WO2023173089A1

WO2023173089A1 - Compositions and methods for nucleic acid editing

Info

Publication number: WO2023173089A1
Application number: PCT/US2023/064140
Authority: WO
Inventors: Andrew FRALEY; Stephen Su; Duncan Brown
Original assignee: Korro Bio, Inc.
Priority date: 2022-03-10
Filing date: 2023-03-10
Publication date: 2023-09-14

Abstract

The present disclosure provides, in part, compositions and methods comprising nucleic acid editing complexes and uses thereof, as well as methods of treatment for diseases and disorders originating from genetic mutations.

Description

COMPOSITIONS AND METHODS FOR NUCLEIC ACID EDITING

FIELD OF THE INVENTION

[0001] The present disclosure relates to the field of nucleic acid editing. Specifically, the present disclosure provides, in part, compositions and methods comprising nucleic acid editing complexes and uses thereof, as well as methods of treatment for diseases, conditions and disorders originating from genetic mutations.

RELATED APPLICATIONS

[0002] The application claims the benefit of, and priority to, U.S. Provisional Application No. 63/318,609, filed March 10, 2022, U.S. Provisional Application No. 63/318,624, filed March 10, 2022, and U.S. Provisional Application No. 63/318,639, filed March 10, 2022, the contents of each are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable XML format copy of the Sequence Listing (Filename: “KOR- 015PC_Sequence_Listing”; Date recorded: March 10, 2023; File size: 121 ,656 bytes).

BACKGROUND

[0004] The process of RNA editing alters the nucleotide sequences of RNA transcripts (e.g., messenger RNAs) once they are synthesized and thereby changes the coded message the mRNA transcript carries. In animals, two principal types of mRNA editing occur: the deamination of adenosine to produce inosine (A-to- I editing) and, less frequently, the deamination of cytosine to produce uracil (C-to-U editing). In the former, the resulting inosine is read as guanosine by translation machinery. If the edit occurs in a coding region, it can have a profound impact on downstream processes, such as changing the amino acids of the protein or producing a truncated protein by creating a premature stop codon, thereby affecting the protein’s structure and function. Edits that occur outside coding sequences can affect the pattern of pre-mRNA splicing, the transport of mRNA from the nucleus to the cytosol, the efficiency with which the RNA is translated, or the base-pairing between microRNAs (miRNAs) and their mRNA targets.

[0005] The process of A-to-l editing is particularly prevalent in humans, where it occurs in approximately 1 ,000 genes. Enzymes called ADARs (adenosine deaminases acting on RNA) perform this type of editing. Endogenously, these enzymes recognize double-stranded RNA structures that are formed through basepairing between the site to be edited and a complementary sequence located elsewhere on the same RNA molecule, typically in an intron. The structure of the double-stranded RNA specifies whether the mRNA is to be edited, and if so, where the edit should be made.

[0006] Advances in the field have led to the development of technologies that harness the power of nucleic acid-editing enzymes by way of editing or altering specific nucleobases in RNA and DNA of cells. However, there is still a need in the field for gene editing strategies for the purpose of therapeutically targeting and correcting genetic mutations that give rise to genetic diseases, conditions and disorders. Furthermore, there remains the need for nucleic acid-editing processes that yield lower off-target mutations, such as those resulting from technologies involving nuclease activities (e.g. , CRISPR).

[0007] Though site-specific RNA editing strategies have progressed in the field and look promising, challenges remain with regards to various RNA editing systems known in the art. See, Vogel and Stafforst, “Critical review on engineering deaminases for site-directed RNA editing,” Current Opinion in Biotech 55 (2019) at 75-76. Accordingly, there remains the need for enhanced RNA-editing systems.

SUMMARY

[0008] Accordingly, the present disclosure provides, in part, compositions and methods for site-specific editing of a target nucleotide (e.g., within an RNA molecule) in a cell. Such compositions and methods, in embodiments, provide increased efficiency and/or specificity of nucleic acid editing than which is provided in present systems. Further, such compositions and methods, in embodiments, provide for nucleotide changes which are reversible and tunable in nature. Without wishing to be bound by theory, this limited duration of effect reduces a likelihood of danger related to harmful off-site editing, and tunability allows continuous adjustment of the effect in a dose-dependent manner, to mitigate or eliminate adverse effects. Further still, the present disclosure allows for manipulation of cellular pathways and processes of which permanent manipulation would have serious health consequences. Importantly, in embodiments, the present disclosure allows for genetic manipulation that is nuclease-free and spares the genetic information encoded in DNA. Stated another way, in embodiments, the present disclosure allows for genetic manipulation that is not DNA level and therefore less likely to cause a permanent effect (e.g. avoids an adverse outcome at the DNA level).

[0009] In aspects, the present compositions and methods provide for targeted enzymatic functionality that supports editing of a target nucleotide combined with targeted nucleic acid specificity that locates the enzymatic functionality to the target nucleotide. [0010] In aspects, fusion proteins of a deaminase are linked to a mutant A (also referred to as “lambda” herein) N-peptide are provided. In embodiments, these fusion proteins find use in efficient and/or specific base editing. In embodiments, the present disclosure relates to a fusion protein between a deaminase, such as an ADAR, or the catalytic domain thereof, linked to a mutant A N-peptide. In embodiments, the fusion protein is capable of specifically interacting with a hairpin (e.g., a hairpin RNA) oligonucleotide.

[0011] In aspects, methods of the present disclosure comprise contacting a cell with a deaminase linked to a mutant A N-peptide, and an antisense RNA oligonucleotide linked to a hairpin (e.g., a hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the mutant A N-peptide. For example, the cognate hairpin oligonucleotide can be a BoxB hairpin RNA oligonucleotide. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the mutant N-peptide and hairpin oligonucleotide interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0012] The present disclosure further contemplates methods for treating diseases, conditions and/or disorders that manifest from genetic mutation(s). Accordingly, methods of treating a disorder, condition or disease by editing a target nucleotide within an RNA molecule in a cell include contacting a cell with a deaminase linked to a mutant A N-peptide, and an antisense RNA oligonucleotide linked to a hairpin (e.g., a hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the mutant A N-peptide. For example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the mutant A N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0013] In embodiments, fusion proteins of a deaminase herein are linked to a lambdoid bacteriophage N-peptide (also referred to as an “N-peptide” herein) are provided. In embodiments, these fusion proteins find use in efficient and/or specific base editing. In embodiments, the present disclosure relates to a fusion protein between a deaminase, such as an ADAR, or the catalytic domain thereof, linked to a P22 N-peptide, or a variant thereof. In embodiments, the fusion protein is capable of specifically interacting with a hairpin (e.g., a hairpin RNA) oligonucleotide.

[0014] In embodiments, methods of the present disclosure comprise contacting a cell with a deaminase linked to a lambdoid bacteriophage N-peptide, and an antisense RNA oligonucleotide linked to a hairpin (e.g., a hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the N-peptide. For example, the cognate hairpin oligonucleotide can be a BoxB hairpin RNA oligonucleotide. In embodiments, the lambdoid bacteriophage is P22. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the P22 N-peptide and hairpin oligonucleotide interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0015] The present disclosure further contemplates methods for treating diseases, conditions and/or disorders that manifest from genetic mutation(s). Accordingly, methods of treating a disorder, condition or disease by editing a target nucleotide within an RNA molecule in a cell include contacting a cell with a deaminase linked to a lambdoid bacteriophage N-peptide, and an antisense RNA oligonucleotide linked to a hairpin (e.g . , a hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the N-peptide. For example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the lambdoid bacteriophage is P22. In embodiments, the lambdoid bacteriophage is P21 . In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the P22 N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change. In embodiments, the P21 N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0016] In embodiments, the present disclosure relates to a fusion protein between a deaminase, such as an ADAR, or the catalytic domain thereof, linked to a P21 (also known as q>21 or phi 21) N-peptide, or a variant thereof. In embodiments, the fusion protein is capable of specifically interacting with a hairpin (e.g., a hairpin RNA) oligonucleotide.

[0017] In embodiments, the lambdoid bacteriophage is P21. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the P21 N-peptide and hairpin oligonucleotide interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0018] Methods of the present disclosure further contemplate adenosine deaminases, wherein an adenosine deaminase may yield an inosine nucleotide upon inducing an adenosine (A) to inosine (I) nucleotide change. In embodiments, the adenosine deaminase is a fragment comprising the catalytic domain of the adenosine deaminase. In embodiments, the fragment comprises a catalytic domain and one or more mutations. In embodiments, the adenosine deaminase is selected from ADAR1 and ADAR2, or fragments or variants thereof.

[0019] In embodiments, the mutant A N-peptide of the present disclosure is derived from the A lambdoid bacteriophage N protein. In embodiments, the A N-peptide is the wild-type amino-terminal domain A N- peptide and comprises the amino acid sequence of SEQ ID NO: 1. In embodiments, the A N-peptide is a mutant of the wild-type amino-terminal domain A N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1 ). For example, the mutant A N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type A N-peptide. In embodiments, such mutations increase the binding affinity of the mutant A N-peptide to its hairpin, as compared to the binding affinity of the wild-type A N-peptide to its hairpin.

[0020] For example, the mutant A N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type A N-peptide. In embodiments, such mutations increase the binding affinity of the mutant A N-peptide to its hairpin, as compared to the binding affinity of the wild-type A N-peptide to its hairpin.

[0021] In embodiments contemplated by the present disclosure, the deaminase and the mutant A N- peptide are a fusion protein, optionally connected by a chemical linker. By way of non-limiting example, the deaminase can be linked to at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 mutant A N- peptides. In embodiments, the deaminase and the mutant A N-peptide comprise a nuclear localization signal, optionally wherein the RNA molecule comprising the target nucleotide is in the nucleus of the cell. In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal.

[0022] In embodiments, the lambdoid bacteriophage N-peptide of the present disclosure is derived from the P22 lambdoid bacteriophage. In embodiments, the lambdoid bacteriophage N-peptide is the wild-type amino-terminal domain P22 N-peptide and comprises the amino acid sequence of SEQ ID NO: 10. In embodiments, the P22 N-peptide is a variant of the wild-type amino-terminal domain P22 N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain P22 N-peptide. In embodiments, one or more mutations of the wild-type amino-terminal domain P22 N-peptide do not yield the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1).

[0023] For example, the variant P22 N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type N-peptides, respectively. In embodiments, such mutations increase the binding affinities of the variant N-peptides to their respective hairpins, as compared to the binding affinities of the wild-type N-types to their respective hairpins.

[0024] In embodiments contemplated by the present disclosure, the deaminase and the lambdoid bacteriophage N-peptide are a fusion protein, optionally connected by a chemical linker. By way of nonlimiting example, the deaminase can be linked to at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 lambdoid bacteriophage N-peptides. In embodiments, the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal, optionally wherein the RNA molecule comprising the target nucleotide is in the nucleus of the cell. In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal.

[0025] In embodiments, the lambdoid bacteriophage N-peptide of the present disclosure is derived from the P21 (a.k.a. <p21 or phi21 ) lambdoid bacteriophage. In embodiments, the lambdoid bacteriophage N- peptide is the wild-type amino-terminal domain P21 N-peptide and comprises the amino acid sequence of SEQ ID NO: 36. In embodiments, the P21 N-peptide is a variant of the wild-type amino-terminal domain P21 N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain P21 N- peptide. In embodiments, one or more mutations of the wild-type amino-terminal domain P21 N-peptide do not yield the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1).

[0026] For example, the variant P21 N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type N-peptides, respectively. In embodiments, such mutations increase the binding affinities of the variant N-peptides to their respective hairpins, as compared to the binding affinities of the wild-type N-types to their respective hairpins.

[0027] In embodiments contemplated by the present disclosure, the deaminase and the lambdoid bacteriophage N-peptide are a fusion protein, optionally connected by a chemical linker. By way of nonlimiting example, the deaminase can be linked to at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 lambdoid bacteriophage N-peptides. In embodiments, the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal, optionally wherein the RNA molecule comprising the target nucleotide is in the nucleus of the cell. In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal.

[0028] Methods of the present disclosure further contemplate an antisense RNA oligonucleotide that hybridizes with a wild-type or mutant target sequence in an endogenous RNA molecule that comprises a target nucleotide to be edited. In embodiments of the present disclosure, the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence. In embodiments, the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence. For example, the target nucleotide can be a genetic mutation associated with a genetic disease, condition or disorder.

[0029] In embodiments of the present disclosure, the mutant A N-peptide interacts with a hairpin (e.g., a hairpin RNA, where there is hairpin recognition of the K N-peptide) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of N-peptide. By way of non-limitation, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5' of the target mutation. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3’ of the target mutation.

[0030] In embodiments of the present disclosure, the lambdoid bacteriophage N-peptide interacts with a hairpin (e.g., a hairpin RNA, where there is hairpin recognition of the P22 N-peptide) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the N-peptide. By way of nonlimitation, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5’ of the target mutation. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3’ of the target mutation.

[0031] In embodiments of the present disclosure, the lambdoid bacteriophage N-peptide interacts with a hairpin (e.g., a hairpin RNA, where there is hairpin recognition of the P21 N-peptide) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the N-peptide. By way of nonlimitation, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5’ of the target mutation. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3' of the target mutation.

[0032] In embodiments, such components of the present disclosure as previously described are synthetically generated or generated from a plasmid. In embodiments, the components are contained within a plasmid or vector, optionally wherein the vector is a viral vector. Such viral vectors can include, but are not limited to, adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses. In embodiments, the present disclosure contemplates the use of mRNA and/or lipid nanoparticles for delivery of the components of the nucleic acid-editing system to a cell. DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic that depicts an illustrative, non-limiting example of the embodiments of the present disclosure. The figure depicts editing of a target nucleotide within an RNA molecule, mediated by (i) a deaminase linked to a mutant lambda lambdoid bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein the lambdoid bacteriophage N-peptide interacts with the hairpin RNA oligonucleotide. Although only one N-peptide is shown in the schematic, the present disclosure contemplates more than one N-peptide.

[0034] Figure 2 is a schematic that depicts an illustrative, non-limiting example of the embodiments of the present disclosure. The figure depicts editing of a target nucleotide within a disease target, mediated by (i) a deaminase domain linked to a lambdoid bacteriophage N-peptide, and (ii) an antisense 2boxB guide RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein the lambdoid bacteriophage N-peptide interacts with the hairpin RNA oligonucleotide.

[0035] Figure 3 is a graph depicting the percentage of AN-peptide editing in an in vitro editing experiment using the Human SerpinAI E342K gene.

[0036] Figure 4 is a graph depicting the percentage of AN-peptide editing in an in vitro editing experiment using the Human IDUA W402X gene.

[0037] Figure 5 is a graph depicting the percentage of P22 N-peptide editing in an in vitro editing experiment using the Human SerpinAI E342K gene.

[0038] Figure 6 is a graph depicting the in vitro editing efficiency of a mutant ADAR (E488Q) fusion with 4xAN-peptide editing in correcting a Human SerpinAI E342K gene mutation.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure provides, in part, compositions and methods for site-specific editing of a target nucleotide (e.g., within an endogenous RNA molecule) in a cell. Specifically, in embodiments, the fusion of a deaminase, such as (without limitation) an ADAR, or the catalytic domain thereof, with a mutant A N-peptide, allows for nucleic acid editing that is specific and not substantially hindered by off-target effects, while also allowing for a transient and tunable effect.

[0040] In embodiments, the fusion of a deaminase, such as (without limitation) an ADAR, or the catalytic domain thereof, with a lambdoid bacteriophage N-peptide, such as (without limitation) P22, or a variant thereof, allows for nucleic acid editing that is specific and not substantially hindered by off-target effects, while also allowing for a transient and tunable effect.

[0041] In embodiments, the fusion of a deaminase, such as (without limitation) an ADAR, or the catalytic domain thereof, with a lambdoid bacteriophage N-peptide, such as (without limitation) P21 , or a variant thereof, allows for nucleic acid editing that is specific and not substantially hindered by off-target effects, while also allowing for a transient and tunable effect.

[0042] Accordingly, the compositions and methods of the present disclosure permit alteration of the genetic code in a therapeutically beneficial manner.

[0043] In aspects, methods of the present disclosure comprise (a) contacting a cell with (i) a deaminase linked to a mutant A N-peptide, P22 N-peptide, or P21 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of mutant A N-peptide, P22 N-peptide, or P21 N-peptide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the mutant A N-peptide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the P22 N-peptide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the P21 N-peptide. For example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the deaminase is linked to a mutant A N-peptide. In embodiments, the deaminase is linked to a lambdoid bacteriophage is P22 N-peptide. In embodiments, the deaminase is linked to a lambdoid bacteriophage is P21 N-peptide. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the A N-peptide, P22 N-peptide, or P21 N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide.

[0044] The present disclosure further contemplates methods for treating diseases, conditions and/or disorders that manifest from genetic mutation(s). Accordingly, methods of treating a disorder, conditions or disease by editing a target nucleotide within an RNA molecule in a cell include contacting a cell with a deaminase linked to a mutant A N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide, and an antisense RNA oligonucleotide linked to a hairpin (e.g., hairpin RNA) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the A N-peptide. For example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide sequence. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the lambdoid bacteriophage is P22 N-peptide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the lambdoid bacteriophage is P21 N-peptide. In embodiments, the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide. In embodiments, the A N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change. In embodiments, the P22 N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change. In embodiments, the P21 N-peptide and hairpin interact, and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0045] In embodiments, the present disclosure contemplates a method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant A N-peptide, P22 N-peptide, or P21 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the mutant A N-peptide, P22 N-peptide, or P21 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A N-peptide, P22 N-peptide, or P21 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[0046] In embodiments, the present disclosure contemplates a method of treating a disease, condition or disorder by editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant A N-peptide, P22 N-peptide, or P21 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin of the AN-peptide, P22 N-peptide, or P21 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the A N-peptide, P22 N-peptide, or P21 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

Nucleic Acid-Editing Enzymes and Deaminase Domains

[0047] Some embodiments of this disclosure provide strategies, systems, compositions, reagents, and methods that are useful for the targeted editing of nucleic acids, including editing a single site within a subject's genome, e.g., the human genome. In embodiments, nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. Methods of the present disclosure further contemplate the use of adenosine deaminases, wherein an adenosine deaminase may yield an inosine nucleotide/nucleoside upon inducing an adenosine (A) to inosine (I) nucleotide change. [0048] In embodiments, the nucleic acid-editing domain is an RNA-editing domain. In embodiments, the nucleic acid-editing domain is a deaminase domain. In embodiments, the deaminase is an adenosine deaminase. For example, the deaminase can be selected from adenosine deaminase, RNA specific (ADAR), adenosine deaminase, RNA specific 2 (ADAR2), adenosine deaminase, RNA specific B1 (ADARB1 ), adenosine deaminase like (ADAL), adenosine deaminase 2 (ADA2), adenosine monophosphate deaminase 1 (AMPD1), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase domain containing 1 (ADAD1 ), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase, tRNA specific 1 (ADAT 1 ), adenosine deaminase, tRNA specific 2 (ADAT2), adenosine deaminase, tRNA specific 3 (ADAT3), TadA or fragments or variants thereof. In embodiments, the deaminase is an AD AT family deaminase. In embodiments, adenosine deaminase of the present disclosure is a fragment comprising the catalytic domain of the adenosine deaminase. In embodiments, the fragment comprises a catalytic domain and one or more mutations. For example, in embodiments, the fragment or variant of the adenosine deaminase can comprise an amino acid sequence having 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%, or at least 99% sequence identity with the adenosine deaminase amino acid sequences provided herein. In embodiments, the fragment is the catalytic domain of the deaminase. In embodiments, the deaminase comprises any isoform thereof, such as those known in the art (e.g., ADAR comprises two isoforms: ADAR1 p150 and ADAR1 p110). Non-limiting examples of adenosine deaminases can be found throughout Y. Savva et al., Genome Biology (2012) 13:252, which is hereby incorporated by reference in its entirety.

[0049] In embodiments, the adenosine deaminase of the present disclosure is adenosine deaminase, RNA specific 2 (ADAR2). For example, the ADAR2 deaminase can comprise a fragment that comprises a catalytic domain, wherein the catalytic domain is the deaminase domain of human ADAR2 (SEQ ID NO: 103), shown below:

LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHA RRKVLAGWMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAE IISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYIS TSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNA SIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSH LLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQ

FSLTP (SEQ ID NO: 103).

[0050] In embodiments, the ADAR2 fragment comprises a catalytic domain and one or more mutations. For example, in embodiments, the fragment or variant of ADAR2 can comprise an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 103. In embodiments, the catalytic domain is the deaminase domain of human ADAR2 comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 103. In embodiments, the ADAR2 comprises the amino acid sequence of SEQ ID NO: 103.

[0051] In embodiments, the ADAR2 comprises an amino acid sequence comprising a mutation of E488Q with respect to SEQ ID NO: 103. Without wishing to be bound by theory, it is contemplated that the E488Q mutation increase A-to-l editing efficiency by increasing the catalytic rate and the affinity of the catalytic domain for substrate RNAs. In embodiments, the fragment or variant of ADAR2 comprises an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 104, as shown below:

LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHA RRKVLAGWMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAE IISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYIS TSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNA SIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSH LLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQ FSLTP (SEQ ID NO: 104, E488Q emphasis added).

[0052] In embodiments, the catalytic domain is a variant deaminase domain of human ADAR2 comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 104. In embodiments, the ADAR2 comprises the amino acid sequence of SEQ ID NO: 104.

[0053] In embodiments, the deaminase is a nucleic acid or protein construct including a catalytic domain of a variant of a human ADAR comprising an E488Q mutant fused to one or more copies of a A N-peptide. In embodiments, the deaminase construct comprises a nucleic acid (e.g., DNA sequence, or RNA complement thereof) having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 101. In embodiments, the deaminase construct comprises the nucleic acid sequence of SEQ ID NO: 101. In embodiments, the deaminase construct comprises an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 102. In embodiments, the deaminase construct comprises the nucleic acid sequence of SEQ ID NO: 102.

[0054] In embodiments, the ADAR2 comprises an amino acid sequence comprising an addition of amino acid residues LV with respect to SEQ ID NO: 103. In embodiments, the fragment or variant of ADAR2 comprises an amino acid sequence having 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%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 105, as shown below:

LHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHA RRKVLAGWMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAE IISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYIS TSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNA SIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILG SLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNF SVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSH LLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQ FSLTPLV (SEQ ID NO: 105, LV addition emphasis added)

[0055] In embodiments, the catalytic domain is a variant deaminase domain of human ADAR2 comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 105. In embodiments, the ADAR2 comprises the amino acid sequence of SEQ ID NO: 105.

[0056] In embodiments, the adenosine deaminase is a fragment comprising the catalytic domain of the adenosine deaminase. In embodiments, the fragment comprises a catalytic domain and one or more mutations. In embodiments, deaminase enzyme or deaminase domain amino acid sequences of the present disclosure are variants having 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%, or at least 99% similarity to the amino acid sequences provided herein. In embodiments, the adenosine deaminase is ADAR2, or fragments or variants thereof. Additional suitable deaminase enzymes or domains will be apparent to the skilled artisan based on this disclosure.

[0057] In embodiments, the present compositions comprise a nucleic acid sequence having 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%, or at least 99% similarity to the nucleic acid sequences SEQ ID NOs: 68, 70, 72-79, provided in Table 4 or a codon-optimized version thereof.

[0058] In embodiments, the present compositions comprise a nucleic acid, or a codon-optimized version thereof, encoding a protein having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 69, 71 , provided in Table 4.

[0059] In embodiments, the present compositions comprise an amino acid sequence having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 69, 71 , provided in Table 4.

[0060] In embodiments, the present compositions comprise a nucleic acid sequence having 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%, or at least 99% similarity to the nucleic acid sequences SEQ ID NOs: 72-73, 76-79, 81 , 83, 85, 87, 89- 90, provided in Table 8, or a codon-optimized version thereof.

[0061] In embodiments, the present compositions comprise a nucleic acid, or a codon-optimized version thereof, encoding a protein having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 82, 84, 86, 88, provided in Table 8.

[0062] In embodiments, the present compositions comprise an amino acid sequence having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 82, 84, 86, 88, provided in Table 8.

[0063] In embodiments, the present compositions comprise a nucleic acid sequence having 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%, or at least 99% similarity to the nucleic acid sequences SEQ ID NOs: 72-73, 76-79, 92, 94, 96, 98, 100, provided in Table 12, or a codon-optimized version thereof. [0064] In embodiments, the present compositions comprise a nucleic acid, or a codon-optimized version thereof, encoding a protein having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 93, 95, 97, 99, provided in Table 12.

[0065] In embodiments, the present compositions comprise an amino acid sequence having 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%, or at least 99% similarity to the amino acid sequences SEQ ID NOs: 93, 95, 97, 99, provided in Table 12.

A Bacteriophage N-Peptide

[0066] In embodiments, the present disclosure contemplates a N-peptide linked to a deaminase domain, or variant thereof. In embodiments, the N-peptide comprises a helical structure. In embodiments, the helical peptide is a A lambdoid bacteriophage N-peptide. In embodiments, the present disclosure contemplates at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 N-peptides linked to a deaminase domain.

[0067] In embodiments, the mutant A N-peptide of the present disclosure is derived from the A lambdoid bacteriophage N protein. The lambdoid bacteriophage N proteins comprise arginine-rich motifs (ARMs) necessary for transcription antitermination that can bind to and/or interact with hairpin sequences. Without wishing to be bound by theory, in embodiments of the present disclosure contemplate that an arginine-rich motif (ARM) can bind to and/or interact with a RNA hairpin sequence (e.g., boxB RNA hairpin) with a characteristic induced a-induced helical structure. Indeed, the present disclosure contemplates that variants and/or mutant lambdoid bacteriophage N-peptides (e.g., mutant A N-peptide) can exhibit enhanced binding affinity properties for their respective hairpin sequences compared to the wild-type phage N-peptides.

[0068] In embodiments, the A N-peptide is the wild-type amino-terminal domain A N-peptide and comprises the amino acid sequence of SEQ ID NO: 1 :

[0069] MDAQTRRRERRAEKQAQWK (SEQ ID NO: 1).

[0070] In embodiments, a A N-peptide, and/or mutants thereof, comprises an arginine-rich motif (ARM). In embodiments, an amino-terminal domain A N-peptide of the present disclosure comprises an Alanine (Ala) at position 3 (respective of wild-type A N-peptide of SEQ ID NO: 1 ) and an Arginine (Arg) at each of positions 6, 10, and 11 (respective of wild-type A N-peptide of SEQ ID NO: 1 ). In embodiments, the amino-terminal domain A N-peptide is a mutant of the wild-type amino-terminal domain A N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain A N-peptide. Such a mutation can be selected from one or more of an amino acid substitution, amino acid deletion, and amino acid addition. For example, a mutant amino-terminal domain A N-peptide can comprise from 1 to 19 amino acid residue substitutions, as compared to the wild-type A N-peptide (SEQ ID NO: 1 ). In embodiments, the mutant aminoterminal domain A N-peptide comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 amino acid residue substitutions, as compared to the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1). In embodiments, an amino acid substitution of the mutant amino-terminal domain A N-peptide takes place at one or more of amino acid residue positions 1 to 19, relative to the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1 ). In embodiments, the mutant amino-terminal domain A N-peptide comprises an amino acid substitution that takes place at one or more of amino acid residue positions 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19, as compared to the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1 ). In embodiments, a mutant amino-terminal domain A N-peptide of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 9. In embodiments, the mutant amino-terminal domain A N-peptide of the present disclosure can comprise the amino acid sequence of MNAQTRRRERRAEKQAQWK (SEQ ID NO: 2), MNAKTRRRERRAEKQAQWK (SEQ ID NO: 3), MNARTRRRERRAEKQAQWK (SEQ ID NO: 4), GNAKTRRRERRAEKQAQWK (SEQ ID NO: 5), GNARTRRRERRAEKQAQWK (SEQ ID NO: 6), MNAQTRRRLRRAEKQAQWK (SEQ ID NO: 7), MDAQTRARERRAEKQAQWK (SEQ ID NO: 8), or MDAQTRRRERKAEKQAQWK (SEQ ID NO: 9).

[0071] In embodiments, one or more amino acids of SEQ ID NO: 1 is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine (R) or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate (E), or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V), a hydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non- classical amino acid (e.g. selenocysteine, pyrrolysine, A/-formylmethionine P-alanine, GABA and 6- Aminolevulinic acid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, c-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a - methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).

[0072] In embodiments, the amino-terminal domain A N-peptide is a mutant of the wild-type aminoterminal domain A N-peptide and comprises at least one mutation as compared to the wild-type aminoterminal domain A N-peptide (SEQ ID NO: 1 ). In embodiments, the at least one mutation to SEQ ID NO: 1 comprises an amino acid substitution selected from any one of the following amino acid substitutions: Met1 Ala; MetICys; MetlAsp; MetIGIu; MetI Phe; MetIGIy; Met1 His; Met1 lie; MetI Lys; MetI Leu; MetlAsn; MetI Pro; MetIGIn; MetlArg; MetlSer; MetIThr; MetIVal; MetITrp; MetITyr; Asp2Ala; Asp2Cys; Asp2Glu; Asp2Phe; Asp2Gly; Asp2His; Asp2lle; Asp2Lys; Asp2Leu; Asp2Met; Asp2Asn; Asp2Pro; Asp2Gln; Asp2Arg; Asp2Ser; Asp2Thr; Asp2Val; Asp2Trp; Asp2Tyr; Ala3Cys; Ala3Asp; Ala3Glu; Ala3Phe; Ala3Gly; Ala3His; Ala3lle; Ala3Lys; Ala3Leu; Ala3Met; Ala3Asn; Ala3Pro; Ala3Gln; Ala3Arg; Ala3Ser; Ala3Thr; Ala3Val; Ala3Trp; Ala3Tyr; Gln4Ala; Gln4Cys; Gln4Asp; Gln4Glu; Gln4Phe; Gln4Gly; Gln4His; Gln4lle; Gln4Lys; Gln4Leu; Gln4Met; Gln4Asn; Gln4Pro; Gln4Arg; Gln4Ser; Gln4Thr; Gln4Val; Gln4Trp; Gln4Tyr; Thr5Ala; Thr5Cys; Thr5Asp; Thr5Glu; Thr5Phe; Thr5Gly; Thr5His; Thr5lle; Thr5Lys; Thr5Leu; Thr5Met; Thr5Asn; Thr5Pro; Thr5Gln; Thr5Arg; Thr5Ser; Thr5Val; Thr5Trp; Thr5Tyr; Arg6Ala; Arg6Cys; Arg6Asp; Arg6Glu; Arg6Phe; Arg6Gly; Arg6His; Arg6lle; Arg6Lys; Arg6Leu; Arg6Met; Arg6Asn; Arg6Pro; Arg6Gln; Arg6Ser; Arg6Thr; Arg6Val; Arg6Trp; Arg6Tyr; Arg7Ala; Arg7Cys; Arg7Asp; Arg7Glu; Arg7Phe; Arg7Gly; Arg7His; Arg7lle; Arg7Lys; Arg7Leu; Arg7Met; Arg7Asn; Arg7Pro; Arg/GIn; Arg7Ser; Arg7Thr; Arg7Val; Arg7Trp; Arg7Tyr; Arg8Ala; Arg8Cys; Arg8Asp; Arg8Glu; Arg8Phe; Arg8Gly; Arg8His; Arg8lle; Arg8Lys; Arg8Leu; Arg8Met; Arg8Asn; Arg8Pro; Arg8Gln; Arg8Ser; Arg8Thr; Arg8Val; Arg8Trp; Arg8Tyr; Glu9Ala; Glu9Cys; Glu9Asp; Glu9Phe; Glu9Gly; Glu9His; Glu9lle; Glu9Lys; Glu9Leu; Glu9Met; Glu9Asn; Glu9Pro; Glu9Gln; Glu9Arg; Glu9Ser; Glu9Thr; Glu9Val; Glu9Trp; Glu9Tyr; Argl OAIa; Argl OCys; Argl OAsp; Argl OGIu; ArglOPhe; Argl OGIy; ArglOHis; ArgWIle; ArglOLys; Argl OLeu; ArglOMet; Argl OAsn; ArglOPro; Argl OGIn; ArglOSer; Arg Thr; Argl OVal; ArglOTrp; ArgWTyr; Arg11Ala; Arg11 Cys; Arg11Asp; Arg11 Glu; Arg11 Phe; Arg11 Gly; Arg11 His; Arg11 lie; Arg11 Lys; Arg11 Leu; Arg11 Met; Arg11Asn; Arg11 Pro; Arg11 Gin; Arg11Ser; Arg11Thr; Arg11Val; Arg11Trp; Arg11Tyr; Ala12Cys; Ala12Asp; Ala12Glu; Ala12Phe; Ala12Gly; Ala12His; Ala12lle; Ala12Lys; Ala12Leu; Ala12Met; Ala12Asn; Ala12Pro; Ala12Gln; Ala12Arg; Ala12Ser; Ala12Thr; Ala12Val; Ala12Trp; Ala12Tyr; Glu13Ala; Glu13Cys; Glu13Asp; Glu13Phe; Glu13Gly; Glu13His; Glu13lle; Glu13Lys; Glu13Leu; Glu13Met; Glu13Asn; Glu13Pro; Glu13Gln; Glu13Arg; Glu13Ser; Glu13Thr; Glu13Val; Glu13Trp; Glu13Tyr; Lys14Ala; Lys14Cys; Lys14Asp; Lys14Glu; Lys14Phe; Lys14Gly; Lys14His; Lys14lle; Lys14Leu; Lys14Met; Lys14Asn; Lys14Pro; Lys14Gln; Lys14Arg; Lys14Ser; Lys14Thr; Lys14Val; Lys14Trp; Lys14Tyr; Gln15Ala; Gln15Cys; Gln15Asp; Gln15Glu; Gln15Phe; Gln15Gly; Gln15His; Gln15lle; Gln15Lys; Gln15Leu; Gln15Met; Gln15Asn; Gln15Pro; Gln15Arg; Gln15Ser; Gln15Thr; Gln15Val; Gln15Trp; Gln15Tyr; Ala16Cys; Ala16Asp; Ala16Glu; Ala16Phe; Ala16Gly; Ala16His; Ala16lle; Ala16Lys; Ala16Leu; Ala16Met; Ala16Asn; Ala16Pro; Ala16Gln; Ala16Arg; Ala16Ser; Ala16Thr; Ala16Val; Ala16Trp; Ala16Tyr; Gln17Ala; Gln17Cys; Gln17Asp; Gln17Glu; Gln17Phe; Gln17Gly; Gln17His; Gln17lle; Gln17Lys; Gln17Leu; Gln17Met; Gln17Asn; Gln17Pro; Gln17Arg; Gln17Ser; Gln17Thr; Gln17Val; Gln17Trp; Gln17Tyr; Trp18Ala; Trp18Cys; Trp18Asp; Trp18Glu; Trp18Phe; Trp18Gly; Trp18His; Trp18lle; Trp18Lys; Trp18Leu; Trp18Met; Trp18Asn; Trp18Pro; Trp18Gln; Trp18Arg; Trp18Ser; Trp18Thr; Trp18Val; Trp18Tyr; Lys19Ala; Lys19Cys; Lys19Asp; Lys19Glu; Lys19Phe; Lys19Gly; Lys19His; Lys19lle; Lys19Leu; Lys19Met; Lys19Asn; Lys19Pro; Lys19Gln; Lys19Arg; Lys19Ser; Lys19Thr; Lys19Val; Lys19Trp; and Lys19Tyr.

[0073] In embodiments, the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 1.

[0074] In embodiments, the mutant A N-peptide sequence comprises an amino acid substitution selected from Table 1 , with respect to SEQ ID NO: 1 . In embodiments, the mutant A N-peptide sequence comprises 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19 amino acid substitutions selected from Table 1 , with respect to SEQ ID NO: 1. In embodiments, the mutant A N-peptide sequence comprises 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1 -4, or 1-3, or 1-2 amino acid substitutions selected from Table 1 , with respect to SEQ ID NO: 1 .

Table t

Table 1 (continued).

Table 1 (continued).

[0075] In embodiments, such mutations increase the binding affinity of the mutant A N-peptide to its respective hairpin RNA oligonucleotide, as compared to the binding affinity of the wild-type A N-peptide to its respective hairpin RNA oligonucleotide. In embodiments, the binding affinity of the inventive contemplated mutant A N-peptide for its hairpin RNA oligonucleotide (e.g., A BoxB hairpin RNA oligonucleotide) is greater than the affinity of the wild-type A N-peptide for its hairpin RNA oligonucleotide (e.g., wild-type A BoxB hairpin RNA oligonucleotide). The present disclosure contemplates mutant A bacteriophage amino-terminal domain N-peptides having binding affinities for respective hairpin oligonucleotides that are greater than the wild-type A bacteriophage N-peptide binding affinities for respective hairpin oligonucleotides by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[0076] Consequentially, in embodiments, a high affinity interaction between the mutant A N-peptide and the respective hairpin oligonucleotide allows for low dose uses of the present agents. Further, without wishing to be bound by theory, such high affinity interaction allows for reduced frequency of dosing and reduced off- target effects. Stated another way the tight interaction between the present mutant A N-peptides and respective hairpin oligonucleotides favors the occurrence of the beneficial interaction which drives editing and disfavors interactions that do not support, or even hinder, a therapeutic effect.

Lambdoid Bacteriophage N-Peptide (P22)

[0077] In embodiments, the present disclosure contemplates a N-peptide linked to a deaminase domain, or variant thereof. In embodiments, the N-peptide comprises a helical structure. In embodiments, the helical peptide is a lambdoid bacteriophage N-peptide (e.g., P22). In embodiments, the present disclosure contemplates at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 N-peptides linked to a deaminase domain.

[0078] In embodiments, the lambdoid bacteriophage N-peptide of the present disclosure is derived from the P22 or lambdoid bacteriophage. In embodiments, the lambdoid bacteriophage N-peptide of the present disclosure is not the wild-type A N-peptide (SEQ ID NO: 1 ). The lambdoid bacteriophages comprise arginine- rich motifs (ARMs) necessary for transcription antitermination that can bind to and/or interact with hairpin oligonucleotides. Without wishing to be bound by theory, in embodiments, the present disclosure contemplates that an arginine-rich motif (ARM) can bind to and/or interact with a hairpin RNA oligonucleotide (e.g., BoxB hairpin RNA oligonucleotide) with a characteristic induced a-induced helical structure. Indeed, the present disclosure contemplates that variants and/or mutant lambdoid bacteriophage N-peptides can exhibit enhanced binding affinity properties for their respective hairpins compared to the wild-type phage N- peptides.

P22

[0079] In embodiments, the lambdoid bacteriophage N-peptide is the wild-type amino-terminal domain P22 N-peptide and comprises the amino acid sequence of SEQ ID NO: 10:

[0080] FAGNAKTRRHERRRKLAIERD (SEQ ID NO: 10).

[0081] In embodiments, a P22 N-peptide, and/or variants thereof, comprises an arginine-rich motif (ARM). In embodiments, an amino-terminal domain P22 N-peptide of the present disclosure comprises an Alanine (Ala) at position 5 (respective of wild-type P22 N-peptide of SEQ ID NO: 10) and an Arginine (Arg) at each of positions 8,12, and 13 (respective of wild-type P22 N-peptide of SEQ ID NO: 10). In embodiments, the amino-terminal domain P22 N-peptide is a variant of the wild-type amino-terminal domain P22 N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain P22 N-peptide. Such a mutation can be selected from one or more of an amino acid substitution, amino acid deletion, and amino acid addition. For example, a variant amino-terminal domain P22 N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type P22 N-peptide (SEQ ID NO: 10). In embodiments, the variant amino-terminal domain P22 N-peptide comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 amino acid residue substitutions, as compared to the wild-type amino-terminal domain P22 N-peptide (SEQ ID NO: 10). In embodiments, an amino acid substitution of the variant amino-terminal domain P22 N-peptide takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild-type amino-terminal domain P22 N-peptide (SEQ ID NO: 10). In embodiments, the variant amino-terminal domain P22 N-peptide comprises an amino acid substitution that takes place at one or more of amino acid residue positions 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 , as compared to the wild-type amino-terminal domain P22 N-peptide (SEQ ID NO: 10). In embodiments, an amino-terminal domain P22 N-peptide variant of the present disclosure comprises the amino acid sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 35. In embodiments, the amino-terminal domain P22 N-peptide variant of the present disclosure can comprise the amino acid sequence of FAGTAKTRRHERRRKLAIERD (SEQ ID NO: 11 ), FAGNARTRRHERRRKLAIERD (SEQ ID NO: 12), FAGNAKSRRHERRRKLAIERD (SEQ ID NO: 13), FAGNAKWRRHERRRKLAIERD (SEQ ID NO: 14), FAGNAKYRRHERRRKLAIERD (SEQ ID NO: 15), FAGNAKARRHERRRKLAIERD (SEQ ID NO: 16), FAGNAKRRRHERRRKLAIERD (SEQ ID NO: 17), FAGNAKTRRFERRRKLAIERD (SEQ ID NO: 18), FAGNAKTRRWERRRKLAIERD (SEQ ID NO: 19), FAGNAKTRRHLRRRKLAIERD (SEQ ID NO: 20),

FAGNAKTRRHERRRQLAIERD (SEQ ID NO: 21 ), FAGNAKTRRHERRRRLAIERD (SEQ ID NO: 22),

FAGNAKTRRHERRRSLAIERD (SEQ ID NO: 23), FAGNAKTRRHERRRKAAIERD (SEQ ID NO: 24),

FAGNAKTRRHERRRKKAIERD (SEQ ID NO: 25), FAGNAKTRRHERRRKLSIERD (SEQ ID NO: 26),

FAGNAKTRRHERRRKLALERD (SEQ ID NO: 27), FAGNAKTRRHERRRKLAMERD (SEQ ID NO: 28), FAGNAKTRRHERRRKLATERD (SEQ ID NO: 29), FAGNAKTRRHERRRKLAVERD (SEQ ID NO: 30),

FAGNAKTRRHERRRKLAISRD (SEQ ID NO: 31 ), FAGNAKTRRHERRRKLAILRD (SEQ ID NO: 32),

FAGNAKTRRHERRRKLAIEKD (SEQ ID NO: 33), FAGNAKTRRHERRRKLAIEFD (SEQ ID NO: 34), or FAGNAKTRRHERRRKLAIEWD (SEQ ID NO: 35). In embodiments, one or more mutations of the wild-type amino-terminal domain P22 N-peptide do not yield the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1 ).

[0082] In embodiments, one or more amino acids of SEQ ID NO: 10 is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine (R) or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate (E), or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V), a hydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non classical amino acid (e.g. selenocysteine, pyrrolysine, W-formylmethionine p-alanine, GABA and 5- Aminolevulinic acid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-am i nobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as methyl amino acids, C a - methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).

[0083] In embodiments, the amino-terminal domain P22 N-peptide is a variant of the wild-type aminoterminal domain P22 N-peptide and comprises at least one mutation as compared to the wild-type aminoterminal domain P22 N-peptide (SEQ ID NO: 10). In embodiments, the at least one mutation to SEQ ID NO: 10 comprises an amino acid substitution selected from any one of the following amino acid substitutions: PhelAla; Phel Cys; PhelAsp; Phel Glu; Phel Phe; PhelGly; Phel His; Phe1 lie; Phel Lys; Phel Leu; Phel Met; PhelAsn; Phel Pro; Phel GIn; PhelArg; Phel Ser; PhelThr; PhelVal; PhelTrp; PhelTyr; Ala2Ala; Ala2Cys; Ala2Asp; Ala2Glu; Ala2Phe; Ala2Gly; Ala2His; Ala2lle; Ala2Lys; Ala2Leu; Ala2Met; Ala2Asn; Ala2Pro; Ala2Gln; Ala2Arg; Ala2Ser; Ala2Thr; Ala2Val; Ala2Trp; Ala2Tyr; Gly3Ala; Gly3Cys; Gly3Asp; Gly3Glu; Gly3Phe; Gly3Gly; Gly3His; Gly3lle; Gly3Lys; Gly3Leu; Gly3Met; Gly3Asn; Gly3Pro; Gly3Gln; Gly3Arg; Gly3Ser; Gly3Thr; Gly3Val; Gly3Trp; Gly3Tyr; Asn4Ala; Asn4Cys; Asn4Asp; Asn4Glu; Asn4Phe; Asn4Gly; Asn4His; Asn4lle; Asn4Lys; Asn4Leu; Asn4Met; Asn4Asn; Asn4Pro; Asn4Gln; Asn4Arg; Asn4Ser; Asn4Thr; Asn4Val; Asn4Trp; Asn4Tyr; Ala5Ala; Ala5Cys; Ala5Asp; Ala5Glu; Ala5Phe; Ala5Gly; Ala5His; Ala5lle; Ala5Lys; Ala5Leu; Ala5Met; Ala5Asn; Ala5Pro; Ala5Gln; Ala5Arg; Ala5Ser; Ala5Thr; Ala5Val; Ala5Trp; Ala5Tyr; Lys6Ala; Lys6Cys; Lys6Asp; Lys6Glu; Lys6Phe; Lys6Gly; Lys6His; Lys6lle; Lys6Lys; Lys6Leu; Lys6Met; Lys6Asn; Lys6Pro; Lys6Gln; Lys6Arg; Lys6Ser; Lys6Thr; Lys6Val; Lys6Trp; Lys6Tyr; Thr7Ala; Thr7Cys; Thr7Asp; Thr7Glu; Thr7Phe; Thr7Gly; Thr7His; Thr7lle; Thr7Lys; Thr7Leu; Thr7Met; Thr7Asn; Thr7Pro; Thr7Gln; Thr7Arg; Thr7Ser; Thr7Thr; Thr7Val; Thr7Trp; Thr7Tyr; Arg8Ala; Arg8Cys; Arg8Asp; Arg8Glu; Arg8Phe; Arg8Gly; Arg8His; ArgSlle; Arg8Lys; Arg8Leu; Arg8Met; Arg8Asn; Arg8Pro; Arg8Gln; Arg8Arg; Arg8Ser; Arg8Thr; Arg8Val; ArgSTrp; Arg8Tyr; Arg9Ala; Arg9Cys; Arg9Asp; Arg9Glu; Arg9Phe; Arg9Gly; Arg9His; Arg9lle; Arg9Lys; Arg9Leu; Arg9Met; Arg9Asn; Arg9Pro; Arg9Gln; Arg9Ser; Arg9Thr; Arg9Val; Arg9Trp; Arg9Tyr; HislOAIa; Hisl OCys; HisWAsp; HislOGIu; Hisl OPhe; HislOGIy; HislOHis; Hisl Olle; HislOLys; Hisl OLeu; HislOMet; Hisl OAsn; Hisl OPro; Hisl OGIn; HislOArg; Hisl OSer; HislOThr; HislOVal; Hisl OTrp; Hisl OTyr; Glu11Ala; Glu11 Cys; Glu11Asp; Glu11Glu; Glu11 Phe; Glu11Gly; Glu11 His; Glu11 lle; Glu11 Lys; Glu11 Leu; Glu11 Met; Glu11Asn; Glu11 Pro; Glu11 Gln; Glu11Arg; Glu11Ser; Glu11Thr; Glu11Val; Glu11Trp; Glu11Tyr; Arg12Ala; Arg12Cys; Arg12Asp; Arg12Glu; Arg12Phe; Arg12Gly; Arg12His; Arg12lle; Arg12Lys; Arg12Leu; Arg12Met; Arg12Asn; Arg12Pro; Arg12Gln; Arg12Arg; Arg12Ser; Arg12Thr; Arg12Val; Arg12Trp; Arg12Tyr; Arg13Ala; Arg13Cys; Arg13Asp; Arg13Glu; Arg13Phe; Arg13Gly; Arg13His; Arg13lle; Arg13Lys; Arg13Leu; Arg13Met; Arg13Asn; Arg13Pro; Arg13Gln; Arg13Arg; Arg13Ser; Arg13Thr; Arg13Val; Arg13Trp; Arg13Tyr; Arg14Ala; Arg14Cys; Arg14Asp; Arg14Glu; Arg14Phe; Arg14Gly; Arg14His; Arg14lle; Arg14Lys; Arg14Leu; Arg14Met; Arg14Asn; Arg14Pro; Arg14Gln; Arg14Arg; Arg14Ser; Arg14Thr; Arg14Val; Arg14Trp; Arg14Tyr; Lys15Ala; Lys15Cys; Lys15Asp; Lys15Glu; Lys15Phe; Lys15Gly; Lys15His; Lys15lle; Lys15Lys; Lys15Leu; Lys15Met; Lys15Asn; Lys15Pro; Lys15Gln; Lys15Arg; Lys15Ser; Lys15Thr; Lys15Val; Lys15Trp; Lys15Tyr; Leu16Ala; Leu16Cys; Leu16Asp; Leu16Glu; Leu16Phe; Leu16Gly; Leu16His; Leu16lle; Leu16Lys; Leu16Leu; Leu16Met; Leu16Asn; Leu16Pro; Leu16Gln; Leu16Arg; Leu16Ser; Leu16Thr; Leu16Val; Leu16Trp; Leu16Tyr; Alai 7 Ala; Ala17Cys; Ala17Asp; Ala17Glu; Ala17Phe; Ala17Gly; Ala17His; Ala17lle; Ala17Lys; Ala17Leu; Ala17Met; Ala17Asn; Ala17Pro; Ala17Gln; Ala17Arg; Ala17Ser; Ala17Thr; Ala17Val; Ala17Trp; Ala17Tyr; lle18Ala; lle18Cys; lle18Asp; lle18Glu; lle18Phe; lle18Gly; lle18His; Ile18lle; lle18Lys; lle18Leu; He18Met; lle18Asn; lle18Pro; He18Gln; lle18Arg; lle18Ser; lle18Thr; lle18Val; lle18Trp; lle18Tyr; Glu19Ala; Glu19Cys; Glu19Asp; Glu19Glu; Glu19Phe; Glu19Gly; Glu19His; Glu19lle; Glu19Lys; Glu19Leu; Glu19Met; Glu19Asn; Glu19Pro; Glu19Gln; Glu19Arg; Glu19Ser; Glu19Thr; Glu19Val; Glu19Trp; Glu19Tyr; Arg20Ala; Arg20Cys; Arg20Asp; Arg20Glu; Arg20Phe; Arg20Gly; Arg20His; Arg20lle; Arg20Lys; Arg20Leu; Arg20Met; Arg20Asn; Arg20Pro; Arg20Gln; Arg20Arg; Arg20Ser; Arg20Thr; Arg20Val; Arg20Trp; Arg20Tyr; Asp21Ala; Asp21 Cys; Asp21Asp; Asp21Glu; Asp21 Phe; Asp21 Gly; Asp21 His; Asp21 lle; Asp21 Lys; Asp21 Leu; Asp21 Met; Asp21Asn; Asp21Pro; Asp21Gln; Asp21Arg; Asp21 Ser; Asp21Thr; Asp21Val; Asp21Trp; and Asp21Tyr. In embodiments, the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 2.

[0084] In embodiments, the P22 N-peptide sequence comprises an amino acid substitution selected from Table 2, with respect to SEQ ID NO: 10. In embodiments, the P22 N-peptide sequence comprises 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 1 1 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 amino acid substitutions selected from Table 2, with respect to SEQ ID NO: 10. In embodiments, the P22 N-peptide sequence comprises 1-20, or 1-10, or 1-5, or 1-4, or 1-3, or 1-2 amino acid substitutions selected from Table 2, with respect to SEQ ID NO: 10.

Table 2.

Table 2 (continued)

Table 2 (continued)

[0085] In embodiments, such mutations increase the binding affinities of the variant N-peptides to their respective hairpin RNA oligonucleotides, as compared to the binding affinities of the wild-type N-peptides to their respective hairpin RNA oligonucleotides. In embodiments, the affinity of any one of the inventive contemplated P22 N-peptide variants for their hairpin RNA oligonucleotide (e.g., P22 BoxB hairpin RNA oligonucleotide) is greater than the affinity of the wild-type P22 N-peptide for its hairpin RNA oligonucleotide (e.g., wild-type P22 BoxB hairpin RNA oligonucleotide). The present disclosure contemplates variant bacteriophage amino-terminal domain N-peptides having binding affinities for respective hairpin oligonucleotides that are greater than the wild-type bacteriophage N-peptide binding affinities for respective hairpin oligonucleotides by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[0086] Consequentially, in embodiments, a high affinity interaction between the bacteriophage P22 N- peptide and the respective hairpin oligonucleotide allows for low dose uses of the present agents. Further, without wishing to be bound by theory, in embodiments, such high affinity interaction allows for reduced frequency of dosing and reduced off-target effects. Stated another way the tight interaction between the present bacteriophage P22 N-peptides and respective hairpin oligonucleotides favors the occurrence of the beneficial interaction which drives editing and disfavors interactions that do not support, or even hinder, a therapeutic effect.

Lambdoid Bacteriophage N-Peptide (P21)

[0087] In embodiments, the lambdoid bacteriophage N-peptide of the present disclosure is derived from the P21 (a.k.a. q>21 or phi21) lambdoid bacteriophage.

P21

[0088] In embodiments, the lambdoid bacteriophage N-peptide is the wild-type amino-terminal domain P21 N-peptide and comprises the amino acid sequence of SEQ ID NO: 36:

[0089] SKGTAKSRYKARRAELIAERR (SEQ ID NO: 36).

[0090] In embodiments, a P21 N-peptide, and/or variants thereof, comprises an arginine-rich motif (ARM). In embodiments, an amino-terminal domain P21 N-peptide of the present disclosure comprises an Alanine (Ala) at position 5 (respective of wild-type P21 N-peptide of SEQ ID NO: 36) and an Arginine (Arg) at each of positions 8,12, and 13 (respective of wild-type P21 N-peptide of SEQ ID NO: 36). In embodiments, the amino-terminal domain P21 N-peptide is a variant of the wild-type amino-terminal domain P21 N-peptide and comprises at least one mutation as compared to the wild-type amino-terminal domain P21 N-peptide. Such a mutation can be selected from one or more of an amino acid substitution, amino acid deletion, and amino acid addition. For example, a variant amino-terminal domain P21 N-peptide can comprise from 1 to 21 amino acid residue substitutions, as compared to the wild-type P21 N-peptide (SEQ ID NO: 36). In embodiments, the variant amino-terminal domain P21 N-peptide comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 amino acid residue substitutions, as compared to the wild-type amino-terminal domain P21 N-peptide (SEQ ID NO: 36). In embodiments, an amino acid substitution of the variant amino-terminal domain P21 N-peptide takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild-type amino-terminal domain P21 N-peptide (SEQ ID NO: 36). In embodiments, the variant amino-terminal domain P21 N-peptide comprises an amino acid substitution that takes place at one or more of amino acid residue positions 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 , as compared to the wild-type amino-terminal domain P21 N-peptide (SEQ ID NO: 36). In embodiments, the variant amino-terminal domain P21 N-peptide comprises one or more mutations at position R13 and/or 117, with respect to SEQ ID NO: 36. In embodiments, the variant amino-terminal domain P21 N-peptide is not mutated at position R13 and/or 117, with respect to SEQ ID NO: 36. In embodiments, one or more mutations of the wild-type amino-terminal domain P21 N-peptide do not yield the wild-type amino-terminal domain X N-peptide (SEQ ID NO: 1).

[0091] In embodiments, one or more amino acids of SEQ ID NO: 36 is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine (R) or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate (E), or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V), a hydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non- classical amino acid (e.g. selenocysteine, pyrrolysine, A/-formylmethionine P-alanine, GABA and 6- Aminolevulinic acid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-am I nobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a - methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).

[0092] In embodiments, the amino-terminal domain P21 N-peptide is a variant of the wild-type aminoterminal domain P21 N-peptide and comprises at least one mutation as compared to the wild-type aminoterminal domain P21 N-peptide (SEQ ID NO: 36). In embodiments, the at least one mutation to SEQ ID NO: 36 comprises an amino acid substitution selected from any one of the following amino acid substitutions: SerlAla; Serl Cys; SerlAsp; SerIGIu; Serl Phe; SerIGIy; Serl His; Seri lie; SerI Lys; SerI Leu; Serl Met; SerlAsn; Serl Pro; SerIGIn; SerlArg; SerlThr; SerlVal; SerlTrp; SerlTyr; Lys2Ala; Lys2Cys; Lys2Asp; Lys2Glu; Lys2Phe; Lys2Gly; Lys2His; Lys2lle; Lys2Leu; Lys2Met; Lys2Asn; Lys2Pro; Lys2Gln; Lys2Arg;

Lys2Ser; Lys2Thr; Lys2Val; Lys2Trp; Lys2Tyr; Gly3Ala; Gly3Cys; Gly3Asp; Gly3Glu; Gly3Phe; Gly3His;

Gly3lle; Gly3Lys; Gly3Leu; Gly3Met; Gly3Asn; Gly3Pro; Gly3Gln; Gly3Arg; Gly3Ser; Gly3Thr; Gly3Val;

Gly3Trp; Gly3Tyr; Thr4Ala; Thr4Cys; Thr4Asp; Thr4Glu; Thr4Phe; Thr4Gly; Thr4His; Thr4lle; Thr4Lys;

Thr4Leu; Thr4Met; Thr4Asn; Thr4Pro; Thr4Gln; Thr4Arg; Thr4Ser; Thr4Val; Thr4Trp; Thr4Tyr; Ala5Cys; Ala5Asp; Ala5Glu; Ala5Phe; Ala5Gly; Ala5His; Ala5lle; Ala5Lys; Ala5Leu; Ala5Met; Ala5Asn; Ala5Pro;

Ala5Gln; Ala5Arg; Ala5Ser; Ala5Thr; Ala5Val; Ala5Trp; Ala5Tyr; Lys6Ala; Lys6Cys; Lys6Asp; Lys6Glu;

Lys6Phe; Lys6Gly; Lys6His; Lys6lle; Lys6Leu; Lys6Met; Lys6Asn; Lys6Pro; Lys6Gln; Lys6Arg; Lys6Ser;

Lys6Thr; Lys6Val; Lys6Trp; Lys6Tyr; Ser7Ala; Ser7Cys; Ser7Asp; Ser7Glu; Ser7Phe; Ser7Gly; Ser7His;

Ser7lle; Ser7Lys; Ser7Leu; Ser7Met; Ser7Asn; Ser7Pro; Ser7Gln; Ser7Arg; Ser7Thr; Ser7Val; Ser7Trp; Ser7Tyr; Arg8Ala; Arg8Cys; Arg8Asp; Arg8Glu; Arg8Phe; Arg8Gly; Arg8His; Arg8lle; Arg8Lys; Arg8Leu; Arg8Met; Arg8Asn; Arg8Pro; Arg8Gln; Arg8Arg; Arg8Ser; Arg8Thr; Arg8Val; Arg8Trp; Arg8Tyr; Tyr9Ala; Tyr9Cys; Tyr9Asp; Tyr9Glu; Tyr9Phe; Tyr9Gly; Tyr9His; Tyr9lle; Tyr9Lys; Tyr9Leu; Tyr9Met; Tyr9Asn; Tyr9Pro; Tyr9Gln; Tyr9Arg; Tyr9Ser; Tyr9Thr; Tyr9Val; Tyr9Trp; LyslOAIa; LysWCys; LyslOAsp; Lysl OGIu; LyslOPhe; Lysl OGIy; LyslOHis; Lys Ile; LyslOLeu; Lysl OMet; LyslOAsn; Lysl OPro; LyslOGIn; Lysl OArg; LyslOSer; Lysl OThr; LyslOVal; Lysl OTrp; LyslOTyr; Alai 1 Cys; Alai 1 Asp; Ala11 Glu; Alai 1 Phe; Alai 1Gly; Ala11 His; Alai 1 lle; Alai 1 Lys; Ala11 Leu; Ala11 Met; Ala11Asn; Ala11 Pro; Ala11 Gln; Ala11Arg; Ala11Ser; Alai 1Thr; Alai 1 Vai; Ala11Trp; Ala11Tyr; Arg12Ala; Arg12Cys; Arg12Asp; Arg12Glu; Arg12Phe; Arg12Gly; Arg12His; Arg12lle; Arg12Lys; Arg12Leu; Arg12Met; Arg12Asn; Arg12Pro; Arg 12Gln; Arg12Ser; Arg12Thr; Arg12Val; Arg12Trp; Arg12Tyr; Arg13Ala; Arg13Cys; Arg13Asp; Arg13Glu; Arg13Phe; Arg13Gly; Arg13His; Arg13lle; Arg13Lys; Arg13Leu; Arg13Met; Arg13Asn; Arg13Pro; Arg13Gln; Arg13Ser; Arg13Thr; Arg13Val; Arg13Trp; Arg13Tyr; Ala14Cys; Ala14Asp; Ala14Glu; Ala14Phe; Ala14Gly; AlaMHis; Ala14lle; Ala14Lys; Ala14Leu; Ala14Met; Ala14Asn; Ala14Pro; Ala14Gln; Ala14Arg; Ala14Ser; Ala14Thr; AlaMVal; Ala14Trp; Ala14Tyr; Glu15Ala; Glu15Cys; Glu15Asp; Glu15Phe; Glu15Gly; Glu15His; Glu15lle; Glu15Lys; Glu15Leu; Glu15Met; Glu15Asn; Glu15Pro; Glu15Gln; Glu15Arg; Glu15Ser; Glu15Thr; Glu15Val; Glu15Trp; Glu15Tyr; Leu16Ala; Leu16Cys; Leu16Asp; Leu16Glu; Leu16Phe; Leu16Gly; Leu16His; Leu16lle; Leu16Lys; Leu16Met; Leu16Asn; Leu16Pro; Leu16Gln; Leu16Arg; Leu16Ser; Leu16Thr; Leu16Val; Leu16Trp; Leu16Tyr; Ile17Ala; Ile17Cys; lle17Asp; Ile17Glu; Ile17Phe; Ile17Gly; lle17His; Ile17Lys; Ile17Leu; Ile17Met; lle17Asn; lle17Pro; lle17Gln; lle17Arg; lle17Ser; lle17Thr; lle17Val; lle17Trp; lle17Tyr; Ala18Cys; Ala18Asp; Ala18Glu; Ala Phe; Ala18Gly; Ala His; Ala18lle; Ala18Lys; Ala18Leu; Ala18Met; Ala18Asn; Ala18Pro; Ala18Gln; Ala18Arg; Ala18Ser; Alai 8Thr; Ala18Val; Ala18Trp; Ala18Tyr; Glu19Ala; Glu19Cys; Glu19Asp; Glu19Phe; Glu19Gly; Glu19His; Glu19lle; Glu19Lys; Glu19Leu; Glu19Met; Glu19Asn; Glu19Pro; Glu19Gln; Glu19Arg; Glu19Ser; Glu19Thr; Glu19Val; Glu19Trp; Glu19Tyr; Arg20Ala; Arg20Cys; Arg20Asp; Arg20Glu; Arg20Phe; Arg20Gly; Arg20His; Arg20lle; Arg20Lys; Arg20Leu; Arg20Met; Arg20Asn; Arg20Pro; Arg20Gln; Arg20Ser; Arg20Thr; Arg20Val; Arg20Trp; Arg20Tyr; Arg21 Ala; Arg21 Cys; Arg21Asp; Arg21 Glu; Arg21 Phe; Arg21 Gly; Arg21 His; Arg21 lie; Arg21 Lys; Arg21 Leu; Arg21 Met; Arg21Asn; Arg21 Pro; Arg21 Gin; Arg21Ser; Arg21 Thr; Arg21 Vai; Arg21 Trp; and Arg21 Tyr. In embodiments, the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 3.

[0093] In embodiments, the P21 N-peptide sequence comprises an amino acid substitution selected from Table 3, with respect to SEQ ID NO: 36. In embodiments, the P21 N-peptide sequence comprises 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 1 1 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 amino acid substitutions selected from Table 3, with respect to SEQ ID NO: 36. In embodiments, the P21 N-peptide sequence comprises 1-20, or 1-10, or 1-5, or 1-4, or 1-3, or 1-2 amino acid substitutions selected from Table 3, with respect to SEQ ID NO: 36.

Table 3.

Table 3 (continued)

Table 3 (continued)

[0094] In embodiments, such mutations increase the binding affinities of the variant N-peptides to their respective hairpin RNA oligonucleotides, as compared to the binding affinities of the wild-type N-peptides to their respective hairpin RNA oligonucleotides. In embodiments, the affinity of any one of the inventive contemplated P21 N-peptide variants for their hairpin RNA oligonucleotides (e.g., P21 BoxB hairpin RNA oligonucleotide) is greater than the affinity of the wild-type P21 N-peptide for its hairpin RNA oligonucleotide (e.g., wild-type P21 BoxB hairpin RNA oligonucleotide). The present disclosure contemplates variant bacteriophage amino-terminal domain N-peptides having binding affinities for respective hairpin oligonucleotides that are greater than the wild-type bacteriophage N-peptide binding affinities for respective hairpin oligonucleotides by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[0095] Consequentially, in embodiments, a high affinity interaction between the bacteriophage P21 N- peptide and the respective hairpin oligonucleotide allows for low dose uses of the present agents. Further, without wishing to be bound by theory, in embodiments, such high affinity interaction allows for reduced frequency of dosing and reduced off-target effects. Stated another way the tight interaction between the present bacteriophage P21 N-peptides and respective hairpin oligonucleotides favors the occurrence of the beneficial interaction which drives editing and disfavors interactions that do not support, or even hinder, a therapeutic effect.

[0096] Methods of assaying binding affinity are known in the art. Non-limiting examples include optical assays, such as monitoring the change in fluorescence of a 2-aminopurine (2AP) base analogue substituted at different adenine positions within the target hairpin in order to measure kinetics; chemical assays, such as gel electrophoresis, ELISA, and immunoblots; and other methods studying RNA-protein interactions, such as electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP). Pollard, Mol Biol Cell. (2010) 21 (23): 4061-4067 and Popova et al. Mol. Biol. (2015) 49(3): 418-426, are hereby incorporated by reference in their entireties.

Antisense RNA Oligonucleotide

[0097] In aspects, the target RNA sequence comprises a sequence associated with a disease, condition or disorder, and wherein the deamination of the nucleotide base results in an RNA sequence that is not associated with a disease, condition or disorder. In embodiments, the target RNA sequence comprises a wild-type sequence, wherein the deamination of a particular nucleotide base therein results in a mutation that induces a beneficial and/or therapeutic effect.

[0098] Accordingly, in embodiments, methods of the present disclosure further contemplate an antisense RNA oligonucleotide that hybridizes with a wild-type or mutant target sequence in an endogenous RNA molecule that comprises a target nucleotide to be edited. In embodiments, the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence. For example, the target nucleotide can be a genetic mutation associated with a genetic disease, condition or disorder. By way of non-limiting example, a genetic mutation of the target nucleotide may cause a premature termination codon (PTC) or an in-frame stop codon. In embodiments, the genetic disorder or condition is caused by a G-to-A nucleotide mutation. The present disclosure contemplates a nucleic acid-editing system that can edit any target nucleotide and/or genetic mutation (e.g., by facilitating splice modulation). In embodiments, the target RNA sequence relates to a target gene of interest. For example, in non-limiting examples, the target gene of interest can include, but is not limited to, Human SerpinAI E342K gene (NM_000295.4) and human IDUA gene (NM_000203.5) that contains the W402X mutation.

[0099] In embodiments, the antisense RNA oligonucleotide hybridizes with an RNA molecule comprising the target nucleotide in endogenous RNA of the cell. In embodiments, the antisense RNA oligonucleotide has perfect base-pairing complementarity with the RNA molecule comprising the target nucleotide. In embodiments, the antisense RNA oligonucleotide comprises one or more mismatches with the RNA molecule comprising the target nucleotide. For example, the antisense RNA oligonucleotide sequence can comprise from 1 to 15 mismatches, e.g., about 1 , or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11 , or about 12, or about 13, or about 14, or about 15 mismatches. In embodiments, a mismatch is a non-complementary match hybridization between the antisense RNA oligonucleotide and the RNA molecule comprising the target nucleotide. In embodiments, the antisense RNA oligonucleotide sequence is at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary with the target sequence. In embodiments, the 5' end of the antisense RNA oligonucleotide begins before the target nucleotide to be edited. In embodiments, the 3' end of the oligonucleotide extends from 2-22, from 2-20, from 5-20, from 5-15, or from 5-10 nucleotides 5' from the target nucleotide to be edited.

[00100] The present disclosure further contemplates an antisense RNA oligonucleotide that hybridizes with a wild-type target sequence in an endogenous RNA molecule that comprises a target nucleotide to be edited. In embodiments of the present disclosure, the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence. In embodiments, an antisense RNA oligonucleotide hybridizes with a target sequence comprising a target nucleotide to be edited under stringent conditions.

[00101] In embodiments, the present disclosure contemplates methods of editing RNA in any cell type of a subject. For example, the cell containing RNA to be edited can be a cell of the central nervous system (e.g., neurons) or a liver cell. In embodiments, the cells to be edited are in a subject so that the methods of the present disclosure are in vivo methods of editing. In embodiments, cells may be edited in vitro and subsequently delivered to a subject as ex vivo treatment.

Hairpin Interaction

[00102] According to the present disclosure, the term “hairpin” refers to a particular oligonucleotide and/or nucleic acid structure (e.g., RNA oligonucleotide and/or RNA structure) that is recognized by and interacts with a A lambdoid bacteriophage N-peptide (e.g., amino-terminal domain N-peptide derived from A N protein), a lambdoid bacteriophage N-peptide (e.g., amino-terminal domain N-peptide derived from P21 or P22), or another helical peptide. In embodiments, a particular example of such a hairpin is a BoxB hairpin RNA oligonucleotide that is the cognate hairpin of the A N-peptide, P22 N-peptide, or P21 N-peptide, or mutants thereof. In embodiments, a particular example of such a hairpin is a BoxB hairpin RNA oligonucleotide that is the cognate hairpin of the A N-peptide, or mutant thereof. In embodiments, a particular example of such a hairpin is a BoxB hairpin RNA oligonucleotide that is the cognate hairpin of the P22 N- peptide, or mutant thereof. In embodiments, a particular example of such a hairpin is a BoxB hairpin RNA oligonucleotide that is the cognate hairpin of the P21 N-peptide, or mutant thereof.

[00103] In embodiments of the present disclosure the A bacteriophage N-peptide, or mutant thereof, interacts with a hairpin RNA (e.g., there is hairpin recognition of the A N-peptide) oligonucleotide. In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the A N-peptide. For example, by way of non-limiting example, the cognate hairpin can be a boxB hairpin RNA oligonucleotide. In embodiments, the boxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5' of the target mutation. In embodiments, the boxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3’ of the target mutation.

[00104] In embodiments, the antisense RNA oligonucleotide of the present disclosure (described herein) is linked to one or more hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to 1 , 2, 3, or 4 hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to at least 2, at least 3, or at least 4 hairpin RNA oligonucleotides.

[00105] In embodiments, a BoxB hairpin RNA oligonucleotide of the present disclosure comprises a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or mutants thereof. Specifically, in embodiments, a BoxB hairpin RNA oligonucleotide can comprise a nucleic acid sequence selected from the wild-type A BoxB hairpin RNA oligonucleotide (e.g., nutR^A BoxB hairpin RNA oligonucleotide), right: GCCCUGAAAAAGGGC (SEQ ID NO: 37), and the wild-type A BoxB hairpin RNA oligonucleotide (e.g., nutl_^A BoxB hairpin RNA oligonucleotide), left: GCCCUGAAGAAGGGC (SEQ ID NO: 38).

[00106] In embodiments, the A N-peptide, or mutant thereof, interacts with a A BoxB hairpin RNA oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or mutant thereof. In embodiments the A N-peptide, or mutant thereof, interacts with a mutant A BoxB hairpin RNA oligonucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 37-38. In embodiments, the A N-peptide, or mutant thereof, and A BoxB hairpin RNA oligonucleotide, or mutant thereof, interaction adopts a 4-out GNRA-like pentaloop. A tetraloop is a type of four-base hairpin loop motif in RNA secondary structures that cap many double helices. In ribosomal RNA, the N could be either uracil, adenine, cytosine, or guanine, and the R is either guanine or adenine. The GNRA tetraloop, specifically, has a guanine-adenine base pair, where the guanine is 5’ to the helix and the adenine is 3’ to the helix.

[00107] In embodiments, a hairpin oligonucleotide of the present disclosure comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion. By way of non-limiting example, such mutations can be made with respect to a hairpin oligonucleotide comprising one or more of SEQ ID NO: 37 or SEQ ID NO: 38. For example, in embodiments, the A BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence having sequence identity to SEQ ID NO: 37 or SEQ ID NO: 38, and further comprises from 1 to 15 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions. In embodiments, the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 15, relative to wild-type A BoxB hairpin RNA oligonucleotide. In embodiments, a mutant A BoxB hairpin RNA oligonucleotide comprises a nucleotide sequence of any one of SEQ ID NOs: 39-44:

[00108] SEQ ID NO: 39: GCCCUAAAAAAGGGC (AboxBR:G6A);

[00109] SEQ ID NO: 40: GCCCUGAUAAAGGGC (AboxBR:A8U);

[00110] SEQ ID NO: 41 : GCCCGGAAAACGGGC (AboxBR:U5G,A11C);

[00111] SEQ ID NO: 42: GCCCUGAACAAGGGC (AboxBR:A9C);

[00112] SEQ ID NO: 43: GCCCUGAAACAGGGC (AboxBR:A10C); and

[00113] SEQ ID NO: 44: GCCCUGAAAAGGGGC (AboxBR:A11 G).

[00114] In embodiments of the present disclosure the lambdoid bacteriophage N-peptide interacts with a hairpin RNA oligonucleotide (e.g., there is hairpin recognition of the P22 N-peptide). In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the P22 N-peptide. For example, by way of non-limiting example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5’ of the target mutation. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3’ of the target mutation.

[00115] In embodiments, the antisense RNA oligonucleotide of the present disclosure (described herein) is linked to one or more hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to 1 , 2, 3, or 4 hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to at least 2, at least 3, or at least 4 hairpin RNA oligonucleotides.

[00116] In embodiments, a BoxB hairpin RNA oligonucleotide of the present disclosure comprises a nucleic acid sequence of any one of SEQ ID NOs: 45-46, or variants thereof. Specifically, a BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence selected from the wild-type P22 BoxB RNA hairpin, right: ACCGCCGACAACGCGGU (SEQ ID NO: 45); the wild-type P22 BoxB RNA hairpin, left: UGCGCUGACAAAGCGCG (SEQ ID NO: 46).

[00117] In embodiments, the P22 N-peptide, or variant thereof, interacts with a P22 BoxB hairpin RNA oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, or variant thereof. In embodiments, the P22 N-peptide and P22 BoxB hairpin RNA oligonucleotide interaction adopts a 3-out GNRA-like pentaloop. A tetraloop is a type of four-base hairpin loop motif in RNA secondary structures that cap many double helices. In ribosomal RNA, the N could be either uracil, adenine, cytosine, or guanine, and the R is either guanine or adenine. The GNRA tetraloop, specifically, has a guanine-adenine base pair, where the guanine is 5’ to the helix and the adenine is 3’ to the helix.

[00118] In embodiments, a hairpin oligonucleotide of the present disclosure comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion. By way of non-limitation, such mutations can be made with respect to a hairpin oligonucleotide comprising one or more of nucleic acid sequences selected from SEQ ID NOs: 45- 46. For example, in embodiments, the P22 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, and further comprises from 1 to 17 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions. In embodiments, the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 17, relative to wild-type P22 BoxB hairpin RNA oligonucleotide.

[00119] In embodiments of the present disclosure the lambdoid bacteriophage N-peptide interacts with a hairpin RNA oligonucleotide (e.g., there is hairpin recognition of the P21 N-peptide). In embodiments, the hairpin RNA oligonucleotide is the cognate hairpin of the P21 N-peptide. For example, by way of non-limiting example, the cognate hairpin can be a BoxB hairpin RNA oligonucleotide. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5’ of the target mutation. In embodiments, the BoxB hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3’ of the target mutation.

[00120] In embodiments, the antisense RNA oligonucleotide of the present disclosure (described herein) is linked to one or more hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to 1 , 2, 3, or 4 hairpin RNA oligonucleotides. In embodiments, the antisense RNA oligonucleotide is linked to at least 2, at least 3, or at least 4 hairpin RNA oligonucleotides.

[00121] In embodiments, a BoxB hairpin RNA oligonucleotide of the present disclosure comprises a nucleic acid sequence of any one of SEQ ID NOs: 47-48, or variants thereof. Specifically, a BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence selected from the wild-type P21 BoxB RNA hairpin, right: UUCACCUCUAACCGGGUGAG (SEQ ID NO: 47); and the wild-type P21 BoxB RNA hairpin, left: UCUCAACCUAACCGUUGAGA (SEQ ID NO: 48). [00122] In embodiments, the P21 N-peptide, or variant thereof, interacts with a P21 BoxB hairpin RNA oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, or variant thereof. In embodiments, the P21 N-peptide and P21 BoxB hairpin RNA oligonucleotide interaction adopts a non- GNRA U-turn with respect to the apical four nucleotides of the P21 BoxB hairpin RNA oligonucleotide. A tetraloop is a type of four-base hairpin loop motif in RNA secondary structures that cap many double helices. In ribosomal RNA, the N could be either uracil, adenine, cytosine, or guanine, and the R is either guanine or adenine. The GNRA tetraloop, specifically, has a guanine-adenine base pair, where the guanine is 5’ to the helix and the adenine is 3’ to the helix.

[00123] In embodiments, a hairpin oligonucleotide of the present disclosure comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion. By way of non-limitation, such mutations can be made with respect to a hairpin oligonucleotide comprising one or more of nucleic acid sequences selected from SEQ ID NOs: 47- 48. For example, in embodiments, the P21 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, and further comprises from 1 to 20 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions. In embodiments, the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 20, relative to wild-type P21 BoxB hairpin RNA oligonucleotide.

Fusion Proteins

[00124] In embodiments contemplated by the present disclosure, fusion proteins are provided comprising a deaminase, or fragment thereof, and a A bacteriophage N-peptide, optionally connected by a chemical linker. By way of non-limiting example, the deaminase, or fragment thereof can be linked to at least 2, at least 3, or at least 4 A N-peptides. Alternatively, the A N-peptide can be linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. Some aspects of the disclosure provide fusion proteins that comprise one or more deaminase domains (e.g., adenosine deaminase). For example, in embodiments, the fusion protein comprises at least two deaminase domains. Without wishing to be bound by any particular theory, dimerization of deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to edit a nucleic acid base and/or nucleotide, for example to deaminate adenosine. In embodiments, any of the fusion proteins may comprise at least 2, at least 3, at least 4 or at least 5 adenosine deaminase domains. In embodiments, any of the fusion proteins provided herein comprise two deaminases. In embodiments, any of the fusion proteins provided herein contain only two deaminases. In embodiments, the deaminases are the same. In embodiments, the deaminases are any of the deaminases provided herein. In embodiments, the deaminases are different. In embodiments, the first deaminase is any of the adenosine deaminases provided herein, and the second deaminase is any of the adenosine deaminases provided herein, but is not identical to the first deaminase.

[00125] In embodiments, the deaminase and A N-peptide fusion protein comprises one or more nuclear localization signals (N LS) , optionally wherein the RNA molecule comprising the target nucleotide to be edited is located in the nucleus of the cell. In embodiments, the fusion protein of the present disclosure comprises 1 , 2, 3, 4, or 5 nuclear localization signals (e.g., nuclear localization sequences). In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal (SEQ ID NO: 49).

[00126] In embodiments, the term "nuclear localization signal" or "NLS," in embodiments, refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. 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., International PCT application, PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31 , 2001 ; and in Kosugi et al., J. Biol. Chem. (2009) 284: 478-485, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences.

[00127] In embodiments, the deaminase and A N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nuclear localization sequence of the SV40 virus large T-antigen the minimal functional unit of which is the seven amino acid sequence PKKKRKV (SEQ ID NO: 50). In embodiments, the deaminase and A N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nucleoplasmin bipartite NLS with the sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 51 ); the c-myct nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID NO: 52) or RQRRNELKRSF (SEQ ID NO: 53); and the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 54). In embodiments, further nuclear localization sequences include: the sequence

RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 55) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 56) and PPKKARED (SEQ ID NO: 57) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 58) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 59) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 60) and PKQKKRK (SEQ ID NO: 61) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO: 62) of the Hepatitis virus delta antigen; and the sequence REKKKFLKRR (SEQ ID NO: 63) of the mouse Mx1 protein. In embodiments, nuclear localization sequences include the use of bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 64) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 65) of the steroid hormone receptors (human) glucocorticoid. In embodiments, a NLS of the present disclosure comprises the amino acid sequence SV40 Large T-antigen NLS comprising the amino acid sequence:

[00128] DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 49).

[00129] In embodiments, the NLS contemplated by the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and/or insertions) with respect to any one of SEQ ID NOs: 49-65.

[00130] In embodiments, the fusion proteins provided herein further comprise one or more nuclear targeting sequences, for example, a nuclear localization signal/sequence (NLS). In embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus. In embodiments, the NLS is fused to the N-terminus of the fusion protein. In embodiments, the NLS is fused to the C-terminus of the fusion protein. In embodiments, the NLS is fused to the N-terminus of the deaminase. In embodiments, the NLS is fused to the C-terminus of the deaminase. In embodiments, the NLS is fused to the N-terminus of the A N-peptide. In embodiments, the NLS is fused to the C-terminus of the A N-peptide. In embodiments, the NLS is fused to the fusion protein via one or more linkers. In embodiments, the NLS is fused to the fusion protein without a linker. In embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 49-65, or variants thereof. 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 illustrative nuclear localization sequences.

[00131] In embodiments contemplated by the present disclosure, fusion proteins are provided comprising a deaminase, or fragment thereof, and a lambdoid bacteriophage P22 N-peptide, optionally connected by a chemical linker. By way of non-limiting example, the deaminase, or fragment thereof can be linked to at least 2, at least 3, or at least 4 lambdoid bacteriophage N-peptides. Alternatively, the bacteriophage N-peptide can be linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. Some aspects of the disclosure provide fusion proteins that comprise one or more deaminase domains (e.g., adenosine deaminase). For example, in embodiments, the fusion protein comprises at least two deaminase domains. Without wishing to be bound by any particular theory, dimerization of deaminases (e.g., in cis or in trans) improve the ability (e.g., efficiency) of the fusion protein to edit a nucleic acid base and/or nucleotide, for example to deaminate adenosine. In embodiments, any of the fusion proteins comprise at least 2, at least 3, at least 4 or at least 5 adenosine deaminase domains. In embodiments, any of the fusion proteins provided herein comprise two deaminases. In embodiments, any of the fusion proteins provided herein contain only two deaminases. In embodiments, the deaminases are the same. In embodiments, the deaminases are any of the deaminases provided herein. In embodiments, the deaminases are different. In embodiments, the first deaminase is any of the adenosine deaminases provided herein, and the second deaminase is any of the adenosine deaminases provided herein, but is not identical to the first deaminase.

[00132] In embodiments, the deaminase and lambdoid bacteriophage P22 N-peptide fusion protein comprises one or more nuclear localization signals (NLS), optionally wherein the RNA molecule comprising the target nucleotide to be edited is located in the nucleus of the cell. In embodiments, the fusion protein of the present disclosure comprises 1 , 2, 3, 4, or 5 nuclear localization signals (e.g., nuclear localization sequences). In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal (SEQ ID NO: 49).

[00133] In embodiments, the deaminase and P22 N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nuclear localization sequence of the SV40 virus large T- antigen the minimal functional unit of which is the seven amino acid sequence PKKKRKV (SEQ ID NO: 50). In embodiments, the deaminase and P22 N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nucleoplasmin bipartite NLS with the sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 51 ); the c-myct nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID NO: 52) or RQRRNELKRSF (SEQ ID NO: 53); and the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 54). In embodiments, further examples of nuclear localization sequences include: the sequence

[00134] DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 49).

[00135] In embodiments, the NLS contemplated by the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and/or insertions) with respect to any one of SEQ ID NOs: 49-65.

[00136] In embodiments, the fusion proteins provided herein further comprise one or more nuclear targeting sequences, for example, a nuclear localization signal/sequence (NLS). In embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus. In embodiments, the NLS is fused to the N-terminus of the fusion protein. In embodiments, the NLS is fused to the C-terminus of the fusion protein. In embodiments, the NLS is fused to the N-terminus of the deaminase. In embodiments, the NLS is fused to the C-terminus of the deaminase. In embodiments, the NLS is fused to the N-terminus of the lambdoid P22 bacteriophage N-peptide. In embodiments, the NLS is fused to the C-terminus of the lambdoid P22 bacteriophage N-peptide. In embodiments, the NLS is fused to the fusion protein via one or more linkers. In embodiments, the NLS is fused to the fusion protein without a linker. In embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 49-65, or variants thereof.

[00137] In embodiments contemplated by the present disclosure, fusion proteins are provided comprising a deaminase, or fragment thereof, and a lambdoid bacteriophage P21 N-peptide, optionally connected by a chemical linker. By way of non-limiting example, the deaminase, or fragment thereof can be linked to at least 2, at least 3, or at least 4 lambdoid bacteriophage N-peptides. Alternatively, the bacteriophage N-peptide can be linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. Some aspects of the disclosure provide fusion proteins that comprise one or more deaminase domains (e.g., adenosine deaminase). For example, in embodiments, the fusion protein comprises at least two deaminase domains. Without wishing to be bound by any particular theory, dimerization of deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to edit a nucleic acid base and/or nucleotide, for example to deaminate adenosine. In embodiments, any of the fusion proteins may comprise at least 2, at least 3, at least 4 or at least 5 adenosine deaminase domains. In embodiments, any of the fusion proteins provided herein comprise two deaminases. In embodiments, any of the fusion proteins provided herein contain only two deaminases. In embodiments, the deaminases are the same. In embodiments, the deaminases are any of the deaminases provided herein. In embodiments, the deaminases are different. In embodiments, the first deaminase is any of the adenosine deaminases provided herein, and the second deaminase is any of the adenosine deaminases provided herein, but is not identical to the first deaminase.

[00138] In embodiments, the deaminase and lambdoid bacteriophage P21 N-peptide fusion protein comprises one or more nuclear localization signals (NLS), optionally wherein the RNA molecule comprising the target nucleotide to be edited is located in the nucleus of the cell. In embodiments, the fusion protein of the present disclosure comprises 1 , 2, 3, 4, or 5 nuclear localization signals (e.g., nuclear localization sequences). In embodiments, the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal (SEQ ID NO: 49).

[00139] In embodiments, the deaminase and P21 N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nuclear localization sequence of the SV40 virus large T- antigen the minimal functional unit of which is the seven amino acid sequence PKKKRKV (SEQ ID NO: 50). In embodiments, the deaminase and P21 N-peptide fusion protein comprises one or more nuclear localization signals (NLS) including the nucleoplasmin bipartite NLS with the sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 51 ); the c-myct nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID NO: 52) or RQRRNELKRSF (SEQ ID NO: 53); and the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 54). In embodiments, further examples of nuclear localization sequences include: the sequence

RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 55) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 56) and PPKKARED (SEQ ID NO: 57) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 58) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 59) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 60) and PKQKKRK (SEQ ID NO: 61) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO: 62) of the Hepatitis virus delta antigen; and the sequence REKKKFLKRR (SEQ ID NO: 63) of the mouse Mx1 protein. In embodiments, nuclear localization sequences include the use of use bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 64) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 65) of the steroid hormone receptors (human) glucocorticoid. In embodiments, a NLS of the present disclosure comprises the amino acid sequence SV40 Large T-antigen NLS comprising the amino acid sequence:

[00140] DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 49).

[00141] In embodiments, the NLS contemplated by the present disclosure comprises one or more mutations (e.g., amino acid substitutions, deletions, and/or insertions) with respect to any one of SEQ ID NOs: 49-65.

[00142] In embodiments, the fusion proteins provided herein further comprise one or more nuclear targeting sequences, for example, a nuclear localization signal/sequence (NLS). In embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus. In embodiments, the NLS is fused to the N-terminus of the fusion protein. In embodiments, the NLS is fused to the C-terminus of the fusion protein. In embodiments, the NLS is fused to the N-terminus of the deaminase. In embodiments, the NLS is fused to the C-terminus of the deaminase. In embodiments, the NLS is fused to the N-terminus of the lambdoid P21 bacteriophage N-peptide. In embodiments, the NLS is fused to the C-terminus of the lambdoid P21 bacteriophage N-peptide. In embodiments, the NLS is fused to the fusion protein via one or more linkers. In embodiments, the NLS is fused to the fusion protein without a linker. In embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 49-65, or variants thereof.

Vector Delivery

[00143] In embodiments, components (i) a deaminase linked to a mutant A N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure are contained within various delivery vehicles.

[00144] In embodiments, components (i) a deaminase linked to a lambdoid P22 bacteriophage N- peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure are contained within various delivery vehicles.

[00145] In embodiments, components (i) a deaminase linked to a lambdoid P21 bacteriophage N- peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure are contained within various delivery vehicles. [00146] For example, in embodiments, the components of the present disclosure are contained within a plasmid or vector. In embodiments, components (i) and (ii) of the present disclosure are contained within a viral vector. In embodiments, such components of the present disclosure as previously described are synthetically generated or generated from a plasmid. In embodiments, the components are contained within a plasmid or vector, optionally wherein the vector is a viral vector. In embodiments, viral vectors include, but are not limited to, adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses. In embodiments, the viral vector is an adeno-associated virus (AAV) vector. In embodiments, the AAV is selected from AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, or AAV11 , and AAV12.

[00147] In embodiments, components of the present disclosure are contained within mRNA for delivery. For example, in embodiments, a synthetic oligonucleotide encoding the present compositions is provided. The synthetic oligonucleotide can have non-canonical nucleotides that, without wishing to be bound by theory, reduce innate immune responses. In embodiments, the synthetic oligonucleotide is an mRNA comprising one or more non-canonical nucleotides. In embodiments, the mRNA comprises one or more non- canonical nucleotides found in U.S. Patent No. 8,278,036, the entire contents of which are hereby incorporated by reference. In embodiments, the mRNA comprises one or more of m5C, m5U, m6A, s2U, 4^J, and 2'-O-methyl-U.

[00148] In embodiments, the components of the present disclosure are contained in lipid nanoparticles for gene delivery. In particular, an excipient may be used that aids in enhancing the stability, solubility, absorption, bioavailability, activity, pharmacokinetics, pharmacodynamics and/or delivery of the components of the present disclosure to a cell and into a cell. In embodiments, particular excipients capable of forming complexes, vesicles, nanoparticles, microparticles, nanotubes, nanogels, hydrogels, poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes and/or liposomes, that deliver components complexed or trapped in the vesicles or liposomes through a cell membrane find use. Examples of nanoparticles that are useful in embodiments include gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, protein nanoparticles and peptide nanoparticles. Another group of nanoparticles that are useful in embodiments are polymeric nanoparticles. Many of these polymeric substances are known in the art. Suitable substances that are useful in embodiments comprise e.g. polyethylenimine (PEI), ExGen 500, polypropyleneimine (PPI), poly(2-hydroxypropylenimine (pHP)), dextran derivatives (e.g. polycations such like diethyl amino ethyl amino ethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver said compound across cell membranes into cells), butylcyanoacrylate (PBCA), hexylcyanoacrylate (PHCA), poly(lactic-co-gly colic acid) (PLGA), polyamines (e.g. spermine, spermidine, putrescine, cadaverine), chitosan, poly(amido amines) (PAMAM), poly(ester amine), polyvinyl ether, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) cyclodextrins, hyaluronic acid, colominic acid, and derivatives thereof), dendrimers (e.g. poly(amidoamine), lipids (e.g. 1 ,2-dioleoyl-3- dimethylammonium propane (DODAP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholine derivatives (e.g. 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), lyso-phosphatidylcholine derivatives (e.g. 1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)), sphingomyeline, 2-{3-[bis-(3-amino- propyl)-amino]-propylamino}-N-ditetracedyl carbamoyl methylacetamide (RPR209120), phosphoglycerol derivatives (e.g. 1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na), phosphaticid acid derivatives (1 ,2-distearoyl-sn-glycero-3-phosphaticid acid, sodium salt (DSPA), phosphatidylethanolamine derivatives (e.g. dioleoyl-J-R-phosphatidylethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE)), JV-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium (DOTAP), 1 ,3-di-oleoyloxy-2-(6-carboxy-spermyl)- propylamid (DOSPER), (1 ,2-dimyristyolxypropyl-3-dimethylhydroxy ethyl ammonium (DMRIE), (N1- cholesteryloxycarbonyl-3,7-diazanonane-1 ,9-diamine (CD AN), dimethyldioctadecylammonium bromide (DDAB), 1 -palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), (b-L-Arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-olelyl-amide trihydrochloride (AtuFECTOI), N,N-dimethyl-3-aminopropane derivatives (e.g. 1 ,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA), 1 ,2-dilinoleyloxy-N,N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4- dimethylaminomethyl[1 ,3]-dioxolane (DLin-K-DMA), phosphatidylserine derivatives [1 ,2-dioleyl-sn-glycero-3- phospho-L-serine, sodium salt (DOPS)], cholesterol), synthetic amphiphils (SAINT-18), lipofectin, proteins (e.g. albumin, gelatins, atellocollagen), peptides (e.g. PepFects, NickFects, polyarginine, polylysine, CADY, MPG) combinations thereof and/or viral capsid proteins that are capable of self-assembly into particles that can deliver said components of the disclosure to a cell.

[00149] LIPOFECTIN is an illustrative liposomal transfection agent that finds use in embodiments. It consists of at least two lipid components, a cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release.

[00150] In addition to these nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate components of the present disclosure as colloids. This colloidal nanoparticle system can form so called “proticles,” which can be prepared by a simple self-assembly process to package and mediate intracellular release of a component as defined herein. The skilled person may select and adapt any of the above or other commercially available or not commercially available alternative excipients and delivery systems.

[00151] In embodiments of the disclosure, the components of the present disclosure are contained within the same or separate vectors, plasmids, mRNAs, and/or lipid nanoparticles, thus allowing for consecutive (e.g., before or after) or concurrent (e.g., at the same time) delivery to the target cells in the same composition or in separate compositions.

[00152] In embodiments, the deaminase and lambdoid bacteriophage N-peptide are not linked and/or fused. In embodiments, the present disclosure contemplates the use of an endogenous deaminase. For example, the deaminase may be recruited by the nucleic acid-editing system that includes some or all of the previously mentioned components.

Pharmaceutically Acceptable Carriers

[00153] In embodiments, compositions of the present disclosure (e.g., compositions comprising plasmids and/or viral vectors) further comprise a pharmaceutically acceptable carrier. In embodiments, the composition is formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects.

[00154] In embodiments, some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21 ) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

Methods of Use

[00155] The present compositions and methods find use in embodiments in altering nucleotides within an RNA molecule to provide a medically-beneficial effect. In embodiments, the present compositions and methods are useful in reducing the prevalence of, or ablating, a mutation. In embodiments, the present compositions and methods are useful in introducing a mutation that beneficially affects a subject’s health. The compositions and methods described herein for the purpose of site-specific editing of a target nucleotide (e.g., within an RNA molecule) in a cell may be administered to a subject in need thereof in a therapeutically effective amount to treat and/or prevent a disease, condition or disorder the subject is suffering from. Alternatively, the subject may not be suffering from a disease, condition or disorder, and the site-specific editing of a target nucleotide (e.g., within an RNA molecule) in a cell may induce a beneficial mutation in the wild-type target sequence.

[00156] In embodiments, methods of use herein include methods of treating diseases and/or disorders associated with various genetic mutations, variants, single nucleotide polymorphisms (SNPs), and the like, for example diseases and/or disorders caused by, or in part by, mutations, variants, or SNPs in the SerpinAI gene. The human SerpinAI gene encodes the enzyme, alpha-1 -antitrypsin (a1AT), which is a protein expressed in several tissues/fluids of the body (e.g., liver, lung, blood, etc.) that functions as a trypsin inhibitor. The a1AT enzyme finds use in modulating several processes in the body, including protecting cells and tissues from aberrant inflammatory responses, such as by neutrophil elastase. In embodiments, deficiencies in a1AT function results in a wide range of disorders and diseases, including in non-limiting examples, pulmonary emphysema, liver disease, anemia, chronic obstructive pulmonary disorder (COPD), etc., which can be corrected, or at least addressed in part, by nucleic acid by compositions and methods described herein. For example, in embodiments, the E342K variant of SerpinAI is one of the most common loss of function variants, which can be edited to a functional wild-type sequence using the deaminase fusions described herein (e.g., as shown in Figures 3, and 5-6).

[00157] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type X N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type A N-peptide, and (II) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the A N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the A N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00158] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type A N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type A N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the A N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the A N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00159] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type P22 N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type P22 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the P22 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the P22 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00160] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type P22 N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type P22 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the P22 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the P22 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00161] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type P21 N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within a SerpinAI gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type P21 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the P21 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the P21 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00162] In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell using the compositions and methods described herein (including, without limitation, a wild-type P21 N-peptide). In embodiments, the present disclosure contemplates a method of site-specific base editing of a target nucleotide within an IDUA gene in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a mutant or wild-type P21 N-peptide, and (ii) an antisense RNA oligonucleotide linked to a BoxB hairpin RNA oligonucleotide, the BoxB hairpin RNA oligonucleotide being the cognate hairpin oligonucleotide of the P21 N-peptide, and wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the P21 N-peptide and BoxB hairpin RNA oligonucleotide interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00163] In embodiments, the compositions of this disclosure may be administered or packaged as a unit dose, for example. The term "unit dose" when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e. , a carrier or vehicle.

[00164] In embodiments, treatment of a disease, condition or disorder includes delaying the development or progression of the disease, or reducing disease severity. Treating the disease or condition does not necessarily require curative results.

[00165] In embodiments, as used therein, "delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[00166] In embodiments, "development" or "progression" ofa disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset.

[00167] In embodiments, as used herein "onset" or "occurrence" of a disease includes initial onset and/or recurrence. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease.

Host Cells

[00168] In embodiments, cells containing any of the compositions described herein include prokaryotic cells and eukaryotic cells. In embodiments, the methods described herein are used to deliver a deaminase and other components contemplated herein into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In embodiments, the host cell is in vitro (e.g., cultured cell). In embodiments, the host cell is in vivo (e.g., in a subject such as a human subject). In embodiments, the host cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject). [00169] Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells. In embodiments, plasmids or viral vectors (e.g., adeno-associated virus (AAV) vectors) are delivered into human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In embodiments, AAV vectors are delivered into stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)). A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. A human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein). Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).

[00170] In embodiments, additional non-limiting examples of cell lines that are used in accordance with the present disclosure include 293-T, 293-T, 3T3, 4T1 , 721 , 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431 , ALC, B 16, B35, BCP-1 , BEAS-2B, bEnd.3, BHK-21 , BR 293, BxPC3, C2C12, C3H-10T1/2, C6, C6/36, Cal-27, CGR8, CHO, CML Tl, CMT, COR-L23, CGR-L23/5010, COR-L23/CPR, COR-L23/R23, COS-7, COV-434, CT26, D17, DH82, DU145, DuCaP, E14Tg2a, EL4, EM2, EM3, EMT6/AR1 , EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepalclc7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1 , Ku812, KYOI, LNCap, Ma-Mel 1 , 2, 3....48, MC-38, MCF-IOA, MCF-7, MDA-MB-231 , MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR/0.2R, MRC5, MTD-1A, MyEnd, NALM-1 , NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NW-145, OPCN/OPCT Peer, PNT-1A/PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA/RMAS, S2, Saos-2 cells, Sf21 , Sf9, Si Ha, SKBR3, SKOV-3, T-47D, T2, T84, THP1 , U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1 and YAR cells. Compositions and Pharmaceutical Compositions

[00171] In embodiments, the composition and/or pharmaceutical composition includes any site-specific RNA editing system described herein.

[00172] In embodiments, the composition and/or pharmaceutical composition includes (I) a deaminase linked to a mutant A N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure, optionally contained within various delivery vehicles and/or admixed with one or more pharmaceutically acceptable excipients.

[00173] In embodiments, the composition and/or pharmaceutical composition includes (I) a deaminase linked to a lambdoid P22 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure, optionally contained within various delivery vehicles and/or admixed with one or more pharmaceutically acceptable excipients.

[00174] In embodiments, the composition and/or pharmaceutical composition includes (I) a deaminase linked to a lambdoid P21 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin (e.g., a BoxB hairpin RNA) oligonucleotide, of the present disclosure, optionally contained within various delivery vehicles and/or admixed with one or more pharmaceutically acceptable excipients.

[00175] In embodiments, the composition and/or pharmaceutical composition includes the fusion protein and/or the one or more cognate RNA molecules. In embodiments, the composition and/or pharmaceutical composition includes one or more nucleic acids for expressing the fusion protein and/or the one or more cognate RNA molecules.

Kits

[00176] In embodiments, the compositions (e.g., nucleic acids/vectors, host cells, etc.) of the present disclosure are assembled into a kit. In embodiments, the kit comprises nucleic acid vectors for the expression of the components of the nucleic acid-editing systems described herein.

[00177] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kit described herein may further comprise components needed for performing the assay methods. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In embodiments, some of the components are reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[00178] In embodiments, the kits may optionally include instructions and/or promotion for use of the components provided. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, "promoted" includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[00179] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container.

[00180] The kits may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. [00181] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in anyway whatsoever.

EXAMPLES

Example 1: Construction of nucleic acid-editing system

[00182] A nucleic acid-editing system of the present disclosure was constructed. A nucleic acid construct, e.g., a plasmid, was created composed of a nucleic acid sequence encoding a mutant A N-peptide linked to a deaminase domain, described herein, according to methods known to those skilled in the art.

A to G nucleic acid-editing system using bacteriophage lambda

To build the A to G AN-deaminase nucleic acid-editing system, the wildtype sequence of deaminase domain of human ADAR2 sequence (NM_001112.4) corresponding to amino acid 299-701 was placed in-frame downstream of 4 copies of the bacteriophage AN peptide. The sequence of the AN peptide used was NARTRRRERRAEKQAQWKAAN-GGGGSGGGGSGGGGS (SEQ ID NO: 66), where the GGGGSGGGGSGGGGS (SEQ ID NO: 67) sequence denotes a linker sequence. This linker sequence separates the 4 AN peptides. An amino-terminus HA-tagged nuclear localization signal was appended to the N-terminus of the first copy of the N peptide. This fusion gene was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, USA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as “4AN_DD wild type” and its nucleic acid sequence is SEQ ID NO: 68. To generate the hyper-editing deaminase, we created a mutation at amino acid position 359 of 4AN_DD wild type polypeptide construct, resulting in a change from glutamic acid to glutamate. This mutant is denoted as “4AN_DD E359Q,” and its nucleic acid sequence is SEQ ID NO: 69.

[00183] A further nucleic acid construct, e.g., the same plasmid or a different plasmid, composed of a nucleic acid sequence encoding an antisense RNA oligonucleotide, which is complementary to a targeted nucleic acid allele sequence, linked to a hairpin RNA oligonucleotide, as described herein. Specifically, the deaminase domain is the human ADAR2 catalytic domain, optionally mutated (e.g., without limitation, with the E448Q mutation). The construct can be modified to include a NLS sequence (e.g. SV40). [00184] The antisense oligonucleotide linked to a hairpin RNA oligonucleotide was made by obtaining a DNA oligonucleotide encoding a U6 (RNA Polymerase III) promoter at the 5' end followed by a sequence comprising the antisense oligonucleotide linked to the hairpin RNA oligonucleotide. A U6 RNA polymerase promoter from the pENTR plasmid was used for plasmids that encode the antisense RNA oligonucleotide used for substrate specificity and selectivity because a U6 promoter gives a very defined 5’-end transcription start site that confers the editing specificity and selectivity. The antisense oligonucleotide sequence corresponds to a variable length of antisense sequence, complementary to the RNA molecule that was targeted. A hairpin RNA oligonucleotide of the present disclosure was used. An antisense version of the same DNA oligonucleotide was also synthesized. The two were then hybridized together and used as a template to make RNA with an RNA polymerase (e.g. U6).

[00185] Human SerpinAI E342K gene (NM_000295.4) was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, Berkeley, CA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as “SerpinA1/E342K/pcDNA3.1 ” and is nucleic acid sequence is SEQ ID NO: 72.

[00186] The heterologous IDUA genetic fusion was designed to express Td Tomato gene fused a 2A peptide that was fused in frame to a small region of the human IDUA gene (NMJJ00203.5) that contains the W402X mutation. This was then fused in frame to eGFP. This entire genetic sequence was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Life Technologies). This plasmid henceforth will be denoted as “TdJDUA W402X_eGFP/pcDNA3.1 ” and the correct insert was sequence verified as SEQ ID NO: 73.

[00187] The sequence for bacteriophage lambda 2boxBLambda guide RNA designed for the SerpinAI E342K allele is SEQ ID NO: 74 and was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBLambda SerpinAI E342K.” The sequence for bacteriophage lambda 2boxBLambda guide RNA designed for the TdJDUA W402X_eGFP mRNA is SEQ ID NO: 75 and was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBLambda IDUA W402X.”

[00188] If desired, AAV vectors containing the plasmids and/or synthetic oligonucleotides are then prepared according to knowledge of those skilled in the art.

[00189] The proper construction of the nucleic acid-editing system is verified via sequence analysis. [00190] The following tables provides the above-mentioned nucleic acid and/or amino acid sequences:

Table 4. Exemplary Sequences of Editase (i.e., nucleic acid-editing system)

Table 5. Sequence of exemplary Target Substrates

Table 6. Exemplary lambda 2boxB sequence and annotation

Table 7. Exemplary primer sequences

Example 2: In vitro RNA editing in a model cell to test activity of the nucleic acid-editinci system

[00191] The nucleic acid-editing system was tested for its ability to specifically edit RNA. The test gene contains a specific nucleotide(s) that is to be targeted for editing. In typical editing experiments 3.64 ng of nucleic acid-editing system plasmid, either 44 ng or 66 ng of BoxB guide RNA expressing plasmid and 0.91 ng of target plasmid were transfected into 2x10⁵ HEK293T cells (ATCC) using LIPOFECTAMINE 3000 (0.2 pL/per well) in a 96-well format. All transfections using LIPOFECTAMINE 3000 were performed according to manufacturer’s instructions. 24 to 120 hours later, the cells were washed once with ice cold PBS and total mRNA isolation was performed using Dyna Beads mRNA Direct Kit (Life Technologies) adapted for KingFisher Flex Purification (Life Technologies). This was done according to manufacturer’s instructions. The samples were treated with TURBO DNase (Life Technologies) prior to elution. The resultant isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer’s instructions (Life Technologies). Two to six pl of the cDNA was used for as template for PCR (Platinum II Hot-Start PCR Master Mix; Life Technologies) using gene specific primers to generate an amplicon for Sanger sequencing (SEQ ID NOs: 76-79 were used for primers in RT PCR). Sanger sequencing was performed by QuintaraBio (USA) or Psomagen (USA). Deamination of base and subsequent editing yields were quantified by measuring the peak height of G and dividing the edited nucleotide peak height by the total peak height measurements of non-edited and edited nucleotide combined.

[00192] The results of an in vitro editing experiment using the Human SerpinAI E342K gene are shown in Figure 3. These results show that the editing system is efficacious, and at least 73% editing occurs using the 4AN_DD wild type construct. The results of an in vitro editing experiment using the Human IDUA W402X gene are shown in Figure 4. These results show that the editing system is efficacious, and at least 65% editing occurs using the 4AN_DD wild type construct.

[00193] In another experiment, the antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide complex is developed according to the method of Example 1. The antisense portion of the RNA oligonucleotide is complementary to specific nucleotides of the test gene mRNA. The hairpin RNA oligonucleotide is inserted in the antisense oligonucleotide as desired. In these experiments, the position and number (1 to 2) of the hairpin RNA oligonucleotide is varied and still has a functional interaction. This antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide is combined with a test gene mRNA in vitro and a bacteriophage N-peptide linked to a deaminase domain protein, where they are expressed via the same or a separate plasmid promotor system, and brought in contact with the cell. After incubation, the test gene mRNA is converted into cDNA. Direct sequencing of RT-PCR products is undertaken to reveal editing at the specific nucleotide positions previously mentioned, directed by the antisense oligonucleotide and adjacent hairpin RNA oligonucleotide. Control experiments that lack either the antisense oligonucleotide linked to the hairpin RNA oligonucleotide or the A N-peptide linked to a deaminase domain protein are expected to show no editing. A further control is to incorporate scrambled RNA sequence in place of the antisense sequence, which would be expected to show no editing and further demonstrates specificity. Taken together, these data are expected to show the hairpin RNA oligonucleotide-A N-peptide interaction is required to target the catalytic domain of the deaminase to the nucleotide positions to be edited.

[00194] Based on the above results, the system is further tested to correct a genetic mutation or sequence expressed endogenously in vitro (the type of genetic mutation described herein is illustrative of any of the various genetic mutations or wild type nucleotide that can be edited). A specific nucleotide of an mRNA is targeted in a human disease model (e.g. cell line). For example, a mutation can cause codons to change so that instead of an mRNA encoding and translating to an amino acid, a premature termination codon (PTC) is read, which can lead to these humans developing a disease or condition. Using the aforementioned nucleic acid-editing system and method of editing, a specific adenosine is converted to inosine by the acting deamination domain, thereby converting the PTC to a codon encoding tryptophan. The A N-peptide linked to a deaminase domain protein will edit at a specific position based on being guided by the antisense RNA oligonucleotide linked to an inserted hairpin RNA oligonucleotide. Accordingly, construction of the antisense oligonucleotide linked to a hairpin RNA oligonucleotide will include selecting a sequence complementary to site to be edited with various lengths of nucleotides 5’ of the position. [00195] When this antisense oligonucleotide linked to the hairpin RNA oligonucleotide is combined with the X N-peptide linked to a deaminase domain protein and the mutated mRNA in vitro, a percentage of the mutant mRNA is corrected, with the adenosine nucleotide being changed to inosine and thus correcting the codon. Editing is expected to be specific to the adenosine.

Example 3: In vivo RNA editing in various genetic disorder models corrects mutation and recovers wild-type function

[00196] The system and methods of Examples 1 and 2 can be tested in living cells or cell lysates. Any suitable model for the desired editing may be used. For instance, if genetically altered mice (e.g. wild type mice in which the target mutation is knocked in) are available, cells or tissues from such mice can be used to study the editing (either in vivo or ex vivo, e.g. by harvesting cells from the mice). Secondly, primary diseased human cell models in which the cells are procured from a human patient afflicted with the disease can be used to study the editing. Additionally, in vivo model can be employed to examine editing using wild type mice when targeting a wild-type mRNA.

[00197] Other suitable methods that may be employed in these Examples are described in PNAS November 5, 2013 110 (45) 18285-18290; PNAS October 31 , 2017 114 (44) E9395-E9402; and Nature Methods February 8, 2019 16:239-242, the entire contents of which are incorporated by reference in their entireties.

Example 4: Construction of nucleic acid-editing system using bacteriophage P22 N-peptide

[00198] A nucleic acid-editing system of the present disclosure was constructed. A nucleic acid construct, e.g., a plasmid, was created composed of a nucleic acid sequence encoding a P22 N-peptide linked to a deaminase domain, described herein, according to methods known to those skilled in the art.

A to G nucleic acid-editing system using bacteriophage P22

To build the A to G P22N-deaminase nucleic acid-editing system, the wildtype sequence of deaminase domain of human ADAR2 sequence (NM_001112.4) corresponding to amino acid 299-701 is placed in-frame downstream of either 1 or 4 copies of the P22N peptides from bacteriophage P22. For the enzyme where there are 4 P22 N-peptides, each peptide sequence is GNAKTRRHERRRKLAIERDTI (SEQ ID NO: 80), and the 4 peptide sequences are separated by a linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 67). For the enzyme where there is only 1 P22N peptide, no linker sequence is used, and the peptide sequence is GNAKTRRHERRRKLAIERDTI (SEQ ID NO: 80). An amino-terminus HA-tagged nuclear localization signal was appended to the N-terminus of the first copy of N peptide. This fusion gene was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, USA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as “either 1 P22N_DD wild type” or “4P22N_DD wild type” and its nucleic acid sequence is SEQ ID NO: 85 (e.g., as listed in Table 8). A hyper-editing mutation is created at amino acid position 253 of 1P22N_DD wild type enzyme and amino acid position 267 of 4P22NJ3D wild type enzyme, in both cases changing a glutamic acid to glutamate. These enzymes are denoted as 1P22N_DD E253Q and 4P22N_DD E267Q, and are sequence verified. The sequences are listed in Table 8.

[00199] A further nucleic acid construct, e.g., the same plasmid or a different plasmid, composed of a nucleic acid sequence encoding an antisense RNA oligonucleotide, which is complementary to a targeted nucleic acid allele sequence, linked to a hairpin RNA oligonucleotide, as described herein. Specifically, the deaminase domain is the human ADAR2 catalytic domain, optionally mutated (e.g., without limitation, with the E448Q mutation). The construct can be modified to include a NLS sequence (e.g. SV40).

[00200] The antisense oligonucleotide linked to a hairpin RNA oligonucleotide was made by obtaining a DNA oligonucleotide encoding a U6 (RNA Polymerase III) promoter at the 5' end followed by a sequence comprising the antisense oligonucleotide linked to the hairpin RNA oligonucleotide. A U6 RNA polymerase promoter from the pENTR plasmid was used for plasmids that encode the antisense RNA oligonucleotide used for substrate specificity and selectivity because a U6 promoter gives a very defined 5’-end transcription start site that confers the editing specificity and selectivity. The antisense oligonucleotide sequence corresponds to a variable length of antisense sequence, complementary to the RNA molecule that was targeted. A hairpin RNA oligonucleotide of the present disclosure was used. An antisense version of the same DNA oligonucleotide was also synthesized. The two were then hybridized together and used as a template to make RNA with an RNA polymerase (e.g. U6).

[00201] Human SerpinAI E342K gene (NM_000295.4) was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, Berkeley, CA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as “SerpinA1/E342K/pcDNA3.1 ” and is nucleic acid sequence is SEQ ID NO: 72.

[00202] The heterologous IDUA genetic fusion was designed to express Td Tomato gene fused a 2A peptide that was fused in frame to a small region of the human IDUA gene (NM_000203.5) that contains the W402X mutation. This was then fused in frame to eGFP. This entire genetic sequence was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Life Technologies). This plasmid henceforth will be denoted as “TdJDUA W402X_eGFP/pcDNA3.1 ” and the correct insert was sequence verified as SEQ ID NO: 73.

[00203] The sequence for bacteriophage P22 2boxB guide RNA designed for the Serpi nA1 E342K allele is SEQ ID NO: 89 and was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBP22 SerpinAI E342K.” The sequence for bacteriophage P22 2boxB guide RNA designed for the TdJDUA W402X_eGFP mRNA is SEQ ID NO: 92 and was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBP22 IDUA W402X.”

[00204] If desired, AAV vectors containing the plasmids and/or synthetic oligonucleotides are then prepared according to knowledge of those skilled in the art.

[00205] The proper construction of the nucleic acid-editing system is verified via sequence analysis.

[00206] The following tables provides the above-mentioned nucleic acid and/or amino acid sequences:

Table 8. Exemplary Sequence of Editase (i.e., nucleic acid-editing system)

Table 9. Exemplary Sequence of Target Substrate

Table 10. Exemplary P22 2boxB sequence and annotation

Table 11. Exemplary Primer sequences

Example 5: In vitro RNA editing in a model cell to test activity of the nucleic acid-editing system

[00207] The nucleic acid-editing system was tested for its ability to specifically edit RNA. The test gene contains a specific nucleotide(s) that is to be targeted for editing. In typical editing experiments 3.64 ng of nucleic acid-editing system plasmid, 50 ng of BoxB guide RNA expressing plasmid and 0.91 ng of target plasmid were transfected into 2x10⁵ HEK293T cells (ATCC) using Lipofectamine 3000 (0.2 L/per well) in a 96-well format. All transfections using Lipofectamine 3000 were performed according to manufacturer’s instructions. 24 to 120 hours later, the cells were washed once with ice cold PBS and total mRNA isolation was performed using Dyna Beads mRNA Direct Kit (Life Technologies) adapted for KingFisher Flex Purification (Life Technologies). This was done according to manufacturer’s instructions. The samples were treated with TURBO DNase (Life Technologies) prior to elution. The resultant isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer’s instructions (Life Technologies). Two to six pl of the cDNA was used for as template for PCR (Platinum II Hot-Start PCR Master Mix; Life Technologies) using gene specific primers to generate an amplicon for Sanger sequencing (SEQ ID NOs: 76-79 were used for primers in RT PCR). Sanger sequencing was performed by QuintaraBio (USA) or Psomagen (USA). Deamination of base and subsequent editing yields were quantified by measuring the peak height of G and dividing the edited nucleotide peak height by the total peak height measurements of non-edited and edited nucleotide combined.

[00208] The results of an in vitro P22 N-peptide fusion editing experiment using the Human SerpinAI E342K gene are shown in Figure 5. These results show that the editing system is efficacious and at least 41% editing occurs using the 4P22N_DD wild type construct and at least 56% editing occurs using the 1 P22N_DD wild type construct. The single-copy P22 N-peptide construct (1 P22NJ3D) showed improved editing compared to the four-copy P22 N-peptide construct (4P22N_DD).

[00209] In another experiment, the antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide complex is developed according to the method of Example 4. The antisense portion of the RNA oligonucleotide is complementary to specific nucleotides of the test gene mRNA. The hairpin RNA oligonucleotide is inserted in the antisense oligonucleotide as desired. In these experiments, the position and number (1 to 2) of the hairpin RNA oligonucleotide is varied and still has a functional interaction. This antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide is combined with a test gene mRNA in vitro and a bacteriophage N-peptide linked to a deaminase domain protein, where they are expressed via a plasmid promoter system, and brought in contact with the cell. After incubation, the test gene mRNA is converted into cDNA. Direct sequencing of RT-PCR products is undertaken to reveal editing at the specific nucleotide positions previously mentioned, directed by the antisense oligonucleotide and adjacent hairpin RNA oligonucleotide. Control experiments that lack either the antisense oligonucleotide linked to the hairpin RNA oligonucleotide, or the bacteriophage N-peptide linked to a deaminase domain protein are expected to show no editing. A further control is to incorporate scrambled RNA sequence in place of the antisense sequence, which would be expected to show no editing and further demonstrates specificity. Taken together, these data are expected to show the hairpin RNA-bacteriophage N-peptide interaction is required to target the catalytic domain of the deaminase to the nucleotide positions to be edited.

[00210] Based on the above results, the system is further tested to correct a genetic mutation or sequence expressed endogenously in vitro (the type of genetic mutation described herein is illustrative of any of the various genetic mutations or wild type nucleotide that can be edited). A specific nucleotide of an mRNA is targeted in a human disease model (e.g. cell line). For example, a mutation can cause codons to change so that instead of an mRNA encoding and translating to an amino acid, a premature termination codon (PTC) is read, which can lead to these humans developing a disease or condition. Using the aforementioned nucleic acid-editing system and method of editing, a specific adenosine is converted to inosine by the acting deamination domain, thereby converting the PTC to a codon encoding tryptophan. The bacteriophage N-peptide linked to a deaminase domain protein will edit at a specific position based on being guided by the antisense RNA oligonucleotide linked to an inserted hairpin RNA oligonucleotide. Accordingly, construction of the antisense oligonucleotide linked to a hairpin RNA oligonucleotide will include selecting a sequence complementary to site to be edited with various lengths of nucleotides 5’ of the position.

[00211] When this antisense oligonucleotide linked to the hairpin RNA oligonucleotide is combined with the bacteriophage N-peptide linked to a deaminase domain protein and the mutated mRNA in vitro, a percentage of the mutant mRNA is corrected, with the adenosine nucleotide being changed to inosine and thus correcting the codon. Editing is expected to be specific to the adenosine.

Example 6: In vivo RNA editing in various genetic disorder models corrects mutation and recovers wild-type function

[00212] The system and methods of Examples 4 and 5 can be tested in living cells or cell lysates. Any suitable model for the desired editing may be used. For instance, if genetically altered mice (e.g. wild type mice in which the target mutation is knocked in) are available, cells or tissues from such mice can be used to study the editing (either in vivo or ex vivo, e.g. by harvesting cells from the mice). Secondly, primary diseased human cell models in which the cells are procured from a human patient afflicted with the disease can be used to study the editing. Additionally, in vivo model can be employed to examine editing using wild type mice when targeting a wild type mRNA. [00213] Other suitable methods that may be employed in these Examples are described in PNAS November 5, 2013 110 (45) 18285-18290; PNAS October 31 , 2017 114 (44) E9395-E9402; and Nature Methods February 8, 2019 16:239-242, the entire contents of which are incorporated by reference in their entireties.

Example 7: Construction of nucleic acid-editing system using bacteriophage P21

[00214] A nucleic acid-editing system of the present disclosure was constructed. A nucleic acid construct, e.g., a plasmid, was created composed of a nucleic acid sequence encoding a P21 N-peptide linked to a deaminase domain, described herein, according to methods known to those skilled in the art.

A to G nucleic acid-editing system using bacteriophage P21

To build the A to G P21 N-deaminase nucleic acid-editing system, the wildtype sequence of deaminase domain of human ADAR2 sequence (NM_001112.4) corresponding to amino acid 299-701 is placed in-frame downstream of 1 or 4 copies of the P21 N peptide from bacteriophage P21 . For the enzyme where there are 4 P22N peptides, each peptide sequence is IVWKESKGTAKSRYKARRAELIAERRSNEA (SEQ ID NO: 91 ), and the 4 peptide sequences are separated by a linker sequence, GGGGSGGGGSGGGGS (SEQ ID NO: 67). For the enzyme where there is only 1 P21 N peptide, no linker sequence is used, and the peptide sequence is IVWKESKGTAKSRYKARRAELIAERRSNEA (SEQ ID NO: 91 ). An amino-terminus HA-tagged nuclear localization signal was appended to the N-terminus of the first copy of the P21 N peptide. This fusion gene was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, USA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as either 1 P21 N_DD wild type or 4P21 N_DD wild type and its nucleic acid sequence is listed in Table 12. A hyper-editing mutation is created at amino acid position 260 of the 1 P21 N_DD wild type enzyme and amino acid position 295 of 4P21 N_DD wild type enzyme, in both cases changing a glutamic acid to glutamate. These enzymes are denoted as 1 P21 N_DD E260Q and 4P21 N_DD E295Q, and are sequence verified. The sequences are listed in Table 12.

[00215] A further nucleic acid construct, e.g., the same plasmid or a different plasmid, composed of a nucleic acid sequence encoding an antisense RNA oligonucleotide, which is complementary to a targeted nucleic acid allele sequence, linked to a hairpin RNA oligonucleotide, as described herein. Specifically, the deaminase domain is the human ADAR2 catalytic domain, optionally mutated (e.g., without limitation, with the E448Q mutation). The construct can be modified to include a NLS sequence (e.g. SV40). [00216] The antisense oligonucleotide linked to a hairpin RNA oligonucleotide was made by obtaining a DNA oligonucleotide encoding a U6 (RNA Polymerase III) promoter at the 5' end followed by a sequence comprising the antisense oligonucleotide linked to the hairpin RNA oligonucleotide. A U6 RNA polymerase promoter from the pENTR plasmid was used for plasmids that encode the antisense RNA oligonucleotide used for substrate specificity and selectivity because a U6 promoter gives a very defined 5’-end transcription start site that confers the editing specificity and selectivity. The antisense oligonucleotide sequence corresponds to a variable length of antisense sequence, complementary to the RNA molecule that was targeted. A hairpin RNA oligonucleotide of the present disclosure was used. An antisense version of the same DNA oligonucleotide was also synthesized. The two were then hybridized together and used as a template to make RNA with an RNA polymerase (e.g. U6).

[00217] Human SerpinAI E342K gene (NM_000295.4) was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, Berkeley, CA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as “SerpinA1/E342K/pcDNA3.1 ” and is nucleic acid sequence is SEQ ID NO: 72.

[00218] The heterologous IDUA genetic fusion was designed to express Td Tomato gene fused a 2A peptide that was fused in frame to a small region of the human IDUA gene (NMJJ00203.5) that contains the W402X mutation. This was then fused in frame to eGFP. This entire genetic sequence was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Life Technologies). This plasmid henceforth will be denoted as “TdJDUA W402X_eGFP/pcDNA3.1 ” and the correct insert was sequence verified as SEQ ID NO: 73.

[00219] The sequence for bacteriophage P21 2boxB guide RNA designed for the SerpinAI E342K allele is SEQ ID NO: 100 and was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBP21 SerpinAI E342K.” The sequence for bacteriophage P21 2boxB guide RNA designed for the TdJDUA W402X_eGFP mRNA was cloned into pENTR/U6 plasmid. The correct insert was sequence verified. This plasmid is denoted as “2boxBP21 IDUA W402X.”

[00220] If desired, AAV vectors containing the plasmids and/or synthetic oligonucleotides are then prepared according to knowledge of those skilled in the art.

[00221] The proper construction of the nucleic acid-editing system is verified via sequence analysis.

[00222] The following tables provides the above-mentioned nucleic acid and/or amino acid sequences: Table 12. Exemplary sequence of Editase (i.e., nucleic acid-editing system)

Table 13. Exemplary sequence of Target Substrate

Table 14. Exemplary P21 2boxB sequence and annotation

Table 15. Exemplary Primer Sequences

Example 8: In vitro RNA editing in a model cell to test activity of the nucleic acid-editinci system

[00223] The nucleic acid-editing system is tested for its ability to specifically edit RNA. The test gene contains a specific nucleotide(s) that is to be targeted for editing. In typical editing experiments 3.64 ng of nucleic acid-editing system plasmid, 50 ng of BoxB guide RNA expressing plasmid and 0.91 ng of target plasmid are transfected into 2x10⁵ HEK293T cells (ATCC) using Lipofectamine 3000 (0.2 L/per well) in a 96-well format. All transfections using Lipofectamine 3000 are performed according to manufacturer’s instructions. 24 to 120 hours later, the cells are washed once with ice cold PBS and total mRNA isolation is performed using Dyna Beads mRNA Direct Kit (Life Technologies) adapted for KingFisher Flex Purification (Life Technologies). This is done according to manufacturer’s instructions. The samples are treated with TURBO DNase (Life Technologies) prior to elution. The resultant isolated mRNA is used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer’s instructions (Life Technologies). Two to six l of the cDNA is used for as template for PCR (Platinum II Hot-Start PCR Master Mix; Life Technologies) using gene specific primers to generate an amplicon for Sanger sequencing (SEQ ID NOs: 76-79 are used for primers in RT PCR). Sanger sequencing is performed by QuintaraBio (USA) or Psomagen (USA). Deamination of base and subsequent editing yields were quantified by measuring the peak height of G and dividing the edited nucleotide peak height by the total peak height measurements of non-edited and edited nucleotide combined. [00224] In another experiment, the antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide complex is developed according to the method of Example 7. The antisense portion of the RNA oligonucleotide is complementary to specific nucleotides of the test gene mRNA. The hairpin RNA oligonucleotide is inserted in the antisense oligonucleotide as desired. In these experiments, the position and number (1 to 2) of the hairpin RNA oligonucleotide is varied and still has a functional interaction. This antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide is combined with a test gene mRNA in vitro and a bacteriophage N-peptide linked to a deaminase domain protein, where they are expressed via a plasmid promotor system, and brought in contact with the cell. After incubation, the test gene mRNA is converted into cDNA. Direct sequencing of RT-PCR products is undertaken to reveal editing at the specific nucleotide positions previously mentioned, directed by the antisense oligonucleotide and adjacent hairpin RNA oligonucleotide. Control experiments that lack either the antisense oligonucleotide linked to the hairpin RNA oligonucleotide, or the bacteriophage N-peptide linked to a deaminase domain protein are expected to show no editing. A further control is to incorporate scrambled RNA sequence in place of the antisense sequence, which would be expected to show no editing and further demonstrates specificity. Taken together, these data are expected to show the hairpin RNA-bacteriophage N-peptide interaction is required to target the catalytic domain of the deaminase to the nucleotide positions to be edited.

[00225] Based on the above results, the system is further tested to correct a genetic mutation or sequence expressed endogenously in vitro (the type of genetic mutation described herein is illustrative of any of the various genetic mutations or wild type nucleotide that can be edited). A specific nucleotide of an mRNA is targeted in a human disease model (e.g. cell line). For example, a mutation can cause codons to change so that instead of an mRNA encoding and translating to an amino acid, a premature termination codon (PTC) is read, which can lead to these humans developing a disease or condition. Using the aforementioned nucleic acid-editing system and method of editing, a specific adenosine is converted to inosine by the acting deamination domain, thereby converting the PTC to a codon encoding tryptophan. The bacteriophage N-peptide linked to a deaminase domain protein will edit at a specific position based on being guided by the antisense RNA oligonucleotide linked to an inserted hairpin RNA oligonucleotide. Accordingly, construction of the antisense oligonucleotide linked to a hairpin RNA oligonucleotide will include selecting a sequence complementary to site to be edited with various lengths of nucleotides 5’ of the position.

[00226] When this antisense oligonucleotide linked to the hairpin RNA oligonucleotide is combined with the bacteriophage N-peptide linked to a deaminase domain protein and the mutated mRNA in vitro, a percentage of the mutant mRNA is corrected, with the adenosine nucleotide being changed to inosine and thus correcting the codon. Editing is expected to be specific to the adenosine.

Example 9: In vivo RNA editing in various genetic disorder models corrects mutation and recovers wild-type function

[00227] The system and methods of Examples 7 and 8 can be tested in living cells or cell lysates. Any suitable model for the desired editing may be used. For instance, if genetically altered mice (e.g. wild type mice in which the target mutation is knocked in) are available, cells or tissues from such mice can be used to study the editing (either in vivo or ex vivo, e.g. by harvesting cells from the mice). Secondly, primary diseased human cell models in which the cells are procured from a human patient afflicted with the disease can be used to study the editing. Additionally, in vivo model can be employed to examine editing using wild type mice when targeting a wild-type mRNA.

[00228] Other suitable methods that may be employed in these Examples are described in PNAS November 5, 2013 110 (45) 18285-18290; PNAS October 31 , 2017 114 (44) E9395-E9402; and Nature Methods February 8, 2019 16:239-242, the entire contents of which are incorporated by reference in their entireties.

Example 10: In vitro RNA editing to correct SerpinAI E342K variant with 4AN DP E488Q mutant

[00229] An in vitro gene editing experiment was performed to correct a SerpinAI gene mutation, E342K, as a model genomic mutation to evaluate the efficiency of a hyper-editing mutant deaminase, ADAR (E488Q) fusion to 4x copy A N-peptide. This mutant is denoted as “4AN_DD E488Q,” with a nucleic acid sequence of SEQ ID NO: 101 and amino acid sequence of SEQ ID NO: 102, e.g., as summarized in Table 16.

Table 16: Exemplary ADAR-N-peptide fusion DNA and amino acid sequences for nucleic acid editing; the amino acid sequence highlights an E488Q mutant made relative to a wild-type sequence.

[00230] Human SerpinAI E342K gene (NM_000295.4) was synthesized and cloned into pcDNA3.1 plasmid under the control of the CMV promoter, as described in Examples 1 , 4, and 7. This plasmid henceforth will be denoted as “SerpinA1/E342K/pcDNA3.1 ” and is nucleic acid sequence is SEQ ID NO: 72.

[00231] The nucleic acid-editing system was tested for its ability to specifically edit RNA. The test gene contains a specific nucleotide(s) that is to be targeted for editing. In typical editing experiments 3.64 ng of nucleic acid-editing system plasmid, and a dose escalation of the BoxB guide RNA-expressing plasmid (0.18 ng, 0.54 ng, 1.64 ng, 4.9 ng, 14.81 ng, 44.44 ng, 133.33 ng, and 400 ng) and 0.91 ng of target plasmid were transfected into 2x10⁵ HEK293T cells (ATCC) using LIPOFECTAMINE 3000 (0.2 pL/per well) in a 96-well format. All transfections using LIPOFECTAMINE 3000 were performed according to manufacturer’s instructions. 24 to 120 hours later, the cells were washed once with ice cold PBS and total mRNA isolation was performed using Dyna Beads mRNA Direct Kit (Life Technologies) adapted for KingFisher Flex Purification (Life Technologies). This was done according to manufacturer’s instructions. The samples were treated with TURBO DNase (Life Technologies) prior to elution. The resultant isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer’s instructions (Life Technologies). Two to six l of the cDNA was used for as template for PCR (Platinum II Hot-Start PCR Master Mix; Life Technologies) using gene specific primers to generate an amplicon for Sanger sequencing (SEQ ID NOs: 76-79 were used for primers in RT PCR). Sanger sequencing was performed by QuintaraBio (USA) or Psomagen (USA). Deamination of base and subsequent editing yields were quantified by measuring the peak height of G and dividing the edited nucleotide peak height by the total peak height measurements of non-edited and edited nucleotide combined.

[00232] The results of an in vitro editing experiment using the Human SerpinAI E342K gene as a model are shown in Figure 6 and Table 17. These results show that the editing system is efficacious, and at least 44% editing occurs using the Human SerpinAI E342K gene using 4AN_DD E488Q construct.

Table 17: Exemplary ADAR-N-peptide fusion DNA and amino acid sequences for nucleic acid editing; the amino acid sequence highlights an E488Q mutant made relative to a wild-type sequence.

DEFINITIONS

[00233] The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this disclosure belongs.

[00234] As used herein, “a,” “an,” or “the” can mean one or more than one.

[00235] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

[00236] In embodiments, the term "deaminase¹' refers to an enzyme that catalyzes a deamination reaction. In embodiments, the deaminase is an adenosine deaminase, catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively.

[00237] In embodiments, an “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest, or optionally “effective amount” refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in embodiments, an effective amount of a nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a target site specifically bound and cleaved by the nuclease. In embodiments, an effective amount of a fusion protein provided herein, e.g., of a fusion protein comprising a deaminase (e.g. , a nucleic acid-editing domain) and a lambdoid bacteriophage N-peptide may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a deaminase, a recombinase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used. [00238] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[00239] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

[00240] In embodiments, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[00241] In embodiments, “homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[00242] In embodiments, “hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequencespecific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[00243] In embodiments, examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C.; hybridization buffer concentrations of about 6><SSC to about 1 OSSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4*SSC to about 8xSSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C.; buffer concentrations of about 9*SSC to about 2xSSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5xSSC to about 2xSSC. Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1 xSSC to about 0.1 xSSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 xSSC, 0.1 xSSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1 , 2, or more washing steps, and wash incubation times are about 1 , 2, or 15 minutes. SSC is 0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[00244] In embodiments, as used herein, the term “specifically binds” refers to the binding specificity of a specific binding pair. Hybridization by a target-specific nucleic acid sequence of a particular target polynucleotide sequence in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different nucleic acid molecules wherein one of the nucleic acid molecules specifically hybridizes with the second nucleic acid molecule through chemical or physical means. The two nucleic acid molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the binding component pair are referred to as ligand and receptor (anti-ligand), specific binding pair (SBP) member and SBP partner, and the like.

[00245] In embodiments, the term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a deaminase domain (e.g., a nucleic acid-editing domain) and N-peptide. In embodiments, a linker joins an antisense RNA oligonucleotide and a hairpin RNA oligonucleotide. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In embodiments, the linker is 5-100 amino acids in length, for example, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[00246] In embodiments, according to the present disclosure a "mismatch" is found at any position where no direct Watson-Crick base pair (A/T, G/C, C/G, T/A) correspondence exists between the oligonucleotide and the target in the region of complementarity between the two strands. An artificial mismatch is typically provided at one or more single nucleotide positions in an oligonucleotide, but can include more extensive changes. A true mismatch in a duplex formed between an oligonucleotide and a variant target can include a substitution, an insertion, a deletion, and a rearrangement of oligonucleotide nucleic acid relative to the target. Substitution can be at one or more positions in the oligonucleotide.

[00247] In embodiments, 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 (4th ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). According to embodiments of the present disclosure, a substituted amino acid residue is any naturally-occurring amino acid, such as a hydrophilic, hydrophobic, or non-classical amino acid. The hydrophilic amino acid can be selected from a polar and positively charged hydrophilic amino acid, a polar and neutral of charge hydrophilic amino acid, and polar and negatively charged hydrophilic amino acid, and an aromatic, polar and positively charged hydrophilic amino acid. A polar and positively charged hydrophilic amino acid can be selected from arginine (R) and lysine (K). A polar and neutral of charge hydrophilic amino acid can be selected from asparagine (N), glutamine (Q), serine (S), threonine (T), praline proline (P), and cysteine (C). A polar and negatively charged hydrophilic amino acid can be selected from aspartate (D) and glutamate (E). An aromatic, polar and positively charged hydrophilic amino acid can be histidine. A hydrophobic amino acid can be selected from a hydrophobic, aliphatic amino acid and a hydrophobic, aromatic amino acid. The hydrophobic, aliphatic amino acid can be glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). A hydrophobic, aromatic amino acid can be phenylalanine (F), tryptophan (W), or tyrosine (Y). A non-classical amino acid can be selected from selenocysteine, pyrrolysine, N-formylmethionine 0-alanine, GABA, 6- Aminolevulinic acid, 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, 0-alanine, fluoroamino acids, designer amino acids such as 0 methyl amino acids, C a- methyl amino acids, N a -methyl amino acids, and amino acid analogs in general. [00248] 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., 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.

[00249] In embodiments, 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 embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In 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 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 can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5¹ to 3' direction unless otherwise indicated. In embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); 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, 0(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).

[00250] In embodiments, the term "pharmaceutically-acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, orsteric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g. , the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

[00251] In embodiments, as used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[00252] In embodiments, the term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human primate. In embodiments, the subject is a rodent. In embodiments, the subject is a sheep, a goat, cattle, a cat, or a dog. In embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In embodiments, the subject is a research animal. In embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

[00253] In embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. In embodiments, the effect will result in a quantifiable change of two-fold, or three-fold, orfour-fold, orfive-fold, or ten-fold. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder or reduction in toxicity, regardless of whether improvement is realized.

[00254] In embodiments, the disclosure is directed to the following embodiments: [00255] Embodiment 1 . A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising (a) contacting the cell with: (i) a deaminase linked to a mutant A bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00256] Embodiment 2. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising contacting the cell with: (i) a deaminase linked to a mutant A bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00257] Embodiment 3. The method of embodiment 1 or 2, wherein the deaminase is adenosine deaminase.

[00258] Embodiment 4. The method of any one of embodiments 1 -3, wherein the adenosine deaminase yields an inosine (I nucleotide.

[00259] Embodiment 5. The method of any one of embodiments 1 -4, wherein the adenosine deaminase is selected from adenosine deaminase RNA specific 1 (ADAR1 ), adenosine deaminase RNA specific 2 (ADAR2), adenosine deaminase RNA specific B1 (ADARB1 ), adenosine deaminase like (ADAL), adenosine deaminase 2 (ADA2), adenosine monophosphate deaminase 1 (AMPD1), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase domain containing 1 (ADAD1 ), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase tRNA specific 1 (ADAT1 ), adenosine deaminase tRNA specific 2 (ADAT2), adenosine deaminase tRNA specific 3 (ADAT3), TadA or fragments or variants thereof.

[00260] Embodiment 6. The method of any one of embodiments 1 -5, wherein the adenosine deaminase is ADARI or ADAR2.

[00261] Embodiment 7. The method of any one of embodiments 1 -6, wherein the adenosine deaminase is a fragment, the fragment comprising the catalytic domain of the adenosine deaminase. [00262] Embodiment 8. The method of any one of embodiments 1 -7, wherein the adenosine deaminase is a fragment, the fragment comprising a catalytic domain and one or more mutations.

[00263] Embodiment 9. The method of any one of embodiments 1-8, wherein the ADAR1 or ADAR2 comprises a fragment that comprises a catalytic domain.

[00264] Embodiment 10. The method of any one of embodiments 1-9, wherein the catalytic domain is the deaminase domain of human ADAR2, comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 103.

[00265] Embodiment 11. The method of any one of embodiments 1-10, wherein the ADAR2 comprises the amino acid sequence of SEQ ID NO: 104.

[00266] Embodiment 12. The method of any one of embodiments 1-11 , wherein the mutant aminoterminal domain A N-peptide is not the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1).

[00267] Embodiment 13. The method of any one of embodiments 1-12, wherein the mutant aminoterminal domain A N-peptide comprises an arginine-rich motif.

[00268] Embodiment 14. The method of any one of embodiments 1-13, wherein the mutant aminoterminal domain A N-peptide comprises an Ala at position 3 and an arginine at each of positions 6, 10, and 11 of SEQ ID NO: 1.

[00269] Embodiment 15. The method of any one of embodiments 1-14, wherein the mutant aminoterminal domain A N-peptide variant comprises at least one mutation as compared to the wild-type aminoterminal domain A N-peptide (SEQ ID NO: 1 ).

[00270] Embodiment 16. The method of any one of embodiments 1-15, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition.

[00271] Embodiment 17. The method of any one of embodiments 1-16, wherein the mutant aminoterminal domain A N-peptide comprises from 1 to 19 amino acid residue substitutions.

[00272] Embodiment 18. The method of any one of embodiments 1-17, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 19, relative to the wild-type aminoterminal domain A N-peptide sequence (SEQ ID NO: 1 ).

[00273] Embodiment 19. The method of any one of embodiments 1-18, wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 1 . [00274] Embodiment 20. The method of any one of embodiments 1-19, wherein the mutant aminoterminal domain A N-peptide comprises the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 9.

[00275] Embodiment 21 . The method of any one of embodiments 1 -20, wherein the affinity of the mutant A N-peptide for the BoxB hairpin RNA oligonucleotide is greater than the affinity of the wild-type A N-peptide for the BoxB hairpin RNA oligonucleotide.

[00276] Embodiment 22. The method of any one of embodiments 1-21 , wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35- fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[00277] Embodiment 23. The method of any one of embodiments 1 -22, wherein the mutant A N-peptide comprises a substituted amino acid residue that is any naturally-occurring amino acid.

[00278] Embodiment 24. The method of any one of embodiments 1 -23, wherein the naturally-occurring amino acid is hydrophilic, hydrophobic, or non-classical.

[00279] Embodiment 25. The method of any one of embodiments 1-24, wherein the hydrophilic amino acid is selected from a polar and positively charged hydrophilic amino acid, a polar and neutral of charge hydrophilic amino acid, and polar and negatively charged hydrophilic amino acid, and an aromatic, polar and positively charged hydrophilic amino acid.

[00280] Embodiment 26. The method of any one of embodiments 1-25, wherein the polar and positively charged hydrophilic amino acid is arginine (R) or lysine (K).

[00281] Embodiment 27. The method of any one of embodiments 1-26, wherein the polar and neutral of charge hydrophilic amino acid is asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), or cysteine (C).

[00282] Embodiment 28. The method of any one of embodiments 1 -27, wherein the polar and negatively charged hydrophilic amino acid is aspartate (D) or glutamate (E).

[00283] Embodiment 29. The method of any one of embodiments 1 -28, wherein the aromatic, polar and positively charged hydrophilic amino acid is histidine.

[00284] Embodiment 30. The method of any one of embodiments 1-29, wherein the hydrophobic amino acid is selected from a hydrophobic, aliphatic amino acid and a hydrophobic, aromatic amino acid. [00285] Embodiment 31. The method of any one of embodiments 1-30, wherein the hydrophobic, aliphatic amino acid is glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).

[00286] Embodiment 32. The method of any one of embodiments 1-31 , wherein the hydrophobic, aromatic amino acid is phenylalanine (F), tryptophan (W), or tyrosine (Y).

[00287] Embodiment 33. The method of any one of embodiments 1-32, wherein the non-classical amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA, 6-Aminolevulinic acid, 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-d iaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, [3- alanine, fluoroamino acids, designer amino acids such as f> methyl amino acids, C o-methyl amino acids, N a -methyl amino acids, and amino acid analogs in general.

[00288] Embodiment 34. The method of any one of embodiments 1 -33, wherein the deaminase and the mutant A N-peptide are a fusion protein.

[00289] Embodiment 35. The method of any one of embodiments 1 -34, wherein the deaminase and the mutant A N-peptide are a connected by a chemical linker.

[00290] Embodiment 36. The method of any one of embodiments 1-35, wherein the deaminase is linked to at least 2, at least 3, or at least 4 A N-peptides.

[00291] Embodiment 37. The method of any one of embodiments 1 -36, wherein the mutant A N-peptide is linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof.

[00292] Embodiment 38. The method of any one of embodiments 1 -37, wherein the deaminase and the mutant A N-peptide comprise a nuclear localization signal.

[00293] Embodiment 39. The method of any one of embodiments 1 -38, wherein the deaminase and the mutant A N-peptide comprise a nuclear localization signal and the RNA molecule comprising the target nucleotide is in the nucleus of the cell.

[00294] Embodiment 40. The method of any one of embodiments 1 -39, wherein the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal. [00295] Embodiment 41. The method of any one of embodiments 1-40, wherein SV40 Large T-antigen nuclear localization signal comprises the amino acid sequence of SEQ ID NO: 49.

[00296] Embodiment 42. The method of any one of embodiments 1-41 , wherein the antisense RNA nucleotide hybridizes with a wild-type target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00297] Embodiment 43. The method of any one of embodiments 1 -42, wherein the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence.

[00298] Embodiment 44. The method of any one of embodiments 1-43, wherein the antisense RNA nucleotide hybridizes with a mutant target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00299] Embodiment 45. The method of any one of embodiments 1 -44, wherein the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence.

[00300] Embodiment 46. The method of any one of embodiments 1 -45, wherein the target nucleotide is a genetic mutation associated with a genetic disease or disorder.

[00301] Embodiment 47. The method of any one of embodiments 1 -46, wherein the genetic mutation of the target nucleotide causes a premature termination codon (PTC) or an in-frame stop codon.

[00302] Embodiment 48. The method of any one of embodiments 1 -47, wherein the genetic disease or disorder is caused by a G-to-A nucleotide mutation.

[00303] Embodiment 49. The method of any one of embodiments 1-48, wherein the antisense RNA oligonucleotide hybridizes with an RNA molecule comprising the target nucleotide in endogenous RNA of the cell.

[00304] Embodiment 50. The method of any one of embodiments 1-49, wherein the antisense RNA oligonucleotide has perfect base-pairing complementarity with the RNA molecule comprising the target nucleotide.

[00305] Embodiment 51. The method of any one of embodiments 1-50, wherein the antisense RNA oligonucleotide comprises a one or more mismatches with the RNA molecule comprising the target nucleotide. [00306] Embodiment 52. The method of any one of embodiments 1-51 , wherein the antisense RNA oligonucleotide sequence comprises from 1 to 15 mismatches, e.g. about 1 , or about 2, or about 3, or about 4, or about 5, or about 7, or about 10, or about 15 mismatches.

[00307] Embodiment 53. The method of any one of embodiments 1-52, wherein the antisense RNA oligonucleotide sequence is at least 80% complementary with the target sequence.

[00308] Embodiment 54. The method of any one of embodiments 1-53, wherein the 5' end of the antisense RNA oligonucleotide begins before the target nucleotide to be edited.

[00309] Embodiment 55. The method of any one of embodiments 1-54, wherein the 3' end of the oligonucleotide extends from 2-22 nucleotides 5' from the target nucleotide to be edited.

[00310] Embodiment 56. The method of any one of embodiments 1-55, wherein the antisense RNA oligonucleotide is linked to at least 2, 3, or 4 hairpin RNA oligonucleotides.

[00311] Embodiment 57. The method of any one of embodiments 1-56, wherein the hairpin RNA oligonucleotide comprises a nucleic acid sequence located from about 2-20 nucleotide residues 5' of the target mutation.

[00312] Embodiment 58. The method of any one of embodiments 1-57, wherein the hairpin RNA oligonucleotide comprises a nucleic acid sequence located from about 2-20 nucleotide residues 3' of the target mutation.

[00313] Embodiment 59. The method of any one of embodiments 1-58, wherein the hairpin RNA oligonucleotide is a BoxB hairpin RNA oligonucleotide.

[00314] Embodiment 60. The method of any one of embodiments 1 -59, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the mutant A N-peptide.

[00315] Embodiment 61 . The method of any one of embodiments 1 -60, wherein the BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or mutants thereof.

[00316] Embodiment 62. The method of any one of embodiments 1-61 , wherein the mutant A N peptide interacts with a A BoxB hairpin RNA oligonucleotide comprising a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or mutant thereof.

[00317] Embodiment 63. The method of any one of embodiments 1-62, wherein the mutant A N peptide and A BoxB hairpin RNA oligonucleotide interaction adopts a 4-out GNRA-like pentaloop. [00318] Embodiment 64. The method of any one of embodiments 1-63, wherein the nucleic acid sequence comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion.

[00319] Embodiment 65. The method of any one of embodiments 1-64, wherein the A BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence having sequence identity to SEQ ID NO: 37 or SEQ ID NO: 38, and further comprises from 1 to 15 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions.

[00320] Embodiment 66. The method of any one of embodiments 1-65, wherein the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 15, relative to the wildtype A BoxB hairpin RNA oligonucleotide.

[00321] Embodiment 67. The method of any one of embodiments 1-66, wherein the A BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of one or more of SEQ ID NOs: 39-44.

[00322] Embodiment 68. The method of any one of embodiments 1-67, wherein components (i) and (ii) are within a plasmid, vector, mRNA, or lipid nanoparticle.

[00323] Embodiment 69. The method of any one of embodiments 1-68, wherein components (i) and (ii) are within a viral vector.

[00324] Embodiment 70. The method of any one of embodiments 1-69, wherein the viral vector is an adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses vector.

[00325] Embodiment 71. The method of any one of embodiments 1-70, wherein the viral vector is an adeno-associated virus (AAV) vector.

[00326] Embodiment 72. The method of any one of embodiments 1-71 , wherein the AAV vector is selected from AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, or AAV11 , and AAV12.

[00327] Embodiment 1001. A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a lambdoid P22 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. [00328] Embodiment 1002. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising contacting the cell with: (i) a deaminase linked to a lambdoid P22 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00329] Embodiment 1003. The method of embodiment 1001 or embodiment 1002, wherein the deaminase is an adenosine deaminase.

[00330] Embodiment 1004. The method of any one of embodiments 1001-1003, wherein the adenosine deaminase yields an inosine (I) nucleotide.

[00331] Embodiment 1005. The method of any one of embodiments 1001-1004, wherein the adenosine deaminase is selected from adenosine deaminase RNA specific 1 (ADAR1), adenosine deaminase RNA specific 2 (ADAR2), adenosine deaminase RNA specific B1 (ADARB1 ), adenosine deaminase like (ADAL), adenosine deaminase 2 (ADA2), adenosine monophosphate deaminase 1 (AMPD1 ), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase domain containing 1 (ADAD1), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase tRNA specific 1 (ADAT 1 ), adenosine deaminase tRNA specific 2 (ADAT2), adenosine deaminase tRNA specific 3 (ADAT3), TadA or fragments or variants thereof.

[00332] Embodiment 1006. The method of any one of embodiments 1001-1005, wherein the adenosine deaminase is ADAR1 or ADAR2.

[00333] Embodiment 1007. The method of any one of embodiments 1001-1006, wherein the adenosine deaminase is a fragment, the fragment comprising the catalytic domain of the adenosine deaminase.

[00334] Embodiment 1008. The method of any one of embodiments 1001-1007, wherein the adenosine deaminase is a fragment, the fragment comprising a catalytic domain and one or more mutations.

[00335] Embodiment 1009. The method of any one of embodiments 1001-1008, wherein the ADAR1 or ADAR2 comprises a fragment that comprises a catalytic domain.

[00336] Embodiment 1010. The method of any one of embodiments 1001-1009, wherein the catalytic domain is the deaminase domain of human ADAR2, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 103. [00337] Embodiment 1011. The method of any one of embodiments 1001-1010, wherein the ADAR2 comprises the amino acid sequence of SEQ ID NO: 104.

[00338] Embodiment 1012. The method of any one of embodiments 1001-1011 , wherein the lambdoid bacteriophage N-peptide is derived from the P22 lambdoid bacteriophage.

[00339] Embodiment 1013. The method of any one of embodiments 1001-1012, wherein the P22 N- peptide is a variant that is the wild-type amino-terminal domain P22 N-peptide and comprises the amino acid sequence of SEQ ID NO: 10.

[00340] Embodiment 1014. The method of any one of embodiments 1001-1013, wherein the aminoterminal domain P22 N-peptide comprises an arginine-rich motif.

[00341] Embodiment 1015. The method of any one of embodiments 1001-1014, wherein the aminoterminal domain P22 N-peptide comprises an Ala at position 5 and an arginine at each of positions 8, 12, and 13 of SEQ ID NO: 2.

[00342] Embodiment 1016. The method of any one of embodiments 1001-1015, wherein the aminoterminal domain P22 N-peptide variant comprises at least one mutation as compared to the wild type aminoterminal domain P22 N-peptide.

[00343] Embodiment 1017. The method of any one of embodiments 1001-1016, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition.

[00344] Embodiment 1018. The method of any one of embodiments 1001-1017, wherein the aminoterminal domain P22 N-peptide variant comprises from 1 to 21 amino acid residue substitutions.

[00345] Embodiment 1109. The method of any one of embodiments 1001-1018, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild type aminoterminal domain P22 N-peptide sequence (SEQ ID NO: 2).

[00346] Embodiment 1020. The method of any one of embodiments 1001-1019, wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 2.

[00347] Embodiment 1021. The method of any one of embodiments 1001-1020, wherein the aminoterminal domain P22 N-peptide variant comprises the amino acid sequence of any one of SEQ ID NO: 10 to SEQ ID NO: 35. [00348] Embodiment 1022. The method of any one of embodiments 1001-1021 , wherein the affinity of the P22 N-peptide variant for the BoxB hairpin RNA is greater than the affinity of the wild type P22 N-peptide for the BoxB hairpin RNA.

[00349] Embodiment 1023. The method of any one of embodiments 1001-1022, wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[00350] Embodiment 1024. The method of any one of embodiments 1001-1023, wherein the substituted amino acid residue of the lambdoid bacteriophage N-peptide is any naturally-occurring amino acid.

[00351] Embodiment 1025. The method of any one of embodiments 1001-1024, wherein the naturally- occurring amino acid is hydrophilic, hydrophobic, or non-classical.

[00352] Embodiment 1026. The method of any one of embodiments 1001-1025, wherein the hydrophilic amino acid is selected from a polar and positively charged hydrophilic amino acid, a polar and neutral of charge hydrophilic amino acid, and polar and negatively charged hydrophilic amino acid, and an aromatic, polar and positively charged hydrophilic amino acid.

[00353] Embodiment 1027. The method of any one of embodiments 1001-1026, wherein the polar and positively charged hydrophilic amino acid is arginine (R) or lysine (K).

[00354] Embodiment 1028. The method of any one of embodiments 1001-1027, wherein the polar and neutral of charge hydrophilic amino acid is asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), or cysteine (C).

[00355] Embodiment 1029. The method of any one of embodiments 1001-1028, wherein the polar and negatively charged hydrophilic amino acid is aspartate (D) or glutamate (E).

[00356] Embodiment 1030. The method of any one of embodiments 1001-1029, wherein the aromatic, polar and positively charged hydrophilic amino acid is histidine.

[00357] Embodiment 1031 . The method of any one of embodiments 1001-1030, wherein the hydrophobic amino acid is selected from a hydrophobic, aliphatic amino acid and a hydrophobic, aromatic amino acid.

[00358] Embodiment 1032. The method of any one of embodiments 1001-1031 , wherein the hydrophobic, aliphatic amino acid is glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). [00359] Embodiment 1033. The method of any one of embodiments 1001-1032, wherein the hydrophobic, aromatic amino acid is phenylalanine (F), tryptophan (W), or tyrosine (Y).

[00360] Embodiment 1034. The method of any one of embodiments 1001-1033, wherein the non- classical amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA, 6- Aminolevulinic acid, 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoroamino acids, designer amino acids such as p methyl amino acids, C a- methyl amino acids, N a -methyl amino acids, and amino acid analogs in general.

[00361] Embodiment 1035. The method of any one of embodiments 1001-1034, wherein the deaminase and the lambdoid bacteriophage N-peptide are a fusion protein.

[00362] Embodiment 1036. The method of any one of embodiments 1001-1035, wherein the deaminase and the lambdoid bacteriophage N-peptide are a connected by a chemical linker.

[00363] Embodiment 1037. The method of any one of embodiments 1001-1036, wherein the deaminase is linked to at least 2, at least 3, or at least 4 lambdoid bacteriophage N-peptides.

[00364] Embodiment 1038. The method of any one of embodiments 1001-1037, wherein the bacteriophage N-peptide is linked to at least 2, at least 3, or at least 4 deaminases.

[00365] Embodiment 1039. The method of any one of embodiments 1001-1038, wherein the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal.

[00366] Embodiment 1040. The method of any one of embodiments 1001-1039, wherein the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal and the RNA molecule comprising the target nucleotide is in the nucleus of the cell.

[00367] Embodiment 1041. The method of any one of embodiments 1001-1040, wherein the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal.

[00368] Embodiment 1042. The method of any one of embodiments 1001-1041 , wherein SV40 Large T- antigen nuclear localization signal comprises the amino acid sequence of SEQ ID NO: 49. [00369] Embodiment 1043. The method of any one of embodiments 1001-1042, wherein the antisense RNA nucleotide hybridizes with a wild-type target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00370] Embodiment 1044. The method of any one of embodiments 1001-1043, wherein the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence.

[00371] Embodiment 1045. The method of any one of embodiments 1001-1044, wherein the antisense RNA nucleotide hybridizes with a mutant target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00372] Embodiment 1046. The method of any one of embodiments 1001-1045, wherein the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence.

[00373] Embodiment 1047. The method of any one of embodiments 1001-1046, wherein the target nucleotide is a genetic mutation associated with a genetic disease or disorder.

[00374] Embodiment 1048. The method of any one of embodiments 1001-1047, wherein the genetic mutation of the target nucleotide causes a premature termination codon (PTC) or an in-frame stop codon.

[00375] Embodiment 1049. The method of any one of embodiments 1001-1058, wherein the genetic disease or disorder is caused by a G-to-A nucleotide mutation.

[00376] Embodiment 1050. The method of any one of embodiments 1001-1049, wherein the antisense RNA oligonucleotide hybridizes with an RNA molecule comprising the target nucleotide in endogenous RNA of the cell.

[00377] Embodiment 1051 . The method of any one of embodiments 1001-1050, wherein the antisense RNA oligonucleotide has perfect base-pairing complementarity with the RNA molecule comprising the target nucleotide.

[00378] Embodiment 1052. The method of any one of embodiments 1001-1051 , wherein the antisense RNA oligonucleotide comprises a one or more mismatches with the RNA molecule comprising the target nucleotide.

[00379] Embodiment 1053. The method of any one of embodiments 1001-1052, wherein the antisense RNA oligonucleotide sequence comprises from 1 to 15 mismatches, or about 1 , or about 2, or about 3, or about 4, or about 5, or about 7, or about 10, or about 15 mismatches. [00380] Embodiment 1054. The method of any one of embodiments 1001-1053, wherein the antisense RNA oligonucleotide sequence is at least 80% complementary with the target sequence.

[00381] Embodiment 1055. The method of any one of embodiments 1001-1054, wherein the 5' end of the antisense RNA oligonucleotide begins before the target nucleotide to be edited.

[00382] Embodiment 1056. The method of any one of embodiments 1001-1055, wherein the 3' end of the antisense RNA oligonucleotide extends from 2-22 nucleotides 5' from the target nucleotide to be edited.

[00383] Embodiment 1057. The method of any one of embodiments 1001-1056, wherein the antisense RNA oligonucleotide is linked to at least 2, 3, or 4 hairpin RNA oligonucleotides.

[00384] Embodiment 1058. The method of any one of embodiments 1001-1057, wherein the hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5' of the target mutation.

[00385] Embodiment 1059. The method of any one of embodiments 1001-1058, wherein the hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3' of the target mutation.

[00386] Embodiment 1060. The method of any one of embodiments 1001-1059, wherein the hairpin RNA oligonucleotide is a BoxB hairpin RNA oligonucleotide.

[00387] Embodiment 1061. The method of any one of embodiments 1001-1060, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the N-peptide.

[00388] Embodiment 1062. The method of any one of embodiments 1001-1061 , wherein the BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 6-7, or variants thereof.

[00389] Embodiment 1063. The method of any one of embodiments 1001-1062, wherein the P22 N peptide interacts with a P22 BoxB hairpin RNA oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, or variant thereof.

[00390] Embodiment 1064. The method of any one of embodiments 1001-1063, wherein the P22 N peptide and P22 BoxB hairpin RNA oligonucleotide interaction adopts a 3-out GNRA-like pentaloop.

[00391] Embodiment 1065. The method of any one of embodiments 1001-1064, wherein the hairpin RNA oligonucleotide comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion. [00392] Embodiment 1066. The method of any one of embodiments 1001 -1065, wherein the P22 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, and further comprises from 1 to 17 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions.

[00393] Embodiment 1067. The method of any one of embodiments 1001-1066, wherein the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 17, relative to the wild type P22 BoxB hairpin RNA oligonucleotide.

[00394] Embodiment 1068. The method of any one of embodiments 1001-1067, wherein components (i) and (ii) are within a plasmid, vector, mRNA, or lipid nanoparticle.

[00395] Embodiment 1069. The method of any one of embodiments 1001-1068, wherein components (I) and (ii) are within a viral vector.

[00396] Embodiment 1070. The method of any one of embodiments 1001-1069, wherein the viral vector is an adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses vector.

[00397] Embodiment 1071. The method of any one of embodiments 1001-1070, wherein the viral vector is an adeno-associated virus (AAV) vector.

[00398] Embodiment 1072. The method of any one of embodiments 1001-1071 , wherein the AAV vector is selected from AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, orAAV11 , and AAV12.

[00399] Embodiment 2001 . A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with: (i) a deaminase linked to a lambdoid P21 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00400] Embodiment 2002. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising contacting the cell with: (i) a deaminase linked to a lambdoid P21 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.

[00401] Embodiment 2003. The method of embodiment 2001 or embodiment 2002, wherein the deaminase is an adenosine deaminase.

[00402] Embodiment 2004. The method of any one of embodiments 2001-2003, wherein the adenosine deaminase yields an inosine (I) nucleotide.

[00403] Embodiment 2005. The method of any one of embodiments 2001-2004, wherein the adenosine deaminase is selected from adenosine deaminase RNA specific 1 (ADAR1), adenosine deaminase RNA specific 2 (ADAR2), adenosine deaminase RNA specific B1 (ADARB1 ), adenosine deaminase like (ADAL), adenosine deaminase 2 (ADA2), adenosine monophosphate deaminase 1 (AMPD1 ), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase domain containing 1 (ADAD1), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase tRNA specific 1 (ADAT 1 ), adenosine deaminase tRNA specific 2 (ADAT2), adenosine deaminase tRNA specific 3 (ADAT3), TadA or fragments or variants thereof.

[00404] Embodiment 2006. The method of any one of embodiments 2001-2005, wherein the adenosine deaminase is ADAR1 or ADAR2.

[00405] Embodiment 2007. The method of any one of embodiments 2001-2006, wherein the adenosine deaminase is a fragment, the fragment comprising the catalytic domain of the adenosine deaminase.

[00406] Embodiment 2008. The method of any one of embodiments 2001-2007, wherein the adenosine deaminase is a fragment, the fragment comprising a catalytic domain and one or more mutations.

[00407] Embodiment 2009. The method of any one of embodiments 2001-2008, wherein the ADAR1 or ADAR2 comprises a fragment that comprises a catalytic domain.

[00408] Embodiment 2010. The method of any one of embodiments 2001-2009, wherein the catalytic domain is the deaminase domain of human ADAR2, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 103.

[00409] Embodiment 2011. The method of any one of embodiments 2001-2010, wherein the ADAR2 comprises the amino acid sequence of SEQ ID NO: 104. [00410] Embodiment 2012. The method of any one of embodiments 2001-2011 , wherein the ADAR2 comprises the amino acid sequence of SEQ ID NO: 105.

[00411] Embodiment 2013. The method of any one of embodiments 2001-2012, wherein the lambdoid bacteriophage N-peptide is derived from the P21 lambdoid bacteriophage.

[00412] Embodiment 2014. The method of any one of embodiments 2001-2013, wherein the P21 N- peptide is the wild-type amino-terminal domain P21 N-peptide and comprises the amino acid sequence of SEQ ID NO: 36.

[00413] Embodiment 2015. The method of any one of embodiments 2001-2014, wherein the aminoterminal domain P21 N-peptide comprises an arginine-rich motif.

[00414] Embodiment 2016. The method of any one of embodiments 2001-2015, wherein the aminoterminal domain P21 N-peptide comprises an Ala at position 5 and an arginine at each of positions 8, 12, and 13 of SEQ ID NO: 36.

[00415] Embodiment 2017. The method of any one of embodiments 2001-2016, wherein the aminoterminal domain P21 N-peptide variant comprises at least one mutation as compared to the wild type aminoterminal domain P21 N-peptide.

[00416] Embodiment 2018. The method of any one of embodiments 2001 -2017, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition.

[00417] Embodiment 2019. The method of any one of embodiments 2001-2018, wherein the aminoterminal domain P21 N-peptide variant comprises from 1 to 21 amino acid residue substitutions.

[00418] Embodiment 2020. The method of any one of embodiments 2001 -2019, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild type aminoterminal domain P21 N-peptide sequence (SEQ ID NO: 36).

[00419] Embodiment 2021. The method of any one of embodiments 2001-2020, wherein the aminoterminal domain P21 N-peptide variant comprises one or mutations at positions R13 and 117 with reference to SEQ ID NO: 36.

[00420] Embodiment 2022. The method of any one of embodiments 2001-2021 , wherein the aminoterminal domain P21 N-peptide variant is not mutated at position R13 and/or 117 with reference to SEQ ID NO: 36. [00421] Embodiment 2023. The method of any one of embodiments 2001 -2022, wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 3.

[00422] Embodiment 2024. The method of any one of embodiments 2001-2023, wherein the affinity of the P21 N-peptide variant for the BoxB hairpin RNA is greater than the affinity of the wild type P21 N-peptide for the BoxB hairpin RNA.

[00423] Embodiment 2025. The method of any one of embodiments 2001-2024, wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[00424] Embodiment 2026. The method of any one of embodiments 2001-2025, wherein the substituted amino acid residue of the lambdoid bacteriophage N-peptide is any naturally-occurring amino acid.

[00425] Embodiment 2027. The method of any one of embodiments 2001-2026, wherein the naturally- occurring amino acid is hydrophilic, hydrophobic, or non-classical.

[00426] Embodiment 2028. The method of any one of embodiments 2001-2027, wherein the hydrophilic amino acid is selected from a polar and positively charged hydrophilic amino acid, a polar and neutral of charge hydrophilic amino acid, and polar and negatively charged hydrophilic amino acid, and an aromatic, polar and positively charged hydrophilic amino acid.

[00427] Embodiment 2029. The method of any one of embodiments 2001-2028, wherein the polar and positively charged hydrophilic amino acid is arginine (R) or lysine (K).

[00428] Embodiment 2030. The method of any one of embodiments 2001-2029, wherein the polar and neutral of charge hydrophilic amino acid is asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), or cysteine (C).

[00429] Embodiment 2031 . The method of any one of embodiments 2001-2030, wherein the polar and negatively charged hydrophilic amino acid is aspartate (D) or glutamate (E).

[00430] Embodiment 2032. The method of any one of embodiments 2001-2031 , wherein the aromatic, polar and positively charged hydrophilic amino acid is histidine.

[00431] Embodiment 2033. The method of any one of embodiments 2001 -2032, wherein the hydrophobic amino acid is selected from a hydrophobic, aliphatic amino acid and a hydrophobic, aromatic amino acid. [00432] Embodiment 2034. The method of any one of embodiments 2001-2033, wherein the hydrophobic, aliphatic amino acid is glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).

[00433] Embodiment 2035. The method of any one of embodiments 2001-2034, wherein the hydrophobic, aromatic amino acid is phenylalanine (F), tryptophan (W), or tyrosine (Y).

[00434] Embodiment 2036. The method of any one of embodiments 2001-2035, wherein the non- classical amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine 0-alanine, GABA, 5- Aminolevulinic acid, 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, c-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, 0-alanine, fluoroamino acids, designer amino acids such as 0 methyl amino acids, C a- methyl amino acids, N a -methyl amino acids, and amino acid analogs in general.

[00435] Embodiment 2037. The method of any one of embodiments 2001-2036, wherein the deaminase and the lambdoid bacteriophage N-peptide are a fusion protein.

[00436] Embodiment 2038. The method of any one of embodiments 2001-2037, wherein the deaminase and the lambdoid bacteriophage N-peptide are a connected by a chemical linker.

[00437] Embodiment 2039. The method of any one of embodiments 2001-2038, wherein the deaminase is linked to at least 2, at least 3, or at least 4 lambdoid bacteriophage N-peptides.

[00438] Embodiment 2040. The method of any one of embodiments 2001-2039, wherein the bacteriophage N-peptide is linked to at least 2, at least 3, or at least 4 deaminases.

[00439] Embodiment 2041. The method of any one of embodiments 2001-2040, wherein the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal.

[00440] Embodiment 2042. The method of any one of embodiments 2001-2041 , wherein the deaminase and the lambdoid bacteriophage N-peptide comprise a nuclear localization signal and the RNA molecule comprising the target nucleotide is in the nucleus of the cell.

[00441] Embodiment 2043. The method of any one of embodiments 2001-2042, wherein the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal. [00442] Embodiment 2044. The method of any one of embodiments 2001-2043, wherein SV40 Large T- antigen nuclear localization signal comprises the amino acid sequence of SEQ ID NO: 49.

[00443] Embodiment 2045. The method of any one of embodiments 2001-2044, wherein the antisense RNA nucleotide hybridizes with a wild-type target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00444] Embodiment 2046. The method of any one of embodiments 2001-2045, wherein the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence.

[00445] Embodiment 2047. The method of any one of embodiments 2001-2046, wherein the antisense RNA nucleotide hybridizes with a mutant target sequence in an endogenous RNA and comprises a target nucleotide to be edited.

[00446] Embodiment 2048. The method of any one of embodiments 2001-2047, wherein the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence.

[00447] Embodiment 2049. The method of any one of embodiments 2001-2048, wherein the target nucleotide is a genetic mutation associated with a genetic disease or disorder.

[00448] Embodiment 2050. The method of any one of embodiments 2001-2049, wherein the genetic mutation of the target nucleotide causes a premature termination codon (PTC) or an in-frame stop codon.

[00449] Embodiment 2051. The method of any one of embodiments 2001-2050, wherein the genetic disease or disorder is caused by a G-to-A nucleotide mutation.

[00450] Embodiment 2052. The method of any one of embodiments 2001-2051 , wherein the antisense RNA oligonucleotide hybridizes with an RNA molecule comprising the target nucleotide in endogenous RNA of the cell.

[00451] Embodiment 2053. The method of any one of embodiments 2001-2052, wherein the antisense RNA oligonucleotide has perfect base-pairing complementarity with the RNA molecule comprising the target nucleotide.

[00452] Embodiment 2054. The method of any one of embodiments 2001-2053, wherein the antisense RNA oligonucleotide comprises a one or more mismatches with the RNA molecule comprising the target nucleotide. [00453] Embodiment 2055. The method of any one of embodiments 2001-2054, wherein the antisense RNA oligonucleotide sequence comprises from 1 to 15 mismatches, or about 1 , or about 2, or about 3, or about 4, or about 5, or about 7, or about 10, or about 15 mismatches.

[00454] Embodiment 2056. The method of any one of embodiments 2001-2055, wherein the antisense RNA oligonucleotide sequence is at least 80% complementary with the target sequence.

[00455] Embodiment 2057. The method of any one of embodiments 2001-2056, wherein the 5' end of the antisense RNA oligonucleotide begins before the target nucleotide to be edited.

[00456] Embodiment 2058. The method of any one of embodiments 2001-2057, wherein the 3' end of the antisense RNA oligonucleotide extends from 2-22 nucleotides 5' from the target nucleotide to be edited.

[00457] Embodiment 2059. The method of any one of embodiments 2001-2058, wherein the antisense RNA oligonucleotide is linked to at least 2, 3, or 4 hairpin RNA oligonucleotides.

[00458] Embodiment 2060. The method of any one of embodiments 2001-2059, wherein the hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 5' of the target mutation.

[00459] Embodiment 2061 . The method of any one of embodiments 2001-2060, wherein the hairpin RNA oligonucleotide is located from about 2-20 nucleotide residues 3' of the target mutation.

[00460] Embodiment 2062. The method of any one of embodiments 2001-2061 , wherein the hairpin RNA oligonucleotide is a BoxB hairpin RNA oligonucleotide.

[00461] Embodiment 2063. The method of any one of embodiments 2001-2062, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the N-peptide.

[00462] Embodiment 2064. The method of any one of embodiments 2001-2063, wherein the BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 47-48, or variants thereof.

[00463] Embodiment 2065. The method of any one of embodiments 2001-2064, wherein the P21 N peptide interacts with a P21 BoxB hairpin RNA oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, or variant thereof.

[00464] Embodiment 2066. The method of any one of embodiments 2001-2065, wherein the P21 N peptide and P21 BoxB hairpin RNA oligonucleotide interaction adopts a non-GNRA U-turn with respect to the apical four nucleotides of the P21 BoxB hairpin RNA oligonucleotide. [00465] Embodiment 2067. The method of any one of embodiments 2001-2066, wherein the hairpin RNA oligonucleotide comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion.

[00466] Embodiment 2068. The method of any one of embodiments 2001-2067, wherein the P21 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, and further comprises from 1 to 20 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions.

[00467] Embodiment 2069. The method of any one of embodiments 2001-2068, wherein the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 20, relative to the wild type P21 BoxB hairpin RNA oligonucleotide.

[00468] Embodiment 2070. The method of any one of embodiments 2001-2069, wherein components (i) and (ii) are within a plasmid, vector, mRNA, or lipid nanoparticle.

[00469] Embodiment 2071. The method of any one of embodiments 2001-2070, wherein components (i) and (ii) are within a viral vector.

[00470] Embodiment 2072. The method of any one of embodiments 2001-2071 , wherein the viral vector is an adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses vector.

[00471] Embodiment 2073. The method of any one of embodiments 2001-2072, wherein the viral vector is an adeno-associated virus (AAV) vector.

[00472] Embodiment 2074. The method of any one of embodiments 2001-2073, wherein the AAV vector is selected from AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, orAAV11 , and AAV12.

EQUIVALENTS

[00473] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. [00474] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

[00475] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[00476] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[00477] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:

1 . A composition comprising a site-specific RNA editing system comprising:

(i) a deaminase linked to one of a mutant A bacteriophage N-peptide, lambdoid P22 bacteriophage

N-peptide, or lambdoid P21 bacteriophage N-peptide; and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising a target nucleotide.

2. A composition comprising a nucleic acid encoding a site-specific RNA editing system comprising:

N-peptide, or lambdoid P21 bacteriophage N-peptide; and

3. The composition of claim 1 or 2, wherein the deaminase is linked to a mutant A bacteriophage N- peptide.

4. The composition of claim 1 or 2, wherein the deaminase is linked to a lambdoid P22 bacteriophage N-peptide.

5. The composition of claim 1 or 2, wherein the deaminase is linked to a lambdoid P21 bacteriophage N-peptide.

6. A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with:

(i) a deaminase linked to a mutant A bacteriophage N-peptide, lambdoid P22 bacteriophage

N-peptide, or lambdoid P21 bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising:

(a) contacting the cell with:

N-peptide, or lambdoid P21 bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. The method of claim 6 or 7, wherein the deaminase is linked to a mutant A bacteriophage N-peptide. The method of claim 6 or 7, wherein the deaminase is linked to a lambdoid P22 bacteriophage N- peptide. The method of claim 6 or 7, wherein the deaminase is linked to a lambdoid P21 bacteriophage N- peptide. The method of any one of claims 6-10, wherein the deaminase is adenosine deaminase. The method of any one of claims 6-11 , wherein the adenosine deaminase yields an inosine (I) nucleotide. The method of any one of claims 6-12, wherein the adenosine deaminase is selected from adenosine deaminase RNA specific 1 (ADAR1), adenosine deaminase RNA specific 2 (ADAR2), adenosine deaminase RNA specific B1 (ADARB1 ), adenosine deaminase like (ADAL), adenosine deaminase 2 (ADA2), adenosine monophosphate deaminase 1 (AMPD1 ), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase domain containing 1 (ADAD1 ), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase tRNA specific 1 (ADAT1), adenosine deaminase tRNA specific 2 (ADAT2), adenosine deaminase tRNA specific 3 (ADAT3), TadA or fragments or variants thereof. The method of any one of claims 6-13, wherein the adenosine deaminase is ADAR1 or ADAR2. The method of any one of claims 6-14, wherein the adenosine deaminase is a fragment, the fragment comprising the catalytic domain of the adenosine deaminase. The method of any one of claims 6-15, wherein the adenosine deaminase is a fragment, the fragment comprising a catalytic domain and one or more mutations. The method of any one of claims 6-16, wherein the ADAR1 or ADAR2 comprises a fragment that comprises a catalytic domain. The method of any one of claims 6-17, wherein the catalytic domain is the deaminase domain of human ADAR2, comprising an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 103. The method of any one of claims 6-18, wherein the ADAR2 comprises the amino acid sequence of SEQ ID NO: 104. The method of any one of claims 6-19, wherein the mutant amino-terminal domain A N-peptide is not the wild-type amino-terminal domain A N-peptide (SEQ ID NO: 1 ). The method of any one of claims 6-20, wherein the mutant amino-terminal domain A N-peptide comprises an arginine-rich motif. The method of any one of claims 6-21 , wherein the mutant amino-terminal domain A N-peptide comprises an Ala at position 3 and an arginine at each of positions 6, 10, and 11 of SEQ ID NO: 1. The method of any one of claims 6-21 , wherein the mutant amino-terminal domain A N-peptide variant comprises at least one mutation as compared to the wild-type amino-terminal domain A N- peptide (SEQ ID NO: 1 ). The method of any one of claims 6-23, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition. The method of any one of claims 6-24, wherein the mutant amino-terminal domain A N-peptide comprises from 1 to 19 amino acid residue substitutions. The method of any one of claims 6-25, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 19, relative to the wild-type amino-terminal domain A N- peptide sequence (SEQ ID NO: 1 ). The method of any one of claims 6-26, wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 1 , Table 2, and/or Table 3. The method of any one of claims 6-27, wherein the mutant amino-terminal domain A N-peptide comprises the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 9. The method of any one of claims 6-28, wherein the affinity of the mutant A bacteriophage N-peptide for the BoxB hairpin RNA oligonucleotide is greater than the affinity of the wild-type A N-peptide for the BoxB hairpin RNA oligonucleotide. The method of claim 29, wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold The method of any one of the preceding claims, wherein the lambdoid bacteriophage N-peptide is derived from the P22 lambdoid bacteriophage. The method of any one of claims 6-30, wherein the P22 N-peptide is a variant that is the wild-type amino-terminal domain P22 N-peptide and comprises the amino acid sequence of SEQ ID NO: 10. The method of any one of claims 6-31 , wherein the amino-terminal domain P22 N-peptide comprises an arginine-rich motif. The method of any one of claims 6-32, wherein the amino-terminal domain P22 N-peptide comprises an Ala at position 5 and an arginine at each of positions 8, 12, and 13 of SEQ ID NO: 10. The method of any one of claims 6-33, wherein the amino-terminal domain P22 N-peptide variant comprises at least one mutation as compared to the wild type amino-terminal domain P22 N-peptide. The method of any one of claims 6-34, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition. The method of any one of claims 6-35, wherein the amino-terminal domain P22 N-peptide variant comprises from 1 to 21 amino acid residue substitutions. The method of any one of claims 6-36, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild type amino-terminal domain P22 N- peptide sequence (SEQ ID NO: 10). The method of any one of claims 6-37, wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 2. The method of any one of claims 6-38, wherein the amino-terminal domain P22 N-peptide variant comprises the amino acid sequence of any one of SEQ ID NO: 11 to SEQ ID NO: 35. The method of any one of claims 6-39, wherein the affinity of the P22 N-peptide variant for the BoxB hairpin RNA is greater than the affinity of the wild type P22 N-peptide for the BoxB hairpin RNA. The method of claims 40, wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold. The method of any one of claims 6-41 , wherein the lambdoid bacteriophage N-peptide is derived from the P21 lambdoid bacteriophage. The method of any one of claims 6-42, wherein the P21 N-peptide is the wild-type amino-terminal domain P21 N-peptide and comprises the amino acid sequence of SEQ ID NO: 36. The method of any one of claims 6-43, wherein the amino-terminal domain P21 N-peptide comprises an arginine-rich motif. The method of any one of claims 6-44, wherein the amino-terminal domain P21 N-peptide comprises an Ala at position 5 and an arginine at each of positions 8, 12, and 13 of SEQ ID NO: 36. The method of any one of claims 6-45, wherein the amino-terminal domain P21 N-peptide variant comprises at least one mutation as compared to the wild type amino-terminal domain P21 N-peptide. The method of any one of claims 6-46, wherein the mutation is selected from one or more of an amino acid substitution, deletion, and addition. The method of any one of claims 6-47, wherein the amino-terminal domain P21 N-peptide variant comprises from 1 to 21 amino acid residue substitutions. The method of any one of claims 6-48, wherein the amino acid substitution takes place at one or more of amino acid residue positions 1 to 21 , relative to the wild type amino-terminal domain P21 N- peptide sequence (SEQ ID NO: 36). The method of any one of claims 6-49, wherein the amino-terminal domain P21 N-peptide variant comprises one or mutations at positions R13 and 117 with reference to SEQ ID NO: 36. The method of any one of claims 6-50, wherein the amino-terminal domain P21 N-peptide variant is not mutated at position R13 and/or 117 with reference to SEQ ID NO: 36. The method of any one of claims 6-51 , wherein the amino acid substitution is selected from any one of the amino acid substitutions listed in Table 2. The method of any one of claims 6-52, wherein the affinity of the P21 N-peptide variant for the BoxB hairpin RNA is greater than the affinity of the wild type P21 N-peptide for the BoxB hairpin RNA. The method of claim 53, wherein the affinity is greater by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold. The method of any one of claims 6-54, wherein the mutant A bacteriophage N-peptide comprises a substituted amino acid residue that is any naturally-occurring amino acid. The method of any one of claims 6-55, wherein the lambdoid bacteriophage N-peptide comprises a substituted amino acid residue that is any naturally-occurring amino acid. The method of claim 55 or 56, wherein the naturally-occurring amino acid is hydrophilic, hydrophobic, or non-classical. The method of any one of claims 57, wherein the hydrophilic amino acid is selected from a polar and positively charged hydrophilic amino acid, a polar and neutral of charge hydrophilic amino acid, and polar and negatively charged hydrophilic amino acid, and an aromatic, polar and positively charged hydrophilic amino acid. The method of claim 58, wherein the polar and positively charged hydrophilic amino acid is arginine (R) or lysine (K). The method of claim 58, wherein the polar and neutral of charge hydrophilic amino acid is asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), or cysteine (C). The method of claim 558, wherein the polar and negatively charged hydrophilic amino acid is aspartate (D) or glutamate (E). The method of claim 58, wherein the aromatic, polar and positively charged hydrophilic amino acid is histidine. The method of claim 57, wherein the hydrophobic amino acid is selected from a hydrophobic, aliphatic amino acid and a hydrophobic, aromatic amino acid. The method of claim 63, wherein the hydrophobic, aliphatic amino acid is glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). The method of claim 63, wherein the hydrophobic, aromatic amino acid is phenylalanine (F), tryptophan (W), or tyrosine (Y). The method of any one of claims 57, wherein the non-classical amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA, 5-Aminolevulinic acid, 4- Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, E-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoroamino acids, designer amino acids such as methyl amino acids, C a-methyl amino acids, N a -methyl amino acids, and amino acid analogs in general. The method of any one of claims 6-66, wherein the deaminase and the mutant A bacteriophage N- peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide are a fusion protein. The method of claim 67, wherein the deaminase and mutant A bacteriophage N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide are a connected by a chemical linker. The method of claim 67 or 68, wherein the deaminase is linked to at least 2, at least 3, or at least 4 mutant A bacteriophage N-peptides. The method of claim 67 or 68, wherein the A N-peptide is linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. The method of claim 67 or 68, wherein the deaminase is linked to at least 2, at least 3, or at least 4 lambdoid P22 bacteriophage N-peptide. The method of claim 67 or 68, wherein the lambdoid P22 bacteriophage N-peptide is linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. The method of claim 67 or 68, wherein the deaminase is linked to at least 2, at least 3, or at least 4 lambdoid P21 bacteriophage N-peptide. The method of claim 67 or 68, wherein the lambdoid P21 bacteriophage N-peptide is linked to at least 2, at least 3, or at least 4 deaminases, or fragments thereof. The method of any one of the preceding claims, wherein the deaminase and the mutant X bacteriophage N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide comprise a nuclear localization signal. The method of any one of the preceding claims, wherein the deaminase and the mutant X bacteriophage N-peptide, lambdoid P22 bacteriophage N-peptide, or lambdoid P21 bacteriophage N-peptide comprise a nuclear localization signal and the RNA molecule comprising the target nucleotide is in the nucleus of the cell. The method of claim 75 or 76, wherein the nuclear localization signal is the SV40 Large T-antigen nuclear localization signal. The method of claim 77, wherein SV40 Large T-antigen nuclear localization signal comprises the amino acid sequence of SEQ ID NO: 49. The method of any one of claims 6-78, wherein the antisense RNA nucleotide hybridizes with a wildtype target sequence in an endogenous RNA and comprises a target nucleotide to be edited. The method of any one of claims 6-79, wherein the target nucleotide to be edited induces a beneficial mutation in the wild-type target sequence. The method of any one of claims 6-80, wherein the antisense RNA nucleotide hybridizes with a mutant target sequence in an endogenous RNA and comprises a target nucleotide to be edited. The method of any one of claims 6-81 , wherein the target nucleotide to be edited eliminates a detrimental mutation in the mutant target sequence. The method of any one of claims 6-82, wherein the target nucleotide is a genetic mutation associated with a genetic disease or disorder. The method of any one of claims 6-83, wherein the genetic mutation of the target nucleotide causes a premature termination codon (PTC) or an in-frame stop codon. The method of any one of claims 6-84, wherein the genetic disease or disorder is caused by a G-to- A nucleotide mutation. The method of any one of claims 6-85, wherein the antisense RNA oligonucleotide hybridizes with an RNA molecule comprising the target nucleotide in endogenous RNA of the cell. The method of any one of claims 6-86, wherein the antisense RNA oligonucleotide has perfect basepairing complementarity with the RNA molecule comprising the target nucleotide. The method of any one of claims 6-87, wherein the antisense RNA oligonucleotide comprises a one or more mismatches with the RNA molecule comprising the target nucleotide. The method of any one of claims 6-88, wherein the antisense RNA oligonucleotide sequence comprises from 1 to 15 mismatches, or about 1 , or about 2, or about 3, or about 4, or about 5, or about 7, or about 10, or about 15 mismatches. The method of any one of claims 6-89, wherein the antisense RNA oligonucleotide sequence is at least 80% complementary with the target sequence. The method of any one of claims 6-90, wherein the 5' end of the antisense RNA oligonucleotide begins before the target nucleotide to be edited. The method of any one of claims 6-91 , wherein the 3' end of the oligonucleotide extends from 2-22 nucleotides 5' from the target nucleotide to be edited. The method of any one of claims 6-92, wherein the antisense RNA oligonucleotide is linked to at least 2, 3, or 4 hairpin RNA oligonucleotides. The method of any one of claims 6-93, wherein the hairpin RNA oligonucleotide comprises a nucleic acid sequence located from about 2-20 nucleotide residues 5' of the target mutation. The method of any one of claims 6-94, wherein the hairpin RNA oligonucleotide comprises a nucleic acid sequence located from about 2-20 nucleotide residues 3' of the target mutation. The method of any one of claims 6-95, wherein the hairpin RNA oligonucleotide is a BoxB hairpin RNA oligonucleotide. The method of any one of claims 6-96, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the mutant A bacteriophage N-peptide. The method of any one of claims 6-97, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the lambdoid P22 bacteriophage N-peptide. The method of any one of claims 6-98, wherein the BoxB hairpin RNA oligonucleotide is the cognate hairpin oligonucleotide of the lambdoid P21 bacteriophage N-peptide. The method of any one of claims 97-99, wherein the BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of one or more of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or mutants thereof, . The method of any one of claims 6-100, wherein the mutant A N peptide interacts with a A BoxB hairpin RNA oligonucleotide comprising a nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or a mutant thereof; the P22 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, or a mutant thereof; or the P21 BoxB hairpin RNA oligonucleotide comprises the nucleic acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, or a mutant thereof. The method of any one of claims 6-102, wherein the mutant A N peptide and A BoxB hairpin RNA oligonucleotide interaction adopts a 4-out GNRA-like pentaloop. The method of any one of claims 6-103, wherein the P22 N peptide and P22 BoxB hairpin RNA oligonucleotide interaction adopts a 3-out GNRA-like pentaloop. The method of any one of claims 6-104, wherein the P21 N peptide and P21 BoxB hairpin RNA oligonucleotide interaction adopts a non-GNRA U-turn with respect to the apical four nucleotides of the P21 BoxB hairpin RNA oligonucleotide. The method of any one of claims 6-105, wherein the nucleic acid sequence comprises at least one mutation selected from missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, and repeat expansion. The method of any one of claims 6-106, wherein the A BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence having sequence identity to SEQ ID NO: 37 or SEQ ID NO: 38, and further comprises from 1 to 15 nucleotide mutations selected from one or more of nucleotide substitutions, nucleotide deletions, and nucleotide additions. The method of any one of claims 6-107, wherein the one or more nucleotide mutations take(s) place at one or more of nucleotide residue positions 1 to 15, relative to the wild-type A BoxB hairpin RNA oligonucleotide. The method of any one of claims 6-108, wherein the A BoxB hairpin RNA oligonucleotide comprises a nucleic acid sequence of one or more of SEQ ID NOs: 37-44. The method of any one of claims 6-109, wherein components (i) and (ii) are within a plasmid, vector, mRNA, or lipid nanoparticle. The method of any one of claims 6-110, wherein components (i) and (ii) are within a viral vector. The method of any one of claims 6-111 , wherein the viral vector is an adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses vector. The method of any one of claims 6-112, wherein the viral vector is an adeno-associated virus (AAV) vector. The method of any one of claims 6-113, wherein the AAV vector is selected from AAV1 , or AAV2, or AAV3, or AAV4, or AAV5, or AAV6, or AAV7, or AAV8, or AAV9, or AAV10, or AAV11 , and AAV12. A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with:

(i) a deaminase linked to a mutant A bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising:

(a) contacting the cell with:

(i) a deaminase linked to a mutant A bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the mutant A bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising: (a) contacting the cell with:

(i) a deaminase linked to a mutant lambdoid P22 bacteriophage N-peptide, and (ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the lambdoid P22 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising:

(a) contacting the cell with:

(i) a deaminase linked to a lambdoid P22 bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the lambdoid P22 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of site-specific editing of a target nucleotide within an RNA molecule in a cell comprising:

(a) contacting the cell with:

(i) a deaminase linked to a lambdoid P21 bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the lambdoid P21 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change. A method of treating a disease or disorder by editing a target nucleotide within an RNA molecule in a cell, the method comprising:

(a) contacting the cell with:

(i) a deaminase linked to a lambdoid P21 bacteriophage N-peptide, and

(ii) an antisense RNA oligonucleotide linked to a hairpin RNA oligonucleotide, wherein: the antisense RNA oligonucleotide comprises a region complementary to an RNA molecule comprising the target nucleotide; the lambdoid P21 bacteriophage N-peptide and hairpin interact; and the deaminase domain causes editing of the target nucleotide to induce a base change.