gRNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, the composition comprises multiple gRNAs that all target the same exon, or multiple gRNAs that target different exons. It can target exons and / or introns of a gene. The gRNA or guide polynucleotide can target nucleic acid sequences of about 20 nucleotides or less (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides), or any length between about 1 and 100 nucleotides (e.g., 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100). The target nucleic acid sequence can be or can be about 20 bases immediately adjacent to the 5′ end of the first nucleotide of the PAM. The gRNA can target the nucleic acid sequence. The target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90 or 1-100 nucleotides.

[0332] Guided polynucleotides may contain standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs.

[0333] In some implementations, the base editor system may comprise multiple guide polynucleotides, such as gRNAs. For example, the gRNAs may target one or more target sites contained in the base editor system (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs). The multiple gRNA sequences may be arranged in tandem and may be separated by direct repeat sequences.

[0334] Modified polynucleotides To enhance expression, stability, and / or genome / base editing efficiency, and / or reduce potential toxicity, base editor coding sequences (e.g., mRNA) and / or guide polynucleotides (e.g., gRNA) can be modified to include one or more modified nucleotides and / or chemical modifications, such as pseudouridine, 5-methylcytosine, 2′-O-methyl-3′-phosphonoacetate, 2′-O-methylthio-PACE (MSP), 2′-O-methyl-PACE (MP), 2′-fluoroRNA (2′-F-RNA), constrained ethyl (S-cEt), 2′-O-methyl ('M'), 2′-O-methyl-3′-thiophosphate ('MS'), 2′-O-methyl-3′-thiophosphonoacetate ('MSP'), 5-methoxyuridine, thiophosphate, and N1-methylpseudouridine. Chemically protected gRNAs can enhance stability and editing efficiency both in vivo and in vitro. Methods using chemically modified mRNAs and guide RNAs are known in the art and are described, for example, by Jiang et al., Chemical modifications of adeninebase editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020); Callum et al., N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, p. e35; and Andries et al., Journal of Controlled Release, Volume 217, 10 November 2015, pp. 337-344, each of which is incorporated herein by reference in its entirety.

[0335] In some embodiments, the guide polynucleotide includes one or more modifying nucleotides at the 5′ and / or 3′ end of the guide. In some embodiments, the guide polynucleotide includes two, three, four or more modifying nucleotides at the 5′ and / or 3′ end of the guide. In some embodiments, the guide polynucleotide includes two, three, four or more modifying nucleotides at the 5′ and / or 3′ end of the guide.

[0336] In some embodiments, the guide contains at least about 50%-75% modified nucleotides. In some embodiments, the guide contains at least about 85% or more modified nucleotides. In some embodiments, at least about 1-5 nucleotides at the 5′ end of the gRNA are modified, and at least about 1-5 nucleotides at the 3′ end of the gRNA are modified. In some embodiments, at least about 3-5 consecutive nucleotides at each of the 5′ and 3′ ends of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present in the direct repeat sequence or inverted direct repeat sequence are modified. In some embodiments, at least about 50% of the nucleotides present in the direct repeat sequence or inverted direct repeat sequence are modified. In some embodiments, at least about 50-75% of the nucleotides present in the direct repeat sequence or inverted direct repeat sequence are modified. In some embodiments, at least about 100 nucleotides present in the direct repeat sequence or inverted direct repeat sequence are modified. In some embodiments, at least about 20% or more of the nucleotides present in the hairpin structure in the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides in the hairpin structure present in the gRNA scaffold are modified. In some embodiments, the guide comprises a spacer sequence of variable length. In some embodiments, the guide comprises a spacer sequence of 20-40 nucleotides. In some embodiments, the guide comprises a spacer sequence comprising at least about 20-25 nucleotides or at least about 30-35 nucleotides. In some embodiments, the spacer sequence comprises modified nucleotides. In some embodiments, the guide comprises two or more of the following: ● At least about 1-5 nucleotides at the 5′ end of the gRNA are modified, and at least about 1-5 nucleotides at the 3′ end of the gRNA are modified; ● At least about 20% of the nucleotides present in direct repeats or antidirect repeats are modified; ● At least 50-75% of the nucleotides present in direct repeat sequences or inverted direct repeat sequences are modified; ● At least about 20% or more of the nucleotides present in the hairpin structure within the gRNA scaffold are modified; ● Variable-length spacer basis; and ● Spacer sequences containing modified nucleotides.

[0337] In one embodiment, the gRNA contains a number of modified nucleotides and / or chemical modifications. Such modifications can increase base editing by approximately 2-fold in vivo or in vitro. In another embodiment, the gRNA contains a 2′-O-methyl or phosphate thioester modification. In one embodiment, the gRNA contains both 2′-O-methyl and phosphate thioester modifications. In one embodiment, the modification increases base editing by at least approximately 2-fold.

[0338] Guided polynucleotides may contain one or more modifications to provide nucleic acids with novel or enhanced characteristics. Guided polynucleotides may contain a nucleic acid affinity tag. Guided polynucleotides may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0339] gRNA or guide polynucleotides can also be modified through the following: 5′ adenosine, 5′ guanosine triphosphate cap, 5′ N7-methylguanosine triphosphate cap, 5′ triphosphate cap, 3′ phosphate, 3′ thiophosphate, 5′ phosphate, 5′ thiophosphate, cis-syn thymine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3′-3′ modification, 2′-O-methylthio-PACE (MSP), 2′-O-methyl-PACE (MP) and conformationally restricted ethyl (S-cEt), 5′-5′ modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desulfurized biotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, Dibiotin, PC-Biotin, Psoralen C2, Psoralen C6, TINA, 3′DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2′-deoxyribonucleoside analog purine, 2′-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methylribonucleoside analog, sugar-modified analog, wobbling / universal base, fluorescent dye labeling, 2′-fluoroRNA, 2′-O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, thiophosphate DNA, thiophosphate RNA, UNA, pseudouridine-5′-triphosphate, 5′-methylcytidine-5′-triphosphate, or any combination thereof.

[0340] In some cases, phosphate-thioester-enhanced RNA gRNAs can inhibit RNase A, RNase T1, fetal bovine serum nuclease, or any combination thereof. These properties allow PS-RNA gRNAs to be used in applications where there is a high probability of exposure to nucleases, either in vivo or in vitro. For example, a phosphate-thioester (PS) bond can be introduced between the last 3-5 nucleotides of the 5′ or 3′ end of the gRNA, which can inhibit exonuclease degradation. In some cases, a phosphate-thioester bond can be added to the entire gRNA to reduce endonuclease attack.

[0341] Fusion proteins or complexes containing nuclear localization sequences (NLS) In some embodiments, the fusion protein or complex provided herein further comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In one embodiment, a bityped NLS is used. In some embodiments, the NLS comprises an amino acid sequence that facilitates the entry of the NLS-containing protein into the nucleus (e.g., via nuclear transport). In some embodiments, the NLS is fused to the N-terminus or C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of the nCas9 or dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the Cas12 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of cytidine deaminase or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided or cited herein. Additional nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al.'s PCT / EP2000 / 011690, the contents of which are incorporated herein by reference to disclose exemplary nuclear localization sequences.

[0342] In some embodiments, the NLS is present in a linker, or the NLS is flanked by linkers, such as those described herein. A bifid NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (hence bipartite—two parts, unlike a monopartite NLS). The nucleoplasmic protein NLS—KR[PAATKKAGQA]KKKK (SEQ ID NO: 191)—is a prototype of a ubiquitous bifid signal: two basic amino acid clusters separated by a spacer sequence of approximately 10 amino acids. An exemplary bifid NLS sequence is as follows: PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328).

[0343] In some embodiments, any fusion protein or complex provided herein comprises an NLS containing the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, any adenosine base editor provided herein comprises the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, the NLS is located at the C-terminal portion of the adenosine base editor. In some embodiments, the NLS is located at the C-terminus of the adenosine base editor.

[0344] Additional domains The base editor described herein may include any domain that facilitates the editing, modification, or alteration of the nucleobases of a polynucleotide. In some embodiments, the base editor includes a polynucleotide programmable nucleotide-binding domain (e.g., Cas9), a nucleobase editing domain (e.g., a deaminase domain), and one or more additional domains. In some embodiments, the additional domains may facilitate the enzymatic or catalytic function of the base editor, the binding function of the base editor, or may be an inhibitor of a cellular mechanism (e.g., an enzyme) that may interfere with the desired base editing outcome. In some embodiments, the base editor includes nuclease, nickase, recombinase, deaminase, methyltransferase, methyltransferase, acetyltransferase, acetyltransferase, transcription activator, or transcription repressor domains.

[0345] In some embodiments, the base editor comprises a uracil glycosidase inhibitor (UGI) domain. In some cases, the base editor is trans-expressed in cells with a UGI peptide. In some embodiments, cellular DNA repair responses in the presence of U:G heteroduplex DNA may be responsible for reducing the efficiency of nuclear base editing in cells. In such embodiments, uracil DNA glycosidase (UDG) catalyzes the removal of U from DNA in cells, which can initiate base excision repair (BER), primarily resulting in the restoration of U:G pairs to C:G pairs. In such embodiments, BER can be inhibited by a base editor comprising one or more domains that bind single strands, block edited bases, inhibit UGI, inhibit BER, protect edited bases, and / or promote the repair of non-edited strands. Therefore, this disclosure covers base editor fusion proteins or complexes comprising a UGI domain and / or a uracil stable protein (USP) domain.

[0346] Base Editor System This document provides systems, compositions, and methods for editing nucleosides using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) including a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleosides; and (2) a guide polynucleotide (e.g., guide RNA) that binds to the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE) or an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA or RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, the deaminase domain may be cytidine deaminase or cytosine deaminase. In some embodiments, the deaminase domain may be adenine deaminase or adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some implementations, the base editor is capable of deaminating cytidine in DNA.

[0347] The method of using the base editor system provided herein includes the following steps: (a) contacting a target nucleotide sequence of a subject's polynucleotide (e.g., double-stranded or single-stranded DNA or RNA) with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., gRNA), wherein the target nucleotide sequence contains a target nucleobase pair; (b) inducing strand separation of the target region; (c) converting a first nucleobase of the target nucleobase pair in the single strand of the target region to a second nucleobase; and (d) cleaving the target region into no more than one strand, wherein a third nucleobase complementary to the first nucleobase is replaced by a fourth nucleobase complementary to the second nucleobase. It should be understood that in some embodiments, step (b) is omitted. In some embodiments, the target nucleobase pair is multiple nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of multiple nucleobase pairs in one or more genes. In some embodiments, the multiple nucleobase pairs are located in the same gene. In some embodiments, the plurality of nucleobase pairs are located in one or more genes, wherein at least one gene is located at a different site.

[0348] Components of a base editor system (e.g., deaminase domains, guide RNA, and / or polynucleotide programmable nucleotide-binding domains) can be covalently or nonvalently associated with each other. For example, in some embodiments, the deaminase domain can target a target nucleotide sequence via a polynucleotide programmable nucleotide-binding domain, optionally wherein the polynucleotide programmable nucleotide-binding domain is complexed with a polynucleotide (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide-binding domain can be fused to or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide-binding domain can target the deaminase domain to a target nucleotide sequence through nonvalent interaction or association with the deaminase domain. For example, in some embodiments, the nucleobase editing component (e.g., the deaminase component) includes an additional heterologous moiety or domain capable of interacting, associating with, or forming a complex with a corresponding heterologous moiety, antigen, or domain, which is part of the polynucleotide programmable nucleotide-binding domain and / or a guide polynucleotide (e.g., guide RNA) complexed therewith. In some embodiments, the polynucleotide programmable nucleotide-binding domain and / or the guide polynucleotide (e.g., guide RNA) complexed therewith includes an additional heterologous moiety or domain capable of interacting, associating, or forming a complex with a corresponding heterologous moiety, antigen, or domain, which is part of a nucleobase editing domain (e.g., a deaminase component). In some embodiments, the additional heterologous moiety may be capable of binding, interacting, associating, or forming a complex with a peptide. In some embodiments, the additional heterologous moiety may be capable of binding, interacting, associating, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety may be capable of binding to a peptide linker. In some embodiments, the additional heterologous moiety is capable of binding to a polynucleotide linker. The additional heterologous moiety may be a protein domain.In some embodiments, the additional heterologous portion comprises a polypeptide, such as the 22-amino acid RNA-binding domain (N22p) of λ phage anti-termination protein N, a 2G12 IgG homodimer domain, ABI, an antibody (e.g., an antibody that binds to a component of a base editor system or its heterologous portion) or a fragment thereof (e.g., heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), the Fc region of immunoglobulin, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab, Fab2, microantibodies and / or ZIP antibodies), a barnase-barstar dimer domain, a Bcl-xL domain, a calcineurin A (CAN) domain, a transmembrane pentamer domain of myocardial phosphatase, a collagen domain, or a Com RNA-binding protein domain (e.g., the SfMu Com shell protein domain and SfMu Com-binding protein domain), Cyp-Fas fusion protein (CyP-Fas) domain, Fab domain, Fe domain, foldon domain, FK506-binding protein (FKBP) domain, mTOR FKBP-binding domain (FRB) domain, foldon domain, fragment X domain, GAI domain, GID1 domain, transmembrane domain of blood group glycoprotein A, GyrB domain, Halo tag, HIV Gp41 trimerization domain, HPV45 oncoprotein E7 C-terminal dimer domain, hydrophobic peptide, K homology (KH) domain, Ku protein domain (e.g., Ku heterodimer), leucine zipper, LOV domain, mitochondrial antiviral signaling protein CARD filament domain, MS2 capsid protein domain (MCP), non-natural RNA aptamer ligands binding corresponding RNA motifs / aptamers, parathyroid hormone dimerization domain, PP7 capsid protein (PCP) domain, PSD95-Dlgl-zo-1 (PDZ) domain, PYL domain, SNAP tag, SpyCatcher portion, SpyTag portion, streptavidin domain, streptavidin-binding protein domain, streptavidin-binding protein (SBP) domain, telomerase Sm7 protein domain (e.g., Sm7 homoheptamer or monomeric Sm-like protein) and / or fragments thereof. In the implementation, the additional heterologous portion comprises a polynucleotide (e.g., an RNA motif), such as an MS2 phage operon stem loop (e.g., MS2, MS2 C-5 mutant, or MS2 F-5 mutant), a non-natural RNA motif, a PP7 operon stem loop, an SfMu phage Com stem loop, a sterile α motif, a telomerase Ku-binding motif, a telomerase Sm7-binding motif, and / or fragments thereof.Non-limiting examples of additional heterologous portions include polypeptides or fragments thereof having at least about 85% sequence identity with any one or more of SEQ ID NO: 380, 382, ​​384, 386-388. Non-limiting examples of additional heterologous portions include polynucleotides or fragments thereof having at least about 85% sequence identity with any one or more of SEQ ID NO: 379, 381, 383, 385.

[0349] In some cases, the components of the base editing system are associated with each other through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In other cases, the components of the base editing system are associated with each other through polypeptide domains (e.g., FokI domains), which bind to form protein complexes comprising about, at least about, or no more than about 1, 2 (i.e., dimerized), 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide domain units, optionally including alterations that reduce or eliminate their activity.

[0350] In some instances, the components of the base editing system are associated with each other through interactions between polyantibodies or fragments thereof, such as heavy chain domain 2 (CH2) of IgG, IgD, IgA, IgM, IgE, IgM (MHD2) or IgE (EHD2), the Fc region of immunoglobulin, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab, and Fab2. In some instances, the antibodies are dimers, trimers, or tetramers. In embodiments, the dimer antibody binds to the polypeptide or polynucleotide component of the base editing system.

[0351] In some cases, components of a base editing system are associated with each other through interactions between one or more polynucleotide-binding protein domains and one or more polynucleotides. In some instances, components of a base editing system are associated with each other through interactions between one or more polynucleotide-binding protein domains and multiple polynucleotides, which are self-complementary and / or complementary to each other, such that the complementary binding of the polynucleotides to each other associates their respective bound polynucleotide-binding protein domains.

[0352] In some cases, the components of a base editing system are associated with each other through interactions between one or more polypeptide domains and one or more small molecules, such as chemically induced dimerizing factors (CIDs), also known as “dimerizing agents.” Non-limiting examples of CIDs include those disclosed by Amara et al., “A versatile synthetic dimerizer for the regulation of protein-protein interactions,” PNAS, 94:10618-10623 (1997); and Voß et al., “Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells,” Current Opinion in Chemical Biology, 28:194-201 (2015), the contents of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds to the unedited strand. In some embodiments, the base editor contains UGI activity or USP activity. In some embodiments, the base editor contains an inosine-specific nuclease that catalytically inactivates the inosine.

[0353] The base editor disclosed herein may contain any domain, feature, or amino acid sequence that facilitates the editing of target polynucleotide sequences. For example, in some embodiments, the base editor contains a nuclear localization sequence (NLS). In some embodiments, the NLS of the base editor is located between a deaminase domain and a polynucleotide-programmable nucleotide-binding domain. In some embodiments, the NLS of the base editor is located at the C-terminus of the polynucleotide-programmable nucleotide-binding domain.

[0354] The protein domains contained in a fusion protein can be heterologous functional domains. Non-limiting examples of protein domains that can be included in a fusion protein include deaminase domains (e.g., cytidine deaminase and / or adenosine deaminase), uracil glycosylation inhibitor (UGI) domains, epitope tags, and reporter gene sequences.

[0355] In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, the ABE is generated by replacing the APOBEC1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, the ABE contains an evolved variant of TadA. In some embodiments, the base editor is ABE8.1, which contains or is substantially composed of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are provided in the accompanying sequence listing (SEQ ID NOs: 332-354).

[0356] In some embodiments, the base editor comprises an adenosine deaminase variant containing an amino acid sequence, as described herein, that contains alterations relative to the ABE 7*10 reference sequence. The term "monomer" as used in Table 7 refers to the monomeric form of TadA*7.10 containing the alterations. The term "heterodimer" as used in Table 7 refers to a heterodimer formed by fusing a specified wild-type E. coli TadA adenosine deaminase with TadA*7.10 containing the alterations.

[0357] Table 7. Variants of adenosine deaminase base editor

[0358] In some embodiments, the base editor includes a domain that contains all or a portion (e.g., a functional portion) of a uracil glycosidase inhibitor (UGI) or uracil stable protein (USP) domain.

[0359] Connecting base In some embodiments, the linker can be used to link any peptide or peptide domain disclosed herein. The linker can be as simple as a covalent bond, or it can be a polymeric linker with a length of many atoms. In some embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In some embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).

[0360] In some embodiments, any fusion protein provided herein comprises a cytidine or adenosine deaminase and a Cas9 domain fused together by a linker. Various linker lengths and flexibilityes can be employed between the cytidine or adenosine deaminase and the Cas9 domain (e.g., ranging from the form (GGGS)). n (SEQ ID NO: 246), (GGGGS) n(SEQ ID NO: 247) and (G) n The highly flexible connecting base is in the form of (EAAAK). n (SEQ ID NO: 248), (SGGS) n (SEQ ID NO:355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, for example, Guilinger JP et al., Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents of which are incorporated herein by reference) and (XP) n A more rigid linker is used to achieve the optimal length for cytidine or adenosine deaminase nucleobase editor activity. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises (GGS). n The motif, where n is 1, 3, or 7. In some embodiments, the cytidine deaminase or adenosine deaminase and the Cas9 domain of any fusion protein provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as the XTEN linker.

[0361] In some embodiments, the domain of the base editor is fused via a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358).

[0362] In some embodiments, the base editor domain is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids long. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids long. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids long. In some embodiments, the linker comprises the amino acid sequence: PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362).

[0363] In some embodiments, the linker contains multiple proline residues and has a length of 5-21, 5-14, 5-9, or 5-7 amino acids, for example, PAPAP (SEQ ID NO: 363), PAPAPA (SEQ ID NO: 364), PAPAPAP (SEQ ID NO: 365), PAPAPAPA (SEQ ID NO: 366), P(AP)4 (SEQ ID NO: 367), P(AP)7 (SEQ ID NO: 368), P(AP) 10(SEQ ID NO: 369) (See, for example, Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25;10(1):439; the entire contents of which are incorporated herein by reference). Such proline-rich linkers are also known as “rigid” linkers.

[0364] Nucleic acid programmable DNA-binding proteins with guide RNA This document provides compositions and methods for base editing in cells. It further provides compositions comprising a guide polynucleotide sequence (e.g., a guide RNA sequence) or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide RNAs provided herein. In some embodiments, the compositions for base editing provided herein further comprise a polynucleotide encoding a base editor (e.g., a C base editor or an A base editor). For example, the compositions for base editing may comprise an mRNA sequence encoding BE, BE4, ABE, and a combination of one or more guide RNAs provided herein. The compositions for base editing may comprise a base editor polypeptide and a combination of any one or more guide RNAs provided herein. Such compositions can be used to achieve base editing in cells via various delivery methods (e.g., electroporation, nuclear transfection, viral transduction, or transfection). In some embodiments, the compositions for base editing comprise an mRNA sequence encoding a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.

[0365] Some aspects of this disclosure provide systems comprising any fusion protein or complex provided herein, and a guide RNA that binds to a nucleic acid programmable DNA-binding protein (napDNAbp) domain (e.g., Cas9 (e.g., dCas9, nuclease-active Cas9, or Cas9 nickase) or Cas12) of the fusion protein or complex. These complexes are also referred to as ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides in length and comprises a sequence of at least 10 consecutive nucleotides complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of bacteria, yeast, fungi, insects, plants, or animals. In some embodiments, the target sequence is a sequence in the human genome. In some embodiments, the 3′ end of the target sequence is adjacent to a classical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is adjacent to a non-classical PAM sequence (e.g., sequences listed in Table 3 or 5′-NAA-3′). In some implementations, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in the target gene (e.g., a gene associated with a disease or condition).

[0366] Some aspects of this disclosure provide methods for using the fusion proteins or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any fusion protein or complex provided herein and at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides in length and comprises a sequence of at least 10 consecutive nucleotides complementary to a target sequence.

[0367] The structural domains of the base editor disclosed in this invention can be arranged in any order.

[0368] The defined target region can be a deamination window. The deamination window can be a defined region where the base editor acts and deaminates the target nucleotide. In some embodiments, the deamination window is located within a region of 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the deamination window is located 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.

[0369] The base editor disclosed herein can contain any domain, feature, or amino acid sequence that facilitates the editing of target polynucleotide sequences.

[0370] Methods using fusion proteins or complexes containing cytidine or adenosine deaminase and a Cas9 domain. Some aspects of this disclosure provide methods for using the fusion proteins or complexes provided herein. For example, some aspects of this disclosure provide methods that include contacting a DNA molecule with any of the fusion proteins or complexes provided herein, as well as at least one guide RNA described herein.

[0371] In some embodiments, the fusion protein or complex of this disclosure is used to edit a target gene. Specifically, the cytidine deaminase or adenosine deaminase base editor described herein is capable of generating multiple mutations within the target sequence. These mutations may affect the function of the target. For example, when the cytidine deaminase or adenosine deaminase base editor is used to target a regulatory region, the function of the regulatory region is altered, and the expression of downstream proteins is reduced or eliminated.

[0372] Base editor efficiency In some embodiments, the methods provided herein are intended to alter genes and / or gene products through gene editing. The nucleobase editing proteins provided herein can be used for in vitro or in vivo gene-editing-based human therapies. Those skilled in the art will understand that the nucleobase editing proteins provided herein, such as fusion proteins or complexes comprising a polynucleotide programmable nucleotide-binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain), can be used to edit nucleotides from A to G or from C to T.

[0373] Advantageously, the base editing system provided herein provides genome editing without producing double-strand DNA breaks, without requiring a donor DNA template, and without inducing excessive random insertions and deletions as CRISPR might produce. In some embodiments, this disclosure provides a base editor that efficiently produces desired mutations, such as stop codons, in nucleic acids (e.g., nucleic acids within the subject's genome) without producing a significant number of unintended mutations, such as unintended point mutations.

[0374] The base editor disclosed herein advantageously modifies specific nucleotide bases encoding proteins without producing a significant proportion of insertions or deletions (i.e., insertions or deletions). Such insertions or deletions can lead to frameshift mutations within gene coding regions.

[0375] In some implementations, the base editor provided herein can generate a ratio of expected mutations to insertions and deletions (i.e., expected point mutations: unexpected point mutations) greater than 1:1. In some implementations, the base editor provided herein is capable of producing a ratio of expected mutations to insertions / deletions of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of expected mutations and insertions / deletions can be determined using any suitable method.

[0376] In some embodiments, the base editors provided herein can restrict the formation of insertions and deletions in nucleic acid regions. In some embodiments, the regions are located at the nucleotide targeted by the base editor, or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor. In some embodiments, any base editor provided herein can restrict the formation of insertions and deletions in nucleic acid regions to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%.

[0377] Base editing is often referred to as "modification," such as genetic modification, gene modification, and nucleic acid sequence modification, and the context clearly indicates that this modification is a base editing modification. Therefore, base editing modification is a modification at the nucleotide base level, such as due to deaminase activity discussed throughout this disclosure, which subsequently leads to changes in the gene sequence and may affect the gene product.

[0378] In some implementations, the modification (e.g., single-base editing) results in a reduction of target gene expression by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or to an undetectable level.

[0379] This disclosure provides adenosine deaminase variants (e.g., the ABE8 variant) with improved efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit desired bases within polynucleotides and less likely to edit bases that are not intended to be changed (e.g., "bystanders").

[0380] In some implementations, bystander edits or mutations are reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in any base editing system containing one of the ABE7 base editors (e.g., ABE7.10).

[0381] In some embodiments, any ABE8 base editor variant described herein has higher base editing efficiency compared to the ABE7 base editor. In some embodiments, any ABE8 base editor variant described herein has at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135% efficiency compared to the ABE7 base editor (e.g., ABE7.10). %, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 450%, or 500% higher base editing efficiency.

[0382] The ABE8 base editor variants described herein may be delivered to host cells via plasmids, vectors, LNP complexes, or mRNA. In some embodiments, any of the ABE8 base editor variants described herein are delivered to host cells in the form of mRNA.

[0383] In some embodiments, the methods described herein (e.g., base editing methods) have minimal to no off-target effects. In some embodiments, the methods described herein (e.g., base editing methods) have minimal to no chromosomal translocations.

[0384] In some implementations, the base editing methods described herein resulted in approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cell population being successfully edited.

[0385] In some embodiments, the percentage of surviving cells in the cell population after base editing intervention is greater than at least 60%, 70%, 80%, or 90% of the initial cell population at the time of the base editing event. In some embodiments, the percentage of surviving cells in the post-edited cell population is approximately 70%. In some embodiments, the percentage of surviving cells in the post-edited cell population is approximately 75%. In some embodiments, the percentage of surviving cells in the post-edited cell population is approximately 80%. In some embodiments, the percentage of surviving cells in the cell population described above is approximately 85%. In some embodiments, the percentage of surviving cells in the cell population described above is approximately 90% of the cells in the population at the time of the base editing event, or approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population at the time of the base editing event.

[0386] In an embodiment, the cell population is a cell population that comes into contact with the base editor, complex, or base editor system of this disclosure.

[0387] The expected number of mutations and insertions / deletions can be determined using any suitable method, for example, as described in international PCT applications PCT / US2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632); Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved baseexcision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:Abase editors with higher efficiency and product purity” Science. Advances 3:eaao4774 (2017); the entire contents of the above reference are incorporated herein by reference.

[0388] In some implementations, to calculate the insertion / deletion frequency, sequencing reads are scanned to find exact matches between two 10-bp sequences flanking the window where insertions / deletions may occur. If no exact match is found, the read is excluded from analysis. If the length of the insertion / deletion window perfectly matches the reference sequence, the read is classified as not containing an insertion / deletion. If the insertion / deletion window is two or more bases longer or shorter than the reference sequence, the sequencing read is classified as an insertion or a deletion, respectively. In some implementations, the base editor provided herein can restrict the formation of insertions / deletions in nucleic acid regions. In some implementations, the region is located at the nucleotide targeted by the base editor, or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor.

[0389] Multiple editing In some embodiments, the base editor systems provided herein are capable of multiplexing multiple nucleobase pairs in one or more genes or polynucleotide sequences. In some embodiments, the multiple nucleobase pairs are located in the same gene or in one or more genes, wherein at least one gene is located at a different site. In some embodiments, the multiplexing comprises one or more guide polynucleotides. In some embodiments, the multiplexing comprises one or more base editor systems. In some embodiments, the multiplexing comprises one or more base editor systems having a single guide polynucleotide or multiple guide polynucleotides. In some embodiments, the multiplexing comprises one or more guide polynucleotides having a single base editor system. It should be understood that the features of multiplexing using any base editor described herein can be applied to any combination of methods using any base editor provided herein. It should also be understood that multiplexing using any base editor described herein can involve sequential editing of multiple nucleobase pairs.

[0390] In some implementations, a base editor system capable of multiplexing multiple nucleobase pairs in one or more genes includes one of the ABE7, ABE8, and / or ABE9 base editors.

[0391] Delivery system Nucleic acid-based base editor system delivery Nucleic acid molecules encoding the base editor system according to this disclosure can be administered to a subject or delivered to cells in vitro or in vivo by methods known in the art or as described herein. For example, base editor systems containing deaminases (e.g., cytidine or adenine deaminase) can be delivered via vectors (e.g., viral or non-viral vectors), naked DNA, DNA complexes, lipid nanoparticles, or combinations of the foregoing. Base editor systems can be delivered to cells using any method available in the art, including but not limited to physical methods (e.g., electroporation, gene gun, calcium phosphate transfection), viral methods, non-viral methods (e.g., liposomes, cationic methods, lipid nanoparticles, polymer nanoparticles), or biological non-viral methods (e.g., attenuated bacteria, engineered bacteriophages, mammalian virus-like particles, biological liposomes, erythrocyte shadows, exosomes).

[0392] Nanoparticles can be organic or inorganic and can be used to deliver base editor systems or components thereof. Nanoparticles are well known in the art, and any suitable nanoparticle can be used to deliver base editor systems or components thereof, or nucleic acid molecules encoding such components. In one instance, organic (e.g., lipid and / or polymer) nanoparticles are suitable as delivery carriers in certain embodiments of this disclosure. Non-limiting examples of lipid nanoparticles suitable for the methods of this disclosure include those described in International Patent Application Publications WO2022140239, WO2022140252, WO2022140238, WO2022159421, WO2022159472, WO2022159475, WO2022159463, WO2021113365, and WO2021141969, and those described in International Patent Application PCT / US23 / 27741, the entire disclosure of which is incorporated herein by reference for all purposes.

[0393] Lipid nanoparticle (LNP) composition The pharmaceutical compositions for gene modification described herein can be encapsulated in lipid nanoparticles (LNPs). LNP compositions or formulations contemplated herein typically have a size on the order of micrometers or smaller and may comprise a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, nanoparticle compositions or formulations contemplated herein may be liposomes comprising a lipid bilayer with a diameter of 500 nm or smaller. The LNP described herein may have an average diameter of 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 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. The LNP described herein may have an average diameter of approximately 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 greater. In one embodiment, the average diameter of the LNP is approximately 70 nm + / - 10 nm, 70 nm + / - 5 nm, 65 nm + / - 10 nm, 65 nm + / - 5 nm, 60 nm + / - 10 nm, or 60 nm + / - 5 nm.

[0394] This document describes LNP compositions comprising ionizable lipids, phospholipids, conjugated-linker lipids (e.g., PEG lipids), sterols (e.g., cholesterol) or derivatives thereof, payloads, or any combination thereof. Each component is described in more detail below.

[0395] In preparing LNP compositions comprising ionizable lipids, phospholipids, conjugated-linker lipids, and sterols, four excipients in de...

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