WO2025159966A1

WO2025159966A1 - Compositions and methods for altering a nucleobase in a transthyretin polynucleotide

Info

Publication number: WO2025159966A1
Application number: PCT/US2025/011842
Authority: WO
Inventors: Luis Brito; Delai Chen
Original assignee: Beam Therapeutics Inc
Current assignee: Beam Therapeutics Inc
Priority date: 2024-01-23
Filing date: 2025-01-16
Publication date: 2025-07-31
Anticipated expiration: 2026-07-23

Abstract

Compositions for modifying or editing a polynucleotide (e.g., a gene) and methods of using the same to treat or prevent conditions associated with the extracellular deposition in various tissues of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) proteins. Such conditions include, but are not limited to, polyneuropathy due to hereditary transthyretin amyloidosis (hATTR-PN) and hereditary cardiomyopathy due to transthyretin amyloidosis (hATTR-CM), both associated with autosomal dominant mutations of the TTR gene, and an age-related cardiomyopathy associated with wild-type TTR proteins (ATTRwt), also known as senile cardiac amyloidosis are disclosed. Compositions and methods for editing TTR polynucleotides using base editor systems are disclosed.

Description

COMPOSITIONS AND METHODS FOR ALTERING A NUCLEOBASE IN A TRANSTHYRETIN POLYNUCLEOTIDE

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application No. 63/624,030, filed January 23, 2024, the entire contents of which are hereby incorporated by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on January 13, 2025, is named 180802-056202PCT_SL.xml, and is 1,550,753 bytes in size.

BACKGROUND

Amyloidosis is a condition characterized by the buildup of abnormal deposits of amyloid protein in the body's organs and tissues. These protein deposits can occur in the peripheral nervous system, which is made up of nerves connecting the brain and spinal cord to muscles and sensory cells that detect sensations such as touch, pain, heat, and sound. Protein deposits in these nerves can result in a loss of sensation in the extremities (peripheral neuropathy). The autonomic nervous system, which controls involuntary body 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 affected. Mutations in the transthyretin (TTR) gene are associated with transthyretin amyloidosis. Furthermore, patients expressing wild-type TTR may also develop amyloidosis. Liver transplant remains the gold standard for treating transthyretin amyloidosis. However, there are a limited number of organ donors, and patients may wait years for an available organ. Accordingly, there is a need for compositions and methods for treating amyloidosis.

SUMMARY

Provided herein are compositions for modifying or editing a polynucleotide (e.g., DNA, RNA, genomic DNA, gene) and methods of using the same to treat or prevent conditions associated with the extracellular deposition in various tissues of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) proteins. Such conditions include, but are not limited to, polyneuropathy due to hereditary transthyretin amyloidosis (hATTR-PN) and hereditary cardiomyopathy due to transthyretin amyloidosis (hATTR-CM), both of which are associated with autosomal dominant mutations of the TTR gene, and an age-related cardiomyopathy associated with wild-type TTR proteins (ATTRwt), also known as senile cardiac amyloidosis. Compositions and methods for editing TTR polynucleotides using base editor systems are disclosed herein.

In one aspect, the disclosure features a lipid nanoparticle (LNP) containing components A), B), and C). Component A) is a guide RNA, or a polynucleotide encoding the guide RNA, where the guide RNA contains a spacer containing a nucleotide sequence selected from GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472) and those listed in any of Tables IB, 1C, ID, IE, 2A, 2B, 2D, or 2E. Component B) is an mRNA molecule, or a polynucleotide encoding the mRNA molecule, where the mRNA molecule encodes a base editor polypeptide containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain contains an amino acid sequence having at least 90% identity to the following TadA*7. 10 amino acid sequence, or a truncation thereof lacking only the first M: MSEVE FSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE IMA LRQGGLVMQNYRLI DATLYVT FE PCVMCAGAMI HSRIGRVVFGVRNAKTGAAGSLMDVLHY P GMNHRVE ITEGILADECAALLCY FFRMPRQVFNAQKKAQS STD (SEQ ID NO: 1). The adenosine deaminase also contains a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from one or more of: 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 one of the following formulas, or a pharmaceutically acceptable salt thereof: i) a compound of Formula (Ih): where: each L¹ and L¹ is independently -C(O)- or -OC(O)-; each L² and L² is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

R¹ is optionally substituted C1-20 aliphatic,

L^CyA is a covalent bond or an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain; each R^a and R¹ is independently optionally substituted C1-20 aliphatic;

Y¹ is -C(O)- or -C(O)O-;

Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or optionally substituted C1-6 aliphatic; ii) a compound of Formula (la): where: R¹ is C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation;

X¹ and X² are each independently absent or selected from -O-, -NR²- and , where each R² is independently hydrogen or C₁-C₆ alkyl; each a is independently an integer between 1 and 6;

X³ and X⁴ are each independently absent or selected from one or more of: 4- to 8-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6-membered aryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 4- to 7-membered cycloalkyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, -O- and -NR³-, where each R³ is a independently a hydrogen atom or C₁-C₆ alkyl and where X⁴-X²-X³-X⁴ does not contain any oxygenoxygen, oxygen-nitrogen or nitrogen-nitrogen bonds;

X⁵ is -(CH2)b-, where b is an integer between 0 and 6;

X⁶ is hydrogen, C₁-C₆ alkyl, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, or -NR⁴R⁵, where R⁴ and R⁵ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, where the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; each X⁷ is independently hydrogen, hydroxyl or -NR⁶R⁷, where R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, where the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; at least one of X¹, X², X³, X⁴, and X⁵ is present;

A¹ and A² are each independently selected from one or more of: C₅-C₁₂haloalkyl, C5-C12 alkenyl, C5-C12 alkynyl, (C5-C12 alkoxy)-(CH2)n2-, (C5-C10 aryl)-(CH2)n3- optionally ring substituted with one or two halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkoxy groups, and (C3-C8 cycloalkyl)-(CH2)n4- optionally ring substituted with 1 or 2 C₁-C₆ alkyl groups; or alternatively A¹ and A² join together with the atoms to which they are bound to form a 5- to 6-membered cyclic acetal substituted with 1 or 2 C4-C10 alkyl groups; nl, n2 and n3 are each individually an integer between 1 and 4; and n4 is an integer between zero and 4; iii) A compound of Formula (lb):

where:

R¹ is C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation;

X¹ and X² are each independently absent or selected from -O-, NR², and where R² is C₁-C₆ alkyl, and where X¹ and X² are not both -O- or NR²; a is an integer between 1 and 6;

X³ and X⁴ are each independently absent or selected from one or more of: 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, and -NR³-, where each R³ is a hydrogen atom or C₁-C₆ alkyl;

X⁵ is -(CH2)b-, where b is an integer between 0 and 6;

X⁶ is hydrogen, C₁-C₆ alkyl, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, or -NR⁴R⁵, where R⁴ and R⁵ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, where the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen;

X⁷ is hydrogen or -NR⁶R⁷, where R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, where the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; at least one of X¹, X², X³, X⁴, and X⁵ is present; and provided that when either X¹ or X² is -O-, neither X³ nor X⁴ is , and when either X¹ or X² is -O-, R⁴ and R⁵ are not both ethyl; iv) A compound of Formula (Ic):

Ic or its N-oxide, or a salt thereof, where

LI is Cl -6 alkylenyl, or C2-6 heteroalkylenyl; each L² is independently C2-10 alkylenyl, or C3-10 heteroalkylenyl;

L is absent, C1-10 alkylenyl, or C2-10 heteroalkylenyl;

L³ is absent, C1-10 alkylenyl, or C2-10 heteroalkylenyl;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-; each R is independently hydrogen, optionally substituted group selected from C6-20 aliphatic, C6-20 haloaliphatic, a 3 - to 7-membered cycloaliphatic ring, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl;

R¹ is hydrogen, a 3- to 7-membered cycloaliphatic ring, a 3- to 7-membered heterocyclic ring containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -NR ²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)₂, -NR²C(S)N(R²)₂, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O) OR², -N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, - N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², - each R² is independently hydrogen, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n-R⁴, or an optionally substituted group selected from Ci-6 aliphatic, a 3- to 7-membered cycloaliphatic ring, and a 3- to 7-membered heterocyclic ring containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or two occurrences of R², taken together with the atom(s) to which they are attached, form an optionally substituted 4- to 7-membered heterocyclic ring containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴, or two occurrences of R³, taken together with the atoms to which they are attached, form an optionally substituted 5- to 6-membered heterocyclic ring containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)₂, -OC(O)R⁵, -OC(O)OR⁵, -CN, -C(O)N(R⁵)₂,-NR⁵C(O)R⁵, -OC(O)N( R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, -NR⁵C(S)N(R⁵)₂, -NR⁵C(NR⁵)N(R⁵)₂, each R⁵ is independently hydrogen, optionally substituted Ci-6 aliphatic, or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form an optionally substituted 4- to 7-membered heterocyclic ring containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently Cr-i₂ aliphatic; and n is 0 to 4; v) A compound of Formula (Id):

Id or its N-oxide, or a pharmaceutically acceptable salt thereof, where

LI is absent, Cl -6 alkylenyl, or C2-6 heteroalkylenyl; each L2 is independently optionally substituted C2-15 alkylenyl, or optionally substituted C3-15 heteroalkylenyl;

L³ is absent, optionally substituted C1-10 alkylenyl, or optionally substituted C2-10 heteroalkylenyl; X is absent, -0C(0)-, -C(0)0-, or -OC(O)O-; each R’ is independently an optionally substituted group selected from C4-12 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

R is hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², - NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)₂, - N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, - C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)₂, -OP(O)(OR²)₂, or - ring selected from 3- to 7-membered cycloaliphatic and 3- to 7-membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n- R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-memberedheterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, - C(O)N(R⁵)2, -NR⁵C(O)R⁵, -OC(O)N(R⁵)2, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)2, each R⁵ is independently hydrogen, or optionally substituted Ci-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4; vi) A compound of Formula (le): or a pharmaceutically acceptable salt thereof, where:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L² is a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or ;

Cy^A is an optionally substituted ring selected from phenylene and 3- to 7-membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-;

R , is H-H ^p »^B , or an optionally substituted saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain where 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

Cy^B is an optionally substituted ring selected from 3- to 12-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, , sterolyl, and phenyl; p is 0, 1, 2, or 3;

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^c-;

Cy^c is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclene, phenylene, 3- to 7-membered saturated or partially unsaturated heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group;

Z¹ is a covalent bond or -O-;

Z² is an optionally substituted group selected from 4- to 12-membered saturated or partially unsaturated carbocyclyl, phenyl, 1-adamantyl, and 2-adamantyl;

Z³ is hydrogen, or an optionally substituted group selected from C1-C10 aliphatic, and 4- to 12-membered saturated or partially unsaturated carbocyclyl; and d is 0, 1, 2, 3, 4, 5, or 6; provided that when L is a covalent bond, then R must be ; vii) A compound of Formula (If): or a pharmaceutically acceptable salt thereof, where: each L¹ and L¹ is independently -C(O)- or -C(O)O-; each L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or ; each Cy^A is independently an optionally substituted ring selected from phenylene or a 3- to 7- membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each L³ and L³ is independently a covalent bond, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; each R¹ and R¹ is independently an optionally substituted group selected from a saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 12-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, and , O-A¹

HA ₂

O-A² - each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵; or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: where x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 5 - to 10-membered aryl ring and a 3- to 8-membered carbocyclic ring ;

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X³ is hydrogen or -Cy^B;

Cy^B is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; provided that when X³ is hydrogen, at least one of R¹ or R¹ is viii) a compound of Formula (Ig): or a pharmaceutically acceptable salt thereof, where: each of L¹ and L¹ is independently a covalent bond, -C(O)-, or -OC(O)-; each of L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or each Cy^A is independently an optionally substituted ring selected from phenylene or 3- to 7- membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each of L³ and L³ is independently a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; each of R¹ and R¹ is independently an optionally substituted group selected from saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain where 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, or each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵, or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: where x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 6- to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring;

Y¹ is a covalent bond, -C(O)-, or -C(O)O-;

Y² is a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain, where 1-2 methylene units are optionally and independently replaced with cyclopropylene, - O-, or -NR-;

Y³ is an optionally substituted group selected from saturated or unsaturated, straight or branched C1-C14 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, or phenyl;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^B-; each Cy^B is independently an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclene, phenylene, 3- to 7-membered heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6- membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; ix) a compound of formula A’ : or its N-oxide, or a pharmaceutically acceptable salt thereof, where

L¹ is absent, Ci-6 alkylenyl, or C2-6 heteroalkylenyl; each L² is independently optionally substituted C2-15 alkylenyl, or optionally substituted C3-15 heteroalkylenyl;

L is Ci-10 alkylenyl, or C2-10 heteroalkylenyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-;

R” is hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl; each of R and R^a is independently hydrogen, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl each of L³ and L^3a is independently absent, optionally substituted Ci-io alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-memberedheterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)2, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², - NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)₂, - N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, - C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)₂, -OP(O)(OR²)₂, or - P(O)(OR²)₂; or ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n- R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-memberedheterocyclyl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, - C(O)N(R⁵)2, -NR⁵C(O)R⁵, -OC(O)N(R⁵)2, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)2, each R⁵ is independently hydrogen, or optionally substituted Ci-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl containing 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4; or x) a compound of Formula I: or a pharmaceutically acceptable salt thereof, where:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L² is a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; an optionally substituted saturated or unsaturated, straight or branched

C1-C20 hydrocarbon chain where 1-3 methylene units are optionally and independently replaced with -O- or -NR-, or

Cy^B is an optionally substituted ring selected from 3- to 12-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, , sterolyl, and phenyl; p is 0, 1, 2, or 3; each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵; or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: where x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, where 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 5 - to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring ;

X¹ is a covalent bond, -O-, or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group;

₃ , provided that when L is a covalent bond, then R must be In another aspect, the disclosure features a lipid nanoparticle (LNP) containing components A), B), and C). Component A) is a guide RNA, or a polynucleotide encoding the guide RNA, where the guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a transthyretin (TTR) polynucleotide. Component B) is an mRNA molecule, or a polynucleotide encoding the mRNA molecule, where the mRNA molecule encodes a base editor polypeptide containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain contains i) an amino acid sequence having at least 90% identity to the following TadA*7. 10 amino acid sequence, or a truncation thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain also contains a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from one or more of: 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, Q154R; h) I76Y, Y147R, and Q154R; i) Y147R, Q154R, and T166R; j) Y147T and Q154R; k) Y147T and Q 154S;1) 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 according to Formula (I), or a pharmaceutically acceptable salt thereof: where: each L¹ and L¹ is independently -C(O)- or -OC(O)-; each L² and L² is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; R¹ is optionally substituted C1-20 aliphatic, L^CyA is a covalent bond or an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or optionally substituted C1-6 aliphatic.

In another aspect, the disclosure features a lipid nanoparticle (LNP) containing components A), B), and C). Component A) is a guide RNA having the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smU smUsmU (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 s is a phosphorothioate (PS) backbone linkage. Component B) is a base editor polypeptide, or a polynucleotide encoding the base editor polypeptide, where the base editor polypeptide contains a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain contains an amino acid sequence with at least 90% identity to the following amino TadA*7. 10 amino acid sequence, or a truncation thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA

LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain also contains the following combination of amino acid alterations 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:

In another aspect, the disclosure features a base editor system containing components A), and B). Component A) is a guide RNA, or a polynucleotide encoding the guide RNA, where the guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a transthyretin (TTR) polynucleotide. Component B) is a base editor polypeptide, or one or more polynucleotide encoding the base editor polypeptide, where the base editor polypeptide contains a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain. The adenosine deaminase domain contains an amino acid sequence with at least 90% identity to the following amino TadA*7. 10 amino acid sequence, or a truncation thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1). The adenosine deaminase domain also contains a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from one or more of: 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. In another aspect, the disclosure features a polynucleotide or set of polynucleotides encoding the base editor system of any aspect of the disclosure, or embodiments thereof.

In another aspect, the disclosure features a cell containing the base editor system or the polynucleotide or set of polynucleotides of any aspect of the disclosure, or embodiments thereof.

In another aspect, the disclosure features a pharmaceutical composition containing the lipid nanoparticle, the base editor system, the polynucleotide or set of polynucleotides, or the cell of any aspect of the disclosure, or embodiments thereof, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the disclosure features a kit containing the lipid nanoparticle, the base editor system, the polynucleotide or set of polynucleotides, the cell, or the pharmaceutical composition of any aspect of the disclosure, or embodiments thereof, and a container.

In another aspect, the disclosure features a method for modifying a target nucleobase in a transthyretin (TTR) polynucleotide in a cell. The method involves contacting the cell with the lipid nanoparticle or the base editor system of any aspect of the disclosure, or embodiments thereof, thereby modifying the target nucleobase in the TTR polynucleotide.

In another aspect, the disclosure features a method of treating a disease in a subject in need thereof. The disease is associated with a pathogenic mutation in a transthyretin (TTR) polynucleotide in the subject. The method involves administering to the subject the lipid nanoparticle or the base editor system of any aspect of the disclosure, or embodiments thereof, thereby altering a target nucleobase in the TTR polynucleotide.

In any aspect of the disclosure, or embodiments thereof, the lipid nanoparticle contains an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

(IZ4).

In any aspect of the disclosure, or embodiments thereof, the LNP has a molar ratio of ionizable nitrogen atoms in an ionizable lipid to the total negative charge in the payload (N:P ratio) of between about 30: 1 to about 1: 1. In any aspect of the disclosure, or embodiments thereof, the LNP has an N:P ratio of about 6: 1.

In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a scaffold with the following nucleotide sequence: gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugG caccgagucggugcuususus (SEQ ID NO: 478), where 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 phosphorothioate (PS) backbone linkage. In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a spacer containing the following nucleotide sequence: GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In any aspect of the disclosure, or embodiments thereof, the guide RNA contains the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm

AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smU smUsmU (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 s is a phosphorothioate (PS) backbone linkage. In any aspect of the disclosure, or embodiments thereof, the guide RNA contains a spacer selected from those listed in any of Tables IB, 1C, ID, IE, 2A, 2B, 2D, or 2E. In any aspect of the disclosure, or embodiments thereof, the guide RNA contains 2-5 contiguous 2’-O-methylated nucleobases at the 3’ end and at the 5’ end. In any aspect of the disclosure, or embodiments thereof, the guide RNA contains 2-5 contiguous nucleobases at the 3 ’ end and at the 5 ’ end that contain phosphorothioate intemucleotide linkages. In any aspect of the disclosure, or embodiments thereof, the napDNAbp domain contains a Cas9 polypeptide. In any aspect of the disclosure, or embodiments thereof, the napDNAbp is a nickase.

In any aspect of the disclosure, or embodiments thereof, the base editor polypeptide contains an amino acid sequence with at least about 90% identity to the following amino acid sequence: ABE9.51 ( Q )

In any aspect of the disclosure, or embodiments thereof, the cell is in a subject. In any aspect of the disclosure, or embodiments thereof, an ionizable lipid of the lipid nanoparticle has a half-life in the liver of the subject that is less than 14 days.

In any aspect of the disclosure, or embodiments thereof, the target nucleobase is altered with an editing efficiency of at least about 50% or 60% in in the cell and/or in the liver of the subject. In any aspect of the disclosure, or embodiments thereof, altering the target nucleobase results in a reduction in TTR polypeptide levels in the cell and/or in the subject. In any aspect of the disclosure, or embodiments thereof, TTR polypeptide levels are reduced by at least about 80% or 90%. In any aspect of the disclosure, or embodiments thereof, the method is associated with only a transient increase or no increase in alkaline phosphatase (ALP), alanine transaminase (ALT), and/or aspartate aminotransferase (AST) levels in the subject. In any aspect of the disclosure, or embodiments thereof, the method is associated with no increase in ALP levels in the subject.

In any aspect of the disclosure, or embodiments thereof, the disease is selected from one or more of amyloidosis, cardiomyopathy, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), familial transthyretin amyloidosis (FTA), senile systemic amyloidosis (SSA), and transthyretin amyloidosis.

In any aspect of the disclosure, or embodiments thereof, the LNP has a mean diameter of from about 20 nm to about 1000 nm, from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 50 nm to 90 nm, from about 55 nm to 85 nm, from about 55 nm to 75 nm, from about 50 nm to about 80 nm, from about 60 nm to about 80 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, or from about 70 nm to about 80 nm. In any aspect of the disclosure, or embodiments thereof, the LNP has a mean diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. In any aspect of the disclosure, or embodiments thereof, the LNP has a mean diameter of 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.

In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in the lipid nanoparticle (LNP) at from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in the lipid nanoparticle (LNP) at from about 33 mole percent to about 60 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, die ionizable lipid is present in the lipid nanoparticle (LNP) at from about 34 mole percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in die lipid nanoparticle (LNP) at from about 33 mole percent to about 51 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in the lipid nanoparticle ( LNP) at about 34.7 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in the lipid nanoparticle (LNP) at about 50 mole percent, based on total moles of components of the lipid nanoparticle. In any aspect of the disclosure, or embodiments thereof, the ionizable lipid is present in the lipid nanoparticle (LNP) at about 40 mole percent to about 60 mole percent or about 45 mole percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle.

In any aspect of the disclosure, or embodiments thereof, the LNP contains from about 0 mole percent to about 5 mole percent conjugate-linker lipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 1.5 mole percent conjugatelinker lipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 2.0 mole percent conjugate-linker lipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 2.5 mole percent conjugate-linker lipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 3 mole percent conjugate-linker lipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 3.5 mole percent conjugate -linker lipid.

In any aspect of the disclosure, or embodiments thereof, the LNP contains from about 5 mole percent to 25 mole percent of phospholipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 5 mole percent to 15 mole percent of phospholipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 9 mole percent to 11 mole percent of phospholipid. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 10 mole percent of phospholipid.

In any aspect of the disclosure, or embodiments thereof, the LNP contains from about 25 mole percent to about 45 mole percent sterol. In any aspect of the disclosure, or embodiments thereof, the LNP contains from about 30 mole percent to about 45 mole percent sterol. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 40 mole percent sterol. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 35.75 mole percent sterol. In any aspect of the disclosure, or embodiments thereof, the LNP contains about 32.5 mole percent cholesterol.

In any aspect of the disclosure, or embodiments thereof, the LNP about 30 mole percent to about 70 mole percent ionizable lipid, about 5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent sterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 47.5 mole percent ionizable lipid, about 10 mole percent phospholipid, about 40 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 51.25 mole percent ionizable lipid, about 10 mole percent phospholipid, about 35.75 mole percent sterol, and about 3 mole percent conjugate-linker lipid.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 55 mole percent ionizable lipid, about 10 mole percent phospholipid, about 32.5 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In any aspect of the disclosure, or embodiments thereof, the LNP about 45 mole percent to about 60 mole percent ionizable lipid of any provided compound, about 9 mole percent to about 11 mole percent 1 -2 -distearoyl-sn-glycero-3 -phosphocholine (DSPC), about 1 mole percent to about 5 mole percent PEG2000-DMG , and about 30 mole percent to about 45 mole percent cholesterol, based on the total moles of these four ingredients.

In any aspect of the disclosure, or embodiments thereof, the LNP is the formulation LNP1, LNP2, LNP3, or LNP4 in TablelOA.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 47.5 mole percent ionizable lipid IZ1, about 10 mole percent 1-2 -distearoyl-sn-glycero-3 - phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 40 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload containing mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and a guide RNA containing a spacer containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table IB.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 55 mole percent ionizable lipid IZ2, about 10 mole percent 1-2 -distearoyl-sn-glycero-3 - phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 32.5 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload containing mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and a guide RNA containing a spacer containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table IB.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 51.25 mole percent ionizable lipid IZ3, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 3 mole percent PEG2000-DMG , and about 35.75 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload containing mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and a guide RNA containing a spacer containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table IB.

In any aspect of the disclosure, or embodiments thereof, the LNP contains about 47.5 mole percent ionizable lipid IZ4, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 40 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload containing mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and a guide RNA containing a spacer containing the nucleotide sequence GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472). In some embodiments, the guide RNA is GA521 of Table IB.

In any aspect of the disclosure, or embodiments thereof, the N/P ratio of the lipid nanoparticle is from about 1 to about 30. In any aspect of the disclosure, or embodiments thereof, the N/P ratio of the lipid nanoparticle 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 the disclosure, or embodiments thereof, the N/P ratio of the lipid nanoparticle is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, or about 10. In any aspect of the disclosure, or embodiments thereof, the N/P ratio of the lipid nanoparticle is N/P ratio is from about 5 to about 7. In any aspect of the disclosure, or embodiments thereof, the N/P ratio of the lipid nanoparticle is about 6.

In any aspect provided herein, or embodiments thereof, the method is not a process for modifying the germline genetic identity of human beings.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By “TadA*8.8 polypeptide” is meant an adenosine deaminase domain having deaminase activity, comprising the alterations Y123H, Y147R, and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some embodiments, TadA*8.8 comprises further alterations (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 alterations) relative to the reference sequence.

By “TadA*8.8 polynucleotide” is meant a polynucleotide encoding a TadA*8.8 polypeptide.

By “TadA*8.13 polypeptide” is meant an adenosine deaminase domain having deaminase activity, comprising the alterations I76Y, Y123H, Y147R, and Q154R relative to the following reference sequence and having at least about 85% amino acid sequence identity to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1; TadA*7.10). In some embodiments, TadA*8.13 comprises 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 alterations relative to the reference sequence.

By “TadA* 8.13 polynucleotide” is meant a polynucleotide encoding a TadA* 8.13 polypeptide.

By “TadA* 8. 17 polypeptide” is meant an adenosine deaminase domain having deaminase activity, comprising the alterations V82S and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1;

TadA* 7.10). In some embodiments, TadA* 8.17 comprises 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 alterations relative to the reference sequence.

By “TadA* 8.17 polynucleotide” is meant a polynucleotide encoding a TadA* 8.17 polypeptide.

By “TadA* 8.20 polypeptide” is meant an adenosine deaminase domain having deaminase activity, comprising the alterations I76Y, V82S, Y123H, Y147R, and Q154R relative to the following reference sequence, and having at least about 85% amino acid sequence identity to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1;

TadA* 7.10). In some embodiments, TadA* 8.20 comprises 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 alterations relative to the reference sequence.

By “TadA* 8.20 polynucleotide” is meant a polynucleotide encoding a TadA* 8.20 polypeptide.

In various embodiments, any adenosine deaminase domain provided herein (e.g., TadA*8.8, TadA*8.17, or TadA*8.20, and variants thereof) comprises the following alterations relative to the TadA*7.10 reference sequence: V82T; or V82T, Y147T, and Q154S. By “ABE9.51 polypeptide” is meant an adenosine deaminase base editor comprising a TadA*8.20 adenosine deaminase polypeptide variant having the amino acid alteration V82T, and a nucleic acid programmable DNA binding protein (napDNAbp) domain. In some embodiments, ABE9.51 comprises an amino acid sequence with at least about 85% sequence identity to the following polypeptide sequence, or a functional fragment thereof capable of deaminating an adenosine nucleotide: In embodiments, ABE9.51 contains one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, ABE9.51 contains two UGI domains.

By “ABE9.51 polynucleotide” is meant a polynucleotide encoding an ABE9.51 polypeptide.

By “adenine” or “9H-Purin-6-amine” is meant a purine nucleobase with the molecular formula C5H5N5, having the structure , and corresponding to CAS No. 73-24-5. a ribose sugar via a glycosidic bond, having the structure corresponding to CAS No. 65-46-3. Its molecular formula is C10H13N5O4.

By “adenosine deaminase” or “adenine deaminase” is meant 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 catalyzing the hydrolytic deamination of adenosine to inosine or deoxy adenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism (e.g., eukaryotic, prokaryotic), 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 an adenosine deaminase variant with one or more alterations and is capable of deaminating both adenine and cytosine in a target polynucleotide (e.g. , DNA, RNA) and may be referred to as a “dual deaminase”. Non-limiting examples of dual deaminases include those described in PCT/US22/22050. In some embodiments, the target polynucleotide is single 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 PCI7US2020/018192, PCT/US2020/049975, PCT/US2017/045381, and

PCT7US2020/028568, the full contents of which are each incorporated herein by reference in their entireties for all purposes.

By “adenosine deaminase activity” is meant catalyzing the deamination of adenine or adenosine to guanine in a polynucleotide.

By “Adenosine Base Editor (ABE)” is meant a base editor comprising an adenosine deaminase.

By “Adenosine Base Editor (ABE) polynucleotide” is meant a polynucleotide encoding an ABE.

By “Adenosine Base Editor 8 (ABE8) polypeptide” or “ABE8” is meant a base editor as defined herein comprising an adenosine deaminase or adenosine deaminase variant comprising one or more of the alterations listed in Table 5B, one of the combinations of alterations listed in Table 5B, or an alteration at one or more of the amino acid positions listed in Table 5B, such alterations are relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or a corresponding position in another adenosine deaminase. In embodiments, ABE8 comprises alterations at amino acids 82 and/or 166 of SEQ ID NO: 1. In some embodiments, ABE8 comprises further alterations, as described herein, relative to the reference sequence.

By “Adenosine Base Editor 8 (ABE8) polynucleotide” is meant a polynucleotide encoding an ABE8 polypeptide.

“Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and without limitation, composition administration (e.g., injection) can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrastemally. Alternatively, or concurrently, administration can be by the oral route.

By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, lipid nanoparticle, polypeptide, polypeptide complex, or fragments thereof. In some embodiments, the agent is a lipid nanoparticle of the disclosure. In some embodiments, the lipid nanoparticle contains a base editor system of the disclosure. In some cases, the agent is a base editor system of the disclosure, or a component thereof. The base editor system contains 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.

The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “carbocyclic”, “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms. In some embodiments, aliphatic groups contain 1-3 carbon atoms, and in some embodiments, aliphatic groups contain 1-2 carbon atoms. In some embodiments, “carbocyclic” (or “cycloaliphatic” or “carbocycle” or “cycloalkyl”) refers to an optionally substituted monocyclic Ca-Cs hydrocarbon, or an optionally substituted C6-C12 bicyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “alkenyl” refers to an alkyl group, as defined herein, having one or more double bonds. In some embodiments, the term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched hydrocarbon chain having at least one double bond and having (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., C2-20, C2-18, C2-16, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C20 for branched chain), and alternatively, about 1-10. In some embodiments, a cycloalkyl ring has from about 3-10 carbon atoms in their ring structure where such rings are monocyclic or bicyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls).

The term “alkylenyl” or “alkylene” refers to a bivalent alkyl group (i.e., a bivalent saturated hydrocarbon chain) that is a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the above mentioned monovalent alkyl groups may be an alkylenyl by abstraction of a second hydrogen atom from the alkyl. In some embodiments, an “alkylenyl” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, preferably from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 5, or from 4 to 8. A substituted alkylenyl is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

As used herein, the term “alkynyl” refers to an alkyl group, as defined herein, having one or more triple bonds. In some embodiments, the term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (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., C2-20, C2-18, C2-16, C2-14, C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. By “alteration” is meant a change in the level, structure, or activity of an analyte, gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a change (e.g., increase or reduction) in expression levels. In embodiments, the increase or reduction in expression levels is by 10%, 25%, 40%, 50% or greater. In some embodiments, an alteration includes an insertion, deletion, or substitution of a nucleobase or amino acid (by, e.g., genetic engineering). In some embodiments, a base editor described herein alters the sequence of a TTR polynucleotide.

By “ameliorate” is meant reduce, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In embodiments, the disease is amyloidosis, and a base editor system described herein reduces the accumulation of amyloid in a tissue of a subject.

By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., Ce-u), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons.

By “base editor (BE),” or “nucleobase editor polypeptide (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying 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 Cpfl). Representative nucleic acid and protein sequences of base editors include those sequences having about or at least about 85% sequence identity to any base editor sequence provided in the sequence listing, such as those corresponding to SEQ ID NOs: 2-11.

By “BE4 cytidine deaminase (BE4) polypeptide,” is meant a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain, a cytidine deaminase domain, and two uracil glycosylase inhibitor domains (UGIs). In embodiments, the napDNAbp is a Cas9n (D10A) polypeptide. Non-limiting examples of cytidine deaminase domains include rAPOBEC, ppAPOBEC, RrA3F, AmAPOBECl, and SsAPOBEC3B.

By “BE4 cytidine deaminase (BE4) polynucleotide,” is meant a polynucleotide encoding a BE4 polypeptide.

By “base editing activity” is meant acting to chemically alter 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, e.g., converting target C*G to T«A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A«T to G*C.

The term “base editor system” or “base editing system” refers to an intermolecular complex for editing a nucleobase of a target nucleotide sequence. In various embodiments, the 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 the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from an adenosine deaminase or a cytidine deaminase, and a domain having nucleic acid sequence specific binding activity. In some embodiments, the 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 in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the 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 a cytidine deaminase) comprises a uracil glycosylase inhibitor or other agent or peptide (e.g., a uracil stabilizing protein such as provided in W02022015969, the disclosure of which is incorporated herein by reference in its entirety for all purposes) that inhibits the inosine base excision repair system.

As used herein, the term “bivalent” refers to a chemical moiety with two points of attachment. For example, a “bivalent Ci-8 (or Ci-e) saturated or unsaturated, straight or branched, hydrocarbon chain”, refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a 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 set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include but are not limited to:

The terms “carbocyclyl,” “carbocycle,” and “carbocyclic ring” as used herein, refer to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbomyl, adamantyl, and cyclooctadienyl. In some embodiments, “carbocyclyl” (or “cycloaliphatic”) refers to an optionally substituted monocyclic Ca-Cs hydrocarbon, or an optionally substituted C6-C12 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. The term “cycloalkyl” refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, cycloalkyl groups have 3-6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic 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.

The term “Cas9” or “Cas9 domain” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease.

The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.

Amino acids generally can be grouped into classes according to the following common side- chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. Coding sequences can also be referred to as open reading frames. The region or sequence is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. Stop codons useful with the base editors described herein include the following: TAG, TAA, and TGA.

By “complex” is meant a combination of two or more molecules whose interaction relies on inter-molecular forces. Non-limiting examples of inter-molecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonding, ionic bonding, halogen bonding, hydrophobic bonding, van der Waals interactions (e.g., dipole-dipole interactions, dipole -induced dipole interactions, and London dispersion forces), and a-cffccts. In an embodiment, a complex comprises polypeptides, polynucleotides, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, a complex comprises one or more polypeptides that associate to form a base editor (e.g., base editor comprising a nucleic acid programmable DNA binding protein, such as Cas9, and a deaminase) and a polynucleotide (e.g., a guide RNA). In an embodiment, the complex is held together by hydrogen bonds. It should be appreciated that one or more components of a base editor (e.g., a deaminase, or a nucleic acid programmable DNA binding protein) may associate covalently or non-covalently. As one example, a base editor may include a deaminase covalently linked to a nucleic acid programmable DNA binding protein (e.g., by a peptide bond). Alternatively, a base editor may include a deaminase and a nucleic acid programmable DNA binding protein that associate noncovalently (e.g., where one or more components of the base editor are supplied in trans and associate directly or via another molecule such as a protein or nucleic acid). In an embodiment, one or more components of the complex are held together by hydrogen bonds. By “cytosine” or ”4-Aminopyrimidin-2( l//)-onc" is meant a purine nucleobase with the molecular formula C4H5N3O, having the structure corresponding to CAS No. 71-30-7.

By “cytidine” is meant a cytosine molecule attached to a ribose sugar via a glycosidic bond, having the structure , and corresponding to CAS No. 65-46-3.

Its molecular formula is C9H13N3O5.

By “Cytidine Base Editor (CBE)” is meant a base editor comprising a cytidine deaminase.

By “Cytidine Base Editor (CBE) polynucleotide” is meant a polynucleotide encoding a CBE.

By “cytidine deaminase” or “cytosine deaminase” is meant 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 5 -methylcytosine to thymine. The terms “cytidine deaminase” and “cytosine deaminase” are used interchangeably throughout the application. Petromyzon marinus cytosine deaminase 1 (PmCDAl) (SEQ ID NO: 13-14), Activation-induced 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. Nonlimiting examples of cytidine deaminases include those described in PCT/US20/16288, PCT/US2018/021878, 180802-021804/PCT, PCT/US2018/048969, and PCT/US2016/058344.

By “cytosine deaminase activity” is meant catalyzing the deamination of cytosine or cytidine. In one embodiment, a polypeptide having cytosine deaminase activity converts an amino group to a carbonyl group. In an embodiment, a cytosine deaminase converts cytosine to uracil (z.e., C to U) or 5-methylcytosine to thymine (z.e., 5mC to T). In some embodiments, a cytosine deaminase as provided herein has increased cytosine deaminase activity (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) relative to a reference cytosine deaminase.

The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or fragment thereof that catalyzes a deamination reaction.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Exemplary diseases include diseases amenable to treatment using the methods and/or compositions of the present disclosure include as nonlimiting examples amyloidosis, cardiomyopathy, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), familial transthyretin amyloidosis (FTA), senile systemic amyloidosis (SSA), transthyretin amyloidosis, and the like. The disease can be any disease associated with a mutation in a transthyretin (TTR) polynucleotide sequence.

By “dual editing activity” or “dual deaminase activity” is meant having adenosine deaminase and cytidine deaminase activity. In one embodiment, a base editor having dual editing activity has both A->G and C->T activity, wherein the two activities are approximately equal or are within about 10% or 20% of each other. In another embodiment, a dual editor has A->G activity that no more than about 10% or 20% greater than C->T activity. In another embodiment, a dual editor has A->G activity that is no more than about 10% or 20% less than C->T activity. 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 no detectable adenosine deaminase activity.

By “effective amount” is meant the amount of an agent (e.g., a base editor, cell) as described herein, that is required to ameliorate the symptoms of a disease relative to an untreated patient or an individual without disease, z.e., a healthy individual, or is the amount of the agent sufficient to elicit a desired biological response. The effective amount of active compound(s) used to practice embodiments of the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount is the amount of a base editor of the disclosure sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect. Such therapeutic effect need not be sufficient to alter a pathogenic gene in all cells of a subject, tissue or organ, but only to alter the pathogenic gene in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of a disease. In one embodiment, an effective amount of a base editor is sufficient to alter the sequence of a TTR polynucleotide in 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more cells of a tissue.

The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material. In an embodiment, an mRNA molecule encoding a base editor and/or a guide polynucleotide is encapsulated by a lipid nanoparticle of the disclosure.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A 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.

By “guide polynucleotide” is meant a polynucleotide or polynucleotide complex which is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.

The term “haloaliphatic” refers to an aliphatic group substituted by one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halo, such as fluoro, iodo, bromo, or chloro). In some embodiments, haloaliphatic groups contain 1-7 halogen atoms. In some embodiments, haloaliphatic groups contain 1-5 halogen atoms. In some embodiments, haloaliphatic groups contain 1-3 halogen atoms.

The term “haloalkyl” refers to an alkyl group substituted by one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halo, such as fluoro, iodo, bromo, or chloro). In some embodiments, haloalkyl groups contain 1-7 halogen atoms. In some embodiments, haloalkyl groups contain 1-5 halogen atoms. In some embodiments, haloalkyl groups contain 1-3 halogen atoms.

The term “heteroalkylenyl” or “heteroalkylene”, as used herein, denotes an optionally substituted straight-chain (i.e., unbranched), or branched bivalent alkyl group (i.e., bivalent saturated hydrocarbon chain) having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” is described below. In some embodiments, heteroalkylenyl groups contain 2-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 2-8 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 4-8 carbon atoms, wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroalkylenyl groups contain 2-5 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroalkylenyl groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroalkylenyl 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-, -O(CH₂)4-. Unless otherwise specified, Cx heteroalkylenyl refers to heteroalkylenyl having x number of carbon atoms prior to replacement with heteroatoms.

The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 71 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Exemplary heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l,2- a]pyrimidinyl, imidazo[l,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H- -quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin- 3(4H)-one, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N- substituted pyrrolidinyl)).

The terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably herein, and refer to a stable 3- to 8-membered monocyclic, a 7- to 12-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR⁺ (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and . A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11 -membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11 -membered bridged heterocyclic ring having one, two, or three bridging atoms.

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By “increases” is meant a positive alteration of at least 10%, 25%, 50%, 75%, or 100%, or about 1.5 fold, about 2 fold, about 3 -fold, about 4-fold, about 5 -fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold.

The terms “inhibitor of base repair”, “base repair inhibitor”, “IBR” or their grammatical equivalents refer to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein 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 by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide.

By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

The term “linker”, as used herein, refers to a molecule that links two moieties. In one embodiment, the term “linker” refers to a covalent linker (e.g., covalent bond) or a non- covalent linker. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element “L”’ has an overall structure of the general form Sl-L’-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123).

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

The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

As used herein, the “N/P ratio” is the molar ratio of ionizable nitrogen atoms in a lipid(s) to phosphate groups in a nucleic acid molecular entity(ies). In some embodiments, the nitrogen atoms are ionizable within a physiological pH range. In some embodiments, the molecular entities in a nanoparticle composition include a lipid component and an RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH range of different cellular compartments (such as organs, tissues, and cells) and bodily fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, the physiological pH range refers to the pH range of blood in a mammal, for example, from about 7.35 to about 7.45. Similarly, for phosphate charge neutralizers that have one or more ionizable nitrogen atoms, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizer to the phosphate groups in a nucleic acid. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable within a pH range between 5 and 14.

For a payload that does not contain a phosphate group, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in a lipid to the total negative charge in the payload. For example, the N/P ratio of an LNP composition can refer to a molar ratio of the total ionizable nitrogen atoms in the LNP composition to the total negative charge in the payload that is present in the composition.

As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter from about 30 to about 170 nm. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter that 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, 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 having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 100 nm. For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have an average hydrodynamic diameter from between 50 nm to 90 nm. In some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from about 60 to about 80 nm. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between 50 nm to 70 nm.

As used herein, the term “nanoparticle composition” refers to a composition that contains at least one nanoparticle and at least one additional agent or ingredient. In some embodiments, a nanoparticle composition contains a substantially uniform collection of nanoparticles as described herein.

The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g. , a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double -stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5- propynyl-uridine, C5-propynyl -cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 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., phosphorothioates and 5'-N- phosphoramidite linkages).

The term “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al., International PCT application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan et al. , Nature Biotech. 2018 doi: 10.1038/nbt.4172. In some embodiments, an NLS comprises the amino acid sequence KRTADGSE FE SPKKKRKV (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), PKKKRKVEGADKRTADGSE FE SPKKKRKV (SEQ ID NO: 328), or RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 329).

The term “nucleobase,” “nitrogenous base,” or “base,” used interchangeably herein, refers to a nitrogen-containing biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to form base pairs and to stack one upon another leads directly to 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 called primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6- dihydrouracil, 5 -methylcytosine (m5C), and 5 -hydromethylcytosine. Hypoxanthine and xanthine can be created through mutagen presence, both of them through deamination (replacement of the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5- methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxy cytidine. Examples of a nucleoside with a modified nucleobase includes inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5 -methylcytidine (m5C), and pseudouridine ( ). A “nucleotide” consists of a nucleobase, a five carbon sugar (either ribose or deoxyribose), and at least one phosphate group. Non-limiting examples of modified nucleobases and/or chemical modifications that a modified nucleobase may include are the following: pseudo-uridine, 5-Methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, T-O- methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-O-methyl (‘M’), 2'-O-methyl-3'-phosphorothioate (‘MS’), 2'-O-methyl-3'- thiophosphonoacetate (‘MSP’), 5 -methoxyuridine, phosphorothioate, and Nl- Methylpseudouridine .

The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g. , DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that 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. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active 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), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, and Casl2j/Cas<b (Casl2j/Casphi). Non-limiting examples of Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Casl2j/Cas<b, Cpfl, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, 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 in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct;l:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference. Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the Sequence Listing as SEQ ID NOs: 197-245, 254-260, and 378. In some embodiments, the napDNAbp is a (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (e.g., SEQ ID NO: 197), Cas9 from Neisseria meningitidis (NmeCas9; SEQ ID NO: 208), Nme2Cas9 (SEQ ID NO: 209), Streptococcus constellatus (ScoCas9), or derivatives thereof (e.g., a sequence with at least about 85% sequence identity to a Cas9, such as Nme2Cas9 or spCas9). The terms “nucleobase editing domain” or “nucleobase editing protein,” as used herein, refers to a protein or enzyme that can catalyze a nucleobase modification in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine) deaminations, as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or an adenosine deaminase; or a cytidine deaminase or a cytosine deaminase).

As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

“Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with, at risk or having, predetermined to have, or suspected of having a disease or disorder.

The terms “pathogenic mutation”, “pathogenic variant”, “disease causing mutation”, “disease causing variant”, “deleterious mutation”, or “predisposing mutation” refers to a genetic alteration or mutation that is associated with a disease or disorder or that increases an individual’s susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid in a protein encoded by a gene. In some embodiments, the pathogenic mutation is in a terminating region (e.g., stop codon). In some embodiments, the pathogenic mutation is in a non-coding region (e.g., intron, promoter, etc.).

The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, z.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

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

The terms “protein”, “peptide”, “polypeptide”, and their grammatical equivalents are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof.

The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.

The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, a reference is an untreated cell that is not subjected to a test condition, or is subjected to placebo or normal saline, medium, buffer, and/or a control vector that does not harbor a polynucleotide of interest. The reference can be a cell or subject with a pathogenic mutation in a transthyretin (TTR) polynucleotide sequence and/or a transthyretin (TTR) polypeptide sequence. A reference can be a subject or cell with an amyloidosis (e.g., a transthyretin amyloidosis) or a subject or cell without an amyloidosis. In one embodiment, the level of amyloidosis in a cell or tissue treated with a base editor system described herein is compared to the level of amyloidosis in a corresponding untreated cell or tissue.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally 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. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein.

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

By “subject” or “patient” is meant a mammal, including, but not limited to, a human or non-human mammal. In embodiments, the mammal is a bovine, equine, canine, ovine, rabbit, rodent, nonhuman primate, or feline. In an embodiment, “patient” refers to a mammalian subject with a higher than average likelihood of developing a disease or a disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and/or female.

As described herein, compounds of this disclosure may be described as “substituted” or “optionally substituted”. That is, compounds may contain optionally substituted and/or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow fortheir production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

Suitable monovalent substituents include halogen; -(CH₂)o 4R⁰; -(CH₂)o 4OR⁰; - 0(CH₂)O-4R°, -0-(CH₂)O ₄C(0)0R^O; -(CH₂)O ₄CH(0R^O)₂; -(CH₂)O ₄Ph, which may be substituted with R°; -(CH₂)o 40(CH₂)o iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH₂)o-40(CH₂)o-i-pyridyl which may be substituted with R°; -NO₂; -CN; -N₃; -(CH₂)O ₄N(R^O)₂; -(CH₂)O ₄N(R°)C(0)R^O; -N(R°)C(S)R°; - (CH₂)O ₄N(R°)C(0)NR^O ₂; -N(R^O)C(S)NR°₂; -(CH₂)O ₄N(R°)C(0)0R^O; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°₂; -N(R°)N(R°)C(O)OR°; -(CH₂)o ₄C(O)R°; - C(S)R°; -(CH₂)O ₄C(O)OR°; -(CH₂)O ₄C(O)SR°; -(CH₂)O 4C(O)OSiR°₃; -(CH₂)o ₄OC(O)R°; -OC(0)(CH₂)O ₄SR^O-, -SC(S)SR°; -(CH₂)O ₄SC(O)R°; -(CH₂)O ₄C(0)NR^O ₂; -C(S)NR^O ₂; - C(S)SR°; -SC(S)SR°, -(CH₂)o ₄OC(O)NR°₂; -C(O)N(OR°)R°; -C(O)C(O)R°; - C(O)CH₂C(O)R°; -C(NOR°)R°; -(CH₂)O ₄SSR^O; -(CH₂)O ₄S(0)₂R^O; -(CH₂)O ₄S(O)₂OR°; - (CH₂)O ₄OS(O)₂R°; -S(0)₂NR^O ₂; -(CH₂)O ₄S(O)R°; -N(R°)S(0)₂NR^O ₂; -N(R°)S(O)₂R°; - N(OR°)R°; -C(NH)NR°₂; -P(O)₂R°; -P(O)R°₂; -OP(O)R°₂; -0P(0)(0R^O)₂; -SiR°₃; - OSiR°₃; -(Ci-4 straight or branched alkylene)O-N(R°)₂; or -(Ci-4 straight or branched alkylene)C(O)O-N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-6 aliphatic, -CH₂Ph, -0(CH₂)o iPh, -CH₂-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, - (CH₂)O 2R*, -(haloR*), -(CH₂)o ₂OH, -(CH₂)o ₂OR*, -(CH₂)o ₂CH(OR*)₂; -O(haloR’), -CN, -N₃, -(CH₂)O ₂C(O)R*, -(CH₂)O ₂C(O)OH, -(CH₂)O ₂C(O)OR*, -(CH₂)O ₂C(O)NH₂, -(CH₂)O ₂C (O)NHR‘. -(CH₂)O ₂C(O)NR*₂, -(CH₂)O ₂SR*, -(CH₂)O ₂SH, -(CH₂)O ₂NH₂, -(CH₂)O ₂NHR*. -(CH₂)O ₂NR*₂, -NO₂, -SiR*3, -OSiR*3, -C(O)SR* -(Ci-4 straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci- 4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

Suitable divalent substituents include the following: =0, =S, =NNR*₂, =NNHC(0)R*, =NNHC(0)0R*, =NNHS(O)₂R*, =NR*, =N0R*, -O(C(R*₂))_{2 3}O-, or -S(C(R*₂))₂ 3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*₂)₂ 3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), - OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH₂, -NHR*, -NR*₂, or -NO₂, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, suitable substituents on a substitutable nitrogen include -R'. - NR^T2, -C(O)R^T, -C(O)OR^T, -C(O)C(O)R^T, -C(O)CH₂C(O)R^T, -S(O)₂R^T, -S(O)₂NR^T2, - C(S)NR'₂. -C(NH)NR'₂. or -N(R')S(O)₂R' : wherein each R' is independently hydrogen, Ci- 6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R ' . taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R ' are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH₂, -NHR*, -NR*₂, or -NO₂, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci-4 aliphatic, -CH₂Ph, -0(CH₂)o iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

The term “stable,” when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure (e.g. , size range and/or distribution of particles) over a period of time. In some embodiments, a stable nanoparticle composition is one for which the average particle size, the maximum particle size, the range of particle sizes, and/or the distribution of particle sizes (i. e. , the percentage of particles above a designated size and/or outside a designated range of sizes) is maintained for a period of time under specified conditions. In some embodiments, a stable provided composition is one for which a biologically relevant activity is maintained for a period of time. In some embodiments, the period of time is at least about one hour; in some embodiments the period of time 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 period of time is within 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 population of nanoparticles is subjected to prolonged storage, temperature changes, and/or pH changes, and a majority of the nanoparticles in the composition maintain a diameter within a stated range, the nanoparticle composition is stable. In some embodiments, a stable composition is stable at ambient conditions. In some embodiments, a stable composition is stable under biologic conditions (i.e. 37 °C in phosphate buffered saline).

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence. In one embodiment, a reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, a reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99%, identical at the amino acid level or nucleic acid level to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BUAST, BESTFIT, GAP, or PIEEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double -stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). The term “target site” refers to a nucleotide sequence or nucleobase of interest within a nucleic acid molecule that is modified. In embodiments, the modification is deamination of a base. The deaminase can be a cytidine or an adenine deaminase. The fusion protein or base editing complex comprising a deaminase may comprise a dCas9-adenosine deaminase fusion protein, a Casl2b-adenosine deaminase fusion, or a base editor disclosed herein.

By “transthyretin (TTR) polypeptide” is meant a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to an amino acid sequence provided at NCBI Reference Sequence No. NP_000362.1, or a fragment thereof, that binds an anti- TTR antibody. In some embodiments, a TTR polypeptide or fragment thereof has holo- retinol-binding protein (RBP) and/or thyroxine (T4) transport activity. In embodiments, TTR is capable of forming a tetramer. An exemplary TTR polypeptide sequence follows (the signal peptide sequence is in bold; therefore, the mature TTR polypeptide corresponds to amino acids 21 to 147 of the following sequence): MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWE

PFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRR YTIAALLSPYSYSTTAVVTNPKE (SEQ ID NO: 464).

By “transthyretin (TTR) polynucleotide” is meant a nucleic acid molecule that encodes a TTR polypeptide, as well as the introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, the regulatory sequence is a promoter region. In embodiments, a TTR polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for TTR expression. An exemplary TTR polynucleotide sequence (corresponding to Consensus Coding Sequence (CCDS) No. 11899. 1) is provided below. Further exemplary TTR polynucleotide sequences include Gene Ensembl ID: ENSG00000118271 and Transcript Ensembl ID: ENST00000237014.8.

ATGGCTTCTCATCGTCTGCTCCTCCTCTGCCTTGCTGGACTGGTATTTGTGTCTGAGGCTGG CCCTACGGGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAG GCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAG CCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGA ATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCA TCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGC TACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCC CAAGGAATGA (SEQ ID NO: 465). A further exemplary TTR polynucleotide sequence is provided at NCBI Reference Sequence No. NG_009490.1 and follows (where exons encoding the TTR polypeptide are in bold, introns are in italics, and exemplary promoter regions are indicated by the combined underlined and bold-underlined text (promoter positions -1 to -177) and by the bold- underlined text (promoter positions -106 to -176):

In the above TTR polynucleotide sequence provided at NCBI Reference Sequence No. NG_009490. 1, exons encoding the TTR polypeptide correspond to the union of nucleotides 5137..5205, 6130..6260, 8354..8489, and 11802..11909, and the intervening sequences correspond to intron sequences. The union of nucleotides 5137..5205, 6130..6260, 8354..8489, and 11802..11909 corresponds to Consensus Coding Sequence (CCDS) No.

11899.1.

By “transthyretin amyloidosis” is meant a disease associated with an accumulation of amyloid in a tissue of a subject.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, z.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, reduces the intensity of, or cures a disease and/or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, z.e., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

By “uracil glycosylase inhibitor” or “UGI” is meant an agent that inhibits the uracil- excision repair system. Base editors comprising a cytidine deaminase convert cytosine to uracil, which is then converted to thymine through DNA replication or repair. In various embodiments, a uracil DNA glycosylase (UGI) prevent base excision repair which changes the U back to a C. In some instances, contacting a cell and/or polynucleotide with a UGI and a base editor prevents base excision repair which changes the U back to a C. An exemplary UGI comprises an amino acid sequence as follows: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDI IEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDA PEYKPWALVIQDSNGENKIKML (SEQ ID NO: 231).

In some embodiments, the agent inhibiting the uracil-excision repair system is a uracil stabilizing protein (USP). See, e g., WO 2022015969 Al, incorporated herein by reference.

As used herein, the term "vector" refers to a means of introducing a nucleic acid molecule into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains

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

As used in this specification and claim(s), the words “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”) are inclusive or open-ended. This wording indicates that specified elements, features, components, and/or method steps are present, but does not exclude the presence of other elements, features, components, and/or method steps. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A and IB provide plots and charts showing that, under similar conditions, base editor systems containing a guide RNA targeting a human TTR polynucleotide sequence for base editing (i.e., human gRNA or “human”) and administered to primary human hepatocytes were associated with higher levels of base editing than base editor systems containing a guide RNA targeting a non-human primate TTR polynucleotide sequence (i.e., cyno gRNA or “cyno”) and administered to primary cyno hepatocytes. The base editor systems contained mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”). FIG. 1A provides a plot and chart showing base editing rates measured for base editing of a synthetic polynucleotide sequence (“AAVS1 Cassette”) in human embryonic kidney (HEK293) cells that contained a polynucleotide having the target sites for both the human gRNA and for the cyno gRNA. The base editor systems were delivered to the HEK293T cells using a commercially available lipid transfection reagent, Lipofectamine® MessengerMAX mRNA Transfection Reagent (LipoMM). FIG. IB provides plots and a chart showing base editing rates measured in primary human hepatocytes (PHH) and primary cyno hepatocytes (PCH) at the TTR gene (left panel) or at the ALAS 1 gene control (right panel). The ALAS 1 gene (right panel) was base edited using a base editor system containing the control guide RNA sg23 known to be effective for use in targeting base editors to alter a nucleobase within the ALAS 1 gene and an mRNA molecule encoding a base editor containing a TadA*8.8 adenosine deaminase domain. The base editor systems of FIG. IB were delivered using lipid nanoparticles containing the ionizable lipid IZ1. In FIGs. 1A and IB, the terms PHH-A, PHH-B, and PHH-C indicate three different human donors from which primary human hepatocytes (PHH) were obtained, “cTTR” indicates a cyno TTR gene, and “Conserved” indicates the ALAS1 gene.

FIGs. 2A and 2B provide plots and charts showing that, under similar conditions, base editor systems containing a guide RNA targeting a human TTR polynucleotide sequence for base editing (i.e., human gRNA or “human”) and administered to primary human hepatocytes were associated with higher levels of base editing than base editor systems containing a guide RNA targeting a non-human primate TTR polynucleotide sequence (i.e., cyno gRNA or “cyno”) and administered to primary cyno hepatocytes (PCH). The base editor systems were administered to the cells using lipid nanoparticles containing the ionizable lipid IZ4. FIG. 2A provides a plot and a chart showing base editing rates measured in primary human hepatocytes (PHH) and primary cyno hepatocytes (PCH) at the TTR gene. The base editor systems administered to the PHH contained the guide RNA gRNA2944, and the base editor systems administered to the PCH contained the guide RNA gRNA2945. The base editor systems contained mRNA encoding a base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”). FIG. 2B provides a plot and a chart showing base editing rates measured for base editing measured in primary human hepatocytes (PHH) and primary cyno hepatocytes (PCH) at the ALAS 1 gene. The ALAS 1 gene was base edited using a base editor system containing the control guide RNA sg23 known to be effective for use in targeting base editors to alter a nucleobase within the ALAS 1 gene and an mRNA molecule encoding a base editor containing a TadA*8.8 adenosine deaminase domain. In FIGs. 2A and 2B, the terms PHH1, PHH2, and PHH3 indicate primary human hepatocytes (PHH) from three different human donors. Throughout the figures the term “surrogacy” refers to evaluating or using base editing measurements for a non-human primate polynucleotide sequence or cell to infer base editing rates achieved under similar or analogous conditions (e.g., using a base editor system containing a guide RNA targeting a human polynucleotide rather than a non-human primate polynucleotide for base editing) in a human polynucleotide sequence or cell.

FIGs. 3A to 3F provide bar graphs showing liver enzyme data for male (FIGs. 3A- 3C) and female (FIGs. 3D-3F) non-human primates administered the indicated doses of lipid nanoparticles containing the lipid IZ 1 and a base editor system containing 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 TadA*8.20 adenosine deaminase domain variant with a V82T amino acid alteration. The groupings of bars from left-to-right in FIGs. 3A to 3F correspond to Control, 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, respectively. In FIGs. 3A to 3F, the term “AST” represents “aspartate transaminase,” the term “ALT” represents “alanine transaminase,” and the term “ALP” represents “alkaline phosphatase.” The x-axes in FIGs. 3A to 3F represent the number of days following lipid nanoparticle administration (day 0). This experiment indicates that LNP administration of a base editor system had no sustained deleterious effect. FIG. 4 provides bar graphs showing rates of on-target (TTR gene A>G nucleotide alterations) and off-target (EIF3E gene A>G nucleotide alteration) base editing measured in primary human hepatocytes from donors PHH Donor 1 (PHH1 of FIGs. 2A and 2B) and PHH Donor 2 (PHH2 of FIGs. 2A and 2B) administered lipid nanoparticles containing base editor systems containing a 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 throughout the figures as “ABE9.51”) or a TadA*8.20 adenosine deaminase domain with the amino acid alterations V82T, Y147T, and Q154S (referred to throughout the figures as “ABE9.52”). For each pair of bars of FIG. 4, the left bar corresponds to “TTR, A>G” and the right bar represents “EIF3E, A>G.”

FIGs. 5A and 5B provide images of primary human hepatocyte cells. FIG. 5A provides an image of primary cyno hepatocyte cells that have not been administered any base editor system. FIG. 5B provides an image of primary cyno hepatocyte cells one day after having been administered lipid nanoparticles containing a base editor system containing a guide RNA targeting a non-human primate TTR gene for base editing and mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”).

FIGs. 6A and 6B provide plots and charts showing that, under similar conditions, base editor systems containing a guide RNA targeting a human TTR polynucleotide sequence for base editing (i.e., human gRNA or “human”) and administered to primary human hepatocytes were associated with higher levels of base editing than base editor systems containing a guide RNA targeting a non-human primate TTR polynucleotide sequence (i.e., cyno gRNA or “cyno”) and administered to primary cyno hepatocytes (PCH). The base editor systems were administered to the cells using lipid nanoparticles containing the ionizable lipid IZ4. FIG. 6A provides a plot and a chart showing base editing rates measured in primary human hepatocytes (PHH) and primary cyno hepatocytes (PCH) at the TTR gene. The base editor systems administered to the PHH contained the guide RNA gRNA2944, and the base editor systems administered to the PCH contained the guide RNA gRNA2945. The base editor systems contained mRNA encoding a base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”). FIG. 6B provides a plot and a chart showing base editing rates measured for base editing measured in primary human hepatocytes (PHH) and primary cyno hepatocytes (PCH) at the ALAS1 gene. The ALAS1 gene was base edited using a base editor system containing the control guide RNA sg23 known to be effective for use in targeting base editors to alter a nucleobase within the ALAS 1 gene and an mRNA molecule encoding a base editor containing a TadA*8.8 adenosine deaminase domain. In FIGs. 6A and 6B, the terms PHH1, PHH2, and PHH3 indicate primary human hepatocytes (PHH) from three different human donors, where the three donors were the same donors as those of FIGs. 2A and 2B.

DETAILED DESCRIPTION

Provided herein are compositions for polynucleotide (e.g., a gene) modification or editing and methods of using the same to treat or prevent conditions associated with the extracellular deposition in various tissues of amyloid fibrils formed by the aggregation of misfolded transthyretin (TTR) proteins. Such conditions include, but are not limited to, polyneuropathy due to hereditary transthyretin amyloidosis (hATTR-PN) and hereditary cardiomyopathy due to transthyretin amyloidosis (hATTR-CM), both associated with autosomal dominant mutations of the TTR gene, and an age-related cardiomyopathy associated with wild-type TTR proteins (ATTRwt), also known as senile cardiac amyloidosis. Compositions and methods directed to editing a TTR polynucleotide using an editing system, such as one comprising a base editor and guide RNAs are disclosed.

The invention is based, at least in part, on the discovery that base editor systems can be used to disrupt expression of a transthyretin polypeptide or to edit a pathogenic mutation in a transthyretin polypeptide and that the base editor systems can be effectively delivered to cells using lipid nanoparticles of the disclosure. In one embodiment, the disclosure provides guide RNA sequences that are effective for use in conjunction with a base editing system for editing a transthyretin (TTR) gene sequence to disrupt splicing or correct a pathogenic mutation. In another embodiment, the invention provides guide RNA sequences that target a Casl2b nuclease to edit a TTR gene sequence, thereby disrupting TTR polypeptide expression.

Accordingly, the disclosure provides guide RNA sequences suitable for use with an ABE and/or a BE4 for transthyretin (TTR) gene splice site disruption and guide RNA sequences suitable for use with bhCasl2b nucleases for disruption of the transthyretin (TTR) gene. In embodiments, the compositions and methods of the present invention can be used for editing a TTR gene in a hepatocyte. The methods provided herein can include reducing or eliminating expression of TTR in a hepatocyte cell to treat an amyloidosis. TRANSTHYRETIN PROTEIN AND GENE

Transthyretin (TTR), originally known as prealbumin, is a 55-kDa transport protein for both thyroxine (T4) and retinol-binding protein, that circulates in soluble form in the serum and cerebrospinal fluid (CSF) of healthy humans. 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 amino acid sequence below: MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWE

PFASGKTSESGELHGLTTEEE FVEGIYKVE IDTKSYWKALGI SPFHEHAEVVFTANDSGPRR YT IAALLSPYSYSTTAVVTNPKE (SEQ ID NO: 464).

The TTR gene, composed of four exons, is located on chromosome 18 at 18q 12.1. The full sequence of the human TTR gene is available at UniProtKB - P02766 (TTHY HUMAN). Over 120 TTR variants have so far been identified, the great majority of which are pathogenic. The most common pathogenic variant consists of a point mutation leading to replacement of valine by methionine at position 30 of the mature protein. This Val30Met mutation is responsible for hATTR amyloidosis and is the most frequent amyloidogenic mutation worldwide, accounting for about 50% of TTR variants.

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

When there is clinical suspicion for hATTR-PN, diagnosis is typically done by tissue biopsy with staining for amyloid, amyloid typing (using immunohistochemistry or mass spectrometry), and/or TTR gene sequencing. When there is clinical suspicion for ATTR-CM, the key diagnostic tools are either endomyocardial biopsy (with tissue staining and amyloid typing by immunohistochemistry or mass spectrometry) or 99m technetium-pyrophosphate scan. Both of these approaches can provide a diagnosis of ATTR-CM. TTR gene sequencing can be used to differentiate between the hATTR-CM (mutation positive) and ATTRwt-CM (mutation negative). In some embodiments, the compositions described herein include guide polynucleotides containing a spacer having a nucleotide sequence that functions as a guide to direct a gene editing protein (e.g., a base editor) to alter a TTR gene, for example by introducing one or more nucleobase alterations in the TTR gene. These point mutations may be used to disrupt gene function, by the introduction of a missense mutation(s) that results in production of a less functional, or non-functional protein, thus silencing the TTR gene. Alternatively, it is contemplated herein that corrections to one or more point mutation(s) may be made using a gene editing protein to alter a mutated gene to correct the underlying mutation causing the dysfunction in the TTR gene or otherwise mitigate against dysfunction of the gene.

AMYLOIDOSIS

Amyloidosis is a disorder that involves extracellular deposition of amyloid in an organ or tissue (e.g., the liver). Amyloidosis may occur when mutant transthyretin polypeptides aggregate (e.g., as fibrils). An amyloidosis caused by a mutation to the transthyretin gene can be referred to as a “transthyretin amyloidosis”. Some forms of transthyretin amyloidosis are not associated with a mutation to the transthyretin gene. Nonlimiting examples of mutations to the mature transthyretin (TTR) protein that can lead to amyloidosis include the alterations T60A, V30M, V30A, V30G, V30L, V122I, V122A, and V122(-). One method for treatment of transthyretin amyloidosis includes disrupting expression or activity of transthyretin in a cell of a subject, optionally a hepatocyte cell. Accordingly, provided herein are methods for reducing or eliminating expression of transthyretin in a cell. The transthyretin in the cell can be a pathogenic variant. Expression of transthyretin in a cell can be disrupted by disrupting splicing of a transthyretin transcript.

Transthyretin amyloidosis is a progressive condition characterized by the buildup of protein deposits in organs and/or tissues. These protein deposits can occur in the peripheral nervous system, which is made up of nerves connecting the brain and spinal cord to muscles and sensory cells that detect sensations such as touch, pain, heat, and sound. Protein deposits in these nerves result in a loss of sensation in the extremities (peripheral neuropathy). The autonomic nervous system, which controls involuntary body functions such as blood pressure, heart rate, and digestion, may also be affected by amyloidosis. In some cases, the brain and spinal cord (i.e., central nervous system) are affected. Other areas of amyloidosis include the heart, kidneys, eyes, liver, and gastrointestinal tract. The age at which symptoms begin to develop can be between the ages of 20 and 70. There are three major forms of transthyretin amyloidosis, which are distinguished by their symptoms and the body systems they effect: neuropathic, leptomeningeal, and cardiac.

The neuropathic form of transthyretin amyloidosis primarily affects the peripheral and autonomic nervous systems, resulting in peripheral neuropathy and difficulty controlling bodily functions. Impairments in bodily functions can include sexual impotence, diarrhea, constipation, problems with urination, and a sharp drop in blood pressure upon standing (orthostatic hypotension). Some people experience heart and kidney problems as well. Various eye problems may occur, such as cloudiness of the clear gel that fills the eyeball (vitreous opacity), dry eyes, increased pressure in the eyes (glaucoma), or pupils with an irregular or ’’scalloped” 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.

The leptomeningeal form of transthyretin amyloidosis primarily affects the central nervous system. In people with this form, amyloidosis occurs in the leptomeninges, which are two thin layers of tissue that cover the brain and spinal cord. A buildup of protein in this tissue can cause stroke and bleeding in the brain, an accumulation of fluid in the brain (hydrocephalus), difficulty coordinating movements (ataxia), muscle stiffness and weakness (spastic paralysis), seizures, and loss of intellectual function (dementia). Eye problems similar to those in the neuropathic form may also occur. When people with leptomeningeal transthyretin amyloidosis have associated eye problems, they are said to have the oculoleptomeningeal form.

The cardiac form of transthyretin amyloidosis affects the heart. People with cardiac amyloidosis may have an abnormal heartbeat (arrhythmia), an enlarged heart (cardiomegaly), or orthostatic hypertension. These abnormalities can lead to progressive heart failure and death. Occasionally, people with the cardiac form of transthyretin amyloidosis have mild peripheral neuropathy.

Mutations in the transthyretin (TTR) gene cause transthyretin amyloidosis. Transthyretin transports vitamin A (retinol) and a hormone called thyroxine throughout the body. Not being bound by theory, to transport retinol and thyroxine, transthyretin must form a tetramer. Transthyretin is produced primarily in the liver (i.e., in hepatic cells). A small amount of transthyretin (TTR) is produced in an area of the brain called the choroid plexus and in the retina.

TTR gene mutations can alter the structure of transthyretin, impairing its ability to bind to other transthyretin proteins. The TTR gene mutation can be autosomal dominant. SPLICE SITES

Gene splice sites and splice site motifs are well known in the art and it is within the skill of a practitioner to identify splice sites in sequence (see, e.g., 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)).

Canonical splice donors contain the DNA sequence GT on the sense strand, whereas canonical splice acceptors comprise the DNA sequence AG. Alteration of the sequence disrupts normal splicing. Splice donors can be disrupted by adenine base editing of the complementary base in the second position in the antisense strand (GT^GC), and splice acceptors can be disrupted by adenine base editing of the first position in the sense strand (AG^GG).

EDITING OF TARGET GENES

To edit the transthyretin (TTR) gene, a cell (e.g., a hepatocyte) is contacted with a guide RNA and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase or adenosine deaminase to edit a base of a gene sequence. Editing of the base can result in disruption of a splice site (e.g., through alteration of a splice-site motif nucleobase). Editing of the base can result in replacement of a pathogenic variant amino acid with a non-pathogenic variant amino acid. As a non-limiting example, editing of the base can result in replacing a T60A, V30M, V30A, V30G, V30L, V122I, V122A, or a V122(-) alteration in the mature transthyretin (TTR) polypeptide with a non-pathogenic variant or the wild-type valine residue. The cytidine deaminase can be BE4 (e.g., saBE4). The adenosine deaminase can 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 a cell with a nuclease and a guide RNA to introduce an indel into a gene sequence. The indel can be associated with a reduction or elimination of expression of the gene. The nuclease can be Casl2b (e.g., bhCasl2b). The cells can be edited in vivo or ex vivo. The guide RNA can be a single guide or a dual guide. In some embodiments, cells to be edited are contacted with at least one nucleic acid, wherein 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., an adenosine or a cytidine deaminase. In some embodiments, the gRNA comprises nucleotide analogs. These nucleotide analogs can inhibit degradation of the gRNA by cellular processes. Exemplary single guide RNA (sgRNA) sequences are provided in Tables 1A to IE and exemplary spacer sequences and target sequences (e.g., protospacer sequences) are provided in Tables IB, 1C, and 2A to 2E.

With the present disclosure, guide polynucleotides and spacer sequences suitable for use therein for targeting a base editor to alter a nucleobase within the human TTR gene to either disrupt the start codon, or disrupt splice sites, whether donors or acceptors, via A^G editing within an editing window (roughly positions 4 to 7 in the 20-nt protospacer region corresponding to the spacer of the guide polynucleotide).

Protospacer, corresponding to guide RNA GA457, has the sequence 5’- GCCATCCTGCCAAGAATGAG-3’ (SEQ ID NO: 467) and is located at 34,879 to 34,898 bp of the human TTR gene.

Protospacer, corresponding to guide RNA GA459, has the sequence 5’- GCAACTTACCCAGAGGCAAA-3’ (SEQ ID NO: 468) and is located at 36,007 to 36,026 bp of the human TTR gene.

Protospacer, corresponding to guide RNA GA460, has the sequence 5’- TATAGGAAAACCAGTGAGTC-3’ (SEQ ID NO: 469) and is located at 38,106-38,125 bp of the human TTR gene.

Protospacer, corresponding to guide RNA GA461, has the sequence 5’- TACTCACCTCTGCATGCTCA-3’ (SEQ ID NO: 470) and is located at 38,234-38253 of the human TTR gene.

Protospacer, corresponding to guide RNA GA458, has the sequence 5’- GCCATCCTGCCAAGAACGAG-3’ (SEQ ID NO: 471) represents the sequence within the cynomolgus macaque TTR gene corresponding to the human protospacer sequence corresponding to guide RNA GA459.

The present disclosure includes a guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-GCCAUCCUGCCAAGAAUGAG-3’ (SEQ ID NO: 472) (GA457). The present disclosure includes a guide polynucleotide having the sequence 5’- GCCAUCCUGCCAAGAAU GAG-3’ (SEQ ID NO: 472) (GA457). The present disclosure includes a modified guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-GCCAUCCUGCCAAGAAUGAG-3’ (SEQ ID NO: 472), wherein the nucleotides GCC shown in bold are modified by methylation (GA521) (C is modified to 2’-O-methylcytidine, G is modified to 2’-O-methylguanosine). The present 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: phosphorothioate (PS) backbone linkage.

The present disclosure includes a guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-GCCAUCCUGCCAAGAACGAG-3’ (SEQ ID NO: 473) (GA458). The present disclosure includes a guide polynucleotide having the sequence 5’- GCCAUCCUGCCAAGAACGAG-3’ (SEQ ID NO: 473) (GA458).

The present disclosure includes a guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-GCAACUUACCCAGAGGCAAA-3’ (SEQ ID NO: 474) (GA459). The present disclosure includes a guide polynucleotide having the sequence 5’- GCAACUUACCCAGAGGCAAA-3’ (SEQ ID NO: 474) (GA459).

The present disclosure includes a guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-UAUAGGAAAACCAGUGAGUC-3’ (SEQ ID NO: 475) (GA460). The present disclosure includes a guide polynucleotide having the sequence 5’- UAUAGGAAAACCAGUGAGUC-3’ (SEQ ID NO: 475) (GA460).

The present disclosure includes a guide polynucleotide having a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the sequence 5’-UACUCACCUCUGCAUGCUCA-3’ (SEQ ID NO: 476) (GA461). The present disclosure includes a guide polynucleotide having the sequence 5’- UACUCACCUCUGCAUGCUCA-3’ (SEQ ID NO: 476) (GA461).

In some aspects, provided herein is a guide RNA, comprising a sequence defined by mG *mC *mC * AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm

AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (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’-0- methyluridine, and wherein nucleotides represented in bold are linked by a phosphorothioate (PS) backbone linkage.

Alternatively, GA521 is represented as mG*smC* smC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmGsmCsmUsmA sGsmAsmAsmAsmUsmAsmGsmCssmAsmAsGsUsUsmAsAsmAsAsmUsAsmAsmGsmGsm CsmUsmAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAs mGsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsmUsmGsmCsmU* smU*sm U* 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’-0- methylguanosine, mU* is 2’-O-methyluridine, and wherein nucleotides are linked by a phosphorothioate (PS) backbone linkage represented by the letter ‘s’.

In some embodiments, mG *mC *mC * AUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (GA521, SEQ ID NO: 477), wherein A is adenosine, C is cytidine, G is guanosine, U is uridine, mA* is 2’-0- methyladenosine, mC* is 2’-O-methylcytidine, mG* is 2’-O-methylguanosine, mU* is 2’-0- methyluridine, and wherein nucleotides represented in bold are linked by a phosphorothioate (PS) backbone linkage.

Alternatively, GA521 is represented as mG*mC*mC*AUCCUGCCAAGAAUGAGmGsUsUsUsUsAsGsmAsmsGsmCsmUsmAs GsmAsmAsmAsmUsmAsmGsmCs smAsmAsGsUsUsmAsAsmAsAsmUsAsmAsmGsmGsmC smUsmAsGsUsmCsmCsGsUsUsAsmUsmCsAsAsmCsmUsmUsGsmAsmAsmAsmAsmAsm GsmUsmGsGsmCsmAsmCsmCsmGsmAsmGsmUsmCsmGsmGsmUsmGsmCsmU*mU*mU*m U (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’-0- methylguanosine, mU* is 2’-O-methyluridine, and wherein nucleotides represented in bold are linked by a phosphorothioate (PS) backbone linkage represented by the letter ‘s’.

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

In various instances, it is advantageous for a spacer sequence to include a 5' and/or a 3' “G” nucleotide. In some cases, for example, any spacer sequence or guide polynucleotide provided herein comprises or further comprises a 5' “G”, where, in some embodiments, the 5' “G” is or is not complementary to a target sequence. In some embodiments, the 5' “G” is added to a spacer sequence that does not already contain a 5' “G ” For example, it can be advantageous for a guide RNA to include a 5' terminal “G” when the guide RNA is expressed under the control of a U6 promoter or the like 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. l231143). In some cases, a 5' terminal “G” is added to a guide polynucleotide that is to be expressed under the control of a promoter, but is optionally not added to the guide polynucleotide if or when the guide polynucleotide is not expressed under the control of a promoter.

Variants of the spacer sequences provided herein comprising 1, 2, 3, 4, or 5 nucleobase alterations are contemplated. For example, variation of a target polynucleotide sequence within a population (e.g., single nucleotide polymorphisms) may require said alterations to a spacer sequence to allow the spacer to better bind a variant of a target sequence in a subject.

In embodiments, a guide RNA comprises a sequence complementary to a promoter region of a TTR polynucleotide sequence. In embodiments, the promoter 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 position +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 of the start codon (ATG) of the TTR polynucleotide sequence.

In various embodiments of the disclosure, a guide RNA contains the following polynucleotide sequence, where the sequence may be modified by adding or removing one or more of the “Ns*’ (i.e., the spacer sequence, shown in bold, can be extended or truncated by, e.g., 1, 2, 3, 4, or 5 nucleotides): nsnsnsNNNNNNNNNNNNNNNNNgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGU ccGUUAucAAcuuGaaaaagugGcaccgagucggugcuususus (SEQ ID NO: 1235). Where, “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 phosphorothioate (PS) backbone linkage and wherein bold type represents the spacer sequence. Table 1A. Exemplary guide RNAs for editing transthyretin (TTR) splice sites and/or introducing indels into the TTR gene (e.g., using bhCas!2b)

Lowercase m indicates 2’-O-methylated nucleobases (e.g., mA, mC, mG, mU), and “s” indicates phosphorothioates.

Table IB. Exemplary guide RNAs for editing a TTR polynucleotide (spacer sequences within a gRNA sequence are in bold)

Leters in the sequences:- 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; and s or *: phosphorothioate (PS) backbone linkage. Bold type in gRNA sequence denotes spacer sequence corresponding to Protospacer. GA460 and GA520 have the same protospacer sequence but have different chemical modifications in the gRNA sequence.

Table 1C. Exemplary guide RNAs for editing a TTR polynucleotide (spacer sequences within gRNA sequences are shown in bold)

¹ The region of the TTR gene targeted by this base editor, either in humans or non-human primates, is referred to as “HM17.” Letters in the sequences: A = adenosine; C = cytidine; G = guanosine; U = uridine; a = 2’-O- methyladenosine; c = 2 ’-0 -methylcytidine; g = 2’-O-methylguanosine; u = 2’-O- methyluridine; s = phosphorothioate (PS) backbone linkage. C = nucleotide that differs in NHP from human TTR sequence. Bold type in gRNA sequence denotes spacer sequence corresponding to Protospacer.

Table ID. Exemplary guide RNA sequences (spacer sequences are in bold)

A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2 ’-0 -methyladenosine; c is

2’-O-methylcytidine; g is 2’-O-methylguanosine; u is 2’-O-methyluridine and s is phosphorothioate (PS) backbone linkage and wherein bold type represents the spacer sequence.

Table IE. Exemplary guide RNA sequences (spacer sequences are in bold)

Table 2A. Exemplary Spacer and Target Site Sequences.²

² One of skill in the art will understand that some of the target site sequences correspond to a reversecomplement to the above-provided transthyretin polynucleotide sequence; i.e., the target sequences may correspond to either strand of a dsDNA molecule encoding a transthyretin polynucleotide. Further, it is to be understood that a C base can be targeted by a cytidine deaminase and that an A base can be targeted by an adenine deaminase.

Table 2B. Exemplary Spacer and Target Site Sequences.

Table 2C. Exemplary human TTR target site sequences and base editor + guide RNA combinations.

Table 2C (CONTINUED)

Table 2D. Exemplary gRNA spacer sequences with PS linkage at 5’ end

A is adenosine; C is cytidine; G is guanosine; U is uridine; a is 2 ’-0 -methyladenosine; c is 2’-O-methylcytidine; g is 2’-O-methylguanosine; u is 2’-O-methyluridine and s is phosphorothioate (PS) backbone linkage.

Table 2E. Exemplary gRNA spacer sequence without PS linkage

A is a modified or unmodified adenosine; C is a modified or unmodified cytidine; G is modified or unmodified guanosine; and U is a modified or unmodified uridine.

The guide polynucleotide sg23 was used throughout the disclosure as a control guide RNA known to be effective in targeting base editor systems to alter a nucleobase in an ALAS1 gene. The sg23 guide RNA contained the following spacer sequence: CAGGAUCCGCACAGACUCCA (SEQ ID NO: 480). The sg23 guide RNA targeted the following sequence for base editing, where the PAM sequence is shown in bold: CAGGATCCGCACAGACTCCAGGG (SEQ ID NO: 481).

The spacer sequences in Table 2A corresponding to sgRNAs sgRNA_361, sgRNA_362, sgRNA_363, sgRNA_364, sgRNA_365, sgRNA_366, and sgRNA_367 may be used for targeting a base editor to alter a nucleobase of a splice site of the transthyretin polynucleotide. The spacer sequences in Table 2A corresponding to sgRNAs sgRNA_368, sgRNA_369, sgRNA_370, sgRNA_37I, sgRNA_372, sgRNA_373, and sgRNA_374 may be used for targeting an endonuclease to a transthyretin (TTR) polynucleotide sequence. The three spacer sequences in Table 2A corresponding to sgRNA_375, sgRNA_376, and sgRNA_377 may be used to alter a nucleobase of a transthyretin (TTR) polynucleotide. The alteration of the nucleobase may result in an alteration of an isoleucine (I) to a valine (V) (e.g., to correct a V122I mutation in a transthyretin polypeptide encoded by the transthyretin polynucleotide). In embodiments, a transthyretin polynucleotide may be edited using the following combinations of base editors and sgRNA sequences (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-bhCasl2b 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 of spCas9-VRQR-ABE can be GGA. The PAM sequence of saCas9-ABE can be AGGAAT.

In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-methylated) strand opposite the targeted nucleobase. Mutation of the catalytic residue (e.g., DIO to Al 0) prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants can generate a single-strand DNA break (nick) at a specific location based on the gRNA-defmed target sequence, leading to repair of the non-edited strand, ultimately resulting in a nucleobase change on the non-edited strand.

NUCLEOBASE EDITORS

Useful in the methods and compositions described herein are nucleobase editors that edit, modify or alter a 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 a dual deaminase). A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g. , gRNA), can specifically bind to a target polynucleotide sequence and thereby localize the base editor to the target nucleic acid sequence desired to be edited.

Polynucleotide Programmable Nucleotide Binding Domain

Polynucleotide programmable nucleotide binding domains bind polynucleotides (e.g., RNA, DNA). A polynucleotide programmable nucleotide binding domain of a base editor can itself comprise one or more domains (e.g, one or more nuclease domains). In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease.

Disclosed herein are base editors 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 as a domain all or a portion (e.g., a functional portion) of a CRISPR protein (e.g., a Cas protein), also referred to as a “CRISPR protein- derived domain” of the base editor). A CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of the CRISPR protein. A CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements and/or recombinations relative to a wild-type or natural version of the CRISPR protein.

Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Casl2a/Cpfl, Casl2b/C2cl (e.g, SEQ ID NO: 232), Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Cas 12g, Casl2h, Casl2i, and Cas 12j/Cas<b. CARF, DinG, homologues thereof, or modified versions thereof. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 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 a target sequence.

A vector that encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. A Cas protein (e.g., Cas9, Casl2) or a Cas domain (e.g., Cas9, Casl2) can refer to a polypeptide or domain with 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 to a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Casl2) can refer to the wild-type or a modified form of the Cas protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

In some embodiments, a CRISPR protein-derived domain of a base editor can include all or a portion (e.g., a functional portion) of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1);

Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

Some aspects of the disclosure provide high fidelity Cas9 domains. High fidelity Cas9 domains are known in the art and described, for example, in Kleinstiver, B.P., et al. “High- fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I.M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each of which are incorporated herein by reference. An Exemplary high fidelity Cas9 domain is provided in the Sequence Listing as SEQ ID NO: 233.

In some embodiments, any of the Cas9 fusion proteins or complexes provided herein comprise one or more of a D10A, N497X, a R661X, a Q695X, and/or a Q926X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. .

Typically, Cas9 proteins, such as Cas9 from .S', pyogenes (spCas9), require a “protospacer 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 a particular nucleic acid region, where the “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and the G is guanosine. In some embodiments, any of the fusion proteins or complexes provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

In some embodiments, the napDNAbp is a circular permutant (e.g., SEQ ID NO: 238).

In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D.

In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9; SEQ ID NO: 201). The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase comprises an amino acid sequence that is 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% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure.

Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (i. e. , incapable of cleaving a target polynucleotide sequence). For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion (e.g., a functional portion) of a nuclease domain. dCas9 domains are known in the art and 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.

The term “protospacer 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 can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM i.e., located downstream of the 5' end of the protospacer). 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, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T. A base editor provided herein can comprise a CRISPR protein-derived domain that is capable of binding a nucleotide sequence that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence.

In some embodiments, the PAM is an “NRN” PAM where the “N” in “NRN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the R is adenine (A) or guanine (G); or the PAM is an “NYN” PAM, wherein the “N” in NYN is adenine (A), thymine (T), guanine (G), or cytosine (C), and the Y is cytidine (C) or thymine (T), for example, as described in R.T. Walton et al., 2020, Science, 10.1126/science.aba8853 (2020), the entire contents of which are incorporated herein by reference. Several PAM variants are described in Table 3 below.

Table 3. Cas9 proteins and corresponding PAM sequences. N is A, C, T, or G; and V is A, C, or G. In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively termed “MQKFRAER”) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335Q, and T1337R (collectively termed VRQR) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from DI 135V, G1218R, R1335E, and T1337R (collectively termed VRER) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant contains one or more amino acid substitutions selected from E782K, N968K, and R1015H (collectively termed KHH) of saCas9 (SEQ ID NO: 218). In some embodiments, the Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively termed “MQKSER”) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9. In some embodiments, the Cas9 variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R (collectively termed “MQKSER”) of spCas9 (SEQ ID No: 197), or a corresponding mutation in another Cas9.

In some embodiments, a CRISPR protein-derived domain of a base editor comprises all or a portion (e.g., a functional portion) of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor can employ a non- canonical PAM sequence. Such sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); R.T. Walton 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 entire contents of each are hereby incorporated by reference. Fusion Proteins or Complexes Comprising a NapDNAbp and a Cytidine Deaminase and/or Adenosine Deaminase

Some aspects of the disclosure provide fusion proteins or complexes comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Casl2) and one or more cytidine deaminase, adenosine deaminase, or cytidine adenosine deaminase domains. It should be appreciated that the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g. , dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the cytidine deaminases and/or adenosine deaminases provided herein. The domains of the base editors disclosed herein can be arranged in any order.

In some embodiments, the fusion proteins or complexes comprising a cytidine deaminase or adenosine deaminase and a napDNAbp (e.g., Cas9 or Casl2 domain) do not include a linker sequence. In some embodiments, a linker is present between the cytidine or adenosine deaminase and the napDNAbp. In some embodiments, cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.

It should be appreciated that the fusion proteins or complexes of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein or complex may comprise inhibitors, cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins or complexes. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to 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. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein or complex comprises one or more His tags.

Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/US2017/045381, PCT/US2019/044935, and PCT/US2020/016288, each of which is incorporated herein by reference for its entirety. Fusion Proteins or Complexes with Internal Insertions

Provided herein are fusion proteins or complexes comprising a heterologous polypeptide fused to a nucleic acid programmable nucleic acid binding protein, for example, a napDNAbp. The heterologous polypeptide can be fused to the napDNAbp at a C-terminal end of the napDNAbp, an N-terminal end of the napDNAbp, or inserted at an internal location of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine or adenosine deaminase) or a functional fragment thereof. For example, a fusion protein can comprise a deaminase flanked by an N- terminal fragment and a C-terminal fragment of a Cas9 or Casl2 (e.g., Casl2b/C2cl), polypeptide.

The deaminase can be a circular permutant deaminase. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 116, 136, or 65 as numbered in a TadA reference sequence.

The fusion protein or complexes can comprise more than one deaminase. The fusion protein or complex can comprise, for example, 1, 2, 3, 4, 5 or more deaminases. The deaminases in a fusion protein or complex can be adenosine deaminases, cytidine deaminases, or a combination thereof.

In some embodiments, the napDNAbp in the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. The Cas9 polypeptide can be a circularly permuted Cas9 protein.

The heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp (e.g., Cas9 or Casl2 (e.g., Casl2b/C2cl)) at a suitable location, for example, such that the napDNAbp retains its ability to bind the target polynucleotide and a guide nucleic acid. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase (dual deaminase)) can be inserted into a napDNAbp without compromising function of the deaminase (e.g, base editing activity) or the napDNAbp (e.g., ability to bind to target nucleic acid and guide nucleic acid).

In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted in regions of the Cas9 polypeptide comprising higher than average B-factors (e.g., higher B factors compared to the total protein or the protein domain comprising the disordered region). Cas9 polypeptide positions comprising a higher than average B-factor can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in the above Cas9 reference sequence. Cas9 polypeptide regions comprising a higher than average B-factor can include, for example, residues 792-872, 792-906, and 2-791 as numbered in the above Cas9 reference sequence.

In some embodiments, a heterologous polypeptide (e.g., deaminase) is inserted in a flexible loop of a Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of: 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.

A heterologous polypeptide (e.g, adenine deaminase) can be inserted into a Cas9 polypeptide region corresponding to amino acid residues: 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 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.

A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of a deleted region of a Cas9 polypeptide. The deleted region can correspond to an N-terminal or C-terminal portion of the Cas9 polypeptide. Exemplary internal fusions base editors are provided in Table 4A below:

Table 4A: Insertion loci in Cas9 proteins

A heterologous polypeptide (e.g., deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. The structural or functional domains of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Reel, Rec2, PI, or HNH.

A fusion protein can comprise a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or a non-peptide linker. For example, the linker can be an 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, SGSETPGTSESAT PES (SEQ ID NO: 249). In some embodiments, the fusion protein comprises a linker between the N- terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are joined to the deaminase domain without a linker. In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase but does not comprise a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase.

In some embodiments, the napDNAbp in the fusion protein or complex is a Casl2 polypeptide, e.g., Casl2b/C2cl, or a functional fragment thereof capable of associating with a nucleic acid (e.g., a gRNA) that guides the Casl2 to a specific nucleic acid sequence. The Casl2 polypeptide can be a variant Casl2 polypeptide. In other embodiments, the N- or C- terminal fragments of the Casl2 polypeptide comprise a nucleic acid programmable DNA binding domain or a RuvC domain. In other embodiments, the fusion protein contains a linker between the Casl2 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 250) or GS SGSETPGTSESAT PES SG (SEQ ID NO: 251). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

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 Casl2b polypeptide contains a mutation that silences the catalytic activity of a RuvC domain. In other embodiments, the Casl2b polypeptide contains D574A, D829A and/or D952A mutations.

In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., Casl2-derived domain) with an internally fused nucleobase editing domain (e.g., all or a portion (e.g., a functional portion) of a deaminase domain, e.g., an adenosine deaminase domain). In some embodiments, the napDNAbp is a Casl2b. In some embodiments, the base editor comprises a BhCasl2b domain with an internally fused TadA*8 domain inserted at the loci provided in Table 4B below.

Table 4B: Insertion loci in Casl2b proteins

In some embodiments, the base editing system described herein is an ABE with TadA inserted into a Cas9. Polypeptide sequences of relevant ABEs with TadA inserted into a Cas9 are provided in the attached Sequence Listing as SEQ ID NOs: 263-308.

Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/US2020/016285 and U.S. Provisional Application Nos. 62/852,228 and 62/852,224, the contents of which are incorporated by reference herein in their entireties.

A to G Editing

In some embodiments, a base editor described herein comprises an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. In some embodiments, an A-to- G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor.

A base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In an embodiment an adenosine deaminase domain of a base editor comprises all or a portion (e.g., a functional portion) of an AD AT comprising one or more mutations which permit the AD AT to deaminate a target A in DNA. For example, the base editor can comprise all or a portion (e.g., a functional portion) of an AD AT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A 106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase. Exemplary AD AT homolog polypeptide sequences are provided in the Sequence Listing as SEQ ID NOs: 1 and 309-315.

The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulohacter crescentus, or Bacillus suhtilis. In some embodiments, the adenine deaminase is a naturally- occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g, mutations in ecTadA). The corresponding residue in any homologous protein can be identified by e.g., sequence alignment and determination of homologous residues. The mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that correspond to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly.

In some embodiments, the adenosine deaminase comprises an amino acid sequence that is 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% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identify plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 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 of the adenosine deaminases provided herein.

It should be appreciated that any of the 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 E. coli TadA (ecTadA), .S', 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 would be apparent to the skilled artisan that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any of the mutations identified in a TadA reference sequence can be made in other adenosine deaminases (e.g., ecTada) that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein can be made individually or in any combination in a TadA reference sequence or another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an alteration or set of alterations selected from those listed in Tables 5A-5E below: Table 5A. Adenosine Deaminase Variants. Residue positions in the E. coli TadA variant (TadA*) are indicated.

Table 5B. TadA*8 Adenosine Deaminase Variants. Residue positions in the E. coli TadA variant (TadA*) are indicated. Alterations are referenced to TadA*7.10 (first row). Table 5C. TadA*9 Adenosine Deaminase Variants. Alterations are referenced to TadA*7.10. Additional details of TadA*9 adenosine deaminases are described in International PCT Application No. PCT/US2020/049975, which is incorporated herein by reference in its entirety for all purposes.

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

In some embodiments, the adenosine deaminase comprises one or more of Mil, MIS, 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, HUN, A15D, A15V, A15L, A15H, T17T, TUA, T17W, T17L, T17F, TUR, T17S, L18A, L18E, L18N, L18L, L18S, A I 9N. A19H, A19K, A19A, A19D, A19G, A19M, R21N, K20K, K20A, K20R, K20E, K20G, K20C, K20Q R21A, R21R, R21N, R21Y, R21C G22P, 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, E27K, 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, N38N, 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, 1491, 149M, I49N, I49K, I49Q, I49T, G50L, G50S, G50R, G50G, R51H, R5 IL, R5 IN, 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, 1761, 176N, 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, 195 S, I95G, I95L, I95H, 195 V, 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, G1OOS, G100M, G1OOI, R101V, R101R, R1O1S, R101C, V102A, V102F, V102I, V102V, D103A, V103A, V103G, V1O3F, VI O3V, F104G, D104N, Fl 04V, Fl 041, 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, KI 10G, KI 1OH, KI 101, KI 10R, KI 10T, KI 10K, KI 10A, KI 101, T111A, T111G, T111H, T111R, T11 IT, T11 IK, G112A, G112G, G112H, G112T, G112R, Al 13N, Al 14G, Al 14H, Al 14V, Al 14C, Al 14S, Al 14A, G115S, G115G, G115M, G115L, G115A, G115F, LI 17M, LI 17L, LI 17W, LI 17A, LI 17S, LI 17N, LI 17V, Ml 18D, Ml 18G, 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, I1361 , 1137A, 1137D, 1137E, L137M, 1137S, 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, Q 1541, Q154A, Q154K, E155F, E155G, E155I, E155K, E155P, E155V, E155D, E155E, E155L, E155Q, I156V, I156A, I156I, I156L, I156F, I156D, I156K, I156N, I156R, I156Y, E157A, E157F, E157I, E157P, E157T, E157V, N157K, K157N, K157V, K157P, K157I, K157F, K157F, K157T, K157A, K157S, K157R, A158Q, A158K, A158V, A158A, A158D, A158S, A158T, A158N, Q159S, Q159Q, Q159A, Q159F, Q159K, Q159L, Q159N, K160A, K160S, K160E, 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, S165P, S165Q, S165A, S165D, S 1651, S165T, S165Y, T166T, T166Q, T166E, T166S, T166D, T166K, T166I, T166N, T166P, T166R, D167S D167D, DI 671, D167G, D167T, D167A and/or DI 67N mutation in a TadA reference sequence (e.g., TadA*7.10,ecTadA, or TadA8e), and any alternative mutation at the corresponding positioner one or more corresponding mutations in another adenosine deaminase. Additional mutations are described in U.S. Patent Application Publication No. 2022/0307003 Al U.S. Patent No. 11,155,803, and International Patent Application Publications No. WO 2023/288304 A2, PCT/CN2022/143408, WO 2018/027078 Al, WO 2021/158921 Al and WO 2023/034959 A2, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the disclosure provides TadA variants comprising a V82T, Y147T, and/or a Q154S mutation. In some embodiments, the disclosure provides TadA variants comprising a V82T, Y147T, and/or a Q154S mutation. In some embodiments, the disclosure provides TadA* 8.8 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA*8.8 further comprising a V82T, a Y147T, and a Q154S mutation. In some embodiments, the disclosure provides TadA* 8.17 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA* 8. 17 further comprising a V82T, a Y147T, and a Q154S mutation. In some embodiments, the disclosure provides TadA* 8.20 further comprising a V82T mutation. In some embodiments, the disclosure provides TadA*8.20 further comprising a V82T, a Y147T, and a Q154S mutation.

In embodiments, a variant of TadA*7.10 comprises one or more alterations selected from any of those alterations provided herein.

In particular embodiments, an adenosine deaminase heterodimer comprises a TadA* 8 domain and an adenosine deaminase domain selected from Staphylococcus aureus (S. aureus) TadA, Bacillus suhtilis (B. subtilis) TadA, Salmonella typhimurium (S. typhimurium) TadA, Shewanella putrefaciens (S. putrefaciens) TadA, Haemophilus influenzae F3031 (H. influenzae) TadA, Caulobacter crescentus (C. crescentus) TadA, Geobacter sulfurreducens (G. sulfurreducens TadA, or TadA*7.10.

In some embodiments, the TadA* 8 is a variant as shown in Table 5D. Table 5D shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in those positions in the TadA-7.10 adenosine deaminase. Table 5D also shows amino acid changes in TadA variants relative to TadA-7. 10 following phage-assisted non- continuous 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 by reference herein. 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, an adenosine deaminase is a TadA* 8 that comprises or consists essentially of SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity.

Table 5D. Select TadA*8 Variants

In some embodiments, the TadA variant is a variant as shown in Table 5E. Table 5E shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in those positions in the 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.

Table 5E. TadA Variants

In particular embodiments, the fusion proteins or complexes comprise a single (e.g., provided as a monomer) TadA* (e.g., TadA*8 or TadA*9). Throughout the present disclosure, an adenosine deaminase base editor that comprises a single TadA* domain is indicates using the terminology ABEm or ABE#m, where is an identifying number (e.g., ABE8.20m), where “m” indicates “monomer.” In some embodiments, the TadA* is linked to a Cas9 nickase. In some embodiments, the fusion proteins or complexes of the disclosure comprise as a heterodimer of a wild-type TadA (TadA(wt)) linked to a TadA* . Throughout the present disclosure, an adenosine deaminase base editor that comprises a single TadA* domain and a TadA(wt) domain is indicates using the terminology ABEd or ABE#d, where is an identifying number (e.g., ABE8.20d), where “d” indicates “dimer.” In other embodiments, the fusion proteins or complexes of the disclosure comprise as a heterodimer of a TadA*7.10 linked to a TadA*. In some embodiments, the base editor is ABE8 comprising a TadA* variant monomer. In some embodiments, the base editor is ABE comprising a heterodimer of a TadA* and a TadA(wt). In some embodiments, the base editor is ABE comprising a heterodimer of a TadA* and TadA*7.10. In some embodiments, the base editor is ABE comprising a heterodimer of a TadA* . In some embodiments, the TadA* is selected from Tables 5A-5E.

In some embodiments, the adenosine deaminase is expressed as a monomer. In other embodiments, the adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks a methionine, for example when included as a component of a fusion protein. This can alter the numbering of positions. However, the skilled person will understand that such corresponding mutations refer to the same mutation.

Any of the mutations provided herein and any additional mutations (e.g., based on the ecTadA amino acid sequence) can be introduced into any other adenosine deaminases. Any of the mutations provided herein can be made individually or in any combination in a TadA reference sequence or another adenosine deaminase (e.g., ecTadA). Details of A to G nucleobase editing proteins are described in International PCT Application No. PCT/US2017/045381 (WO2018/027078) and Gaudelli, N.M., etal., “Programmable base editing of A«T to G*C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.

C to T Editing

In some embodiments, a base editor disclosed herein comprises a fusion protein or complex comprising cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C:G to a T:A transition. In other embodiments, deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T.

The deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be substituted (e.g, by base repair machinery) with another base, such as a C, by for example a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions (e.g., A, G or T) can also occur.

Accordingly, in some embodiments a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C-to-T base editing event. In another example, the base editor can comprise a uracil stabilizing protein as described herein. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (z.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event).

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

In some embodiments, a cytidine deaminase of a base editor comprises all or a portion (e.g., a functional portion) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is a family of evolutionarily conserved 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 a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC 1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase.

Other exemplary deaminases that can be fused to Cas9 according to aspects of this disclosure are provided below. In embodiments, the deaminases are activation-induced deaminases (AID). It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

Some aspects of the present disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins or complexes described herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors) or complexes. For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein or complexes can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to specific target residues, which can reduce or prevent off- target effects.

In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBECl; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; and any alternative mutation at the corresponding position, or one or more corresponding mutations in another APOBEC deaminase.

A number 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, 85177). In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC 1 deaminase.

In some embodiments, the fusion proteins or complexes of the disclosure comprise 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 deaminase may be derived from any suitable organism. In some embodiments, the cytidine deaminase is a naturally-occurring cytidine deaminase that includes one or more mutations corresponding to any of the mutations provided herein. One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring cytidine deaminase that corresponds to any of the mutations described herein. In some embodiments, the cytidine deaminase is from a prokaryote. In some embodiments, the cytidine deaminase is from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., human).

In some embodiments, the cytidine deaminase comprises an amino acid sequence that is 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% identical to any one of the cytidine deaminase amino acid sequences set forth herein. It should be appreciated that cytidine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). Some embodiments provide a polynucleotide molecule encoding the cytidine deaminase nucleobase editor polypeptide of any previous aspect or as delineated herein. In some embodiments, the polynucleotide is codon optimized.

In embodiments, a fusion protein of the disclosure comprises two or more nucleic acid editing domains. Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT/US2016/058344 (WO2017/070632) and Komor, A.C., etal., “Programmable editing of a target base in genomic DNA without double -stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference.

Cytidine Adenosine Base Editors (CABEs)

In some embodiments, a base editor described herein comprises an adenosine deaminase variant that has increased cytidine deaminase activity. Such base editors may be referred to as “cytidine adenosine base editors (CABEs)” or “cytosine base editors derived from TadA* (CBE-Ts),” and their corresponding deaminase domains may be referred to as “TadA* acting on DNA cytosine (TADC)” domains. In some instances, an adenosine deaminase variant has both adenine and cytosine deaminase activity (i.e., is a dual deaminase). In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant predominantly deaminates cytosine in DNA and/or RNA (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deaminations catalyzed by the adenosine deaminase variant, or the number of cytosine deaminations catalyzed by the variant is about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 500-fold, or 1,000-fold greater than the number adenine deaminations catalyzed by the variant). In some embodiments, the adenosine deaminase variant has approximately equal cytosine and adenosine deaminase activity (e.g., the two activities are within about 10% or 20% of each other). 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 no detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in a cell in vitro or in vivo. In some embodiments, the cell is a bacteria, yeast, fungi, insect, plant, or mammalian cell.

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.

In some embodiments, an adenosine deaminase variant of the 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 increase) 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 instances, the adenosine deaminase variant comprises 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 increase) relative to the activity of a reference adenosine deaminase and comprise undetectable adenosine deaminase activity or adenosine deaminase activity that is less than 30%, 20%, 10%, or 5% of that of a reference adenosine deaminase. In some embodiments, the reference adenosine deaminase is TadA* 8.20 or TadA*8.19.

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising two or more alterations at an amino acid position selected from the group consisting of 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, of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or greater identity to SEQ ID NO: 1, or a corresponding alteration in another deaminase. I

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising one or more alterations selected from the group consisting of 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, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, Al 14C, G115M, Ml 18L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or greater identity to SEQ ID NO: 1, or a corresponding alteration in another deaminase.

In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising an amino acid alteration or combination of amino acid alterations selected from those listed in any of Tables 6A-6F.

The residue identity of exemplary adenosine deaminase variants that are capable of deaminating adenine and/or cytidine in a target polynucleotide (e.g., DNA) is provided in Tables 6A-6F below. Further examples of adenosine deaminase variants include the following variants of 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 of the amino acid alterations provided herein are substituted with a conservative amino acid. Additional mutations known in the art can be further added to any of the adenosine deaminase variants provided herein.

In some embodiments, the base editor systems comprising a CABE provided herein have at least about a 30%, 40%, 50%, 60%, 70% or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, a base editor system comprising a CABE as provided herein has an increased C to T base editing activity (e.g., increased 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).

Table 6A. Adenosine Deaminase Variants. Mutations are indicated with reference to TadA*8.20. “S” indicates “Surface,” and “NAS” indicates “Near Active Site.” 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.”

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

Table 6C. (CONTINUED)

Table 6D. Adenosine deaminase variants. Mutations are indicated with reference to TadA*8.20. | | i

Table 6E. Hybrid constructs. Mutations are indicated with reference to TadA*7.10.

Table 6F. Base editor variants. Mutations are indicated with reference to TadA*8.19/8.20.

Guide Polynucleotides

A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g. , gRNA), can specifically bind to a target polynucleotide sequence (z. e. , via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single -stranded DNA 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.

In an embodiment, a guide polynucleotide described herein can be RNA or DNA. In one embodiment, the guide polynucleotide is a gRNA.

In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g, a dual guide polynucleotide, dual gRNA). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) or can comprise one or more trans-activating CRISPR RNA (tracrRNA).

A guide polynucleotide may include natural or non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the targeting region of a guide nucleic acid sequence (e.g., a spacer) can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. Exemplary gRNA scaffold sequences are provided in the sequence listing as SEQ ID NOs: 317-327 and 425. Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. In embodiments, the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. The spacer of a gRNA can be or can be about 19, 20, or 21 nucleotides in length.

A gRNA or a guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a composition comprises multiple gRNAs that all target the same exon or multiple gRNAs that target different exons. An exon and/or an intron of a gene can be targeted. A gRNA or a guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides or less than about 20 nucleotides (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides), or anywhere between about 1-100 nucleotides (e.g, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100). A target nucleic acid sequence can be or can be about 20 bases immediately 5' of the first nucleotide of the PAM. A gRNA can target a nucleic acid sequence. A 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.

The guide polynucleotides can comprise standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and/or ribonucleotide analogs.

In some embodiments, a base editor system may comprise multiple guide polynucleotides, e.g., gRNAs. For example, the gRNAs may target to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a base editor system. The multiple gRNA sequences can be tandemly arranged and may be separated by a direct repeat.

Modified Polynucleotides

To enhance expression, stability, and/or genomic/base editing efficiency, and/or reduce possible toxicity, the base editor-coding sequence (e.g., mRNA) and/or the guide polynucleotide (e.g., gRNA) can be modified to include one or more modified nucleotides and/or chemical modifications, e.g. pseudo-uridine, 5-Methyl-cytosine, 2'-O-methyl-3'- phosphonoacetate, 2'-O-methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), =constrained ethyl (S-cEt), 2'-O-methyl (‘M’), 2'-O-methyl-3'-phosphorothioate (‘MS’), 2'-O-methyl-3'-thiophosphonoacetate (‘MSP’), 5 -methoxyuridine, phosphorothioate, and N1 -Methylpseudouridine. Chemically protected gRNAs can enhance stability and editing efficiency in vivo and ex vivo. Methods for using chemically modified mRNAs and guide RNAs are known in the art and described, for example, by Jiang et al., Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). doi.org/10.1038/s41467-020-15892-8, Callum et al., A1- Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e35, and Andries et al., Journal of Controlled Release, Volume 217, 10 November 2015, Pages 337-344, each of which is incorporated herein by reference in its entirety.

In some embodiments, the guide polynucleotide comprises one or more modified nucleotides at the 5' end and/or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and/or the 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, four or more modified nucleosides at the 5' end and/or the 3' end of the guide.

In some embodiments, the guide comprises at least about 50%-75% modified nucleotides. In some embodiments, the guide comprises 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 contiguous nucleotides at each of the 5' and 3' termini of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present in a direct repeat or anti -direct repeat are modified. In some embodiments, at least about 50% of the nucleotides present in a direct repeat or anti -direct repeat are modified. In some embodiments, at least about 50-75% of the nucleotides present in a direct repeat or antidirect repeat are modified. In some embodiments, at least about 100 of the nucleotides present in a direct repeat or anti -direct repeat are modified. In some embodiments, at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified. In some embodiments, the guide comprises a variable length spacer. In some embodiments, the guide comprises a 20-40 nucleotide spacer. In some embodiments, the guide comprises a spacer comprising at least about 20-25 nucleotides or at least about 30-35 nucleotides. In some embodiments, the spacer 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 a direct repeat or anti-direct repeat are modified;

• at least about 50-75% of the nucleotides present in a direct repeat or anti-direct repeat are modified; • at least about 20% or more of the nucleotides present in a hairpin present in the gRNA scaffold are modified;

• a variable length spacer; and

• a spacer comprising modified nucleotides.

In embodiments, the gRNA contains numerous modified nucleotides and/or chemical modifications. Such modifications can increase base editing ~2 fold in vivo or in vitro. In embodiments, the gRNA comprises 2'-O-methyl or phosphorothioate modifications. In an embodiment, the gRNA comprises 2'-O-methyl and phosphorothioate modifications. In an embodiment, the modifications increase base editing by at least about 2 fold.

A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.

A gRNA or a guide polynucleotide can also be modified by 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-Methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3 '-3' modifications, 2'-O-methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), and constrained ethyl (S-cEt), 5 '-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, 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 linkers, 2'-deoxyribonucleoside analog purine, 2'- deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 '-triphosphate, 5'- methylcytidine-5 '-triphosphate, or any combination thereof.

In some cases, a phosphorothioate enhanced RNA gRNA can inhibit RNase A, RNase Tl, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA gRNAs to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5'- or 3'-end of a gRNA which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases.

Fusion Proteins or Complexes Comprising a Nuclear Localization Sequence (NLS)

In some embodiments, the fusion proteins or complexes provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, the NLS is fused to the N-terminus or the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the Casl2 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the cytidine 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 an amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences.

In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, for example described herein. A bipartite NLS comprises two basic amino acid clusters, which are separated by a relatively short spacer sequence (hence bipartite - 2 parts, while monopartite NLSs are not). The NLS of nucleoplasmin, KR [ PAATKKAGQA] KKKK (SEQ ID NO: 191), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. The sequence of an exemplary bipartite NLS follows: PKKKRKVEGADKRTADGSE FE SPKKKRKV (SEQ ID NO: 328).

In some embodiments, any of the fusion proteins or complexes provided herein comprise an NLS comprising the amino acid sequence EGADKRTADGSE FES PKKKRKV (amino acids 8 to 29 of SEQ ID NO 328). In some embodiments, any of the adenosine base editors provided herein comprise the amino acid sequence EGADKRTADGSE FES PKKKRKV (amino acids 8 to 29 of SEQ ID NO: 328). In some embodiments, the NLS is at a C-terminal portion of the adenosine base editor. In some embodiemtns, the NLS is at the C-terminus of the adenosine base editor.

Additional Domains

A base editor described herein can include any domain which helps to facilitate the nucleobase editing, modification or altering of a nucleobase of a polynucleotide. In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain can facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that could interfere with the desired base editing result. In some embodiments, a base editor comprises a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.

In some embodiments, a base editor comprises an uracil glycosylase inhibitor (UGI) domain. In some cases, a base editor is expressed in a cell in trans with a UGI polypeptide. In some embodiments, cellular DNA repair response to the presence of U: G heteroduplex DNA can be responsible for a reduction in nucleobase editing efficiency in cells. In such embodiments, uracil DNA glycosylase (UDG) can catalyze removal of U from DNA in cells, which can initiate base excision repair (BER), mostly resulting in reversion of the U:G pair to a C:G pair. In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and /or promote repairing of the non-edited strand. Thus, this disclosure contemplates a base editor fusion protein or complex comprising a UGI domain and/or a uracil stabilizing protein (USP) domain.

BASE EDITOR SYSTEM

Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g., guide RNA) in conjunction with 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, a deaminase domain can be a cytidine deaminase or an cytosine deaminase. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor is capable of deaminating a cytidine in DNA.

Use of the base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g., double- or single stranded DNA or RNA) of a subject 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 comprises a targeted nucleobase pair; (b) inducing strand separation of said target region; (c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. It should be appreciated that in some embodiments, step (b) is omitted. In some embodiments, said targeted nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more genes, wherein at least one gene is located in a different locus.

The components of a base editor system (e.g., a deaminase domain, a guide RNA, and/or a polynucleotide programmable nucleotide binding domain) may be associated with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain, optionally where the polynucleotide programmable nucleotide binding domain is complexed with a polynucleotide (e.g., a guide RNA). In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component (e.g., the deaminase component) comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a polynucleotide programmable nucleotide binding domain and/or a guide polynucleotide (e.g., a guide RNA) complexed therewith. In some embodiments, the polynucleotide programmable nucleotide binding domain, and/or a guide polynucleotide (e.g., a guide RNA) complexed therewith, comprises an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with a corresponding heterologous portion, antigen, or domain that is part of a nucleobase editing domain (e.g., the deaminase component). In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion is capable of binding to a polynucleotide linker. An additional heterologous portion may be a protein domain. In some embodiments, an additional heterologous portion comprises a polypeptide, such as a 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (N22p), a 2G12 IgG homodimer domain, an ABI, an antibody (e.g. an antibody that binds a component of the base editor system or a heterologous portion thereof) or fragment thereof (e.g. heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, an Fab2, miniantibodies, and/or ZIP antibodies), a bamase-barstar dimer domain, a Bcl-xL domain, a Calcineurin A (CAN) domain, a Cardiac phospholamban transmembrane pentamer domain, a collagen domain, a Com RNA binding protein domain (e.g. SfMu Com coat protein domain, and SfMu Com binding protein domain), a Cyclophilin-Fas fusion protein (CyP-Fas) domain, a Fab domain, an Fe domain, a fibritin foldon domain, an FK506 binding protein (FKBP) domain, an FKBP binding domain (FRB) domain of mTOR, a foldon domain, a fragment X domain, a GAI domain, a GID 1 domain, a Glycophorin A transmembrane domain, a GyrB domain, a Halo tag, an HIV Gp41 trimerisation domain, an HPV45 oncoprotein E7 C-terminal dimer domain, a hydrophobic polypeptide, a K Homology (KH) domain, a Ku protein domain (e.g., a Ku heterodimer), a leucine zipper, a LOV domain, a mitochondrial antiviral-signaling protein CARD fdament domain, an MS2 coat protein domain (MCP), a non-natural RNA aptamer ligand that binds a corresponding RNA motif/aptamer, a parathyroid hormone dimerization domain, a PP7 coat protein (PCP) domain, a PSD95-Dlgl-zo-l (PDZ) domain, a PYL domain, a SNAP tag, a SpyCatcher moiety, a SpyTag moiety, a streptavidin domain, a streptavidin-binding protein domain, a streptavidin binding protein (SBP) domain, a telomerase Sm7 protein domain (e.g. Sm7 homoheptamer or a monomeric Sm-like protein), and/or fragments thereof. In embodiments, an additional heterologous portion comprises a polynucleotide (e.g., an RNA motif), such as an MS2 phage operator stem-loop (e.g., an MS2, an MS2 C-5 mutant, or an MS2 F-5 mutant), a non-natural RNA motif, a PP7 operator stem-loop, an SfMu phate Com stem-loop, a steril alpha motif, a telomerase Ku binding motif, a telomerase Sm7 binding motif,, and/or fragments thereof . Non-limiting examples of additional heterologous portions include polypeptides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 380, 382, 384, 386-388, or fragments thereof. Non-limiting examples of additional heterologous portions include polynucleotides with at least about 85% sequence identity to any one or more of SEQ ID NOs: 379, 381, 383, 385, or fragments thereof.

In some instances, components of the base editing system are associated with one another through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In some cases, components of the base editing system are associated with one another through polypeptide domains (e.g., FokI domains) that associate to form protein complexes containing about, at least about, or no more than about 1, 2 (i.e., dimerize), 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units, optionally the polypeptide domains may include alterations that reduce or eliminate an activity thereof.

In some instances, components of the base editing system are associated with one another through the interaction of multimeric antibodies or fragments thereof (e.g., IgG, IgD, IgA, IgM, IgE, a heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), an immunoglobulin Fc region, a heavy chain domain 3 (CH3) of IgG or IgA, a heavy chain domain 4 (CH4) of IgM or IgE, an Fab, and an Fab2). In some instances, the antibodies are dimeric, trimeric, or tetrameric. In embodiments, the dimeric antibodies bind a polypeptide or polynucleotide component of the base editing system.

In some cases, components of the base editing system are associated with one another through the interaction of a polynucleotide-binding protein domain(s) with a polynucleotide (s). In some instances, components of the base editing system are associated with one another through the interaction of one or more polynucleotide-binding protein domains with polynucleotides that are self-complementary and/or complementary to one another so that complementary binding of the polynucleotides to one another brings into association their respective bound polynucleotide-binding protein domain(s).

In some instances, components of the base editing system are associated with one another through the interaction of a polypeptide domain(s) with a small molecule(s) (e.g., chemical inducers of dimerization (CIDs), also known as “dimerizers”). Non-limiting examples of CIDs include those disclosed in Amara, et al., “A versatile synthetic dimerizer for the regulation of protein-protein interactions,” PNAS, 94: 10618-10623 (1997); and VoB, et al. “Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells,” Current Opinion in Chemical Biology, 28: 194-201 (2015), the disclosures of each of which are incorporated herein by reference in their entireties 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 the non-edited strand. In some embodiments, the base editor comprises UGI activity or USP activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease.

The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a polynucleotide programmable nucleotide binding domain. In some embodiments, an NLS of the base editor is localized C-terminal to a polynucleotide programmable nucleotide binding domain.

Protein domains included in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains which can be included in the fusion protein include a deaminase domain (e.g., cytidine deaminase and/or adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences. In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC 1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE comprises an evolved TadA variant. In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are provided in the attached sequence listing (SEQ ID NOs: 332-354). In some embodiments, the base editor includes an adenosine deaminase variant comprising an amino acid sequence, which contains alterations relative to an ABE 7* 10 reference sequence, as described herein. The term “monomer” as used in Table 7 refers to a monomeric form of TadA*7.10 comprising the alterations described. The term “heterodimer” as used in Table 7 refers to the specified wild-type E. coli TadA adenosine deaminase fused to a TadA*7. 10 comprising the alterations as described.

Table 7. Adenosine Deaminase Base Editor Variants In some embodiments, the base editor comprises a domain comprising all or a portion

(e.g., a functional portion) of a uracil glycosylase inhibitor (UGI) or a uracil stabilizing protein (USP) domain. Linkers

In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the disclosure. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).

In some embodiments, any of the fusion proteins provided herein, comprise a cytidine or adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be employed (e.g. , ranging from very flexible linkers of the form ( GGGS ) _n (SEQ ID NO: 246), (GGGGS)_n (SEQ ID NO: 247), and (G)n to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 248), (SGGS)n (SEQ ID NO: 355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, e.g., 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 are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the cytidine or adenosine deaminase nucleobase editor. 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 a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, cytidine deaminase or adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which can also be referred to as the XTEN linker.

In some embodiments, the domains of the base editor are fused via a linker that comprises the amino acid sequence of: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358).

In some embodiments, domains of the base editor 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. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSG GS (SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence: PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362).

In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5- 9, 5-7 amino acids in length, e.g., 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, e.g., 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 are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers.

Nucleic Acid Programmable DNA Binding Proteins with Guide RNAs

Provided herein are compositions and methods for base editing in cells. Further provided herein are 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 as provided herein. In some embodiments, a composition for base editing as provided herein further comprises a polynucleotide that encodes a base editor, e.g., a C-base editor or an A-base editor. For example, a composition for base editing may comprise a mRNA sequence encoding a BE, a BE4, an ABE, and a combination of one or more guide RNAs as provided. A composition for base editing may comprise a base editor polypeptide and a combination of one or more of any guide RNAs provided herein. Such a composition may be used to effect base editing in a cell through different delivery approaches, for example, electroporation, nucleofection, viral transduction or transfection. In some embodiments, the composition for base editing comprises an mRNA sequence that encodes a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.

Some aspects of this disclosure provide systems comprising any of the fusion proteins or complexes provided herein, and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain (e.g., a Cas9 (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) or Casl2) of the fusion protein or complex. These complexes are also termed ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is 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 a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 3 or 5'-NAA-3'). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder).

Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.

The domains of the base editor disclosed herein can be arranged in any order.

A defined target region can be a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 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.

The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. Methods of Using Fusion Proteins or Complexes Comprising a Cytidine or Adenosine Deaminase and a Cas9 Domain

Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein, and with at least one guide RNA described herein.

In some embodiments, a fusion protein or complex of the disclosure is used for editing a target gene of interest. In particular, a cytidine deaminase or adenosine deaminase nucleobase editor described herein is capable of making multiple mutations within a target sequence. These mutations may affect the function of the target. For example, when a cytidine deaminase or adenosine deaminase nucleobase editor is used to target a regulatory region the function of the regulatory region is altered and the expression of the downstream protein is reduced or eliminated.

Base Editor Efficiency

In some embodiments, the purpose of the methods provided herein is to alter a gene and/or gene product via gene editing. The nucleobase editing proteins provided herein can be used for gene editing -based human therapeutics in vitro or in vivo. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g. , the 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 a nucleotide from A to G or C to T.

Advantageously, base editing systems as provided herein provide genome editing without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions as CRISPR may do. In some embodiments, the present disclosure provides base editors that efficiently generate an intended mutation, such as a STOP codon, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations.

The base editors of the disclosure advantageously modify a specific nucleotide base encoding a protein without generating a significant proportion of indels (i.e., insertions or deletions). Such indels can lead to frame shift mutations within a coding region of a gene.

In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels i.e., intended point mutations: unintended point mutations) that is greater than 1 : 1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is 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 intended mutations and indels may be determined using any suitable method.

In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels at a region of a nucleic acid 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%.

Base editing is often referred to as a “modification”, such as, a genetic modification, a gene modification and modification of the nucleic acid sequence and is clearly understandable based on the context that the modification is a base editing modification. A base editing modification is therefore a modification at the nucleotide base level, for example as a result of the deaminase activity discussed throughout the disclosure, which then results in a change in the gene sequence and may affect the gene product.

In some embodiments, the modification, e.g., single base edit results in about or at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% reduction, or reduction to an undetectable level, of the gene targeted expression.

The disclosure provides adenosine deaminase variants (e.g., ABE8 variants) that have increased efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide and are less likely to edit bases that are not intended to be altered (e.g., “bystanders”).

In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations 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% compared to a base editor system comprising an ABE7 base editor, e.g, ABE7.10.

In some embodiments, any of the ABE8 base editor variants described herein has higher base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have 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%, 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 compared to an ABE7 base editor, e.g, ABE7.10.

The ABE8 base editor variants described herein may be delivered to a host cell via a plasmid, a vector, a LNP complex, or an mRNA. In some embodiments, any of the ABE8 base editor variants described herein is delivered to a host cell as an mRNA.

In some embodiments, the method described herein, for example, the base editing methods has minimum to no off-target effects. In some embodiments, the method described herein, for example, the base editing methods, has minimal to no chromosomal translocations.

In some embodiments, the base editing method described herein results in about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of a cell population that have been successfully edited.

In some embodiments, the percent of viable cells in a cell population following a base editing intervention is greater than at least 60%, 70%, 80%, or 90% of the starting cell population at the time of the base editing event. In some embodiments, the percent of viable cells in a cell population following editing is about 70%. In some embodiments, the percent of viable cells in a cell population following editing is about 75%. In some embodiments, the percent of viable cells in a cell population following editing is about 80%. In some embodiments, the percent of viable cells in a cell population as described above is about 85%. In some embodiments, the percent of viable cells in a cell population as described above is about 90%, or about 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.

In embodiments, the cell population is a population of cells contacted with a base editor, complex, or base editor system of the present disclosure. The number of intended mutations and indels can be determined using any suitable method, for example, as described in International PCT Application

Nos. PCT/US2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632); Komor, A.C., et al. , “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N.M., etal., “Programmable base editing of A«T to G*C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A.C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.

In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels can occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor.

Multiplex Editing

In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes or polynucleotide sequences. In some embodiments, the plurality of nucleobase pairs is located in the same gene or in one or more genes, wherein at least one gene is located in a different locus. In some embodiments, the multiplex editing comprises one or more guide polynucleotides. In some embodiments, the multiplex editing comprises one or more base editor systems. In some embodiments, the multiplex editing comprises one or more base editor systems with a single guide polynucleotide or a plurality of guide polynucleotides. In some embodiments, the multiplex editing comprises one or more guide polynucleotides with a single base editor system. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any combination of methods using any base editor provided herein. It should also be appreciated that the multiplex editing using any of the base editors as described herein can comprise a sequential editing of a plurality of nucleobase pairs.

In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of ABE7, ABE8, and/or ABE9 base editors.

DELIVERY SYSTEMS

Nucleic Acid-Based Delivery of Base Editor Systems

Nucleic acid molecules encoding a base editor system according to the present disclosure can be administered to subjects or delivered into cells in vitro or in vivo by art- known methods or as described herein. For example, a base editor system comprising a deaminase (e.g., cytidine or adenine deaminase) can be delivered by vectors (e.g., viral or non-viral vectors), or by naked DNA, DNA complexes, lipid nanoparticles, or a combination of the aforementioned compositions. A base editor system may be delivered to a cell using any methods available in the art including, but not limited to, physical methods (e.g., electroporation, particle gun, calcium phosphate transfection), viral methods, non-viral methods (e.g., liposomes, cationic methods, lipid nanoparticles, polymeric nanoparticles), or biological non-viral methods (e.g., attenuated bacterial, engineered bacteriophages, mammalian virus-like particles, biological liposomes, erythrocyte ghosts, exosomes).

Nanoparticles, which can be organic or inorganic, are useful for delivering a base editor system or component thereof. Nanoparticles are well known in the art and any suitable nanoparticle can be used to deliver a base editor system or component thereof, or a nucleic acid molecule encoding such components. In one example, organic (e.g., lipid and/or polymer) nanoparticles are suitable for use as delivery vehicles in certain embodiments of this disclosure. Non-limiting examples of lipid nanoparticles suitable for use in the methods of the present disclosure include those described in International Patent Application Publications No. WO2022140239, WO2022140252, WO2022140238, WO2022159421, WO2022159472, WO2022159475, WO2022159463, WO2021113365, and WO2021141969, and in International patent Application No. PCT/US23/27741, the disclosures of each of which is incorporated herein by reference in its entirety for all purposes.

Lipid Nanoparticle (LNP) Compositions

The pharmaceutical compositions for gene modification described herein may be encapsulated in a lipid nanoparticle (LNP). LNP compositions or formulations, as contemplated herein, are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition or formulation as contemplated herein may be a liposome having a lipid bilayer with a diameter of 500 nm or less. A LNP as described herein may have a mean diameter of from about 20 nm to about 1000 nm, from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 50 nm to 90 nm, from about 55 nm to 85 nm, from about 55 nm to 75 nm, from about 50 nm to about 80 nm, from about 60 nm to about 80 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, or from about 70 nm to about 80 nm. The LNPs described herein can have a mean diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. In one embodiment the mean 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.

Described herein are LNP compositions comprising an ionizable lipid, a phospholipid, a conjugate-linker lipid (e.g., PEG lipid), a sterol (e.g., cholesterol), or a derivative thereof, a payload, or any combination thereof. Each component is described in more detail below.

In the preparation of LNP compositions comprising the ionizable lipid, phospholipid, conjugate -linker lipid, and sterol, a desired molar ratio of the four excipients may be dissolved in a water miscible organic solvent, such as ethanol. The homogenous lipid solution may then be rapidly in-line mixed with an aqueous buffer containing nucleic acid payload to form the lipid nanoparticle encapsulating the nucleic acid payload(s). After rapid in-line mixing, the LNPs thus formed may undergo further downstream processing including concentration and buffer exchange to achieve the final LNP pharmaceutical composition with near neutral pH for administration into cell line or animal diseases model for evaluation, or to administer to human subjects.

The LNP payload comprises a guide RNA targeting the TTR gene and an mRNA encoding a base editor protein. In some embodiments, the guide RNA to mRNA ratio in the acidic aqueous buffer and in the final formulation is 6: 1, 5: 1, 4: 1, 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1: 1, 1: 1.5, 1:2, 1:2.5, 1:3, 1:5 or 1:6 by weight. In some embodiments, the guide RNA to mRNA ratio in the acidic aqueous buffer and in the final formulation is about 1 : 1 by weight. In some embodiment the mRNA encodes an adenosine base editor protein. In some other embodiments the mRNA encodes cytosine or a cytidine base editor protein.

Exemplary LNP compositions are described in Examples 1, 4, and 5 below.

Ionizable lipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more ionizable lipids as described herein. An ionizable lipid as used herein comprises one or more ionizable nitrogen atoms or amine containing groups. In some embodiments, the ionizable nitrogen atom or amine containing group is on the head group of the ionizable lipid.

For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomatenals having an ionizable lipid that is at about 50 mole percent or less, based on total moles of components of the lipid nanoparticle, may be useful and/or critical to functional activity of lipid nanoparticles such as desired tropisms, stabilization, and drug delivery efficacy as described herein.

In some embodiments, an ionizable lipid is or comprises a compound described herein. In some embodiments, an ionizable lipid is present in a lipid nanoparticle (LNP) preparation from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mole percent to about 60 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 34 mole percent to about 55 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present from about 33 mole percent to about 51 mole percent, based on total moles of components of the lipid nanopartide. In some embodiments, an ionizable lipid is present at about 34.7 mole percent, based on total moles of components of the lipid nanoparticle. In some embodiments, an ionizable lipid is present at about 50 mole percent, based on total moles of components of the lipid nanopartide. In some embodiments, an ionizable lipid is present at about 40 mole percent to about 60 mole percent or about 45 mole percent to about 55 mole percent, based on total moles of components of the lipid nanopartide.

Exemplary, non-limiting ionizable lipids suitable for the compositions described herein include those described herein. Exemplary Lipids of WO2022140252

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022140252, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae A’, A, I”, I’, I, II”, IF, II, III’, III, I”-a, r-a, I-a, I”-a-z, I”-a-zz, I”-a-zzz, I”-b, F-b, I-b, F’-b-z, I”-b-zz, I”-b-zzz, I”-c, I’-c, I-c, I”-c-z, F’-c-zz, I”-c-zzz, F-d, I-d, F-d-z, Il-a, Il-a-z, Ill-a, and III-a-z of WO2022140252, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids of Table 1 ofWO2022140252, including any of the lipids represented by Examples 7-1 to 7-253 and Examples 8-1 to 8-106, or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, an ionizable lipid is according to Formula A’ of WO2022140252:

A’ or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

L is Ci-10 alkylenyl, or C2-10 heteroalkylenyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-;

R” is hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; each of R and R^a is independently hydrogen, or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl each of L³ and L^3a is independently absent, optionally substituted Ci-io alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10- membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, - NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², - N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)₂, -OP(O)(OR²)₂, or -P(O)(OR²)₂; or ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n- R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7- membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, - C(O)N(R⁵)₂,

-NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, or optionally substituted Ci-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4.

In some embodiments, an ionizable lipid is according to Formula Ill-a of

WO2022140252: or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein each of R, R¹, L, L¹, L², L³ is as defined therein for any of Formulae A’, A, III’, and III, and described in classes and subclasses above and herein, both singly and in combination. In embodiments of Formula III -a, each of R, R¹, L, L¹, L², L³ is as defined herein for Formula A’ above. In some embodiments, an ionizable lipid is according to Formula Ill-a-z of WO2022140252: or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein each of R, R¹, L, L¹, and L² is as defined therein for any of Formulae A’, A, III’, and III, and described in classes and subclasses above and herein, both singly and in combination. In embodiments of Formula Ill-a-z, each of R, R¹, L, L¹, and L² is as defined herein for Formula A’ above.

In some embodiments, an ionizable lipid is selected from any of the lipids described in Table 1 of WO2022140252, or its N-oxide, or a pharmaceutically acceptable salt thereof.

In embodiments, an ionizable lipid is selected from the group consisting of:

In some embodiments, an ionizable lipid is Example 7-1, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-2, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example

7-19, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-20, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-22, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-24, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-25, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-1, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example

8-2, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-3, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-4, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-5, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-13, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-14, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-17, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-18, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-19, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-20, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-55, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-57, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-58, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-59, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-60, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-61, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-62, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-63, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-232, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-233, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-234, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-235, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-236, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-237, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example

7-238, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-239, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-67, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-68, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-69, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-70, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example

8-71, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 8-72, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-243, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-244, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-245, or a pharmaceutically acceptable salt thereof. In some embodiments, an ionizable lipid is Example 7-246, or a pharmaceutically acceptable salt thereof. Exemplary Lipids of WO2022159472

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022159472, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I, II, III, III A, IIIB, IIIC, IV, V, VA, VI, VIA, VII, and VIIA ofWO2022159472, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids of Table 1 of WO2022159472, including any of the lipids represented by Examples 4-1 to 4-86, or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, an ionizable lipid is according to Formula I of

WO2022159472: or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; an optionally substituted saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, or

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 5 - to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring ;

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^c-;

₃ , provided that when L is a covalent bond, then R must be In some embodiments, an ionizable lipid is according to Formula VI of

WO2022159472: or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3 or 4, and L², R¹, A¹, A², X¹, X², and X³ are as defined therein for Formula I and also described in classes and subclasses therein, both singly and in combination. In embodiments, L², R¹, A¹, A², X¹, X², and X³ are as defined herein for Formula I above.

In some embodiments, an ionizable lipid is according to Formula VIA of

WO2022159472: or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3 or 4, and L², R¹, A¹, A², X², and X³ are as defined therein for Formula I and also described in classes and subclasses therein, both singly and in combination. In embodiments, L², R¹, A¹, A², X², and X³ are as defined herein for Formula I above.

In some embodiments, an ionizable lipid is selected from any of the lipids described in Table 1 ofWO2022159472, or a pharmaceutically acceptable salt thereof. In embodiments, an ionizable lipid is selected from the group consisting of:

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

Exemplary Lipids of WO 2021141969

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2021141969, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO2021141969, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO 2021141969.

In some embodiments, an ionizable lipid is according to a compound of Formula I of WO2021141969:

In various embodiments, the compound of Formula (I) is an ionizable lipid as described elsewhere herein. In various embodiments, R¹ in Formula (I) is C9-C20 alkyl or C$>- C20 alkenyl with 1-3 units of unsaturation. For example, in some embodiments R¹ in Formula (I) is C9-C20 alkenyl with 2 units of unsaturation, such as a C17 alkenyl group of the formula In various embodiments, X¹ and X² in Formula (I) are each independently absent or selected from -O-, -NR²-, and X⁷ , wherein R² is hydrogen or C₁-C₆ alkyl, a is an integer between 1 and 6, X⁷ is independently hydrogen, hydroxyl or -NR⁶R⁷, and R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen. In some embodiments, X¹ is absent, X² is absent, or both X¹ and X² are absent. As described elsewhere herein, X⁴-X²- X³-X⁴ does not contain any oxygen-oxygen, oxygen-nitrogen or nitrogen-nitrogen bonds to one another. Accordingly, X¹ and X² cannot both be -O- and cannot both be -NR²-. Similarly, X¹ and X² cannot be -O- and -NR²-, respectively, nor -NR²- and -O-, respectively.

In various embodiments, X¹ is -O-. In various embodiments, X² is -O-. In some embodiments, each a is independently 1, 2, 3, 4, 5 or 6. In various embodiments, X¹ is - NR⁶R⁷. In various embodiments, X² is -NR⁶R⁷. In some embodiments, R⁶ is hydrogen or C₁-C₆ alkyl. In some embodiments, R⁷ is hydrogen or C₁-C₆ alkyl. In other embodiments, R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups. In some embodiments, the 4- to 7-membered heterocyclyl formed by the joining together of R⁶ and R⁷ optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen.

In various embodiments, X³ and X⁴ in Formula (I) are each independently absent or selected from:

(1) 4- to 8-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups;

(2) 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups;

(3) 5- to 6-membered aryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups;

(4) 4- to 7-membered cycloalkyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups; (5) -0-; or

(6) -NR³-, wherein each R³ is independently a hydrogen atom or C₁-C₆ alkyl.

In some embodiments, X³ is absent, X⁴ is absent, or both X³ and X⁴ are absent. As described elsewhere herein, X'-X²-X³-X⁴ does not contain any oxygen-oxygen, oxygennitrogen or nitrogen-nitrogen bonds to one another. Accordingly, X² and X³ cannot both be - O-. When X² is -O- or -NR²- then X³ cannot be -NR³-. Similarly, when X³ is -O- or - NR³- then X² cannot be -NR²-. Likewise, X³ and X⁴ cannot both be -O- and cannot both be -NR³-. Similarly, X³ and X⁴ cannot be -O- and -NR³-, respectively, nor -NR³- and -O-, respectively.

In various embodiments, X³ and X⁴ in Formula (I) are each independently a 4- to 8- membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ or C1-C3 alkyl groups. For example, in various embodiments X³ and X⁴ are each independently azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, methyldiazepanyl, octahydro-2H-quinolizinyl, azabicyclo[3.2.1]octyl, methyl- azabicyclo[3.2. l]octyl, diazaspiro [3 ,5]nonyl or methyldiazaspiro [3 ,5]nonyl.

In various embodiments, X³ and X⁴ in Formula (I) are each independently a 5- to 6- membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ or C1-C3 alkyl groups. For example, in various embodiments X³ and X⁴ are each independently pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl, or methylpyridinyl.

In various embodiments, X³ and X⁴ in Formula (I) are each independently a 5- to 6- membered aryl optionally substituted with 1 or 2 C₁-C₆ or C1-C3 alkyl groups. For example, in various embodiments X³ and X⁴ are each independently phenyl, methylphenyl, naphthyl or methylnaphthyl .

In various embodiments, X³ and X⁴ in Formula (I) are each independently a 4- to 7- membered cycloalkyl optionally substituted with 1 or 2 C₁-C₆ or C1-C3 alkyl groups. For example, in various embodiments X³ and X⁴ are each independently cyclopentyl, methylcyclopentyl, cyclohexyl, or methylcyclohexyl.

In various embodiments, X³ in Formula (I) is -O-. In other embodiments, X⁴ in Formula (I) is -O-. In various embodiments, X³ is -NR³-, wherein R³ is a hydrogen atom or C₁-C₆ alkyl, such as a C1-C3 alkyl. For example, in various embodiments X³ is -N(CHs)-, - N CFLCFL)-, or N(CH₂CH₂CH3)-. In other embodiments, X⁴ is -NR³-, wherein R³ is a hydrogen atom or C₁-C₆ alkyl, such as a C1-C3 alkyl. For example, in various embodiments X⁴ is -N(CH₃)-, -N(CH₂CH3)-, orN(CH₂CH₂CH₃)-. In various embodiments, X⁵ in Formula (I) is -(CHzjb-, wherein b is an integer between 0 and 6. In some embodiments, b is 0, in which case X⁵ is absent. In other embodiments, b is 1, 2, 3, 4, 5 or 6.

In various embodiments, X⁶ in Formula (I) is hydrogen, C₁-C₆ alkyl, 5- to 6- membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, or -NR⁴R⁵. In some embodiments, R⁴ and R⁵ are each independently hydrogen or C₁-C₆ alkyl. Alternatively, in other embodiments R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the 4- to 7-membered heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen.

In various embodiments of Formula (I), at least one of X¹, X², X³, X⁴, and X⁵ is present. For example, in various embodiments at least two of X¹, X², X³, X⁴, and X⁵ are present in Formula (I). In other embodiments, at least three of X¹, X², X³, X⁴, and X⁵ are present in Formula (I). For example, in some embodiments, at least four of X¹, X², X³, X⁴, and X⁵ are present in Formula (I). In other embodiments, all of X¹, X², X³, X⁴, and X⁵ are present in Formula (I).

In some embodiments, X⁶ is hydrogen. In other embodiments, X⁶ is C₁-C₆ alkyl, such as C1-C3 alkyl (e.g., methyl, ethyl or propyl). In other embodiments, X⁶ is 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups. For example, in various embodiments X⁶ is pyrrolyl, methylpyrrolyl, imidazolyl, methylimidazolyl, pyridinyl, or methylpyridinyl. In other embodiments, X⁶ is -NR⁴R⁵. For example, in some embodiments X⁶ is -NH₂, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -N(CH₃)2, -N(CH₂CH₃)2, or - N(CH2CH₂CH3)2. Alternatively, in other embodiments, R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl. The 4- to 7- membered heterocyclyl can be optionally substituted with 1 or 2 C₁-C₆ alkyl groups, such as C1-C3 alkyl, and/or the 4- to 7-membered heterocyclyl can optionally include an additional heteroatom selected from oxygen, sulfur, and nitrogen. For example, in some embodiments X⁶ is azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, or methyldiazepanyl.

In various embodiments, each X⁷ in Formula (I) is hydrogen. In other embodiments, each X⁷ is hydroxyl. In other embodiments, each X⁷ is -NR⁶R⁷. For embodiments in which a is between 2 and 6, each X⁷ can be the same or different. For example, in various embodiments X⁷ is -(CH2)a-iCH(X⁷)-, where a is 2, 3, 4, 5 or 6. In some embodiments for which X⁷ is -NR⁶R⁷, R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl, such as Ci- C3 alkyl. For example, in some embodiments X⁷ is -NH2, -NHCH3, -NHCH2CH3, - NHCH2CH2CH3, -N(CH₃)2, -N(CH₂CH3)2, or -N(CH₂CH₂CH3)2. Alternatively, in some embodiments for which X⁷ is -NR⁶R⁷, R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 Ci- Ce alkyl groups. Alternatively, in other embodiments for which X⁷ is -NR⁶R⁷, the R⁶ and R⁷ can join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl. The 4- to 7-membered heterocyclyl can be optionally substituted with 1 or 2 Ci- Ce alkyl groups, such as C1-C3 alkyl, and/or the 4- to 7-membered heterocyclyl can optionally include an additional heteroatom selected from oxygen, sulfur, and nitrogen. For example, in some embodiments X⁶ is azetidinyl, methylazetidinyl, pyrrolidinyl, methylpyrrolidinyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, dimethylpiperazinyl, morpholinyl, diazepanyl, or methyldiazepanyl.

In various embodiments, A¹ and A² in Formula (I) are each independently selected from:

(1) C5-C12 haloalkyl;

(2) C5-C12 alkenyl;

(3) C5-C12 alkynyl;

(4) (C5-C12 alkoxy)-(CH₂)n2-;

(5) (C5-C10 aryl)-(CH2)n3- optionally ring substituted with one or two halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkoxy groups; and

(6) (C3-C8 cycloalkyl)-(CH2)n4- optionally ring substituted with 1 or 2 C₁-C₆ alkyl groups; or alternatively A¹ and A² join together with the atoms to which they are bound to form a 5- to 6-membered cyclic acetal substituted with 1 or 2 C4-C10 alkyl groups.

In various embodiments of Formula (I), nl, n2 and n3 are each individually an integer between 1 and 4 (i.e., 1, 2, 3 or 4), and n4 is an integer between zero and 4 (i.e., 0, 1, 2 , 3 or 4). In various embodiments, A¹ and A² have the same chemical structure.

In various embodiments of Formula (I), A¹ and A² are each independently a C5-C12 haloalkyl. For example, in various embodiments the C5-C12 haloalkyl is a C5-C12 fluoroalkyl such as a Ce fluoroalkyl, a C7 fluoroalkyl, a Cs fluoroalkyl, a C9 fluoroalkyl, a C10 fluoroalkyl, a C11 fluoroalkyl, or a C12 fluoroalkyl. The number of halogen atoms attached to the C5-C12 haloalkyl can vary over a broad range, depending on the length of the alkyl chain and the degree of halogenation. For example, in various embodiments the C5-C12 haloalkyl contains between 1 and 25 halogen atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 halogen atoms. In various embodiments, the Cs-Cnhaloalkyl is a C5-C12 fluoroalkyl that comprises a fluorinated end group such as CF3(CF2)n5-, where n5 is an integer in the range of 0 to 5. For example in various embodiments the C5-C12 fluoroalkyl is CF3(CF2)ns(CH2)n6-, where n5 is an integer in the range of 0 to 5, n6 is an integer in the range of 0 to 11, and n5 + n6 + 1 is equal to number of carbons in the C5-C12 fluoroalkyl.

In various embodiments of Formula (I), A¹ and A² are each independently a C5-C12 alkenyl. The position of the alkenyl double bond(s) can vary. For example, in various embodiments the C5-C12 alkenyl is CH3CH2CH=CH(CH2)n7-, where n7 is an integer in the range of 1 to 8, such as CH3CH2CH=CH(CH2)4-. In some embodiments, the C5-C12 alkenyl is branched, such as, for example, (CH3)2C=CH(CFb)n8-CH(CH3)-(CH2)n9- wherein n8 and n9 are each independently 1, 2 or 3.

In various embodiments of Formula (I), A¹ and A² are each independently a C5-C12 alkynyl. The position of the alkynyl triple bond(s) can vary. For example, in various embodiments the C5-C12 alkynyl is CH3CH2C=C(CH2)nio-, where nlO is an integer in the range of 1 to 8, such as CH3CH2C=C(CH2)4-. In some embodiments, the C5-C12 alkynyl is branched, such as, for example, (CH3)2CHC=C(CH2)nii-CH(CH3)-(CH2)ni2- wherein ni l and n 12 are each independently 1, 2 or 3 and nl 1 + nl2 is in the range of 2 to 5.

In various embodiments of Formula (I), A¹ and A² are each independently a (C5-C12 alkoxy)-(CH2)n2-. In various embodiments, each n2 is independently an integer in the range of 1 to 4 (i.e., 1, 2, 3 or 4). The position of the oxygen(s) can vary. For example, in various embodiments the (C5-C12 alkoxy) -(CH2)n2- is CH3O(CH2)ni3-(CH2)n2-, where nl3 is an integer in the range of 1 to 11, such as CFFOICH₂)₇-. In other embodiments the (C5-C12 alkoxy)-(CH2)n2- is CH3(CH2)ni4-O-(CH2)nis-(CH2)n2-, wherein nl4 and nl5 are each independently integers between 1 and 8, and nl4 + nl5 is an integer in the range of 4 to 11, such as CH3(CH2)7-O-(CH2)2— (CH2)n2-. In some embodiments, the C5-C12 alkoxy is branched, such as, for example, CH3O(CH2)ni6-CH(CH3)-(CH2)ni7— (CH2)n2-, wherein nl6 and nl7 are each independently 1, 2, 3, 4 or 5 and nl6 + nl7 is an integer in the range of 2 to 9.

In various embodiments of Formula (I), A¹ and A² are each independently a (C5-C10 aryl)-(CH2)n3- optionally ring substituted with one or two halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkoxy groups. In various embodiments, each n3 is independently an integer between 1 and 4 (i.e., 1, 2, 3 or 4). In some embodiments, the C5-C10 aryl is a phenyl. For example, in various embodiments the (C5-C10 aryl)-(CH2)n3- is C6Hs-(CH2)n3- optionally ring substituted with one or two halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkoxy groups. In an embodiment, the optionally ring substituted (Cs-Cio aryl)-(CH2)n3- is CF3-C6H4- (CH2)n3- , such as CF3-C6H4-CH2- or CF3-CeH4-(CH2)2-. In another embodiment, the optionally ring substituted (C5-C10 aryl)-(CH2)n3- is CH3-(CH2)ni8-CeH4-(CH2)n2-, wherein nl8 is 1, 2 or 3 and n2 is 1, 2, 3 or 4, such as CH3(CH2)3-CeH4-CH2- or CH3(CH2)3-CeH4- (CH₂)2-.

In various embodiments of Formula (I), A¹ and A² are each independently a (C3-C8

Alternatively, in other embodiments of Formula (I), A¹ and A² join together with the atoms to which they are bound to form a 5- to 6-membered cyclic acetal substituted with 1 or 2 C4-C10 alkyl groups. For example, in an embodiment, A¹ and A² join together with the atoms to which they are bound to form a 6-membered cyclic acetal that is ring substituted with 2 Cs alkyl groups as follows: . In another embodiment,

A¹ and A² join together with the atoms to which they are bound to form a 5-membered cyclic acetal that is ring substituted with 2 Cs alkyl groups as follows.

Exemplary Lipids of WO2021113365

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2021113365, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO2021113365, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2021113365. In some embodiments, an ionizable lipid is according to a compound of Formula I of WO2021113365: wherein:

R¹ is C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation;

X¹ and X² are each independently absent or selected from -O-, NR², and wherein R² is C₁-C₆ alkyl, and wherein X¹ and X² are not both -O- or NR²; a is an integer between 1 and 6;

X³ and X⁴ are each independently absent or selected from the group consisting of: 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6- membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, and -NR³-, wherein each R³ is a hydrogen atom or C₁-C₆ alkyl;

X⁵ is — (CHzjb— , wherein b is an integer between 0 and 6;

X⁶ is hydrogen, C₁-C₆ alkyl, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, or -NR⁴R⁵, wherein R⁴ and R⁵ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen;

X⁷ is hydrogen or -NR⁶R⁷, wherein R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; at least one of X¹, X², X³, X⁴, and X⁵ is present; and provided that when either X¹ or X² is -O-, neither X³ nor X⁴ is , and when either X¹ or X² is -O-, R⁴ and R⁵ are not both ethyl.

Exemplary Lipids of WO2022140239

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022140239, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I’ of WO2022140239, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2022140239.

In some embodiments, an ionizable lipid is according to a compound of Formula I' of WO2022140239: r or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

L is absent, optionally substituted C1-10 alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

L³ is absent, optionally substituted C1-10 alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-; each R is independently hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10- membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)2, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N( R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)₂, -OP(O)(OR²)₂, or -P(O)(OR²)₂; or ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)₂N(R⁴)₂, -(CH2)n-R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7-membered cycloaliphatic, 5- to 6- membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)₂, -OC(O)R⁵, -OC(O)OR⁵, -CN, -C(O)N(R⁵)₂, -NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, or optionally substituted Ci-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4.

Exemplary Lipids of WO2022140238

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022140238, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I’ of WO2022140238, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2022140238.

In some embodiments, an ionizable lipid is according to a compound of Formula F of WO2022140238: or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein L¹ is absent, Ci-6 alkylenyl, or C₂-6 heteroalkylenyl; each L² is independently optionally substituted C2-15 alkylenyl, or optionally substituted C3-15 heteroalkylenyl;

X is absent, -OC(O)-, -C(O)O-, or -OC(O)O-; each R’ is independently an optionally substituted group selected from C4-12 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2- adamantyl, sterolyl, and phenyl;

R is hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl;

-NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, or optionally substituted C1-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4. Exemplary Lipids of WO2022159421

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022159421, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO2022159421, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2022159421.

In some embodiments, an ionizable lipid is according to a compound of Formula I of WO2022159421: or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-;

R , is IH- ^p cy⁸ , or an optionally substituted saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X¹ is a covalent bond, -O-, or -NR-; X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^c-;

Z¹ is a covalent bond or -O-;

Z³ is hydrogen, or an optionally substituted group selected from C1-C10 aliphatic, and 4- to 12-membered saturated or partially unsaturated carbocyclyl; and d is 0, 1, 2, 3, 4, 5, or 6;

₃ , provided that when L is a covalent bond, then R must be

Exemplary lipids of WO2022159475

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022159475, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO2022159475, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2022159475.

In some embodiments, an ionizable lipid is according to a compound of Formula I of WO2022159475: or a pharmaceutically acceptable salt thereof, wherein: each L¹ and L¹ is independently -C(0)- or -C(O)O-; each L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or each Cy^A is independently an optionally substituted ring selected from phenylene and a 3 - to 7-membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each L³ and L³ is independently a covalent bond, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; each R¹ and R¹ is independently an optionally substituted group selected from a saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 12-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, and each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵; or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: wherein x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 5 - to 10-membered aryl ring and a 3- to 8 -membered carbocyclic ring ;

X¹ is a covalent bond, -O-, or -NR-;

X² is a covalent bond or an optionally substituted, bivalent saturated or unsaturated, straight or branched, C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X³ is hydrogen or -Cy^B;

Cy^B is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; provided that when X³ is hydrogen, at least one of R¹ or R¹ is

In some embodiments, the present disclosure provides a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein: each L¹ and L¹ is independently -C(O)- or -C(O)O-; each L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or each Cy^A is independently an optionally substituted ring selected from phenylene and a 3 - to 7-membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each L³ and L³ is independently a covalent bond, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; each R¹ and R¹ is independently an optionally substituted group selected from a saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, and each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵; or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: wherein x is selected from 1 or 2; and

X¹ is a covalent bond, -O-, or -NR-;

X³ is hydrogen or -Cy^B;

Exemplary Lipids of WO2022159463

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Publication No. WO2022159463, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of WO2022159463, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of WO2022159463.

In some embodiments, an ionizable lipid is according to a compound of Formula I of WO2022159463: or a pharmaceutically acceptable salt thereof, wherein: each of L¹ and L¹ is independently a covalent bond, -C(O)-, or -OC(O)-; each of L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or each Cy^A is independently an optionally substituted ring selected from phenylene or 3- to 7- membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each of L³ and L³ is independently a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-; each of R¹ and R¹ is independently an optionally substituted group selected from saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, or each L⁴ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵, or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: wherein x is selected from 1 or 2; and # represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 6- to 10-membered aryl ring or a 3- to 8-membered carbocyclic ring;

Y¹ is a covalent bond, -C(O)-, or -C(O)O-;

Y² is a bivalent saturated or unsaturated, straight or branched C₁-C₆ hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced with cyclopropylene, -O-, or -NR-;

Y³ is an optionally substituted group selected from saturated or unsaturated, straight or branched C1-C14 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, or phenyl;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^B-; each Cy^B is independently an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclene, phenylene, 3- to 7-membered heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6- membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group.

Exemplary Lipids of PCT/US23/27741

In another aspect, an ionizable lipid is according to any formula or structure, or a pharmaceutically acceptable salt or solvate thereof, as described in International Patent Application No. PCT/US23/27741, which is hereby incorporated by reference in its entirety. In some embodiments, an ionizable lipid has a structure according to any of Formulae I of PCT/US23/27741, or a pharmaceutically acceptable salt or solvate thereof. Exemplary ionizable lipids also include any of the lipids represented by the Examples of PCT/US23/27741.

In some embodiments, an ionizable lipid is according to a compound of Formula I of PCI7US23/27741: or a pharmaceutically acceptable salt thereof, wherein: each L¹ and L¹ is independently -C(O)- or -OC(O)-; each L² and L² is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O

R¹ is optionally substituted C1-20 aliphatic,

Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, I-adamantyl, 2-adamantyl, sterolyl, and phenyl;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or optionally substituted Ci-6 aliphatic.

In some embodiments, the present disclosure provides a compound of Formula I of PCT7US23/27741, wherein: each L¹ and L¹ is -C(O)-; each L² and L² is independently a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -C(O)O-, or -OC(O)-;

R¹ is C1-20 alkyl, C2-20 alkenyl, each L^Ra and R¹ is independently C1-20 alkyl or C2-20 alkenyl;

Y¹ is -C(O)- or -C(O)O-;

Y² is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain;

Y³ is Ci -20 alkyl or C2-20 alkenyl;

X¹ is -O- or -NR-;

X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -NR-;

X³ is hydrogen or a 3 - to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with C1-6 alkyl or C2-6 alkenyl; and each R is independently hydrogen or C1-6 alkyl or C2-6 alkenyl.

In some embodiments, the present disclosure provides a compound of Formula II of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y¹, Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III of PCT7US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III -A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula III-B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IV of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IV-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IV-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IV-B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula V of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula V-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula V-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula V-B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VI of PCI7US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VI-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination. In some embodiments, the present disclosure provides a compound of Formula VI-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VI-B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L¹, L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VII of PCI7US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VII-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination. In some embodiments, the present disclosure provides a compound of Formula VII-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VII-B- z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VIII of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VIII -A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VIII-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula VIII-

B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , L³, R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IX of

PCI7US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination. In some embodiments, the present disclosure provides a compound of Formula IX-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IX-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula IX-B-z of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula X of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X¹, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula X-A of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula X-B of PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , R, Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments, the present disclosure provides a compound of Formula X-B-z PCT/US23/27741: or a pharmaceutically acceptable salt thereof, wherein each of L², L² , R¹, R¹ , Y², Y³, X², and X³ is as described above and in classes and subclasses herein, both singly and in combination.

In some embodiments of any Formulae of PCT/US23/27741 described herein, L¹ is - C(O)- or -OC(O)-. In some embodiments, L¹ is -C(O)-. In some embodiments, L¹ is - OC(O)-.

In some embodiments, the present disclosure provides a compound of Formula I of PCT/US23/27741, wherein: each L¹ and L¹ is -C(O)-.

In some embodiments, the present disclosure provides a compound of Formula I of PCT/US23/27741, wherein: each L² and L² is independently a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain.

In some embodiments of any Formulae of PCT/US23/27741 described herein, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched Ci-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C4-8 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is -CH2-. In some embodiments, L² is -(CH2)4- or -(CH2)s-. In some embodiments, L² is -(CH2)₆- or -(CH2)7-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L² is -(CH2)2-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L³ is a covalent bond, -C(O)O-, or -OC(O)-. In some embodiments, L³ is a covalent bond. In some embodiments, L³ is -O-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L³ is - C(O)O- or -OC(O)-. In some embodiments, L³ is -O-. In some embodiments, L³ is -C(O)O-. In some embodiments, L³ is -OC(O)-. In some embodiments, L³ is -OC(O)O-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, R¹ is some embodiments, R¹ is . In some embodiments, R¹ is C1-20 aliphatic or . In some embodiments, R¹ is optionally substituted C6-12 aliphatic. In some embodiments, R¹ is optionally substituted C6-12 alkyl. In some embodiments, R¹ is optionally substituted C6-12 alkenyl. In some embodiments, R¹ is optionally substituted C12-20 aliphatic. In some embodiments, R¹ is optionally substituted C12-20 alkyl. In some embodiments, R¹ is optionally substituted C12-20 alkenyl. In some embodiments, R¹ is C1-20 aliphatic. In some embodiments, R¹ is C6-12 aliphatic. In some embodiments, R¹ is C6-12 alkyl. In some embodiments, R¹ is C6-12 alkenyl. In some embodiments, R¹ is C12-20 aliphatic. In some embodiments, R¹ is C12-20 alkyl. In some embodiments, R¹ is C12-20 alkenyl. In some

In some embodiments of any Formulae of PCT/US23/27741 described herein, L^CyA is a covalent bond or an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain.

In some embodiments of any Formulae of PCT/US23/27741 described herein, Cy^A is an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl.

In some embodiments of any Formulae of PCT/US23/27741 described herein, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, L^Ra is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L^Ra is a bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, L^Ra is -CH2- or -(CH2)2-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, each R^a is independently optionally substituted C1-20 aliphatic. In some embodiments, each R^a is independently optionally substituted C1-12 aliphatic. In some embodiments, each R^a is independently optionally substituted C1-12 alkyl. In some embodiments, each R^a is independently optionally substituted C1-12 alkenyl. In some embodiments, each R^a is independently optionally substituted C4-10 aliphatic. In some embodiments, R^a is optionally substituted C6-12 aliphatic. In some embodiments, R^a is optionally substituted C6-12 alkyl. In some embodiments, R^a is optionally substituted C6-12 alkenyl. In some embodiments, R^a is optionally substituted C7-9 aliphatic. In some embodiments, R^a is optionally substituted C7-9 alkyl. In some embodiments, R^a is optionally substituted C7-9 alkenyl. In some embodiments, each R^a is independently C1-20 aliphatic. In some embodiments, R^a is C6-12 aliphatic. In some embodiments, R^a is C6-12 alkyl. In some embodiments, R^a is C6-12 alkenyl. In some embodiments, R^a is C7-9 aliphatic. In some embodiments, R^a is C7-9 alkyl. In some embodiments, R^a is C7-9 alkenyl. In some embodiments, R^a is

In some embodiments of any Formulae of PCT/US23/27741 described herein, each R¹ is independently optionally substituted C1-20 aliphatic. In some embodiments, R¹ is optionally substituted C6-12 aliphatic. In some embodiments, R¹ is optionally substituted Ce- 12 alkyl. In some embodiments, R¹ is optionally substituted C6-12 alkenyl. In some embodiments, R¹ is optionally substituted C7-9 aliphatic. In some embodiments, R¹ is optionally substituted C7-9 alkyl. In some embodiments, R¹ is optionally substituted C7-9 alkenyl. In some embodiments, each R¹ is independently C1-20 aliphatic. In some embodiments, R¹ is C6-12 aliphatic. In some embodiments, R¹ is C6-12 alkyl. In some embodiments, R¹ is C6-12 alkenyl. In some embodiments, R¹ is C7-9 aliphatic. In some embodiments, R¹ is C7-9 alkyl. In some embodiments, R¹ is C7-9 alkenyl. In some embodiments,

In some embodiments of any Formulae of PCT/US23/27741 described herein, Y¹ is - C(O)- or -C(O)O-. In some embodiments, Y¹ is -C(O)-. In some embodiments, Y¹ is - C(O)O-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched Ci-6 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is an optionally substituted bivalent saturated, straight or branched Ci- 3 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, Y² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, Y² is -CH2- or -(CH₂)₂-. In some embodiments of any Formulae of PCT/US23/27741 described herein, Y³ is optionally substituted C1-20 aliphatic. In some embodiments, Y³ is C1-20 aliphatic. In some embodiments, Y³ is optionally substituted C1-12 aliphatic. In some embodiments, Y³ is optionally substituted C1-12 alkyl. In some embodiments, Y³ is optionally substituted C4-8 aliphatic. In some embodiments, Y³ is optionally substituted C4-8 alkyl. In some embodiments, Y³ is optionally substituted C1-6 alkyl. In some embodiments, Y³ is -CH3, - CH2CH3, -(CH₂)₂CH3, -(CH2)₃CH₃, -(CH₂)4CH₃, -(CH2)₅CH₃, -(CH₂)6CH₃, or -(CH₂)7CH3. In some embodiments, Y³ is -CH3. In some embodiments, Y³ is -CH2CH3. In some embodiments, Y³ is -(CH2)2CH3. In some embodiments, Y³ is -(CH2)3CH3. In some embodiments, Y³ is -(CH2)4CH3. In some embodiments, Y³ is -(CH2)sCH3. In some embodiments, Y³ is -(CH2)eCH3. In some embodiments, Y³ is -(CH2)?CH3.

In some embodiments of any Formulae of PCT/US23/27741 described herein, X¹ is a covalent bond, -O-, or -NR-. In some embodiments, X¹ is a covalent bond. In some embodiments, X¹ is -O- or -NR-. In some embodiments, X¹ is -O-. In some embodiments, X¹ is -NR-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-12 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-6 hydrocarbon chain. In some embodiments, X² is a bivalent saturated, straight or branched C1-3 hydrocarbon chain. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1-2 methylene units are independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 2 methylene units are independently replaced with -NR-. In some embodiments, X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-2 methylene units are optionally and independently replaced with -NR-. In some embodiments, X² is a bivalent saturated or unsaturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are independently replaced with -O- or -NR- . In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1-2 methylene units are independently replaced with -O- or -NR-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 2 methylene units are independently replaced with -NR-. In some embodiments, X² is an optionally substituted bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-. In some embodiments, X² is a bivalent saturated, straight or branched C4-8 hydrocarbon chain, wherein 1 methylene unit is replaced with -NR-.

In some embodiments of any Formulae of PCT/US23/27741 described herein, X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is hydrogen or a 3 - to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with C1-6 aliphatic. In some embodiments, X³ is hydrogen. In some embodiments, X³ is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is optionally substituted 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is optionally substituted 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5- to 6-membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted 5-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5- membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 5 -membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted 5 -membered heterocyclyl having 1-2 nitrogen atoms. In some embodiments, X³ is 5-membered heterocyclyl having 1-2 nitrogen atoms. In some embodiments, X³ is 5- membered heterocyclyl, having 1-2 nitrogen atoms, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted 6- membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, X³ is 6-membered heterocyclyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl). In some embodiments, X³ is optionally substituted pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. In some embodiments, X³ is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. In some embodiments, X³ is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl substituted with -R°, wherein R° is Ci-6 aliphatic (e.g., methyl or ethyl).

In some embodiments, at least one of X² and X³ comprises at least one ionizable nitrogen atom.

In some embodiments of any Formulae of PCT/US23/27741 described herein, each R is independently hydrogen or optionally substituted Ci-6 aliphatic. In some embodiments, each R is independently hydrogen or Ci-6 aliphatic. In some embodiments, R is hydrogen. In some embodiments, R is optionally substituted Ci-6 aliphatic. In some embodiments, R is Ci- 6 aliphatic. In some embodiments, R is optionally substituted C1-3 aliphatic. In some embodiments, R is Ci-3 aliphatic. In some embodiments, R is -CH3.

In some embodiments, R° is methyl. In some embodiments, R° is ethyl.

In some embodiments of any Formulae of PCT/US23/27741 described herein, the present disclosure provides compounds comprising an ionizable nitrogen atom, wherein the pKa of the conjugate acid thereof is between about 4 and about 12.

In some embodiments, the present disclosure provides a compound selected from Table 1 of PCT/US23/27741. In various embodiments, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

It will be understood that, unless otherwise specified or prohibited by the foregoing definition of any of Formulae I, II, III, III-A, III-B, III-B-z, IV, IV-A, IV-B, IV-B-z, V, V-A, V-B, V-B-z, VI, VI-A, VI-B, VI-B-z, VII, VII-A, VII-B, VII-B-z, VIII, VIII-A, VIII-B, VIII- B-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of PCT/US23/27741, embodiments of variables L¹, L¹’, L², L² , L³, L^CyA, Cy^A, L^Ra, R^a, R¹, R¹’, R, Y¹, Y², Y³, X¹, X², and X³ as defined above and described in classes and subclasses herein, apply to compounds of any of Formulae I, II, III, III-A, III-B, III-B-z, IV, IV-A, IV-B, IV-B-z, V, V-A, V-B, V-B-z, VI, VI- A, VI-B, VI-B-z, VII, VII-A, VII-B, VII-B-z, VIII, VIII-A, VIII-B, VIII-B-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of PCT/US23/27741, both singly and in combination.

In some embodiments, provided compounds are provided and/or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated.

It will be appreciated that throughout the present disclosure, unless otherwise indicated, reference to a compound of Formula I of PCT/US23/27741 is intended to also include any of Formulae II, III, III-A, III-B, III-B-z, IV, IV-A, IV-B, IV-B-z, V, V-A, V-B, V- B-z, VI, VI-A, VI-B, VI-B-z, VII, VII-A, VII-B, VII-B-z, VIII, VIII-A, VIII-B, VIII-B-z, IX, IX-A, IX-B, IX-B-z, X, X-A, X-B, and X-B-z of PCT/US23/27741, and compound species of such formulae disclosed herein.

Conjugate-linker lipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more conjugate-linker lipids as described herein.

In some embodiments, a conjugate-linker lipid is or comprises a polyethylene glycol (PEG)-lipid or PEG-modified lipid. In some embodiments, PEG or PEG-modified lipids may be alternately referred to as PEGylated lipids or PEG-lipids. Inclusion of a PEGylating lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in blood circulation. Exemplary PEG-lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20. PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEGDAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, in some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE lipid.

In some embodiments, a conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises DiMystyrlGlycerol (DMG), 1,2- Dipalmitoyl-rac-glycerol, methoxypolyethylene Glycol (DPG-PEG), or 1,2-Distearoyl-rac- glycero-3 -methylpolyoxyethylene (DSG - PEG). In some embodiments, a conjugate-linker lipid has an average molecular mass from about 500 Da to about 5000 Da. In some embodiments, a conjugate -linker lipid has an average molecular mass of about 2000 Da.

In some embodiments, a LNP preparation comprises from about 0 mole percent to about 5 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 1.5 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 2.0 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 2.5 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 3 mole percent conjugate-linker lipid. In some embodiments, a LNP preparation comprises about 3.5 mole percent conjugate-linker lipid.

Phospholipids

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more phospholipids as described herein. In some embodiments, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more (poly)unsaturated lipids.

In some embodiments, one or more phospholipids may assemble into one or more lipid bilayers. In some embodiments, one or more phospholipids may include a phospholipid moiety. In some embodiments, one or more phospholipids may include one or more fatty acid moieties. In some embodiments, one or more phospholipids may include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, a phospholipid moiety includes but is not limited to phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, a fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alphalinolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Exemplary phospholipids include but are not limited to l,2-distearoyl-5«-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-5«-glycero-3 -phosphoethanolamine (DOPE), 1,2- dilinoleoyl-5«-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-5«-glycerophosphocholine (DMPC), l .2-diolcoyl-s77-glyccro-3-phosphocholinc (DOPC), l .2-dipalmitoyl-.s77-glyccro-3- phosphocholine (DPPC), l,2-diundecanoyl-5«-glycerophosphocholine (DUPC), 1-palmitoyl- 2-olcoyl-s7 -glyccro-3-phosphocholinc (POPC), l.2-di-O-octadcccnyl-s77-glyccro-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoy l-sw-glycero-3- phosphocholine (OChemsPC), 1 -hexadecyl snglycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2- dilinolenoyl-5«-glycero-3-phosphocholine, l .2-diarachidonoyl-s77-glyccro-3-phosphocholinc. l,2-didocosahexaenoyl-5«-glycero-3 -phosphocholine, l .2-diphytano l-s77-glyccro-3- phosphoethanolamine (ME 16.0 PE), l .2-distcaroyl-s77-glyccro-3-phosphocthanolaminc. 1,2- dilinoleoyl-5«-glycero-3-phosphoethanolamine, l.2-dilinolcnoyl-s77-glyccro-3- phosphoethanolamine, l .2-diarachidonoyl-s77-glyccro-3-phosphocthanolaminc. 1,2- didocosahexaenoyl-5«-glycero-3-phosphoethanolamine, I,2-dioleoyl-s77-glycero-3-phospho- rac-(l -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1 -stearoyl -2 -oleoyl-phosphatidy ethanolamine (SOPE), 1 -stearoyl -2 oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In some embodiments, a phospholipid is DSPC. In some embodiments, a phospholipid is DMPC.

In some embodiments, the phospholipid comprises l,2-distearoyl-5«-glycero-3- phosphocholine (DSPC), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), or a combination thereof.

In some embodiments, a LNP preparation comprises from about 5 mole percent to 25 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 5 mole percent to 15 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 9 mole percent to 11 mole percent of phospholipid. In some embodiments, a LNP preparation comprises about 10 mole percent of phospholipid.

Sterols

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that comprise one or more sterols as described herein.

In some embodiments, a sterol is a cholesterol, or a variant or derivative thereof. In some embodiments, a cholesterol is modified. In some embodiments, a cholesterol is an oxidized cholesterol. In some embodiments, a cholesterol is esterified cholesterol. Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized. In some embodiments, a cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure. In some embodiments, a sterol is a phytosterol. Exemplary sterols that are considered for use in the disclosed lipid nanoparticles include but are not limited to 25-hydroxycholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27- hydroxycholesterol, 6-keto-5a-hydroxy cholesterol, 7-ketocholesterol, 7p-hydroxy cholesterol, 7 a-hydroxy cholesterol, 7p-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, a side-chain oxidized cholesterol can enhance payload delivery relative to other cholesterol variants. In some embodiments, a cholesterol is an unmodified cholesterol.

In some embodiments, a LNP preparation comprises from about 25 mole percent to about 45 mole percent sterol. In some embodiments, a LNP preparation comprises from about 30 mole percent to about 45 mole percent sterol. In some embodiments, a LNP preparation comprises about 40 mole percent sterol. In some embodiments, a LNP preparation comprises about 35.75 mole percent sterol. In some embodiments, a LNP preparation comprises about 32.5 mole percent cholesterol.

LNP Formulations

Particular formulation of a nanoparticle composition comprising one or more described lipids is described herein. The present invention provides for compositions, preparations, nanoparticles, and/or nanomaterials that comprise lipid nanoparticles.

In some embodiments, a lipid nanoparticle preparation comprises about 30 mole percent to about 70 mole percent ionizable lipid, about 5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent sterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 47.5 mole percent ionizable lipid, about 10 mole percent phospholipid, about 40 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 51.25 mole percent ionizable lipid, about 10 mole percent phospholipid, about 35.75 mole percent sterol, and about 3 mole percent conjugate-linker lipid.

In some embodiments, the lipid nanoparticle preparation comprises about 55 mole percent ionizable lipid, about 10 mole percent phospholipid, about 32.5 mole percent sterol, and about 2.5 mole percent conjugate-linker lipid.

In some embodiments, a lipid nanoparticle preparation comprises about 45 mole percent to about 60 mole percent ionizable lipid of any provided compound, about 9 mole percent to about 11 mole percent 1 -2 -distearoyl-sn-glycero-3 -phosphocholine (DSPC), about 1 mole percent to about 5 mole percent PEG2000-DMG , and about 30 mole percent to about 45 mole percent cholesterol, based on the total moles of these four ingredients. In some embodiments, a lipid nanoparticle preparation is the formulation LNP1, LNP2, LNP3, or LNP4 in TablelOA.

In some embodiments, a lipid nanoparticle preparation comprises about 47.5 mole percent ionizable lipid IZ1, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 40 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload comprising mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and guide RNA GA521 of Table IB.

In some embodiments, a lipid nanoparticle preparation comprises about 55 mole percent ionizable lipid IZ2, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 32.5 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload comprising mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and guide RNA GA521 of Table IB.

In some embodiments, a lipid nanoparticle preparation comprises about 51.25 mole percent ionizable lipid IZ3, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 3 mole percent PEG2000-DMG , and about 35.75 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload comprising mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and guide RNA GA521 of Table IB.

In some embodiments, a lipid nanoparticle preparation comprises about 47.5 mole percent ionizable lipid IZ4, about 10 mole percent l-2-distearoyl-sn-glycero-3- phosphocholine (DSPC), about 2.5 mole percent PEG2000-DMG , and about 40 mole percent cholesterol, based on the total moles of these four ingredients, an N/P ratio of 6, and a payload comprising mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T or the combination of the amino acid alterations V82T, Y147T, and Q154S, and guide RNA GA521 of Table IB. The described lipid nanoparticle compositions are capable of delivering a therapeutic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs.

Weight Ratio of Lipid Component to Therapeutic Agent

The amount of a therapeutic agent or drug substance (e.g., the mRNA that encodes for the base editor and the guide RNA) in an LNP composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition. For example, the amount of an RNA comprised in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5 : 1 to about 60: 1, such as about 5: 1. 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10: 1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. The amount of a therapeutic agent in a nanoparticle composition can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

N/P Ratio

In some embodiments, an LNP formulation comprises one or more nucleic acids such as RNAs. In some embodiments, the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N/P ratio. The N/P ratio can be selected from about 1 to about 30. In some embodiments, the N/P ratio 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 some embodiments, the N/P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9, or about 10. In some embodiments, the N/P ratio is from about 5 to about 7. In some embodiments, the N/P ratio is about 6. In each of the above N/P ratios and any other recitation of a single number for an N/P ratio, the moles or parts P is 1 and is not recited for brevity.

Polydispersity

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that have a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

Encapsulation efficiency

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials, wherein encapsulation efficiency of provided compositions, preparations, nanoparticles, and/or nanomaterials is from about 80% to about 100%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is from about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and/or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. pKa

Among other things, the present disclosure describes compositions, preparations, nanoparticles, and/or nanomaterials that have a pKa from about 5 to about 9. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a pKathat is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the aforementioned values. In some embodiments, compositions, preparations, nanoparticles, and/or nanomaterials described herein have a pKa that is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the aforementioned values.

Payload

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

In some embodiments, the payload comprises one or more nucleic acid(s) (i.e., one or more nucleic acid molecular entities). In some embodiments, the nucleic acid is a singlestranded nucleic acid. In some embodiments, single-stranded nucleic acid is an RNA. In some embodiments, the nucleic acid is a double -stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is a DNA. In some embodiments, the nucleic acid is a messenger RNA (mRNA).

Methods of manufacturing LNPs

Methods of manufacturing lipid nanoparticles are known in the art. In some embodiments, the described compositions, preparations, nanoparticles, and/or nanomaterials are manufactured using microfluidics. For instance, exemplary methods of using microfluidics to form lipid nanoparticles are described by Leung, A.K.K, et al., J Phys ('hem. 116: 18440-18450 (2012), Chen, D„ et al., J Am Chem Soc, 134:6947-6951 (2012), and Belliveau, N.M., et al., Molecular Therapy- Nucleic Acids, 1 : e37 (2012), the disclosures of which are hereby incorporated by reference in their entireties. Briefly, a payload, such as a payload described herein, is prepared in a first buffer solution. The other lipid nanoparticle components (such as ionizable lipid, conjugate -linker lipids, cholesterol, and phospholipid) are prepared in a second buffer solution. In some embodiments, a syringe pump introduces the two solutions into a microfluidic device. The two solutions come into contact within the microfluidic device to form lipid nanoparticles encapsulating the payload.

PHARMACEUTICAL COMPOSITIONS

In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the cells, LNPs, polynucleotides, vectors, base editors, base editor systems, guide polynucleotides, fusion proteins, complexes, or the fusion protein-guide polynucleotide complexes described herein.

The pharmaceutical compositions of the present disclosure can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed. 2005). In general, the cell, or population thereof is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., a liver). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins or complexes provided herein. In some embodiments pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. In embodiments, pharmaceutical compositions comprise a lipid nanoparticle and a pharmaceutically acceptable excipient. In embodiments, the lipid nanoparticle contains a gRNA, a base editor, a complex, a base editor system, or a component thereof of the present disclosure, and/or one or more polynucleotides encoding the same. Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances.

The compositions, as described above, can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.

In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and/or conditions.

METHODS OF TREATMENT

Some aspects of the present invention provide methods of treating a subject having or having a propensity to develop amyloidosis, the method comprising administering to a subject in need an effective therapeutic amount of a pharmaceutical composition as described herein. In some embodiments, the methods of the invention comprise expressing or introducing into a cell of a subject a base editor polypeptide and one or more guide RNAs capable of targeting a nucleic acid molecule encoding a transthyretin polypeptide comprising a pathogenic mutation. One of ordinary skill in the art would recognize that multiple administrations of the pharmaceutical compositions contemplated in particular embodiments may be required to affect the desired therapy. For example, a composition may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

In any of such methods, the methods may comprise administering to the subject an effective amount of an edited cell or a base editor system or polynucleotide encoding such system. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the edited cells per day. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the modified cells per day. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the edited cells per day. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the edited cells per week. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the edited cells per week. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the edited cells per week. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the edited cells per month. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the edited cells per month. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the edited cells per month.

Administration of the pharmaceutical compositions contemplated herein may be carried out using conventional techniques including, but not limited to, infusion, transfusion, or parenterally. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrastemally.

In some embodiments, the composition is administered over a period of 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h. In another embodiment, the composition is administered over a period of 0.25-2 h. In another embodiment, the composition is gradually administered over a period of 1 h. In another embodiment, the composition is gradually administered over a period of 2 h. KITS

The invention provides kits for the treatment of amyloidosis in a subject. In some embodiments, the kit further includes a base editor system or a polynucleotide encoding a base editor system, wherein the base editor polypeptide system a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and a guide RNA. In some embodiments, the napDNAbp is Cas9 or Casl2. In some embodiments, the polynucleotide encoding the base editor is a mRNA sequence. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the kit comprises an edited cell and instructions regarding the use of such cell.

The kits may further comprise written instructions for using the base editor system and/or edited cell. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization. The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for use.

The practice of embodiments of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing embodiments of the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

Throughout the Examples, all of the guide polynucleotides contained the following polynucleotide sequence: Where, “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 phosphorothioate (PS) backbone linkage and wherein bold type represents the spacer sequence.

EXAMPLES

Example 1: 15-Day In Vivo Non-human Primate (NHP) Study of Biodistribution and Base Editing

In this example, sgRNA (GA519; SEQ ID NO: 631) targeting a non-human primate transthyretin (TTR) gene site (i.e., “NHP surrogate sgRNA) corresponding to the human TTR gene site targeted for base editing by sgRNA GA457 (SEQ ID NO: 637) was prepared and formulated with mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V82T (referred to throughout the figures as “ABE9.51”), encapsulated in lipid nanoparticles (LNP1, LNP2, LNP3, and LNP4), and intravenously dosed to non-human primates (NHPs).

The objectives of this study were to determine the targeted biodistribution and base editing of lipid nanoparticles (LNPs) encapsulating mRNA and a single guide RNA when given intravenously once on Day 1 to cynomolgus monkeys.

Each of LNP1 through LNP4 encapsulate the same sgRNA and ABE9.51 mRNA in a weight-to-weight ratio of 1 : 1. In other words, the LNPs were formulated with an equal amount by weight of guide RNA as mRNA. The resulting LNPs encapsulating the sgRNA and mRNA were filtered using 0.22 micron filters. The N/P ratio of the LNPs was 6. While the ratios of ionizable lipid, PEG-lipid, and sterol lipid differed between various LNP formulations, the most notable difference was in the ionizable lipid component in the LNP formulation, where LNP1 comprised Ionizable Lipid 1 (IZ 1) and LNP2, LNP3, and LNP4 comprised IZ2, IZ3, and IZ4, respectively. The components of LNP1 through LNP4 are described in Tables 10A and 10B below. Table 10A. LNP Components

Table 10B. Ionizable Lipids

It should be understood that the mol % of components in Tables 10A may be adjusted and that the mol % included in Table 10A are targeted excipient percentages of the LNP, which is intended to represent the aggregate mol % of all the LNPs formulated in a given batch and that specific LNPs within a batch may have varying mol %. Thus, it is contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 10A may be adjusted, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%. It is further contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 10A with respect to a specific LNP formulated in a given batch of LNPs formulated in accordance with desired target excipient percentages, may vary from the targeted mol %, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%, or even greater than +/- 20%. Further, it should be understood that additional LNP components, including nonlipid components, may be added to the LNP components set-forth in Table 10A.

Physical characteristics of the formulated LNPs are summarized in Table 11 below.

Table 11. LNP Characterization

One of ordinary skill in the art would understand that the average LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 11 are subject to measurement error or accuracy. It is also contemplated herein that the LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 11 may be varied by +/- 1-5%, +/- 5-10%, or +/- 10-20%.

NHP Study Design

Naive female cynomolgus monkeys of Cambodian origin were used as study animals. A premedication regimen comprising dexamethasone and antihistamines (diphenylhydramine and famotidine) was administered to all animals on day -1 and day 1 (predose), at 30 to 60 minutes prior to test article dose administration.

Four groups of three monkeys each were dosed with LNP1 through LNP4 via a single IV infusion at a dose level of 1 mg of combined sgRNA and mRNA per kg of animal body weight, at a dose concentration of 0.167 mg per kg of animal body weight, and at a dose volume of 6 mL per kg of animal body weight.

Blood samples were collected from all animals predose for baseline measurements, including genotyping of the TTR target site via next-generation sequencing, and post-dose at various time points on days 1 through 15 to assess biomarkers, cytokines, pharmacokinetics, and serum safety parameters. Necropsies were performed on all animals at day 15. Liver biopsy samples were collected to assess TTR gene editing.

Analysis of Editing Efficiency

The amount of gene editing in liver tissue of LNPs 1-4 was evaluated by nextgeneration sequencing (NGS) of the TTR target site. The NGS assay included the majority of Exon 1 of the TTR gene, which corresponded to the first 22 amino acids of the TTR protein. The editing target was in the start codon of the gene, specifically the second base on the reverse strand, which was an A.

Editing was assessed through single-plex, NGS amplicon sequencing using a two-step PCR process on genomic DNA isolated from liver tissue from each study animal. Analysis begins with isolation and QC of genomic DNA. Post isolation, first round PCR amplified the genomic specific target with primers carrying partial Illumina adapter sequences. PCR1 products were then used in a second PCR reaction which added on full length adapters as well as dual index sequences. Purified, final products were pooled for NGS data acquisition on Illumina instrumentation. Final editing analysis was performed using the laboratory’s existing computational pipeline.

In Table 12, editing was reported as the percent measure of the editing rate of the second base in the ATG start codon on the reverse strand (A to G conversion).

Table 12. Hepatic TTR Editing Efficiency

Quantification of TTR protein expression in plasma

TTR protein expression in plasma was quantitated using LC-MS/MS. Plasma samples were denatured and proteolytically digested to generate proteotypic peptides, and then spiked with isotopically labeled peptides as internal standards for quantitation. Samples were analyzed with a targeted LC-MS/MS method that monitors four unique TTR peptide sequences. The measured TTR abundance at each timepoint for each animal was normalized against the pre-dose average TTR abundance for the same animal. Normalized percent plasma cTTR (4-peptide average) at terminal (day 15) vs. pre-dose levels is provided in

Table 13 below.

Table 13. cTTR Plasma Levels at Terminal vs. Pre-dose

Thus, infusion of LNP1 through LNP4 in NHPs resulted in editing of the TTR gene in the liver. LNP4 demonstrated greater editing than LNP1, LNP2, and LNP3, and LNP3 demonstrated greater reduction in plasma TTR concentrations than LNP1, LNP2 and LNP4.

Example 2: Base editing of the human or non-human primate transthyretin (TTR) gene using base editor systems

Experiments were undertaken to demonstrate that higher base editing rates were observed in human cells administered a base editor system for altering the TTR gene than in non-human primate cells administered a base editor system for altering the TTR gene. The base editor system contained a guide RNA targeting the TTR gene for base editing and mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration. The amino acid sequence for the base editor (referred to as ABE9.51 in the figures of the disclosure) is provided below. The human TTR gene was targeted for base editing using the guide RNA gRNA2944 (human gRNA) and the cyno TTR gene was targeted for base editing using a cyno guide RNA (cyno gRNA; gRNA2945).

Amino acid sequence for the “ABE9.51” adenosine deaminase base editor used in the Examples of the present disclosure: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSES ATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEI FSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL

NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLD NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 463).

An experiment was undertaken to measure any difference in base editing rates for the base editor systems that correlated with the gRNA polynucleotide used. HEK293T cells containing a polynucleotide containing both the human gRNA and cyno gRNA target sequences were administered base editor systems containing either the cyno gRNA or the human gRNA. The base editor systems were administered to the cells using the Lipofectamine™ MessangerMAX™ transfection reagent. The human gRNA was associated with base editing rates that were between about 1.28- and 2.65-fold (FIG. 1A) greater than those associated with the cyno gRNA.

An experiment was undertaken to compare base editing rates measured in primary human hepatocyte co-cultures and primary cyno hepatocyte co-cultures administered the base editor systems. Base editing was measured in the cells using 2-3 technical replicates. The base editor systems were administered to the primary human hepatocyte cells (PHH) or the primary cyno hepatocyte cells (PCH) using lipid nanoparticles containing the ionizable lipid IZ1 (FIGs. IB) or the ionizable lipid IZ4 (FIGs. 2A, 2B, 6A, and 6B). To alter a nucleobase in the TTR gene, the PHH were administered base editor systems containing the guide gRNA2944, and the PCH were administered base editor systems containing the guide gRNA2945. As a control, the cells were administered a base editor system containing the guide RNA sg23, which is known to be effective in targeting base editor systems to alter a nucleotide in an ALAS1 gene, and mRNA encoding the base editor ABE8.8 (FIG. IB, right panel; FIG. 2B, and FIG. 6B). The base editing rates measured in the PHH at the TTR or ALAS 1 gene locus were at least about 10- to 100-fold higher than those measured in the PCH. The base editor systems administered to the PHH were observed to be consistently more potent than base editor systems administered to the PCH (e.g., the EC50 value for the base editor system administered to the PHH was between 20- and 30-fold lower than that for the base editor system administered to the PCH (FIGs. 2A and 6A)). Similar differences in base editing rates were observed between the PHH and PCH cells across two different target loci and three different donors. Only minor and comparatively negligible differences in base editing rates were observed between cells from different donors. Representative images of the primary cyno hepatocytes used in the experiments are provided in FIGs. 5A and 5B. Without intending to be bound by theory, it is believed that the difference in base editing rates observed between the PHH and PCH is primarily driven mainly by differences between the two species from which the cells were derived.

Example 3: Base editing of the transthyretin (TTR) gene in non-human primates using base editor systems administered using lipid nanoparticles

Experiments were undertaken to investigate base editing in non-human primates carried out using base editor systems administered using lipid nanoparticles and to investigate the pharmacology and toxicity of the lipid nanoparticles. The base editor systems contained a guide RNA targeting the TTR gene for base editing (cyno gRNA; gRNA2945) and mRNA encoding an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration. The lipid nanoparticles contained the ionizable lipid IZ1 and were administered to the non-human primates at doses of 0.5 mg/kg, 1 mg/kg, and 2 mg/kg.

Liver enzyme levels were measured in the non-human primates at different timepoints before and after administration of the lipid nanoparticles. Administration of the lipid nanoparticles was not associated with any changes in alkaline phosphatase (ALP) levels in male or female non-human primates (FIGs. 3C and 3F). Administration of the lipid nanoparticles was associated with minimal, transient, and generally non-dose dependent increases in alanine transaminase (ALT) and aspartate aminotransferase (AST) levels at day 2 or day 8 post-administration, and all ALT and AST levels were back to baseline by day 28 (FIGs. 3A, 3B, 3D, and 3E). Changes were observed in AST and ALT in control animals at a higher magnitude. There were also no changes in body weight or clinical signs in the nonhuman primates. All doses evaluated were well tolerated.

Example 4: A 4 Week In Vivo Non-human Primate (NHP) Study of Biodistribution and Base Editing

In this example, sgRNA (GA519; SEQ ID NO: 631) targeting a non-human primate transthyretin (TTR) gene site (i.e., “NHP surrogate sgRNA) corresponding to the human TTR gene site targeted for base editing by sgRNA GA457 (SEQ ID NO: 637) was prepared and formulated with mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alteration V 82T (referred to throughout the figures as “ABE9.51”), encapsulated in a lipid nanoparticle (LNP1), and intravenously dosed to NHPs.

The objectives of this study were to determine the pharmacokinetics, pharmacodynamics and biodistribution of LNP1 for the treatment of ATTR amyloidosis when given intravenously (60 minutes infusion) as a single dose to cynomolgus monkeys.

LNP1 encapsulated the sgRNA and ABE9.51 mRNA in a weight-to-weight ratio of 1 : 1. In other words, the LNP was formulated with an equal amount by weight of guide RNA as mRNA. The resulting LNPs encapsulating the sgRNA and mRNA were filtered using 0.22 micron filters. The N/P ratio of the LNPs was 6. The components of LNP1 are described in Tables 14 and 15 below.

Table 14. LNP1 Components

Table 15. Ionizable Lipid

It should be understood that the mol % of components in Table 14 may be adjusted and that the mol % included in Table 14 are targeted excipient percentages of the LNP, which is intended to represent the aggregate mol % of all the LNPs formulated in a given batch and that specific LNPs within a batch may have varying mol %. Thus, it is contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 14 may be adjusted, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%. It is further contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 14 with respect to a specific LNP formulated in a given batch of LNPs formulated in accordance with desired target excipient percentages, may vary from the targeted mol %, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%, or even greater than +/- 20%. Further, it should be understood that additional LNP components, including nonlipid components, may be added to the LNP components set-forth in Table 14.

Physical characteristics of the formulation LNP are summarized in Table 16 below.

Table 16. LNP Characterization

One of ordinary skill in the art would understand that the average LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 16 are subject to measurement error or accuracy. It is also contemplated herein that the LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 16 may be varied by +/- 1-5%, +/- 5-10%, or +/- 10-20%.

NHP Study Design

Naive male and female cynomolgus monkeys of Cambodian origin were used as study animals. A premedication regimen comprising dexamethasone and antihistamines (diphenylhydramine and famotidine) was administered to all animals on day -1 and day 1 (predose), at 30 to 60 minutes prior to test article dose administration.

Three groups of two monkeys each (one male and one female) were dosed. As described in Table 17, one group was dosed with vehicle as control, and the remaining three groups were dosed with LNP1 via a single IV infusion at a dose levels of 0.5 mg, 1 mg, and 2 mg of combined sgRNA and mRNA per kg of animal body weight, at dose concentrations of 0.083 mg, 0. 167 mg, and 0.333 mg per kg of animal body weight, respectively, and at a dose volume of 6 mL per kg of animal body weight.

Table 17. Dosing regimens.

Blood samples were collected from all animals predose for baseline measurements, including genotyping of the TTR target site via next generation sequencing (NGS), and postdose at various time points to assess biomarkers, cytokines, pharmacokinetics, and serum safety parameters. Necropsies were performed on all animals at day 28. Biopsy and other samples were collected to assess TTR gene editing as well as other aspects.

Analysis of Editing

The amount of gene editing in liver tissue was evaluated by NGS sequencing of targeted PCR amplicons at the TTR target site. The NGS assay included the majority of Exon 1 of the TTR gene, which corresponded to the first 22 amino acids of the TTR protein. The editing target was in the start codon of the gene, specifically the second base on the reverse strand, which was an A.

Editing was assessed through single-plex, NGS amplicon sequencing using a two-step PCR process on genomic DNA isolated from liver tissue from each study animal. Analysis began with isolation and QC of genomic DNA. Post isolation, first round PCR amplified the genomic specific target with primers carrying partial Illumina adapter sequences. PCR1 products were then used in a second PCR reaction which added on full length adapters as well as dual index sequences. Purified, final products were pooled for NGS data acquisition on Illumina instrumentation. Final editing analysis was performed using the laboratory’s existing computational pipeline.

In Table 18, editing was reported as the percent measure of the editing rate of the second base in the ATG start codon on the reverse strand (A to G conversion).

Table 18. Hepatic TTR Editing of LNP1 at Various Doses

Quantification of TTR protein expression in plasma

TTR protein expression in plasma was quantitated using LC-MS/MS. Plasma samples were denatured and proteolytically digested to generate proteotypic peptides, and then spiked with isotopically labeled peptides as internal standards for quantitation. Samples were analyzed with a targeted LC-MS/MS method that monitors four unique TTR peptide sequences. The measured TTR abundance at each timepoint for each animal was normalized against the pre-dose average TTR abundance for the same animal. Normalized percent plasma cTTR (4-peptide average) at terminal timepoint is provided in Table 19 below. Table 19. cTTR Normalized Plasma Levels at Terminal Timepoint (Day 28)

Example 5: A 28-Day In Vivo Non-human Primate (NHP) Study of Biodistribution, and Base Editing

In this example, NHP surrogate sgRNA (GA519; SEQ ID NO: 631) targeting a non- human primate transthyretin (TTR) gene site (i.e., “NHP surrogate sgRNA) corresponding to the human TTR gene site targeted for base editing by sgRNA GA457 (SEQ ID NO: 637)) was prepared and formulated with mRNA encoding an adenosine base editor (ABE) containing a TadA*8.20 adenosine deaminase domain with the amino acid alterations V82T, Y147T, and Q154S (referred to throughout the figures as “ABE9.52”), encapsulated in a lipid nanoparticle (LNP1), and intravenously dosed to NHPs.

The objectives of this study were to determine the pharmacokinetics, pharmacodynamics and biodistribution of LNP 1 when given intravenously (60 minutes infusion) as a single dose to cynomolgus monkeys.

LNP1 encapsulated the sgRNA and mRNA encoding the ABE in a weight-to-weight ratio of 1 : 1. In other words, the LNP was formulated with an equal amount by weight of guide RNA as mRNA. The resulting LNP formulation was filtered using 0.22 micron filters. The N/P ratio of the LNP1 was 6. The components of LNP 1 are described below in Tables 20 and 21.

Table 20. LNP1 Components

Table 21. Ionizable Lipid

It should be understood that the mol % of components in Table 20 may be adjusted and that the mol % included in Table 20 are targeted excipient percentages of the LNP, which is intended to represent the aggregate mol % of all the LNPs formulated in a given batch and that specific LNPs within a batch may have varying mol %. Thus, it is contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 20 may be adjusted, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%. It is further contemplated herein that the mol % of one or more, or all of the LNP components set forth in Table 20 with respect to a specific LNP formulated in a given batch of LNPs formulated in accordance with desired target excipient percentages, may vary from the targeted mol %, for example, by +/- 1-5%, +/- 5-10%, or +/- 10%-20%, or even greater than +/- 20%. Further, it should be understood that additional LNP components, including nonlipid components, may be added to the LNP components set-forth in Table 20.

Physical characteristics of the LNP1 formulation are summarized in Table 22 below.

Table 22. LNP1 Characterization

One of ordinary skill in the art would understand that the average LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 22 are subject to measurement error or accuracy. It is also contemplated herein that the LNP size, PDI, RNA encapsulation efficiency, and pH values set forth in Table 22 may be varied by +/- 1-5%, +/- 5-10%, or +/- 10-20%.

NHP Study Design

Three groups of monkeys each were dosed. As described in Table 23 below, one group was dosed with vehicle as control, and the remaining two groups were dosed with LNP1 via a single IV infusion at a dose levels of 0.5 mg and 1 mg of combined sgRNA and mRNA per kg of animal body weight, at a dose concentrations of 0.083 mg and 0.167 mg per kg of animal body weight, respectively, and at a dose volume of 6 mL per kg of animal body weight.

Table 23. Dosing regimens.

Blood samples were collected from all animals predose for baseline measurements, including genotyping of the TTR target site via next generation sequencing, and post-dose at various time points to assess biomarkers, cytokines, pharmacokinetics, and serum safety parameters. Necropsies were performed on all animals at day 28. Biopsy and other samples were collected to assess TTR gene editing as well as other aspects.

Analysis of Editing

The amount of gene editing in liver tissue was evaluated by next-generation sequencing (NGS) of the TTR target site. The NGS assay included the majority of Exon 1 of the TTR gene, which corresponded to the first 22 amino acids of the TTR protein. The editing target was in the start codon of the gene, specifically the second base on the reverse strand, which was an A. Editing was assessed through single-plex, NGS amplicon sequencing using a two-step PCR process on genomic DNA isolated from liver tissue from each study animal. Analysis began with isolation and QC of genomic DNA. Post isolation, first round PCR amplified the genomic specific target with primers carrying partial Illumina adapter sequences. PCR1 products were then used in a second PCR reaction which added on full length adapters as well as dual index sequences. Purified, final products were pooled for NGS data acquisition on Illumina instrumentation.

In Table 24, editing was reported as the percent measure of the editing rate of the second base in the ATG start codon on the reverse strand (A to G conversion).

Table 24. Hepatic TTR Editing of LNP1 At Various Doses

Quantification of TTR protein expression in plasma

TTR protein expression in plasma was measured using quantitative peptide mapping LC-MS/MS. Plasma samples were denatured and proteolytically digested to generate proteotypic peptides, and then spiked with isotopically labeled peptides as internal standards for quantitation. Samples were analyzed with a targeted LC-MS/MS method that monitors four unique TTR peptide sequences. The measured TTR abundance at each timepoint for each animal was normalized against the pre-dose average TTR abundance for the same animal. Normalized percent plasma cTTR (4-peptide average) at the terminal timepoint is provided in Table 25 below. Group 2 showed plasma TTR reduction of -41% at day 28 relative to pre-dose baseline, and Group 3 showed plasma TTR reduction of -70% at day 28 relative to pre-dose baseline.

Table 25. cTTR Normalized Plasma Levels at Terminal Timepoint (Day 28) Example 6: Assessment of off-target editing using ABE9.51

Experiments were undertaken to measure levels of gRNA-dependent off-target editing for base editor systems containing an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”) and a guide polynucleotide targeting the TTR gene for base editing, where the base editor systems were administered to cells using lipid nanoparticles at an EC90editin_g dose.

Only one gRNA-dependent off-target site was identified, namely location chr8: 108362156-108362179 in the human genome, which corresponds to the middle of a 125kb intron in the EIF3E gene. The off-target site had the sequence CCCATCA- GCCAAGAATGAGAGG (SEQ ID NO: 483), where the nucleotides in bold differ from the TTR target site, the term indicates a deletion of a nucleotide relative to the TTR target site, and underlined text corresponds to the PAM sequence corresponding to the TTR HM17 target site. The ETF3E gene is expressed in the liver and spleen at > 1 transcript per million (TMP), and humans are tolerant of loss of function variants of the gene. Intentional base editing at the EIF3E off-target site did not affect EIF3E gene expression of splicing patterns in HepG2 cells.

Example 7: Assessment of on- and off-target editing for two different base editor systems in primary human hepatocytes

Experiments were undertaken to compare base editing rates associated with base editor systems containing an adenosine deaminase base editor containing a TadA*8.20 adenosine deaminase domain variant with a V82T amino acid alteration (referred to throughout the figures as “ABE9.51”) or a TadA*8.20 adenosine deaminase domain with the amino acid alterations V82T, Y147T, and Q154S (referred to throughout the figures as “ABE9.52”) and a guide polynucleotide targeting the human TTR gene for base editing (i.e., GA521). Primary human hepatocytes were contacted with mRNA encoding the adenosine deaminase base editor and the guide polynucleotide. The two base editor systems showed similar on- and off- target editing activity (FIG. 4). On- and off-target editing was assessed by amplicon-seq at the TTR (on-target) and EIF3E (off-target) loci. OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations and modifications may be made to the aspects or embodiments described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. The disclosure may be related to International Patent Applications No. PCT/US2022/030359, filed May 20, 2022, or PCT/US2022/029278, filed May 13, 2022, the disclosures of which are each incorporated herein by reference in their entireties for all purposes. The disclosure may be related to U.S. Provisional Patent Applications No. 63/383,394, filed November 11, 2022, or to 63/385,004, filed November 25, 2022, the disclosures of which are each incorporated herein by reference in their entireties for all purposes.

Claims

CLAIMS What is claimed:

1. A lipid nanoparticle (LNP) comprising:

A) a guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA comprises a spacer comprising a nucleotide sequence selected from GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472) and those listed in any of Tables IB, 1C, ID, IE, 2A, 2B, 2D, or 2E;

B) an mRNA molecule, or a polynucleotide encoding the mRNA molecule, wherein the mRNA molecule encodes a base editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises: i) an amino acid sequence having at least 90% identity to the following TadA*7. 10 amino acid sequence, or a truncation thereof lacking only the first M: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH

AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), and ii) a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from the group consisting of: 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; and

C) an ionizable lipid according to any one of the following formulas, or a pharmaceutically acceptable salt thereof: i) a compound of Formula (Ih): wherein: each L¹ and L¹ is independently -C(0)- or -OC(O)-; each L² and L² is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

R¹ is optionally substituted C1-20 aliphatic,

L^CyA is a covalent bond or an optionally sub stituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or optionally substituted C1-6 aliphatic; ii) a compound of Formula (la): wherein:

R¹ is C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation;

X¹ and X² are each independently absent or selected from -O-, -NR²- and wherein each R² is independently hydrogen or C₁-C₆ alkyl; each a is independently an integer between 1 and 6;

X³ and X⁴ are each independently absent or selected from the group consisting of: 4- to 8-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6- membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 5- to 6-membered aryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, 4- to 7-membered cycloalkyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, -O- and -NR³-, wherein each R³ is a independently a hydrogen atom or C₁-C₆ alkyl and wherein X¹-X²-X³-X⁴ does not contain any oxygen-oxygen, oxygen-nitrogen or nitrogen-nitrogen bonds;

X⁵ is -(CH2)b-, wherein b is an integer between 0 and 6;

X⁶ is hydrogen, C₁-C₆ alkyl, 5- to 6-membered heteroaryl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, or -NR⁴R⁵, wherein R⁴ and R⁵ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁴ and R⁵ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; each X⁷ is independently hydrogen, hydroxyl or -NR⁶R⁷, wherein R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; at least one of X¹, X², X³, X⁴, and X⁵ is present;

A¹ and A² are each independently selected from the group consisting of: C5-C12 haloalkyl, C5-C12 alkenyl, C5-C12 alkynyl, (C5-C12 alkoxy)-(CH2)n2-, (C5-C10 aryl)-(CH2)n3- optionally ring substituted with one or two halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, or C₁-C₆ alkoxy groups, and (Ca-Cs cycloalkyl)-(CH2)n4- optionally ring substituted with 1 or 2 C₁-C₆ alkyl groups; or alternatively A¹ and A² join together with the atoms to which they are bound to form a 5- to 6-membered cyclic acetal substituted with 1 or 2 C4-C10 alkyl groups; nl, n2 and n3 are each individually an integer between 1 and 4; and n4 is an integer between zero and 4; iii) A compound of Formula (lb): wherein:

R¹ is C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation;

X⁵ is — (CH jb— , wherein b is an integer between 0 and 6;

X⁷ is hydrogen or -NR⁶R⁷, wherein R⁶ and R⁷ are each independently hydrogen or C₁-C₆ alkyl; or alternatively R⁶ and R⁷ join together with the nitrogen to which they are bound to form a 4- to 7-membered heterocyclyl optionally substituted with 1 or 2 C₁-C₆ alkyl groups, wherein the heterocyclyl optionally includes an additional heteroatom selected from oxygen, sulfur, and nitrogen; at least one of X¹, X², X³, X⁴, and X⁵ is present; and provided that when either X¹ or X² is -O-, neither X³ nor X⁴ is , and when either X¹ or X² is -O-, R⁴ and R⁵ are not both ethyl; iv) A compound of Formula (Ic): or its N-oxide, or a salt thereof, wherein

L is absent, C1-10 alkylenyl, or C2-10 heteroalkylenyl;

L³ is absent, C1-10 alkylenyl, or C2-10 heteroalkylenyl;

R¹ is hydrogen, a 3- to 7-membered cycloaliphatic ring, a 3- to 7-membered heterocyclic ring comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)₂, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)2, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², each R² is independently hydrogen, -CN, -NO2, -OR⁴, -S(O)₂R⁴, -S(O)₂N(R⁴)₂, -(CH₂)_n-R⁴, or an optionally substituted group selected from C1-6 aliphatic, a 3- to 7-membered cycloaliphatic ring, and a 3- to 7- membered heterocyclic ring comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or two occurrences of R², taken together with the atom(s) to which they are attached, form an optionally substituted 4- to 7-membered heterocyclic ring comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH₂)_n-R⁴, or two occurrences of R³, taken together with the atoms to which they are attached, form an optionally substituted 5- to 6-membered heterocyclic ring comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)₂, -OC(O)R⁵, -OC(O)OR⁵, -CN, -C(O)N(R⁵)₂,

-NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, optionally substituted C1-6 aliphatic, or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form an optionally substituted 4- to 7-membered heterocyclic ring comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and n is 0 to 4; v) A compound of Formula (Id): or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

R is hydrogen, optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)₂, -C(O)N(R²)₂, -S(O)₂N(R²)₂, -NR²C(O)R², -OC(O)N(R²)₂, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, -NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², -N(OR²)C(O)N(R²)₂, -N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)2, -OP(O)(OR²)2, or -P(O)(OR²)₂; or ring selected from 3- to 7-membered cycloaliphatic and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, - (CH2)n-R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7- membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, - C(O)N(R⁵)₂,

-NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, or optionally substituted C1-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4; vi) A compound of Formula (le): or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

L² is a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, o

L³ is a covalent bond, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-; , or an optionally substituted saturated or unsaturated, straight or branched

C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-;

X¹ is a covalent bond, -O-, or -NR-;

Z¹ is a covalent bond or -O-;

Z³ is hydrogen, or an optionally substituted group selected from Ci-Cio aliphatic, and 4- to 12-membered saturated or partially unsaturated carbocyclyl; and d is 0, 1, 2, 3, 4, 5, or 6;

, , provided that when L is a covalent bond, then R must b vii) A compound of Formula (If): or a pharmaceutically acceptable salt thereof, wherein: each L¹ and L¹ is independently -C(O)- or -C(O)O-; each L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, or ; each Cy^A is independently an optionally substituted ring selected from phenylene or a 3- to 7-membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each L³ and L³ is independently a covalent bond, -C(O)O-, -OC(O)-, -O-, or -

OC(O)O-; each R¹ and R¹ is independently an optionally substituted group selected from a saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 12- membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, each L⁴ is independently a bivalent saturated or unsaturated, straight or branched Ci- Ce hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵; or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: where x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched Ci- C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 5 - to 10- membered aryl ring and a 3- to 8-membered carbocyclic ring ;

X¹ is a covalent bond, -O-, or -NR-;

X³ is hydrogen or -Cy^B;

Cy^B is an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; provided that when X³ is hydrogen, at least one of R¹ or R¹ is viii) a compound of Formula (Ig): or a pharmaceutically acceptable salt thereof, wherein: each of L¹ and L¹ is independently a covalent bond, -C(O)-, or -OC(O)-; each of L² and L² is independently a covalent bond, an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, each Cy^A is independently an optionally substituted ring selected from phenylene or 3- to 7-membered saturated or partially unsaturated carbocyclene; each m is independently 0, 1, or 2; each of L³ and L³ is independently a covalent bond, -O-, -C(O)O-, -OC(O)-, or - OC(O)O-; each of R¹ and R¹ is independently an optionally substituted group selected from saturated or unsaturated, straight or branched C1-C20 hydrocarbon chain wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-, a 3- to 7- membered saturated or partially unsaturated carbocyclic ring, 1-adamantyl, 2-adamantyl, sterolyl, phenyl, each L⁴ is independently a bivalent saturated or unsaturated, straight or branched Ci- C20 hydrocarbon chain; each A¹ and A² is independently an optionally substituted C1-C20 aliphatic or -L⁵-R⁵, or A¹ and A², together with their intervening atoms, may form an optionally substituted ring: wherein x is selected from 1 or 2; and

# represents the point of attachment to L⁴; each L⁵ is independently a bivalent saturated or unsaturated, straight or branched Ci- C20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O- or -NR-; each R⁵ is independently an optionally substituted group selected from a 6- to 10- membered aryl ring or a 3- to 8-membered carbocyclic ring;

Y¹ is a covalent bond, -C(O)-, or -C(O)O-;

X¹ is a covalent bond, -O-, or -NR-;

X² is an optionally substituted bivalent saturated or unsaturated, straight or branched C1-C12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced with -O-, -NR-, or -Cy^B-; each Cy^B is independently an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclene, phenylene, 3- to 7-membered heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7- membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6- membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; ix) a compound of formula A’:

A’ or its N-oxide, or a pharmaceutically acceptable salt thereof, wherein

L¹ is absent, C1-6 alkylenyl, or C2-6 heteroalkylenyl; each L² is independently optionally substituted C2-15 alkylenyl, or optionally substituted C3-15 heteroalkylenyl;

L is Ci-10 alkylenyl, or C2-10 heteroalkylenyl;

X² is -OC(O)-, -C(O)O-, or -OC(O)O-;

X is absent, -0C(0)-, -C(0)0-, or -OC(O)O-;

R” is hydrogen , or an optionally substituted group selected from C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; each of R and R^a is independently hydrogen, or an optionally substituted group selected from Ce-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic comprising 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1- adamantyl, 2-adamantyl, sterolyl, and phenyl each of L³ and L^3a is independently absent, optionally substituted Ci-io alkylenyl, or optionally substituted C2-10 heteroalkylenyl;

R¹ is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered cycloaliphatic, optionally substituted 3- to 7-membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10- membered bicyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR², -C(O)OR², -C(O)SR², -OC(O)R², -OC(O)OR², -CN, -N(R²)2, -C(O)N(R²)2, -S(O)₂N(R²)2, -NR²C(O)R², -OC(O)N(R²)2, -N(R²)C(O)OR², -NR²S(O)₂R², -NR²C(O)N(R²)2, -NR²C(S)N(R²)2, -NR²C(NR²)N(R²)2, - NR²C(CHR²)N(R²)2, -N(OR²)C(O)R², -N(OR²)S(O)₂R², -N(OR²)C(O)OR², - N(OR²)C(O)N(R²)2,

-N(OR²)C(S)N(R²)2, -N(OR²)C(NR²)N(R²)2, -N(OR²)C(CHR²)N(R²)2, -C(NR²)N(R²)2, -C(NR²)R², -C(O)N(R²)OR², -C(R²)N(R²)₂C(O)OR², -CR²(R³)₂, -OP(O)(OR²)₂, or -P(O)(OR²)₂; or ring selected from 3- to 7-membered cycloaliphatic and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the cycloaliphatic or heterocyclyl ring is optionally substituted with 1-4 R² or R³ groups; each R² is independently hydrogen, oxo, -CN, -NO2, -OR⁴, -S(O)2R⁴, -S(O)2N(R⁴)2, -(CH2)n- R⁴, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 3- to 7- membered cycloaliphatic, 5- to 6-membered monocyclic heteroaryl comprising 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 3- to 7- membered heterocyclyl comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two occurrences of R², taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R³ is independently -(CH2)n-R⁴; or two occurrences of R³, taken together with the atom(s) to which they are attached, form optionally substituted 5- to 6-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁴ is independently hydrogen, -OR⁵, -N(R⁵)2, -OC(O)R⁵, -OC(O)OR⁵, -CN, - C(O)N(R⁵)₂,

-NR⁵C(O)R⁵, -OC(O)N(R⁵)₂, -N(R⁵)C(O)OR⁵, -NR⁵S(O)₂R⁵, -NR⁵C(O)N(R⁵)₂, each R⁵ is independently hydrogen, or optionally substituted Ci-6 aliphatic; or two occurrences of R⁵, taken together with the atom(s) to which they are attached, form optionally substituted 4- to 7-membered heterocyclyl comprising 0-1 additional heteroatom selected from nitrogen, oxygen, and sulfur; each R⁶ is independently C4-12 aliphatic; and each n is independently 0 to 4; or x) a compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein:

L¹ is a covalent bond, -C(O)-, or -OC(O)-;

X¹ is a covalent bond, -O-, or -NR-;

X³ is hydrogen or an optionally substituted ring selected from 3- to 7-membered saturated or partially unsaturated carbocyclyl, phenyl, 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and each R is independently hydrogen or an optionally substituted C₁-C₆ aliphatic group; provided that when L 3 is a covalent bond, then R i must be

2. A lipid nanoparticle (LNP) comprising:

A) a guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a transthyretin (TTR) polynucleotide; B) an mRNA molecule, or a polynucleotide encoding the mRNA molecule, wherein the mRNA molecule encodes a base editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises: i) an amino acid sequence having at least 90% identity to the following

TadA*7.10 amino acid sequence, or a truncation thereof lacking only the first M:

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH

AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA

GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 1), and ii) a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from the group consisting of: 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, 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; and

C) an ionizable lipid according to Formula (I), or a pharmaceutically acceptable salt thereof: wherein: each L¹ and L¹ is independently -C(0)- or -OC(O)-; each L² and L² is independently an optionally substituted bivalent saturated or unsaturated, straight or branched C1-12 hydrocarbon chain;

L³ is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-;

R¹ is optionally substituted C1-20 aliphatic,

Y¹ is -C(O)- or -C(O)O-;

Y³ is optionally substituted C1-20 aliphatic;

X¹ is a covalent bond, -O-, or -NR-;

3. The LNP of claim 1 or claim 2, wherein the lipid nanoparticle comprises an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

4. The LNP of claim 1 or claim 2, wherein the lipid nanoparticle comprises an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

5. The LNP of claim 1 or claim 2, wherein the guide RNA comprises a scaffold with the following nucleotide sequence: gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugG caccgagucggugcuususus (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 phosphorothioate (PS) backbone linkage.

6. The LNP of claim 1, 2, or 5, wherein the guide RNA comprises a spacer comprising the following nucleotide sequence: GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472).

7. The LNP of claim 6, wherein the guide RNA comprises the following nucleotide sequence: 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 s is a phosphorothioate (PS) backbone linkage.

8. The LNP of claim 2 or claim 5, wherein the guide RNA comprises a spacer selected from those listed in any of Tables IB, 1C, ID, IE, 2A, 2B, 2D, or 2E.

9. The LNP of claim 1 or claim 2, wherein the guide RNA comprises 2-5 contiguous 2’- O-methylated nucleobases at the 3’ end and at the 5’ end.

10. The LNP of claim 1 or claim 2, wherein the guide RNA comprises 2-5 contiguous nucleobases at the 3 ’ end and at the 5 ’ end that comprise phosphorothioate intemucleotide linkages.

11. The LNP of claim 1 or claim 2, wherein the napDNAbp domain comprises a Cas9 polypeptide.

12. The LNP of claim 1 or claim 2, wherein the napDNAbp is a nickase.

13. The LNP of claim 1 or claim 2, wherein the base editor polypeptide comprises an amino acid sequence with at least about 90% identity to the following amino acid sequence: ABE9.51

14. A lipid nanoparticle (LNP) comprising:

A) a guide RNA having the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm

AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smU smUsmU (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 s is a phosphorothioate (PS) backbone linkage;

B) 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, wherein the adenosine deaminase domain comprises an amino acid sequence with at least 90% identity to the following amino TadA*7.10 amino acid sequence, or a truncation thereof lacking only the first M:

MSEVE FSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE IMA LRQGGLVMQNYRLIDATLYVT FEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVE ITEGILADECAALLCY FFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), and comprises the following combination of amino acid alterations relative to the TadA*7.10 amino acid sequence: I76Y, V82T, Y123H, Y147R, and Q154R; and C) an ionizable lipid having the following structure, or a pharmaceutically acceptable salt thereof:

(IZ4).

15. The lipid nanoparticle of claim 14, wherein the base editor polypeptide comprises an amino acid sequence with at least about 90% identity to the following amino acid sequence: ABE9.51 PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 463).

16. A base editor system comprising:

A) a guide RNA, or a polynucleotide encoding the guide RNA, wherein the guide RNA is capable of directing a base editor polypeptide to alter a nucleotide in a transthyretin (TTR) polynucleotide; and

B) a base editor polypeptide, or one or more 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, wherein the adenosine deaminase domain comprises: i) an amino acid sequence with at least 90% identity to the following amino

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH

AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 1), and ii) a combination of amino acid alterations relative to the TadA*7.10 amino acid sequence selected from the group consisting of: 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.

17. The base editor system of claim 16, wherein the guide RNA comprises a spacer selected from those listed in any of Tables IB, 1C, ID, IE, 2A, 2B, 2D, or 2E.

18. The base editor system of claim 16, wherein the guide RNA comprises a scaffold with the following nucleotide sequence: gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugG caccgagucggugcuususus (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 phosphorothioate (PS) backbone linkage.

19. The base editor system of claim 16 or claim 18, wherein the guide RNA comprises a spacer with the following nucleotide sequence: GCCAUCCUGCCAAGAAUGAG (SEQ ID NO: 472).

20. The base editor system of claim 16, wherein the guide RNA comprises 2-5 contiguous 2’-O-methylated nucleobases at the 3’ end and at the 5’ end.

21. The base editor system of claim 16, wherein the guide RNA comprises 2-5 contiguous nucleobases at the 3 ’ end and at the 5 ’ end that comprise phosphorothioate intemucleotide linkages.

22. The base editor system of claim 16, wherein the guide RNA comprises the following nucleotide sequence: mGsmCsmCsAUCCUGCCAAGAAUGAGmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAm AGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmU mGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU smU smUsmU (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 s is a phosphorothioate (PS) backbone linkage.

23. The base editor system of claim 16, wherein the napDNAbp domain comprises a Cas9 polypeptide.

24. The base editor system of claim 16, wherein the napDNAbp is a nickase.

25. The base editor system of claim 16, wherein the base editor polypeptide comprises an amino acid sequence with at least about 90% identity to the following amino acid sequence: ABE9.51

MSEVE FSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE IMA LRQGGLVMQNYRLYDATLYTT FEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP

26. A polynucleotide or set of polynucleotides encoding the base editor system of any one of claims 16-25.

27. A cell comprising the base editor system of any one of claims 16-25 or the polynucleotide or set of polynucleotides of claim 26.

28. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-15, the base editor system of any one of claims 16-25, the polynucleotide or set of polynucleotides of claim 26, or the cell of claim 27, and a pharmaceutically acceptable carrier or excipient.

29. A kit containing the lipid nanoparticle of any one of claims 1-15, the base editor system of any one of claims 16-25, the polynucleotide or set of polynucleotides of claim 26, the cell of claim 27, or the pharmaceutical composition of claim 28, and a container.

30. A method for modifying a target nucleobase in a transthyretin (TTR) polynucleotide in a cell, the method comprising contacting the cell with the lipid nanoparticle of any one of claims 1-15 or the base editor system of any one of claims 16-25, thereby modifying the target nucleobase in the TTR polynucleotide.

31. The method of claim 30, wherein the cell is in a subject.

32. A method of treating a disease in a subject in need thereof, wherein the disease is associated with a pathogenic mutation in a transthyretin (TTR) polynucleotide in the subject, the method comprising administering to the subject the lipid nanoparticle of any one of claims 1-15 or the base editor system of any one of claims 16-25, thereby altering a target nucleobase in the TTR polynucleotide.

33. The method of claim 31 or claim 32, wherein an ionizable lipid of the lipid nanoparticle has a half-life in the liver of the subject that is less than 14 days.

34. The method of claim 31 or claim 32, wherein the target nucleobase is altered with an editing efficiency of at least about 50% or 60% in in the cell and/or in the liver of the subject.

35. The method of claim 31 or claim 32, wherein altering the target nucleobase results in a reduction in TTR polypeptide levels in the cell and/or in the subject.

36. The method of claim 35, wherein TTR polypeptide levels are reduced by at least about 80% or 90%.

37. The method of claim 32, wherein the method is associated with only a transient increase or no increase in alkaline phosphatase (ALP), alanine transaminase (ALT), and/or aspartate aminotransferase (AST) levels in the subject.

38. The method of claim 37, wherein the method is associated with no increase in ALP levels in the subject.

39. The method of claim 32, wherein the disease is selected from the group consisting of amyloidosis, cardiomyopathy, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), familial transthyretin amyloidosis (FTA), senile systemic amyloidosis (SSA), and transthyretin amyloidosis.