RNA and DNA base editing via recruitment of engineered ADARs

Abstract

Disclosed herein is a system for recruiting ADAR to catalyze therapeutic editing of point mutations through the use of an engineered RNA scaffold, an engineered DNA scaffold, or a DNA-RNA hybrid scaffold. The system comprises an engineered ADAR2 guide RNA (adRNA) having 20-100 bp of complementarity with a domain that recruits ADAR2 from a target RNA and GluR2 mRNA at either or both of its 5'-end or 3'-end.

Description

cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 728,007, filed September 6, 2018, U.S. Provisional Application No. 62 / 766,433, filed October 17, 2018, and U.S. Provisional Application No. 62 / 780,241, filed December 15, 2018, all of which are incorporated herein by reference in their entireties. STATEMENT REGARDING GOVERNMENT SUPPORT

[0002] This disclosure was made with government support under Grant Nos. CA222826, GM123313, and HG009285 awarded by the National Institutes of Health. The government has certain rights in this invention. Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on October 18, 2019, is named 00015-365WO1_SL.txt and is 163,723 bytes in size. Abstract

[0003] One embodiment of the present disclosure provides a vector. In some cases, the vector can include a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity-recruiting domain, wherein (a) the polynucleotide sequence encoding the at least one RNA editing entity-recruiting domain does not form a secondary structure including a stem-loop, or (b) the polynucleotide sequence encoding the at least one RNA editing entity-recruiting domain comprises at least about 80% sequence identity with at least one sequence selected from a sequence encoding an Alu domain, a sequence encoding an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-recruiting domain, and any combination thereof. In some cases, the polynucleotide sequence encoding the RNA editing entity-recruiting domain can comprise at least about 80% sequence identity with an Alu domain sequence. In some cases, the polynucleotide sequence encoding the RNA editing entity-recruiting domain can comprise at least about 80% sequence identity with a sequence encoding an APOBEC-recruiting domain. In some cases, the vector can be a viral vector. In some cases, the vector may be a liposome. In some cases, the vector may be a nanoparticle. In some cases, the domain that recruits the RNA editing entity may be configured to recruit an ADAR protein. In some cases, the ADAR protein may be an ADAR1, ADAR2, or ADAR3 protein. In some cases, the ADAR protein may be a human ADAR protein. In some cases, the ADAR protein may be a recombinant ADAR protein. In some cases, the ADAR protein may be a modified ADAR protein. In some cases, the domain that recruits the RNA editing entity may be configured to recruit an APOBEC protein.In some cases, the APOBEC protein may be an APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4 protein. In some cases, the ADAR protein may be a human ADAR protein. In some cases, the ADAR protein may be a recombinant ADAR protein. In some cases, the ADAR protein may be a modified ADAR protein. In some cases, the at least one RNA editing entity recruiting domain may not form a secondary structure comprising a stem-loop. In some cases, the polynucleotide sequence may encode at least two RNA editing recruiting domains. In some cases, at least one of the at least two RNA editing recruiting domains may be an Alu domain. In some cases, the Alu domain sequence may form a secondary structure comprising at least one stem-loop. In some cases, the sequence encoding the Alu domain may comprise multiple Alu repeats. In some cases, the sequence encoding the Alu domain may be at least partially single-stranded. In some cases, at least one of the at least two RNA editing recruiting domains may be an APOBEC recruiting domain. In some cases, at least one of the sequences encoding the at least two RNA editing recruiting domains may comprise at least about 80% sequence identity with a sequence encoding a GluR2 domain. In some cases, at least one of the at least two RNA editing recruiting domains may be a GluR2 domain. In some cases, at least one of the at least two RNA editing recruiting domains may be a Cas13 domain.In some cases, the at least two RNA editing entity-recruiting domains may be the Alu domain and the APOBEC-recruiting domain. In some cases, the vector may further comprise a nucleic acid encoding an RNA that may be at least partially complementary to a target RNA. In some cases, the nucleic acid encoding the RNA that may be at least partially complementary to the target RNA may be about 10 base pairs (bp) to about 1000 bp in length. In some cases, the nucleic acid encoding the at least one RNA editing entity-recruiting domain and the nucleic acid encoding the RNA that may be complementary to at least a portion of the target RNA may comprise a contiguous nucleic acid of at least about 200 bp in length. In some cases, the nucleic acid may be chemically synthesized. In some cases, the nucleic acid may be genetically encoded. In some cases, the vector may comprise DNA. In some cases, the DNA may be double-stranded. In some cases, the DNA may be single-stranded. In some cases, the vector may comprise RNA. In some cases, the RNA may comprise a base modification. In some cases, the vector may be an adeno-associated virus (AAV) vector. In some cases, the AAV may be a recombinant AAV (rAAV). In some cases, the AAV may be selected from the group consisting of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, any derivative thereof, and any combination thereof. In some cases, the AAV may be an AAV5 serotype or a derivative thereof. In some cases, the AAV derivative may comprise a modified VP1 protein.In some cases, the APOBEC-recruiting domain may be selected from the group consisting of an APOBEC1-recruiting domain, an APOBEC2-recruiting domain, an APOBEC3A-recruiting domain, an APOBEC3B-recruiting domain, an APOBEC3C-recruiting domain, an APOBEC3E-recruiting domain, an APOBEC3F-recruiting domain, an APOBEC3G-recruiting domain, an APOBEC3H-recruiting domain, an APOBEC4-recruiting domain, and any combination thereof. In some cases, the RNA editing entity-recruiting domain can recruit at least two RNA editing entities, wherein at least one of the polynucleotide sequences encoding the at least two RNA editing entities comprises at least about 80% identity to a sequence encoding an APOBEC protein. In some cases, the domain that recruits the RNA editing entity can recruit the at least two RNA editing entities, and at least one of the at least two polynucleotide sequences that encode the RNA editing entities comprises at least about 80% identity with the sequence that encodes ADAR protein.In some cases, the domain that recruits the RNA encoded by the nucleic acid can comprise at least one stem-loop.In some cases, the polynucleotide sequence that encodes the domain that recruits the RNA editing can comprise a secondary structure that can be substantially cruciform.In some cases, the polynucleotide sequence that encodes the domain that recruits the RNA editing can comprise at least two secondary structures that are substantially cruciform.In some cases, the polynucleotide sequence that encodes the domain that recruits the RNA editing entity can be located between the polynucleotide sequences that form at least two secondary structures that are substantially cruciform.In some cases, the cruciform secondary structure can comprise a stem-loop that is adjacent to at least one pair of at least partially complementary strands of the cruciform secondary structure.In some cases, the polynucleotide sequence encoding the RNA editing recruiting domain may comprise a secondary structure that may be substantially toe-folded. In some cases, the non-naturally occurring RNA may be encoded by the vector. In some cases, the kit may comprise the vector in a container. In some cases, the kit may further comprise a syringe. In some cases, the container may be a syringe. In some cases, the isolated cell may comprise the vector. In some cases, the pharmaceutical composition may comprise the vector in a unit dose form. In some cases, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition may comprise a second active ingredient. In some cases, a method for treating a disease or condition in a subject comprises administering the vector to the subject. In some cases, the administration may be by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further comprise providing a secondary treatment to the subject. In some cases, the disease or condition may be selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferative disorder (e.g., a neoplasm), and any combination thereof. In some cases, the disease or condition may be Alzheimer's disease. In some cases, the disease or condition may be muscular dystrophy. In some cases, the disease or condition may be retinitis pigmentosa. In some cases, the disease or condition may be Parkinson's disease. In some cases, the disease or condition may be pain. In some cases, the disease or condition may be Stargardt's macular dystrophy. In some cases, the disease or condition may be Charcot-Marie dental disease. In some cases, the disease or condition may be Rett syndrome.In some cases, the administration may be sufficient to reduce gene expression relative to before administration. In some cases, the administration may be sufficient to edit at least one point mutation in the subject. In some cases, the administration may be sufficient to edit at least one stop codon in the subject, thereby generating readthrough of the stop codon. In some cases, the administration may be sufficient to generate exon skipping in the subject.

[0004] Another aspect of the present disclosure provides a vector. In some cases, the vector can include a nucleic acid encoding an RNA having a two-dimensional shape that may be substantially cruciform, wherein the RNA includes at least one sequence encoding a domain that recruits an RNA editing entity. In some cases, the vector can further include a nucleic acid encoding an RNA having a sequence encoding at least one targeting domain that may be at least partially complementary to a target RNA sequence. In some cases, the nucleic acid encoding the RNA having at least one targeting domain that may be at least partially complementary to the target RNA sequence can include a substantially linear two-dimensional structure. In some cases, a non-naturally occurring RNA can be encoded by the vector. In some cases, a kit can include the vector in a container. In some cases, the kit can further include a syringe. In some cases, the container can be a syringe. In some cases, isolated cells can include the vector. In some cases, a pharmaceutical composition can include the vector in a unit dose form. In some cases, the pharmaceutical composition can further include a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition may include a second active ingredient. In some cases, a method for treating a disease or condition in a subject includes administering the vector to the subject. In some cases, the administration may be by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further include providing a secondary treatment to the subject. In some cases, the disease or condition may be selected from the group consisting of neurodegenerative disorders, muscle disorders, metabolic disorders, eye disorders, cell proliferative disorders (e.g., neoplasms), and any combination thereof. In some cases, the disease or condition may be Alzheimer's disease. In some cases, the disease or condition may be muscular dystrophy.In some cases, the disease or condition may be retinitis pigmentosa. In some cases, the disease or condition may be Parkinson's disease. In some cases, the disease or condition may be pain. In some cases, the disease or condition may be Stargardt macular dystrophy. In some cases, the disease or condition may be Charcot-Marie dental disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce gene expression relative to before administration. In some cases, the administration may be sufficient to edit at least one point mutation in the subject. In some cases, the administration may be sufficient to edit at least one stop codon in the subject, thereby generating readthrough of the stop codon. In some cases, the administration may be sufficient to generate exon skipping in the subject.

[0005] Another aspect of the present disclosure provides a non-naturally occurring RNA. In some cases, the non-naturally occurring RNA may comprise a first domain sequence having a substantially cruciform two-dimensional shape and a second domain sequence having a substantially linear two-dimensional structure connected to the first domain sequence. Here, the first domain sequence encodes a domain that recruits an RNA editing entity, and the second domain sequence encodes a targeting domain. The second domain sequence may also be complementary to at least a portion of a target RNA. In some cases, the non-naturally occurring RNA may further comprise a third domain sequence linked to the second domain sequence. In some cases, the third domain sequence may comprise a sequence encoding a domain that recruits an RNA editing entity that forms a secondary structure having a substantially cruciform two-dimensional shape. In some cases, at least one base of the non-naturally occurring RNA may comprise a chemical modification. In some cases, at least one sugar of the non-naturally occurring RNA may comprise a chemical modification. In some cases, a kit may comprise the non-naturally occurring RNA in a container. In some cases, the kit may further comprise a syringe. In some cases, the container may be a syringe. In some cases, the isolated cells may contain the non-natural RNA. In some cases, the pharmaceutical composition may contain the non-natural RNA in a unit dose. In some cases, the pharmaceutical composition may further contain a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition may contain a second active ingredient. In some cases, the method for treating a disease or condition in a subject comprises administering the non-natural RNA to the subject. In some cases, the administration may be by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further comprise providing a secondary treatment to the subject.In some cases, the disease or condition may be selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferative disorder (e.g., a neoplasm), and any combination thereof. In some cases, the disease or condition may be Alzheimer's disease. In some cases, the disease or condition may be muscular dystrophy. In some cases, the disease or condition may be retinitis pigmentosa. In some cases, the disease or condition may be Parkinson's disease. In some cases, the disease or condition may be pain. In some cases, the disease or condition may be Stargardt's macular dystrophy. In some cases, the disease or condition may be Charcot-Marie dental disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce expression of the gene relative to before administration. In some cases, the administration may be sufficient to edit at least one point mutation in the subject. In some cases, the administration may be sufficient to edit at least one stop codon in the subject, thereby producing readthrough of the stop codon. In some cases, the administration may be sufficient to produce exon skipping in the subject.

[0006] Another aspect of the present disclosure provides nucleic acids. In some cases, the nucleic acid can include a domain that recruits an RNA editing entity and an antisense domain sequence. When the nucleic acid is accessible to an RNA editing entity, a target nucleic acid complementary to at least a portion of the antisense domain modifies at least one base pair of the target nucleic acid with at least about four times greater efficiency than a comparable nucleic acid complexed with a Cas13b protein or an active fragment thereof, as determined by Sanger sequencing of the target nucleic acid.

[0007] Another aspect of the present disclosure provides a nucleic acid. In some cases, the nucleic acid may comprise a domain that recruits an RNA editing entity and an antisense domain. When the nucleic acid is accessible to an RNA editing entity, a target nucleic acid complementary to at least a portion of the antisense domain modifies at least one base pair of the target nucleic acid with at least about four times greater efficiency than an equivalent nucleic acid complexed with a GluR2 domain and the antisense domain, as determined by Sanger sequencing of the target nucleic acid. In some cases, the nucleic acid may comprise RNA. In some cases, the target nucleic acid may comprise RNA. In some cases, the RNA may be mRNA. In some cases, the mRNA may encode a protein or a portion thereof. In some cases, dysfunction of the protein or a portion thereof may be involved in a disease or condition. In some cases, the disease or condition may be selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferative disorder (e.g., a neoplasm), and any combination thereof. In some cases, the RNA may be a small interfering RNA (siRNA). In some cases, the domain that recruits the RNA editing entity can comprise at least about 80% identity with the GluR2 domain. In some cases, the domain that recruits the RNA editing entity can comprise at least about 80% identity with the Alu domain. In some cases, the domain that recruits the RNA editing entity can comprise at least about 80% identity with the domain that recruits APOBEC. In some cases, the domain that recruits the RNA editing entity can be configured to recruit an ADAR protein. In some cases, the ADAR protein can be an ADAR1, ADAR2, or ADAR3 protein. In some cases, the ADAR protein can be a human ADAR protein. In some cases, the ADAR protein can be a recombinant ADAR protein.In some cases, the ADAR protein may be a modified ADAR protein. In some cases, the RNA editing entity recruiting domain may be configured to recruit an APOBEC protein. In some cases, the APOBEC protein may be an APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4 protein. In some cases, the ADAR protein may be a human ADAR protein. In some cases, the ADAR protein may be a recombinant ADAR protein. In some cases, the ADAR protein may be a modified ADAR protein. In some cases, the nucleic acid may be chemically synthesized. In some cases, the nucleic acid may be genetically encoded. In some cases, the kit may include the nucleic acid in a container. In some cases, the kit may further include a syringe. In some cases, the container may be a syringe. In some cases, an isolated cell may include the nucleic acid. In some cases, the pharmaceutical composition may comprise the nucleic acid in a unit dose form. In some cases, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition may comprise a second active ingredient. In some cases, a method for treating a disease or condition in a subject comprises administering the nucleic acid to the subject. In some cases, the administration may be by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further comprise providing a secondary treatment to the subject. In some cases, the disease or condition may be selected from the group consisting of neurodegenerative disorders, muscle disorders, metabolic disorders, eye disorders, cell proliferative disorders (e.g., neoplasms), and any combination thereof. In some cases, the disease or condition may be Alzheimer's disease.In some cases, the disease or condition may be muscular dystrophy. In some cases, the disease or condition may be retinitis pigmentosa. In some cases, the disease or condition may be Parkinson's disease. In some cases, the disease or condition may be pain. In some cases, the disease or condition may be Stargardt's macular dystrophy. In some cases, the disease or condition may be Charcot-Marie dental disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce gene expression relative to before administration. In some cases, the administration may be sufficient to edit at least one point mutation in the subject. In some cases, the administration may be sufficient to edit at least one stop codon in the subject, thereby generating readthrough of the stop codon. In some cases, the administration may be sufficient to generate exon skipping in the subject.

[0008] Another aspect of the present disclosure can provide a nucleic acid. In some cases, the nucleic acid can include a sequence including an antisense domain, a sequence forming a first stem-loop, and a sequence forming a second stem-loop. When the nucleic acid contacts (a) a first polypeptide including a domain that binds to a first polynucleotide configured to bind to a first portion of an RNA editing entity and the sequence forming the first stem-loop, (b) a domain that binds to a second polynucleotide including a second portion of an RNA editing entity and the sequence forming the second stem-loop, and (c) a target nucleic acid complementary to at least a portion of the antisense domain, it modifies at least one base pair of the target nucleic acid. In some cases, the first stem-loop or the second stem-loop can be an MS2 stem-loop. In some cases, the first stem-loop or the second stem-loop can be a BoxB stem-loop. In some cases, the first stem-loop or the second stem-loop can be a UlA stem-loop. In some cases, the first part of the RNA editing entity or the second part of the RNA editing entity can comprise an N-terminal fragment of a sequence encoding an ADAR deaminase domain. In some cases, the first part of the RNA editing entity or the second part of the RNA editing entity can comprise a C-terminal fragment of a sequence encoding an ADAR deaminase domain. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain can comprise MS2 coat protein. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain can comprise lambda N peptide. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain can comprise a human nucleic acid binding protein. In some cases, the human nucleic acid binding protein can be a UlA protein, a TBP6.7 protein, a human histone stem-loop binding protein, or a DNA binding domain of a glucocorticoid receptor.In some cases, the RNA editing entity can perform an adenosine to inosine mutation in the target nucleic acid. In some cases, the RNA editing entity can perform a cytosine to thymine mutation in the target nucleic acid. In some cases, the kit can include the nucleic acid in a container. In some cases, the kit can further include a syringe. In some cases, the container can be a syringe. In some cases, the isolated cell can include the nucleic acid. In some cases, the pharmaceutical composition can include the nucleic acid in a unit dose form. In some cases, the pharmaceutical composition can further include a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutical composition can include a second active ingredient. In some cases, a method for treating a disease or condition in a subject includes administering the nucleic acid to the subject. In some cases, the administration can be by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method can further include providing a secondary treatment to the subject. In some cases, the disease or condition may be selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferative disorder (e.g., a neoplasm), and any combination thereof. In some cases, the disease or condition may be Alzheimer's disease. In some cases, the disease or condition may be muscular dystrophy. In some cases, the disease or condition may be retinitis pigmentosa. In some cases, the disease or condition may be Parkinson's disease. In some cases, the disease or condition may be pain. In some cases, the disease or condition may be Stargardt macular dystrophy. In some cases, the disease or condition may be Charcot-Marie dental disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce expression of the gene relative to before administration.In some cases, the administration may be sufficient to edit at least one point mutation in the subject. In some cases, the administration may be sufficient to edit at least one stop codon in the subject, thereby producing readthrough of the stop codon. In some cases, the administration may be sufficient to produce exon skipping in the subject.

[0009] Another aspect of the present disclosure provides a method for treating muscular dystrophy in a subject. In some cases, the method can include administering to the subject a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a first nucleic acid encoding a second nucleic acid. The second nucleic acid comprises (a) an antisense region at least partially complementary to an RNA sequence involved in muscular dystrophy, and (b) at least one RNA editing entity recruiting domain. The at least one RNA editing entity recruiting domain does not contain a stem-loop, or the at least one RNA editing entity recruiting domain comprises at least about 80% sequence identity with at least one of an Alu domain, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruiting domain, and any combination thereof. In some cases, the pharmaceutical composition can be in a unit dose form. In some cases, the administration can be at least weekly. In some cases, the administration can be at least monthly. In some cases, the administration can be by injection. In some cases, the injection may be subcutaneous, intravenous, intravenous, intramuscular, intrathecal, or intraperitoneal. In some cases, the administration may be transdermal, transmucosal, oral, or pulmonary. In some cases, the method may further comprise providing the subject with a secondary treatment.

[0010] Another aspect of the present disclosure may provide a method for producing a vector. In some cases, the method may include cloning at least one copy of a nucleic acid into the vector, wherein the nucleic acid encodes a domain that recruits at least one RNA editing entity, and the sequence encoding the domain that recruits at least one RNA editing entity does not form a secondary structure containing a stem-loop, or the nucleic acid encoding the domain that recruits at least one RNA editing entity comprises at least about 80% sequence identity with a sequence selected from a sequence encoding an Alu domain, a sequence encoding an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-recruiting domain, and any combination thereof. In some cases, the vector may be a viral vector. In some cases, the viral vector may be an AAV vector. In some cases, the viral vector may comprise a modified VP1 protein. In some cases, the vector may be a liposome. In some cases, the vector may be a nanoparticle. In some cases, the method may further comprise transfecting or transducing the vector into isolated human cells.

[0011] Aspects of the present disclosure relate to engineered ADAR1 or ADAR2 guide RNAs ("adRNAs") that comprise, consist essentially of, or consist of a sequence complementary to a target RNA, optionally comprising, consisting essentially of, or consisting of, an ADAR2-recruiting domain derived from engineered ADAR2 and GluR2 mRNA. In some embodiments, the sequence complementary to the target RNA comprises, consists essentially of, or consists of about 15-200, or 20-100 base pairs. In one aspect, the engineered adRNA does not comprise, consist essentially of, or consist of an ADAR-recruiting domain. In some embodiments, the ADAR-recruiting domain comprises, consists essentially of, or consists of GluR2 mRNA, an Alu repeat element, or other RNA motif to which ADARs bind. In one aspect, the engineered adRNA comprises, consists essentially of, or consists of about 1-10 ADAR-recruiting domains. In another aspect, the ADAR2-recruiting domain may be derived from GluR2 mRNA and may be located at the 5' or 3' end of the engineered adRNA. In yet another embodiment, the engineered adRNA comprises, consists essentially of, or consists of GluR2 mRNA at both the 5' and 3' ends. In a further aspect, the engineered adRNA of the present disclosure further comprises, consists essentially of, or consists of two MS2 hairpins flanking the sequence complementary to the target RNA.

[0012] In some embodiments, the target RNA may be ornithine transcarbamylase. Also disclosed herein is a complex comprising, consisting essentially of, or further consisting of an AdRNA hybridized to a complementary polynucleotide under high stringency conditions. In one aspect, the polynucleotide may be DNA. In another aspect, the polynucleotide may be RNA.

[0013] In one embodiment, the engineered adRNA of the present disclosure further comprises, consists essentially of, or further consists of an editing-inducing element.

[0014] A further embodiment relates to an engineered ADAR2 guide RNA ("adRNA") encoded by a sequence selected from the group of sequences provided in Table 1 or Figure 2. The adRNA can be combined with a carrier, such as a pharmaceutically acceptable carrier, examples of which are provided herein.

[0015] Also disclosed herein is a genetically engineered adRNA-snRNA (small nuclear RNA) fusion. In one embodiment, the genetically engineered adRNA further comprises, essentially consists of, or further consists of an N-terminal mitochondrial targeting sequence (MTS), which facilitates the localization of the genetically engineered adRNA to the mitochondria. In another embodiment, the present invention provides a genetically engineered construct further comprises, essentially consists of, or further consists of a cis-acting zipper code, which can facilitate the localization of the genetically engineered adRNA to peroxisomes, endosomes, and exosomes.

[0016] Furthermore, the present specification provides small molecule regulable engineered adRNA.In one embodiment, the present specification discloses engineered adRNA-aptamer fusion.The example of the aptamer that can be used for this purpose includes but is not limited to the aptamer that binds to flavin mononucleotide, guanine, other natural metabolites or sugar.Also, the present specification discloses the U1A-ADAR fusion that is completely derived from human.

[0017] Also disclosed herein are complexes comprising, consisting essentially of, or further consisting of an engineered adRNA of the present disclosure hybridized to a complementary polynucleotide under high stringency conditions.

[0018] Also disclosed herein are vectors comprising, consisting essentially of, or further consisting of an isolated polynucleotide sequence encoding the engineered adRNA of the present disclosure, and optionally one or more regulatory sequences operably linked to the isolated polynucleotide, including, but not limited to, a plasmid or a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0019] Further disclosed herein are recombinant cells further comprising, consisting essentially of, or further consisting of the above-described vectors, wherein the engineered adRNA may be recombinantly expressed.

[0020] Compositions are provided that include one or more of the above compounds and carriers. In one embodiment, the compositions may be pharmaceutical compositions, and therefore further include at least a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. The compositions are formulated according to various administration methods, such as systemic (oral) or topical.

[0021] Also provided herein are methods for modifying protein expression, the methods comprising, consisting essentially of, or further consisting of contacting a polynucleotide encoding a protein whose expression is to be modified with an engineered adRNA of the disclosure.

[0022] Yet another embodiment relates to a method for treating a disease or disorder associated with abnormal protein expression, which method comprises, consists essentially of, or further consists of administering to a subject in need thereof an effective amount of any one or more of the engineered adRNAs disclosed herein and / or using an effective amount of any one or more of the engineered adRNAs disclosed herein to treat the disease or disorder associated with abnormal protein expression. In one particular embodiment, provided herein is a method for treating Duchenne muscular dystrophy, which method comprises, consists essentially of, or further consists of administering to a subject in need thereof an effective amount of one or more of the engineered adRNAs disclosed herein.

[0023] This disclosure demonstrates the efficacy of this approach in vivo in the spf-ash mouse model of ornithine transcarbamylase deficiency, which harbors a G to A point mutation at the last nucleotide of exon 4. When adRNA alone is delivered via AAV, point mutations are observed to occur with a 1% or lower error in the absence of ADAR enzyme overexpression.

[0024] Additional aspects relate to the same or similar structures comprising, consisting essentially of, or further consisting of DNA or a combination of DNA and RNA. Further aspects relate to kits comprising any one or more of the above embodiments and instructions for use in vitro and / or in vivo.

[0025] Aspects of the present disclosure may relate to ADAR and APOBEC systems for gene editing. Some aspects relate to an ADAR system for exon skipping, comprising an adRNA targeting a splice acceptor and / or branch point in an intron, and optionally an ADAR enzyme. In some embodiments, the ADAR enzyme may be ADAR1, ADAR2, or a mutant or variant thereof. In some embodiments, the mutant or variant may be selected from ADAR1(E1008Q) and ADAR2(E488Q). In some embodiments, the intron may be contained in a gene selected from dystrophin, SCN9A, or ornithine transcarbamylase. In some cases, the adRNA may be selected from Sequence Set 1. A further aspect relates to a method for treating a disease, disorder, or condition characterized by abnormal gene expression, comprising administering the disclosed ADAR system. In some embodiments, the disease, disorder, or condition may be selected from Duchenne muscular dystrophy or ornithine transcarbamylase deficiency. In some embodiments, the disease, disorder, or condition may be associated with pain.

[0026] An additional aspect relates to an APOBEC system for cytosine to thymine editing, which comprises a pair of gRNAs that produce an alipoprotein B mRNA-like structure, and optionally an APOBEC enzyme. In some embodiments, the pair of gRNAs may be the pair of sequences provided in Sequence Set 2. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram showing endogenous recruitment of ADARs.

[0028] [Figure 2] 1 shows a list of stabilized scaffolds that have been evaluated for (1) improved efficiency and (2) ability to recruit ADARl (SEQ ID NOs: 2-9).

[0029] [Figure 3] 1 shows the results of screening exemplary adRNAs in vitro and in vivo.

[0030] [Figure 4] This diagram shows a schematic of the recruitment of MCP-APOBEC fusions or MCP-ACF-APOBEC fusions via MS2-RNA with two MS2 stem-loops. As shown in this diagram, the target cytosine can be kept single-stranded, making it accessible to APOBEC-mediated editing via the creation of a bulge. The bulge was created using either the exact target sequence (20-30 base pairs) in the MS2-RNA between the MS2 stem-loops (blue) or the sequence ACATATATGATACAATTTGATCAGTATATT (SEQ ID NO: 175), along with complementary sequences (20-30 base pairs) on either side of the MS2 stem-loop (green) that bind to the mRNA of interest. The sequence ACATATATGATACAATTTGATCAGTATATT (SEQ ID NO: 176) is taken from the naturally occurring apoB substrate that APOBEC edits. The MS2 stem-loop sequence used in the design is aACATGAGGATCACCCATGTc (SEQ ID NO: 177).

[0031] [Figure 5] The percentage of mRNA editing by constructs containing the Alu domain compared to editing by constructs containing the GluR2 domain is shown.

[0032] [Figure 6] An example of an Alu domain-containing construct (SEQ ID NO: 10) used to obtain the data in FIG. 5 is shown.

[0033] [Figure 7] An example of a construct containing two cruciforms linked by an antisense domain and containing an Alu domain is shown. Both an exemplary structure (SEQ ID NO:11) and an exemplary sequence (SEQ ID NO:1) are shown.

[0034] [Figure 8] Figures 8-1, 8-2 and 8-3 show various construct designs (including those containing the GluR2 domain) (SEQ ID NOs: 12-15, 252 and 16-34) and the percentage of mRNA editing for each.

[0035] [Figure 9] The target preference of the mapping enzyme for each base (e.g., A, C, G, and T) for both ADAR1 and ADAR2 is shown.

[0036] [Figure 10] Figure 10 shows the engineering of next-generation adRNAs with enhanced ADAR1 and ADAR2 recruitment potential. The first series of columns shows relative activity without adRNA. The second series of columns shows relative activity with a construct containing a GluR2 domain. The third series of columns shows relative activity with a construct containing an Alu domain associated with two cruciform structures. Figure 10 discloses SEQ ID NO: 11.

[0037] [Figure 11A-D] Different construct designs are shown. Figure 11A illustrates an antisense domain linked to two GluR2 domains. Figure 11B illustrates the antisense domain alone. Figure 11C illustrates the antisense domain linked to two cruciforms. Figure 11D illustrates a toehold.

[0038] [Figure 12] The percentage of editing yield between different construct designs is shown.

[0039] [Figure 13] A comparison of a short antisense oligonucleotide (AON) with a mismatched bulge and a longer construct containing a hairpin structure is shown.

[0040] [Figure 14] Exemplary adRNA designs (SEQ ID NOs: 250 and 251) are shown.

[0041] [Figure 15] Exemplary adRNA structures (SEQ ID NOs: 35-37) are shown with parameters (d, 1, m), where d = number of GluR2 domains, 1 = length of antisense domain, and m = position of mismatch.

[0042] [Figure 16] 1 shows a schematic diagram of RNA editing by recruiting endogenous ADARs in the presence of adRNA.

[0043] [Figure 17] U6 promoter-transcribed adRNAs with increasing antisense domain lengths combined with zero, one, or two GluR2 domains were evaluated for their ability to induce target RNA editing with or without exogenous ADAR2 expression. Values represent mean + / - SEM (n=3). Longer adRNAs can recruit endogenous ADARs for RNA editing.

[0044] [Figure 18] Figure 1 shows chemically synthesized adRNA versions tested against a panel of mRNAs with or without exogenous ADAR2 expression. Chemical modifications are identified along with the source of the adRNA. Values represent the mean + / - SEM (n=3).

[0045] [Figure 19] Figure 1 shows the in vivo RNA correction efficiency of correctly spliced OTC mRNA in the liver of treated adult spfash mice (retro-orbital injection). A 0.6% RNA editing level is observed in mice injected with U6-transcribed short adRNA.

[0046] [Figure 20]Figure 1 shows the design of Alu adRNA. Left: Structure of the Alu element. Center: Design described herein, containing a locus-specific antisense sequence with a C mismatch opposite the target A. Right: Recruitment of the RNA-editing enzyme ADAR to the target.

[0047] [Figure 21] Exemplary Alu guide sequences (SEQ ID NOs: 38-41) are shown.

[0048] [Figure 22] A schematic diagram of the split ADAR2 DD system is shown.

[0049] [Figure 23] An exemplary sequence of a split ADAR2 DD (SEQ ID NO: 42) is shown with potential sites of split highlighted.

[0050] [Figure 24] The pairs of fragments 1 to 16 tested via the Cypridina luciferase reporter (Cluc W85X) are shown.

[0051] [Figure 25] Fragments 9 and 10 tested against the Cluc reporter are shown.

[0052] [Figure 26] Figures 26-1, 26-2 and 26-3 show exemplary sequences (SEQ ID NOs: 43 to 55).

[0053] [Figure 27] 1 shows a schematic diagram of ADAR recruitment via U1A (SEQ ID NO: 56).

[0054] [Figure 28] Figures 28-1 to 28-6 show exemplary sequences (SEQ ID NOs: 57 to 68) for several fusion constructs or one or more APOBEC family members.

[0055] [Figure 29] 1 shows exemplary sequences (SEQ ID NOs: 69-73) of engineered apRNAs configured to recruit APOBEC3A.

[0056] [Figure 30] 1 shows exemplary sequences (SEQ ID NOs: 74-78) of engineered MS2-apRNAs configured to recruit MCP-APOBEC3A.

[0057] [Figure 31] Two different scenarios are shown: no ADAR recruitment and ADAR recruitment allowing ribosomal read-through resulting in normal luciferase expression.

[0058] [Figure 32A-C] Characterization of the genetic engineering and specificity profile of programmable RNA editing is shown: (Figure 32A) Schematic of RNA editing via engineered adRNAs derived from full-length ADAR2 and GluR2 transcripts, or constructs utilizing MS2 coat protein (MCP) fusions to the ADAR1 / 2 deaminase domain and the corresponding MS2 hairpin-bearing adRNA. (Figure 32B) Comparison of RNA editing efficiency of endogenous RAB7A transcripts by different RNA editing constructs, quantified by Sanger sequencing (efficiency calculated as the Sanger peak height ratio G / (A+G)). Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n=3). (Figure 32C) Violin plot showing the distribution of A-to-G editing yields observed at reference sites where at least one treated sample showed a significant change in editing yield (Fisher's exact test, FDR=1%) compared to the control sample. Blue circles indicate editing yields at the targeted A site within the RAB7A transcript. Black dots represent median off-target editing yields. To better visualize the shape of the distributions, their maximum range along the y-axis was equalized across all plots and truncated at 60% yield.

[0059] [Figure 33A-E] Figure 33A shows in vivo RNA editing in mouse models of human disease. (Figure 33A) Schematic of DNA and RNA targeting approaches to restore dystrophin expression in the mdx mouse model of Duchenne muscular dystrophy: (i) a dual gRNA-CRISPR-based approach leading to in-frame excision of exon 23, and (ii) ADAR2- and MCP-ADAR1-based editing of the ochre codon. (Figure 33B) Immunofluorescence staining of dystrophin in TA muscle shows partial restoration of expression in treated samples (intramuscular injection of AAV8-ADAR2, AAV8-ADAR2(E488Q), and AAV8-CRISPR). Partial restoration of nNOS localization is also seen in treated samples (scale bar: 250 μm). (Figure 33C) In vivo TAA->TGG / TAG / TGA RNA editing efficiency in corresponding treated adult mdx mice. Values represent the mean + / - SEM (n = 4, 3, 7, 3, 3, 10, 3, 4 single TA muscles, respectively). (Figure 33D) Schematic diagram of the OTC locus in the spfash mouse model of ornithine transcarbamylase deficiency, which harbors a G-to-A point mutation at the donor splice site in the last nucleotide of exon 4, and the approach for correction of mutant OTC mRNA by ADAR2-mediated RNA editing. (Figure 33E) In vivo RNA correction efficiency of correctly spliced OTC mRNA in the liver of treated adult spfash mice (retro-orbital injection of AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Values represent the mean + / - SEM (n = 4, 4, 3, 3, 4, 5 single animals, respectively).

[0060] [Figure 34A-C]Antisense domain engineering is shown. (Figure 34A) Optimization of the adRNA antisense region using adRNA scaffold 2: The length and distance from the ADAR2 recruitment region were systematically varied. Values represent the mean + / - SEM (n = 3) (SEQ ID NOs: 253 and 79-102). (Figure 34B) U6 promoter-transcribed adRNAs with increasing antisense domain lengths combined with zero, one, or two GluR2 domains were evaluated for their ability to induce target RNA editing with or without exogenous ADAR2 expression. Values represent the mean + / - SEM (n = 3). All the above experiments were performed in HEK 293T cells. (Figure 34C) Experimental confirmation of endogenous ADAR1 and ADAR2 expression (relative to GAPDH) in HEK 293T and HeLa cell lines. The observed levels were similar to those described in the human protein atlas (see proteinatlas.org on the world-wide web (www)).

[0061] [Figure 35A-B] Figure 35 shows the engineering of MS2 adRNA. (Figure 35A) Systematic evaluation of the antisense RNA targeting domain of MS2 adRNA (SEQ ID NOs: 103-110). Values represent mean + / - SEM (n=3). (Figure 35B) On-target RNA editing by MCP-ADAR2 DD-NLS requires co-expression of MS2 adRNA. Values represent mean + / - SEM (n=3). All experiments were performed in HEK 293T cells.

[0062] [Figure 36A-C]Analysis of RNA editing yields across a panel of targets is shown. (Figure 36A) Comparison of RNA editing efficiency of an OTC reporter transcript by GluR2 adRNA, an MS2 adRNA-guided RNA editing construct, and a Cas13b-based repair construct. Values represent the mean + / - SEM (n = 6 for reporter and Cas13b-based constructs, n = 3 for all other constructs). (Figure 36B) Chemically synthesized adRNA versions were tested against a panel of mRNAs with or without exogenous ADAR2. The exact chemical modifications are noted in the figure, along with the source of the adRNA. Values represent the mean + / - SEM (n = 3). (Figure 36C) Analysis of RNA editing yields across a spectrum of endogenous targets selected to cover a range of expression levels. U6-transcribed long adRNAs with zero or two GluR2 domains were also evaluated against multiple endogenous mRNA targets with or without exogenous ADAR2 expression. Editing is observed at all tested loci, even in the absence of exogenous ADAR2 expression. Values represent mean + / - SEM (n=3). All experiments were performed in HEK 293T cells.

[0063] [Figure 37A-D]ADAR2 mutants and their effects on editing and specificity are shown. (Figure 37A) Comparison of on-target RNA editing and adjacent adenosine editing of RAB7A transcripts by GluR2 adRNA, MS2adRNA-guided RNA editing constructs, and Cas13b-based repair constructs. Average (n=3) editing yields are shown (SEQ ID NO: 111). All experiments were performed in HEK 293T cells. Editing efficiency was calculated as the Sanger peak height ratio G / (A+G). (Figure 37B) ADAR2(E488Q) exhibits higher efficiency than ADAR2 in in vitro editing of the spfash OTC reporter transcript (p=0.037, unpaired t-test, two-tailed). Values represent mean + / - SEM (n=3). (Figure 37C) mdx DMD reporter transcript (p=0.048, p=0.012, unpaired t-test, two-tailed); values represent mean + / - SEM (n=3). (Figure 37D) Comparison of editing efficiency and specificity profiles of ADAR2, ADAR2(E488Q), and ADAR2(Δ1-138) for the OTC reporter transcript (upper panel) and the endogenous RAB7A transcript (lower panel). Heatmaps show A-to-G editing near the target (arrow). Values represent mean + / - SEM (n=3). All experiments were performed in HEK 293T cells. Editing efficiency was calculated as the Sanger peak height ratio G / (A+G). Figure 37D discloses SEQ ID NOs: 254-255, respectively, in order of appearance.

[0064] [Figure 38] Figure 1 shows the transcriptome-scale specificity profile of the RNA editing approach (Cas13b-ADAR repair + / - gRNA).

[0065] [Figure 39] We show the transcriptome-scale specificity profile of the RNA editing approach (ADAR2+ / - adRNA). The version used in these studies is GluR2 adRNA (1,20,6).

[0066] [Figure 40]1 shows the transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR1 DD+ / - adRNA).

[0067] [Figure 41] 1 shows the transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR2 DD+ / - adRNA).

[0068] [Figure 42-B] The change in transcriptome-scale editing specificity is shown by construct. (Figure 42A) Each point in the figure corresponds to the fraction of editing sites in one of the MCP-ADAR constructs listed in Figure 32. The fraction of editing sites in each construct was calculated by dividing the number of reference sites with significantly altered A to G editing yield (see Table 3) by the total number of reference sites examined (8,729,464). Constructs shown on the horizontal axis were compared using a Mann-Whitney U test. The resulting p-values were 0.16 for NLS vs. NES, 0.0070 for ADAR1 vs. ADAR2, 0.72 for -adRNA vs. +adRNA, and 0.038 for ADAR WT vs. ADAR E>Q (n=8 for all conditions). (Figure 42B) 2D histogram showing the transcriptome-wide A to G editing yield observed with each construct (y-axis) compared to that observed in the control sample (x-axis). The inset shows a violin plot representing the distribution of A to GG editing yields observed at reference sites where at least one treated sample was found to have a significant change (Fisher's exact test, FDR = 1%) compared to the control sample. Blue circles indicate editing yields at target A sites within the RAB7A transcript. To better visualize the shape of the distributions, their maximum range along the y-axis was equalized across all plots and truncated at 60% yield. The samples here correspond to 293T cells transduced with a long antisense domain-containing adRNA that enables RNA editing via exogenous and / or endogenous ADAR recruitment.

[0069] [Figure 43A-E]Figure 43 shows the optimization and evaluation of in vitro and in vivo dystrophin editing experiments in mdx mice. (Figure 43A) Schematic of RNA editing using full-length ADAR2 with engineered adRNA or reverse adRNA (raRNA); (ii) RNA editing efficiency of amber and ochre stop codons in one and two steps. Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n = 3). (Figure 43B) RNA editing of the ochre codon requires two cytosine mismatches in the antisense RNA targeting domain of adRNA or radRNA (SEQ ID NOs: 112-116) to restore GFP expression. Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n = 3). (Figure 43C) Schematic diagram of the AAV vector used for in vivo delivery of adRNA and ADAR2, and in vitro optimization of RNA editing of amber and ochre stop codons in the presence of one or two copies of adRNA delivered via AAV vectors (p = 0.0003, p = 0.0001, p = 0.0015, respectively, unpaired t-test, two-tailed). Experiments were performed in HEK 293T cells. Values represent the mean + / - SEM (n = 3 for reporter, n = 6 for all other conditions). (Figure 43D) Representative Sanger sequencing plot showing editing of the ochre stop codon (TAA->TGG) in the mdx DMD reporter transcript (quantified by NGS) (SEQ ID NOs: 117-118). Experiments were performed in HEK 293T cells (n = 3). (Figure 43E) Representative example of in vivo RNA editing analysis of treated mdx mice (quantified using NGS) (SEQ ID NOs: 119-130).

[0070] [Figure 44A-C]Immunofluorescence and Western blot analyses of in vivo dystrophin RNA editing experiments in mdx mice are shown. (Figure 44A) Immunofluorescence staining of dystrophin in TA muscle shows partial restoration of expression in treated samples (intramuscular injection of AAV8-ADAR2, AAV8-ADAR2(E488Q), and AAV8-MCP-ADAR1(E1008Q)NLS). Partial restoration of nNOS localization is also seen in treated samples (scale bar: 250 μm). (Figure 44B) Western blot showing partial restoration of dystrophin expression (1-2.5%) in TA muscle of mdx mice injected with both components of the editing machinery, the enzyme and adRNA, and stable ADAR2 expression in the injected TA muscle up to 8 weeks after injection. (Figure 44C) Western blot showing partial restoration of dystrophin expression (10%) using AAV8-CRISPR.

[0071] [Figure 45A-E]Optimization and validation of in vitro and in vivo OTC RNA editing experiments in spfash mice are shown. (Figure 45A) Representative Sanger sequencing plot showing correction of point mutations in the spfash OTC reporter transcript (quantified using NGS) (SEQ ID NOs: 131-132). Experiments were performed in HEK 293T cells (n=3). (Figure 45B) Representative example of in vivo RNA editing analysis of treated spfash mice showing correction of point mutations in correctly spliced OTC mRNA (quantified using NGS) (SEQ ID NOs: 133-139). (Figure 45C) In vivo RNA correction efficiency in OTC pre-mRNA in the liver of treated adult spfash mice (retro-orbitally injected with AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Values represent mean + / - SEM (n=4, 4, 3, 3, 4, 5 single animals, respectively). (Figure 45D) PCR products showing correctly and incorrectly spliced OTC mRNA. The incorrectly spliced mRNA is extended by 48 base pairs. In mice treated with adRNA + ADAR2(E488Q), the fraction of incorrectly spliced mRNA is reduced. (Figure 45E) Western blot of OTC shows partial restoration of expression (2.5% to 5%) in treated adult spfash mice and stable ADAR2(E488Q) expression 3 weeks after injection.

[0072] [Figure 46] Toxicity analysis of in vivo RNA editing experiments.

[0073] [Figure 47] FIG. 1 is a schematic diagram showing exon skipping via the generation of splice acceptor and / or branchpoint mutations.

[0074] [Figure 48] Schematic diagram of APOBEC-mediated C→T editing.

[0075] [Fig. 49A-D]A schematic diagram of both strand editing of DNA and DNA / RNA hybrids is shown.

[0076] [Fig. 50A-D] Figure 50A shows the results of a study in a model of ornithine transcarbamylase deficiency. Figure 50A shows the in vivo RNA correction efficiency in the livers of adult spfash mice treated (retro-orbitally injected with AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Each data point represents a single animal. Editing efficiency measured in spliced OTC mRNA. Error bars represent + / - SEM. Figure 50B shows the in vivo RNA correction efficiency in OTC pre-mRNA in the livers of adult spfash mice treated (retro-orbitally injected with AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Each data point represents a single animal. Figure 50C shows the in vivo RNA correction efficiency in OTC pre-mRNA in the livers of adult spfash mice treated (retro-orbitally injected with AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Each data point represents a single animal. Figure 50D shows a PCR product representing the OTC mRNA. The incorrectly spliced mRNA is extended by 48 base pairs. The fraction of incorrectly spliced mRNA is reduced in mice treated with adRNA + ADAR2(E488Q). Figure 50D is a Western blot of OTC showing partial restoration (2.5% to 5%) of expression in treated adult spfash mice.

[0077] [Figure 51A-B] Figure 51 shows the results of a study in a model of Duchenne muscular dystrophy. Figure 51A shows in vivo TAA->TGG / TAG / TGA RNA editing efficiency in corresponding treated adult mdx mice. Each data point represents a single TA muscle. Error bars represent + / - SEM. Figure 51B is a Western blot of dystrophin showing partial restoration of expression (1-2.5%) in corresponding treated adult mdx mice.

[0078] [Figure 52] Provides further information regarding potential branch point locations. Detailed Description

[0079] Aspects of the present disclosure provide nucleic acids, non-naturally occurring RNAs, vectors comprising nucleic acids, compositions, and pharmaceutical compositions for RNA editing. Any of the above or described herein can be configured for A (adenosine) → I (inosine) editing, C (cytosine) → T (thymine) editing, or a combination thereof. In some cases, an A → I editing can be interpreted or read as a C → U mutation. In some cases, an A → I editing can be interpreted or read as an A → G mutation. The nucleic acids, non-naturally occurring RNAs, vectors comprising nucleic acids, compositions, and pharmaceutical compositions described herein can provide enhanced editing efficiency, off-target editing, enhanced stability or in vivo half-life, or any combination thereof, compared to natural systems.

[0080] An embodiment of the present disclosure provides a vector. The vector can include a nucleic acid having a polynucleotide sequence encoding (i) a domain that recruits an RNA editing entity, (ii) a targeting domain that is complementary to at least a portion of a target RNA, (iii) two or more of any of the domains, or (iv) any combination thereof. In some cases, the vector can be administered to a subject, such as a subject in need thereof. In some cases, the vector can be administered to a subject, such as a subject in need thereof, as part of a pharmaceutical composition.

[0081] Embodiments of the present disclosure provide non-naturally occurring RNA. The non-naturally occurring RNA may include (i) a domain that recruits an RNA editing entity, (ii) a targeting domain complementary to at least a portion of a target RNA, (iii) two or more of any of these domains, or (iv) any combination thereof. In some cases, the non-naturally occurring RNA may be administered to a subject, such as a subject in need thereof. In some cases, the non-naturally occurring RNA may be administered to a subject, such as a subject in need thereof, as part of a pharmaceutical composition. In some cases, the non-naturally occurring RNA may be incorporated into a vector for administration. The vector may include a viral vector, a liposome, a nanoparticle, or any combination thereof. In some cases, the non-naturally occurring RNA may include at least one base, at least one sugar, two or more of which have a modification, such as a chemical modification, or a combination thereof.

[0082] An embodiment of the present disclosure provides a nucleic acid. The nucleic acid may include (i) a domain that recruits an RNA editing entity, (ii) a targeting domain that is complementary to at least a portion of a target RNA, (iii) two or more of any of these domains, or (iv) any combination thereof. In some cases, the nucleic acid may be administered to a subject, such as a subject in need thereof. In some cases, the nucleic acid may be administered to a subject, such as a subject in need thereof, as part of a pharmaceutical composition. In some cases, the nucleic acid may be formulated in a vector for administration. The vector may include a viral vector, a liposome, a nanoparticle, or any combination thereof. The nucleic acid may be genetically encoded. The nucleic acid may be chemically synthesized.

[0083] A nucleic acid can include one or more domains, such as one, two, three, four, five, or more domains. In some cases, a nucleic acid can include a recruitment domain, a targeting domain, two or more, or a combination thereof. In some cases, a nucleic acid can include a targeting domain and a recruitment domain. In some cases, a nucleic acid can include one targeting domain and two recruitment domains.

[0084] The domain can form a two-dimensional shape or secondary structure. For example, the targeting domain, the recruitment domain, or a combination thereof can form a secondary structure that can include a linear region, a cruciform or a portion thereof, a toehold, a stem loop, or any combination thereof. The domain itself can form a substantially linear two-dimensional structure. The domain can form a secondary structure that can include a cruciform. The domain can form a secondary structure that can include a stem loop. The domain can form a secondary structure that can include a toehold.

[0085] In some cases, the targeting domain can be located adjacent to the recruitment domain, either directly adjacent or adjacent but separated by a number of nucleotides. In some cases, the targeting domain can be adjacent to two recruitment domains. In some cases, two or more recruitment domains can be adjacent to each other.

[0086] Embodiments of the present disclosure include reducing off-target editing. One approach described herein involves limiting the catalytic activity of ADARs or APOBECs through a split reassembly approach. In such a design, the first domain (e.g., recruitment domain) may be catalytically inactive by itself, and the second domain may be catalytically inactive by itself, but the two domains together can provide catalytic activity to recruit ADARs or APOBECs. A nucleic acid comprising two domains can be split at any number of locations, such as between the two domains. In some cases, the first domain or the second domain can comprise an MS2 stem-loop, a BoxB stem-loop, a U1A stem-loop, a modified version of any of these, or any combination thereof.

[0087] The two-dimensional shape or secondary structure of a domain can affect the efficiency of editing, targeting, or a combination thereof, compared to nucleic acids that can form different two-dimensional shapes or secondary structures. Accordingly, aspects of the present disclosure include modifications of nucleic acids such that the two-dimensional shape is advantageously designed to improve editing efficiency and reduce targeting. Modifications to sequences containing naturally occurring recruitment domains can also improve editing efficiency and reduce targeting. Accordingly, aspects of the present disclosure include modifications of nucleic acids such that the sequence (e.g., a synthetic sequence) is advantageously designed to improve editing efficiency and reduce targeting. Modifications can include changing the length of the domain (e.g., extending the length), changing the native sequence to result in a change in secondary structure, adding chemical modifications, or any combination thereof. The nucleic acids described herein can provide these advantages.

[0088] In some cases, the nucleic acids described herein can modify at least one base pair of a target nucleic acid with at least about 3, 4, or 5 times greater efficiency than an equivalent nucleic acid complexed with a naturally occurring recruitment domain and an antisense domain (complementary to the target nucleic acid). In some cases, the nucleic acids described herein can modify at least one base pair of a target nucleic acid with at least about 3, 4, or 5 times greater efficiency than an equivalent nucleic acid complex with a GluR2 domain and an antisense domain (complementary to the target nucleic acid). In some cases, the nucleic acids described herein can modify at least one base pair of a target nucleic acid with at least about 3, 4, or 5 times greater efficiency than an equivalent nucleic acid complex with a Cas13b protein, or an active fragment thereof, and an antisense domain (complementary to the target nucleic acid). The increased efficiency can be measured by sequencing methods such as Sanger sequencing.

[0089] An embodiment of the present disclosure provides a vector. The vector can include a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity recruiting domain. In some cases, the polynucleotide sequence may not form a secondary structure including a stem-loop. In some cases, the polynucleotide sequence can form one or more stem-loops. In some cases, the polynucleotide sequence can form a secondary structure including a cruciform. In some cases, the polynucleotide sequence can form a secondary structure that may be substantially linear. In some cases, the polynucleotide sequence can include at least about 80% sequence identity with one or more sequences including an Alu domain-encoding sequence, a sequence encoding an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruiting domain, and any combination thereof. In some cases, the nucleic acid can be genetically encoded. In some cases, the nucleic acid can be chemically synthesized.

[0090] In some cases, the polynucleotide sequence may comprise at least about 80% sequence identity with a sequence encoding an Alu domain. In some cases, the polynucleotide sequence may comprise at least about 85% sequence identity with a sequence encoding an Alu domain. In some cases, the polynucleotide sequence may comprise at least about 90% sequence identity with a sequence encoding an Alu domain. In some cases, the polynucleotide sequence may comprise at least about 95% sequence identity with a sequence encoding an Alu domain. In some cases, the sequence encoding the Alu domain may be a non-naturally occurring sequence. In some cases, the sequence encoding the Alu domain may comprise a modified portion. In some cases, the sequence encoding the Alu domain may comprise a portion of a naturally occurring Alu domain sequence.

[0091] In some cases, the polynucleotide sequence may comprise at least about 80% sequence identity with the sequence encoding the APOBEC domain. In some cases, the polynucleotide sequence may comprise at least about 85% sequence identity with the sequence encoding the APOBEC domain. In some cases, the polynucleotide sequence may comprise at least about 90% sequence identity with the sequence encoding the APOBEC domain. In some cases, the polynucleotide sequence may comprise at least about 95% sequence identity with the sequence encoding the APOBEC domain. In some cases, the sequence encoding the APOBEC domain may be a non-naturally occurring sequence. In some cases, the sequence encoding the APOBEC domain may comprise a modified portion. In some cases, the sequence encoding the APOBEC domain may comprise a portion of a naturally occurring APOBEC domain sequence.

[0092] In some cases, the polynucleotide sequence may comprise at least about 80% sequence identity with the sequence encoding the GluR2 domain. In some cases, the polynucleotide sequence may comprise at least about 85% sequence identity with the sequence encoding the GluR2 domain. In some cases, the polynucleotide sequence may comprise at least about 90% sequence identity with the sequence encoding the GluR2 domain. In some cases, the polynucleotide sequence may comprise at least about 95% sequence identity with the sequence encoding the GluR2 domain. In some cases, the sequence encoding the GluR2 domain may be a non-naturally occurring sequence. In some cases, the sequence encoding the GluR2 domain may comprise a modified portion. In some cases, the sequence encoding the GluR2 domain may comprise a portion of a naturally occurring GluR2 domain sequence.

[0093] In some cases, the polynucleotide sequence can comprise at least about 80% sequence identity with the coding sequence that recruits ADAR. The polynucleotide sequence encoding the domain that recruits at least one RNA editing entity can be isolated, purified, or synthesized. Such a polynucleotide sequence can be specifically configured to recruit ADAR to a target site. The recruitment can include exogenous ADAR recruitment (i.e., can be delivered simultaneously or separately), endogenous ADAR recruitment, or a combination thereof. In some cases, the polynucleotide sequence can be specifically configured to enhance ADAR recruitment or enhance the specificity of ADAR recruitment to a specific site compared to naturally occurring recruitment domains. In some cases, the coding sequence can be a non-natural sequence. In some cases, the coding sequence can comprise a modified portion. In some cases, the coding sequence can comprise a portion of a naturally occurring ADAR recruitment domain sequence. It can be envisioned that either a natural or synthetic sequence that recruits ADAR can be included in the polynucleotide sequence. In some cases, the polynucleotide sequence can include example sequences described herein. Exemplary sequences are included in Figures 2, 6, 7, 8, 14, 15, 21, 26, 28, 29, 30, and Table 1. The sequences provided herein include sequences having at least a portion that can encode a domain that recruits at least one RNA editing entity.

[0094] (Table 1) DNA encoding adRNA sequences. The adRNA sequences produced from these sequences are identical to the DNA sequences, but with T replaced by U. JPEG0007720623000001.jpg19139JPEG0007720623000002.jpg213139JPEG0007720623000003.jpg213139JPEG0007720623000004.jpg212139

[0095] The polynucleotide sequence encoding the domain that recruits the RNA editing entity can include the recruitment of any ADAR protein (e.g., ADAR1, ADAR2, ADAR3, or any combination thereof), any APOBEC protein (e.g., APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or any combination thereof), or a combination thereof. In some cases, the recruited ADAR or APOBEC protein can be mammalian. In some cases, the recruited ADAR or APOBEC protein can be human. In some cases, the recruited ADAR or APOBEC protein can be recombinant (e.g., an exogenously delivered ADAR or APOBEC), modified (e.g., an exogenously delivered ADAR or APOBEC), endogenous (e.g., an endogenous ADAR or APOBEC), or any combination thereof.

[0096] In some cases, the domain that recruits at least one RNA editing entity does not form a secondary structure that includes a stem-loop. In some cases, the domain that recruits at least one RNA editing entity forms a secondary structure that includes a stem-loop. In some cases, the domain that recruits at least one RNA editing entity forms a secondary structure that does not include a stem-loop. In some cases, the domain that recruits at least one RNA editing entity forms a secondary structure that includes a linear portion. In some cases, the domain that recruits at least one RNA editing entity forms a secondary structure that includes a cruciform or a portion thereof.

[0097] The polynucleotide sequence can encode two or more RNA editing recruitment domains. In some cases, the polynucleotide sequence can encode multiple recruitment domains. In some cases, the polynucleotide sequence can encode two, three, four, five, six, or more recruitment domains. The multiple recruitment domains can include Alu domains, APOBEC domains, GluR2 domains, Cas13 domains, or any combination thereof. In some cases, the Alu domains, APOBEC domains, Cas13 domains, or GluR2 domains can be naturally occurring recruitment domains. In some cases, the Alu domains, APOBEC domains, Cas13 domains, or GluR2 domains can be non-naturally occurring, modified from a naturally occurring sequence, or recombinant. At least one of the multiple recruitment domains can include a single-stranded sequence. At least one of the multiple recruitment domains can include multiple Alu repeats. At least one of the multiple recruitment domains can form a secondary structure including a stem-loop. At least one of the plurality of recruitment domains can form a secondary structure that does not include a stem-loop, at least one of the plurality of recruitment domains can form a secondary structure that includes a cruciform or a portion thereof, or at least one of the plurality of recruitment domains can form a secondary structure that includes a toehold.

[0098] In some cases, the nucleic acid can encode a domain that recruits at least one RNA editing entity. In some cases, the nucleic acid can encode an RNA complementary to at least a portion of the target RNA. In some cases, the nucleic acid can encode a recruitment domain and a targeting domain. In some cases, the nucleic acid can encode a recruitment domain and a targeting domain. A portion of the target RNA can comprise a single base. A portion of the target RNA can comprise multiple bases. A portion of the target RNA can comprise about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more base pairs. In some cases, the target RNA can comprise about 1 bps to about 10 bps. In some cases, the target RNA can comprise about 10 bps to about 100 bps. In some cases, the target RNA can comprise about 10 bps to about 500 bps. In some cases, the target RNA can comprise about 10 bps to about 1000 bps. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 200 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 150 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 250 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 275 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 300 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 400 bp in length. A nucleic acid comprising a targeting domain and a recruitment domain can comprise a contiguous sequence at least about 500 bp in length.

[0099] A vector can be used to deliver a nucleic acid. The vector can include DNA, such as double-stranded DNA or single-stranded DNA. The vector can include RNA. In some cases, the RNA can include base modifications. The vector can include a recombinant vector. The vector can be a vector modified from a natural vector. The vector can include at least a portion of a non-naturally occurring vector. Any vector can be utilized. In some cases, the vector can include a viral vector, a liposome, a nanoparticle, an exosome, an extracellular vesicle, or any combination thereof. In some cases, the viral vector can include an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a retroviral vector, a portion of any of these, or any combination thereof. In some cases, the nanoparticle vector can include a polymer-based nanoparticle, an aminolipid-based nanoparticle, a metal nanoparticle (e.g., a gold-based nanoparticle), a portion of any of these, or any combination thereof. In some cases, the vector can include an AAV vector. The vector can be modified to include a modified VP1 protein (e.g., an AAV vector modified to include a VP1 protein, etc.). The AAV can include serotypes such as AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, any derivative thereof, or any combination thereof.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in a 5' to 3' direction unless otherwise defined. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods, devices, and materials are described herein. All technical and patent literature cited herein is incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to such disclosure by virtue of prior disclosure.

[0101] Unless otherwise indicated, the practice of the present technology employs conventional tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA techniques.Green and Sambrook (eds.), (2012) Molecular Cloning: A Laboratory Manual, 4th Edition; Ausubel et al. (eds.), (2015) Current Protocols in Molecular Biology: Methods in Enzymology Series (Academic Press, Inc., NY); MacPherson et al., (2015) PCR1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al., (1995) PCR2: A Practical Approach; Macpherson et al., (2006) PCR: The Basics (Garland Science); Harlow and Lane (eds.), (1999) Antibodies, A Laboratory Manual; Greenfield (eds.), (2014) Antibodies, A Laboratory Manual; Freshney (2010) Animal Cell Culture: A Manual of Basic Techniques, 6th Edition; Gait (ed.), (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins (eds.), (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn (eds.), (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins (eds.), (1984) Transcription and Translation; Buzdin and Lukyanov (eds.), (2007) Nucleic Acid Hybridization: Current Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi (eds.), (2007) in In vitro transcription and translation protocols, 2nd edition; Guisan (ed.), (2006) Enzymes and cell fixation; Perbal (1988) Practical guide to molecular cloning, 2nd edition; Miller and Calos (eds.), (1987) Gene transfer vectors for mammalian cells (Cold Spring Harbor Laboratory); Markrides (eds.), (2003) Gene transfer and expression in mammalian cells; Mayer and Walker (eds.), (1987) Immunochemical methods in cell and molecular biology (Academic Press, London); Lundblad and Macdonald (eds.), (2010) Handbook of biochemistry and molecular biology, 4th edition; Herzenberg et al. (eds.), (1996) Weir's Handbook of experimental immunology, 5th edition; and / or their latest editions.

[0102] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0103] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight (including ranges), are approximate. They may vary by increments of 1.0 or 0.1, appropriately or alternatively by + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are well known in the art.

[0104] Unless otherwise indicated by context, it is specifically contemplated that the various features of the disclosure described herein can be used in any combination. Furthermore, the present disclosure also contemplates, in some embodiments, the exclusion or omission of any feature or combination of features described herein. Specifically, the specification contemplates that if a composite is described as comprising components A, B, and C, it specifically contemplates that any of A, B, or C, or combinations thereof, singly or in any combination, can be omitted or disclaimed.

[0105] Unless otherwise specified, all specific embodiments, features, and terms are intended to encompass both the described embodiment, feature, or term and their biological equivalents. definition

[0106] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a polypeptide" includes multiple polypeptides, including mixtures thereof. Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0107] As used herein, the term "about" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art. This may depend, in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean plus or minus 10% per single measurement by one of ordinary skill in the art. Alternatively, "about" can mean within a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, such as within 5-fold or 2-fold. When specific values are described in the present application and claims, unless otherwise specified, the term "about" can be assumed to mean within that acceptable error range. Also, when ranges and / or subranges of values are presented, the ranges and / or subranges can include the endpoints of the ranges and / or subranges. In some cases, variations can include amounts or concentrations of 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the given amount.

[0108] For purposes of describing numerical ranges herein, each intervening value to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the values 7 and 8 are contemplated in addition to 6 and 9. In the range 6.0 to 7.0, the values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0109] As used herein, the terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" (nucleobases within inosine) refer to such nucleobases.

[0110] The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to a nucleobase linked to a (deoxy)ribosyl sugar.

[0111] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses belonging to the genus Dependoparvovirus in the family Parvoviridae. Multiple serotypes of the virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells from a variety of tissue types. At least eleven have been disclosed in the prior art. Non-limiting exemplary serotypes useful for the purposes disclosed herein include any of the eleven serotypes, e.g., AAV2 and AAV8. The term "lentivirus" as used herein refers to a member of the class of viruses belonging to the genus Lentivirus in the family Retroviridae. Some lentiviruses are known to cause disease, while others are known to be suitable for gene delivery. See, for example, Tomas et al. (2013) Biochemistry, Genetics and Molecular Biology: "Gene Therapy - Tools and Potential Applications" ISBN 978-953-51-1014-9, DOI: 10.5772 / 52534.

[0112] As used herein, the term "adenosine deaminase acting on RNA" or "ADAR" can refer to an adenosine deaminase that can convert adenosine (A) in an RNA sequence to inosine (I). ADAR 1 and ADAR 2 are two exemplary species of ADAR involved in mRNA editing in vivo. Examples of ADAR1 sequences can be found, but are not limited to, in the following reference numbers: HGNC:225; Entrez Gene:103; Ensembl:ENSG 00000160710; OMIM:146920; UniProtKB:P55265; and GeneCards:GC01M154554, and their biological equivalents. Examples of ADAR2 sequences, including but not limited to, can be found in the following reference numbers: HGNC:226; Entrez Gene:104; Ensembl:ENG00000197381; OMIM:601218; UniProtKB:P78563; and GeneCards:GC21P045073, and biological equivalents thereof. Further, non-limiting examples of catalytic domain sequences are provided above. The forward and reverse RNAs used in site-specific ADAR editing are known as "adRNA" and "radRNA," respectively. The catalytic domains of ADAR1 and ADAR2 are contained in the sequences set forth herein below.

[0113] ADAR1 catalytic domain: KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVE NGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV (SEQ ID NO: 140)

[0114] ADAR2 catalytic domain:

[0115] QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT (SEQ ID NO: 141)

[0116] The double-stranded RNA binding domain (dsRBD) of ADAR is contained in the sequence shown herein below.

[0117] ADAR dsRBD:

[0118] MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN (SEQ ID NO: 142)

[0119] It should be understood that further mutations can be made to the sequences of ADAR and / or its various domains. For example, the present disclosure provides E488Q and E1008Q mutants of both ADAR1 and ADAR2, as well as a "promiscuous" mutant of ADAR2 with a C-terminal deletion. The "promiscuous" mutant is known as such because it exhibits promiscuity in the edited readthrough (confirmed across two different loci) with several A's near the target sequence showing an A-to-G transition. The sequences of the mutants are shown herein below.

[0120] "Promiscuous" ADAR2 mutants

[0121] *(SEQ ID NO: 143)

[0122] Without being bound by theory, C-terminal deletions in ADAR1 may produce the same or similar effects.

[0123] The term "Alu domain" can refer to a sequence obtained from an Alu transposable element ("Alu element"). Typically, the Alu element is about 300 base pairs in length. An Alu element typically comprises a cruciform-polyA5-TAC-polyA6-cruciform-polyA tail, where the two cruciform domains are similar in nucleotide sequence. An "Alu domain" can comprise the cruciform portion of the Alu element. In some embodiments, two Alu domains comprising a cruciform structure are linked by a sequence complementary to a target RNA sequence.

[0124] As used herein, the term "APOBEC" can refer to any protein in the family of evolutionarily conserved cytidine deaminases involved in mRNA editing, catalyzing C-to-T editing. This can be interpreted as C-to-U conversion and its equivalents. In some aspects, the term APOBEC can refer to APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or any of their equivalents. Non-limiting example sequences of fusion proteins containing one or more APOBEC domains fused to an ADAR domain or fused to an alternative domain suitable for use in an RNA editing system are provided herein. For this purpose, APOBECs are considered equivalent to ADAR-catalyzed editing through different conversions. Thus, without being bound by theory, all embodiments of ADAR-based editing systems contemplated herein can be adapted for use in APOBEC-based RNA editing systems. In some cases, the use of APOBECs can include specific modifications. For example, but not limited to, specific guide RNAs or "gRNAs" are used to recruit enzymes.

[0125] "Aptamer" can refer to a short, single-stranded oligonucleotide that can bind various molecules with high affinity and specificity. Non-limiting examples of aptamers are described in Lakhin, AV et al. (2013). Acta naturae, 5(4), 34-43.

[0126] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. Unless otherwise indicated, "contain," "containing," "including," "comprising," and the like mean "comprising." When used to define compositions and methods, "consisting essentially of" means excluding significant other elements essential to the combination for the intended use. Thus, a composition essentially comprising elements as defined herein may not exclude trace amounts of contaminants from isolation and purification methods, and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Consisting of" will mean excluding more than trace amounts of other ingredients, and substantial method steps for administering the compositions of the present disclosure. Embodiments defined by any of these transition terms are within the scope of the present disclosure.

[0127] "Non-canonical amino acids" refers to the 20 amino acids found naturally in the human body, which are shown in the table below with their three-letter abbreviations, one-letter abbreviations, structures, and corresponding codons. JPEG0007720623000005.jpg66139JPEG0007720623000006.jpg147139JPEG0007720623000007.jpg56141JPEG0007720623000008.jpg191141

[0128] The term "Cas9" can refer to the CRISPR-associated endonuclease referred to therein. Examples of Cas9 include, but are not limited to, Staphylococcus aureus Cas9, nuclease-dead Cas9, and their orthologs and biological equivalents. Orthologs include, but are not limited to, Streptococcus pyogenes Cas9 ("spCas9"), Cas9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida, and Cpf1 (which performs a cleavage function similar to cas9) from various bacterial species, including Acidaminococcus spp. and Francisella novicida U112. For example, UniProtKB G3ECR1 (Cas9_STRTR) can be used, as well as dead Cas9 or dCas9 lacking nuclease activity (e.g., with mutations in both the RuvC and HNH domains). The term "Cas9" can further refer to equivalents of the referenced Cas9 having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, including, but not limited to, other large Cas9 proteins. In some embodiments, the Cas9 is derived from Campylobacter jejuni or other Cas9 orthologues up to 1000 amino acids in length.

[0129] As used herein, the term "CRISPR" can refer to a sequence-specific gene manipulation technology using clustered regularly interspaced short palindromic repeats. CRISPR can be used to perform gene editing and / or gene regulation, as well as simply targeting target proteins to specific genomic locations. "Gene editing" can refer to a type of genetic engineering in which the nucleotide sequence of a target polynucleotide is changed through deletion, insertion, introduction of single- or double-strand breaks, or base substitutions into the polynucleotide sequence. In some embodiments, CRISPR-mediated gene editing utilizes the pathways of non-homologous end joining (NHEJ) or homologous recombination to perform the editing. Gene regulation can refer to increasing or decreasing the production of a specific gene product, such as a protein or RNA.

[0130] As used herein, the term "deficiency" can refer to a lower than normal (physiologically acceptable) level of a particular agent. In the context of a protein, deficiency can refer to a lower than normal level of the full-length protein.

[0131] As used herein, the term "detectable marker" can refer to at least one marker capable of directly or indirectly producing a detectable signal, including, for example, enzymes that produce a detectable signal by colorimetry, fluorescence, or luminescence, e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent or luminescent dyes; electron-dense functional groups detected by electron microscopy or electrical properties such as conductivity, amperometry, voltammetry, or impedance; detectable functional groups whose molecules are of a size sufficient to induce a detectable modification of their physical and / or chemical properties (such detection can be achieved by optical methods such as diffraction, surface plasmon resonance, surface alteration, etc.); physical methods such as contact angle changes or atomic force spectroscopy, tunneling effect, or the like. 32 P, 35 S or 125Radioactive molecules such as I, are not limited to radioactive molecules.

[0132] As used herein, the term "domain" can refer to a region of a protein or polypeptide that is associated with a particular function. For example, a "domain associated with an RNA hairpin motif" can refer to a domain of a protein that binds to one or more RNA hairpins. The binding can optionally be specific to a particular hairpin.

[0133] As used herein, the term "dystrophin" refers to the protein corresponding to its name and encoded by the gene Dmd, non-limiting examples of which can be found in Uniprot under the reference numbers P11532 (for humans) and P11531 (for mice).

[0134] "Editing induction element" can refer to a large double-stranded RNA structure required for efficient RNA editing. Non-limiting examples of editing induction elements are described in Daniel, C. et al. (2017) Genome Biol. 18, 195. A further non-limiting example of an editing induction element is provided by the following structure (SEQ ID NO: 15): JPEG0007720623000009.jpg32120 Implementation Model of the Disclosure

[0135] ADARs are naturally occurring RNA editing enzymes that catalyze the hydrolytic deamination of adenosine, converting it to inosine, which is biochemically recognized as guanosine. As explained herein above, APOBECs are enzymes that can perform a similar function, converting cytosine to thymine.

[0136] The term "encoding," as applied to a polynucleotide, can refer to a polynucleotide that, in its natural state or when manipulated by methods well known to those of skill in the art, is said to encode a polypeptide, which can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0137] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecular, biological, or cellular substance and are intended to have minimal homology while maintaining the desired structure or function.

[0138] "Eukaryotic cells" include all kingdoms of life except the prokaryotic kingdom, which can be easily distinguished through their membrane-bound nuclei. Animals, plants, fungi, and prokaryotes are eukaryotic organisms or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" encompasses eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans.

[0139] As used herein, "expression" can refer to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0140] As used herein, the term "functional" is used to mean modifying any molecule, organism, or cellular material to achieve a particular effect.

[0141] As used herein, the term "Glur2 mRNA" can refer to an mRNA encoding ionotropic AMPA glutamate receptor 2 ("Glur2") that undergoes adenosine to inosine (A to I) editing. The mRNA site-selectively recruits ADARs.

[0142] As used herein, the term "gRNA" or "guide RNA" can refer to a guide RNA sequence used to target a specific polynucleotide sequence for gene editing using CRISPR technology. Techniques for designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. See, for example, Doench, J. et al., Nature Biotechnology 2014;32(12):1262-7; Mohr, S. et al. (2016) FEBS Journal 283:3232-38; and Graham, D. et al., Genome Biol. 2015;16:260. A gRNA may comprise, consist essentially of, or further consist of a fusion polynucleotide comprising a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), or a polynucleotide comprising a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In some embodiments, the gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83).

[0143] The terms "hairpin," "hairpin loop," "stem loop," and / or "loop," used alone or in conjunction with "motif," are used in the context of oligonucleotides to refer to a structure formed in a single-stranded oligonucleotide when sequences within the single strand that are complementary when read in opposite directions base pair to form a region whose conformation resembles a hairpin or loop.

[0144] "Homology" or "identity" or "similarity" can refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be measured by comparing positions within each sequence, which can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The 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 this disclosure.

[0145] Homology refers to a% sequence identity with the reference sequence.In practice, whether a specific sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical with any sequence described herein (which may correspond to the specific nucleic acid sequence described herein), this specific polypeptide sequence can be measured using a conventionally known computer program, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 5711).When using Bestfit or any other sequence alignment program to measure whether a specific sequence is, for example, 95% identical with a reference sequence, the percentage identity is calculated over the entire length of the reference sequence, and parameters can be set to allow a homology gap of up to 5% of the entire reference sequence.

[0146] For example, in certain embodiments, the identity between a reference sequence (query sequence, i.e., a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be measured using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some cases, for certain embodiments in which identity is narrowly constructed, the parameters used in the FASTDB amino acid alignment can include: scoring scheme = PAM (percentage of accepted mutations) 0, k-tuple = 2, mismatch penalty = 1, joining penalty = 20, randomization group length = 0, cutoff score = 1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500, or a shorter subject sequence length. According to such embodiments, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the results can be manually corrected to account for the fact that the FASTDB program does not consider N- and C-terminal truncations of the subject sequence when calculating the global percent identity. For subject sequences truncated at the N- and C-termini, the percentage identity can be corrected by calculating the number of query sequence residues flanking the N- and C-termini of the subject sequence that are not matched / aligned with the corresponding subject residues in the query sequence as a percentage of the total bases in the query sequence. The results of the FASTDB sequence alignment provide a measure of whether residues are matched / aligned. This percentage can then be subtracted from the percent identity calculated by the FASTDB program using specific parameters to arrive at a final percent identity score. This final percent identity score can be used for purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are considered for purposes of manually adjusting the percent identity score. That is, for this manual correction, only query residue positions outside the farthest N-terminal residue of the subject sequence are considered. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity.Because this deletion occurs at the N-terminus of the subject sequence, the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since the 10 unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90-residue subject sequence is compared with a 100-residue query sequence. This time, because the deletion is an internal deletion, there are no residues at the N- or C-termini of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, manual correction is required to ensure that only residues located outside the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are displayed in the FASTDB alignment.

[0147] "Hybridization" can refer to a reaction in which one or more polynucleotides react to form a stabilized complex through hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex can include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can 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.

[0148] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C, a hybridization buffer concentration of about 6x SSC to about 10x SSC, a formamide concentration of about 0% to about 25%, and a wash solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C, a buffer concentration of about 9x SSC to about 2x SSC, a formamide concentration of about 30% to about 50%, and a wash solution of about 5x SSC to about 2x SSC. Examples of highly stringent hybridization conditions include an incubation temperature of about 55°C to about 68°C, a buffer concentration of about 1x SSC to about 0.1x SSC, a formamide concentration of about 55% to about 75%, and a wash solution of about 1x SSC to 0.1x SSC, or deionized water. Generally, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps. Wash incubation times are approximately 1, 2, or 15 minutes. SSC is a 0.15 M NaCl and 15 mM citrate buffer. It should be understood that equivalents of SSC using other buffer systems can be used.

[0149] As used herein, "inhibit" means the ability to substantially antagonize, prohibit, block, suppress, slow, hinder, alter, eliminate, halt, or reverse the progression or severity of the activity of a particular pathogen (e.g., an infectious pathogen) or disease.

[0150] As used herein, the term "interferon" can refer to a group of signaling proteins known to be involved in immune responses. In the context of this application, interferons of interest are those that enhance ADAR expression. The correlation between interferon alpha and ADAR1 is well known, and therefore, the present disclosure contemplates the use of interferon alpha as a means of increasing endogenous ADAR1 expression. Commercial sources of isolated or recombinant interferons include, but are not limited to, Sigma-Aldrich, R&D Systems, Abcam, and Thermo Fisher Scientific. Alternatively, interferon alpha can be produced using known vectors and predetermined protein sequences, such as Q6QNB6 (human IFNA).

[0151] As used herein, the term "isolated" can refer to a molecule or biological substance or cellular material that is substantially free of other substances. In one embodiment, the term "isolated" can refer to a nucleic acid, such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or its derivative), or a cell or organelle, or tissue or organ, separated from other DNA or RNA, or a protein or polypeptide, or a cell or organelle, or tissue or organ, that is present in a natural source. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. "Isolated nucleic acid" also refers to nucleic acid fragments that are not naturally occurring as fragments and would not be found in their natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins and is meant to include both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues and is meant to include both cultured and genetically engineered cells or tissues.

[0152] "Transfer RNA" or "mRNA" is a nucleic acid molecule that has been transcribed from DNA and then processed to remove non-coding portions known as introns. The resulting mRNA is exported from the nucleus (or another locus where the DNA resides) and translated into protein. The term "pre-mRNA" can refer to the strand before it has been processed to remove the non-coding portions.

[0153] As used herein, the term "mutation" can refer to an alteration to a nucleic acid sequence encoding a protein relative to the consensus sequence of that protein. A "missense" mutation results in the substitution of one codon for another. A "nonsense" mutation changes a codon from one encoding a specific amino acid to a stop codon. Nonsense mutations often result in truncated translation of a protein. A "silent mutation" is one that does not affect the resulting protein. As used herein, the term "point mutation" can refer to a mutation that affects only one nucleotide in a gene sequence. A "splice site mutation" is a mutation present in pre-mRNA (before processing to remove introns) that results in mistranslation and often a truncated protein due to incorrect delineation of the splice site. A mutation can include a single nucleotide variation (SNV). A mutation can include a sequence variant, sequence variation, sequence modification, or allelic variant. A reference DNA sequence can be obtained from a reference database. A mutation can affect function. A mutation can have no effect on function. Mutations can occur at the DNA level in one or more nucleotides, at the ribonucleic acid (RNA) level in one or more nucleotides, at the protein level in one or more amino acids, or any combination thereof. Reference sequences can be obtained from databases such as the NCBI Reference Sequence Database (RefSeq) database. Specific changes that can constitute mutations can include substitutions, deletions, insertions, inversions, or transversions in one or more nucleotides or one or more amino acids. Mutations can be point mutations. Mutations can also be fusion genes. Fusion pairs or fusion genes can result from mutations such as translocations, interstitial deletions, chromosomal inversions, or any combination thereof. Mutations can constitute variability in the number of repeat sequences, such as triplicates, quadruplicates, etc. For example, mutations can be an increase or decrease in the copy number associated with a given sequence (i.e., copy number variation, or CNV). Mutations can include two or more sequence changes in different alleles or two or more sequence changes in a single allele.The mutation can include two different nucleotides at one position of one allele, such as a mosaic. The mutation can include two different nucleotides at one position of one allele, such as a chimera. The mutation can be present in malignant tissue. The presence or absence of the mutation can indicate an increased risk of developing a disease or condition. The presence or absence of the mutation can indicate the presence of a disease or condition. The mutation can be present in benign tissue. The absence of the mutation can indicate that the tissue or sample is benign. Alternatively, the absence of the mutation may not indicate that the tissue or sample is benign. The methods described herein can include identifying the presence of the mutation in a sample.

[0154] The term "non-canonical amino acid" can refer to synthetic or modified amino acids that do not belong to this group, typically produced by chemical synthesis or modification from a canonical amino acid (e.g., an amino acid analog). The present disclosure employs proteinogenic non-canonical amino acids in some of the methods and vectors disclosed herein. An example of a non-canonical amino acid is, but is not limited to, pyrrolysine (Pyl or O), the chemical structure of which is shown below: JPEG0007720623000010.jpg3697

[0155] Inosine (I) is another example of a non-canonical amino acid. It is found in tRNA and is essential for proper translation through "wobble base pairing." The structure of inosine is shown above.

[0156] As used herein, the term "ornithine transcarbamylase" or "OTC" can refer to the protein encoded by the gene Otc, corresponding to its name. Examples are found in UniProt reference numbers P00480 (for humans) and P11725 (for mice). OTC deficiency is an X-linked genetic condition that results in high levels of ammonia in the blood. In some cases, OTC deficiency is caused by a G-to-A splice site mutation at the donor splice site of exon 4, which results in mis-splicing of the pre-mRNA. The mutation results in the formation of an elongated or point-mutated protein. There is a 15- to 20-fold decrease in OTC protein levels. See, e.g., Hodges, PE and Rosenberg, LE. spf ash Mouse: Missense mutations in the ornithine transcarbamylase gene also cause aberrant mRNA splicing. Proc. Natl. Acad. Sci. USA 86, 4142-4146 (1989) (showing alternative forms of OTC produced). These sequences are shown below. JPEG0007720623000011.jpg62118

[0157] As noted above, when mutations are present, correct splice variants can be produced, but such production can also result in missense mutations and contribute to OTC deficiency.

[0158] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by a peptide bond. In alternative embodiments, the subunits can be linked by other bonds, such as esters, ethers, etc. A protein or peptide can contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. As used herein, the term "amino acid" can refer to any natural and / or unnatural or synthetic amino acid, amino acid analog, and peptidomimetic, including glycine and its D and L optical isomers. As used herein, the term "fusion protein" can refer to a protein composed of domains from one or more naturally occurring or recombinantly produced proteins, where generally each domain performs a different function. In this regard, the term "linker" can refer to a protein fragment used to link these domains together, preserve the conformation of the fusion protein domains, and / or prevent undesirable interactions between the fusion protein domains that would compromise their respective functions.

[0159] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, transfer RNA (mRNA), transcribed RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated RNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Furthermore, polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term can refer to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide encompasses both the double-stranded form and any of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0160] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. In some embodiments, a polynucleotide can contain one or more other nucleotide bases. For example, inosine (I), which is a nucleoside formed when hypoxanthine is linked to ribofuranose via a β-N9-glycosidic bond, resulting in the following chemical structure: JPEG0007720623000012.jpg7071

[0161] Inosine is read as guanine (G) by the translational machinery.

[0162] The term "polynucleotide sequence" refers to the alphabetical representation of a polynucleotide molecule that can be entered into a database in a computer having a central processing unit and used in bioinformatic applications such as functional genomics and homology searching.

[0163] As used herein, the term "purification marker" can refer to at least one marker useful for purification or identification. A non-exhaustive list of such markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, Cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag1, Softag3, Strep, or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, Cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC, or any other fluorescent dye or hapten.

[0164] As used herein, the term "recombinant expression system" refers to a genetic construct or construct for the expression of certain recombinantly formed genetic material. In this regard, the term "construct" is interchangeable with the term "vector," as defined herein.

[0165] As used herein, the term "recombinant protein" can refer to a polypeptide produced by recombinant DNA techniques, where, generally, DNA encoding the polypeptide is inserted into an appropriate expression vector, which is then used to transform a host cell to produce the heterologous protein.

[0166] As used herein, the term "repair" in relation to protein expression can refer to the ability to establish full-length protein expression where a mutation previously truncated the expression of the protein. In the context of "restoring activity," the term encompasses affecting protein expression to normal, unmutated levels where the mutation caused aberrant expression (e.g., too low or too high).

[0167] As used herein, the term "sample" generally refers to any sample from a subject (e.g., a blood sample or a tissue sample). The sample, or a portion thereof, may contain stem cells. The sample, or a portion thereof, may be enriched for stem cells. Stem cells may be isolated from the sample. The sample may include tissue, cells, serum, plasma, exosomes, bodily fluid, or any combination thereof. The bodily fluid may include urine, blood, serum, plasma, saliva, mucus, spinal fluid, tears, semen, bile, amniotic fluid, or any combination thereof. The sample, or a portion thereof, may include extracellular fluid obtained from a subject. The sample, or a portion thereof, may include cell-free nucleic acid, DNA, or RNA. The sample, or a portion thereof, may be analyzed for the presence or absence of one or more mutations. Genomic data may be obtained from the sample, or a portion thereof. The sample may be a sample for suspecting or confirming a disease or condition. The sample may be a sample removed from a subject using non-invasive, minimally invasive, or invasive techniques. The sample or a portion thereof can be obtained by tissue brushing, swabbing, tissue biopsy, tissue resection, fine needle aspiration, tissue washing, cytological preparation, surgical resection, or any combination thereof. The sample or a portion thereof can include tissue or cells from any tissue type. For example, the sample can include nasal tissue, tracheal tissue, lung tissue, pharyngeal tissue, laryngeal tissue, bronchial tissue, pleural tissue, alveolar tissue, breast tissue, bladder tissue, kidney tissue, liver tissue, colon tissue, thyroid tissue, cervical tissue, prostate tissue, heart tissue, muscle tissue, pancreatic tissue, anal tissue, bile duct tissue, bone tissue, brain tissue, spinal cord tissue, uterine tissue, ovarian tissue, endometrial tissue, vaginal tissue, vulva tissue, uterus tissue, stomach tissue, eye tissue, sinus tissue, penile tissue, salivary gland tissue, intestinal tissue, gallbladder tissue, gastrointestinal tissue, bladder tissue, brain tissue, spinal cord tissue, blood sample, or any combination thereof.

[0168] As used herein, the term "sequencing" can include bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, super-similarity signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, bombardment sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore sequencing, shotgun sequencing, RNA sequencing, Enigma sequencing, or any combination thereof.

[0169] The term "stop codon" refers to a consecutive sequence of three nucleotides in transfer RNA that signals the end of translation. Non-limiting examples include in RNA UAG, UAA, UGA, and in DNA TAG, TAA, or TGA. Unless otherwise specified, the term also includes nonsense mutations in DNA or RNA that introduce a premature stop codon, causing the resulting protein to be abnormally truncated. tRNAs corresponding to various stop codons are known by specific names: amber (UAG), ochre (UAA), and opal (UGA).

[0170] A "transfer ribonucleic acid" or "tRNA" is a nucleic acid molecule that aids in the translation of mRNA into protein. tRNAs have a unique folded structure containing three hairpin loops. One of these loops contains a "stem" portion that encodes an anticodon. The anticodon recognizes the corresponding codon on the mRNA. Each tRNA is "charged" with the amino acid corresponding to the mRNA codon. This "charging" is achieved by a tRNA synthetase. Once the codon corresponding to the anticodon is recognized by the tRNA, the tRNA transfers the charged amino acid to a growing amino acid chain to form a polypeptide or protein. Endogenous tRNAs can be charged by endogenous tRNA synthetases. Thus, endogenous tRNAs are typically charged with canonical amino acids. Orthogonal tRNAs derived from external sources require the corresponding orthogonal tRNA synthetases. Such orthogonal tRNAs can be charged with both canonical and non-canonical amino acids. In some embodiments, the amino acid to which the tRNA is charged can be detectably labeled to enable in vivo detection. Labeling techniques are known in the art and include, but are not limited to, click chemistry, in which an azide / alkyne containing unnatural amino acid is added by an orthogonal tRNA / synthetase pair and can therefore be detected using an alkyne / azide containing fluorophore or other such molecule.

[0171] As used herein, the terms "treat," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease, disorder, or condition or its signs or symptoms are completely or partially prevented, and / or the effect may be therapeutic, in that a disorder and / or adverse effects resulting from the disorder are partially or completely cured.

[0172] As used herein, the term "vector" can refer to a nucleic acid construct engineered for transmission between different hosts, including, but not limited to, a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. A "viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. In some embodiments, a plasmid vector can be prepared from a commercially available vector. In other embodiments, a viral vector can be produced from a baculovirus, a retrovirus, an adenovirus, an AAV, etc., according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, etc. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, the vector construct can refer to a polynucleotide comprising the retroviral genome or a portion thereof, and a gene of interest. Further details on the latest methods of vectors for use in gene transfer can be found, for example, in Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Perspectives in Biomedical Engineering Annual Review 17.Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In one embodiment, the promoter is a pol III promoter.

[0173] The pharmaceutical composition for administering adRNA can be conveniently presented in dosage unit form and can be prepared by any method known in the pharmaceutical field.For example, the pharmaceutical composition can be prepared by mixing the compound provided herein with a liquid carrier, a finely divided solid carrier, or both, and then optionally forming it into a desired dosage form.In the pharmaceutical composition, the compound provided herein is contained in an amount sufficient to achieve the desired therapeutic effect.For example, the pharmaceutical composition of the technology can be in a form suitable for virtually any form of administration, including, for example, a form suitable for topical, ophthalmic, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal administration, or for inhalation or insufflation administration.

[0174] For topical administration, the compounds may be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art.

[0175] Formulations for systemic administration include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0176] Useful injectable preparations include sterile suspensions, solutions, or emulsions of compounds provided in aqueous or oily media.The composition may also contain compounding agents such as suspending agents, stabilizers, and / or dispersing agents.Injectable preparations may be presented in unit dosage form, for example, ampoules, or in multi-dose containers.They may also contain added preservatives.

[0177] Alternatively, injectable formulations can be provided in powder form for reconstitution with a suitable vehicle before use. Such vehicles include, but are not limited to, sterile pyrogen-free water, buffer solution, and dextrose solution. For this purpose, the compounds provided herein can be dried by any known technique, such as lyophilization, and reconstituted before use.

[0178] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0179] For oral administration, the pharmaceutical compositions can take the form of lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art, for example, with sugar coatings, films, or enteric coatings.

[0180] Compositions for oral use can be prepared by any method known in the art for preparing pharmaceutical compositions. To provide an elegant and palatable preparation, such compositions can contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives. Tablets contain the compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents (e.g., cornstarch or alginic acid); binders (e.g., starch, gelatin, or acacia); and lubricants (e.g., magnesium stearate, stearic acid, or talc). The tablets can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated by techniques well known to those skilled in the art. The pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions.

[0181] Liquid preparations for oral administration can take the form of, for example, elixirs, solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared using, for example, suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore); TM or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring agents, coloring agents, and sweetening agents as appropriate.

[0182] "Administration" can be in a single dose, continuous or intermittent administration throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those of skill in the art and may vary depending on the therapeutic composition, the therapeutic objectives, the target cells being treated, and the subject being treated. Single or repeated administration can be carried out, with the dosage level and pattern selected by the attending physician. Suitable dosage formulations and methods for administering agents are known in the art. The route of administration can also be determined. Methods for determining the most effective route of administration are also known to those of skill in the art and may vary depending on the therapeutic composition, the therapeutic objectives, the health or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical administration.

[0183] Administration can refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological action (e.g., DNA construct, viral vector, etc.). These methods can include topical administration (e.g., lotion, cream, ointment, etc.) on an external surface such as the skin. These methods can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular, or infusion), oral administration, inhalation administration, intraductal administration, and rectal administration. In some instances, a subject may administer the composition without supervision. In some instances, a subject may administer the composition under the supervision of a medical professional (e.g., a doctor, nurse, physician's assistant, hospice worker, etc.). In some cases, a medical professional may administer the composition. In some cases, a cosmetic professional may administer the composition.

[0184] Administration or application of the compositions disclosed herein may result in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 days of treatment, which can be administered continuously or non-continuously. In some cases, the treatment period is about 1 to about 30 days, about 2 to about 30 days, about 3 to about 30 days, about 4 to about 30 days, about 5 to about 30 days, about 6 to about 30 days, about 7 to about 30 days, about 8 to about 30 days, about 9 to about 30 days, about 10 to about 30 days, about 11 to about 30 days, about 12 to about 30 days, about 13 to about 30 days, about 14 to about 30 days, about 15 to about 30 days, or about 16 to about 30 days. The period may be about 1 to about 30 days, about 16 to about 30 days, about 17 to about 30 days, about 18 to about 30 days, about 19 to about 30 days, about 20 to about 30 days, about 21 to about 30 days, about 22 to about 30 days, about 23 to about 30 days, about 24 to about 30 days, about 25 to about 30 days, about 26 to about 30 days, about 27 to about 30 days, about 28 to about 30 days, or about 29 to about 30 days.

[0185] Administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day. In some cases, administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some cases, the administration or application of a composition disclosed herein is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times.

[0186] In some cases, the compositions may be administered / applied as a single dose or in divided doses. In some cases, the compositions described herein may be administered at a first time point and a second time point. In some cases, the compositions may be administered with a time interval of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or more, with the first administration preceding the other administration.

[0187] The term "effective amount" can refer to an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition at the time of use, as well as individual subject characteristics such as general physical condition, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of immunotherapeutic compositions, in some embodiments, the effective amount is an amount sufficient to produce a protective response against a pathogen. In other embodiments, an effective amount of an immunotherapeutic composition is an amount sufficient to produce antibodies against an antigen. In some embodiments, the effective amount is an amount necessary to confer passive immunity to a subject in need thereof. With respect to immunotherapeutic compositions, in some embodiments, the effective amount may depend on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health status / immune system responsiveness of the subject, in addition to the factors described above. One of skill in the art can determine the appropriate amount depending on these and other factors.

[0188] When used in vitro, in some embodiments, the effective amount can depend on the size and nature of the application. It can also depend on the nature and sensitivity of the in vitro target and method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount can, depending on the embodiment, include one or more administrations of the composition.

[0189] Unless expressly stated and unless otherwise intended, when this disclosure refers to a polypeptide, protein, polynucleotide, or antibody, their equivalents or biological equivalents are intended within the scope of this disclosure. As used herein, the term "biological equivalent thereof," when referring to a reference protein, antibody, polypeptide, or nucleic acid, is intended to be synonymous with "equivalent thereof," and is intended to be synonymous with "equivalent thereof," and to be intended to be one that has minimal homology while still maintaining the desired structure or function. Unless specifically stated herein, any polynucleotide, polypeptide, or protein referred to herein is also intended to encompass their equivalents. For example, an equivalent is intended to be at least about 70% homology or identity, or at least 80% homology or identity, or at least about 85%, or at least about 90%, or alternatively at least about 95%, or alternatively 98% homology or identity, and exhibits substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes to the reference polynucleotide or its complement under stringent conditions.

[0190] The present disclosure provides polypeptide and / or polynucleotide sequences for use in the gene and protein editing techniques described below. It should be understood that the sequences provided herein may be used to provide expression products similar to substantially identical sequences that produce proteins with the same biological properties. These "biologically equivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides as described herein. When compared using sequence identity methods performed under default conditions, they can have an amino acid primary sequence that is at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical to a reference polypeptide. Specific polypeptide sequences are provided as examples of specific embodiments. Modifications to the amino acid sequence alter amino acids with similar charges. Furthermore, an equivalent polynucleotide is one that hybridizes under stringent conditions to a reference polynucleotide or its complement, or, with respect to polypeptides, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference encoding polynucleotide or its complement. Alternatively, an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.

[0191] A "pharmaceutical composition" typically contemplates a combination of an active agent (e.g., an adRNA of the present disclosure), compound, or composition, and a natural or non-natural carrier, an inert substance (e.g., a detectable agent or label), or an active substance (e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc.), and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical additives and additives such as proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), alone or in combination, in amounts ranging from 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acids / antibody components that also function in a buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also contemplated within the scope of the present technology, examples of which include, but are not limited to, monosaccharides such as lucetose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0192] Compositions used in accordance with the present disclosure, including cells, treatments, therapies, agents, drugs, and pharmaceutical preparations, may be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" can refer to a physically discrete unit suitable for use in a subject. Each unit contains a predetermined quantity of the composition calculated to produce a desired response in association with such administration (e.g., a suitable route and regimen). The amount administered depends on the desired outcome and / or protection, depending on the number of treatments and unit dose. The precise amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the subject's physical and clinical condition, the route of administration, the intended therapeutic goal (relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular composition. Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solution types described herein.

[0193] As used herein, the term "reducing or eliminating expression and / or function" can refer to reducing or eliminating transcription of the polynucleotide into mRNA, or reducing or eliminating translation of the mRNA into a peptide, or reducing or eliminating function of the peptide, polypeptide, or protein. In a non-limiting example, transcription of the polynucleotide into mRNA is reduced to at least half of its normal level found in a wild-type cell.

[0194] The terms "first-line," "second-line," or "third-line" can refer to the order of treatments a patient receives. A first-line therapy regimen is the treatment given first, while a second- or third-line therapy is given after a first- or second-line therapy, respectively. The National Cancer Institute defines first-line therapy as "the first treatment for a disease or condition." In cancer patients, the first-line treatment may be surgery, chemotherapy, radiation therapy, or a combination of these treatments. First-line therapy is also referred to by those skilled in the art as "primary therapy and primary treatment." See the National Cancer Institute's website, cancer.gov, as of November 15, 2017. Typically, a patient is given a subsequent chemotherapy regimen because they have not shown a positive clinical or paraclinical response to the first-line therapy or because the first-line therapy has been discontinued.

[0195] The term "contacting" refers to a direct or indirect binding or interaction between two or more entities. A particular example of a direct interaction is binding. A particular example of an indirect interaction is when one entity acts on an intermediate molecule, which in turn acts on a second, referenced entity. As used herein, contacting includes contacting in solution, in solid phase, in vitro, ex vivo, in vivo, in a cell, and in vivo. In vivo contacting can be referred to as administering or administration.

[0196] "Cryoprotectants" are known in the art and include, but are not limited to, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are commonly used.

[0197] Disclosed herein are adRNAs for site-specific RNA editing in the absence of overexpression of ADAR enzymes. Also provided herein are A to G engineered editing of DNA. Also provided herein are screening for ADAR2 mutants that enable site-specific C to T editing of RNA and DNA. Also provided herein are C to T engineered editing of RNA via the use of APOBEC expressed with ACF.

[0198] Compared with other ADAR2 systems, this disclosure is unique because it presents a novel method for recruiting endogenous ADAR to catalyze therapeutic RNA editing.Also, prior art systems do not provide a means for using ADAR enzymes for C>T gene editing.Finally, they do not disclose the use of APOBEC for programmable site-specific RNA editing.

[0199] Exemplary adRNA disclosed herein comprises an RNA targeting domain complementary to target RNA and one or more ADAR recruiting domains.When bound to its target, adRNA can recruit ADAR enzyme to target RNA.Then, this ADAR enzyme can catalyze the conversion of target adenosine to inosine.Without being bound by theory, it is believed that adRNA can also be used to recruit one of ADAR2 variants or APOBEC1 to affect C->T RNA editing.

[0200] Also, as disclosed herein, both in vitro and in vivo experiments have been performed using engineered adRNAs to recruit endogenous ADAR enzymes, and experiments demonstrating the efficiency of C to T editing of ADAR mutants and APOBEC1 / ACF constructs have also been disclosed.

[0201] As described herein, the viral vector can include a nucleic acid sequence encoding at least one RNA editing entity recruiting domain. In some cases, the nucleic acid sequence can encode two or more RNA editing entity recruiting domains, such as two, three, four, or more. The RNA editing entity recruiting domain can include at least about 80% sequence identity with at least one of an Alu domain, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruiting domain, a Cas13 domain, a GluR2 domain, or any combination thereof. The recruitment domain can include one or more GluR2 domains, one or more Alu domains, one or more APOBEC domains, or any combination thereof. The recruitment domain can include two or more GluR2 domains, two or more Alu domains, two or more APOBEC domains, a Cas13 domain, or any combination thereof. The recruitment domain can exclude an Alu domain. The recruitment domain can exclude a GluR2 domain. The recruitment domain may not include an APOBEC domain. The recruitment domain may not include a Cas13 domain.

[0202] The APOBEC recruiting domain can include an APOBEC1 recruiting domain, an APOBEC2 recruiting domain, an APOBEC3A recruiting domain, an APOBEC3B recruiting domain, an APOBEC3C recruiting domain, an APOBEC3D recruiting domain, an APOBEC3E recruiting domain, an APOBEC3F recruiting domain, an APOBEC3G recruiting domain, an APOBEC3H recruiting domain, an APOBEC4 recruiting domain, a derivative of any of these, or any combination thereof.

[0203] The recruitment domain can comprise at least about 80% sequence identity to any one of the Alu domains described herein, hi some cases, the recruitment domain can comprise at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of the Alu domains described herein.

[0204] The recruitment domain can comprise at least about 80% sequence identity with any one of the APOBEC domains described herein, hi some cases, the recruitment domain can comprise at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of the APOBEC domains described herein.

[0205] The recruitment domain can comprise at least about 80% sequence identity to any one of the GluR2 domains described herein, hi some cases, the recruitment domain can comprise at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of the GluR2 domains described herein.

[0206] The recruitment domain can comprise at least about 80% sequence identity to any one of the Cas13 domains described herein. In some cases, the recruitment domain can comprise at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of the Cas13 domains described herein.

[0207] The nucleic acid sequence can encode at least one, two, three, four, or five RNA editing recruitment domains. The nucleic acid sequence can encode at least two RNA editing recruitment domains, one of which is an Alu domain. The nucleic acid sequence can encode at least two RNA editing recruitment domains, one of which is an APOBEC domain. The nucleic acid sequence can encode at least two RNA editing recruitment domains, one of which is a GluR2 domain. The nucleic acid sequence can encode at least two RNA editing recruitment domains, one of which is a Cas13 domain.

[0208] The recruitment domain can comprise one or more stem-loop structures. The recruitment domain can comprise at least two stem-loop structures. The recruitment domain can comprise at least three stem-loop structures. The recruitment domain can also comprise no stem-loop structures. The recruitment domain comprising at least one stem-loop structure can be an Alu domain, an APOBEC domain, a GluR2 domain, a Cas13 domain, or any combination thereof.

[0209] At least a portion of the recruitment domain may be single-stranded. In some cases, the Alu domain may be at least partially single-stranded. In some cases, the APOBEC domain may be at least partially single-stranded. In some cases, the GluR2 domain may be at least partially single-stranded. In some cases, the Cas13 domain may be at least partially single-stranded.

[0210] The recruitment domain can comprise multiple repeats. The recruitment domain can comprise multiple Alu repeats.

[0211] In some cases, the viral vector can include one or more RNA editing recruiting domains. In some cases, the viral vector can include two or more RNA editing recruiting domains. In some cases, the viral vector can include two, three, four, five, or more RNA editing recruiting domains. The nucleic acid sequence can encode one or more RNA editing recruiting domains. The nucleic acid sequence can encode two or more RNA editing recruiting domains. The nucleic acid sequence can encode two, three, four, five, or more RNA editing recruiting domains. The nucleic acid sequence can encode at least one Alu domain and one GluR2 domain. The nucleic acid sequence can encode at least one Alu domain and one Cas13 domain. The nucleic acid sequence can encode at least one Alu domain and one APOBEC domain. The nucleic acid sequence can encode at least one GluR2 domain and one APOBEC domain. The nucleic acid sequence can encode at least one GluR2 domain and one Cas13 domain. The nucleic acid sequence can encode at least one Cas13 domain and one APOBEC domain.

[0212] The nucleic acid sequence can encode a target RNA that may be complementary to at least a portion of the target RNA. This may be complementary to at least a portion of the target RNA. The length of the portion that may be complementary may be about 50 base pairs (bp) to about 200 bp. The length of the portion that may be complementary may be about 20 bp to about 100 bp. The length of the portion that may be complementary may be about 10 bp to about 50 bp. The length of the portion that may be complementary may be about 50 bp to about 300 bp. Modifying the length of the complementary portion can increase the efficiency of editing. In some cases, a longer length of the portion can increase the efficiency of editing compared to a shorter length.

[0213] The nucleic acid sequence can encode a domain that recruits at least one RNA editing entity and a nucleic acid sequence that encodes an RNA that is at least partially complementary to the target RNA and includes a flanking nucleic acid sequence at least about 200 bp in length. The flanking nucleic acid sequence can have a length of about 100 bp to about 300 bp. The flanking nucleic acid sequence can have a length of about 150 bp to about 400 bp. The flanking nucleic acid sequence can have a length of about 200 bp to about 500 bp. The flanking nucleic acid sequence can have a length of about 50 bp to about 300 bp. Modifying the length of the flanking nucleic acid sequence can increase the efficiency of editing. In some cases, a longer flanking sequence can increase the efficiency of editing compared to a shorter flanking sequence.

[0214] The nucleic acid can include a linker sequence, such as a linker sequence located between the targeting domain and the recruitment domain. In some cases, the nucleic acid can include a sequence such as 5'-X-(Y-X')nLZ-3', where X is complementary to the target RNA sequence downstream of a specific position, X' is complementary to the target RNA sequence upstream of a specific position, Y includes one or more nucleotides that may not be complementary to the target RNA sequence, n is an integer from 1 to 10, L can be a linker sequence containing any number of nucleotides (including zero), and Z can be a sequence recognized and bound by the RNA editing entity. L can also be composed of different chemical bonds, such as an (oligo)peptide bond or a PEG bond.

[0215] A nucleic acid can contain from 20 to several hundred nucleotides. In some cases, a longer targeting portion provides greater specificity for the target site of the RNA sequence to be edited, fewer off-target effects due to unintended (off-target) binding, and more space, mismatches, or wobble bases (due to mismatches with one or more complementary bases in the target RNA sequence at or near the site to be edited) to form secondary structures within the target portion itself, such as stem-loop structures, cruciforms, and toehold structures. In some cases, the targeting portion may be complementary to the target RNA sequence over the entire length of the targeting portion, except for the mismatch opposite the nucleotide to be edited and, optionally, one or two wobble bases.

[0216] Nucleic acids can be modified using a variety of chemical methods and modifications. In some cases, the regular internucleoside linkage between nucleotides can be modified by mono- or di-thiolation of the phosphodiester bond to generate phosphorothioate or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkage can include amidation or peptide linkers. The ribose sugar can be modified by substituting the 2'-O moiety with lower alkyl (C1-4, e.g., 2'-O-Me), alkenyl (C2-4), alkynyl (C2-4), methoxyethyl (2'-MOE), or other substituents. In some cases, the substituent 2'OH group can include a methyl, methoxyethyl, or 3,3'-dimethylallyl group. In some cases, locked nucleic acid sequences (LNAs) containing a 2'-4' intramolecular bridge (e.g., a methylene bridge between the 2' oxygen and the 4' carbon) within the ribose ring can be used. For example, purine and / or pyrimidine nucleobases can be modified by amination or deamination of the heterocycle to change their properties.

[0217] The viral vector may be an adeno-associated viral (AAV) vector. The AAV may be a recombinant AAV. The AAV may include an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, any derivative thereof, or any combination thereof. The AAV may be selected from the group consisting of an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, any derivative thereof, and any combination thereof. The viral vector may be a modified viral vector. The viral vector may be modified to include a protein. In some cases, the viral vector may include a modified VP1 protein.

[0218] The nucleic acid sequence encoding the RNA editing entity recruiting domain, the targeting domain, or a combination thereof can comprise a structure (e.g., a secondary structure) that may be substantially cruciform. The nucleic acid sequence can comprise at least two structures that may be substantially cruciform. The recruitment domain can comprise a structure that may be substantially cruciform. The recruitment domain can comprise at least two structures that may be substantially cruciform. The secondary structure of the nucleic acid sequence (e.g., the portion encoding the recruitment domain) can be modified to enhance ADAR recruitment or binding. The structural modification to enhance ADAR recruitment or binding can include the formation of a cruciform structure.

[0219] The domain that recruits the RNA editing entity can be disposed between at least two structures that may be substantially cruciform. The targeting domain can be disposed between at least two structures that may be substantially cruciform. The domain that recruits the RNA editing entity can be disposed adjacent to at least one structure that may be substantially cruciform. The targeting domain can be disposed adjacent to at least one structure that may be substantially cruciform.

[0220] The cruciform structure may comprise a stem loop adjacent to at least one pair of at least partially complementary strands of the cruciform structure. The cruciform structure may be substantially cruciform. The cruciform structure may comprise substantially one cruciform, for example, three of four stem loops, or two of four stem loops. One or more stem loops capable of forming a cruciform may comprise different lengths. One or more stem loops capable of forming a cruciform may comprise the same length. One or more stem loops capable of forming a cruciform may comprise one or more mismatched bulges.

[0221] The domain that recruits the RNA editing entity may comprise a structure that may be substantially toe-hold. The RNA editing entity may comprise one or more mismatch bulges. The RNA editing entity may not comprise a mismatch bulge. The domain that recruits the RNA editing entity may comprise a substantially toe-hold structure, a substantially cruciform structure, a substantially linear structure, a stem-loop structure, a double stem-loop structure, or a combination thereof.

[0222] The viral vector can comprise a nucleic acid sequence encoding an RNA with a two-dimensional shape. The two-dimensional shape can transmit superior ADAR recruitment or binding to RNA with a different two-dimensional shape. The sequence of the nucleic acid sequence encoding the RNA can be modified so that the RNA comprises a two-dimensional shape that transmits superior ADAR recruitment or binding. The two-dimensional shape can be substantially cross-shaped, toehold, stem-loop, or any combination thereof. The two-dimensional shape can comprise a substantially cross-shaped shape. The two-dimensional shape can comprise toehold. The two-dimensional shape can comprise stem-loop. The two-dimensional shape can be linear.

[0223] The RNA encoded by the nucleic acid sequence can include a first domain and a second domain. The first domain can include a cruciform structure, and the second domain can include a linear structure. The first and second domains can be directly or indirectly connected. The first domain can be a recruitment domain, and the second domain can be a targeting domain. The RNA can include a third domain. The third domain can be directly or indirectly connected to the first or second domain. The third domain can be a recruitment domain. The third domain can include a cruciform structure.

[0224] The RNA encoded by the nucleic acid sequence may be non-natural RNA. The RNA encoded by the nucleic acid sequence may contain at least one base or at least one sugar containing a chemical modification. The RNA may contain two or more chemical modifications. The chemical modification may increase the stability of the RNA, which may increase the half-life of the RNA's biological activity in vivo.

[0225] The nucleic acid may comprise one or more recruitment domains and one or more antisense domains. When the nucleic acid is contacted with an RNA editing entity and a target nucleic acid complementary to at least a portion of the antisense domain, it can modify at least one base pair of the target nucleic acid at least about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, or 6-fold more efficiently than a comparable nucleic acid complexed with a Cas13b protein or an active fragment thereof, as measured by sequencing (e.g., Sanger sequencing). The efficiency may be at least about 3-fold higher. The efficiency may be at least about 4-fold higher. The efficiency may be at least about 5-fold higher.

[0226] The nucleic acid may comprise one or more recruitment domains and one or more antisense domains. When the nucleic acid is contacted with an RNA editing entity and a target nucleic acid complementary to at least a portion of the antisense domain, it can modify at least one base pair of the target nucleic acid at least about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, or 6-fold more efficiently than a comparable nucleic acid complexed with a GluR2 protein or an active fragment thereof, as measured by sequencing (e.g., Sanger sequencing). The efficiency may be at least about 3-fold higher. The efficiency may be at least about 4-fold higher. The efficiency may be at least about 5-fold higher.

[0227] The nucleic acids described herein can provide greater editing efficiency than at least a portion of a naturally occurring recruitment domain, such as a GluR2 domain. The nucleic acids can provide greater editing efficiency than at least a portion of a modified recruitment domain, such as a modified GluR2 domain.

[0228] The target nucleic acid can include RNA. The RNA can be mRNA. The RNA can encode a protein or a portion thereof. Malfunction of the protein or a portion thereof can be associated with a disease or condition. Administration of the compositions, vectors, nucleic acids, and non-naturally occurring RNA described herein can treat, eliminate, cure, or alleviate one or more symptoms of a disease or condition.

[0229] The disease or condition can include a neurodegenerative disease, a muscle disorder, a metabolic disorder, an eye disorder, or any combination thereof. The disease or condition can include cystic fibrosis, albinism, alpha-1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, beta-thalassaemia, Cadasil syndrome, Charcot-Marie dental disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, epidermolysis bullosa dystrophica, epidermolysis bullosa, Fabry disease, V Leiden-related disorders, familial adenomatosis, polyposis, lactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-e The diseases or conditions may include sol-associated cancer, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary hair disease, prothrombin mutation-related disorders (e.g., prothrombin G20210A mutation), pulmonary hypertension, retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, various forms of cancer (e.g., BRCA1 and 2-associated breast and ovarian cancer). The diseases or conditions may include muscular dystrophy, ornithine transcarbamylase deficiency, retinitis pigmentosa, breast cancer, ovarian cancer, Alzheimer's disease, pain, Stargardt macular dystrophy, Charcot-Marie dental disease, Rett syndrome, or any combination thereof.Administration of the composition may be sufficient to (a) reduce expression of the gene relative to expression of the gene prior to administration, (b) edit at least one point mutation in a subject, such as a subject in need thereof, (c) edit at least one stop codon in the subject to produce stop codon readthrough, (d) produce exon skipping in the subject, or (e) any combination thereof.

[0230] The pharmaceutical composition may comprise a first active ingredient. The first active ingredient may comprise a viral vector described herein, a non-naturally occurring RNA described herein, or a nucleic acid described herein. The pharmaceutical composition may be formulated in a unit dose form. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition may comprise a second, third, or fourth active ingredient.

[0231] The compositions described herein can include an excipient. The excipient can be added to the stem cells or co-isolated with the stem cells from their source. The excipient can include a cryopreservative, such as DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. The excipient can include a cryopreservative, such as sucrose, trehalose, starch, a salt of any of these, a derivative of any of these, or any combination thereof. The excipient can include a pH agent (to minimize oxidation or degradation of the components of the composition), a stabilizer (to prevent denaturation or degradation of the components of the composition), a buffer (to improve temperature stability), a solubilizer (to increase protein solubility), or any combination thereof. The excipient can include a surfactant, sugar, amino acid, antioxidant, salt, non-ionic surfactant, solubilizer, neutral fat, alcohol, or any combination thereof. The excipient may include sodium carbonate, acetate, citrate, phosphate, polyethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate ester, HCl, sodium edetate, lecithin, glycerin, xanthan gum, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. The excipient may be an excipient listed in the Pharmaceutical Excipients Handbook, American Pharmaceutical Association (1986).

[0232] Non-limiting examples of suitable excipients can include buffering agents, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and coloring agents.

[0233] In some cases, the excipient may be a buffer. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium carbonate. Buffers include sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, and potassium polyphosphate. Sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts, or combinations thereof, may be used in pharmaceutical formulations.

[0234] In some cases, the excipient may contain a preservative. Non-limiting examples of suitable preservatives include antioxidants such as α-tocopherol and ascorbate, and antibacterial agents such as parabens, chlorobutanol, and phenol. Antioxidants may also include, but are not limited to, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some examples, the preservative can include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antibacterial agents, oxidase inhibitors, or other inhibitors.

[0235] In some cases, the pharmaceutical formulation may include a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, sugar, oligosaccharide, and combinations thereof.

[0236] Binders that can be used in the pharmaceutical formulations can be selected from starches such as potato starch, corn starch, wheat starch, etc.; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin, etc.; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, etc.; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, and water, or combinations thereof.

[0237] In some cases, pharmaceutical formulations can include lubricants as excipients. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, steroid tex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. Lubricants that can be used in pharmaceutical formulations can be selected from metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfates (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0238] In some cases, pharmaceutical formulations can include dispersion enhancers as excipients. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as a high HLB emulsifier surfactant.

[0239] In some cases, the pharmaceutical formulation may include a disintegrant as an excipient. In some cases, the disintegrant may be a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums, such as agar, gaur, locust bean, karaya, pectin, and tragacanth. In some cases, the disintegrant may be an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0240] In some cases, the excipient may include a flavoring agent. The flavoring agent incorporated in the outer layer may be selected from synthetic flavor oils and flavoring aromatics, natural oils, extracts from plants, leaves, flowers, and fruits, and combinations thereof. In some cases, the flavoring agent may be selected from the group consisting of cinnamon oil, winggreen oil, peppermint oil, clover oil, hay oil, anise oil, eucalyptus, vanilla, citrus oils such as lemon oil, orange oil, grape and grapefruit oil, and fruit extracts including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

[0241] In some cases, the excipient can contain a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as sodium salt, dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (stevioside); sucrose chloroderivatives such as sucralose; and sugar alcohols such as sorbitol, mannitol, and sucralitol.

[0242] In one embodiment, the present disclosure discloses that adRNA recruits endogenous ADAR enzymes to target mRNAs and facilitates site-specific A-to-G editing. This has potential applications in gene therapy, where delivery of a single adRNA can potentially correct G-to-A point mutations. This also allows target RNA editing without overexpressing RNA-editing enzymes such as ADAR2. The present disclosure demonstrates the applicability of this technology both in vitro and in vivo. The present disclosure also demonstrates that generating long double-stranded RNAs can recruit endogenous ADARs even in the absence of an ADAR-recruiting domain. These engineered adRNAs can also be used to generate multiple A-to-G edits in mRNAs at target regions. In one embodiment, the system uses adRNAs transcribed with the U6 promoter (pol III) as well as chemically synthesized adRNAs, and has been shown to be efficient at RNA editing. Thus, in one embodiment, the construct further includes a promoter, such as a pol III promoter, to transcribe the adRNA. Transcription from a promoter such as a polIII promoter can improve the efficiency of target RNA editing.The Alu transcript from a polIII promoter is preferentially edited.Also provided herein is an engineered adRNA from the structure of the Alu repeat that is the target of endogenous ADAR.

[0243] The constructs of the present disclosure can be used to localize adRNA to specific cellular compartments. For example, adRNA-snRNA fusions can be used for nuclear localization. Similarly, adding an N-terminal mitochondrial targeting sequence (MTS) can localize adRNA to mitochondria. Thus, in one embodiment, the construct further comprises an N-terminal mitochondrial targeting sequence (MTS). Adding an appropriate cis-acting zipper code can localize adRNA to peroxisomes, endosomes, and exosomes. Thus, in a further embodiment, the construct further comprises an appropriate cis-acting zipper code for localizing adRNA to peroxisomes, endosomes, and exosomes. Localization of adRNA to endosomes may allow them to be transported over long distances in neurons. Localization in exosomes may potentially facilitate the spread of adRNA to neighboring cells. Tethering moieties, such as cholesterol, to adRNA can aid cellular uptake. Thus, in one embodiment, the construct further comprises a targeting moiety, such as cholesterol.

[0244] In one embodiment, as shown in the present disclosure, adRNA-aptamers, such as aptamers that bind flavin mononucleotide, guanine, and other natural metabolites, are used to generate small molecule-regulatable adRNAs, allowing for temporal control of RNA editing. Sugar-binding aptamers can also be used for this purpose.

[0245] In one embodiment, the production of the UlA-ADAR fusion is entirely human in origin. The N-terminal RNA recognition motif of the splicesomal U1A protein binds to its cognate Ul hairpin II RNA with a dissociation constant of 63 nm.

[0246] The present disclosure also provides a construct further comprising a toe hold.

[0247] The constructs of the present disclosure can, in one embodiment, be used in the absence of overexpression of an ADAR enzyme.

[0248] Thus, in certain embodiments, the adRNAs of the present disclosure have specific components: an RNA targeting domain of about 15 to about 200 base pairs in length (and ranges therebetween) that is complementary to the target RNA; 0 to 10 ADAR-recruiting domains, which may be derived from GluR2 mRNA, Alu repeat elements, or other RNA motifs to which ADARs bind; and a cytosine mismatch required to guide ADARs to the target adenosine, which may be present anywhere within the targeting domain. When the adRNA binds to its target RNA, it recruits an ADAR enzyme to the target RNA. The ADAR enzyme can then catalyze the conversion of the target adenosine to inosine. For adRNAs longer than 50 base pairs, when expressed in HEK293T and HeLa cells, the adRNA can recruit ADARs, even in the absence of an ADAR-recruiting domain, enabling significant levels of target RNA editing. A single adRNA can also be used to produce multiple A-to-G edits in the target mRNA. In addition, by using multiple adRNAs, multiple different adRNAs can be edited in the same cell.For example, in the mdx mouse model of Duchenne muscular dystrophy, not only can dystrophin mutation be corrected, but also the mRNA sequence of the gene encoding the protein involved in nonsense-mediated decay can be destroyed.In another application, this technology can be used to obtain loss of function, function or dominant negative mutation.In one embodiment, this technology can be used for cancer screening, tumor progression and immune editing research. Engineered adRNA

[0249] Provided herein are engineered ADAR1 or ADAR2 guide RNAs ("adRNAs") that comprise, consist essentially of, or further consist of a sequence complementary to a target RNA. In one particular embodiment, the engineered adRNA of the present disclosure further comprises, consists essentially of, or further consists of a sequence complementary to ornithine transcarbamylase.

[0250] In one embodiment, the engineered adRNA of the present disclosure further comprises, consists essentially of, or consists of an ADAR2-recruiting domain derived from GluR2 mRNA. In another embodiment, the engineered adRNA of the present disclosure further comprises, consists essentially of, or consists of an ADAR1-recruiting domain derived from an Alu repeat. In a further embodiment, the engineered adRNA of the present disclosure further comprises, consists essentially of, or consists of two MS2 hairpins flanking the sequence complementary to the target RNA. In some embodiments, the sequence complementary to the target RNA in the engineered adRNA of the disclosure comprises, consists essentially of, or consists of about 15-30 base pairs, or about 30-45 base pairs, or about 45-60 base pairs, or about 60-75 base pairs, or about 75-90 base pairs, or about 90-105 base pairs, or about 105-120 base pairs, or about 120-135 base pairs, or about 135-150 base pairs, or about 150-165 base pairs, or about 165-180 base pairs, or about 180-200 base pairs. In further embodiments, it is from about 40 to about 200, or from about 50 to about 200, or from about 60 to about 200, or from about 70 to about 200, or from about 80 to about 200, or from about 90 to about 200, or from about 100 to about 200 base pairs.

[0251] Disclosed herein are engineered adRNAs that comprise, consist essentially of, or further consist of no ADAR-recruiting domain, or about 1-2 ADAR-recruiting domains, or about 2-3 ADAR-recruiting domains, or about 3-4 ADAR-recruiting domains, or about 4-5 ADAR-recruiting domains, or about 5-6 ADAR-recruiting domains, or about 6-7 ADAR-recruiting domains, or about 7-8 ADAR-recruiting domains, or about 8-9 ADAR-recruiting domains, or about 9-10 ADAR-recruiting domains. In some embodiments, the ADAR-recruiting domain comprises, consists essentially of, or further consists of GluR2 mRNA, Alu repeat elements, or other RNA motifs to which ADARs bind. Also provided herein are engineered adRNAs in which the ADAR2-recruiting domain of the engineered adRNA derived from GluR2 mRNA is located at the 5'-end or 3'-end of the engineered adRNA. In some embodiments, the GluR2 mRNA is located at both the 5'-end and the 3'-end of the engineered adRNA.

[0252] In one embodiment, the engineered adRNA of the present disclosure further comprises, essentially consists of, or alternatively comprises an editing induction element. "Editing induction element" can refer to the large double-stranded RNA structure required for efficient RNA editing. Non-limiting examples of editing induction elements are described in Daniel, C. et al. (2017) Genome Biol. 18, 195.

[0253] In one particular embodiment, the engineered adRNA of the present disclosure is encoded by a polynucleotide sequence selected from the group of sequences shown in Table 1 or Figure 2, or its equivalent.

[0254] Also disclosed herein are complexes comprising, consisting essentially of, or further consisting of the engineered adRNAs of the present disclosure that are hybridized with complementary polynucleotides under high stringency conditions.

[0255] The present disclosure also provides polypeptide and / or polynucleotide sequences for use in the gene and protein editing techniques described below. It is understood, although not necessarily explicitly stated, that the sequences provided herein can be used to provide expression products, as well as substantially identical sequences, that produce proteins with the same biological properties. These "biologically equivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides, as described herein. They can have primary amino acid sequences that are at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical to a reference polypeptide when compared using sequence identity methods performed under default conditions. Specific polypeptide sequences are provided as examples of specific embodiments. Modifications to the amino acid sequence utilize alternative amino acids with similar charges. Alternatively, an equivalent polynucleotide is one that hybridizes under stringent conditions to a reference polynucleotide or its complement, or, with respect to polypeptides, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference encoding polynucleotide or its complementary strand, or alternatively, an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.

[0256] Also disclosed herein are engineered adRNA-snRNA (small nuclear RNA) fusions. In one embodiment, the engineered adRNA further comprises, consists essentially of, or consists of an N-terminal mitochondrial targeting sequence (MTS), facilitating localization of the engineered adRNA to mitochondria. In another embodiment, the present specification provides engineered constructs further comprising, consists essentially of, or consists of a cis-acting ZIP code to promote localization of the engineered adRNA to peroxisomes, endosomes, and exosomes. Localization of adRNAs in endosomes can potentially enable their transport over long distances in neurons. Localization in exosomes can potentially aid in the propagation of adRNAs to neighboring cells. Tethering moieties, such as cholesterol, to adRNAs can aid cellular uptake.

[0257] Furthermore, the present specification provides engineered adRNAs that can be regulated by small molecules. In one embodiment, the present specification discloses engineered adRNA-aptamer fusions. Non-limiting examples of aptamers that can be used for this purpose include aptamers that bind to flavin mononucleotide, guanine, other natural metabolites, or sugars. "Aptamer" can refer to short, single-stranded oligonucleotides that can bind various molecules with high affinity and specificity. Non-limiting examples of aptamers are described in Lakhin, AV et al. (2013), Acta, naturae, 5(4), 34-43.

[0258] Also disclosed herein is a fully human UlA-ADAR fusion, an N-terminal RNA recognition motif of the spliceosomal U1A protein that binds to its cognate Ul hairpin II RNA with a dissociation constant of 63 nM. Vectors and recombinant cells expressing genetically engineered adRNA

[0259] Provided herein is a vector comprising, consisting essentially of, or further consisting of one or more isolated polynucleotide sequences encoding the genetically engineered adRNA of the present disclosure, and optionally a regulatory sequence operably linked to the isolated polynucleotide. Non-limiting examples of vectors include plasmids or viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors. The vector may further comprise a targeting sequence, a ZIP code, or a toehold, as known in the art.

[0260] In one aspect, the regulatory sequence comprises, consists essentially of, or further consists of a promoter, a transcriptional enhancer, and / or a reporter. In some embodiments, the promoter is a human U6 promoter, a mouse U6 promoter, a CMV promoter, a pol III promoter, or a pol II promoter. In one aspect, the vector further comprises, consists essentially of, or further consists of a detectable or purification marker.

[0261] Further disclosed herein are recombinant cells comprising, consisting essentially of, or further consisting of the above-described vectors, wherein the engineered adRNA is recombinantly expressed. Composition of engineered adRNA

[0262] Disclosed herein are compositions comprising, consisting essentially of, or consisting of a carrier and one or more of the engineered adRNAs of the present disclosure, an isolated polynucleotide encoding the engineered adRNA of the present disclosure, a vector expressing the engineered adRNA of the present disclosure, or a recombinant cell expressing the engineered adRNA of the present disclosure. In one embodiment, the carrier is a pharmaceutically acceptable carrier or solid support. In a further embodiment, the composition further comprises, consists essentially of, or consists of a chemotherapeutic agent or drug. How to Use Engineered adRNA

[0263] Provided herein are methods for altering protein expression, the methods comprising, consisting essentially of, or further consisting of contacting a polynucleotide encoding a protein whose expression is to be altered with an engineered adRNA of the present disclosure.

[0264] Also provided herein are methods for treating diseases or disorders associated with abnormal protein expression, comprising, consisting essentially of, or further consisting of, administering to a subject in need of such treatment an effective amount of one or more of the genetically engineered adRNAs of the present disclosure. In one particular embodiment, provided herein are methods for treating Duchenne muscular dystrophy, comprising, consisting essentially of, or further consisting of, administering to a subject in need of such treatment an effective amount of one or more of the genetically engineered adRNAs of the present disclosure.

[0265] For in vitro applications, in some embodiments, the effective amount can depend on the size and nature of the application. It can also depend on the nature and sensitivity of the in vitro target and method of use. Those skilled in the art can determine the effective amount based on these and other considerations. The effective amount can include one or more administrations of the composition according to this embodiment.

[0266] The terms "subject," "host," "individual," and "patient" are used interchangeably herein and refer to an animal, typically a mammal. Any suitable mammal can be treated with the methods, cells, or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., adult, teenager, child, infant, or mammal in utero). The mammal may be male or female. The mammal may be a pregnant female. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having cancer or a neoplastic disorder. In other embodiments, the subject has or is suspected of having a disease or disorder associated with aberrant protein expression.

[0267] Referring to Figure 13, in contrast to antisense oligonucleotide (AON) designs (left side of Figure 13) that can include short ssRNAs (e.g., about 35 nt in length), exemplary constructs of the present disclosure (right side of Figure 13) can have lengths of about 60 bp to about 100 bp, providing excellent target specificity and long ssRNAs with total lengths of about 150 nt to about 250 nt. The constructs described herein can include optimized and true hairpin structures, as opposed to mismatched bases that may be added to produce hairpin-like RNA bulges for AON designs, as shown on the left side of Figure 13. Advantages of the true hairpin design of the constructs include optimal and superior ADAR recruitment efficiency and higher target editing yields compared to AON constructs. The hairpin design can be completely independent of the mRNA target sequence and can be easily deployed to any new mRNA target. The target site for deamination is unique and precise, requiring no undesired chemical modifications and eliminating the risk of undesired deamination at other sites. In contrast, AON designs utilizing mismatched hairpin-like RNA bulges (e.g., as shown on the left side of Figure 13) (a) have limited efficacy in recruiting ADARs (they are not true hairpins and are too short), (b) prevent sequence and bulge reuse (requiring unique design for each new target mRNA), (c) reduce specificity for cellular mRNA target sites due to the mismatch (increasing the risk of target damage), and (d) require 2'OMe modifications in the bulge to protect other adenylines from ADAR deamination activity (which can lead to undesired mutations at the incorrect ribonucleotide).

[0268] Recruitment of exogenous and endogenous ADARs via long antisense-adRNA

[0269] Although the CRISPR / Cas9 system has been widely used in research, there are concerns regarding its in vivo application. Two major concerns are the permanent edits made to the genome and the potential immune response that may result from introducing a bacterially derived system. As a result, RNA editing has emerged as a potential solution to both challenges. The first challenge can be easily overcome by applying RNA editing; the RNA transcribed from the gene can be transient, and the edits will not permanently alter the cell. However, this can create new problems in the form of reduced editing efficiency, as many RNAs can be edited to achieve a phenotypic effect. Furthermore, the second challenge of immune response may not necessarily be overcome. The ADAR family of RNA-editing enzymes originates from humans. In particular, ADAR1 is nearly unidirectionally expressed in many cell types; by exploiting its natural preference for RNA A→I (A→G) editing, many potential therapeutic applications are possible. This method also overcomes a major delivery hurdle, as small guide RNAs are easier to transport than large, bulky enzymes. Thus, the objective of the methods and compositions described herein can include engineering guides (called adRNAs) to recruit endogenous ADARs. ADARs may prefer to edit regions of double-stranded RNA, particularly sequences close to those with secondary structure. This can favor engineering guides with longer antisense regions. AdRNAs can have three components: (1) an RNA targeting domain (e.g., about 15 to about 200 base pairs in length) that may be complementary to the target RNA; (2) about 0 to about 10 ADAR-recruiting domains (which may be derived from GluR2 mRNA, Alu repeat elements, or other RNA motifs) to which ADARs bind; and (3) cytosine mismatches that may be required to guide ADARs to the target adenosine, which may be present anywhere within the targeting domain.

[0270] When this adRNA binds to its target RNA, it can recruit ADAR enzymes to the target RNA. The ADAR enzymes can catalyze the conversion of target adenosine to inosine. Interestingly, when expressed in HEK 293T and HeLa cells, long adRNAs longer than 50 base pairs can recruit ADARs even without the presence of an ADAR-recruiting domain, enabling significant levels of target RNA editing. A single adRNA can also be used to produce multiple A-to-G edits in target mRNA. Furthermore, by utilizing multiple adRNAs, multiple different mRNAs can be edited in the same cell. For example, in the mdx mouse model of Duchenne muscular dystrophy, not only can dystrophin mutations be corrected, but also the mRNA sequence of a gene encoding a protein involved in nonsense-mediated decay can be disrupted. Other applications include the use of this technology to produce loss-of-function, gain-of-function, dominant-negative mutations, or any combination thereof. This is extremely important in cancer screening, tumor progression, and immunoediting research.

[0271] Referring to Figure 16, a schematic of RNA editing by recruiting endogenous ADARs in the presence of adRNA is shown. These adRNAs can be delivered as either chemically modified RNA or U6-transcribed RNA. Referring to Figure 17, U6 promoter-transcribed adRNAs with progressively longer antisense domain lengths, combined with zero, one, or two GluR2 domains, are evaluated for their ability to induce target RNA editing with or without exogenous ADAR2 expression. Values represent mean + / - SEM (n=3). Longer adRNAs can recruit endogenous ADARs for RNA editing. Referring to Figure 18, chemically synthesized versions of adRNAs are tested against a panel of mRNAs with or without exogenous ADAR2 expression. The exact chemical modifications are noted in the figure, along with the source of the adRNA. Values represent mean + / - SEM (n=3). Referring to Figure 19, treated adult spf ashIn vivo RNA correction efficiency of correctly spliced OTC mRNA in the liver of mice (retro-orbital injection). Mice injected with U6-transcribed short adRNAs had an RNA editing level of 0.6%.

[0272] Alu-adRNA-mediated recruitment of exogenous and endogenous ADARs

[0273] Alu genes are transposable elements in the genome and natural targets for RNA editing by ADARs. To enable editing by endogenously recruited ADARs, adRNAs can be designed based on the Alu element structure. Various positions on the Alu element structure can be tested, where the native sequence can be replaced with an antisense sequence complementary to the target. A single-stranded linker region can be selected, as shown in Figure 20. The length of the antisense guide can be optimized, varying from about 20 to about 100 bases to target the RAB7A locus. Each guide can be designed to contain one or more mismatches. A mismatch can be placed between the antisense cytosine and the target adenosine base to be edited. The mismatch can be placed midway along each antisense length. Editing efficiency can be compared between a 100-50 antisense guide, which can recruit both ADAR1 and ADAR2, and a GluR2 20-6 guide, which can recruit only ADAR2.

[0274] Referring to Figure 20, the design of Alu adRNAs is shown. Left: Structure of the Alu element. Center: Design incorporating a locus-specific antisense sequence with a C mismatch opposite the target A. Right: Recruitment of the RNA-editing enzyme ADAR to the target.

[0275] These guides were tested in 293FT cells by transfection using Lipofectamine. Each guide was tested for its ability to recruit endogenous ADARs in cells overexpressing either ADARlpl10, ADARlpl50, or ADAR2, or in cells expressing no enzymes at all. 48 hours after transfection, cells were harvested, RNA was extracted, converted to cDNA, and the RAB7A locus was amplified for Sanger sequencing. Editing efficiency was calculated as the peak height ratio. Figure 5 shows the results of the Alu guide length experiment.

[0276] Referring to Figure 5, the long Alu-v2-l00-50 guide demonstrates improved editing in cells where the ADAR enzyme is not overexpressed. Overexpression of ADARlpl50 results in significantly higher editing rates for the Alu construct, while the editing rates are similar to those of the linear 100-50 guide. ADARlpl50 preferentially binds to Z-RNA due to the extra ds-RNA binding domain, which is missing in the short isoform, ADAR1p110. The high GC content of this Alu element, known to form Z-DNA and Z-RNA, aids in the recruitment of ADARlpl50.

[0277] Split-ADAR2 deaminase domain (DD)

[0278] Overexpression of ADARs can result in several transcriptome-wide off-target edits. The ability to restrict the catalytic activity of ADAR2dd to only the target mRNA can reduce the number of off-target edits. The production of split-ADAR2dds could be one potential approach to reduce the number of off-target edits. Split-protein reassembly or protein fragment complementation could be a widely used approach for studying protein-protein interactions. Splitting the ADAR2dd can be designed so that each fragment of the split-ADAR2dd is catalytically inactive by itself. However, in the presence of adRNA, the split halves can dimerize to form a catalytically active enzyme at the intended mRNA target.

[0279] The region for splitting the protein can be identified by studying the crystal structure of the ADAR2 DD in complex with its naturally occurring substrate, by understanding the solvent accessibility score using prediction software, or by any combination thereof. The MS2-MCP system and the boxB-lambda N system (which can efficiently recruit ADAR) can be used alone or in combination to recruit the N- and C-termini of the split ADAR2 DD, respectively. The adRNA can contain one MS2 stem-loop and one boxB hairpin with an antisense domain complementary to the target. This allows the N- and C-termini of the split ADAR2 DD to be recruited to the target, thereby forming a catalytically active DD.

[0280] Referring to Figures 22 and 23, a schematic diagram of the split-ADAR2 DD system is shown, with the split site highlighted in the example sequence of the ADAR2 DD.

[0281] Referring to Figure 24, a pair of fragments 1-16 can be assayed via a Cypridina luciferase reporter (cluc W85X). Fragments 9 and 10 show the highest activity. In Figure 23, the split positions corresponding to fragments 9 and 10 are indicated by blue circles.

[0282] Referring to Figure 25, fragments 9 and 10 tested against the Cluc reporter are shown. Additionally, NES-MCP-AD2-CU mutants can be tested for A to G editing. N: N-terminal fragment, C: C-terminal fragment, MM: MS2-MS2 adRNA, MB: MS2-BoxB adRNA, BB: BoxB-boxB adRNA.

[0283] Further fully humanized versions of these constructs can be created by using (a) U1a, or (b) the evolved variant TBP6.7, which has no known endogenous human hairpin target, or (c) human histone stem-loop binding protein (SLBP), or (d) the DNA-binding domain of the glucocorticoid receptor, or (e) any combination thereof. These proteins can be fused to the N- and C-terminal fragments of ADAR2 to produce a programmable RNA editing toolset that can edit adenoviruses with complete human and desired specificity. Furthermore, chimeric RNAs with two corresponding RNA hairpins can be used to recruit ADAR2 fragments. The sequences of the RNA hairpins are provided herein.

[0284] By creating one or more mutations (e.g., 16 mutations) in the ADAR2 deaminase domain, a C→U RNA editing enzyme can be produced. Even with one or more mutations, the editing enzyme can still exhibit some transcriptome-wide A→G and C→U off-targets. To improve the specificity of the enzyme, it can be split at a residue (e.g., a residue identified in a previous screen), thus producing a split ADAR system for C→U editing.

[0285] Recruitment of exogenous and endogenous APOBECs for C→U editing

[0286] APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) is an RNA editing enzyme that converts cytidine to uracil, creating diversity at the mRNA level. These entities can be recruited for C→U editing of RNA using guide RNAs, such as engineered APOBEC-recruiting guide RNAs (apRNAs). In some cases, fusion constructs can be generated by including one or more APOBEC family members. For example, a fusion construct can include ADAT1 (adenosine deaminase TRNA-specific 1) and AID (activation-induced cytidine deaminase) to MCP (MS2 coat protein). Engineered MS2-apRNAs can be used to recruit one or more MCP fusions. The protein sequences of the constructs, as well as the MS2-apRNA sequences, are shown in Figures 28-30. The protein sequence of a fusion construct, or a sequence utilized in any of the methods described herein, can comprise at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity with at least a portion of any of the sequences in Figure 28.

[0287] In some cases, the methods can include constructs configured to recruit APOBECs, such as APOBEC3A. Recruitment can be endogenous recruitment. Constructs configured to recruit APOBECs can be designed by targeting preferences for primary sequence, secondary structure, or a combination thereof. The design of one or more apRNAs can include a sequence, or a portion thereof, as shown in Figure 29. In some cases, a sequence capable of recruiting APOBECs, such as APOBEC3A, or utilized in any of the methods described herein can include at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more sequence identity to at least a portion of any of the sequences in Figure 29.

[0288] MS2-apRNAs can be designed to recruit MCP-APOBEC3A, and their sequences can include any one or more of the sequences in Figure 30. In some cases, a sequence capable of recruiting MCP-APOBEC3A, or a sequence utilized in any of the methods described herein, can include at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to at least a portion of any of the sequences in Figure 30.

[0289] Luciferase assay

[0290] When ADAR converts the target A in TAG to I (read as G), the ribosome can fully translate the protein as shown in Figure 31. Cells can be transfected and visualized 48 hours later with a luciferase reporter assay. Light readout from the cells can indicate restoration of luciferase activity.

[0291] Referring to Figure 31, the first scenario illustrates an example in which the ribosome reaches a premature TAG stop codon in the luciferase gene and can terminate translation, resulting in a truncated, non-functional luciferase enzyme. AdRNA can recruit ADAR to a site where the TAG stop codon can be edited to a TGG codon at the tryptophan site, allowing ribosome readthrough, resulting in normal luciferase expression. Such a system can enable evaluation of proper ADAR recruitment by adRNA. If luciferase expression is detected, ADAR recruitment by adRNA may have occurred. If luciferase expression is not detected, ADAR recruitment may not have occurred. kit

[0292] Also disclosed herein are kits comprising, consisting essentially of, or further consisting of an engineered adRNA of the present disclosure, an isolated polynucleotide encoding an engineered adRNA of the present disclosure, a vector expressing an engineered adRNA of the present disclosure, a recombinant cell expressing an engineered adRNA of the present disclosure, or a composition disclosed herein, or instructions for use. In one embodiment, the instructions describe how to use the engineered adRNA disclosed herein.

[0293] The kit may include a vector. The vector may be packaged in a container. The kit may include non-naturally occurring RNA. The non-naturally occurring RNA may be packaged in a container. The kit may include a syringe. The syringe may be a container in which the vector, nucleic acid, or non-naturally occurring RNA is packaged. The kit may include a pharmaceutical composition described herein. The kit may include instructions for administering the viral vector, non-naturally occurring RNA, or pharmaceutical composition described herein to a subject in need thereof. Example

[0294] The following examples are non-limiting and illustrative of procedures that may be used in various instances to effectively practice the present disclosure, and all references disclosed herein are incorporated by reference in their entirety. Example 1

[0295] Without being bound by theory, it is believed that ADAR that can be found in mammals can be recruited to catalyze therapeutic editing of point mutations.ADAR1 or ADAR2 can be recruited to target RNA or potentially DNA through the use of genetically engineered RNA scaffold, genetically engineered DNA scaffold or DNA-RNA hybrid scaffold.The tissues that can potentially be targeted using this approach include but are not limited to central and peripheral nervous system, lung, liver, gastrointestinal tract, pancreas, cardiac muscle, kidney and skin.

[0296] An exemplary embodiment proposed herein is an engineered ADAR2 guide RNA (adRNA) that has 20-100 bp of complementarity with a target RNA, and also contains a domain at either the 5' or 3' end that recruits ADAR2 from GluR2 mRNA.

[0297] This is called ornithine transcarbamylase deficiency (SPF) ash This study was carried out in vivo in a mouse model. This model has a G->A point mutation at the last nucleotide of exon 4. When adRNA alone is delivered via AAV, point mutations are observed with an error of less than 1% without overexpression of ADAR enzyme. This disclosure also demonstrates its effectiveness in vitro in HeLa cells, which are known to express ADAR. When adRNA alone is delivered, RNA editing is observed in these cells.

[0298] Moreover, this efficiency can also be applied to other recruitment domains.Accordingly, a further aspect relates to genetically engineered single-stranded ADAR2 guide DNA (adDNA) and adDNA-RNA hybrids that have higher stability than adRNA.Methods of using these enzymes disclosed herein are also provided.

[0299] Without being bound by theory, it is believed that because the ADAR family of enzymes catalyzes the hydrolytic deamination of adenosine to inosine, these enzymes can be used to catalyze the hydrolytic deamination of cytosine to thymine by mutating three specific residues, ADAR2-V351, E396 and C451, which interact with target adenosine.Thus, the way to provide the catalytic potential is provided by mutating these sites.

[0300] All possible amino acid substitutions at the three residues mentioned have been generated and screened to test the hypothesis.

[0301] Experiments are also being conducted to explore the role of other amino acids, such as S486, which may allow for the abrogation of the ADAR-specific preference for the UAG editing site.

[0302] The role of hAPOBECl and rAPOBECl is investigated in conjunction with overexpression of Apobecl complement factor (ACF) to engineer C to T editing. Also provided herein is an MCP-(h / r)APOBECl-ACF fusion protein.

[0303] Additionally, compositions are provided herein that target the ssDNA-displaced strand by current base editors, in contrast to current adenine base editing approaches for Cas9 (or Cpf1)-ADAR deaminase domain fusions (ADAR1, ADAR2, and their catalytically active mutants E1008Q and E488Q). To achieve this, the gRNA-binding strand, ideally with an AC bulge in the first 10 bp proximal to the 5' end of the gRNA, is targeted.

[0304] Other embodiments are illustrated in the accompanying documents, which are incorporated herein by reference. Example 2

[0305] Referring to Figure 6, Alu elements may be the primary target of endogenous ADAR-based RNA editing. Therefore, for programmable RNA editing, the goal is to design ADAR-recruiting RNAs (adRNAs) based on Alu elements, which can potentially enable efficient recruitment of endogenous ADARs. Alu-adRNAs are expressed from the human U6 promoter, and the linker sequences between the Alu repeats are replaced by antisense domains of various lengths that target RAB7A transcripts. Each antisense domain has a mismatched nucleotide in the middle of the antisense region and a cytosine from the target adenosine.

[0306] Alu-adRNA was tested in vitro by transfection of 293FT cells with ADARlpl10, ADARlpl50, and ADAR2, or without overexpressed enzymes, demonstrating recruitment of endogenous ADARs. Cells were harvested 48 hours after transfection, RNA was extracted, and converted to cDNA using either random hexamers or oligo-dT primers. The RAB7A locus was then amplified and subjected to Sanger sequencing. Editing efficiency was calculated as the ratio of Sanger peak heights G / (A+G). Example 3

[0307] Vector design and construction

[0308] Zero, one, or two copies of GluR2 adRNA were cloned into AAV vectors containing the human U6 and mouse U6 promoters along with a CMV promoter driving expression of FP or full-length human ADAR2 enzyme or its highly active mutant ADAR2 (E488Q). Similarly, one or two copies of MS2 adRNA were cloned into AAV vectors carrying MCP-ADAR1 or MCP-ADAR2 deaminase domain fusions and their highly active mutants. To construct GFP reporters—GFP-amber, GFP-ochre, and GFP-opal—the Y39 residue of wild-type GFP was replaced with "TAG," "TAA," and "TGA," respectively. Three gene blocks were synthesized and cloned downstream of a CAG promoter. To construct OTC and DMD reporters, spf containing the target adenosine to be edited was used. ash 200 bp fragments of the OTC and mdxDMD transcripts were cloned downstream of the CAG promoter.

[0309] Mammalian cell culture and gene transfer

[0310] All HEK 293T cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher) in an incubator at 37°C under a 5% CO2 atmosphere. All in vitro transfection experiments were performed in HEK 293T cells using the commercially available transfection reagent Lipofectamine 2000 (Thermo Fisher). All in vitro RNA editing experiments involving reporters were performed in 24-well plates using 400 ng of reporter plasmid and 800 ng of adRNA + enzyme plasmid. All in vitro RNA editing experiments targeting endogenous transcripts were performed in 24-well plates using 800 ng of adRNA / enzyme plasmid. dCasl3b-ADAR2DDE488Q-based RNA editing experiments were performed using 800 ng of enzyme plasmid (Addgene #103864) and 800 ng of gRNA plasmid. Cells were transfected at 25–30% confluence and harvested for editing quantification 60 hours after transfection. Chemically synthesized adRNA (synthesized via IDT or Synthego) was transfected at 20 pmol / well using Lipofectamine 3000 (Thermo Fisher).

[0311] AAV vector production

[0312] AAV8 particles were produced using HEK 293T cells in a triple transfection method and purified through an iodixanol gradient. The cells were approximately 80% confluent at the time of transfection. Two hours before transfection, DMEM supplemented with 10% FBS was added to the HEK 293T cells. Each virus was produced in a 5 x 15 cm plate. Each plate was transfected with 7.5 μg of pXR-8, 7.5 μg of recombinant transcription vector, and 7.5 μg of pHelper vector using PEI (1 μg / μL linear PEI in 1xDPBS pH 4.5, HCl) at a 4:1 PEI:DNA mass ratio. The mixture was incubated at room temperature for 10 minutes and then added dropwise onto the cell culture medium. After 72 hours, the virus was harvested and purified using iodixanol density gradient ultracentrifugation. The virus was then dialyzed using a 50 kDA filter (Millipore) against 1× PBS (pH 7.2) supplemented with 50 m m NaCl and 0.0001% Pluronic F68 (Thermo Fisher) to a final volume of approximately 1 mL and quantified by qPCR against a standard (ATCC VR-1616) using primers specific for the ITR region.

[0313] AAV-ITR-F: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 149), and

[0314] AAV-ITR-R: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 150) Example 4 - In vivo RNA editing of point mutations via RNA-guided adenosine deaminase

[0315] We have designed a sequence-specific RNA base editing system that mediates adenosine deaminase-mediated RNA (ADAR) enzymes associated with ADAR guide RNAs (adRNAs). The system was systematically engineered to utilize ADARs, and its specificity and activity were comprehensively evaluated in vitro and in vivo through two mouse models of human disease. In some cases, the platform can enable tunable and reversible RNA engineering for diverse applications.

[0316] Inosine-to-inosine RNA editing, a post-transcriptional RNA modification, is catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes. Inosine is the deaminated form of adenosine, which is biochemically recognized as guanine. Recently, several studies have demonstrated ADAR-mediated targeted RNA editing. Building on these findings, two orthogonal tool sets were engineered for sequence-specific, programmable RNA-based editing in vitro and in vivo. Specifically, an ADAR1 / 2-mediated targeted RNA editing system with associated ADAR guide RNAs (adRNAs) was utilized (Figure 32A). The adRNAs contain a programmable antisense region complementary to the target RNA sequence, partially offset by a mismatched cytidine opposite the target adenosine. They also contain zero, one, or two ADAR-recruiting domains engineered from the naturally occurring ADAR substrate GluR2 pre-mRNA (herein referred to as GluR2 adRNA), and a second format with two MS2 hairpins flanking an antisense region (herein referred to as MS2 adRNA). By evaluating multiple scaffold variants, including mutagenesis scaffolds based on GC-to-AU pairing, adding editing-inducing elements, and modifying the antisense domain length and mismatch position, the GluR2 adRNAs were systematically optimized to enhance exogenous and / or endogenous ADAR recruitment (Figure 8, Figure 34A-C). Next, we optimized the latter MS2 adRNA versions, utilizing synthetic proteins containing the deaminase domain (DD) of ADAR1 or ADAR2 fused to the MS2 coat protein (MCP) through systematic antisense domain length and mismatch position modifications, along with the use of hyperactive versions of the deaminase domain and versions with nuclear localization (NLS) versus export (NES) signals (Figures 32B, 35A, and 41B).

[0317] We comprehensively evaluated the activity of these two systems and benchmarked them with a recently developed RNA editing system based on Cas13b. These in vitro experiments revealed the following: (1) the engineered constructs were active in target RNA editing with yields comparable to those of the Cas13b-based system (Figure 32B, Figure 36A, Tables 2 and 3), and both U6-transcribed and chemically synthesized adRNAs were effective formats (Figure 36B); (2) adRNAs with long antisense domains, with or without GluR2 domains, were sufficient to recruit exogenously expressed ADARs, to the extent of endogenous ADARs, enabling efficient RNA editing (Figure 32B, Figure 34B, Figure 40C, Figure 42C); (3) MS2 Constructs based on adRNAs and corresponding MCP-ADAR1 / 2 fusions showed the highest and strongest activity across a large panel of endogenous genes selected across a spectrum of different expression levels (Figures 32B, 36C); (4) the use of NES and / or hyperactive deaminase domains in MCP-ADAR1 / 2 fusions consistently resulted in higher RNA editing yields at the target adenosine but was more likely to result in editing at non-targeted adenosines in the flanking sequences (Figures 32B, 37A). To further validate this, deletion of the native NLS domain in ADAR2(Δ1-138) resulted in similar promiscuity (Figures 37B-37D). (5) These two toolsets are operationally orthogonal; specifically, we evaluated the editing efficiency of MCP-ADAR2 deaminase domain fusions with co-expressed MS2 adRNA or GluR2 adRNA, demonstrating targeted editing only via the former. Conversely, full-length ADAR2 was observed to be recruited by GluR2 adRNA, but not by MS2 adRNA (FIG. 35B).

[0318] Although this toolset demonstrated robust activity, its specificity profile was examined in a transcriptome-wide off-target A to G editing analysis performed by the system (Figure 32C). For this purpose, HEK 293T cells were transfected with each construct and analyzed by RNA-seq. Non-transfected cells were included as a control. Approximately 400,000 uniquely aligned sequencing reads were collected from each sample. Fisher's exact test was used to quantify significant changes in A to G editing yields relative to non-transfected cells at each reference adenosine site with sufficient read coverage. The number of sites with at least one A to G editing event detected in any sample was calculated. Among these, the number of sites with statistically significant A to G editing was found to vary widely, with a false discovery rate (FDR) of 1% and a fold change of at least 1.1, being lowest for the MCP-ADAR2 DD-NLS construct and highest for the MCP-ADAR1 DD(E1008Q)-NES construct (Figures 38-41, Tables 4 and 5). To investigate the distribution of editing yields, a Baolin plot was generated considering A sites with significantly altered editing yields in at least one sample (Figure 32). In summary, RNA-seq experiments revealed that extensive off-target editing of the transcriptome was (1) less common in MCP-ADAR constructs with NLSs than in constructs with NESs; (2) more common in MCP-ADAR2 constructs than in MCP-ADAR1 constructs; (3) more prevalent in wild-type MCP-ADAR constructs than in E>Q high-activity mutants (Figure 42A, Table 5); and (4) that off-targets were primarily due to ADAR overexpression, with the use of adRNA alone resulting in at least a few off-targets (Figure 42B).

[0319] Table 2: Sequence list of adRNA and gRNA antisense JPEG0007720623000013.jpg172138

[0320] Table 3. Primer list for next-generation sequencing (NGS) analysis JPEG0007720623000014.jpg183139

[0321] Table 4: Summary of RNA-seq reads from a high-throughput sequencing experiment. Counts given represent read mates, not read pairs, from paired-end sequencing. The terms are as follows: sn, sample name; nt, total number of raw reads after demultiplexing; nu, number of reads in pairs that were uniquely aligned to the reference genome; nd, number of reads in duplicated pairs; nr, number of remaining reads; df, downsampling fraction. Samples designated "293T," "293T L2," "293T L8," and "293T L4" were taken from the same control library but sequenced on different lanes of the Illumina instrument. JPEG0007720623000015.jpg170138

[0322] Table 5: Quantification of A to G editing yield from aligned RNA-seq reads. Items include: Sample Name; Total Sites, the total number of reference sites with a significant change in A to G editing yield in at least one comparison between treated and control samples; Altered Sites, the number of reference A sites found to have a significant change in A to G editing yield when comparing treated samples with the control sample, which is the first sample in Table 4; On-Target Editing Yield, the editing yield observed at the intended target A site within the RAB7A mRNA; Median Editing Yield, the median yield at all sites considered excluding the target site. JPEG0007720623000016.jpg137138

[0323] Following these in vitro studies, we utilized a version of the adRNA and exogenous ADAR expression construct, similar to the one that consistently enabled the highest in vitro RNA editing yields, and evaluated the system for in vivo RNA targeting in gene therapy applications. We first evaluated the mdx mouse model of Duchenne muscular dystrophy (DMD), which contains an ochre stop site in exon 23 of the dystrophin gene. This selection was further motivated by the fact that nonsense mutations may account for approximately 11% of described genetic lesions that commonly cause inherited human diseases, as well as approximately 20% of disease-associated single-base substitutions affecting the coding region of genes. Therefore, the effectiveness of our RNA editing method may have broad therapeutic applications. To this end, we first optimized RNA editing of stop codons in vitro (Figure 43). Notably, we observed that the addition of a second copy of adRNA significantly improved targeting efficiency (Figure 43C). Therefore, a dual adRNA delivery approach was utilized for in vivo studies. The construct was then packaged in AAV8, and the 2E+12 vector genome (vg) was injected into the tibialis anterior (TA) or gastrocnemius muscles of mdx mice. To further evaluate this approach, the mdx mice were simultaneously targeted via CRISPR-Cas9-based excision of exon 23 (Figure 33A). Four or eight weeks after injection, the (TA) and gastrocnemius muscles were harvested from mdx mice, wild-type mice, mice treated with adRNA targeting and non-targeting controls, and CRISPR-Cas9. Immunofluorescence staining revealed clear restoration of dystrophin expression via targeted RNA editing (Figure 33B, Figure 44A). nNOS activity was also restored in the muscle cell membrane (Figure 33B, Figure 44A). In the treated mice, RWe observed a 3.6% or lower yield of NA editing (TAA->TGG / TAG / TGA) and a 2.4% yield of TAA->TGG (Figure 33C, Figure 43E). Western blots of treated muscles confirmed the immunofluorescence observations and showed 1-2.5% protein restoration (Figure 44B). As a baseline, muscles injected with CRISPR-Cas9-carrying vectors also resulted in restoration of dystrophin expression in a subset of muscle cells, as expected (Figure 33B). Western blots of treated muscles confirmed up to 10% protein restoration (Figure 44C).

[0324] Next, to further validate the efficacy of this approach, we tested ADAR-mediated RNA editing in a single mouse model of human disease, ornithine transcarbamylase (OTC) deficiency in male sparse fur fur (spf) mice. ash ) was evaluated in a mouse model. ash Mice carry a G-to-A point mutation in the last nucleotide of the fourth exon of the OTC gene, leading to a lack of OTC mRNA and production of a mutant protein. Recent studies have demonstrated the use of CRISPR-Cas9 and homologous recombination-based methods for independent correction of the mutation in newborn mice. ash To test the effectiveness of the system in editing point mutations in OTC mRNA (Fig. 33D), the construct was evaluated in vitro (Fig. 45A). The construct was packaged into AAV8, which has high liver tropism, and 5E+12vg was transfected into 10-12 week old spf mice. ash Three to four weeks after injection, the mice were infected with SPF. ash Mice, wild-type littermates, spf treated with ADAR2-targeted vector and non-targeted vector ashLiver samples were collected from mice and the corresponding editing efficiencies were assessed via next-generation sequencing (NGS). Notably, delivery of both adRNA and ADAR2 resulted in 0.8–4.7% edited mRNA within correctly spliced OTC mRNA. Interestingly, only adRNA resulted in a low but significant RNA editing yield (Figure 33E). Furthermore, delivery of a hyperactive ADAR2 mutant (E488Q) resulted in a high editing fraction (4.6–33.8%) within correctly spliced OTC mRNA (Figure 33E, Figure 45B), and 4.6–8.2% within OTC pre-mRNA (Figure 45C), confirming a reduction in incorrectly spliced products (Figure 45D). Western blot analysis of treated liver samples confirmed partial restoration of OTC protein (2.5–5%) (Figure 45E).

[0325] In conclusion, these results establish the usefulness of RNA-guided ADAR for in vivo RNA editing of point mutations.In some cases, the sequence selection of ADAR enzymes, RNA folding, intrinsic half-life, localization, translation machinery, and resident RNA-binding proteins can potentially affect the accessibility and editability of target sites in RNA, and may also be important design parameters to consider for effective targeting.For example, in the mdx model, ADAR-based RNA editing approaches can compete with the nonsense-mediated decay of mutant dystrophin mRNA and the need to perform two A->I substitutions in non-ideal adjacent nucleotide contexts to eliminate premature stop codons and their potential impact on RNA stability and function.In addition, spf ashIn our model, the need for transient OTC pre-mRNA targeting may result in rapid target engagement and editing. Further advances are also needed to address important limitations of the system, such as off-targeting induced by endogenous enzyme-RNA binding, processivity, promiscuity, stimulation of the interferon response by the delivery modality itself (lipid, nanoparticle, viral, etc.) leading to increased endogenous ADAR expression, the potential for adRNA to induce RNAi, and off-target hybridization of the antisense domain of adRNA, which could have potentially deleterious effects. In this regard, this study revealed toxicity in mice systemically injected with a highly active ADAR mutant (Figure 46). These studies may be important for systematically improving the specificity and safety of this approach. Another important consideration when considering RNA targeting for gene therapy, particularly via the use of non-integral vectors, is the need for periodic re-administration of the effector construct due to the limited half-life of the edited mRNA and effector. In this regard, compared to CRISPR-based RNA editing approaches, the RNA-guided ADAR method will be directly relevant to human therapeutics, as similar versions utilize effector RNA and human proteins alone. Also, ADARs are widely expressed. For example, ADAR1 is expressed in most human tissues, and ADAR2 is particularly expressed in the lung and brain. Endogenous recruitment via adRNAs with long antisense domains (as shown in Figures 32, 33E, 34, and 36) represents a very attractive method for effective RNA editing. This progressively improved toolset can have broad implications for a variety of basic science and therapeutic applications.

[0326] Figure 32: Genetic engineering and characterization of specificity profiles for programmable RNA editing. (A) Schematic of RNA editing via engineered adRNAs derived from full-length ADAR2 and GluR2 transcripts, or constructs utilizing MS2 coat protein (MCP) fusions to the ADAR1 / 2 deaminase domain and the corresponding MS2 hairpin-bearing adRNA. (B) Comparison of RNA editing efficiency of endogenous RAB7A transcripts by different RNA editing constructs, quantified by Sanger sequencing (efficiency calculated as the ratio of Sanger peak heights G / (A+G)). Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n=3). (C) Violin plot depicting the distribution of A-to-G editing yields observed at reference sites where at least one treated sample was found to have a significant change in editing yield (Fisher's exact test, FDR=1%) compared to the control sample. Blue circles indicate editing yields at the target A site within the RAB7A transcript. Black dots represent the median off-target editing yield. To better visualize the shape of the distributions, their maximum range along the y-axis was equalized across the plot and truncated at 60% yield.

[0327] Figure 33: In vivo RNA editing in mouse models of human disease: (A) Schematic of DNA and RNA-targeted approaches to restore dystrophin expression in the mdx mouse model of Duchenne muscular dystrophy. (i) Dual gRNA-CRISPR-based approach leading to in-frame excision of exon 23, and (ii) ADAR2- and MCP-ADAR1-based editing of the ochre codon. (B) Immunofluorescence staining of dystrophin in the TA muscle shows partial restoration of expression in treated samples (intramuscular injection of AAV8-ADAR2, AAV8-ADAR2(E488Q), and AAV8-CRISPR). Partial restoration of nNOS localization is also seen in treated samples (Scale bar: 250 μm). (C) In vivo TAA->TGG / TAG / TGA RNA editing efficiency in the corresponding treated adult mdx mice. Values represent the mean + / - SEM (n = 4, 3, 7, 3, 3, 10, 3, 4 single TA muscles, respectively). (D) Ornithine transcarbamylase deficiency spf with a G->A point mutation in the donor splice site at the last nucleotide of exon 4. ash Schematic diagram of the OTC locus in the mouse model and the approach for correction of mutant OTC mRNA via ADAR2-mediated RNA editing. (E) Treated adult spf ash In vivo RNA correction efficiency of correctly spliced OTC mRNA in the liver of mice (retro-orbital injection of AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Values represent the mean + / - SEM (n=4, 4, 3, 3, 4, 5 single animals, respectively).

[0328] Vector design and construction

[0329] One or two copies of adRNA were cloned into an AAV vector containing the human U6 or mouse U6 promoter and a CMV promoter driving enzyme expression. To construct the GFP reporters—GFP-amber, GFP-ochre, and GFP-opal—three gene blocks were synthesized with "TAG," "TAA," and "TGA," respectively, replacing the Y39 residue of wild-type GFP, and cloned downstream of the CAG promoter. To construct the OTC and DMD reporters, spf carrying the target adenosine to be edited was used. ash 200 bp fragments of the OTC and mdx DMD transcripts were cloned downstream of the CAG promoter.

[0330] Mammalian cell culture and gene transfer

[0331] All HEK 293T cells were grown in Dulbecco's Modified Eagle's medium supplemented with 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher) in an incubator at 37°C under a 5% CO2 atmosphere. All in vitro transfection experiments were performed in HEK 293T cells using the commercially available transfection reagent Lipofectamine 2000 (Thermo Fisher). All reporter-containing in vitro RNA editing experiments were performed in 24-well plates using 400 ng of reporter plasmid and 800 ng of adRNA + enzyme plasmid. All endogenous transcript-targeting in vitro RNA editing experiments were performed in 24-well plates using 800 ng of adRNA / enzyme plasmid. dCas13b-ADAR2 DD E488Q-based RNA editing experiments were performed using 800 ng of enzyme plasmid (Addgene #103864) and 800 ng of gRNA plasmid. Cells were transfected at 25-30% confluence and harvested for editing quantification 60 hours after transfection. Chemically synthesized adRNA (synthesized via IDT or Synthego) was transfected using Lipofectamine 3000 (Thermo Fisher) at 20 pmol / well.

[0332] AAV vector production

[0333] AAV8 particles were produced using a triple transfection method in HEK 293T cells and purified using an iodixanol gradient. The cells were approximately 80% confluent at the time of transfection. Two hours before transfection, DMEM supplemented with 10% FBS was added to the HEK 293T cells. Each virus was produced in a 5 x 15 cm plate. Each plate was transfected with 7.5 μg of pXR-8, 7.5 μg of recombinant transfer vector, and 7.5 μg of pHelper vector using PEI (1 μg / μL linear PEI in 1x DPBS, pH 4.5, with HCl) at a PEI:DNA mass ratio of 4:1. The mixture was incubated at room temperature for 10 minutes and then added dropwise onto the cell media. After 72 hours, the virus was harvested and purified using iodixanol density gradient ultracentrifugation. The virus was then dialyzed in a 50 kDA filter (Millipore) against 1x PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher) to a final volume of approximately 1 mL and quantified by qPCR using primers specific for the ITR region against a standard (ATCC VR-1616).

[0334] AAV-ITR-F: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 149), and

[0335] AAV-ITR-R: 5-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 150)

[0336] RNA isolation and next-generation sequencing library construction

[0337] RNA was extracted from animal tissues using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. RNA from cells was extracted using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 500 ng of RNA using the Protoscript II First-Strand cDNA Synthesis Kit (NEB). Next-generation sequencing libraries were constructed as follows: Briefly, a lump of the cDNA prepared above was amplified by PCR using primers amplifying approximately 150 bp surrounding the site of interest using the KAPA Hifi HotSart PCR Mix (Kapa Biosystems). The PCR product was purified (Qiagen PCR Purification Kit / Gel Extraction Kit) and by-products were removed. Libraries were constructed using the NEBNext Multiplex Oligo for Illumina kit (NEB). 10 ng of input DNA was amplified with index primers. Samples were then pooled and loaded onto an Illumina Miseq (150 bp single-end run) or Hiseq (100 bp paired-end run). Data analysis was performed using CRISPResso (Pinello, L. et al., 2016). A minimum of 100,000 reads were analyzed for all in vivo experiments. RNA-seq libraries were constructed from 300 ng of RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra RNA Library Construction Kit for Illumina. Samples were pooled and loaded onto an Illumina Miseq (100 bp paired-end run).

[0338] spf ash Quantification of editing yield of OTC mRNA in mice

[0339] spf ashMice have three OTC RNA forms: pre-mRNA, correctly spliced mRNA, and incorrectly spliced and extended mRNA formed by using the 48 base pair negative splice site in intron 4. Therefore, the total number of correctly spliced mRNAs is X, the incorrectly spliced variant is Y, and the pre-mRNA is Z. Xe, Ye, and Ze represent the three forms of A-to-G edited mRNA. The mRNA editing yield can ideally be calculated as (Xe + Ye + Ze) / (X + Y + Z). However, because the spliced and mRNA variants cannot be amplified using the same primers, the results are not shown in Figure 1. 33E shows the fraction of edited transcripts in correctly spliced mRNA (Xe / X), which is then translated to produce the OTC protein. 45C shows the fraction of edited transcripts in the pre-mRNA (Ze / Z). This fraction, upon correct splicing, contributes to the formation of the OTC protein. Finally, incorrectly spliced mRNA leads to the production of a protein elongated by 16 amino acids that is selectively degraded. 45D In the figure, the bands corresponding to X and Y are shown.

[0340] Animal experiments

[0341] All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego. Mice were obtained from Jackson Labs. Mdx mice (C57BL / 10ScSn-Dmd) were cultured at 2E+12vg / muscle. mdx AAV was injected into the abdominal or TA muscles of mice (J / J). AAV was administered via retroorbital injection at 2.5E+12vg / mouse. ash Mouse (B6EiC3Sn a / A-Oct spf-ash Mice with a rough-looking hair coat, sluggish movements, and slight hunger were designated sick mice and were euthanized.

[0342] Immunofluorescence

[0343] Harvested abdominal or TA muscles were placed in molds containing OCT compound (VWR) and flash-frozen in liquid nitrogen. 10 μm sections were cut onto pretreated histological slides. Slides were fixed with 4% paraformaldehyde. Dystrophin and nNOS were detected with rabbit polyclonal antibodies against the C-terminal domain of dystrophin (1:200, Abcam 15277) and the N-terminal domain of nNOS (1:100, Immunostar 24431), respectively, followed by donkey anti-rabbit Alexa 546 secondary antibody (1:400, Thermo Fisher Scientific).

[0344] Western blot

[0345] Muscle biopsy and spf from mdx mice ashLiver biopsies from mice were fragmented with proteinase inhibitor cocktail (Roche) in RIPA buffer (Sigma), vortexed intermittently, and incubated on ice for 1 hour. Samples were centrifuged at 5,500 x g for 30 minutes at 4°C, and the supernatant was isolated and quantified using a Pierce Coomassie Plus (Bradford) assay kit (Thermo Fisher). Protein isolates were mixed with 4x Laemmli loading buffer (Biorad) and 2-mercaptoethanol (Biorad) and boiled at 100°C for 10 minutes. 100 μg of total protein from muscle biopsies or 60 μg from liver biopsies was loaded into each well of a 4-15% Mini Protean TGX gel (Biorad) using Tris-glycine-SDS buffer (Biorad) and electrophoresed at 100 V for 60 minutes. Protein from the muscle biopsies was transferred to a nitrocellulose membrane and electrophoresed overnight at 34 V. Meanwhile, proteins from liver biopsies were transferred at 65V and incubated for 1 hour and 30 minutes in 1X Tris-glycine loading buffer containing 10% methanol and 0.1% SDS. The blots were blocked for 1 hour in 5% milk-TBST. The blots were probed overnight at 4°C with rabbit anti-dystrophin (1:200, Abcam 15277), rabbit anti-GAPDH (1:4000, Cell Signaling 2118S), rabbit anti-OTC (1:800, Abcam 203859), and mouse anti-ADAR2 (1:150, Santa Cruz Biotechnology 73409) in 5% milk-TBST. The blots were washed with TBST and then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) in 5% milk-TBST for 1 hour. After washing with TBST, the blots were visualized using SuperSigneal West Femto Chemiluminescent Substance (Thermo Fisher) and X-ray film.

[0346] Statistics and Reproducibility

[0347] In vitro experiments: In vitro experiments were performed once with a minimum of three independent repeats. In vivo experiments: Experiments based on ADAR2 and MCP-ADAR1(E1008Q) NLS were performed twice for the mdx mouse model. Both rounds of experiments resulted in consistent RNA editing efficiency, dystrophin immunofluorescence, and dystrophin restoration as seen by Western blot. Experiments based on ADAR2(E488Q) and CRISPR-Cas9 were performed once. spf ash For the mouse model, experiments were performed twice due to the availability of mice. The RNA editing efficiency of OTC transcripts, both spliced mRNA and pre-mRNA, was consistent in both rounds of experiments. RT-PCR and Western blot analysis were performed on animals from experimental set 1.

[0348] 1. Quantification of RNA A->G editing

[0349] (a) RNA-seq read orientation

[0350] RNA-seq read pairs with 100 bases per readmate were aligned to the GRCh38 reference genome using Star Exposure version 2.6.0c (Dobin A et al., 2013). The genome index was constructed using primary assembly annotations from GENCODE release 28 (GRCh38.pl2). Star was run using default parameters, with the following exceptions: readMapNumber=-1, alignSJoverhangMin=5, alignSJDBoverhangMin=1, alignEndsType=EndToEnd, outFilterMismatchNmax=10, outFilterMultimapNmax=1, outSAMunmapped=None, outSAMmultNmax=1. The resulting uniquely aligned paired reads were sorted by genome coordinates using samtools sort (Li H. et al., 2009). Duplicate read pairs were marked using samtools markdup and excluded from subsequent analysis. The tabulated, aligned, replicated, and remaining reads (pairs) for each sample are shown in Table 4.

[0351] (b) Selection of reference sites for quantifying editing yield

[0352] Assessment of sites with significant changes in A-to-G editing yield (see below) is sensitive to the number of uniquely aligned reads available for each sample. To minimize potential bias when comparing different samples with respect to significantly edited sites, the uniquely aligned reads for each HEK293T sample were downsampled using samtools view, with the options and downsampling fraction reported in Table 4. These fractions were calculated by dividing the minimum number of uniquely aligned reads between samples by the number of uniquely aligned reads available for a sample to be downsampled. No downsampling was performed on the control sample reads (first in Table 4). The downsampled reads were then processed using samtools mpileup. The output of this tool was parsed to extract counts of each base found in aligned reads at each A-site and T-site in the GRCh38 reference genome sequence. Insertions and deletions were ignored. Reference sites with less than 10 read coverage were omitted from downstream analysis. Across the samples listed in Table 4, the number of remaining reference A- and T-sites with a read coverage of at least 10 varied by approximately 15%. Without downsampling, this number was expected to vary by approximately 50%. From the reference A- and T-sites with a read coverage of at least 10, we selected the final list of total sites (A- and T-sites) by selecting those sites that were common to all samples and in which at least one G or C was observed at the reference A- or T-site, respectively, in the aligned reads of at least one sample. Other sites were discarded if they were not common to all samples or had zero editing events observed in all samples.

[0353] (c) Assessment of significant changes in A→G editing yield

[0354] To reveal significant changes in A→G editing yield, several pairs of control and treated samples were considered. For each pair, the control sample was the first sample listed in Table 4, and the treated sample was one of the samples shown in Figure 32. For each pair of comparison samples and each reference A site selected as above, a Fisher's exact test was performed using a 2x2 discontinuity matrix C with entries defined as follows: C 1.1 = counts of bases other than G observed in the control sample, C 2.1 = counts of G bases observed in the control sample, C 1.2 = counts of bases other than G observed in the test sample, C 2.2 = counts of G bases observed in the test sample. A similar discontinuity matrix was used for each selected reference T-site, except that G was replaced with C in the above definition. For multiple testing, the calculated p-values for all selected reference sites and the calculated p-values for a given comparison of samples were adjusted using the Benjamini-Hochberg method. A-sites and T-sites with adjusted p-values below a false discovery rate (FDR) of 1% and with at least a 1.1-fold change in editing yield were considered to have a significant change in A→G editing yield in the forward and reverse transcripts, respectively. The counts of these sites for each comparison of samples are shown in Figures 38-42. sig The counts are shown as "C" and are shown in the "Altered Sites" column of Table 5. The total number of reference sites with significant changes in A-to-G editing yield was calculated. The editing yields at these sites were used to construct the distribution shown in Figure 32. For each sample, the on-target A-to-G editing yield, shown as blue circles in Figure 32 and Figures 38-42, was calculated using the counts observed at the intended target A site in the RAB7A transcript. 2.2 / (C 1.2 +C 2.2 ) These values are shown in Table 5 under the column "Editing Yield." After selecting the reference assembly hg38, the following sequence was submitted to BLAT, and the single base genomic coordinate of the intended target A site was found to be chr3:128814202. AGCGGCAGTATTCTGTACAGTAGACACAAGAATTATGTACGCCTTTTATCA (SEQ ID NO: 151)

[0355] Figure 8 - Engineering GluR2 adRNA: Engineering the scaffold domain. Sequence information for the adRNA scaffold: ADAR-recruiting domain, antisense RNA targeting domain, and highlighted cytosine mismatch. Base pairs mutated to create the stabilized scaffold are numbered and highlighted in red, and the editing induction element motif is shown in green. Quantification of the editing efficiency of the produced scaffolds for the OTC reporter transcript, quantified by Sanger sequencing, is shown. Values represent the mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0356] Figure 34 - Engineering GluR2 adRNA: Antisense domain engineering. (a) Optimization of the adRNA antisense region using adRNA scaffold 2: length and distance from the ADAR2 recruitment region were systematically varied. Values represent mean + / - SEM (n=3). (b) U6 promoter-transcribed adRNAs with progressively longer antisense domain lengths were combined with zero, one, or two GluR2 domains and evaluated for their ability to induce target RNA editing with or without exogenous ADAR2 expression. Values represent mean + / - SEM (n=3). A portion of the data is shown in Figure 34. 32B All of the above experiments were performed in HEK 293T cells. (c) Experimental confirmation of endogenous ADAR1 and ADAR2 (relative to GAPDH) expression in HEK 293T and HeLa cell lines. The observed levels were similar to those described in the human protein atlas (see proteinatlas.org on the world-wide web).

[0357] Figure 35 - Genetic engineering of MS2 adRNA. (a) Systematic evaluation of the antisense RNA targeting domain of MS2 adRNA. Values represent the mean + / - SEM (n=3). (b) On-target RNA editing by MCP-ADAR2 DD-NLS requires co-expression of MS2 adRNA. Values represent the mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0358] Figure 36—Analysis of RNA editing yield across a target panel. (A) Comparison of RNA editing efficiency of an OTC reporter transcript by GluR2 adRNA- and MS2 adRNA-guided RNA editing constructs with a Cas13b-based repair construct. Values represent the mean + / - SEM (n=6 for reporter and Cas13b-based constructs, n=3 for other constructs). (B) Chemically synthesized adRNA versions were tested against a panel of mRNAs with or without exogenous ADAR2 expression. The exact chemical modifications are noted in the figure, along with the source of the adRNA. Values represent the mean + / - SEM (n=3). (C) Analysis of RNA editing yield across a spectrum of endogenous targets selected to cover a range of expression levels. U6-transcribed long adRNAs with zero or two GluR2 domains were also evaluated against multiple endogenous mRNA targets with or without exogenous ADAR2 expression. Editing at the tested locus sites is observed even in the absence of exogenous ADAR2 expression. Values represent the mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0359] Figure 37 - ADAR2 mutants and their effect on editing and specificity. (A) Comparison of on-target RNA editing and editing in adjacent adenoviruses of the RAB7A transcript by GluR2 adRNA- and MS2 adRNA-guided RNA editing constructs and Cas13b-based repair constructs. Average (n=3) editing yields are shown. Experiments were performed in HEK 293T cells, and editing efficiency was calculated as the ratio of Sanger peak heights, G / (A+G). (B) ADAR2(E488Q) inhibits spf ash(C) The mdx DMD reporter transcript (p = 0.048, r = 0.012, unpaired t-test, two-tailed) shows higher efficiency than ADAR2 in in vitro editing of the OTC reporter transcript. Values represent the mean + / - SEM (n = 3). (D) Comparison of the editing efficiency and specificity profiles of ADAR2, ADAR2(E488Q), and ADAR2(Δ1-138) for the OTC reporter transcript (upper panel) and the endogenous RAB7A transcript (lower panel). Heatmaps show A-to-G editing near the target (red arrow). Values represent the mean + / - SEM (n = 3). Experiments were performed in HEK 293T cells, and editing efficiency was calculated as the Sanger peak height ratio G / (A+G).

[0360] Figure 38—Transcriptome-scale specificity profile of the RNA editing approach (Cas13b-ADAR repair + / - gRNA). 2D histograms comparing the transcriptome-wide A to G editing yield observed with each Cas13b-ADAR2 construct (y-axis) with the yield observed in control samples (x-axis). Each histogram represents the same set of 8,729,464 reference sites with a read coverage of at least 10 and at least one putative editing event detected in at least one sample. Bins highlighted in red contain sites with significant changes in A to G editing yield when comparing treated samples to control samples. Red crosses in each plot indicate the 100 sites with the lowest adjusted p-values. Blue circles indicate the intended target A site within the RAB7A transcript. The large counts in the bins near the bottom left corner likely correspond not only to low editing yields in both test and control samples, but also to sequencing and alignment errors. Large counts in bins near the upper right corner of each plot likely correspond to homozygous single nucleotide polymorphisms (SNPs) as well as other differences between the reference genome and the genome of the HEK 293T cell line used in the experiments.

[0361] Figure 39 - Transcriptome-scale specificity profile of the RNA editing approach (ADAR2+ / - adRNA). The version used in these studies is GluR2 adRNA(l.20,6). 2D histogram comparing the transcriptome-wide A->G editing yield observed with each ADAR construct (y-axis) with that observed in control samples (x-axis). More details are provided in Figure 38.

[0362] Figure 40 - Transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR1 DD+ / - adRNA). 2D histogram comparing the transcriptome-wide A to G editing yield observed with each ADAR construct (y-axis) with that observed in control samples (x-axis). More details are provided in Figure 38.

[0363] Figure 41 - Transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR2 DD+ / - adRNA). 2D histogram comparing the transcriptome-wide A to G editing yield observed with each ADAR construct (y-axis) with that observed in control samples (x-axis). More details are provided in Figure 38.

[0364] Figure 42 - Change in transcriptome-scale editing specificity with construct features. (A) Each point in the box plot corresponds to the fraction of edited sites for one of the MCP-ADAR constructs listed in Figure 32. The fraction of edited sites for each construct was calculated by dividing the number of reference sites with significant changes in A → G editing yield (see Table 3) by the total number of reference sites considered, 8,729,464. Construct features depicted on the horizontal axis were compared using the Mann-Whitney U test. The resulting p-values were 0.16 for NLS vs. NES, 0.0070 for ADAR1 vs. ADAR2, 0.72 for "-adRNA" vs. "+adRNA," and 0.72 for "ADAR WT" vs. "ADAR For E>Q", it is 0.038. (n = 8 for all conditions) (B) 2D histogram comparing the transcriptome-wide A>G editing yield observed with each construct (y-axis) to the yield observed in control samples (x-axis). More details are provided in Figure 38. The inset shows a violin plot representing the distribution of A>G editing yields observed at reference sites where at least one treated sample was found to have a significant change (Fisher's exact test, FDR = 1%) compared to the control sample. Blue circles indicate editing yields at targeted A sites within the RAB7A transcript. To better visualize the shape of the distributions, their maximum range along the y-axis was equalized across the plot and truncated at 60% yield. Here, samples correspond to 293Ts transfected with a long antisense domain-harboring adRNA that can enable RNA editing by exogenous and / or endogenous ADAR recruitment.

[0365] Figure 43 - Optimization and evaluation of dystrophin RNA editing experiments in mdx mice in vitro and in vivo. (A) Schematic of RNA editing using full-length ADAR2 with engineered adRNA or reverse adRNA (raRNA); (ii) RNA editing efficiency of amber and ochre stop codons in one-step and two-step procedures. Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n=3). (B) RNA editing of the ochre codon requires two cytosine mismatches in the antisense RNA targeting domain of adRNA or radRNA to restore GFP expression. Experiments were performed in HEK 293T cells. Values represent mean + / - SEM (n=3). (C) Schematic of the AAV vectors utilized for in vivo delivery of adRNA and ADAR2, and in vitro optimization of RNA editing of amber and ochre stop codons in the presence of one or two copies of adRNA delivered via AAV vectors (p=0.0003, r=0.0001, p=0.0015, respectively, t-test, two-tailed). Experiments were performed in HEK 293T cells. Values represent the mean + / - SEM (n=3 for reporter, n=6 for other conditions). (D) Representative Sanger sequencing plot showing editing of the ochre stop codon (TAA->TGG) in the mdx DMD reporter transcript (quantified by NGS). Experiments were performed in HEK 293T cells (n=3). (E) Representative example of in vivo RNA editing analysis of treated mdx mice (quantified using NGS).

[0366] Figure 44—Immunofluorescence and Western blot analysis of in vivo dystrophin RNA editing experiments in mdx mice. (A) Immunofluorescence staining of dystrophin in TA muscle shows partial restoration of expression in treated samples (intramuscular injection of AAV8-ADAR2, AAV8-ADAR2(E488Q), and AAV8-MCP-ADAR1(E1008Q)NLS). Partial restoration of nNOS is localized and is also seen in treated samples (scale bar: 250 μm). (B) Western blot showing partial restoration (1-2.5%) of dystrophin expression in TA muscle of mdx mice injected with both components of the editing machinery, the enzyme and adRNA, and stable ADAR2 expression in injected TA muscle up to 8 weeks post-injection. (C) Western blot showing partial restoration (10%) of dystrophin expression using AAV8-CRISPR.

[0367] Figure 45-spf ash Optimization and evaluation of OTC RNA editing experiments in vitro and in vivo in mice. (A) spf ash Representative Sanger sequencing plots showing correction of point mutations in the OTC reporter transcript (quantified using NGS). Experiments were performed in HEK 293T cells (n=3). (B) Processed spf mRNA showing correction of point mutations in correctly spliced OTC mRNA (quantified using NGS). ash Representative example of in vivo RNA editing analysis in mice. (C) Treated adult spf ash In vivo RNA correction efficiency of OTC pre-mRNA in the liver of mice (retro-orbital injection of AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Values represent the mean + / - SEM (n=4, 4, 3, 3, 4, 5 single animals, respectively). (D) PCR products showing correctly and incorrectly spliced OTC mRNA. The incorrectly spliced mRNA is extended by 48 base pairs. The fraction of incorrectly spliced mRNA is reduced in mice treated with adRNA+ADAR2(E488Q). (E) Western blot of OTC in treated adult spf mice. ashPartial restoration of expression (2.5%-5%) and stable ADAR2(E488Q) expression in mice 3 weeks after injection are shown.

[0368] Figure 46 - Toxicity analysis of in vivo RNA editing experiments. Summary of animal studies summarizing route of AAV administration, delivered constructs, and health status of injected mice 3 weeks post-injection. Example 5

[0369] Disclosed herein are experimental results in the mdx mouse model of Duchenne muscular dystrophy using the E100Q mutant of ADAR1 (included in MCP-ADAR1(E100Q)). In some cases, this mutant can improve in vivo editing yield compared to ADAR2 and the E488Q mutant of ADAR2. Furthermore, disclosed herein is the application of the ADAR system in a manner that alters splicing patterns by editing splice acceptor sites or branch points in introns, thereby resulting in exon skipping. Also contemplated and exemplified are methods using APOBEC. Provided herein is an example in which the creation of a local structure in alipoprotein B mRNA where the ACF-APOBEC complex binds to the target mRNA allows C→T RNA editing. Furthermore, disclosed herein are methods utilizing ADAR enzymes for programmable editing of both RNA and DNA.

[0370] The ADAR editing system can be used to generate adRNAs with or without exogenous ADAR1 or ADAR2. The examples provide specific adRNAs of interest for a given target. Further, examples of chemically synthesized adRNAs are provided herein with 2'-O-methyl 3' phosphothiolate modifications at the first and last 3, 6, 9, or 12 nucleotides, and / or 2'-O-methyl modifications throughout the antisense region, except for the 3 nucleotides centered at the mismatch site (underlined), that are linked to a targeting moiety such as GalNc, cholesterol, or a cell-penetrating peptide, can be used to engineer targeted RNA editing in cells or in vivo (particularly in the liver, lung, and brain), with or without exogenous ADAR1 / 2 overexpression. General design:

[0371] adRNA22: GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACNNNNN NCN NNNNNNNNNNNNN (SEQ ID NO: 152)

[0372] adRNA32: GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCNNNNN NCN NNNNNNNNNNNNN (SEQ ID NO: 153)

[0373] Examples:

[0374] adRNA22_RAB7A: GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACTGCCG CCA GCTGGATTTCCC (SEQ ID NO: 154)

[0375] adRNA32_RAB7A: GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCTGCCG CCA GCTGGATTTCCC (SEQ ID NO: 155)

[0376] adRNA22_CKDN2A: GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACCTCCT CCA CCCGACCCCGGG (SEQ ID NO: 156)

[0377] adRNA32_CKDN2A: GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCCTCCT CCA CCCGACCCCGGG (SEQ ID NO: 157)

[0378] The examples provided below describe a number of steps taken to further define the embodiments disclosed herein. For example, (1) Delivering the MCP-ADAR construct using MS2-adRNA targeting a premature stop codon in the dystrophin transcript of mdx mice; (2) test exon skipping via the production of point mutations in the mdx mouse model of Duchenne muscular dystrophy using adRNA delivered with ADAR2 / ADAR2(E488Q), or MS-adRNA delivered with MCP-ADAR1 / ADAR(E1008Q), or ms-adRNA delivered with MCP-ADAR2 / ADAR2(E488Q), used to produce an A → G substitution at the splice site; (3) test editing splice site mutations in the OTC transcript in spc-ash mice; (4) testing editing of mutant splice sites to alter splicing patterns in the spf-ash mouse model of ornithine transcarbamylase deficiency; (5) targeting mouse SCN9A transcripts to engineer pain sensitivity by generating an A→G substitution at the splice site to knock down the SCN9A transcript; (6) engineering an APOBEC gRNA to produce a localized structure of alipoprotein B mRNA at the target mRNA of interest; and (7) Testing ADARs in editing RNA and DNA using RNA, DNA, and RNA-DNA hybrids to recruit ADARs.

[0379] In these examples, enhanced RNA yield and protein expression were observed while editing premature stop codons in dystrophin transcripts in mdx mice using MS2-adRNA and MCP-ADAR1(E1008Q). Delivery of ADAR2(E488Q) via adRNA also reduced incorrectly spliced OTC mRNA in the spf-ash mouse model.

[0380] Thus, the present disclosure describes a system that can enable site-specific A to G editing of RNA. Such an approach can be used to edit splice acceptor sites and branch points to alter splicing. This can also enable exon skipping. Further embodiments contemplate the use of A to G editing of DNA. The present disclosure also provides the possibility of C to T editing in RNA through the use of APOBEC1 co-expressed with ACF1.

[0381] This disclosure describes the first site-specific RNA editing in vivo. Indeed, the use of MCP-ADAR1(E1008Q) demonstrates higher editing efficiency than conventional constructs in the mdx mouse model of muscular dystrophy. Further studies will examine the effectiveness of (1) RNA editing to alter splicing, and (2) APOBEC-mediated C to T editing in conjunction with ACF and (3) ADAR overexpression to edit DNA, both in vitro and in vivo.

[0382] Compared to other ADAR2 systems (eg, Stafforst, Zhang, and Rosenthal labs) and Casl3d-based inhibition of splicing, the present ADAR system is unique.

[0383] As described herein, the ADAR system comprises, consists essentially of, or further consists of an RNA targeting domain complementary to the target RNA and one or more ADAR recruiting domains that enable ADAR recruitment. Upon introducing these components, the ADAR enzyme can catalyze the conversion of the target adenosine to inosine, thereby repairing the point mutation. The present disclosure describes the use of ADAR2 or MCP-ADAR1 / 2-NLS or their hyperactive mutants to engineer exon skipping by generating A-to-G edits at splice acceptor sites and / or intron branch points. Skipping the symmetric exon results in the formation of a truncated protein. This method can be used to skip exon 23 in the dystrophin transcript of the mdx mouse model of DMD, which has a premature stop codon in the exon. Skipping exon 23 results in the translation of a functional truncated dystrophin protein. Skipping the asymmetric exon also leads to a frameshift mutation. Thus, gene knockdown can be engineered by skipping essential or asymmetric exons. Editing the ATG initiation codon and Kozak / Shine-Dalgarno sequence can also help alter translation efficiency, resulting in gene knockdown. Using exon skipping, SCN9A transcripts can be targeted to engineer pain sensitivity. Localized structures of apolipoprotein B mRNA that are bound by the ACF-APOBEC complex can be engineered to carefully position the editing C at a location similar to the natural site of apolipoprotein B mRNA at the target mRNA. This is achieved by overexpressing a pair of adRNAs, generating an apolipoprotein B mRNA-like structure that can be edited by overexpressing MCP-ACF1 and APOBEC1. Example 6 - Exon skipping via generation of splice acceptor and / or branch point mutations

[0384] This disclosure demonstrates the use of ADAR2 or MCP-ADAR1 / 2-NLS or their hyperactive mutants to engineer exon skipping by generating A-to-G edits at splice acceptor sites and / or branch points in introns. Skipping the symmetric exon results in the formation of a truncated protein. In this example, this method can be used to skip exon 23 in the dystrophin transcript of the mdx mouse model of DMD, which has a premature stop codon in that exon. Skipping exon 23 results in the translation of a functional truncated dystrophin protein. Skipping the asymmetric exon also leads to a frameshift mutation. In this way, skipping essential or asymmetric exons can engineer gene knockdown. Editing the start codon ATG and Kozak / Shine-Dalgarno sequence also helps alter translation efficiency, resulting in gene knockdown. This method can also be used to engineer pain sensitivity by targeting the SCN9A transcript.

[0385] An exemplary sequence to achieve this goal is shown below.

[0386] Sequence Set 1: Exemplary adRNA Sequences

[0387] adRNA sequence for exon 23 skipping in dystrophin transcripts in mdx mice (SEQ ID NOs: 158-159). JPEG0007720623000017.jpg25140

[0388] MS2-adRNA sequence for exon 23 skipping in dystrophin transcripts in mdx mice (SEQ ID NOs: 160-161) JPEG0007720623000018.jpg25139

[0389] MS2-adRNA sequence for editing a premature stop codon in mdx mice (SEQ ID NO: 162) JPEG0007720623000019.jpg19139

[0390] adRNA sequence for exon skipping in SCN9A transcripts in mice (SEQ ID NOs: 163-167) JPEG0007720623000020.jpg62139

[0391] MS2-adRNA sequence for exon skipping in SCN9A transcripts in mice (SEQ ID NOs: 168-172) JPEG0007720623000021.jpg14139JPEG0007720623000022.jpg50139Example 7 - C->T editing via APOBEC

[0392] The local structure of apolipoprotein B mRNA to which the ACF-APOBEC complex binds can be generated to carefully position the editing C in the mRNA target of interest at a location similar to the naturally occurring site in apolipoprotein B mRNA. This is achieved by overexpressing a pair of gRNAs to generate an apolipoprotein B mRNA-like structure that can be edited by overexpressing one or more of the following combinations: 1. MCP-ACF1 and APOBEC1 2. MCP-ACF1 and MCP-APOBEC1 3. MCP-linker-ACF-linker-APOBEC1 4. MCP-APOBEC1 and ACF1

[0393] Sequence Set 2: Exemplary C→T edited sequences

[0394] MS2-adRNA sequence for C→T editing (SEQ ID NOs: 173-174) JPEG0007720623000023.jpg25139Example 8 - Generation of cancer-associated point mutations

[0395] Several genes involved in cancer pathways have single amino acid substitutions. The generation of dominant-negative, constitutively active, and catalytically inactive mutants is possible by generating A-to-G substitutions in the mRNA sequences of these genes. Some of these genes include KRAS, HRAS, JAK2, GSK3β, β-catenin, SmoM2, capa3, Caspase 8, TGF-β, and p53. Example 9 - Editing the DNA and both strands of a DNA / RNA hybrid.

[0396] Because ADARs have been shown to edit both double-stranded RNA and DNA-RNA hybrids, it is possible to recruit ADARs via single-stranded DNA or DNA-RNA hybrids to edit both DNA and RNA. This could be used to modify the current adenine base editing approach using Cas9 (or Cpf1)-ADAR-deaminase domain fusions (ADAR1, ADAR2, and their catalytically active mutants (E1008Q) and E488Q) to target the gRNA-bound strand with an AC bulge, ideally in the first 10 bp near the 5' end of the gRNA, instead of targeting the ssDNA-displaced strand as current base editors do. Example 10 - Ornithine Transcarbamylase Deficiency

[0397] spf ash In mice, ADAR2(E488Q) was delivered with adRNA. In addition to correctly spliced mRNA, spf ashMice harbored an incorrectly spliced, extended mRNA variant, formed by the use of a cryptogenic splice site, 48 base pairs into exon 4. Delivery of ADAR2(E488Q) with adRNA confirmed a reduction in the incorrectly spliced product. Highly efficient RNA editing yields of up to 33.9% were also observed in spliced mRNA. A maximum pre-mRNA yield of 8% was observed. Protein repair of 2.5-5% was also observed within 3 weeks of injection. This demonstrates the utility of RNA editing in correcting splice site mutations and altering splicing patterns (Figure 50A-D). Example 11 - Duchenne Muscular Dystrophy

[0398] MCP-ADAR1(E008Q) was tested with MS2-adRNA in mdx mice. Enhanced RNA editing yields and restoration of dystrophin expression via the ADAR2 and ADAR2(E488Q) enzymes were observed (Figures 51A-B). equivalent

[0399] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0400] The technology illustratively described herein may be suitably practiced with or without any elements or limitations not specifically disclosed herein. Thus, for example, terms such as "consisting of," "including," and "containing" are intended to be read broadly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation. Furthermore, the use of such terms and expressions is not intended to exclude equivalents of the functions shown and described or portions thereof. However, it is recognized that various modifications are possible within the scope of the claimed technology.

[0401] Accordingly, it should be understood that the materials, methods, and examples provided herein represent preferred embodiments and are exemplary only and are not intended to limit the scope of the technology.

[0402] The present technology is described broadly and generically herein. Each of the narrower species and subgeneric groupings included in the generic disclosure also form part of the present technology. This includes any generic description of the technology with a proviso or negative limitation that removes the subject matter from the genus, regardless of whether the excised material is specifically described herein.

[0403] Additionally, when features or aspects of the technology are described with respect to the Markush group, those skilled in the art will recognize that the technology is also described with respect to any individual member or subgroup of members of the Markush group.

[0404] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. Specific Embodiments

[0405] Numerous compositions, methods, and systems are disclosed herein. Examples of specific embodiments of these compositions, methods, and systems are disclosed below.

[0406] Part 1

[0407] Embodiment 1. An engineered ADAR1 or ADAR2 guide RNA ("adRNA") comprising a sequence complementary to a target RNA.

[0408] Embodiment 2. The engineered adRNA of embodiment 1, further comprising an ADAR2-recruiting domain derived from GluR2 mRNA.

[0409] Embodiment 3. The engineered adRNA of embodiment 1, further comprising two MS2 hairpins flanking the sequence complementary to the target RNA.

[0410] Embodiment 4. The engineered adRNA of any one of embodiments 1-3, wherein the sequence complementary to the target RNA comprises about 20-100 base pairs.

[0411] Embodiment 5. The engineered adRNA of embodiment 2 or 4, wherein the ADAR2-recruiting domain from GluR2 mRNA is positioned at the 5' or 3' end of the engineered adRNA.

[0412] Embodiment 6. The engineered adRNA of embodiment 5, comprising GluR2 mRNA at both the 5' and 3' ends of the engineered adRNA.

[0413] Embodiment 7 The engineered adRNA of embodiment 5 or 6, further comprising an editing induction element.

[0414] Embodiment 8 The engineered adRNA of any preceding embodiment, wherein the target RNA is ornithine transcarbamylase.

[0415] Embodiment 9. An engineered ADAR2 guide RNA ("adRNA") encoded by a polynucleotide sequence selected from the group of sequences shown in Table 1 or Figure 2, or an equivalent thereof.

[0416] Embodiment 10. An isolated polynucleotide encoding the engineered adRNA of any one of embodiments 1-9, or any equivalent thereof.

[0417] Embodiment 11. A vector comprising a polynucleotide sequence encoding one or more of the isolated polynucleotides of embodiment 10 or the engineered adRNA of embodiment 9, and optionally a regulatory sequence operably linked to the isolated polynucleotide.

[0418] Embodiment 12. The vector of embodiment 11, wherein the regulatory sequence comprises a promoter, a facilitating element, and / or a reporter.

[0419] Embodiment 13. The vector of embodiment 12, wherein the promoter is a human U6, mouse U6 promoter, or a CMV promoter.

[0420] Embodiment 14. The vector of any one of embodiments 11 to 13, further comprising a detectable or purification marker.

[0421] Embodiment 15. The vector of embodiment 14, wherein the vector is a plasmid or a viral vector.

[0422] Embodiment 16. The vector of embodiment 15, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0423] Embodiment 17. A recombinant cell further comprising the vector of any one of embodiments 11-16, wherein the engineered adRNA is recombinantly expressed.

[0424] Embodiment 18. A composition comprising a carrier and one or more of the engineered adRNAs of any one of embodiments 1 to 9, the isolated polynucleotide of embodiment 10, the vector of any one of embodiments 11 to 16, or the recombinant cell of embodiment 17.

[0425] Embodiment 19. The composition of embodiment 18, further comprising a chemotherapeutic agent or agents.

[0426] Embodiment 20. The composition of embodiment 18 or 19, wherein the carrier is a pharmaceutically acceptable carrier or solid support.

[0427] Embodiment 21. A method of modifying protein expression, comprising contacting a polynucleotide encoding the protein with the engineered adRNA of any one of embodiments 1 to 9.

[0428] Embodiment 22. The method of embodiment 21, wherein the contacting is in vitro or in vivo.

[0429] Embodiment 23. A method of treating a disease or disorder associated with aberrant protein expression, comprising administering to a subject in need of such treatment an effective amount of one or more engineered adRNAs of any of Embodiments 1-9.

[0430] Embodiment 24. The method of embodiment 23, wherein the disease or disorder is Duchenne muscular dystrophy.

[0431] Embodiment 25 The method of embodiment 23 or 24, wherein the subject is an animal.

[0432] Embodiment 26 The method of embodiment 23 or 24, wherein the animal is a mammal.

[0433] Embodiment 27. Use of an effective amount of one or more engineered adRNAs of any of embodiments 1 to 9 for treating a disease or disorder associated with aberrant protein expression.

[0434] Embodiment 28. The use of embodiment 27, wherein the disease or disorder is Duchenne muscular dystrophy.

[0435] Embodiment 29. A kit comprising the engineered adRNA of any one of embodiments 1 to 9, the isolated polynucleotide of embodiment 10, the vector of any one of embodiments 11 to 16, the recombinant cell of embodiment 17, or the composition of any one of embodiments 18 to 20, and instructions for use.

[0436] Embodiment 30. The kit of embodiment 19, wherein the instructions describe the method of any one of embodiments 21 to 26.

[0437] Embodiment 31. A complex comprising the adRNA of any one of embodiments 1 to 9, which hybridizes to a complementary polynucleotide under high stringency conditions.

[0438] Part 2

[0439] Embodiment 1. An engineered ADAR2 guide RNA ("adRNA") comprising a sequence complementary to a target RNA and a domain that recruits ADAR2 from GluR2 mRNA.

[0440] Embodiment 2. The engineered adRNA of embodiment 1, wherein the sequence complementary to the target RNA comprises about 20-100 base pairs.

[0441] Embodiment 3 The engineered adRNA of Embodiment 1 or Embodiment 2, wherein the ADAR2-recruiting domain from GluR2 mRNA is located at the 5' or 3' end of the engineered adRNA.

[0442] Embodiment 4. The engineered adRNA of embodiment 3, comprising GluR2 mRNA at both the 5' and 3' ends of the engineered adRNA.

[0443] Embodiment 5. The engineered adRNA of any of the above embodiments, wherein the target RNA is ornithine transcarbamylase.

[0444] Embodiment 6. An engineered ADAR2 guide RNA ("adRNA") encoded by a sequence selected from the group of sequences shown in Table 1 or FIG. 2.

[0445] Embodiment 7. A method of modifying protein expression, comprising contacting a polynucleotide encoding the protein with the engineered adRNA of any one of embodiments 1-6.

[0446] Embodiment 8 The method of embodiment 7, wherein said contacting is in vitro or in vivo.

[0447] Embodiment 9. A method of treating a disease or disorder associated with aberrant protein expression, comprising administering to a subject in need of such treatment an effective amount of the engineered adRNA of any one of Embodiments 1-6.

[0448] Embodiment 10. The method of embodiment 9, wherein the subject is an animal.

[0449] Embodiment 11 The method of embodiment 9, wherein the animal is a mammal.

[0450] Embodiment 12. Use of an effective amount of the engineered adRNA of any one of embodiments 1 to 6 for treating a disease or disorder associated with aberrant protein expression.

[0451] Embodiment 13. A kit comprising the engineered adRNA of any one of embodiments 1 to 6 and instructions for use.

[0452] Embodiment 14. The kit of embodiment 13, wherein the instructions describe the method of embodiment 7 or embodiment 9.

[0453] Embodiment 15. A composition comprising the adRNA of any one of embodiments 1 to 6 and a carrier.

[0454] Embodiment 16. The composition of embodiment 15, wherein the carrier is a pharmaceutically acceptable carrier.

[0455] Embodiment 17. A complex comprising the adRNA of any one of embodiments 1 to 6, which hybridizes to a complementary polynucleotide under high stringency conditions.

[0456] Part 3

[0457] Embodiment 1. An ADAR system for exon skipping comprising a splice acceptor that targets an adRNA and / or a branch point in an intron, and optionally an ADAR enzyme.

[0458] Embodiment 2. The ADAR system of embodiment 1, wherein said ADAR enzyme is ADAR1, ADAR2, or a mutant or variant thereof.

[0459] Embodiment 3. The ADAR system of embodiment 2, wherein the mutant or variant is selected from ADAR1(E1008Q) and ADAR2(E488Q).

[0460] Embodiment 4. The ADAR system of any one of Embodiments 1-3, wherein the intron is contained in a gene selected from dystrophin, SCN9A, or ornithine transcarbamylase.

[0461] Embodiment 5. The ADAR system of any one of embodiments 1 to 4, wherein the adRNA is selected from Sequence Set 1.

[0462] Embodiment 6. A method of treating a disease, disorder, or condition characterized by aberrant gene expression, comprising administering the ADAR system of any one of Embodiments 1-5.

[0463] Embodiment 7. The method of embodiment 6, wherein the disease, disorder, or condition is selected from Duchenne muscular dystrophy or ornithine transcarbamylase deficiency.

[0464] Embodiment 8 The method of embodiment 6 or embodiment 7, wherein the disease, disorder, or condition is associated with pain.

[0465] Embodiment 9. An APOBEC system for cytosine to thymine editing comprising an alipoprotein B mRNA-like structure and, optionally, a pair of gRNAs producing an APOBEC enzyme.

[0466] Embodiment 10. The APOBEC system of embodiment 9, wherein the pair of gRNAs is a pair of sequences set forth in Sequence Set 2.

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[0468] Wettengel, J., Reautschnig, P., Geisler, S., Kahle, PJ & Stafforst, T. Equipping human ADAR2 for RNA repair by recoding PINK1 mutations rescues mitophagy. Nucleic Acids Res. 45, 2797-2808 (2017).

[0469] Fukuda, M. et al., Construction of guide RNA for site-directed RNA mutagenesis utilizing intracellular A→I RNA editing. Sci. Rep. 7, 41478 (2017).

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Claims

1. a vector comprising a nucleic acid encoding an engineered guide RNA comprising a sequence of 50-200 nucleotides in length that hybridizes to the target RNA, which, upon hybridization to the target RNA, forms a double-stranded RNA containing a mismatch, wherein the double-stranded RNA recruits ADAR1 or ADAR2, which edits the target RNA; (a) hybridization to the target RNA is configured to form a complex stabilized by hydrogen bonds between the bases of the target RNA and the nucleic acid; (b) editing of the target RNA results in decreased expression of a polypeptide encoded by the target RNA; or (c) both (a) and (b), and The engineered guide RNA does not contain the GluR2 mRNA from the ADAR recruitment domain, but rather a vector.

2. The vector of claim 1 further comprising a U6 promoter.

3. The vector of claim 2 , wherein the U6 promoter is a human U6 promoter or a mouse U6 promoter.

4. The vector of claim 1 , wherein hybridization to the target RNA is configured to form a complex stabilized by hydrogen bonds between the bases of the target RNA and the nucleic acid.

5. The vector of claim 4, wherein editing of the target RNA results in increased expression of a polypeptide encoded by the target RNA.

6. The vector of claim 1 , wherein editing of the target RNA results in decreased expression of a polypeptide encoded by the target RNA.

7. 1) hybridization to the target RNA is configured to form a complex stabilized by hydrogen bonds between the bases of the target RNA and the nucleic acid; and 2) The vector of claim 1, wherein editing of the target RNA results in decreased expression of a polypeptide encoded by the target RNA.

8. The vector of claim 1 , wherein the vector comprises a viral vector.

9. The vector of claim 8 , wherein the viral vector comprises an adeno-associated viral (AAV) vector.

10. 10. The vector of claim 1, wherein the engineered guide RNA further comprises a second sequence that hybridizes to the target RNA.

11. A pharmaceutical composition comprising the vector of any one of claims 1 to 10 and a pharmaceutically acceptable excipient, diluent, or carrier.

12. 12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is in unit dose form.

13. 12. The pharmaceutical composition of claim 11 for use in treating a disease or condition in a subject in need thereof.

14. 14. The pharmaceutical composition of claim 13, wherein the disease or condition is selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, and any combination thereof.

15. 14. The pharmaceutical composition of claim 13, wherein the disease or condition is Alzheimer's disease.

16. The pharmaceutical composition of claim 13, wherein the subject is a human.

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